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A man is as old as his arteries was a favorite axiom of william osler (18491919), sometimes called the father of modern medicine, and to some extent accurately represents the effect of vascular dysfunction on various aging processes . To date, it has been recognized that arterial dysfunction, such as increased arterial stiffness, is closely associated with the pathogenesis of cardiovascular disease, which in turn increases mortality by increasing the risk of events such as myocardial infarction and stroke [24]. A higher physical activity level as well as regular exercise may be effective at diminishing the risk of coronary heart disease [5, 6] and stroke [7, 8]. From the standpoint of exercise physiology, exercise is categorized as aerobic and resistance exercise . Briefly, aerobic exercise is a physical exercise of relatively low intensity that depends primarily on the aerobic energy - generating process, for example, running and leg cycling . In contrast, resistance exercise is also physical exercise of relatively moderate and higher intensity that uses a resistance to the force of muscular contraction, in other words, strength training . Although aerobic exercise may improve arterial function, it has also been reported that aerobic exercise is insufficient to inhibit the loss in muscular strength that comes with advancing age [12, 13]. Resistance exercise is recommended to prevent sarcopenia, age - induced muscular degeneration which often entails reduced activities of daily living (adls). According to the guideline of american college of sports medicine (acsm), a mechanical load greater than 70% of the one - repetition maximum load (1 rm) can produce morphological and functional muscular adaptations . However, these higher - load exercises are frequently associated with orthopedic complications [15, 16]. In addition, it has been reported that high - intensity resistance training (> 80% of 1 rm) reduces central artery compliance . These findings suggest that such a high - intensity resistance exercise should be prescribed carefully, particularly for aged people and patients with cardiovascular disease . Recently, several studies have demonstrated that low - intensity resistance exercise with blood flow restriction (bfr) [1723] and bfr walking dramatically leads to muscle hypertrophy and strength gain and that it results in adaptations equal to those of high - intensity resistance training . Although the effect of resistance exercise with bfr and bfr walking on vascular function is still unclear, there is a possibility that this exercise modality can be an important therapeutic prescription not only for sarcopenia but also for vascular dysfunction because of the lower exercise intensity compared to high - intensity resistance training . In this review, we would like to focus on the impact of such exercise on vascular function in comparison with the effects of aerobic and resistance training alone and in combination . In human studies, as it is almost impossible to evaluate large arterial function directly, various noninvasive methodologies have been used to evaluate arterial function in humans . In this section, we introduced several methodologies, which have investigated the impact of bfr exercise and training . Generally, arterial compliance can be measured by a combination of ultrasound imaging of any artery, for example, carotid artery, with simultaneous applanation of tonometrically obtained arterial pressure from the contralateral artery, permits noninvasive determination of arterial compliance . This methodology can be applied to any artery, which can measure pulse wave, for example, radial and femoral arteries . In addition to arterial compliance, -stiffness index provides an indicator of arterial compliance adjusted for distending pressure . Arterial compliance decreases with advancing age [2729], and these reductions are associated with isolated systolic hypertension, accompanied with left ventricular hypertrophy . Indeed, several studies demonstrated that decreased arterial compliance and/or increased arterial stiffness have been identified as independent risk factors of cardiovascular disease [27, 3136]. Although ankle and brachial blood pressure indicates similar value in healthy humans, under continued occlusion and/or stenosis in lower limbs induced by arteriosclerosis, ankle blood pressure would decrease compared with brachial blood pressure . Therefore, ankle - brachial blood pressure index (abi) is a typical indicator for screening peripheral arterial disease (pad). Indeed, diagnostic accuracy for stenosis above 50% in leg arteries in pad patients showed excellent values, that is, sensitivity is 90% and specificity is 98%, respectively [3739]. However, abi is simple, inexpensive, and noninvasive methodology, and it is also reported that abi is a good predictive factor for coronary arterial disease, suggesting that this indicator can be useful test for arterial dysfunction . Arterial stiffness is defined by a decrease in aortic distensibility . In human clinical studies, the measurement of the pulse wave velocity (pwv) has been broadly used and generally accepted as the gold standard to evaluate aortic distensibility . Pwv is calculated by dividing the distance between any two different arteries, for example, carotid and femoral arteries, by the traveled time in the pulse wave from one site to the other site in arteries [42, 43]. Pwv is inversely related to caliber of a blood vessel and blood viscosity and is proportional to vessel wall thickness and distensibility, indicating that higher speed of pwv reflects lower aortic distensibility . Clinically, the pwv is closely associated with pathogenesis in cardiovascular disease [3236, 4446]. As endothelial dysfunction precedes arteriosclerosis, assessment of endothelium - mediated vasodilator function has also been widely used to evaluate endothelial function [47, 48]. In general, flow - mediated dilation (fmd) can be described by any vasodilatation of an artery following an increase in luminal blood flow and internal - wall shear stress induced by reactive hyperemia . The principles, assessment, and evaluation are stated in some excellent reviews [49, 50], briefly, after several minutes of arterial cuff occlusion at proximal or distal portion in any artery, for example, brachial and popliteal artery, immediate cuff deflation can lead to increase shear stress induced by reactive hyperemia and activate endothelial nitric oxide (no) synthase (enos). This activation leads to a shear - stress - mediated augmented no production in endothelial cells . Fmd is calculated as the difference between the maximum diameter and during reactive hyperemia and baseline diameter, and it is expressed as the relative change (%). Since previous studies have shown that brachial artery fmd is emerging as an independent predictor of future cardiac events [5153], fmd can be a good predictor indicating that this assessment can be a good noninvasive marker of local no bioavailability in the endothelial cell, which is an important factor and predictor in protecting against cardiovascular disease . Aerobic capacity determined by maximal oxygen uptake (vo2max) is strongly associated with the risk of cardiovascular disease [54, 55], and an inverse relationship has been observed between vo2max and pwv . Since aerobic capacity is improved by regular aerobic training, to date, numerous studies have demonstrated the influence of regular aerobic training on vascular function in athletes, sedentary subjects, and aged people . Decreased arterial stiffness has been observed in cyclists, middle- or long - distance runners, and triathletes compared with sedentary subjects . It was also reported that collegiate middle- or long - distance runners had lower arterial stiffness [58, 59] and middle - aged and older athletes had more distensibility in their central arteries compared with age - matched sedentary people [11, 60]. Moreover, in healthy young men and postmenopausal women, regular aerobic exercise reduced arterial stiffness . In addition to these cross - sectional studies, intervention studies of regular aerobic training also revealed an improvement in arterial stiffness and endothelial function . Despite differences in the training period from 4 to 16 weeks, regular aerobic training improved arterial stiffness in young healthy subjects [61, 63] and in middle- and older - aged people . It was also reported that 16 weeks of regular aerobic training improved pwv in middle- and older - aged people and pre- and stage 1 hypertensive patients . Moreover higashi et al . Revealed that regular aerobic exercise reduced resting blood pressure and improved endothelium - dependent vasodilatation in essential hypertensive patients . Similar results were observed in healthy subjects, diabetic patients, and those with coronary arterial disease, respectively . Interestingly, daily physical activity level was also associated with abi, intima - media thickness, and pwv [72, 73]. To the best of our knowledge, one study demonstrated that endurance athletes showed a higher pww compared to recreational active control subjects . Although this mechanism is unclear, it is assumed that repeated and excessive training stress due to lower resting heart rate, resulting in increased stroke volume, imposed on the elastic component, may cause mechanical fatigue of the arterial wall . However, this is clearly insufficient evidence to support that aerobic exercise impairs vascular function because the results emerged from extremely trained athletes and cross - sectional study . Taken together, continued aerobic exercise training may improve vascular function assessed by various methodologies even though with one exception . During resistance exercise, for example, weight lifting, both systolic and diastolic blood pressures increase dramatically [75, 76], whereas only systolic blood pressure increases during aerobic exercise, without an accompanying increase in diastolic blood pressure [77, 78]. Thus, in the classical paradigm, resistance training would not be recommended for aged people, and in particular, for patients with cardiovascular disease . However, recent studies have revealed that aerobic training alone cannot maintain muscle volume and strength, which are needed to prevent sarcopenia [12, 13], the degenerative loss of skeletal muscle with advanced age, often leading to a bed - ridden lifestyle with reduced activities of daily living (adls). Resistance exercise is widely recommended to protect against metabolic syndrome, because such exercise can increase muscle strength and volume and may have a positive effect on glucose metabolism, blood lipids, and basal metabolic rate [13, 14]. Its value for older - aged subjects at risk of sarcopenia or vascular disease thus seems potentially significant . However, because of conventional wisdom regarding the dangers of resistance training, studies about the impact of resistance training on vascular function are ten years behind where they might be today . . Revealed that aortic distensibility in resistance - trained men was lower than that in age - matched control subjects . It was also reported that age - related arterial stiffness is more pronounced in strength - trained men than in age - matched sedentary subjects . However, results of more recent studies, which investigated effects of resistance exercise and training on vascular function, seem to be controversial . Greater arterial stiffness was also observed in strength - exercised athletes than in sedentary people [58, 59, 80]. It was also reported that greater age - related reduction in arterial compliance in resistance - trained men was observed compared to sedentary men . In contrast, arterial stiffness assessed by central / peripheral pwv did not differ between highly resistance trained and sedentary men . It was also reported that ischemic reperfusion injury induced by 20-min cuff occlusion significantly reduced brachial artery fmd in sedentary young men but unchanged in resistance - trained adults . Phillips et al . Demonstrated that resistance and endurance trained subjects showed a similar responses in fmd to acute impairment of endothelial dysfunction induced by single weight lifting . Moreover, fahs et al . Demonstrated that a significant inverse association between muscular strength and central pww, independent of aerobic fitness, suggested that resistance training, which can improve muscular strength, might improve aortic stiffness . In addition to these cross - sectional studies, an intervention study found that four months' resistance training reduced arterial compliance in young men . In contrast, it was reported that resistance training did not decrease arterial compliance in young men, in premenopausal women, and in elderly men . In addition, resistance training did not alter resting and postexercise aortic blood pressure and wave reflection in middle - aged women . It has been suggested that continued resistance exercise did not affect endothelial function assessed by fmd in young men and postmenopausal women . However, shorter period of resistance training, that is, four weeks, improved peak forearm blood flow induced by reactive hyperemia but increased arterial stiffness in pre- and stage-1 hypertensives . . Demonstrated that fmd responses were not altered with resistance training, however, brachial artery vessel diameter increases and postocclusion blood flow increases with this training modality . These results may have an important role in clinical application because resistance training can be a stimulator that may enhance resistance vessel function . Similarly, slow movement resistance training increased basal limb blood flow as well as resistance training at normal speed, and weight training increased forearm blood flow as well as aerobic exercise in healthy males . A recent finding also indicated that resting forearm blood flow and peak blood flow in response to reactive hyperemia significantly increased by resistance training in overweight and obese women . From the point view of comparison in different populations, it was reported that decreased augmentation index was observed in old women, but not in old men . Heffernan et al . Indicated that resistance training leads to increase in microvascular endothelial function in african - american and white men, while it increased brachial stiffness in only african - american . Additionally, resistance training improved endothelium - dependent vasodilatation as well as aerobic training and aerobic plus resistance training in patients with recent myocardial infarction, indicating that these improvements were independent of exercise type . Recently, the effects of combined exercise, that is, aerobic and resistance training on vascular function have been elucidated [100, 101]. Combined training consisting of high - intensity resistance training, followed by 30-min aerobic leg cycling, demonstrated a slight increase in carotid arterial compliance (p = 0.06) in young men; furthermore, moderate - intensity combined circuit resistance exercise and endurance exercise improved arterial stiffness in postmenopausal women . However, from the viewpoint of elderly health, it may not be necessary to use higher loads, for example,> 80% 1 rm . Thus, recent studies have investigated the influence of moderate - intensity resistance exercise on vascular function . It was reported that three months' moderate - intensity resistance exercise training did not alter brachial - ankle (ba) pwv, arterial compliance, or arterial stiffness . Moreover, 1-year resistance training intervention in overweight women significantly improved fmd, and low - intensity resistance training with short interset rest period improved bapwv and fmd in young subjects . Similarly, moderate - intensity resistance training for short period, that is, four weeks training, improved fmd responses in end - stage heart failure patients . Although the physiological mechanisms underlying the vascular function, such as arterial stiffness, compliance, and endothelial function assessed by fmd, associated with resistance training are unclear, it is well known that higher - intensity resistance training may be a potent stimulator to increase sympathetic nervous system activity [108, 109]. Augmented sympathetic nerve activity may act to increase arterial stiffness by providing chronic restraint on the arterial wall via greater sympathetic adrenergic vasoconstrictor tone . During resistance exercise, for example, weight lifting, these acute elevated blood pressures during resistance exercise may alter the arterial structure, and/or arterial load - bearing properties, resulting in arterial stiffness increase and/or impaired reactive hyperemic blood flow with repeated exposure . One interesting and supporting finding was that upper body resistance training group increased arterial stiffness but unchanged with lower limb resistance training group and sedentary control subjects . In their study, norepinephrine concentration (ne) significantly increased in only upper body training group after 10-week training . As it is established that ne release is strongly related to the changes in absolute levels of sympathetic nerve activity, elevated ne concentration suggested that resting sympathetic nerve activity increased by 10-week upper body resistance training, resulted in that increased arterial stiffness caused by alteration arterial structure, and/or arterial load - bearing properties . Given these numerous previous studies, it is still unclear and in controversy whether resistance training increases arterial stiffness and/or diminish endothelial function . It might be possible that effect of resistance exercise on vascular function may be affected by various factors, such as exercise intensity, different populations, and exercise modality . According to the guidelines of the acsm, resistance training at 70% or greater of 1-rm is recommended in order to achieve muscle hypertrophy and increased strength . However, it is difficult for certain individuals, such as the elderly and rehabilitating athletes, to use such a high - intensity load . In recent years, studies on one alternative, namely, low - intensity resistance training with bfr and bfr walking, have provided compelling data that such training leads to muscle hypertrophy and strength increases [17, 1922, 24, 113117] and results in adaptations equal to those of high - intensity resistance training [21, 22]. As the universal way of this exercise modality seems not to be established, applied cuff pressure and exercise intensity are varied . The underlying principle of this unorthodox technique is that occlusive cuff pressure is greater above individual's systolic blood pressure and exercise intensity is 2030% of maximal voluntary contraction . During bfr exercise, cuff pressure occludes venous return and causes arterial blood flow to become turbulent, resulting in the enhanced metabolic stress and fast - twitch fiber recruitment in skeletal muscle . At the end of exercise, ischemic reperfusion induced by cuff deflation stimulates shear stress, followed by greater vasodilatation and/or enhanced blood flow . Although the precise mechanism by which bfr exercise produces muscle hypertrophy is still unclear, it was reported that low - intensity exercise with bfr can increase that rate of muscle protein synthesis and stimulate mammalian target of rapamycin complex 1 (mtorc1) and mapk - mediated anabolic signaling . However, a recent study revealed that reactive hyperemic blood flow is not a primary mechanism for bfr exercise - induced mtorc1 signaling and muscle protein synthesis . Another potential factor to induce muscle hypertrophy may be an intramuscular metabolic stress such as depletion of phosphocreatine, an increase in inorganic phosphate, and a decrease in muscle ph . We recently investigated the effects of short - term resistance training with bfr on muscle mass and strength, and found that elevated metabolic stress may be a crucial factor in obtaining successful results from resistance training with bfr . Taken together, it is likely that bfr training can produce muscle hypertrophy without the effect of reactive hyperemic blood flow . However, since hyperemic blood flow per se may be induced by greater shear stress and this greater shear stress induces vasodilatation, which results in increased nitric oxide [49, 50], moreover, maximal dilation was observed after ischemic exercise, there might be a possibility that bfr exercise and training would have a beneficial effect on vascular function such as arterial compliance and endothelial function, indeed, observed . Tables 1 and 2 summarize studies undertaken to investigate the effects of acute or chronic bfr exercise on vascular function such as arterial compliance, endothelial function, and related biomarkers . It has been difficult to obtain consensus due to the large number of variables such as age, gender, intensity and exercise modality, intervention period, applied cuff pressure and cuff width, and evaluation of vascular function, all of which may influence the training effect . We discuss the influence of bfr exercise and training on vascular function based on current findings in the following session . Reported that acute knee extension and flexion with bfr increased arterial compliance without changes in vascular conductance and that this increase was similar to low - intensity knee extension without bfr . They suggested that this acute increase in arterial compliance may be attributed to augmented regional vasoactive substances, and decreased systemic sympathetic vasoconstrictor tone . Additionally, several interventional studies have indicated the influences of bfr training on arterial compliance . Kim et al . Reported that arterial compliance assessed in the radial artery of young men did not change by low - intensity resistance training with bfr for 3 weeks . Similarly, four - weeks bfr training did not alter arterial stiffness evaluated by the pwv between the femoral and tibial arteries in young male subjects . Extended the training period from 3 to 6 weeks and observed no change in arterial compliance assessed by the same device that had been used in the previous study . Interestingly, one recent study revealed that low - intensity (30% 1 rm) upper - body bfr exercise training, that is, bench press did not diminish carotid artery compliance, while high - intensity bench press training without bfr decreased arterial compliance . The underlying physiological mechanism(s) associated with unaltered arterial compliance, that is, four intervention studies, and with increased arterial compliance, that is, one cross - sectional study, is unclear . Impaired arterial compliance induced by high - intensity resistance training without bfr may be attributed to acute elevated blood pressure via increasing sympathetic nerve activity system [108110], resulting in alteration arterial structure and/or arterial load - bearing properties . These changes may be related to impaired arterial compliance . In the previous studies of bfr training, resting blood pressure did not change after several weeks intervention with bfr resistance training [127, 129]. Moreover, the resistance training with and without bfr - induced carotid arterial compliance changes was associated with changes in systolic blood pressure during training intervention, indicating that elevation blood pressure may play a role to induce arterial compliance changes during bfr training . Conversely, postexercise hypotension did not occur after low - intensity resistance training with bfr, while high - intensity resistance training elicited greater postexercise hypotension . Postexercise hypotension can be considered an important strategy to control resting blood pressure, especially in hypertensive patients [132, 133]. Therefore, it may be reasonable that bfr training did not change arterial compliance based on unobserved postexercise hypotension with acute bfr exercise . In addition to these resistance exercise and training with bfr, effect of chronic bfr walking on arterial compliance has been reported . Ozaki et al . Revealed that carotid arterial compliance significantly improved by 10 weeks of bfr walking in elderly people . The interesting considerations regarding their study are that the training period was longer than in previous studies (10 weeks versus 3 to 6 weeks), and the participants were elderly, with the majority being women (3 men and 10 women). This result might have great clinical relevance in understanding the potential application of bfr training . Recently, age- and sex - related differences in cardiovascular responses both at rest and during exercise have been elegantly reviewed . For example, (1) reducing oxidative stress improves carotid arterial compliance in postmenopausal women but not older men, (2) habitual exercise abolished age - related differences in central arterial stiffness in older women, (3) vasoconstrictor responses to sympathetic stimulation is blunted in women compared to men (see in detail). These gender differences in cardiovascular responses and adaptation to exercise training might affect vascular function in response to bfr exercise and training though only one study showed that carotid arterial compliance was improved by bfr training in elderly women . It is well known that regular aerobic training improves carotid arterial compliance, whereas high - intensity resistance training reduces arterial compliance . Although the mechanism of improved arterial compliance in elderly women is still unclear, there are several possible physiological mechanisms to explain these changes . A previous study demonstrated that bfr training - induced improvement in carotid arterial compliance was approximately 30% . In the previous cross - sectional study, carotid arterial compliance in middle - aged and older men is 2035% higher than that in age - matched recreational and sedentary subjects . They also conducted interventional study and demonstrated that 13.5 weeks of aerobic training produced a 25% increase in carotid arterial compliance . Similarly, carotid artery compliance in postmenopausal women increased about 40% following 3 months of home - based regular aerobic exercise . Reported that 6 weeks of bfr walking training did not improve peak aerobic capacity, but it significantly increased muscular size and strength in elderly subjects (2 men and 9 women). Thus, it might be significant that arterial compliance was improved in elderly women, probably without systemic aerobic capacity improvement . However, only study revealed these results, hence, future studies are needed whether these improvements are specific for elderly women . Taken together, bfr exercise and training did not change arterial compliance, while only two studies showed that acute bfr exercise and chronic bfr walking improved arterial compliance [124, 134]. However, due to insufficient evidence, at least what we can say is that bfr training including resistance exercise and walking may not worse arterial compliance, possibly, associated with unchanged resting blood pressure with bfr resistance training [127, 129] and bfr walking . Endothelium - dependent vasodilatation in conduit artery, for example, brachial and popliteal arteries, can be evaluated by fmd, which is a good in protecting against cardiovascular disease [5153]. Bfr exercise can lead to reactive hyperemic blood flow and increased microvascular filtration capacity induced by ischemic reperfusion . Thus, it might be possible that these physiological responses may lead to an enhanced vascular reactivity such as flow - mediated dilation (fmd) responses and/or improved basal limb blood flow . However, the effect of bfr exercise on vascular reactivity is still controversial . To date, three studies examined about acute effects of bfr walking and resistance training with bfr [140, 141] on endothelial function assessed by fmd . Although exercise modality and target conduit artery vary, two of three studies showed impaired fmd with bfr exercise / training and one study indicated unaltered fmd with four - week training with bfr . . Demonstrated that fmd was impaired after bfr walking compared to walking without bfr . In their study, systemic cardiovascular responses, such as blood pressure, and total peripheral resistance significantly increased and stroke volume / pulse pressure (an index of systemic arterial compliance) significantly decreased during walking compared to those in the control condition while abi showed a similar value . In addition to this cross - sectional study, two studies on chronic bfr training showed different results in fmd responses . Credeur et al . Reported that four - weeks hand - grip training with bfr reduced fmd, whereas hunt et al . Demonstrated that four - weeks hand - grip training with bfr did not alter fmd responses but induced transient adaptations to brachial artery structure, although the mechanism underlying impaired fmd responses after acute bfr walking and chronic bfr training is still unclear, it is assumed that reduced fmd is a product of diminished endothelial function . Moreover, they speculated that oxidative stress induced by ischemic reperfusion injury might be the cause of the reduced fmd [139, 140]. As increased oxidative stress is associated with endothelial dysfunction [142, 143], followed by arteriosclerosis, augmented oxidative stress plays an important role in the pathogenesis and development of cardiovascular disease . It has been reported that high - intensity resistance exercise, that is,> 70% 1 rm, elicits oxidative stress markers [145149], whereas these responses to lower - intensity exercise have been inconsistent [150, 151]. During bfr exercise, as far as we know, only two studies have examined the effect of bfr exercise on oxidative stress markers . Measured serum interleukin-6 (il-6) and lipid peroxides (lp) before and after low - intensity resistance exercise (20% 1 rm) with moderate blood flow restriction and found no significant increases . Although exhaustive exercise in humans has been shown to cause a sustained elevation of muscular xanthine oxidase activity as well as increases in serum il-6 and lp concentrations, it did not seem to produce an excess amount of oxygen - derived free radicals in takarada et al . It was also reported that protein carbonyls and blood glutathione, which are indicators of oxidative stress, did not increase during low - intensity (30% 1 rm) bfr exercise, but did increase in moderate - resistance exercise (up to 70% 1 rm). Since both of previous studies did not measure oxidative stress markers [139, 140], it is unknown whether impaired fmd responses are associated with elevated oxidative stress . However, the study of credeur et al . Used higher - intensity resistance load (60% 1 rm) and applied the cuff for a longer occlusion time (20 min). Thus, there is a possibility that increased oxidative stress may have reduced the fmd in the case of credeur's study because it was reported that the region between 60 and 70% 1 rm may be a critical load marker [145151], in terms of inducing oxidative stress during resistance training without bfr . Another explanation associated with impaired fmd responses after bfr walking is that elevated blood pressure during 20-min bfr walking . The blood pressure of subjects during 20-min walking with blood flow restriction in renzi et al was> 20% higher than controls, whereas takano et al . Observed only slightly higher blood pressure during knee extension with bfr than that without bfr (127 versus 113 mmhg). Since acute elevations in arterial blood pressure are associated with the arterial structure and/or the arterial load - bearing properties of collagen and elastin, the rise in blood pressure during bfr walking might have caused the diminished fmd . Collectively, although the influence of bfr walking and training with small muscle group on endothelial dysfunction assessed by fmd is still controversial, these contradictory results may be dependent on the variety of methodologies used, including exercise intensity and occlusion time during exercise . However, it should be still noted that cuff release - induced hyperemic blood flow may be expected, suggesting that it stimulates shear stress, followed by nitric oxide increase, simultaneously, bfr exercise and training should be carefully prescribed because it is supposed that systemic arterial compliance increase during bfr walking . As large muscle group dynamic exercise may alter not only local vasculature but also systemic cardiovascular responses to either acute or chronic exercise, small muscle group exercise may be useful as a physiological model to observe impact of exercise training on localized vasomotor control in skeletal muscles without marked changes in central hemodynamics during exercise . Thus, to date, several studies have used handgrip exercise training to elucidate vascular function . Accordingly, two studies of handgrip exercise training with bfr have been investigated, furthermore, studies of calf raise with own body weight and planter flexion exercise training with bfr were also carried out . Generally, handgrip exercise training increases forearm blood flow and/or improves fmd with one exceptional unchanged study . Enhanced brachial fmd responses as well as endothelium - dependent vasodilatation were observed after isometric handgrip training in old men, in elderly hypertensives [156, 157], and chronic heart failure subjects [158, 159]. Similarly, forearm blood flow was increased with regular handgrip exercise training in young men [160, 161] and in middle aged . In contrast, improved blood flow was not observed in patients with chronic heart failure . Moreover, mcgowan et al . Reported that isometric handgrip training did not alter fmd with normal blood pressure . One interesting finding was that four - week handgrip exercise training improved peak vasodilator capacity induced by 10-minute ischemic stimulus without influencing endothelium vasodilator system . These results suggested that evaluation for vascular function should be considered from many aspects, such as endothelium - dependent dilation, basal blood flow, and peak vasodilator capacity . In addition to these previous studies of handgrip exercise training without bfr, two studies of handgrip exercise training with bfr have been elucidated, resulted in impaired and unchanged fmd responses . As above stated, exercise intensity, which was used in one previous study, was higher and longer, that is, 60% mvc dynamic handgrip exercise training for 20 min, compared to another previous study with bfr (tables 1 and 2). It is possible that these higher exercise intensity and longer duration may cause oxidative stress, resulted in diminished fmd responses . Interesting findings were that handgrip exercise training with bfr did not improve fmd responses but caused brachial artery structural modifications, that is, increased resting and maximal diameters . Based on the principle of fmd calculation, changes in baseline diameter can influence the magnitude of fmd responses . Moreover, it should be taken a consideration about time course alterations in vessel structure and function . Animal studies showed that prolonged training induces structural changes, namely, arterial remodeling [165, 166], while short - term training improves vascular function via enhanced no bioavailability [167170]. Indeed, in human studies, brachial artery fmd increases after just one week of handgrip exercise training and decreased after two weeks, whereas conduit artery vasodilator capacity showed a progressive increase for eight weeks during training intervention . In one of the bfr study, fmd did not change after four weeks training, however, peak blood flow after ischemia and baseline diameter significantly increased . It may be speculated that four - week bfr handgrip exercise training might cause improvement in functional changes within a few weeks, followed by structural changes after four weeks, resulting in without changes fmd responses normalized by increased basal vessel diameter . It is well known that coagulation factors and fibrinolysis are closely associated with the risk of cardiovascular diseases such as ischemic heart disease, particularly acute coronary syndrome . Several previous studies have demonstrated the influence of bfr training on coagulation factors and fibrinolysis . However, to date, no study has observed a significant increase in either acute [120, 174, 175] or chronic bfr exercise . It was reported that thrombin generation is associated with exercise - induced metabolites [176, 177]. Indeed, bfr exercise produced greater metabolites; hence, there is a possibility that increased metabolites might produce coagulation factors and fibrinolysis . In contrast, hilberg et al . [178, 179] reported that thrombin antithrombin iii complex (tat) and d - dimer increased after 6090 min prolonged exercise but did not increase after high - intensity exercise, from which blood lactate concentrations were significantly increased, suggesting that the tat and d - dimer may be dependent on exercise time . Thus, it is unlikely that accumulated metabolites during bfr would be related to coagulation factors and fibrinolysis . Another possible explanation is that expanded blood flow after cuff release would exclude thrombosis, so that the possibility of thrombosis during bfr could be ruled out . More important findings were that the incidence of serious side effects related to thrombosis was lower in 7 cases (0.055%) based on the national survey for more than 12,000 people in japan . It has been pointed out that endothelial dysfunction may be improved through angiogenesis induced by vascular endothelial growth factors . One study revealed that acute bfr exercise significantly increased vascular endothelial growth factor (vegf) compared to control exercise . In fact, vegf secretion and production are activated under hypoxia [182, 183] and/or under decreases in local muscle oxygen tension during exercise . Increased vegf may produce angiogenesis, followed by enhanced no bioavailability, which might be a beneficial effect for endothelial function . However, since no studies have examined the effects of chronic bfr training on vegf, it is uncertain whether chronic bfr training can increase vefg, resulting in an improvement of endothelial function . In this review, we focused on what is currently known (tables 1 and 2) and hypothesised changes (figure 1) of the influences of bfr exercise and training on vascular function . It is well established that aerobic exercise can improve vascular function but with insufficient muscle hypertrophy, while it is possible that high - intensity resistance training can produce muscle hypertrophy but with impaired vascular function . Higher load of resistance training should be considered carefully to apply aged people, disease patients, and rehabilitaining athletes . Bfr exercise and training might be a novel therapeutic modality because it combines lower exercise intensity than higher - intensity resistance training from the point view of during exercise with enhanced reactive hyperemic blood flow after cuff release . Although accumulating evidence has revealed that bfr training leads to muscle hypertrophy and strength increase as well as high - intensity resistance training, little attention has been given to the impact of bfr training on vascular function . At least the majority of the previous studies have demonstrated that bfr training did not impair arterial compliance . Conversely, the effect of bfr training and bfr walking on endothelial function assessed by fmd is not consistent . Available evidence suggests that acute bfr training did not increase oxidative stress markers or coagulation factors . Also, the effect of bfr training on vascular function may be influenced by various factors, such as, age, sex, exercise type, intensity, applied cuff pressure, and intervention period, not to mention the different evaluative methods for vascular function . However, the majority of bfr exercise and training use lower intensity, that is, 2040% 1 rm, compared to high - intensity resistance training, for example,> 80% 1 rm . Therefore, bfr exercise and training seems to become novel method because bfr exercise and training can obtain muscle hypertrophy as well as high - intensity resistance training even if bfr exercise and training does not affect vascular function . Future studies should be conducted with the aim of elucidating the mechanisms of the influence of bfr training on vascular function, with careful selection of the influencing parameters mentioned above and the potential therapeutic benefits for the elderly and for the rehabilitation of patients with cardiovascular disease and physical injury.
Blunt cerebrovascular injury (bcvi) is caused by neck trauma that predominantly occurs in high - impact injuries including motor vehicle - related accidents, car - pedestrian collisions, falls, and skiing or snowmobile accidents . However, a lucid interval occurs in many cases, and this may lead to a delayed diagnosis . We experienced a case of cervical spinal fracture caused by a direct blunt external low - energy force . Initially, there was no disturbance of consciousness, but 6 hours later loss of consciousness occurred due to traumatic dissection of the carotid artery that resulted in cerebral infarction . This case indicates that bcvi should be considered in a patient with blunt neck trauma, particularly when this is accompanied by a cervical spinal fracture . A 52-year - old man who was working at a factory put his head into a big drum - type washing machine . The door closed suddenly and crushed his neck by sandwiching him between the machine and the door . Upon transfer to our hospital, he was conscious with a glasgow coma scale (gcs) of e4v5m6, and a subcutaneous hematoma was present on the right side of the neck (fig . The patient complained of neck pain and numbness in both hands, but there was no apparent limb paralysis . A spinous process fracture at the c6 and c7 levels was detected in a cervical ct (fig . 2a, b), and there was swelling of the right neck in the ct (fig . After fixation of a cervical collar, the patient was admitted to the intensive care unit . Subcutaneous hematoma (arrow) in the neck cervical ct showed a spinous process fracture (arrow) at the c6 (a) and c7 (b) levels . Swelling was present around the right neck in cervical ct (circles) cervical spondylotic changes observed in cervical spine mri t1 (a) and t2 (b). Six hours after injury, paralysis suddenly developed in the left upper and lower extremities, and a decline in consciousness appeared with a gcs of e1v2m5 . Emergency head mri was performed after consultation with a neurosurgeon, and acute infarction of the right middle cerebral artery perfusion area was found (fig . Mri angiography showed blockage of the right common carotid artery and internal carotid artery, and hypoperfusion of the middle cerebral artery (fig . This led to a diagnosis of right cerebral infarction due to acute vascular occlusion associated with right carotid artery dissection in the neck trauma . Edaravone and glycerol were administered to improve brain edema, and mechanical ventilation after tracheal intubation was performed on the same day . On the next day, extensive brain edema was observed in a head ct, and an emergency craniectomy and internal decompression were performed by a neurosurgeon (fig . 7a, b). However, improvement in the brain edema was insufficient, and the patient died on day 7 . The patient s wife and son gave informed consent for submission of this case study for publication . Acute infarction of the right middle cerebral artery based on perfusion of this area in head diffusion - weighted mri at six hours after injury . (a) disruption of the common carotid artery at six hours after injury (arrow). (b) due to occlusion of the right internal carotid artery (triangles) and poor collateral circulation through the posterior communicating (pcom) artery and anterior communicating (acom) artery, the right middle cerebral artery was disrupted (arrow) at six hours after injury . (a) head ct on the day after injury showed a low - density area in the mca region and compression of the anterior horn of the lateral ventricle due to swelling, and the midline shift was more than 10 mm . (b) two days after injury, swelling was worse, and the low - density area had expanded on head ct after craniectomy and internal decompression . Bcvi may occur due to damage to the vertebral and carotid arteries, mainly by blunt neck trauma . The frequency is between 0.50% and 1.55% of all cases of blunt trauma . Among patients at high risk based on criteria, including an extensive skull base fracture, cervical fracture, cervical soft tissue hematoma, and facial fracture, the incidence of bcvi is much higher, in the range of 2444% . In 185 cases diagnosed with bcvi, bruns et al . Found that a motor vehicle collision (52%) was the primary cause of injury, followed by a fall (20%) and collisions involving a motor cycle (11%), a pedestrian (8%), and a cyclist (3%). Also suggested that bcvi could occur after a minor impact, such as cervical traction and heading a soccer ball, but there are few reports of this condition caused by blunt external force, as described in the present case . In a study of 17,007 admissions for blunt injury, cothren et al . Most fracture patterns were subluxations (56 patients), upper c1 to c3 body fractures (42 patients), and transverse foramen fractures (19 patients), whereas spinous process fractures of the lower cervical spine were rare (2 patients). The denver screening criteria provide a screening protocol for bcvi diagnosis based on injury mechanisms, injury patterns, and symptoms (table 1). This protocol can identify trauma patients at risk for bcvi with a sensitivity of 97% and a specificity of 42% . In our case, " expanding cervical hematoma " and " cervical spinal fracture " were consistent with the denver screening criteria, but bcvi was not initially suspected and the patient was not diagnosed with bcvi until after loss of consciousness . Ultrasonography, ct angiography, and magnetic resonance angiography (mra) are effective noninvasive diagnostic modalities for screening and evaluation of soft tissue in the initial treatment of blunt trauma . Ultrasonography is particularly useful for diagnosis of macrovascular damage and airway narrowing, and is desirable as a secondary examination . Ultrasonography, mri, and mra are all relatively easy to use as screening tools . The pathogenesis of cerebral infarction after vascular injury involves acute occlusion of the major artery by dissection, and embolization by the thrombus around the dissection . In our patient, since there were no infarctions in the pca and aca areas, embolization would have occurred at the base of the mca with the common carotid artery dissection . The main symptom of traumatic carotid artery occlusion is loss of consciousness that occurs after a lucid interval . Neurological deficits, such as aphasia and hemiplegia, and transient ischemic attack may also occur . Found that a lucid interval occurred in 74% of the cases and that the incidence within 24 hours after injury was 77.4% . In our case, initially, consciousness was normal and we did not perform a head mri or mra . In previous reports, onset of cerebral infarction occurred within 24 hours of injury in 80% of the cases . The prognosis is very poor, with a mortality of traumatic carotid artery occlusion of 38.5%, and serious sequelae occur in 50% of the cases . In this case, we experienced a patient with a cerebral infarction caused by a combination of a lower cervical spinal fracture and traumatic injury to the carotid artery . Since there was no loss of consciousness in the acute phase, we missed the bcvi diagnosis that led to the cerebral infarction in the middle cerebral artery lesion resulting in loss of consciousness after 6 hours . An emergency craniectomy and internal decompression were performed by a neurosurgeon, but unfortunately this did not provide a sufficient decompression effect and was unsuccessful . (1) in spite of the cervical hematoma, we did not perform a doppler us or an early ct angiography as a screening test . (2) although the initial level of consciousness corresponded to a lucid interval, we should have paid closer attention to the possibility of a loss of consciousness . (3) although there was a major blunt trauma to the neck, a hematoma, and a cervical fracture, we did not suspect bvci initially . We should have considered the injury mechanism, injury pattern, and symptoms of cvi in the denver screening criteria as described above (table1). Bcvi leads to a high incidence of cerebral infarction, with 41% and 16% observed respectively in injuries to the carotid and vertebral arteries . This case demonstrates that cervical vascular injury should be considered in a patient with blunt neck trauma and that additional imaging (doppler us, ct angiography, mra if possibly) should be performed . In addition, if bcvi were identified early, then antithrombotic treatment heparin (ptt 4050 sec) or antiplatelet therapy would be desirable . If the injury is surgically accessible depending on the degree of damage, bypass surgery and endovascular treatment with a stent should be performed . We predict that if immediate action and aggressive measures were taken, it might have prevented a fatal outcome . None of the authors have a conflict of interest regarding the contents of the manuscript.
Twenty - four percent of canadians are obese and a further 37% are overweight using measured height and weight data from the 20072009 canadian health measures survey . Further, over half (58%) of the canadian women aged 40 to 59 years are considered overweight or obese . Poor eating habits and physical inactivity contribute to the increasing prevalence of overweight and obese individuals . The majority of middle - aged women do not regularly participate in exercise and/or fail to report healthy nutrition practices such as consuming five or more vegetables and fruits per day [4, 5]. These trends mark the need to identify lifestyle interventions that address these healthy behaviours and their contribution to addressing midlife weight gain in women, to minimize the risks for obesity and related cardiometabolic complications . In their midlife, women undergo the biologic transition into menopause . This menopause transition is divided into several stages and clinically has been categorized by international criteria developed to help assess women's reproductive stage . The early menopause transition stage is specifically defined by menstrual cycles varying by seven or more days from regular cycles; the next stage is defined by intervals of skipped cycles or amenorrhea of at least 60 days . The third stage begins following the first year without menstruation and is defined as the end of perimenopause; which is then followed by a stage of early postmenopause and lasting up to six years . During the transition, women can experience vasomotor symptoms such as hot flashes and/or night sweats which may continue for five years or more years after the last menstrual period . These symptoms typically occur in women ages 40 to 65 years and are more prevalent in obese women [9, 10]. Other changes that occur during the menopause transition are changes in body composition and increase in abdominal fat mass as well as associated alterations in cardiometabolic risks due to hormone - related decreases in energy expenditure and fat oxidation [7, 11]. Health conditions (or problems) associated with central obesity in women include coronary heart disease, hypertension, type 2 diabetes, cancer, osteoarthritis, and gall bladder disease . Lifestyle interventions to minimize gains in fat mass and changes in body composition and body fat distribution predominantly include exercise and healthy nutrition [13, 14]. Current guidelines recommend (a) assessing factors contributing to overweight (body mass index (bmi) 2529.9 kgm) and obesity (bmi 30 kgm) in adults and (b) intervening with counseling and treatment of obesity . Specific interventions include encouraging individuals to set realistic lifestyle goals, referral to weight loss programs, pharmacotherapy, or surgical interventions . Despite these guidelines, it is unclear what effect lifestyle interventions, such as exercise and/or healthy nutrition, have on weight gain, body composition, and/or body fat distribution in women, if performed specifically during the menopause transition stage . While systematic reviews of the literature evaluating interventions to prevent weight gain exist for more broadly defined populations, there are limited studies evaluating interventions for women during this critical midlife menopausal transition stage . Furthermore, the sequel of the menopause transition stage are often including increases in fat mass and abdominal adiposity and the associated risks of developing cardiometabolic complications are greater compared to the other periods in a women's life, when there are also significant hormone changes (e.g., menarche and pregnancy). Ultimately, this review was conducted to provide a synthesis of current evidence and identify gaps in the knowledge around effective exercise and nutrition interventions that address body weight and body composition changes in women specifically during the menopause transition stage . To date, only one known literature review on this topic exists and it was not systematic . Further, it focused on the effects of hormonal changes on body weight and body composition during the transition to menopause . The objective of this study was to determine the effectiveness of exercise and/or nutrition interventions on changes in body weight, body composition, and body fat distribution in women specifically during the menopause transition stage . A systematic review was conducted using a protocol developed a priori according to the cochrane handbook for systematic reviews of interventions and the preferred reporting items for systematic reviews and meta - analyses (prisma) statement . Systematic reviews apply explicit methods to conduct an extensive search, select appropriate research studies, critically appraise the studies, and synthesize studies to answer specific questions [22, 23]. The search strategy was developed collaboratively with an academic reference librarian (lau) and researchers investigating the effect of menopause transition on body weight regulation (table 1). Population, intervention, comparator, and outcome (pico) were used for developing the search strategy and focused on key words related to menopause transition, physical activity / exercise, and nutrition interventions (see table 2). The search strategy was designed for medline and adapted for embase, cinahl, amed, psycinfo, global health, and sportdiscus . Grey literature and hand searching were conducted by using included articles to look for related articles in pubmed and checking reference lists of included articles . During the screening process for inclusion / exclusion of studies, if the published information was unclear, authors were contacted by email with requests for additional details on their study . Included studies evaluated exercise, or nutrition interventions targeting specifically women in the menopause transition stage and evaluated impacts on body weight and/or central and abdominal weight gain . The menopause transition is divided into several stages and clinically has been categorized by international criteria developed to help assess women's reproductive stage . All intervention designs were included: randomized controlled trials (rct's), crossover designs, case controlled studies, and pre-/post tests . Screening and selection of each of the included studies was conducted independently by two reviewers (jj, sp; jj, sb) using standardized forms and included three phases: (i) title screen; (ii) abstract screen; and (iii) full article screen, with screening decisions recorded . For the level (i) title screen, the citation titles identified by the search strategy were reviewed to identify any studies about women and body weight and/or abdominal obesity during the menopause transition stage . Titles identified as include or unsure by any reviewer were retained for second level screening . For level (ii) screening, abstracts were evaluated using the pico inclusion / exclusion criteria (table 2). Level (iii) screening consensus on differences was reached in a discussion with a third reviewer (ds). Data extraction was conducted independently by two reviewers (jj, sb) and using standardized forms . Collected data included characteristics of the intervention, characteristics of the study design, characteristics of the participants, and findings . The menopausal status of study participants was described based on the international straw+10 staging system's stages of reproductive aging in women . The cochrane collaboration risk of bias tool was used to appraise study quality for the rct's [21, 25] and the critical appraisal skills programme (casp) checklist was used for other study designs . Differences in extracted data or appraisal ratings between the reviewers were resolved through discussion with consensus reached and reasons for decisions documented . Given the heterogeneity of included study designs and few studies included, a descriptive analysis was conducted . Of the 3,564 unique citations identified, only 3 studies were eligible for inclusion (figure 1). Of the 137 citations in full text review, 134 were excluded: 111 included women whose menopausal status was not explicitly defined as being specifically in the stage of menopause transition (e.g., menopausal status undefined, mixed pre-/peri-/postmenopausal), 7 were not experimental designs (e.g., editorials or summaries discussing evidence on the role of exercise, diet, and diet and exercise), and 16 examined other outcomes (e.g., bone health and quality of life) (in the appendix). Included were two rcts [18, 19] and one pre-/post study published between 2003 and 2011 (table 3). The number of participants ranged from 24 to 535 . The loss of participants due to study withdrawal or lost to follow - up was described in all three included studies [1820]. Participants from the five - year rct ranged in age from 44 to 50 years at the start of enrollment for the five - year study . The other two studies included women in varying menopausal stages; one reported baseline information for perimenopausal and postmenopausal women with ages ranging from 40 to 59 years of age, although the intervention and results were reported as separate perimenopausal and postmenopausal groups of women . The other study identified women during the menopause transition (last menstruation 60 and 365 days) separately from premenopausal (last menstruation <60 days) or postmenopausal women (last menstruation> 365 days) and resulted in a wider age range . The other rct provided insufficient detail to judge risk of bias for allocation concealment, blinding, and selective reporting of outcomes . The pre-/post study had lower risk of bias but did not report adequate detail to judge risk of bias related to confounding factors, follow - up, and results . One study evaluated highly supported (i.e., coaching, education) caloric and dietary restrictions combined with exercise over 20 weeks, followed by ongoing and less intensive dietary and exercise interventions for another 48 months . Another study evaluated a 12-week exercise intervention with participants randomly assigned to a circuit training program or control group . The third study evaluated endurance exercise using a nordic walking program over 12 weeks . In the studies that utilized exercise as an intervention, participants were either not instructed in their dietary habits or were asked to refrain from increasing their daily fat intake and to avoid changing their nutrition routines . The other study focused on supporting significant changes to both exercise and dietary lifestyle habits . One study focused predominantly on changes in bmi and waist - to - hip ratio . The other studies measured overall gains and/or losses in body weight reported as bmi with percentage body fat or total kilograms of fat . Abdominal obesity was measured using waist circumference [18, 20] or waist - to - hip ratio only . Compared to controls, participants randomized to an exercise and dietary intervention at 54 months were more likely to be at or below baseline weight (55% versus 26%) and experienced a decreased bmi and greater loss of percentage of body fat for all three follow - up measures, including decreased waist circumference (p <0.001). While both intervention and control groups were found to have decreased waist circumference measurements at follow - up, the greater changes were measured in the intervention group . (0.1 kg, sd = 5.2 kg) in the intervention group and + 5.2 lbs . (2.4 kg, sd = 4.9 kg) in the control group . Participants randomized to circuit training significantly decreased the mean values of their waist - to - hip ratio (p <0.05) between baseline and week 12, whereas those in the control group saw no significant changes over the 12-week follow - up period . Significant changes in bmi were not observed in either group (p> 0.05) and attributed to the minimal changes in body weight for both groups during the 12 weeks of the study . Overall, participants randomized to a 12-week circuit training exercise program only decreased their waist - to - hip ratio . Compared to baseline, participants exposed to moderate endurance exercise over 12 weeks saw reductions in bmi, total body fat, and waist circumference (p 0.05). This systematic review was focused on determining the effectiveness of exercise and/or nutrition interventions on mitigating changes in body weight, body composition, and body fat distribution in women specifically in menopause transition stage . Despite extensive searching of published and unpublished research, most studies were excluded due to poorly defining the menopausal status of study populations, lacking an intervention study, or lacking assessment outcomes related to body weight or body composition . The included studies described various study environments, participant characteristics, and interventions . Of the three included studies, one higher quality trial with minimal risk of bias showed that compared to usual activities in the control group, women exposed to a program of combined exercise and caloric restriction dietary interventions for 54 weeks had improved body weight and reduced abdominal adiposity . As well, significant reductions in waist circumference and body fat were maintained beyond four years . Despite using a less rigorous study design and having a shorter follow - up period, another study concluded that a nordic walking program might reduce weight gain during the menopause transition stage . Similarly, a study implementing a circuit training program found that the waist - to - hip ratio could be decreased with exercise, even while measures of bmi were not significantly influenced . While limited in number, these studies suggest that exercise or both exercise and caloric reduction interventions may be able to disrupt the process and patterns of weight gain and change in body fat distribution during the menopause transition stage . These findings are consistent with guidelines on the prevention and management of obesity that recommend lifestyle intervention as the first approach for preventing or treating obesity . These recommendations are supported by other studies addressing excess weight and obesity in middle - aged women and obese adults [28, 29]. The paucity of studies that have investigated the effects of lifestyle intervention on weight, body fat, and body fat distribution specifically in women in the menopausal transition stage indicates that better designed studies are needed to support an evidence - based approach to managing and preventing women's body weight changes during this specific important life stage . Of the three included studies, only one study suggested the potential impact of periodic clinical measurements as an intervention to attenuate weight gain during the menopause transition stage . Clinical measurements included weight, waist circumference, blood pressure, lipids, glucose, and level of physical activity and nutrient intake by questionnaire . Of note is that 26% of controls were at or below baseline weight at the 54-month visit and the group showed a significant decrease in waist circumference without being exposed to the intensive lifestyle intervention . Therefore, it is possible that the women in the control group may have been influenced by the periodic clinical measurements at 6, 18, 30, 42, and 54 months and this is consistent with another study . A longitudinal observational study attempting to document the effect of menopause transition in healthy nonobese premenopausal women found no significant weight gain after 5-year follow - up . In this study, there was no structured intervention other than that of yearly clinical measures of body weight, waist circumference, body composition, blood pressure, lipids profile, glucose, physical activity, and a food intake journal . Selection of participants using self - reported menopausal status was described for all three studies and details on characteristics of participants were limited [1820]. Participants included in the studies varied in age from 40 to 62 years and reproductive aging was understood as distinct from generalized physical aging . Of concern was the fact that many of the excluded studies examining weight gain in women from pre- to postmenopause did not describe menopausal status and failed to clearly define the reproductive stage of participants . Reporting of the data in ways that obscure potential variability between women of different reproductive ages is an issue and has been previously identified as contributing to the development of less effective interventions for managing menopausal symptoms . Criteria defining the stages of menopause can better situate studies examining effects of reproductive aging and help women and health professionals to effectively address issues, such as identification of critical stages for increased risk of adiposity . For this systematic review, the menopause transition stage was described based on the international straw+10 staging system's stages of reproductive aging in women . Future studies could benefit from standard reporting of menopausal status using these internationally approved criteria . Several factors have been shown to be influential during women's experience of menopause . While the prevalence of excess weight and obesity is high within the general population; population subgroups such as those with lower socioeconomic status the findings of this systematic review indicate that a narrow segment of the population has been studied (i.e., predominantly white, middle - class women). Other key factors in reproductive aging (e.g., race, ethnicity, culture, geography, and socioeconomic status) are not prominent in the study of body weight changes during the menopause transition, despite longitudinal evidence that socioeconomic status is related to bmi, weight gain, and certain weight control practices within nationally representative samples of women . One included study in this review identified this gap in the literature addressing diversity in research and reporting on women and changes in adiposity associated with menopause and cited this as a rationale for their study . Indeed, the attrition rate for participants in the study was attributed to the socioeconomic structures limiting participation of women in their active lifestyles . Hence, the findings of this systematic review highlight the need to ensure that consistent definitions are applied in defining reproductive aging stage, and that the characteristics of subpopulations of women are included as an integral part of research on interventions that target body weight, body composition, and body fat distribution during the menopause transition stage . The potential limitations of this review included a lack of a standardized definition for the population of women in the menopause transition within the literature as described above; poor indexing of studies in databases; and the limited time frame (<26 weeks) over which two of the three studies were conducted . Given the poor indexing of studies in electronic databases, it is possible that some studies were missed; however, there is transparency in the extensive search strategy used . Sustained weight loss, up to and longer than one year, is considered a standard in studies of management of overweight and obesity . However, only one of the three included studies was conducted for longer than one year with the other two studies being only 12-week duration . During study screening, sensitivity was favoured over specificity, resulting in larger numbers of studies screened and ultimately excluded . As well, the studies relied on self - reports by women on their menopausal status . Strengths of this review included the comprehensive search strategy which was developed in collaboration with an academic librarian, use of two independent reviewers at each screening stage, and the iterative and ongoing consultation with an interprofessional team of researchers having expertise in obesity, reproductive aging, and systematic review methodology . Few studies have measured the effect of exercise and/or dietary interventions on women's body weight or body composition specifically during the menopause transition stage . Evidence from one large higher quality rct indicates that women should exercise and eat a caloric restricted diet during the menopause transition stage to prevent weight gain and abdominal fat gain . The review also identified the need for use of common terminology for defining reproductive age of study participants as described by the straw+10 staging system . Common terminology would facilitate synthesizing results across studies . Finally, further research should focus on more rigorous study methods using durations of one year or longer . Ideally, studies should be sensitive to the potential relationships between cultural norms, socioeconomic status, and body weight changes during the menopause transition stage . Subsequent results would be more likely to help women and their health professionals choose effective interventions for addressing changes in body weight and achieving a body composition defined by women and their care professionals as healthy.
Nanotechnology is an emerging tool as drug delivery system in variety of serious disorders . It is an innovative technique which includes the design, characterization, production, and application of structures, devices, and systems by controlling shape and size at the nanometer scale . Nanoparticles can be classified as polymeric (natural and synthetic), lipoidal (biodegradable), and metal nanoparticles (iron oxide, gold nanoparticles, and silver nanoparticles). Metal nanoparticles using plant or plant extract are a way ahead towards greener approach for application as drug delivery system . Silver nanoparticles show good anti bacterial properties as silver has been widely used as healing and antibacterial agent for many years . Silver nanoparticles (agnps) also possess antifungal, anti - inflammatory, antiviral, and antiplatelet activity . Silver nanoparticles can be synthesized by using any of the following methods, namely, physical methods, chemical reduction (thermal reduction, polyol process, microemulsion techniques), and photochemical reduction (microwave reduction, photoreduction, and x - ray radiolysis). Malus domestica (rosaceae) commonly known as apple is full of high levels of triterpenoids, anthocyanins, and phenolic compounds . The utilization of natural compounds in protection of skin especially topical application of antioxidants indicates that they usefully decrease skin aging [9, 10]. Basically, premature skin ageing is a vast biological phenomenon that mainly occur by the combination of endogenous or intrinsic (genetics, cellular metabolism, hormone, and metabolic processes) and exogenous or extrinsic (chronic light exposure, pollution, ionizing radiation, chemicals, and toxins) factors . The responsible free radicals are oxygen free radicals or generally known as reactive oxygen species (ros). Studies show that apple decreases the presence of ros generated by hydrogen peroxide exposure in lymphocytes . In present investigation, we have tried to investigate the synergistic effect of agnps and flavonoids in apple extract by loading apple extract agnps into hydrogel and characterizing it for antibacterial and antioxidant properties in a unit dose formulation . Apple extract was utilized for the green synthesis of silver nanoparticles via chemical reduction technique . . Moreover, optimized silver nanoparticles were selected and loaded to hydrogel to deliver actives via topical route for the therapy of premature skin ageing . Malus domestica, family rosaceae (apple), was purchased from local market . Methanol, formic acid, silver nitrate, carbopol-934, triethanolamine, glycerine, potassium dihydrogen phosphate, sodium hydroxide, 2,2-diphenyl-2-picrylhydrazyl hydrate (dpph), agar, and nutrient medium were purchased from cdh pvt . Phenolic compounds from apple were extracted using solvent mixture of methanol: formic acid: water in the ratio of 70: 2: 28 . Weighed quantity of freshly cut apple pieces was added to the solution of methanol: formic acid: water (mfw) and homogenized in a blender (polytron pt 1600e, kinematica ag) for 2 min . The mixture was transferred to a beaker, covered with parafilm, and held for 24 h at 4c . Then the mixture was washed with 20 ml of mfw . Extract was collected and then evaporated for dryness at 45c under vacuum with a rotary evaporator . 1 m silver nitrate solution was prepared by adding 1.699 g of agno3 to 1 l distilled water . 30 ml of apple extract was taken in a 100 ml conical flask . 20 ml of freshly prepared 0.1 m agno3 solution was added (drop - wise) to the flask . Apple extract loaded silver nanoparticles were then incorporated into a hydrogel base as given in table 1 . Accurately weighed quantity of carbopol-934 (poly (acrylic) acid or carbomer) was gradually dispersed in distilled water under mild stirring (remi, mumbai) for 30 min and kept for two hours for the proper swelling of polymer . The glycerin - polymeric solution was then neutralized with triethanolamine to form a transparent gel as basic nature of triethanolamine helps in increasing the cross - linking of acidic polymer . Apple extract loaded silver nanoparticulate suspension (equivalent to 310% of apple extract) was then incorporated in hydrogel using slow mechanical mixing (remi rq121/d, mumbai) for 10 min so as to avoid entry of air bubbles . (1) characterization of apple extract preliminary screening for the presence of flavonoids and polyphenols in extract . The prepared apple extract was subjected to ultravoilet - visible spectrophotometry (perkin elmer, waltham, ma) for detecting the presence of flavonoids . The extract was scanned in the region of 200 nm800 nm against solvent mixture (mfw) as blank and peaks were obtained . Few drops of dilute sodium hydroxide were added to 1 ml of apple extract to observe the yellow color which on further addition of few drops of dilute hydrochloric acid makes the solution colorless which confirms the presence of flavonoids . Confirmatory testing (magnesium ribbon test). Magnesium ribbon test is known because of the use of magnesium ribbon in the testing of flavonoids . Concentrated hydrochloric acid and magnesium ribbon were added to 1 ml of apple extract which in turn gives pink - red color for the presence of flavonoids . (2) characterization of apple extract loaded silver nanoparticles ultravoilet - visible spectrophotometry . The optical property of prepared agnps was analyzed via uv - visible absorption spectrophotometer with a deuterium and tungsten iodine lamp in range of 200800 nm at room temperature (rt). Uv - visible spectrophotometer monitors the formation of agnps with the color change . To analyze formation of agnps, samples were withdrawn with the help of pipette at different time intervals, that is, 5, 10, 15, 20, 30, and 60 minutes . Scanning electron microscopic (sem) analysis was done for analyzing particle shape and morphology . The shape and surface morphology of the silver nanoparticles was visualized by scanning electron microscopy (cart zeiss ev018). The samples were prepared by lightly sprinkling nanoparticles on double - sided adhesive tape on an aluminum stub . The stubs were then coated with gold to a thickness of 200 to 500 under an argon atmosphere using a gold sputter module in a high vacuum evaporator . The samples were then randomly scanned and photomicrographs were taken at different magnifications with sem . For the determination of particle size, samples were prepared by tenfold dilution of 1 ml of the nanoparticulate suspension with distilled water . Zeta potential was determined by the electrophoretic mobility of nanoparticles in u - type tube at 25c, using a zetasizer (3000hs malvern instruments, uk). (3) characterization of apple extract loaded silver nanoparticles based hydrogels ph . Ph of prepared hydrogel was measured by dissolving 1 gm of hydrogel in 10 ml of distilled water . Ph meter (systronics, 361-micro ph meter) was used to evaluate the ph of hydrogel . The readings were taken in triplicate and the average ph was calculated . Porosity measurement . For porosity measurement, accurately weighed 1 gm of dried hydrogels was immersed in absolute ethanol and soaked overnight . Afterwards, excess ethanol on the surface of hydrogels was blotted and weighed again . The porosity can be calculated from the following equation. (1)porosity = m2m1v.here, m1 and m2 are the mass of hydrogel before and after the immersion in absolute ethanol, respectively; is the density of absolute ethanol and v is the volume of the hydrogel . Viscosity . To analyze the rheological properties, all the formulated gels were taken in beakers and placed beneath the spindle, and the spindle (rv-7) was rotated at 10 rpm at room temperature (2527c) in brookfield viscometer . The spreadability measurements of the apple extract loaded silver nanoparticle gel were made in triplicate by using glass slide method . The preweighted plate was kept above the gel, and more weights were added on the plate until the gel stop spreading . Final cumulative weight and the total time taken by the gel to spread were measured and noted . Then total weight applied and mass of the gel were compared by the time. (2)s = mlt, where s = spreadability, m = weight tide to upper slide, l = length of glass slide, and t = time taken to separate the slides completely from each other . The in vitro drug release was evaluated using keshary - chien (k - c) diffusion cell to evaluate the diffusion of nanoparticles across the dialysis membrane . Dialysis membrane 70 (hi - media, mumbai, india) having a pore size of 2.4 nm and a molecular weight cutoff between 12,000 and 14,000 da was used and mounted on k - c cells . Phosphate buffer saline (pbs; ph 6.8) was used as the receptor medium (10 ml) being stirred at 100 rpm . 0.5 gm of apple extract loaded silver nanoparticulate based hydrogel (equivalent to 310% of apple extract) was placed in the donor compartment . During the experiments, the solution in receptor side 5 ml of the samples was withdrawn from the receiver compartment and replaced by the same volume of freshly prepared pbs (ph 6.8). The withdrawn samples were analyzed by the uv - visible spectrophotometer at 420 nm . The ex vivo drug release studies were carried out with the help of k - c diffusion cell . The subcutaneous tissue was removed surgically and the dermis side was wiped with isopropyl alcohol to remove adhering fat . The cleaned skin was washed with distilled water and stored at 18c until further use . The skin was mounted between the donor and receiver compartments of the k - c cell where the stratum corneum side was facing the donor compartment and the dermal side was facing the receiver compartment . One gram (equivalent to 310% apple extract) of each gel was placed in a donor compartment . The receptor compartment was filled with 10 ml pbs (ph 6.8), thermoregulated at 37c, and magnetically stirred at 400 rpm . Two milliliters of receptor fluid was withdrawn at an interval of 1, 2, 3, 5, 7, 12, 18, and 24 h. an equal volume of pbs was simultaneously added to the receptor compartment after each sampling to maintain sink conditions . Each sample was filtered and then determined for apple extract content by uv spectrophotometer at 420 nm . The concentrations of all the formulations in withdrawn samples were calculated and then the percent drug release was determined . The antioxidant activity was characterized utilizing dpph (2,2-diphenyl-2-picrylhydrazyl hydrate) assay . A stock solution of dpph in methanol 1 ml of this stock solution was added to 3 ml of hydrogel solution (1 gm of prepared hydrogel in 10 ml of distilled water). The mixture was shaken vigorously and allowed to stand at room temperature for 30 min . Then the absorbance was measured at 517 nm by using a uv - visible spectrophotometer . Antioxidant activity was estimated by calculating the% inhibition by following formula: (3)dpph scavenging effect%=control absorbancesample absorbancecontrol absorbance100 . Antimicrobial activity was performed using well - diffusion technique employing agar and nutrient broth as media . Media was constituted by mixing agar and nutrient broth and then autoclaved at 121c at 15 lbsi for 60 min for sterilization . Sterilized liquid media were then poured into two different petri plates and inoculated with e. coli and s. aureus, respectively . Sterilized cork borer was used to create well of 1 cm diameter in the solidified media . Samples were poured in the well of prepared petri plates and then incubated at 37 5c for 24 h for inhibition to the growth of e. coli and s. aureus . Extract obtained by the process was found to be pinkish red in color; the extract was further screened for the presence of flavonoids and phenols by preliminary screening and confirmatory testing . Silver nanoparticles prepared from apple extract showed the color change after 120 minutes of preparation giving the evidence for the reduction of silver ions and, eventually, formation of silver nanoparticles . The color change of the solution is clearly depicted in figures 1(a) and 1(b). The color change was recorded by visual observation in beaker which contains silver nitrate solution with apple extract . The color of agnps apple extract solution changes from colorless to light brown after 5 min and eventually to dark brown as shown in figures 1(a) and 1(b). The synthesis of agnps was furthermore confirmed by uv - visible spectroscopy and scanning electron microscopy . The uv - vis spectrum of silver nanoparticles was recorded as a function of a reaction time . Aliquots of samples were withdrawn at different time intervals, that is, 10, 15, 30, 45, 60, and 120 min, to monitor the bioreduction of ag ions . The graph obtained by uv spectrophotometer contains significant bell shaped peak indicating the formation of silver nanoparticles as shown in figure 2 . This spr absorption band may be attributed to combined variation of electrons of agnps in resonance with light wave . It is very much familiar that silver nanoparticles in nanorange show absorption at the wavelength from 390 nm to 420 nm due to mie scattering . Therefore, a broad absorption band was observed in the range of 400 nm440 nm which is the characteristic of mie scattering [27, 28]. No other peak was recorded in spectrum which confirms that synthesized nanoparticles are silver particles only . The average size of the particles was found to be below 200 nm and the particles formed were spherical in shape, as shown in figure 3 . From figure 3, it can be illustrated that morphology of agnps is spherical and particles are well dispersed without any aggregation . The particle size of the silver nanoparticles was found to be in the range of 100 nm indicating best suitability for topical delivery as particles on nanoscale can easily permeate through skin . The zeta potential was found to be very negative on higher scale revealing good stability of agnps in dispersion form . The ph of all formulated hydrogel was found in the range of 5.57.5, which mimics the ph of skin; therefore, formulation shows no sign of irritation on skin . The results of rheological studies, namely, viscosity and spreadability of the formulation, were found to be 42000 cps and 5.56 0.15 gcm / sec, respectively, which clearly signify that the hydrogel is easy to spread on skin . Furthermore, hydrogel formulations were then subject to in vitro drug release to compute the rate and extent of drug release . The results of in vitro drug release of all three formulations were in the range of 74.1% 0.28 to 98.01% 0.37 up to 24 h concluding the maximum release was shown by f2 hydrogel formulation as shown in figure 4 . Primary focus was laid on particle size as this will play significant role in nanoparticle permeation from skin . The smaller the particle size is, the easier the permeation will be . Additionally, to confirm the permeation of nanoparticle across the membrane, rat abdominal skin was selected . Ex vivo permeation studies were only conducted for batch f2 as smaller particle size and maximum in vitro release was shown by this formulation . Cumulative percent permeation was found to be 98.81% 0.24 up to 24 h. the permeation studies were best explained by zero - order kinetics (r = 0.96), followed by first - order kinetics (r = 0.884) and higuchi equation (r = 0.803). Ex vivo permeation kinetics through zero - order, first - order, korsmeyer - peppas model, and higuchi model have been explained in figure 5 . The values of r and k for all release kinetics models are tabulated in table 3 . Zero - order process is a constant rate process that is independent of drug concentration . Permeation parameters like flux, permeability coefficient, and lag time are summarized in table 3 . Dpph radical scavenging assay was investigated for the evaluation of antioxidant potential of the prepared hydrogel formulations . The purple solution containing dpph turns yellow on addition of formulation, which indicates the scavenging of free radicals and presence of antioxidant activity . The results are compiled in table 4 and showed that the ratio of agnps with carbopol (2: 1), that is, f1, demonstrated maximum inhibition with a value of 75.16 0.04 . At the same time, the value differs with other ratios, namely, agnps with carbopol (1: 1) f2 and agnps with carbopol (1: 2) f3 . Thus, it can be concluded that maximum concentration of apple extract revealed maximum percent inhibition . Apple extract loaded silver nanoparticles based hydrogels showed higher antimicrobial properties against the bacterial species of e. coli and s. aureus . The inhibitory action of silver compounds may be attributed to the strong interaction of silver with thiol groups present in key respiratory enzymes in bacteria . Figures 6(a) and 6(b) show the zone of inhibition obtained against e. coli and s. aureus, respectively . A good inhibition was shown by silver nanoparticles based hydrogel contrary to plain hydrogel containing apple extract where no zone of inhibition was seen against e. coli as well as for s. aureus . In the present research, green synthesis of apple extract loaded ag - nps was successfully achieved by treatment of silver nitrate with extract of malus domestica (apple). This technique revealed that the apple extracts can be used as an effective stabilizing reducing and capping agent for the synthesis of agnps . This method of reduction utilized here is very simple, easy to perform, inexpensive, eco - friendly, and superior substitute to chemical synthesis . The finally fashioned agnps based hydrogels were highly stable formulations, showing strong antibacterial and antioxidant activity.
Tooth loss is one of the common causes of reduced quality of life in adults . Dental implants have become a widely accepted treatment option for patients with both partially and complete edentulous posterior mandibles . Rehabilitation of edentulous posterior mandibular regions with severe ridge atrophy using implants is subject to anatomical, surgical and biological difficulties and poses a challenge to dental teams . Osseointegrated dental implants are often placed in the posterior mandible, mostly to support fixed restorative prostheses . In many cases, the bone has so severely atrophied that sufficiently long fixtures cannot be placed without encroaching on the inferior alveolar nerve (ian). In this situation, restorative options include the use of short fixtures, onlay bone grafting to increase ridge height, and more complicated and detailed imaging analysis to allow positioning of implants alongside, rather than into, the nerve canal during the procedure . Another option is to move the ian laterally from its canal by either nerve lateralization (ianl). With nerve lateralization, the ian is exposed and traction is used to deflect it laterally while the implants are placed . The ian is then left to fall back into position, against the fixtures . With this procedure, there is no interference with the incisive nerve . With nerve transposition, a corticotomy is done around the mental foramen and the incisive nerve is transacted, such that the mental foramen is repositioned more posteriorly . In the posterior mandible, the bone quality may not be as good as it is in the anterior mandible . In particular, if shorter implants are used to ensure that there is no encroachment on the nerve canal, initial implant stability will be unicortical . In addition, there is a risk to the ian as the operator tries to maximise implant length on the basis of measured available bone height . The advantages of iant include the ability to place longer fixtures and to engage 2 cortices for initial stability . Kan et al . Pointed out that the amount of bone superior to the mandibular canal (mc) is often insufficient for the placement of fixtures of the ideal length . In addition, the existing bone that is superior to the mc is often of poorer quality than its cortical counterpart . These factors and the fact that shorter implants have been associated with higher failure rates have led to the development of methods of ian repositioning that allow placement of longer fixtures; with these methods, the inferior cortex of the mandible is engaged, which leads to greater initial stability . Therefore, the purpose of the present paper was to review the literature concerning the indications, contraindications, advantages, disadvantages and the surgical techniques of the lateralization and transposition of the inferior alveolar nerve, followed by the implants placement in an edentulous atrophic posterior mandible . Ian lateralization (ianl) and transposition (iant) definition ianl and iant are surgical procedures that reposition the ian for the purpose of implant placement without bone augmentation . This procedure raises the risk of neuropathies, such as paresthesia, hypoesthesia, and anaesthesia of the ian . Ianl is defined as the lateral reflection of the ian without incisive nerve traction (preservation of the incisive nerve and lateralization of inferior alveolar neurovascular bundle posterior to the mental foramen); exposure and traction are used to deflect the ian laterally while the implants are placed . The ian is then left to fall back in against the fixtures . During the iant procedure, a corticotomy is done around the mental foramen and the incisive nerve is transacted (incisive neurovascular bundle is sacrificed), to allow transposition of both the mental foramen and the ian such that the mental foramen is repositioned more posteriorly . The first case of ian repositioning was reported by alling (1977) to rehabilitate patients with severe atrophy for dentures . In 1987, jenson and nock carried out iant for the placement of dental implants in posterior mandibular regions . According to subjective criteria, sensory function of the mental nerve returned to normal five weeks post - operative . There is limited information about implants survival rate . In 1992, rosenquist performed the first case series study using iant followed by 26 implants placement on 10 patients . Neurosensory function of the mental nerve was evaluated objectively using the two - point discrimination (2-pd) method . At one year follow - up, he reported an implant survival rate of 96% and implant survival rate of 93.6% for this procedure; therefore, this technique was accepted as the best - practice treatment modality for reconstruction of the dentoalveolar system with dental implants in the posterior mandible . As a result, this technique is constantly undergoing improvement . Iant is a new procedure that needs further refinements in terms of technique and instrumentation to decrease complications . The review included studies, case reports, clinical trials on human subjects that were published in english between january 1997 and july 2014, and included a minimum of 6 months of follow - up . Letters and phd theses were excluded, as well as abstracts and reviews and studies on animals . The information source was the medline (ncbi pubmed and pmc) database and other scientific electronic sources . According to the prisma guidelines, an electronic search was conducted using the medline (ncbi pubmed and pmc) database to locate articles concerning ian lateralization or transposition and implant placement in an edentulous atrophic posterior mandible . The search terms used were: inferior alveolar nerve lateralization, inferior alveolar nerve repositioning, inferior alveolar nerve transposition, implants in atrophic posterior mandible + repositioning, inferior alveolar nerve transposition + mental, implants in atrophic posterior mandible + lateralization, implants in atrophic posterior mandible + transposition, mandibular atrophy + repositioning, and inferior alveolar nerve + minimal bone height . Due to the low number of relevant articles and to ensure the sensitivity of the systemic review process, articles were searched from january 1997 to july 2014 . Titles derived from this broad search were independently screened by two authors based on the inclusion criteria . Full reports were obtained for all the studies that were deemed eligible for inclusion in this paper . Inclusion and exclusion criteria inclusion criteria for the selection were: articles regarding to ianl and iant procedures; all articles in english; clinical reports with minimum 6 months follow - up; information regarding implant osseointegration and survival; studies on adult (between the ages 18 and 80) human beings, with no immunologic diseases, uncontrolled diabetes mellitus, osteoporosis, or other contraindicating systemic conditions . Exclusion criteria for the selection were: clinical reports with no minimum 6 months of follow - up; not enough information regarding the selected topic; no information regarding implant osseointegration and survival; studies of patients with immunologic diseases, uncontrolled diabetes mellitus, osteoporosis or other contraindicating systemic conditions; studies of adolescents (under 18 years of age) and elderly people (over 80). Article review and data extraction article review and data extraction was performed according to a prisma flow diagram (figure 1). The search displayed 876 results from the ncbi pmc and pubmed databases, and 3 results from other sources (dental-tribune.com, acta.tums.ac.ir, hindawi.com/journals). A total of 38 titles and abstracts were selected according to relevancy after the removal of duplications . Another exclusion was made based upon follow - up time (n = 6) and information regarding implant osseointegration and survival (n = 4). Studies of adult human beings between 18 and 80 years of age with minimal residual bone above the mc, in which ianl and iant + implant placement had been performed, were selected . Data collection process data was independently extracted from reports in the form of variables according to the aim and themes of the present review as listed below . The review aims to achieve the following: to describe the purpose (indications and contraindications) of ianl and iant . To describe the current surgical techniques used for ianl and iant with simultaneous implant placement . Risk of bias assessment risk of bias (e.g., lack of information or selective reports on variables of interest) was assessed at the study level . The risks were indicated as lack of precise information of interest in each individual study that can blind the reader from particular information about the examined samples . The cochrane collaboration tool for assessing risk of bias was used to assess bias across the studies that could affect cumulative evidence . The search displayed 876 results from the ncbi pmc and pubmed databases, and 3 results from other sources (dental-tribune.com, acta.tums.ac.ir, hindawi.com/journals). A total of 38 titles and abstracts were selected according to relevancy after duplications removal . Exclusion was made according to information amount regarding selected topic (n = 12). Twenty - six full - text articles were in the end, assessed for eligibility . During the eligibility stage, articles that did not meet the inclusion and exclusion criteria where filtered as follows: no minimum 6 months of follow - up (n = 6) and no information regarding implant osseointegration and survival (n = 4). In the end, 16 articles that included data on 160 patients were utilised for the systematic review (figure 1). Nine were related to iant, 4 to ianl and 3 to both iant and ianl (table 1). Description of studies included in the review risk of bias within studies the cochrane collaboration bias summary for potential bias was used to assess the quality of studies and identify papers with intrinsic flaws in method and design . After analysing the risk of bias (table 2), we found that six authors [5,13 - 17] selected the patients randomly . In four studies [1 - 3,5], the surgical procedure was not performed by the authors; and in two studies, the method of implant osseointegration evaluation was not mentioned . Assesment of the risks of bias results of individual studies results of individual studies of implant treatment using are shown in table 3 . Results of individual studies ianl = inferior alveolar nerve lateralization; iant = inferior alveolar nerve transposition . From the results of individual study data (table 3), we can conclude that the most popular surgical technique for ian repositioning was iant . It was calculated that 51.44% (107/208) of all operations performed utilised iant, and 48.56% (101/208) utilised ianl . Number of implants placed after iant was 211, while number of implants placed after ianl was 95 . In the study by lorean et al . (in which 68 ianl and 11 iant were performed), the authors failed to mention how many implants were placed during each procedure . It was 88%; in ferrigno et al ., it was 95.7%; and kan et al . Presented a 93.8% implant survival rate.the lowest implant survival rates were found in kan et al . With 33.33% and karlis et al . With the most popular method of implant osseointegration evaluation was panoramic radiograph (12/16 studies). In two studies, both of which were performed by the same author (proussaefs), the method of implant osseointegration evaluation was a perio - test unit (siemens, bensheim, germany); while in two other studies (lorean et al ., kan et al . ), the implant osseointegration evaluation method was not mentioned . The purpose (indications and contraindications) of ianl and iant the study of number selected articles [9,22 - 25] revealed the following indications of ianl and iant: the major reason for using this technique is to prevent ian injury during implant placement in edentulous posterior atrophic mandibles . Class iv, v, or vi of cawood and howell with extrusion of the antagonist tooth and reduced prosthetic free space . Class v or vi of cawood and howell with presence of interforaminal teeth (patients were not candidates for interforaminal implant - prosthetic methods). Class v or vi of cawood and howell if the patient desires a fast implant - prosthetic rehabilitation with predictable outcomes . In orthognathic surgeries, such as lower border shaving and total mandibular subapical osteotomy . In the pre - prosthetic surgery . In the anastomosis and repairing of a disrupted ian . Preservation of ian in cancer surgery in the posterior mandible . When placement of short implants is not a viable option (in case of severely atrophic mandibles when the residual bone above mc ranges between 0.5 and 1.5 mm). Less than 10 - 11 mm bone height above the canal, when the quality of the spongy bone does not provide sufficient stability for implant placement . Contraindications if the mandible presents advanced resorption of the alveolar process . If the patient has poor general health, including systemic diseases that may worsen the patient s health condition after the ian reposition procedure . Limitations in accessing the surgical site . People who become easily stressed out and are over sensitive even towards the smallest surgical complications . Such patients do not have tolerance and compatibility skills and, therefore, are not good candidates for nerve transposition surgery . The current surgical techniques utilised for lateralization and transposition of ian with simultaneous implant placement the preoperative work - up included an assessment of the ian using appropriate diagnostic records, such as a panoramic radiograph, a computed tomography (ct) scan, casts, diagnostic wax - up, and surgical templates . During preoperative consultation with the patients, the risk of postoperative neurosensory disturbances (nd) that can result following the ian repositioning is discussed . This possibility gives many patients pause to consider the ramifications of the procedure . To help the patient decide whether this would be tolerable, the clinician can perform a preoperative block with a long - acting local anaesthetic, such as marcaine, which reproduces symptoms lasting 8 to 16 hours that are similar to the postoperative anaesthesia the patient may experience . Two ian repositioning techniques have been developed: lateralization and transposition (table 4). Both procedures include an ian block plus local infiltrating anaesthesia with vasoconstrictors . Intravenous sedation is recommended because of the procedure s technique - sensitive nature and the need for patient cooperation . A crestal incision with anterior- and posterior releasing incisions is made, and a labial mucoperiosteal flap is reflected that exposes the alveolar ridge and buccal cortex . The incision should extend at least 1 cm beyond the anticipated site of the osteotomy . Care is taken during flap reflection to preserve the integrity of the periosteum and the neurovascular bundle where it exits the mental foramen and enters the soft tissue . To increase the flaps relaxation and improve exposure the flaps are raised, one lingual to access the alveolar crest, and another vestibular for subperiosteal dissection of the ian, until sufficient surgical field is obtained . Ct is used to locate the approximate area of the mental foramen, after which blunt dissection is used to identify and isolate the mental nerve . From this point inferior alveolar nerve (ian) replacement division into two distinct surgical procedures, i.e. Transposition and lateralization; the steps are outlined below prf = platelet - rich fibrin . Ian transposition (iant) an osteotomy is performed at the mental foramen, drilling around the orifice to obtain a ring of external cortical bone . A window also may be made about 5 mm ahead of the foramen, in order to avoid damaging the nerve over its anterior loop . An en bloc osteotomy is then made at the external cortical level, or a posterior window is performed in the external cortical layer along the intrabony trajectory of the nerve (figure 2). A round drill is used to create the window, replacing it with a diamond drill while working close to the mc, to minimise the risk of nerve damage . Bone is removed using a round bur number 700 or 701, a straight handpiece and copious normal saline for irrigation or a piezosurgery device . In order to secure complete mobilisation of the ian, the incisor branch, located about 5 mm from the mental foramen, a = before transposition; b = after transposition (courtesy of dr dainius razukevicius, kauno implantologijos centras kaunas, lithuania). Then, with the nerve fully lateralized, the dental implants are placed under direct visualisation - in this case bicortically, taking advantage of the mandibular basal layer . Once the implants have been positioned, the vestibular cortical layer is replaced in those cases where an osteotomy has been performed, or the nerve is passively positioned against the implants in those cases where cortical drilling has been carried out . In either case, the emergence of the nerve becomes more distal . Ian lateralization (ianl) in this case, neither dissection of the terminal branches of the ian nor sectioning of the incisor branch is needed . The technique involves the preparation of a cortical bone window (via osteotomy or drilling) that is located posterior to the mental foramen (figure 3 and figure 4). Intraoperative photography showing the inferior alveolar neurovascular bundle lateralization (courtesy of dr dainius razukevicius, kauno implantologijos centras kaunas, lithuania). Schematic drawing showing the inferior alveolar neurovascular bundle transposition (a) and lateralization (b). Bone is removed using a round bur number 700 or 701, a straight handpiece and copious normal saline for irrigation or a piezosurgery device . After removing the cortical bone, a curette may be used for removal of spongy bone and the cortical layer of the canal in cases where the cortical layer surrounding the canal is not dense or thick . A special instrument (hassani nerve protector, ali hassani, iran) is required to protect the ian while the cortical layer is removed using surgical burs or a piezosurgery device . Bone removal in close vicinity to the neurovascular bundle should be performed patiently and thoroughly . This is usually performed using special curettes parallel to the surface of the nerve bundles in an antero - posterior direction . The area should be thoroughly irrigated so that the nerve bundle can be clearly seen . The neurovascular bundle inside the canal is freed using special curettes and is moved laterally using a nerve hook . Then, a 10 mm wide gauze cord or elastic band is passed below the nerve to retract it from the surgical site in order to decrease the risk of ischemic trauma to the nerve . The second purpose of retracting the nerve from the surgical site during the operation is to reduce the risk of ian damage . After carefully freeing the nerve, the latter is separated using a vessel loop - while applying gentle traction outwards as the implants are positioned, the implant should be long enough to pass the mc and engage the basal body below the canal to achieve sufficient primary stability . Finally, the vessel loop is removed and the nerve is replaced, positioning a resorbable membrane between it and the bone window to avoid direct contact with the implants . The procedure is completed by suturing the mucoperiosteal flap . The decision whether to use iant or ianl depends on the amount of stretching that is needed in order to mobilize the ian . Study, stretching the nerve by 10 - 17% of its original length may result in disruption of the nerve fibers internally . In situations where only small traction is needed, anl may be utilised . Kan et al . Retrospective study demonstrated that the nd for iant were 77.8% (7/9) and for the ianl 33.3% (4/12). As a summary of both surgical techniques (iant and ianl), it can be concluded that ianl produces less side effects than iant . The use of piezosurgery to avoid ian damage some studies recommend piezosurgery for bone removal in nerve transposition surgery . This device causes vibrations in the range of 20 - 200 m and cuts through the mineralized tissue completely and smoothly . If soft tissue or the neurovascular bundle comes in contact with this device, it stops functioning because it is designed to stop working when it contacts unmineralized tissue . This device is especially beneficial when a small osteotomy is going to be performed . Among the disadvantages of this device are the long duration of time that it takes to remove bone . Also, there remains controversy regarding the indications of piezosurgery devices, and some believe that the vibrations they generate may damage the nerve . Repositioning the neurovascular bundle inside the mc before this phase, the surgeon should decide whether or not to place materials between the implant and the inferior alveolar neurovascular bundle . The preference is to place a collagen membrane or bone material between the implant and the inferior alveolar neurovascular bundle . A potential advantage of bone over a membrane is that, if proper healing occurs in the area, the contact area of implant and bone will increase . Before releasing the inferior alveolar neurovascular bundle from the elastic band this way, the inferior alveolar neurovascular bundle will be in a vent that is medially adjacent to the implants and covered by the mucoperiosteal flap . Alternatively, the inferior alveolar neurovascular bundle may be left to lie passively outside of the mc . The bone defect is then covered by several methods: repositioning the bony window that was removed or the bony window can be crushed and mixed with an allograft or xenograft . Postoperative measures antibiotic and corticosteroid prophylaxis is recommended because of the extensiveness and duration of surgery . However, there is no consensus in this regard; since inflammation can be among the causes of nerve dysfunction, corticosteroid therapy can be beneficial . After analysing the current literature concerning ianl and iant, several advantages and disadvantages of the procedures were found and are described below . The advantages of the ianl and iant longer implants can be placed in the same surgical step . Possibility of placement of a greater number of implants, which improves the overall strength of the final prosthesis . Possibility for simultaneous placement of implants during surgery, which allows a reduction in treatment time compared with other techniques as bone grafts that require a long waiting period before implant insertion together with additional surgeries . The option for immediate loading for the enhancement of masticatory function, dramatically improving the patient s quality of life . The evaluation values for implant survival rates are similar to those for standard implantation procedures . As a biomechanical advantage, ian transposition presents an increase in resistance to occlusal forces and promotes a good proportion between the implant and the prosthesis . In addition, with this procedure, all of the following are accomplished: restoration of the correct vertical dimension; prevention of tissues atrophy; replacement of dentures; stabilisation of the anterior dentition; temporomandibular joint and masticatory muscle balance . The disadvantages of the ianl and iant one of the disadvantages of this procedure is that it does not recover alveolar ridge anatomy . Another negative point of this procedure is that it temporarily weakens the mandible due to removal of cortical bone; which, in combination with implant placement, may lead to mandibular fracture at the operation site . According to peleg, cylindrical non - threaded implants are recommended after the ianl procedure, as threaded implants in close contact with the nerve may cause neurosensory problems . This recommendation may be a disadvantage because non - threaded implants have a lower surface area compared to threaded implants, which decreases the stability of the implant . Since this surgical procedure is delicate, it is best performed under a general anesthesia to eliminate patient movement and to maximise access . The general anaesthetic management of patients with myotonic dystrophy (dystrophia myotonica) can, however, be challenging . The most common failures are due to bruxism and poor occlusal relations; therefore, patients with bruxism or poor occlusal relations make, implant stability and survival to less favourable . . The main limitations of this review were that six authors [5,13 - 17] selected the patients randomly; in four studies [1 - 3,5], the surgical procedure was not performed by the authors and, in two studies, the method of implant osseointegration evaluation was not mentioned . Inferior alveolar nerve repositioning is a technique that has been used for more than 20 years with good survival and survival rates . This is sometimes the only possible procedure to help patients to obtain a fixed prosthesis, especially in edentulous atrophic posterior mandibles . There are two main techniques for inferior alveolar nerve repositioning that are relatively safe and offer a high survival rate: inferior alveolar nerve lateralization and inferior alveolar nerve transposition . Increased protection of the neurovascular bundle inferior alveolar nerve lateralization and transposition in combination with the installation of dental implants offer advantages, such as reducing the risk of inferior alveolar nerve damage . With careful pre - operative surgical and prosthetic planning, imaging, and extremely precise surgical technique, this procedure can be successfully used for implant placement in an edentulous atrophic posterior mandible.
Secondary objectives included the evaluation of retinal ganglion cell function by pattern electroretinogram (perg) and optic nerve through visual evoked potential (vep), and tolerability to the eye drops . To exclude any interference of citicoline treatment on animal wellbeing, basic hematological parameters of all animals were investigated and compared with experimental controls and with data already available in the literature . Treatment and experimental handling of animals were done also according with the italian directive (decree 116/92). A veterinary surgeon controlled the health status of the animals in order to avoid physical injury, suffering, and distress . The experiment was performed on five cd1 mice (weight of each mouse approximately 30 g) so divided: group a) two mice treated with a solution of 1% citicoline, 0.2% high molecular weight hyaluronic acid (ha), and 0.01% benzalkonium chloride (bak)group b) two mice treated with a solution of 2% citicoline, 0.2% ha, and 0.01% bak (omk1, omikron italia s.r.l. )group c) one control mouse . Group a) two mice treated with a solution of 1% citicoline, 0.2% high molecular weight hyaluronic acid (ha), and 0.01% benzalkonium chloride (bak) group b) two mice treated with a solution of 2% citicoline, 0.2% ha, and 0.01% bak (omk1, omikron italia s.r.l .) The right eye (re) of each mouse in group a and b was treated with two drops twice - a - day for three days . On the fourth day after sacrificing the animal, after aspirating the vitreous with a sge analytical science 100 l syringe, it was diluted with 20 l of distilled water . Subsequently, the vitreous was centrifuged and the 50 l of solution 50% water and 50% methanol were added to the collected supernatant and passed by vortex . For the sample analysis, liquid chromatography and mass spectrometry (lc - ms / ms) was used . Instrumentation employed consisted of triple - quadruple mass spectrometer (applied biosystems api 3000) equipped with a turbo ion - spray and coupled to an uplc (dionex ultimate 3000 rs). In a preliminary stage, chromatographic separation and mass spectrometry identification has been optimized through the use of a pure grade citicoline standard . Once set all instrumental and the analytical parameters to get the best conditions, the results were evaluated respect to the therapeutic range expected . This was achieved by mean of a calibration curve built by deuterated citicoline standard solutions made at fixed concentrations . The vitreous was added with an equal volume of water / methanol solution, containing deuterated - citicoline . Then, the sample obtained was processed though the previously described lc - ms / ms system . Between tests, the machine had always done white readings to avoid contamination . Inclusion criteria to take part in the study were: age between 20 and 60 years old, best corrected visual acuity 5/10, and diagnosis of progressive open - angle glaucoma (loss of 1 db / year). Glaucoma was defined by glaucomatous visual field defect (humphrey 24 - 2 sita standard, carl zeiss meditec, inc) with mean deviation (md) up to 12 db, glaucomatous optic disc, and iop less than 21 mmhg on hypotensive treatment . Exclusion criteria were considered as follows: ocular surgery in the last three months, argon laser trabeculoplasty in the last six months, cataract or maculopathy, hypersensitivity to citicoline, secondary ocular hypertension (i.e. Due to steroids), history of ocular disease, changes in systemic therapy affecting iop, use of statin, pregnancy or nursing, failure to use contraceptive, diabetes, arthritis, use of anticoagulant and lithium . As these patients were progressing, we decided to perform an interim analysis after three months . During this period, patients underwent three visits, at baseline, after 60 days, and after 90 days . Patients were randomized in two groups, omk1, treated with omk1 one drop three times a day plus hypotensive drops for two months and one month of wash out, and oag, treated only with hypotensive drops for three months . It was made dividing patients into two groups with similar characteristics including age, refraction, and glaucomatous damage . At baseline and at each visit, a comprehensive ophthalmological examination was performed, including corrected visual acuity, biomicroscopy, indirect ophthalmoscopy, iop measurement with goldmann applanation tonometer, perimetry white on white (humphrey 24 - 2 sita standard), recording of perg and vep . The following variables were considered: visual field test: md and pattern standard deviation (psd)perg: latency p50 and amplitude p50-n95pev: latency n75 and p100, amplitude n75-p100 and p100-n145, and retinocortical time (rtc). Visual field test: md and pattern standard deviation (psd) perg: latency p50 and amplitude p50-n95 pev: latency n75 and p100, amplitude n75-p100 and p100-n145, and retinocortical time (rtc). Perg and vep responses between groups a and b were examined by analysis of variance (anova). The anova was also used for repeated measurements of each parameter in the follow - up compared with baseline . A p <0.01 to compensate for multiple comparisons was considered statistically significant . The safety and tolerability of topical citicoline were analyzed evaluating the anterior segment of the patient at each visit . In each patient, the presence of ocular manifestations was evaluated using a score from 0 to 4 (0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe) to indicate the presence and intensity of adverse ocular manifestations observed: conjunctival hyperemia, secretion, inflammation, keratopathy, scleritis, uveitis, cataract, ocular hypertension . Patients were also asked to express an evaluation using the same score (from 0 to 4) to indicate the presence and intensity of ocular symptoms associated with the instillation of the product: burning, itching, foreign body sensation, diffuse pain, blurred vision . Inclusion criteria to take part in the study were: age between 20 and 60 years old, best corrected visual acuity 5/10, and diagnosis of progressive open - angle glaucoma (loss of 1 db / year). Glaucoma was defined by glaucomatous visual field defect (humphrey 24 - 2 sita standard, carl zeiss meditec, inc) with mean deviation (md) up to 12 db, glaucomatous optic disc, and iop less than 21 mmhg on hypotensive treatment . Exclusion criteria were considered as follows: ocular surgery in the last three months, argon laser trabeculoplasty in the last six months, cataract or maculopathy, hypersensitivity to citicoline, secondary ocular hypertension (i.e. Due to steroids), history of ocular disease, changes in systemic therapy affecting iop, use of statin, pregnancy or nursing, failure to use contraceptive, diabetes, arthritis, use of anticoagulant and lithium . As these patients were progressing, we decided to perform an interim analysis after three months . During this period, patients underwent three visits, at baseline, after 60 days, and after 90 days . Patients were randomized in two groups, omk1, treated with omk1 one drop three times a day plus hypotensive drops for two months and one month of wash out, and oag, treated only with hypotensive drops for three months . It was made dividing patients into two groups with similar characteristics including age, refraction, and glaucomatous damage . At baseline and at each visit, a comprehensive ophthalmological examination was performed, including corrected visual acuity, biomicroscopy, indirect ophthalmoscopy, iop measurement with goldmann applanation tonometer, perimetry white on white (humphrey 24 - 2 sita standard), recording of perg and vep . The following variables were considered: visual field test: md and pattern standard deviation (psd)perg: latency p50 and amplitude p50-n95pev: latency n75 and p100, amplitude n75-p100 and p100-n145, and retinocortical time (rtc). Visual field test: md and pattern standard deviation (psd) perg: latency p50 and amplitude p50-n95 pev: latency n75 and p100, amplitude n75-p100 and p100-n145, and retinocortical time (rtc). Perg and vep responses between groups a and b were examined by analysis of variance (anova). The anova was also used for repeated measurements of each parameter in the follow - up compared with baseline . The safety and tolerability of topical citicoline were analyzed evaluating the anterior segment of the patient at each visit . In each patient, the presence of ocular manifestations was evaluated using a score from 0 to 4 (0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe) to indicate the presence and intensity of adverse ocular manifestations observed: conjunctival hyperemia, secretion, inflammation, keratopathy, scleritis, uveitis, cataract, ocular hypertension . Patients were also asked to express an evaluation using the same score (from 0 to 4) to indicate the presence and intensity of ocular symptoms associated with the instillation of the product: burning, itching, foreign body sensation, diffuse pain, blurred vision . The concentration of citicoline has been calculated for comparison with the calibration curve previously acquired, and the following graph was obtained in which the peak corresponds to the presence of citicoline in the vitreous [figure 1]. The graph shows the spectrometry result: the peak corresponds to the presence of citicoline in the vitreous . The machine detected the molecule very well as the signal was present always at the same time and in all four transitions used: the molecule reached the vitreous . There was a difference between the concentrations 1% and 2% regarding the systemic absorption, which was zero for the lower concentration [figure 2]. The graph shows the spectrometry result of the left eye of group a mouse: citicoline is not in the vitreous . We enrolled 34 patients, 16 in the omk1 and 18 in the oag group, with mean age of 51 years and male / female ratio of 1:1 . The mean iop in both groups was similar, and it did nt change significantly during the study . At study inclusion, the mean iop was 14.3 2.5 mmhg in omk1 group and 14.6 0.5 mmhg in oag group, while at the end of the study, it was respectively 15.6 1.5 mmhg (p = 0.55) and 15.3 2.5 mmhg (p = 0.67). In no patients, signs and symptoms indicative of lack of safety and local tolerability were reported . Md and psd values improved [figures 3 and 4], but this change was nt statistically significant (baseline md -4.92 db vs. 60 day md -4.59, p = 0.75; baseline psd 5.14 db vs. 60 day psd 5.01 db, p = 0.91 . At 90 day, md -4.28 db, p = 0.55; psd 4.53 db, p = 0.62). An example of visual field md improvement after 30 and 60 days an example of visual field md improvement after 30 and 60 days after 60 days of therapy with omk1, a significant improvement in ganglion cells function was observed, demonstrated by the reduction of the p50 latency and the increase in the n95-p50 amplitude [tables 1 and 2]. Perg p50 latency (ms = milliseconds) values at baseline and at 60 and 90 day perg p50-n95 amplitude (v = microvolt) values at baseline and at 60 and 90 day the p100 latency reduction observed after 60 days (127.8 ms vs. 117.9 ms, p = 0.01) regressed after 30 days of washout (127.8 ms vs. 124.4 ms, p = 0.34). There was no significant changes of n75-p100 amplitude (7.03 ms vs. 8.63 ms, p = 0.27; 7.03 ms vs. 6.91 ms, p = 0.92). Rct p100 latency did nt show statistically improvement after 60 days (68.9 ms vs. 62.8 ms, p = 0.15), and it got worse after 30 days (62.8 ms vs. 68.1 ms, p = 0.83). Oag group did nt show any significant change in perimetric and electrofunctional parameters during the follow - up, comparing to baseline data . The concentration of citicoline has been calculated for comparison with the calibration curve previously acquired, and the following graph was obtained in which the peak corresponds to the presence of citicoline in the vitreous [figure 1]. The graph shows the spectrometry result: the peak corresponds to the presence of citicoline in the vitreous . The machine detected the molecule very well as the signal was present always at the same time and in all four transitions used: the molecule reached the vitreous . There was a difference between the concentrations 1% and 2% regarding the systemic absorption, which was zero for the lower concentration [figure 2]. The graph shows the spectrometry result of the left eye of group a mouse: citicoline is not in the vitreous . We enrolled 34 patients, 16 in the omk1 and 18 in the oag group, with mean age of 51 years and male / female ratio of 1:1 . The mean iop in both groups was similar, and it did nt change significantly during the study . At study inclusion, the mean iop was 14.3 2.5 mmhg in omk1 group and 14.6 0.5 mmhg in oag group, while at the end of the study, it was respectively 15.6 1.5 mmhg (p = 0.55) and 15.3 2.5 mmhg (p = 0.67). In no patients, signs and symptoms indicative of lack of safety and local tolerability were reported . Md and psd values improved [figures 3 and 4], but this change was nt statistically significant (baseline md -4.92 db vs. 60 day md -4.59, p = 0.75; baseline psd 5.14 db vs. 60 day psd 5.01 db, p = 0.91 . At 90 day, md -4.28 db, p = 0.55; psd 4.53 db, p = 0.62). An example of visual field md improvement after 30 and 60 days an example of visual field md improvement after 30 and 60 days after 60 days of therapy with omk1, a significant improvement in ganglion cells function was observed, demonstrated by the reduction of the p50 latency and the increase in the n95-p50 amplitude [tables 1 and 2]. Perg p50 latency (ms = milliseconds) values at baseline and at 60 and 90 day perg p50-n95 amplitude (v = microvolt) values at baseline and at 60 and 90 day the p100 latency reduction observed after 60 days (127.8 ms vs. 117.9 ms, p = 0.01) regressed after 30 days of washout (127.8 ms vs. 124.4 ms, p = 0.34). There was no significant changes of n75-p100 amplitude (7.03 ms vs. 8.63 ms, p = 0.27; 7.03 ms vs. 6.91 ms, p = 0.92). Rct p100 latency did nt show statistically improvement after 60 days (68.9 ms vs. 62.8 ms, p = 0.15), and it got worse after 30 days (62.8 ms vs. 68.1 ms, p = 0.83). Oag group did nt show any significant change in perimetric and electrofunctional parameters during the follow - up, comparing to baseline data . Citicoline is a natural precursor of the pdc with important neuroprotective features, as demonstrated in the literature . Many studies demonstrate how citicoline have a positive effect on both visual field and the entire visual way, through the use of perg and vep . Confirmed the same result in patients under both hypotensive medical therapy and citicoline administered intramuscularly (1000 mg / day) or orally (1600 mg / day) compared with patients receiving only hypotensive medical therapy, and the need to repeat the treatment to maintain the positive effects on visual function (total period of the study was 8 years). Recently, it has been demonstrated that also citicoline oral solution 500 mg seems to have a neuroprotective effect after a cycle of at least 60 days . Other studies have used mice as laboratory animals to test the neuroprotective effect of citicoline . Oshitari et al . Showed the results of quantitative analysis of tdt - dutp terminal sequence (tunel) on the retina of mice fixed and incubated with four different concentrations of citicoline (0.01, 0.1, 1, 10 mol / l). The tunel sequence was significantly lower in the retina treated with citicoline at all concentrations compared to the retina of controls not incubated with other active agents . In 2009, chan et al . Have published the results of the use of in vivo magnetic resonance spectroscopy (1h mrs proton magnetic resonance spectroscopy) on models of experimental glaucoma in rats, showing that glaucoma is also characterized by altered metabolism of choline in the visual cortex, reflecting the impairment of structural integrity of neuronal membranes . To date, there has not been a neuroprotective drug in eye drops since they are very large and lipophilic molecules, which are not able to penetrate the ocular tissues . To increase its penetration, it is necessary to relax the intercellular junctions of the corneal epithelium, which is possible through the use of bak, used for decades in ophthalmic preparations as drug penetration enhancer . The experimental study has demonstrated that the ophthalmic solution omk1 based on citicoline 2% in combination with high molecular weight hyaluronic acid and bak at low concentration, 0.01%, guarantees the passage of the molecule in the posterior segment, reaching the retina and the optic nerve head: once in the anterior chamber, the molecule diffuses in the vitreous through the zonula and through the uveoscleral route . Hyaluronic acid has the ability to convey the active ingredient and to increase the contact time in the anterior chamber, thus increasing the absorption through the retina, conjunctiva, and sclera . The clinical study showed that treatment with topical citicoline induces, after 60 days of therapy, a significant improvement in the ganglion cell function (increased amplitude and reduced latency of perg). In addition, improvements in the ganglion cells function induced a reduction of the delay of post - retinal nerve conduction (although the improvement of rct was not significant). The simultaneous significant improvement in the ganglion cells function together with a reduction of the delay of the post - retinal nerve conduction results in an improvement of nerve conduction over the entire optical pathway (reduction of the latency of the vep). After 30 days of suspension of the treatment, the residue improvement of ganglion cell function no longer causes the reduction of post - retinal nerve conduction delay, which returns to pre - treatment values (rct values similar to baseline). The loss of this effect on post - retinal nerve conduction is reflected on the entire visual pathway (latency of vep unchanged at 90 days). In individual eyes, we observed an improvement of the visual field parameters (md and psd). However, as the baseline data were highly variable to reach significant values, it is necessary to have a greater sample size, which we will reach at the end of the full study period (24 months). In conclusion, this interim analysis shows that topical citicoline seems to have a neuroprotective effect that is independent of pressure - lowering effect due to any hypotensive drops . Furthermore, topical administration has several advantages: it's easier to use than other forms (intramuscular and oral), and it contains lower concentration of the molecule because it bypasses the hepatic and renal metabolism; therefore, patients can use it several times throughout the day.
These tumors can arise from any nerve covered with a schwann cell sheath, which include the cranial nerves (except for the optic and olfactory), the spinal nerves, and the autonomic nervous system . When the nerve of origin is small, its association with a given tumor may be difficult to demonstrate . On the other hand, if a larger nerve is the site of origin, the nerve fibers are found to be splayed out over the outer aspect of the capsule rather than incorporated within the mass of the tumor . Approximately 112% occur intraorally [4, 5] with the tongue being the most common site [5, 6]. Although several case reports of schwannomas of the tongue exist in the literature, there has been no comprehensive review of the literature since hatziotis et al . . We present two cases of schwannoma of the tongue and review the available literature of the last 51 years (19552006). A pubmed search of the terms tongue schwannoma, lingual schwannoma, tongue neurilemmoma, and schwannomas of the floor of mouth were not included unless the ventral tongue was also involved . All of the case reports had histologically confirmed the identity of the masses as schwannomas . The following elements were extracted from the case reports for data analysis: age, gender, location of schwannoma (anterior one - third vs. posterior two - thirds of tongue), presenting symptoms, size of tumor, and treatment modality . An 19-year - old female presented to the head and neck surgery clinic with complaints of a slowly enlarging painless mass in the back of her throat . Magnetic resonance (mr) imaging was obtained which illustrated a well - circumscribed mass in the right posterolateral tongue that was isointense on t1, hyperintense on t2, and enhanced with gadolinium (fig . The patient was taken to the operating room for a transoral excision under general anesthesia . The mass was submucosal and once a mucosal flap was raised, the tumor was readily shelled out using blunt dissection . The mass had a smooth surface, was tan - white in color, and measured 18 13 11 mm . 1a well - circumscribed mass is located at right posterior tongue as illustrated by the axial t1 (a), sagittal t2 (b), and coronal t1 (c) magnetic resonance imagesfig . A at low power (h&e, 4), both antoni a and antoni b morphologies can be seen . B at high power (h&e, 20), palisading nuclei and verocay bodies are noted . C s-100 is diffusely positive (s-100, 20) a well - circumscribed mass is located at right posterior tongue as illustrated by the axial t1 (a), sagittal t2 (b), and coronal t1 (c) magnetic resonance images schwannoma of the tongue . A at low power (h&e, 4), both antoni a and antoni b morphologies can be seen . B at high power (h&e, 20), palisading nuclei and verocay bodies are noted . C s-100 is diffusely positive (s-100, 20) a 77-year - old male was referred to the head and neck surgery clinic after his dentist noted an 1 cm firm, sessile mass at the right lateral border of his tongue . The patient reported that the mass had been there for approximately 5 years and had not grown significantly over those years . The patient denied any pain associated with the mass, dysphagia, or change in voice . The mass was gray - tan in color, smooth in texture, dome - shaped, and measured 7 5 5 mm . A final diagnosis of schwannoma was ascribed . An 19-year - old female presented to the head and neck surgery clinic with complaints of a slowly enlarging painless mass in the back of her throat . Magnetic resonance (mr) imaging was obtained which illustrated a well - circumscribed mass in the right posterolateral tongue that was isointense on t1, hyperintense on t2, and enhanced with gadolinium (fig . The patient was taken to the operating room for a transoral excision under general anesthesia . The mass was submucosal and once a mucosal flap was raised, the tumor was readily shelled out using blunt dissection . The mass had a smooth surface, was tan - white in color, and measured 18 13 11 mm . 1a well - circumscribed mass is located at right posterior tongue as illustrated by the axial t1 (a), sagittal t2 (b), and coronal t1 (c) magnetic resonance imagesfig . A at low power (h&e, 4), both antoni a and antoni b morphologies can be seen . B at high power (h&e, 20), palisading nuclei and verocay bodies are noted . C s-100 is diffusely positive (s-100, 20) a well - circumscribed mass is located at right posterior tongue as illustrated by the axial t1 (a), sagittal t2 (b), and coronal t1 (c) magnetic resonance images schwannoma of the tongue . A at low power (h&e, 4), both antoni a and antoni b morphologies can be seen . B at high power (h&e, 20), palisading nuclei and verocay bodies are noted . A 77-year - old male was referred to the head and neck surgery clinic after his dentist noted an 1 cm firm, sessile mass at the right lateral border of his tongue . The patient reported that the mass had been there for approximately 5 years and had not grown significantly over those years . The patient denied any pain associated with the mass, dysphagia, or change in voice . The mass was gray - tan in color, smooth in texture, dome - shaped, and measured 7 5 5 mm . A pubmed search from 1955 to 2006 identified 124 cases of schwannoma of the tongue (table 1). Of the 126 cases (including the two patients reported in this paper), 106 identified the gender of the patient which showed an almost equal gender predilection with 50 males (47.2%) and 56 females (52.8%). Tongue schwannomas were also noted at all age ranges, but had a significantly higher incidence between the second and fourth decades of life (fig . 3).table 1patient and tumor characteristics of tongue schwannomasauthoryearage / genderlocation of tumorsize (greatest dimension, mm)presenting symptomssurgical approachcameron 195925manterior15painless masstransoralmercantini et al . 1960report of 5 cases; patient and tumor characteristics not describedchadwick 196420fposterior22globustransoralcraig 19648fposterior30painless masstransoralchhatbar 196529mposterior50throat discomforttransoralpantazopoulos 196525manterior<10painless masstransoral45fposterior45dysphagia, change in voicetransoralfirfer et al . 196725mposteriorhazelnut sizepainless masstransoral60fanteriorpea sizepainless masstransoralreport of 46 more cases (18 males, 28 females); patient and tumor characteristics not describedoles et al . 196824manterior10painless masstransoral23manterior5painless masstransoralbititci 196940manterior25slight discomforttransoraldas gupta 196921fposterior50paintransoralreport of 7 more cases; patient and tumor characteristics not describedcherrick et al . 200626fposterior40dysphagia, snoring, change in voicesuprahyoidcohen and wangcurrent study18fposterior18painless masstransoral77manterior7painless masstransoralcase report of malignant schwannoma of the tonguefig . 3age at presentation of patients with tongue schwannomas patient and tumor characteristics of tongue schwannomas case report of malignant schwannoma of the tongue age at presentation of patients with tongue schwannomas the location of the schwannoma was categorized as either anterior two - thirds or posterior one - third of the tongue . This classification was possible in 56 of the 126 cases with 37 cases occurring anteriorly and 19 cases identified posteriorly . Descriptions of the patient s clinical symptoms were only found in 57 case reports . The majority of patients presented with an asymptomatic mass (39/57; 69.6%). Of the patients that did present with a symptomatic mass, the most common complaints were throat pain or discomfort, dysphagia, and voice change (table 2). When the location of the schwannoma and the presence of symptoms were examined, a correlation was identified . Of the 19 patients with posterior one - third tumors, 12 patients reported symptoms (63.2%). On the other hand, in the anterior two - thirds group, only five patients reported symptoms (13.5%).table 2presenting symptoms of patients with tongue schwannomassymptomnumber of patients (n = 56)painless mass39throat pain / discomfort7dysphagia4change in voice3globus sensation2difficulty breathing2snoring2ulceration with pain1 presenting symptoms of patients with tongue schwannomas the exact size of the schwannoma was documented in 53 patients . The average size of the excised tumor in all documented patients was 24.04 mm . When the size of the tumor was examined between the symptomatic and asymptomatic groups, a clear distinction was noted . The average schwannoma size in the asymptomatic groups was 18.2 versus 33.0 mm in the symptomatic group . The remaining patients had their tumors excised via a transhyoid, suprahyoid, or submandibular approach . It originates from the schwann cell of the peripheral, autonomic, and cranial nerve . It is usually a single, circumscribed, firm, painless lesion of variable size . Approximately 2545% of all schwannomas occur in the head and neck with the parapharyngeal space being the most common site . Less commonly, schwannomas present in the oral cavity, and of this subset, the tongue is the most frequently involved [5, 6]. In this literature review of the past 51 years, the majority of them present as a painless mass . However, as they approach 3 cm in greatest dimension, they are more likely to produce symptoms such as throat discomfort, dysphagia, and voice changes . Moreover, if the schwannomas arises in the posterior two - thirds of the tongue, they are more likely to produce debilitating symptoms . The differentiation between schwannoma and neurofibroma is essential because an apparently solitary neurofibroma may be a manifestation of neurofibromatosis . Approximately 15% of patients with neurofibromatosis will have malignant transformation in one or more lesion, which is in marked contrast to the typical behavior of a schwannoma . Two main patterns are seen intermingled but sharply defined from each other . The first pattern is referred to as antoni type a which consists of closely packed schwann cells that form bundles or are arranged in rows with palisading, elongated nuclei . The second pattern is known as antoni type b and is composed of very loosely arranged schwann cells set in a meshwork of reticulum fibers and microcysts . In addition to these characteristic patterns, diagnosis is aided by immunohistochemical markers, s-100 and leu 7, which support the schwann cell nature of these tumors . These authors, in addition to others, noted extensive degeneration within the schwannoma and attributed this change to the aging process of the schwannoma . Aside from nuclear atypia, additional changes associated with the degenerative process include formation of cysts, stromal edema, xanthomatous change, and fibrosis . This hypothesis is supported by the finding that the degenerative changes correlate with tumor size . It is now generally accepted that all schwannomas can demonstrate some degenerative changes and the ancient variety probably represents the far end of the continuum of histologic appearance . Several cases of malignant transformation of head and neck schwannomas have been reported [2, 17], including one occurrence in the tongue . To support a diagnosis of malignant transformation in benign schwannoma, the following features should be confirmed: (a) the tumor demonstrates, to some extent, benign schwannoma; (b) the tumor contains unequivocal malignant foci as manifested by the presence of increased cellularity, numerous mitoses, anaplastic cells, and invasiveness; (c) transitional features between malignant and benign areas can be seen; and (d) the patient has no evidence of von recklinghausen s disease . The mr image is not degraded by dental amalgam or the beam - hardening artifacts that plague ct scanning of the oral cavity . In addition, mr allows an accurate measurement of tumor size and precise localization in relation to other structures . On mr, tongue schwannomas appear isointense to muscle on t1-weighted images and homogenously hyperintense on t2-weighted images . Moreover, these tumors usually appear smooth, well demarcated, and do not invade the surrounding musculature . This is an obvious choice for approaching these tumors since most are easily accessible via this route . Several other approaches have also been reported to have success including submandibular, suprahyoid pharyngotomy, and transhyoid approaches . All of these approaches were used for base of tongue schwannomas that were deemed difficult to approach by the transoral route . More recently, the use of co2 laser for excision of a base of tongue schwannoma has also been reported . If this is accomplished, recurrence is rare . In evaluating a patient with a slow - growing tongue mass that has been present for a long period of time, in addition to schwannomas, the differential diagnosis should include neurofibromas, granular cell tumors, irritation fibromas, leiomyomas, rhabdomyomas, hemangiomas, lymphangiomas, lipomas, pyogenic granulomas, and benign salivary gland tumors . Malignant etiologies should always be considered, but are unlikely to present with the slow clinical course that is typical of a schwannoma . Transoral resection allows for removal of this tumor in a manner that precludes recurrence, avoids causing morbidity of tongue function, and remains the standard approach for treatment of the vast majority of these tumors.
Obesity is associated with a low - grade proinflammatory state resulting in an increase of circulating cytokines and inflammatory markers . Inflammatory cytokines have been involved in the impairment of insulin signaling, thus providing molecular links between inflammation and insulin resistance . Inflammation reportedly produces metabolic alterations in skeletal muscle with both inflammatory response and insulin resistance being associated with loss of muscle mass by decreased protein synthesis and increased proteolysis [35]. Recently, our group has shown that leptin reverses muscle loss of ob / ob mice by inhibiting the activity of the transcriptional factor forkhead box class o3a (foxo3a). Leptin is an adipocyte - derived peptidic hormone that inhibits food intake and increases thermogenesis by acting through its hypothalamic receptors [8, 9]. Leptin - deficient ob / ob mice are obese, hyperphagic, exhibit type 2 diabetes, decreased body temperature and hypogonadotropic hypogonadism . Leptin is a member of the long - chain helical cytokine family and its receptors, which belong to the class i cytokine receptors, are present in bone marrow and spleen as well as on peripheral monocytes and lymphocytes . Leptin increases in response to acute infection and sepsis and it has been reported to exert a profound influence on the function and proliferation of t lymphocytes and natural killer cells, on the phagocytosis of macrophages / monocytes, and to have a direct effect on the secretion of anti- and proinflammatory cytokines . In this regard, impaired cellular and humoral immunity have been shown in leptin - deficient ob / ob mice as well as in leptin receptor - deficient db / db mice [14, 15]. These studies reflect the molecular nature of leptin as a cytokine and are consistent with leptin signaling playing a pivotal role in the pathogenesis of obesity - associated inflammation and muscle loss . In the present paper, gastrocnemius muscle samples from wild type and ob / ob mice were analyzed for mrna presence of over 41,000 transcripts by microarray analysis to identify genes involved in inflammation and oxidative stress that are affected by leptin deficiency and leptin administration in ob / ob mice . It was shown that leptin increases the gastrocnemius weight and reduces the high expression levels of genes related to the obesity - associated low - grade inflammation in skeletal muscle of ob / ob mice . Ten - week - old male genetically obese ob / ob mice (c57bl/6j) (n = 15) and their lean control littermates wild type (n = 5) supplied by harlan (barcelona, spain) were housed in a room with controlled temperature (22 2c) and a 12:12 light - dark cycle (lights on at 08:00 am). Body weight of ob / ob mice was measured before randomization into control, leptin - treated (1 mg / kg / d) and pair - fed groups (n = 5 per group). The control and pair - fed groups received vehicle (pbs), while leptin - treated mice were intraperitoneally administered with leptin (bachem, bubendorf, switzerland) twice daily at 08:00 am and 08:00 pm for 28 days . Control and leptin - treated groups were provided with water and food ad libitum with a standard rodent chow (2014s teklad, harlan), while daily food intake of the pair - fed group was matched to the amount consumed by the leptin - treated group the day before in order to discriminate the inhibitory effect of leptin on appetite . Animals were sacrificed on the 28th day of treatment by co2 inhalation 20 hours after the last pbs or leptin administration (in order to avoid picking up effects reflecting an acute response) and after 8 hours of fasting . All experimental procedures conformed to the european guidelines for the care and use of laboratory animals (directive 86/609) and were approved by the ethical committee for animal experimentation of the university of navarra (080/05). Serum glucose was analyzed using a sensitive - automatic glucose sensor (ascensia elite, bayer, barcelona, spain). Free fatty acid (ffa) concentrations were measured by a colorimetric determination using the nefa c kit (wako chemicals, neuss, germany). Serum triglycerides (tg) concentrations were spectrophotometrically determined using a commercial kit (infinity, thermo electron, melbourne, australia). Insulin and leptin were determined using specific mouse elisa kits (crystal chem inc ., intra- and interassay coefficients of variation for measurements of insulin and leptin were 3.5% and 6.3%, respectively, for the former, and 2.8% and 5.8%, for the latter . Adiponectin concentrations were also assessed using a mouse elisa kit (biovendor laboratory medicine, inc ., modrice, czech republic). Insulin resistance was calculated using the homeostasis model assessment score (homa; fasting insulin (u / ml) fasting glucose (mmol / l)/22.5). An indirect measure of insulin sensitivity was calculated by using the quantitative insulin sensitivity check index (quicki; 1/[log(fasting insulin mu / ml) + log(fasting glucose mg / dl)]. Lipid peroxidation was analyzed by the measurement of thiobarbituric acid reactive substances (tbars) in serum and gastrocnemius as previously described by conti et al . With some modifications . Since the best - known specific tbars is malondialdehyde (mda), we used serum mda levels, a secondary product of lipid peroxidation, as an indicator of lipid peroxidation and oxidative stress . Gastrocnemius samples (2030 mg) were homogenized in 20 volumes of phosphate buffer ph 7.4 . Serum, muscle homogenates (5 l) or standard (mda) were mixed with 120 l of diethyl thiobarbituric acid (detba) 10 mm and vortexed for 5 seconds . The reaction mixture was then incubated at 95c for 60 minutes . After cooling to room temperature detba - mda adducts were extracted in 360 l n - butanol vortexing for 1 minute and centrifuged at 1,600 g for 10 minutes at room temperature . Then, the chromophore of the detba - mda adduct was quantified in 200 l of the upper butanol phase by fluorescence emission at 535 nm with an excitation at 590 nm . Mda equivalents (tbars) were quantified using a calibration curve prepared using mda standard working solutions and expressed as serum mda m and gastrocnemius mda m / mg protein . Protein concentrations were determined using a bradford protein assay kit (biorad, hercules, ca, usa). Total rna was extracted from 2030 mg of gastrocnemius muscle samples by homogenization with an ultra - turrax t 25 basic (ika werke gmbh, staufen, germany) using trizol reagent (invitrogen, barcelona, spain). Rna was purified using the rneasy mini kit (qiagen, barcelona, spain) and treated with dnase i (rnase - free dnase set, qiagen) in order to remove any trace of genomic dna . Gene expression analyses were conducted using the agilent whole mouse genome array (g4121b, agilent technologies, santa clara, ca, usa) containing ~41, 000 mouse genes and transcripts . Fluorescence - labeled cdna probes were prepared from 1 g of total rna from each sample (5 animals per group) to be subsequently amino - allyl labeled and amplified using the amino allyl messageamp ii arna amplification kit (ambion, austin, tx, usa). Aliquots (1.2 g) of amplified arna were fluorescently labeled using cy3/cy5 (amersham biosciences, buckinghamshire, uk) and then appropriately combined and hybridized to agilent microarrays . Hybridizations were performed following a reference design, where control samples were pools of rna from all individual samples . Two hybridizations with fluor reversal (dye - swap) were performed for each sample . After washing, microarray slides were scanned using a gene pix 4100a scanner (axon instruments, union city, ca, usa) and image quantization was performed using the software genepix pro 6.0 . Gene expression data for all replicate experiments were analyzed using the genespring gx software version 7.3.1 (agilent technologies). In addition, gene ontology database (http://babelomics.bioinfo.cipf.es) and the kegg website (http://www.genome.ad.jp/kegg/pathway) were used in conjunction with genespring (http://www.agilent.com/chem/genespring) to identify pathways and functional groups of genes . More information regarding the microarray experiments can be found at the embl - european bioinformatics institute (http://www.ebi.ac.uk/aerep/login . Arrayexpress accession number: e - mexp-1831). To validate the microarray data, a number of representative differentially expressed genes were selected to be individually studied by real - time pcr (7300 real time pcr system, applied biosystems, foster city, ca, usa) (n = 5 per group) as previously described . Primers and probes were designed using the software primer express 2.0 (applied biosystems) and purchased from genosys (sigma, madrid, spain) (table 1). Differences between groups were assessed by kruskal - wallis followed by mann whitney's u test . As previously outlined, gene ontology groupings were used to identify pathways significantly affected by leptin deficiency as opposed to its administration . Furthermore, statistical comparisons for microarray data to identify differentially expressed genes across different groups were performed using one - way anova and student's t - tests as appropriate . All statistical analyses were performed by using the spss statistical program version 15.0 for windows (spss, chicago, il, usa) and statistical significance was defined as p <.05 . The morphological and biochemical characteristics of wild type and ob / ob mice are reported in table 2 . As expected, leptin treatment corrected the obese and diabetic phenotype of ob / ob mice . Body weight was significantly higher (p <.01) in the control ob / ob group as compared to wild type mice . Leptin - treated mice exhibited a decreased body weight (p <.01) as compared to control and pair - fed ob / ob animals . Importantly, leptin treatment normalized body weight of ob / ob mice as compared to wild type (p = .690). In addition, the gastrocnemius of control ob / ob mice exhibited a lower (p <.01) muscle weight than that of wild type mice and it was increased (p <.01) by leptin administration in comparison with that of control and pair - fed ob / ob rodents . As depicted in table 2, higher fasting glucose (p <.05) and insulin (p <.01) concentrations were observed in the control ob / ob mice compared to wild types . Although no differences in glucose concentrations were observed in pair - fed as compared to leptin - treated ob / ob mice, higher serum insulin concentrations (p <.05) were detected in the pair - fed animals than in the leptin - treated ob / ob group . Furthermore, leptin administration normalized both the glucose and insulin levels in ob / ob mice compared to wild types . These data suggest that leptin increases the insulin sensitivity in peripheral tissues, as evidenced by the lower homa and higher quicki indices (p <.01) in the leptin - treated in comparison with the control ob / ob animals . Serum glycerol was markedly increased (p <.05) in the control ob / ob mice, while ffa and tg levels remained unchanged as compared to wild type mice . Interestingly, leptin not only decreased circulating concentrations of ffa (p <.05) and glycerol (p <.01) levels as compared to control ob / ob mice, but also ffa (p <.01), glycerol (p <.01) and tg (p <.05) concentrations as compared to pair - fed mice . Leptin administration to ob / ob mice reduced serum glycerol concentrations (p = .032) and tended to decrease ffa (p = .095) as compared to wild types . Furthermore, leptin treatment increased the low concentrations of adiponectin of ob / ob mice, but the differences fell out of statistical significance (p = .095). Control ob / ob mice exhibited significantly higher serum tbars than wild type littermates (p <.01), which were significantly reduced after leptin administration as compared to the control (p <.01) and pair - fed (p <.05) ob / ob groups (figure 1(a)). In addition, leptin decreased (p <.01) the high concentrations of mda measured in the gastrocnemius muscle of control ob / ob mice, while this effect was not observed in the pair - fed group (figure 1(b)). Serum and gastrocnemius tbars levels were positively associated with body weight, ffa, insulin, and the homa index . Oppositely, tbars levels were negatively associated with adiponectin and the quicki index both in serum and muscle . Importantly, a high positive relation were found between serum and gastrocnemius concentrations of tbars (= 0.63, p = .003) (table 3). Differential gene expression profiles in gastrocnemius muscle of wild type and ob / ob groups were compared by microarray analysis . Only genes whose mrna levels were changed 1.5-fold or higher and identified as significantly changed by statistical analysis were designated as differentially expressed genes . Applying these criteria, microarray data showed that 7,582 genes were differentially expressed by leptin deficiency and leptin administration in ob / ob mice . In particular, leptin deficiency altered the expression of 1,127 genes between wild type and control ob / ob mice . Of these, 580 were upregulated and 547 were downregulated in ob / ob mice . Leptin treatment modified the expression of 1,546 genes in ob / ob mice, upregulating 512 and repressing 1,034 . In addition, leptin repressed 736 genes that were upregulated in gastrocnemius muscle of control ob / ob and increased the transcript levels of 846 downregulated genes . Functional enrichment analysis using geneontology and kegg databases revealed that the set of genes with altered expression levels induced by leptin deficiency and administration represents a broad spectrum of biological processes . However, for the purpose of the present paper we focused on the effects of leptin on the set of genes encoding proteins involved in oxidative stress and inflammation . Table 4 shows that leptin deficiency and leptin administration altered the expression of a large number of genes involved in oxidative stress and inflammation . The biological processes mainly affected between control ob / ob mice and wild types included furthermore, several processes regulating proliferation, differentiation, and activity of lymphocytes were also significantly affected by leptin deficiency . Importantly, comparison of leptin - treated and control ob / ob groups showed that leptin administration altered the expression of genes implicated in the positive regulation of lymphocyte activation (p = .0187), as well as genes involved in the chaperone cofactor dependent protein folding dna microarray analysis showed that 86 genes encoding proteins related to defense, stress, and inflammatory responses were altered in the gastrocnemius muscle of control ob / ob mice and modified by leptin administration . Leptin reduced the mrna levels of various isoforms of the family of heat shock proteins (hsps) (dnajc16, dnaja4, dnajb4, hspa2, hspa4, and hspb7), metallothioneins (mt2, mt4), crystallins (cryab, crybb1) and rna binding proteins (rbms) (rbm9, rbm22) in ob / ob mice (table 5). In addition, histocompatibility 2, complement component factor b h2-bf and several genes of the acute - phase response or inflammatory processes, such as kallikrein 5 (klk5), and serine (or cysteine) proteinase inhibitor clade c member 1 (serpinc1) and clade b member 1a (serpinb1a), displayed an increased expression in ob / ob mice that was reduced by leptin administration . On the contrary, gene expression of cryl1, hsp105, rbm5, and h2-aa were enhanced in ob / ob mice after treated with leptin . Pair - feeding, which accounts for the decrease in food intake that is independent of the direct action of leptin, altered the expression of 1,960 genes, upregulating 984 while downregulating 976 genes . In the context of a food intake reduction as compared to the simple effect due to the caloric restriction, leptin administration further significantly altered the expression of genes involved in processes encompassing (p = 2.99e), positive regulation of t cell activation (p = .0003) and positive regulation of immune cell mediated cytotoxicity in particular, the gene array analysis provided evidence for elevated hspa4, mt4, crybb1, and serpinb8 mrna levels in the pair - fed group as compared to the leptin - treated ob / ob mice (table 6). On the contrary, leptin increased the gene expression of h2-ab1 and h2-eb1 in ob / ob mice . To confirm the microarray data, the mrna expression of several representative transcripts was analyzed by real - time pcr (figure 2). In this sense, leptin administration reduced the mrna levels of the muscle atrophy - related transcription factor forkhead box o1 (foxo1) and of the e3 ubiquitin - ligases muscle atrophy f - box (mafbx) and muscle ring finger 1 (murf1) in leptin - treated ob / ob mice, while no effect of leptin was evidenced on the mrna levels of the transcriptional coactivator peroxisome proliferator - activated receptor- coactivator-1 (pgc1). Obesity is accompanied by a chronic proinflammatory state associated not only with insulin resistance, but also with muscular atrophy [4, 5]. Our study provides evidence that leptin constitutes a negative regulator of oxidative stress and inflammation in the gastrocnemius, which is a representative skeletal muscle of the whole skeletal musculature . This statement is supported by findings reported herein: (a) leptin deficiency is accompanied by systemic and skeletal muscle oxidative stress, muscle inflammation, and reduced muscle mass; (b) systemic and skeletal muscle oxidative stress, muscle atrophy and inflammation of ob / ob mice are reversed by leptin administration independently of the effects of food intake inhibition . Therefore, leptin is able to prevent the muscle atrophy associated with obese and inflammatory states . Skeletal muscle constitutes an important target for leptin playing a key role on the regulation of lipid and glucose metabolism . Since obese ob / ob mice exhibit an increased oxidative stress and impaired immune response [14, 15] and a reduced skeletal muscle mass compared with their lean littermates, we aimed to identify the genes related to inflammatory processes differentially altered by leptin in the gastrocnemius muscle of obese ob / ob mice . In particular, 86 transcripts encoding inflammation - related proteins were shown to be modified by exogenous leptin administration . However, it has to be taken into account that many of these genes are multifunctional and may have important functions in other biological processes . Among them, leptin repressed the high expression levels of acute - phase reactants and several members of the hsp and rbm families . In addition, confirming a previous study of our group, leptin treatment increased the reduced muscle weight of gastrocnemius muscle of ob / ob mice . Taken together, these data suggest that leptin may prevent the obesity - associated inflammatory state and the muscle mass loss related to inflammatory states in leptin - deficient ob / ob mice . Leptin - deficient ob / ob and leptin receptor - deficient db / db mice display many abnormalities in the immune response similar to those observed in starved animals and malnourished humans [14, 15, 22]. In this respect, exogenous leptin replacement to ob / ob mice modulates t cell responses in mice and prevents starvation - induced immunosuppression, suggesting that lack of leptin is directly involved in these immune system abnormalities [23, 24]. In agreement with these studies, our findings show that leptin deficiency and administration differentially regulate biological processes related to the immune response as well as the t and b cell differentiation and activation in gastrocnemius muscle of ob / ob mice . Oxidative stress is defined as the imbalanced redox state in which prooxidants overwhelm the antioxidant capacity, resulting in an increased production of reactive oxygen species (ros), ultimately leading to oxidative damage of cellular macromolecules . Dismutation of o2 by superoxide dismutase (sod) produces hydrogen peroxide (h2o2), a more stable ros, which, in turn, is converted to water by catalase and glutathione peroxidase (gpx). Oxidative stress is increased in diabetes [26, 27] with leptin administration reportedly improving insulin sensitivity in normal and diabetic rodents [2830]. Leptin stimulates in vitro ros production by inflammatory cells and endothelial cells and the level of systemic oxidative stress in nonobese animals [33, 34], suggesting a prooxidative role of leptin . However, administration of recombinant leptin reduces the oxidative stress induced by a high - fat diet in mice . In this sense, findings of our study show a high oxidative stress in diabetic ob / ob mice, as reflected by increased tbars concentrations in serum and the gastrocemius muscle . These observations are in agreement with a large number of studies related to increased plasma tbars or mda in diabetic rats and humans . Lipid peroxidation is a common index of free radical mediated injury and induction of antioxidant enzyme is a common cellular response . More importantly, leptin administration decreased serum and gastrocnemius tbars concentrations as compared to control ob / ob mice, with tbars levels in gastrocnemius muscle from pair - fed ob / ob animals remaining very similar to those of control ob / ob mice . In this sense, from a molecular perspective, our results further show that transcript levels of sod1, gpx3 and glutathione s - transferase 1 gstp1 are downregulated in control ob / ob mice as compared to wild type controls being upregulated after leptin treatment . Furthermore, leptin administration also upregulated gpx7, glutathione s - transferase 5 (gstm5) and glutathione s - transferase 2 (gstt2). On the contrary, the high expression of catalase (cat) was repressed by the exogenous injection of leptin to ob / ob mice . These findings are in line with previous observations showing the restoration of the defective antioxidant enzyme activity in plasma of ob / ob mice and humans with a leptin gene mutation . Acute - phase reactants have been suggested to contribute to the maintenance of the chronic low - grade inflammation state involved in the progression of obesity and related diseases . Interestingly, our study provides evidence that genes of the acute - phase response were altered in gastrocnemius muscle of ob / ob mice, which were counteracted by exogenous leptin administration . Leptin reduced the elevated gene expression of tissue - type plasminogen activator (plat) and lipocalin-2 (lcn2), which are upregulated in many inflammatory conditions [42, 43], including human obesity . In addition, a pivotal role for oxidative stress in the pathogenesis of muscle wasting in disuse and in a variety of pathological conditions is now being widely recognized . A potential link between oxidative stress and muscle moreover, various inflammatory cytokines induce oxidative stress and muscle atrophy through the activation of the lysosomal [48, 49] and the ubiquitin - proteolysis system . In this context, biological processes related to oxidative stress and inflammatory responses were altered in the gastrocnemius muscle of ob / ob mice and improved following leptin treatment . In spite of the usual upregulation of the e3 ubiquitin - ligases mafbx and murf1 in most conditions associated with atrophy, their gene expression levels in ob / ob were lower as compared to wild type animals, although no statistically significant differences were observed . Contrarily to what would be expected, leptin administration prevented the increase of both mafbx and murf1 mrna expression levels induced by pair - feeding in ob / ob mice . A plausible explanation for this surprising finding may relate to the fact that in extreme conditions the energy homeostasis system is overriden whereby leptin is able to inhibit muscular protein degradation associated to food intake reduction . These data are in accordance with a previous study of our group evidencing that leptin replacement inhibits the ubiquitin proteolysis system activity in leptin - deficient mice . Muscle atrophy is associated with increased expression of genes coding for rbm proteins which facilitate the translation, protection, and restoration of native rna conformations during oxidative stress . It has been suggested that the gene expression of rbm proteins may increase as a compensatory mechanism in response to loss of muscle proteins [51, 52]. Other proteins involved in oxidative stress are metallothioneins, endogenous antioxidants that have been shown to be overexpressed in muscle atrophy in rodents [5456]. In the present work, we have observed that administration of leptin inhibits the gene expression of several members of the rbm (rbm9, rbm22) and metallothioneins (mt2, mt4) families in the gastrocnemius of ob / ob mice, suggesting that leptin may modulate the inflammatory and oxidative stress responses and consequently, the muscle loss related to inflammatory states . Genes involved in the chaperone system were also differentially expressed in ob / ob mice as compared to wild types and modified by leptin treatment . Hsps represent a family of molecular chaperones induced in response to cellular stress, responsible for maintaining the structure of proteins and contributing to the repair of damaged or malformed proteins in highly oxidative and lipotoxic conditions . As a result in fact, hsps are repressed in many rat models of skeletal muscle atrophy [54, 59, 60]. Hsp70 is constitutively expressed in skeletal muscle, but its levels are increased in response to oxidative stress with the induction of hsp70 expression by hyperthermia and during inactivity attenuating muscle atrophy [62, 63]. In this regard, a recent study has shown that hsp70 prevents muscle atrophy induced by physical inactivity through inhibition of the muscle atrophy - related transcription factor foxo3a and the expression of mafbx and murf1 . Among the hsps, hsp70 and b - crystallin in particular, are considered negative regulators of muscle cell apoptosis [65, 66] and may inhibit the loss of nuclei taking place during muscle atrophy . In addition, ros induce the activity of foxo and gene expression of members of the ubiquitin - proteolysis system in myotubes . In this sense, our results provide evidence that leptin inhibits the increased gene expression of different members of the hsps (hspb7, dnajc16, hspa4, cryab, and crybb1) in the gastrocnemius muscle of ob / ob mice . Taken together, the elevated expression of hsps in the control and pair - fed ob / ob groups suggests a high defense and stress response in these mice . Moreover, induction of hsps may confer broader health benefits to patients who are insulin resistant or diabetic . In mammals, caloric restriction has been shown to upregulate hsp induction [70, 71], while expression of hsp72 has been found to be low in skeletal muscle of patients with insulin resistance or type 2 diabetes [72, 73]. Have recently shown that leptin downregulates hsp70 gene expression in chicken liver and hypothalamus but not in muscle, which was independent of food intake restriction . On the contrary, bonior et al . Reported an increase in hsp60 gene expression in pancreatic cells by leptin . Obesity is accompanied by a chronic proinflammatory state resulting in an increase in circulating cytokines and inflammatory markers . In this regard, inflammation produces metabolic alterations in skeletal muscle with both inflammatory response and insulin resistance being associated with muscle mass loss . Findings of our study provide evidence that systemic and skeletal muscle oxidative stress, muscle atrophy and the elevated expression of genes involved in oxidative stress and inflammation of ob / ob mice are reversed by leptin administration . Taken together, these data thereby support that leptin is able to prevent the muscle atrophy associated with obese and inflammatory states in ob / ob mice . Most obese people develop muscle atrophy in spite of exhibiting high leptin circulating concentrations, which may be explained by the leptin resistance present in these patients . Our paper sheds light on the relation between obesity and the loss of muscle mass associated to inflammatory states suggesting that leptin treatment may be an attractive therapeutic approach to prevent muscle loss associated with inflammatory diseases.
It is valuable for general practitioners and specialists alike to include novl in their differential when evaluating patients with visual complaints that are inconsistent with normal examination findings . Nonorganic vision loss (novl), also referred to as functional vision loss, is characterized by the onset of visual deficit or disturbance that cannot be explained by organic pathology . Typically, patients present with subjective visual complaints, but extensive diagnostic imaging, labs, and exams reveal no abnormalities . The first published case of novl was in 1865, however, despite its relatively long history there are few studies and case reports on the topic . This case describes a patient with an unusual presentation of novl, reviews the important features of diagnosis and management, and highlights the need for a heightened awareness of this condition . Prompt diagnosis can prevent extensive workups that are costly to the patient, as well as time - consuming and frustrating for both the patient and the clinician . A diagnosis of novl may be a clue of underlying psychological, academic, or social stress in a pediatric patient . As a consequence, novl offers an opportunity for the ophthalmologist and primary care physician to collaborate and address psychiatric, psychosocial, and emotional issues uniquely presenting as visual deficits . In situations where visual disturbances are the sole clinical manifestation, those psychiatric or social issues may otherwise remain undetected and unaddressed . A 12-year - old girl was referred to a pediatric ophthalmologist for evaluation of abrupt onset of monocular visual field loss . Ten days prior to presentation, she had experienced a headache and blurry vision, and 1 day later half of the vision blacked out . She saw a local optometrist who performed humphrey visual field (hvf) testing and diagnosed her with monocular temporal hemianopsia of her left eye . She underwent magnetic resonance imaging (mri) of the brain and orbit 6 days prior to presentation, which was repeated twice more prior to presentation due to metallic artifacts from dental braces, and all yielded normal results . The patient was given one course of intravenous methylprednisolone with no improvement of her symptoms . The patient was the product of a full - term, uncomplicated pregnancy and had no significant past medical history . She was a seventh - grade student with no recent changes in her academic progress . She lived with her mother, stepfather, brother, sister, brother - in - law, and nephew . She denied sexual activity and any use of illicit substances . On review of systems, the patient denied any other neurological or visual disturbances . Upon examination, she had 20/20 visual acuity without correction in both eyes at distance and near . The pupils were equal, round, reactive to light without an afferent pupillary defect . Objective slit - lamp examination of her lids, lashes, conjunctiva, cornea, anterior chamber, lacrimal system, and vitreous did not reveal any abnormalities in either eye . Optical coherence tomography (oct) was performed to analyze the macula and the retinal nerve fiber layer, and the results were normal . Monocular automated visual field testing revealed clear - cut temporal visual field defect of her left eye respecting the vertical meridian, which is consistent with her prior evaluations (fig.1). Automated humphrey visual field (hvf) test results . (a) an example of a normal hvf result of the right and left eyes with the physiological blind spot . (b) our patient's hvf test showing a temporal visual field defect of her left eye respecting the vertical meridian . (c) our patient's left eye hvf compared side - by - side with her left eye manual perimetry results . (d) of note, binocular visual field testing appeared identical to monocular testing confirming the diagnosis of nonorganic vision loss . The completely intact visual field in her fellow eye made a post - chiasmal lesion very unlikely . An anterior optic tract lesion, optic nerve compression, optic disk pathology, or monocular retinovascular disease can all produce a clinical picture similar to this patient, however, these were unlikely given her lack of afferent pupillary defect, normal color discrimination, and normal retinal exam / oct . Her unremarkable examination and the absence of any structural abnormalities raised some suspicion that the etiology of her visual defect may be nonorganic . The visual fields were tested using manual perimetry, which involves a skilled technician testing each point of the patient's visual field using illuminated targets of different sizes . Although the manual perimetry of the right eye remains normal, the left eye demonstrates a temporal visual field defect with blurred vertical meridian . When tested with both eyes open, the left temporal defect persists, which defies organic explanation . The patient was managed conservatively, primarily with reassurance and follow - up . Within 3 months novl is relatively common, occurring in 15% of pediatric patients in general ophthalmology practice . Many of these patients initially present to their primary care physician before seeing an ophthalmologist, so there is significant value in promoting more awareness about this disease . The most common clinical presentation is reduction in visual acuity with symmetric, bilateral eye involvement . Of novl patients with visual field deficit, patients with novl are frequently subjected to protracted evaluations and workups that are expensive, frustrating, and time - consuming for both the provider and the patient . It is valuable, therefore, for general practitioners and specialists alike to include novl in their differential when evaluating patients with visual complaints that are inconsistent with normal examination findings . Diagnosis of novl requires the absence of findings that are attributable to the vision loss, as well as the demonstration of positive test results that are physiologically inconsistent with the presenting visual complaints . In the example of our patient, persistence of the hemianopsia during binocular visual field testing and the absence of a relative afferent pupillary defect distinguish functional hemianopsia (i.e., nonorganic) from organic etiologies of visual field loss . On binocular visual field testing, a normal - functioning fellow eye will compensate for the defect in the field of the affected eye . Additionally, in the case of organic hemianopsia of this size and depth, one would expect to find an ipsilateral afferent pupillary defect . For example, visual - evoked potential (vep) provides the practitioner with objective assessment of a patient's visual acuity . In some cases, novl may be accompanied by organic disease, but in those instances, patients will rarely have a completely normal examination . Psychological and social stressors are thought to play a role in the development of novl in children . As a result, a diagnosis of novl should prompt clinicians to investigate possible triggers and associated psychopathologies that may have precipitated the child's ophthalmologic symptoms . Psychiatric conditions, such as conversion disorder, are also implicated as a cause of novl, however, psychiatric therapeutic management has not been shown to improve outcome or decrease the period of time the child experiences the visual deficit [2,1315]. In a small percentage of children with novl while this association is not common, it is important to consider it and the possibility should be carefully explored . In our patient, the addition of her nephew to the home was the likely trigger for the novl as there were no signs of physical or sexual abuse . Recognition and diagnosis of novl offers an opportunity for clinicians to identify and address issues in a child's life that may otherwise remain obscured, benefitting both the patient and the provider . The secondary goal associated with malingering may be as benign and simple as a desire to wear glasses . Malingering children are typically less cooperative with the examination and their findings are less likely to fluctuate over time . Often times the distinction between a malingerer and a patient with a somatoform / conversion disorder can be quite difficult . Regardless of the severity of the patient's clinical presentation, several studies have shown that the most effective management of novl is reassurance and follow - up [6,13,1618]. However, in situations where psychotherapy is specifically desired or is likely to confer a clear benefit to the patient, it should be discussed with the family as well as the primary care physician . Overall, the prognosis for these patients is excellent and the vast majority will have full recovery within several months to a year after diagnosis . The clinical approach to a pediatric patient with new - onset visual deficits should begin with referral for a basic ophthalmologic examination that includes visual acuity, slit - lamp, and fundoscopic exams, ocular motility, pupillary function, and cycloplegic refraction . If all of those tests yield normal results, more specific ophthalmologic tests should be undertaken prior to referral for a full neurologic workup . If novl is suspected, it must be positively supported by examination findings that establish normal visual function, and organic lesions should always be suspected and ruled out . This is important because some severe organic disorders can cause concomitant neuropsychiatric and visual manifestations (e.g., central nervous system neoplasms), so caution must be taken to not automatically dismiss visual complaints when psychiatric features are present . If novl is indeed supported by positive test results, the child's pediatrician and ophthalmologist should collaborate to rule out the presence of an underlying psychogenic cause . Referral for psychiatric evaluation may be helpful in cases with a high suspicion of a psychosocial stress or traumatic event, however, reassurance and follow - up are typically sufficient treatment measures in novl.
Obstructive azoospermia (oa) is present in 1020% of infertile men and is fundamentally characterized by normal testicular volume and reproductive hormones indicating normal spermatogenesis . While postvasectomy oa has been extensively reported by many authors, primary oa (unrelated to a prior vasectomy) is present in about 3%6% of all infertile men and most frequently is produced by epididymal obstruction . Primary oa (mainly after sexually transmitted diseases and rarely after iatrogenic lesion of the male genital tract) or congenital oa (bilateral absence of the vas deferens and idiopathic epididymal obstruction) may be acquired . Both microsurgical reconstruction of the spermatic tract and sperm retrieval followed by intracytoplasmic sperm injection (icsi) could be beneficial for such patients . Nowadays, urologist specialized in infertility should be able to accurately predict which patients have normal spermatogenesis and/or are suitable for surgical reconstruction . In the evaluation algorithm of patients with suspected oa normal serum concentration of follicle - stimulating hormone (fsh), a traditional marker for spermatogenesis, is the first step that differentiates primitive oa from non - oa [36]. Also, seminal anti - mllerian hormone and plasma antisemen antibodies are other potential noninvasive predictive markers for the presence of spermatogenesis in patients with suspected oa [79]. Percutaneous epididymal sperm aspirationhas a high rate of sperm retrieval for icsi regardless of the etiology of oa but with the risk of subsequent scaring that might end with a second obstruction located in the epididymal head nonfeasible for microsurgical reconstruction . Testicular fine needle aspiration (tfna) has been traditionally used to retrieve sperm in non - oa . Recently it was established that a primary azoospermic male with proven normal spermatogenesis by tfna and a normal fsh level should be diagnosed as oa and explored for reconstruction . Testicular sperm extraction (tese) is the best invasive procedure to predict which cases are suitable for microsurgical reconstruction but still cannot predict its results . The aim of this study was to assess our results obtained by microsurgical vasoepididymostomy (ves) in the treatment of men diagnosed with primary epididymal oa and to identify the factors associated with natural pregnancy occurring after microsurgical reconstruction . The patients were included in the study, if they had oa secondary to epididymal obstruction and fulfilled all the inclusion criteria (vide infra). The study end point was the occurrence of a natural pregnancy confirmed by appropriate diagnostic tools . In patients without a natural pregnancy, the local ethics committee approved the study protocol, and all the patients gave an informed consent at admission . All consecutive patients with oa secondary to epididymal obstruction admitted to our department between september 2008 and october 2012 in whom microsurgical reconstruction was achievable were considered for inclusion (n = 65). The diagnosis criteria of oa secondary to epididymal obstruction were azoospermia diagnosed on at least 2 semen analyses after centrifugation, vas deferens clinically present, seminal volume 1 ml, semen ph 7, seminal fructose present, normal serum level of follicle stimulating hormone (fsh), luteinizing hormone (lh) and testosterone, at least one testicle with a volume 10 cc, and the width of the epididymis caput 5 mm, as measured by ultrasound . Microsurgical reconstruction was considered feasible in all patients who fulfilled the following criteria: (1) at least one viable spermatozoon in the aspirated fluid by microsurgical epididymal sperm aspiration (mesa), (2) a dilated epididymal tubule located cranially to the level of viable spermatozoa aspirated by mesa, and (3) a patent vas deferens . No exploration of the vas deferens was performed if criteria 1 and 2 were not achieved . A dilated / not dilated tubule was defined as a tubule large / not large enough to allow placement of the needles used in reconstruction . Patients were excluded if they had a history of vasectomy, attempted microsurgical reconstruction and pre / intraoperative diagnostic of bilateral congenital absence of vas deferens, infertile female partner, and lack of desire to have children . The patency was defined as a concentration of more than 0.1 10 sperms / ml . The final study population included 36 patients with primary epididymal oa who underwent microsurgical reconstruction (figure 1). The patient's examined characteristics included age, age of female partners, smoking status, testicular volume, thickness of the right and left epididymis, total motile sperm count (tmsc), and serum reproductive hormones (fsh, lh, testosterone). Other parameters of interest were prior fertility status (natural pregnancy in the past history) and medical history of urological infections . All semen specimens were collected by masturbation at the clinical andrology laboratory after a period of 35 days of abstinence . The patients who underwent microsurgical reconstruction were advised to wait until the end of the postoperative follow - up period before taking any other treatment option for infertility . A microsurgical transversal end - to - side double arm single tubule ves was performed as was previously described by marmar . The tail of the epididymis was identified and a microscope (optical magnification 1230) was brought into the operatory field . The tubule was opened, mesa was performed, and the epididymal fluid was examined in the operating room for viable spermatozoa using light microscopy . If they were present, the fluid was sent for cryopreservation and ves was attempted if the epididymal tubule located 5 mm cranially was dilated . The vas deferens and its pedicle were transected transversally as close as possible to the testicle, but in its straight part, and dissected cranially 3 - 4 cm to obtain a tension - free anastomosis with the epididymis . The vas deferens was considered if the injection was easy and the urine turned green in the collection bag . In case of patent vas deferens, a single epididymal tubule was dissected 5 mm cranially to the level where the viable sperms were identified . Two parallel double needles (6.5 mm) 10.0, 30 cm long sutures (ethicon) were placed in the selected epididymal tubule, oriented transversally . The double - arm needles were placed in - to - out through the vassal lumen finishing the anastomosis . If the motile spermatozoa were not present, more proximal mesa was performed until motile spermatozoa were identified . If viable spermatozoa were not identified along the entire epididymis, a multisite testicular biopsy (tese) was performed and sent for sperm cryopreservation . Continuous variables are presented as mean or median and 95% confidence interval, according to their distribution, and categorical variables as percentages . Group comparisons were performed with student's t - test, test, and mann - whitney u test, as appropriate . Receiver operator characteristic (roc) curve analysis was performed to evaluate the utility and to identify cut - off values for parameters significantly correlated with the natural pregnancy occurrence . A multivariable cox regression analysis was performed in order to determine the factors associated with the natural pregnancy occurrence . Hazard ratios are expressed per natural logarithm unit of tmsc, per unit of age, fsh, lh, and testosterone, and dichotomized for prior fertility status and medical history of urological infections . Analyse - it (analyse - it software, ltd ., leeds, uk) and spss (spss inc . Thirty - six patients with primary oa secondary to epididymal obstruction who underwent microsurgical reconstruction were included . Baseline characteristics of the study population are displayed in table 1 . At the time of study inclusion, the median age of patients was 34 [iqr 3137] years and the median age of female partners was 32 [iqr 2936] years . The total patency rate in the study group was 77.7% (n = 28). In the group of patients with patent anastomosis, 4 (14.3%) patients had biological father status and 15 (53.6%) patients had medical history of infection of the male genital tract . During the follow - up period, no patient was lost and in 8 (28.6%), out of 28 subjects with patent anastomosis, natural pregnancies occurred with an overall live birth rate of 100% . In univariate analysis, as compared to those without natural pregnancy, the patients with natural pregnancy had significantly higher level of the total motile sperm count (tmsc) (p = 0.0001). Also, patients with natural pregnancy during the follow - up period had significantly lower fsh levels when compared with patients with no natural pregnancy (p = 0.02) (table 1). In contrast, baseline lh and testosterone levels were not associated with natural pregnancy (p = 0.75, resp ., p = 0.70). Also, testicular volume and thickness of the right and left epididymis the median age of female partners with and without natural spontaneous pregnancy was 30 and 31.5 years (p = 0.41), respectively . History of an infection of the male genital tract showed an inverse relationship with pregnancy occurrence, although not significant (p = 0.38). Also, a positive prior fertility status showed a direct relationship with pregnancy occurrence, although not significant (p = 0.56). Next we performed a multivariate analysis, including age, age of female partners, fsh, lh, testosterone, tmsc levels, prior fertility status, and medical history of urological infections at the time of study start . The results of multivariable cox regression analysis are shown in table 2 . According to multivariate analysis, two variables were independently related to increased rates of natural pregnancy occurrence after microsurgical reconstruction: higher tmsc and low fsh level at the time of study start . An increase of tmsc by 1 natural logarithm unit was associated with a 1.001-fold increase of spontaneous pregnancy rates (95% ci: 11.001). An increase of fsh by 1 ui / l was associated with a 0.22-fold increase of spontaneous pregnancy rates (95% ci: 0.0520.88) (table 2). Roc curve analysis was performed to identify the cut - off values for tmsc and fsh levels which predict a natural pregnancy occurrence after microsurgical reconstruction . Thus, a tmsc of 37.30 10 has a sensitivity of 87.5% and a specificity of 85% to predict a spontaneous pregnancy occurrence after microsurgical reconstruction (figure 2). Also, a fsh level of 2.6 ui / l has a sensitivity of 85% and a specificity of 62.5% to predict a spontaneous pregnancy occurrence after microsurgical reconstruction (figure 3). Sperm retrieval and icsi have dramatically improved the chances for patients diagnosed with azoospermia to obtain a pregnancy . Although icsi can be used in patients with azoospermia of any etiology, it has already been proven that microsurgical reconstruction of the genital tract in patients with a history of vasectomy is the preferred approach, with reported patency rates of 67%85% [17, 18] and spontaneous pregnancy of 27%49% [18, 19]. Although the results of the reconstructions are essentially the same for patients with primary epididymal obstruction, many gynecologists and endocrinologists are not aware of this data . Kim et al . Reported the results of 43 end - to - side ves in patients with primary epididymal obstruction unrelated to a prior vasectomy . After a mean follow - up period of 42 months, the patency and pregnancy rates were 81% and 37%, respectively . Reported the results of 61 patients with primitive obstructive azoospermia who underwent microsurgical single tubule ves . After a minimum of 24 months follow - up period the overall patency and natural birth rates were 68.9% and 21.3%, respectively . In our study, after a median period of follow - up of 15 months, the total patency and natural birth rates were 77.7% and 22.2%, respectively . The time of follow - up in our study was shorter than that in the cited studies which might affect our pregnancy rates . As it was previously reported, microsurgical reconstruction is not the first option for couples where the male partner is diagnosed with oa and the female partner is> 39 years old and has a low ovarian reserve . In our study, the mean age of female partners in the group of patients with patent anastomosis and no natural pregnancy was 32.5 years . Natural pregnancy in the past history of a man with oa is a strong evidence of normal spermatogenesis before obstruction occurrence . In our study our results show a direct relationship between positive prior fertility status and pregnancy occurrence, although not significant (p = 0.56), very probable because of a low number of patients . After microsurgical reconstruction for azoospermia, if the controls 13 months postoperatively show some sperm, the couple is inclined to perform icsi using fresh ejaculated sperm . Predicting natural pregnancy after reconstruction is understandable because it helps counseling the patients after reconstruction . Icsi not only is costly, but also is invasive for women and carries some risks for offspring . So it would be advisable to know when to choose icsi and when to wait for a natural pregnancy . Our study shows that, in patients with primitive epididymal oa treated with reconstruction, fsh level is a strong predictive factor for obtaining a natural pregnancy . / l has a sensitivity of 85% and a specificity of 62.5% to predict a spontaneous pregnancy occurrence after microsurgical reconstruction . To our knowledge, there are no published studies which report the role of fsh in predicting a natural pregnancy after reconstruction . The potential mechanism of fsh level in impacting spontaneous pregnancy would be the fact that a low fsh level reflects a good spermatogenesis before and after obstruction occurrence . Some studies attempted to find a correlation between the sperm parameter and natural pregnancy in a group of primitive epididymal oa [20, 21]. Kim et al . Found higher sperm density and motility in postoperative semen analyses in patients who obtained natural pregnancy versus no natural pregnancy, but the difference was not significant . Out of 13 live deliveries by natural conception, 9 occurred in a group of patients with postoperative sperm concentration over 20 10 . All cases of live delivery by natural conception occurred in patients with at least 40% motile sperm . In our study, tmsc strongly predicts spontaneous pregnancy after reconstruction in a group of primitive epididymal oa . A tmsc of 37.30 10 has a sensitivity of 87.5% and a specificity of 85% to predict a spontaneous pregnancy occurrence after microsurgical reconstruction . In couples with younger female partners, the baseline fsh level and tmsc after reconstruction could be good tools for counseling the couple to wait for a longer time to obtain natural conception after surgery . If a male with primitive epididymal oa with reconstruction and patency has a baseline low fsh level and/or a tmsc good enough for a natural pregnancy, it is advisable to wait for a natural pregnancy and not to proceed to icsi . First, this was a single - center observational study on a small number of patients, so larger multicenter trials are needed to confirm our results . Our data suggest that microsurgical ves is an effective therapy used in the treatment of men diagnosed with epididymal obstructive azoospermia . Lower fsh levels and higher tmsc at baseline were independent predictors for natural pregnancy occurrence after ves . As the sample size is not large enough, larger multicenter trials are needed to confirm our results.
Obtaining an accurate view of factors that can affect health and wellbeing is of vital importance . In order to implement strategies to improve overall health through better lifestyle choices, it is necessary to first have a clear picture of the types of behaviour that represent a genuine threat to health, while at the same time identifying those in the community who are most likely to develop such types of behaviour . If data gathered on these types of behaviour is not being interpreted correctly, the efficacy of health promotion strategies may be compromised . Physical inactivity is increasingly being viewed as one of the most serious public health problems in the developed countries today . Not only does it contribute heavily to the development of many other risk factors (e.g., obesity and high blood pressure), but it can itself be attributed to a large proportion of noncommunicable deaths . Long - term physical inactivity contributes significantly to the secondary aging of various metabolic systems and reduces average life expectancy . It is estimated to cause around 2125% of breast and colon cancer burden, 27% of diabetes, and about 30% of ischaemic heart disease burden . It was found to be responsible for 1 in 10 deaths in the united states, and in a recent large study of australian adults it was consistently associated with all - cause mortality across a number of different demographic and behavioural factors, including physical activity itself . In australia, about 16,000 people die prematurely every year because they are not active enough . Lives are at risk, and so too is the economy with the cost of physical inactivity in australia estimated at $14 billion dollars annually . Current recommendations in australia suggest that adults should participate in at least 30 minutes of moderate to vigorous physical activity each day and that children should participate in at least 60 minutes . However, recent research indicates that, irrespective of meeting physical activity guidelines, those who spend a significant proportion of their day sedentary are still susceptible to a number of adverse health outcomes such as increased waist circumference, blood pressure and blood glucose, and lipid profiles . It is becoming clearer that not only do we need to encourage people, particularly young people, to get involved in traditional forms of physical activity, but we also need to encourage people to engage in more active forms of entertainment in their leisure time, rather than the more sedentary types of behaviour . Screen time can account for a large proportion of the time awake spent being physically inactive and includes any activity in which a person is viewing images on a screen, whether it is a television, a computer screen, or a hand - held video game device . However, the differing nature of the various types of screen - based activities may make the dynamics of this relationship ambiguous . There is growing evidence that not all screen - based activities are created equal in terms of factors like energy expenditure [1114], and so they should not be treated as such when attempting to determine an accurate picture of levels of physical inactivity in the community . The emergence and growing popularity of exergames (video games in which people are required to be physically active in order to play them) in recent years requires us to be a little more specific when determining levels of sedentary time and physical activity . Many of these games require extended periods of moderate levels of activity, similar to walking in relation to energy expenditure [1517], and currently this is not taken into consideration when calculating time spent being either sedentary or physically active . The aim of this study was to determine what proportion of time spent playing video games was actually spent playing exergames . Using this, it would be possible to calculate estimates within other data sources as to the amount of video game time that involves a moderate degree of physical activity . This would allow us to understand the degree to which current estimates of physical activity and sedentary behaviour in the community are not fully representative . In order to assess the proportion of video game time spent playing exergames, questions were developed and contributed to the may / june 2010 south australian health monitor survey, conducted by population research and outcome studies (pros) within the discipline of medicine, the university of adelaide . Health monitor is a user - pays telephone survey system that conducts large representative surveys of 2000 south australian households two to three times a year . Only one interview was conducted per household . Where more than one person aged 18 or over resided in the household, the respondent was the person who was last to have their birthday . This was a nonreplacement sample, and up to 10 callbacks were made, if needed, to households to interview the selected person . Respondents were informed that participation in the survey was voluntary, that they could choose to not answer any questions they did not want to, and that they were free to end the interview at any time . The may / june 2010 health monitor received ethics approval from the human research ethics committee, sa health (hrec protocol number 356/03/2013). The survey was administered using a cati (computer - aided telephone interviewing) system whereby respondents' answers were entered directly into the computer by the interviewer . From the 4900 households selected, 2026 interviews were conducted with a participation rate of 64.6% and a response rate of 59.2% . Telephone calls were made between 10:00 a.m. and 8:30 p.m., seven days a week . They were then provided with a definition of the term exergames (exergames is the term used to describe video games that incorporate physical activity as part of their gameplay) and were then asked how much time they spent playing those types of games . Respondents that indicated that there was a child between 5 and 17 years in the household were asked about the age, sex, and video game / exergame habits of the last child to have a birthday in that age range . Demographic questions included in the survey were on age, sex, area of residence, dwelling status, country of birth, education level, marital status, gross annual household income, work status, and number of people in the household . Data are weighted by area (metropolitan / country), age, gender, and probability of selection in the household to the most recent south australian population data so that the results are representative of the south australian population . Overall, 2026 adult respondents were asked about their gaming habits (specifically, how many hours per day or per week they spent playing them), and of these 537 gave additional information regarding the gaming habits of a child in the household aged between 5 and 17 years . The demographic characteristics of the respondents are presented in table 1, while the age and sex distribution of the children for whom data regarding video game habits was collected is displayed in table 2 . The mean age of the adult respondents was 47.6 (sd 18.5), while the mean age of the children was 11.0 (sd 3.8). Table 3 presents the proportion of adults and children that spend any time playing video games and the average amount of time spent playing them . Table 4 highlights the proportion of these respondents that spend at least some of this time playing exergames and presents the average amount of time spent playing these types of games per day . The average amount of time spent playing video games per day was 15 minutes (sd 38.9) for adults and 40 minutes (sd 53.9) for children . Of those that spent any time playing video games, 24.1% of adults and 42.1% of children reported spending at least some of that time playing exergames . The average amount of time spent playing exergames per day was 5 minutes (sd 13.1) for adults and 8 minutes (sd 14.7) for children . Based on the means given above, exergames accounted for 19.9% of the total time that children spend playing video games; this equates to 11.9 minutes per hour . Similarly, for adults, exergames accounted for 33.6% of the total time spent playing video games or 20.2 minutes per hour . Results indicate that a quarter of all adults and nearly half of all children that play video games spend at least some of that time playing exergames . On average, it accounts for a third of adult video game time and 20% of children's . The average amount of time per week spent playing exergames was 35 minutes for adults and an hour for children . This study highlights that a substantial proportion of video game time is spent playing games that require a light to moderate amount of physical activity . It could be argued that the standard practice of asking people how much time they spend engaged in any kind of screen activity should be amended to include clarification of what proportion of that time is spent playing exergames . There is a growing body of the literature to support the position that exergames can have a positive effect on the health and wellbeing of children and adolescents that play them . It has been observed to increase heart rate [18, 19], caloric expenditure [2022], and weight loss, particularly in the overweight and obese youth [23, 24]. There has also been evidence to suggest that participation in exergames can increase the amount of other types of physical activity that children undertake, while decreasing the total amount of time spent playing video games overall . The strengths of this study include a sound methodology that enables confidence in the data gathered as being representative of the south australian community . However, it is acknowledged that the method of using proxy respondents to gather data on children's gaming habits is less than ideal . Directly surveying the children themselves or better yet monitoring their gaming habits for a period of time would yield more representative data and increase confidence in the relative exergame time proportions . To the best of our knowledge, no other population - based research has been undertaken to determine what proportion of screen - based time is spent playing exergames . It remains to be seen if the association between screen time and undesirable health outcomes would hold constant if the distinction between exergames and other screen - based activities was factored into calculations . Further research is needed in order to assess whether those who spend their leisure time playing exergames display improved health outcomes compared to those who engage in other, more sedentary, screen - based activities . This can only be achieved if the accuracy in which we measure sedentary behaviour is improved . Historically, campaigns to promote physical activity and also the processes that measure their success have focused almost exclusively on encouraging people to participate in the more traditional types of activities (e.g., group sports, walking, etc . ). While these interventions are, and will continue to be, very important with regard to improving health outcomes for people, current research suggests the necessity to widen the focus of such campaigns and processes to include people's leisure time activities . As previously stated, it is becoming clear that the time people spend engaged in sedentary behaviour is a major contributor to poor health outcomes, regardless of their level of activity at other times . Encouraging them to choose more active types of behaviour in their leisure time
Intestinal parasitic infections are widely distributed throughout the world causing substantial intimidation to the public health, economy, and physical and cognitive development particularly among children in developing countries like ethiopia . The poor personal hygiene, poor environmental hygiene, and poor health system commonly observed in developing countries make the prevalence to be highest among these populations [1, 2]. The consumption of fruits and vegetables helps in protecting human body from a number of diseases by providing nutrients, vitamins, minerals, protein, and fibers . It could also have a positive impact on body - weight regulation and related conditions, including diabetes and hypertension . However, fruits and vegetables, especially, those that are consumed raw and or not properly washed, have been the major way for the transmission of human pathogens [35]. Intestinal parasitic infection may be acquired in different ways like by consumption of contaminated fruits, vegetables, other food stuff, and water . Eating unclean, raw, or undercooked fruits and vegetables is one of the means by which the transmission of intestinal parasitic infections is propagated . Fruits and vegetables act as vehicles for the transmission of parasitic infections when contaminated as a result of various associated factors related to planting, such as while they are still on the field, harvesting, transportation, storage, market chain, and even at home [5, 8]. Despite the fact that intestinal parasitosis is common in jimma town [9, 10], there are no studies conducted to assess the level of contamination of fruits and vegetables with parasites of medical and zoonotic importance . If our target is to control the intestinal parasitic diseases, it is not enough to depend merely on the chemotherapeutic intervention of identified cases, but need the concerted effort to reduce and eliminate the potential sources of infection . To our knowledge there is no published document to attest the level of parasitological contamination of fruits and vegetables in jimma town . Therefore, this study was designed to determine the level of parasitic contamination of selected fruits and vegetables and associated factors in jimma town . The study was conducted in jimma town, which is located at south west of ethiopia, about 352 km from addis ababa, the capital of ethiopia . A cross - sectional study was conducted to determine the level of parasitic contamination of fruits and vegetables sold in selected local markets in jimma town from april 22 to may 14, 2013 . Eight types of fruits and vegetables including lettuce, cabbage, carrot, tomato, green pepper, banana, mango, and salad were purchased from four conveniently selected local markets, namely, bishishe, hirmata merkato, kochi, and agip found in jimma town . Equal numbers of samples (45 each, totally 360 samples) were collected from the selected markets . The samples were collected, put in plastic bags, properly labeled, and brought to the medical parasitology laboratory of jimma university, for parasitological analysis . A portion (200 g) of each fruit and vegetable was washed separately in 500 ml of normal saline for detaching the parasitic stages (ova, larvae, cysts, and oocysts) of helminths and protozoan parasites commonly assumed to be associated with vegetable contamination . After overnight sedimentation of the washing solution, 15 ml of the sediment was then transferred to a centrifuge tube using sieve, to remove undesirable matters . For concentrating the parasitic stages, finally, the sediment was examined under a light microscope using 10 and 40 objectives . Modified zeihl - neelsen staining technique was also used for identification of oocysts of cryptosporidium and cyclospora spp as described elsewhere . Data were entered into, cleaned, and analyzed using spss for windows version 16.0 . The difference between prevalence of intestinal parasites among different categories was compared using pearson chi - square test . Univariate and multivariate logistic regression was used to identify factors associated with parasitic contamination of the fruits and vegetables data collection using questionnaire was done after the purpose of the study was explained to the respondents (vendors of fruits and vegetables) and verbal consent was obtained . A total of 360 samples of fruits and vegetables were collected from the local markets and examined for parasitological contamination . The results of the study showed that 208 samples were identified to be contaminated with at least one type of parasite, which gave rise to the overall contamination rate of 57.8% . These include 53% of green pepper, 68.9% of cabbage, 55.6% of lettuce, 77.8% of salad, 62.2% of carrot, 46.7% of tomato, 51.1% of banana, and 46.7% of mango (table 1). The stages and species of parasites detected include larvae of strongyloides like parasite, ova of ascaris lumbricoides, toxocara spp, hymenolepis nana, and hymenolepis diminuta, oocysts of cyclospora spp and cryptosporidium spp, and cysts of giardia lamblia, and entamoeba histolytica / dispar . Table 2 shows that strongyloides like parasite (21.9%) was the most frequently detected parasitic contaminant followed by toxocara spp (14.7%), cryptosporidium spp (12.8%), h. nana (8.3%), g. lamblia (7.5%), a. lumbricoides (6.7%), e. histolytica / dispar (5.3%), cyclospora spp (5.0%), and h. diminuta (1.4%). The highest frequency of strongyloides like parasite was detected in samples of salad and the least frequency from samples of carrot and tomato . Ova of toxocara spp was detected most frequently from cabbage samples but not recovered from mango samples . Ova of a. lumbricoides was detected from salad samples with highest frequency and not detected from samples of green pepper . 37.5% of the total samples were contaminated with two species of parasites, while 6.25% of the samples with three species of parasites and quadruple parasitic contamination were observed in two samples (table 1). Strongyloides like parasite and toxocara spp combination was the most frequently encountered with 35.48% of the multiple contamination . The parasitic contamination rate among the different fruits and vegetables was significantly different (p = 0.027) table 3 . The highest prevalence of intestinal parasites was recorded in salad (16.8%) followed by cabbage (14.9%), carrot (13.5%), lettuce (12%), green pepper (11.5%), banana (11.1%), and tomato and mango each 10.1% . Further analysis with a binary logistic regression showed that, as - compared to mango, salad was significantly contaminated (aor = 4.5, 95% ci (1.7, 11.9)); see table 4 . In addition to the parasitological investigations, factors associated with contamination of fruits and vegetables were also assessed . These factors were assessed by interviewing the vendors of fruits and vegetables in the selected markets of jimma town . The educational status of the vendors was ascertained and the majority (52%) of the vendors had no formal education, while 36% of the vendors had primary education and only 12% had secondary education . There was no significant association between education level of vendors and parasitic contamination rate of the produces they were selling (p = 0.845), table 3 . Bishishe (28.4%), agip (22.1%), and kochi (19.7%) markets . The percentage contamination rate was significantly different among samples collected from the different markets (p = 0.003), table 3 . Samples from groceries contributed for 15.4% of the positive samples while 84.6% of the contamination was contributed by open markets with no statistically significant difference (p = 0.811), table 3 . Another factor associated with parasitic contamination of fruits and vegetables is the act of washing the produces before displaying for sale . According to this study, majority (73.1%) of the produces were not washed before display for selling while only 26.9% of the produces were washed before displaying for sale . 82.2% of the unwashed produces were contaminated with one or more parasites, while 17.8% of the washed produces were contaminated with intestinal parasites . The cross tabulation of washing the produces before display for sale and result of parasitological analysis showed a significant difference in contamination rate among washed and unwashed produces (p = 0.000), table 3 . As compared to the washed produces, the odds of parasitic contamination for unwashed ones was 3.33 times (aor = 3.3, 95% ci (1.9, 5.6)), table 4 . The sources of water used for washing the produces among the vendors include pipe water (62.5%), well water (30.2%), and river water (7.3%). Well water, and river water were contaminated with at least one parasitic species, respectively . There was no significant difference in contamination rate among produces washed by water from different sources (p = 0.412), table 3 . The means of display for selling is also another factor assessed for association with parasitic contamination of fruits and vegetables . Various means of display were observed among the vendors as follows: 67.5% of the produces are displayed on the floor by the road sides while 23.1% on tables by the road sides . The detection of intestinal parasitic stages from fruits and vegetables is an indicative of the fecal contamination from human and or animal origin . As in many tropical countries, intestinal parasites are widely distributed in ethiopia not only due to the favorable climatic conditions for the survival and dissemination of the parasites but also due to the unsanitary conditions that facilitate fecal pollution of water, food stuffs, and soil . The present study has attempted to assess the level of contamination and prevalence of different intestinal parasites from different fruits and vegetables sold in selected markets of jimma town . The overall parasitic contamination rate was found to be 57.8%, which is in agreement with the findings reported elsewhere [3, 5]. However, it is higher than what was reported in similar studies from other areas [8, 1217]. On the other hand, it is lower when compared with the findings of some studies [18, 19]. The discrepancy between the present study and previous studies might be as a result of the variations in geographical locations, climatic and environmental conditions, the kind of sample and sample size examined, the sampling techniques, methods used for detection of the intestinal parasites, and socioeconomic status . So long as these factors differ, consequently the discrepancy of the results would be expected . Salad (77.8%) was found to be the most frequently contaminated produce followed by cabbage (68.9%), carrot (62.2%), lettuce (55.6%), green pepper (53.3%), and banana (51.1%). This variation among the produces might be due to the fact that salad, cabbage, carrot, and lettuce have uneven surfaces which make the parasitic stages attach more easily to the surface of these vegetables . The smooth surface of green pepper, tomato, and mango might reduce the rate of parasitic attachment hence had lower contamination rate . In this study, larvae of strongyloides like parasite was the most frequently detected parasite with a prevalence of 21.9% . This might be due to the fact that the parasite has a free living state and does not require a host for its proliferation, in addition to its parasitic mode of life . The predominance of strongyloides like parasite is similar with similar studies conducted elsewhere [5, 8, 12, 17]. However, the finding is in contrast with what was reported by other investigators where ascaris lumbricoides, cryptosporidium spp, e. histolytica / dispar, and toxocara spp were the predominant parasites detected [1316]. Ova of toxocara spp was the second most prevalent contaminant next to strongyloides like parasite . This dominance might be attributed to the high fertility of toxocara female adult producing up to 10,000 eggs daily and the resistant nature of the eggs, which may survive for up to ten years resisting harsh conditions in the environment . Cryptosporidium spp was the third most frequently detected parasite in this study with a prevalence of 12.8% . However, majority of researches did not report the parasite as vegetable contaminant [3, 5, 12, 13, 2022]. This might be due to differences in methods used between this study and the previous ones . In the present study, modified acid fast stain was used for detection of the coccidian oocysts, while the majority of the previous studies did not . No ova of hookworm species were detected from the samples examined in the present study . This is in agreement with other studies conducted elsewhere [3, 14, 16, 20, 23]. This might be due to the fact that hookworms have very short life span in the soil . However others have reported the contamination of vegetables with hookworm species [5, 6, 25]. The differences might be attributed to differences in geographical locations, climate conditions, and the type of soil . This might indicate the possibility of high level contamination of the fruits and vegetables, which perhaps results in multiple parasitic infections in human . The contamination rate was significantly different for the samples collected from the different markets in which samples collected from this might be associated with the act of washing of the produces before display; 77.8% of the samples collected from the market were not washed . Majority (69%) of the produces were displayed for sale on the floor where it is exposed to dusts and flies . It is well established that the flies can act as vectors for a number of pathogenic microorganisms including parasites like cryptosporidium parvum and toxoplasma gondii . Besides, there might be bacterial and viral contamination of the produces during display for sale on the floor . The habit of eating raw vegetables like salad and tomato is commonly practiced in the study area . Hence, the findings of the present study are of public health importance, requiring an appropriate intervention to prevent transmission of parasitic diseases that can be acquired through consumption of contaminated fruits and vegetables . However, this study did not address the effect of seasonal variation on the contamination of the fruits and vegetables . The findings of this study could not underscore the infectivity of the parasitic stages detected as viability study was not conducted . In conclusion, this study highlighted the importance of raw fruits and vegetables as the potential source of transmission for intestinal parasites to humans . The fruits and vegetables contamination with the pathogenic parasites poses health risk to the consumers if consumed without proper cleaning and or cooking . A comprehensive health education should be given to vendors and farmers of fruits and vegetables and to the general population on the health risks associated with consumption of contaminated fruits and vegetables . The consumers should always observe the basic principle of food and personal hygiene, that is, thorough washing of the fruits and vegetables before eating and washing hands before meal . The vendors of fruits and vegetables should avoid the contact of the produces with soil while display for selling . Further studies should be conducted on the viability of parasitic contaminants of fruits and vegetables . Also, other researches must be done to evaluate the level of parasitic contamination of farm produces, water, and soil in which fruits and vegetables are cultivated.
Heart rate (hr) and vo2 are widely used to assess aerobic capacities and prescribe exercise programs to clinical, healthy, and athletic populations . However, it is important to be able to differentiate between% maximal oxygen uptake (% vo2max),% oxygen uptake reserve (% vo2r),% heart rate max (% hrmax), and% heart rate reserve (% hrr). This is due to the fact that vo2 and hr do not have absolute zeros, and their max values vary depending on the individual, thus% vo2max and% hrmax cannot be directly correlated . However, the term reserve describes the difference between the maximum and resting value for a given measurement providing more accurate predictive capabilities . Research has supported the finding that% vo2r and% hrr are more closely correlated than% vo2max and% hrmax (5). This association between% vo2r and% hrr was first demonstrated in 1997 with the use of cycle ergometers and it was concluded that% vo2r and% hrr should be used when prescribing exercise intensities . (7). The same relationship was found a year later with the use of treadmills . When exercise intensities were prescribed using a percentage of vo2r, the same percentage of hrr further research in this field has provided evidence that indicates this association to be present on a much larger scope . For example, this association was found to be present in elliptical exercise (4), as well as in individuals with substandard health, such as those suffering from heart disease (1). All of these studies looked at maximal tests, from which, regression lines were constructed to determine the association between% vo2r and% hrr . However, the majority of research has not been conducted at moderate intensities to accurately determine the relationship between% vo2r and% hrr at these levels . Currently exercise programs are prescribed using either% vo2r or% hrr and the american college of sports medicine (acsm) provides guidelines of specific percentages for varying intensities (9). If earlier findings are correct then a subject exercising at a set% vo2r should exhibit the same% hrr, and vice - versa . However, recent research has been contradictory to earlier findings that a 1:1 ratio exists at all exercise intensities . It has been found that as exercise intensity increases,% vo2r and% hrr deviate from the expected 1:1 ratio (3). Therefore, the purpose of this study was to determine if a 1:1 relationship exists between% hrr and% vo2r during moderate (50%) treadmill exercise . 28 college students (11 male, 17 female, age 21.9 1.6 years) participated in this study . The university s institutional review board approved this study and all participants were presented with an informed consent form as well as a physical activity readiness questionnaire (par - q) prior to testing . If potential participants answered yes to any of the questions on the par - q, this resulted in automatic disqualification from the study . The informed consent highlighted the purpose of the study and what was expected of the participants . Subjects were required to perform two separate exercise bouts on two different days with at least 48 hours of rest between successive bouts . Weight (kg) was taken prior to each exercise bout using a befour portable scale (befour, inc . The protocol was the 12 kmh/7.5 mph vo2max test found in the cosmed fitmate user manual, nh edition (cosmed srl - italy) (table 1). The procedure was programmed into a quinton q - stress stress test system that was synced with a tm55 treadmill (quinton instruments, bothell, wa). In order to collect a minute by minute recording of hr, a polar heart rate monitor (polar electro, tempere, finland) was lubricated using spectra 360 electrode gel (scientific digital imaging, llc . Minute - by - minute recordings of vo2 were collected via indirect calorimetry with the use of a metabolic cart (parvo medics true one 2400, sandy ut, usa). To ensure complete analysis of expired gases a nosepiece was worn in addition to a mouthpiece that was held in place using a light headpiece . The subjects were encouraged to perform until absolute exhaustion at which point the participants were instructed to straddle the belt of the treadmill while it was brought to rest . At the completion of the test, vo2max and hrmax were collected from the parvo system s minute - by - minute printout and imported into microsoft excel for future analysis . An rer of> 1.1 was considered to indicate that a true max was achieved . On a separate day, at least 48 hours following the initial test, subjects returned to the lab to complete an exercise bout on a treadmill at an intensity that was predicted to yield 50% of the participants vo2 reserve . This was calculated using the following equation: 50%vo2 = (vo2max 3.5ml / kg / min) 50% + 3.5ml / kg / min . Once 50%vo2 was calculated, exercise intensity was determined using a speed of 3 mph (80.4m / min) with varying degrees of incline, using the following equation: 50%vo2 = (.1ml / kg / min 80.4m / min) + (80.4m / min% grade 1.8ml / kg / min) + (3.5ml / kg / min). The speed of 3 mph was kept constant to ensure that all participants would maintain a walking gait pattern . It has been shown that running requires two times the amount of oxygen as walking (11). Prior to exercising, the participants resting heart rate was palpated for fifteen seconds and multiplied by four to determine beats per minute . The subjects would lie prone for ten minutes prior to palpation to ensure a true rested state . The subjects then warmed up for three minutes at a speed of 3 mph and 0% grade . Following the three - minute warm - up the grade was increased to the calculated value (value expected to yield 50% of reserve) for 5 minutes . Acsm guidelines state that steady state is reached within 14 minutes during light exercise (10); consequently, a 5-minute exercise bout was used to ensure steady state was achieved . After the test, calculations were made to determine what percent of reserves (vo2r and hrr) had been reached . These values were then compared to the pre - test 50% calculation . While subjects performed the exercise bout at the predicted% 50vo2r, vo2 and hr were obtained on a minute - by - minute basis . The mean vo2 (ml / kg / min) and hr (bpm) paired t - tests were performed to determine the difference between the predicted and actual values for vo2 and hr at 50% of their reserves . Correlational analysis via pearson s r was used to compare the percent of vo2r and hrr achieved . Statistical significance was determined at p <0.05, and standard error of estimate (see) was also calculated . 28 college students (11 male, 17 female, age 21.9 1.6 years) participated in this study . The university s institutional review board approved this study and all participants were presented with an informed consent form as well as a physical activity readiness questionnaire (par - q) prior to testing . If potential participants answered yes to any of the questions on the par - q, this resulted in automatic disqualification from the study . The informed consent highlighted the purpose of the study and what was expected of the participants . Subjects were required to perform two separate exercise bouts on two different days with at least 48 hours of rest between successive bouts . Weight (kg) was taken prior to each exercise bout using a befour portable scale (befour, inc . The protocol was the 12 kmh/7.5 mph vo2max test found in the cosmed fitmate user manual, nh edition (cosmed srl - italy) (table 1). The procedure was programmed into a quinton q - stress stress test system that was synced with a tm55 treadmill (quinton instruments, bothell, wa). In order to collect a minute by minute recording of hr, a polar heart rate monitor (polar electro, tempere, finland) was lubricated using spectra 360 electrode gel (scientific digital imaging, llc . Minute - by - minute recordings of vo2 were collected via indirect calorimetry with the use of a metabolic cart (parvo medics true one 2400, sandy ut, usa). To ensure complete analysis of expired gases a nosepiece was worn in addition to a mouthpiece that was held in place using a light headpiece . The subjects were encouraged to perform until absolute exhaustion at which point the participants were instructed to straddle the belt of the treadmill while it was brought to rest . At the completion of the test, vo2max and hrmax were collected from the parvo system s minute - by - minute printout and imported into microsoft excel for future analysis . An rer of> 1.1 was considered to indicate that a true max was achieved . On a separate day, at least 48 hours following the initial test, subjects returned to the lab to complete an exercise bout on a treadmill at an intensity that was predicted to yield 50% of the participants vo2 reserve . This was calculated using the following equation: 50%vo2 = (vo2max 3.5ml / kg / min) 50% + 3.5ml / kg / min . Once 50%vo2 was calculated, exercise intensity was determined using a speed of 3 mph (80.4m / min) with varying degrees of incline, using the following equation: 50%vo2 = (.1ml / kg / min 80.4m / min) + (80.4m / min% grade the speed of 3 mph was kept constant to ensure that all participants would maintain a walking gait pattern . It has been shown that running requires two times the amount of oxygen as walking (11). Prior to exercising, the participants resting heart rate was palpated for fifteen seconds and multiplied by four to determine beats per minute . The subjects would lie prone for ten minutes prior to palpation to ensure a true rested state . The subjects then warmed up for three minutes at a speed of 3 mph and 0% grade . Following the three - minute warm - up the grade was increased to the calculated value (value expected to yield 50% of reserve) for 5 minutes . Acsm guidelines state that steady state is reached within 14 minutes during light exercise (10); consequently, a 5-minute exercise bout was used to ensure steady state was achieved . After the test, calculations were made to determine what percent of reserves (vo2r and hrr) had been reached . While subjects performed the exercise bout at the predicted% 50vo2r, vo2 and hr were obtained on a minute - by - minute basis . The mean vo2 (ml / kg / min) and hr (bpm) were then calculated for each subject between the fifth and eighth minute of exercise . Paired t - tests were performed to determine the difference between the predicted and actual values for vo2 and hr at 50% of their reserves . Correlational analysis via pearson s r was used to compare the percent of vo2r and hrr achieved . Statistical significance was determined at p <0.05, and standard error of estimate (see) was also calculated . Statistical analysis revealed that the predicted 50%vo2 (27.1 3.3) was significantly greater than the actual vo2 achieved (25.6 3.3), t(27) = 8.2, p <.001 (figure 1). It was also found that the mean predicted hr at 50% of the reserve (128.8 7.5) was significantly less than the actual hr achieved (135.6 10.6), t(27) = 5.4, p <.001 (figure 2). The correlation between measured and predicted vo2 at 50%r was r = .96, p <.001, with an see = .94 ml / kg / min . The correlation between measured and predicted hr at 50%r was r = .78, p <.001, with an see = 6.6 bpm . In addition to t - tests, pearson s r was used to analyze the correlation between% vo2r and% hrr during moderate intensity exercise . It was found that a statistically non - significant, weak positive, relationship existed between the two variables (r = .31, p = .105) (figure 3). The primary purpose of this study was to determine the relationship between vo2r and hrr during moderate intensity exercise . Secondly, determining the predictability of vo2 and hr at moderate intensity exercise . It was hypothesized that vo2r and hrr would be directly correlated at moderate intensity exercise (50%r). This study indicated that vo2 could be more accurately predicted than hr during moderate intensity exercise . Also, a weak positive correlation existed between vo2r and hrr . This indicated that it was less reliable to predict one variable given another (i.e., hr cannot be accurately predicted when the vo2 of a given exercise bout is known). On the basis of a 50%r (moderate intensity) prediction, mean% vo2r values were over - predicted (actual% vo2r = 46.9% 2.0%) whereas mean% hrr values were under - predicted (actual% hrr = 55.3% 5.4%). Our results on the prediction of vo2 supported the findings of cunha et al ., which examined the association between vo2r and hrr as the intensity, and duration, of exercise were increased (28 physically active males). Cunha s study concluded that predicted vo2 values were significantly greater than actual vo2 values . Additionally, it was found that as exercise intensity increased the strength of the relationship between% vo2r and% hrr weakened (3). The results of our study were shown to overpredict vo2, however, the see value of .94 ml / kg / min suggests that accurate predictions may still be made in most practical circumstances . This finding supports the research conducted on 26 highly trained, healthy male cyclists by lounana et al . They found that vo2 could be accurately predicted when using reserve values (5). However, the results of this study that were dependent on hr data deviated from the majority of previous research findings . The majority of past research had found a direct correlation between% vo2r and% hrr (i.e., enabling accurate predictions of hr from vo2). Investigated the relationship between% vo2r and% hrr in apparently healthy individuals (26 males, 24 women, 1820 yrs) during treadmill exercise they found that the regression between vo2r and% hrr was r = 0.99 0.002 (7) whereas our results found a weak correlation (r = .31). These differences could have been the result of many different factors, the first of which is elevated levels of catecholamines in the blood circulating throughout the body . Catecholamines are hormones released by the body during exercise and are responsible for elevating blood pressure, breathing rate, and heart rate (6). Many participants in this study had exercised prior to performing the submaximal portion of the study . This would have resulted in elevated levels of these hormones . It was assumed that by lying prone for 10 minutes, an accurate resting hr, as well as an accurate ambulatory hr would be achieved . However, it is plausible that catecholamines were still circulating through the system, causing the participants resting hr to be elevated above that which was predicted . Some other limitations consisted of making the assumption of 3.5ml / kg / min as the value for resting vo2 as well as inconsistency of hr measurements . The value of 3.5ml / kg / min was assumed for all participants as resting vo2 but upon further research it was found that resting vo2 has been shown to vary greatly from subject to subject . Cunha et al . Found that their subjects measured resting vo2 values were significantly greater than the assumed value of 3.5ml / kg / min (4.3 0.9 ml / kg / min, p <.001) (3). That looked at two separate cohorts (593 women, 78 men, 1874 yrs; 49 women 49 men, 38 5 yr) resting vo2 values were found to be significantly lower than 3.5ml / kg / min (2.6 0.4 ml / kg / min) (2). When collecting exercising hr data, a hr monitor was used . However, resting hr s were palpated, using the radial pulse, for fifteen seconds and multiplied by four . This method, while convenient and easy to use, has a large margin for error . The counting of one extra or one less beat would result in a difference of 4 beats per minute . By using a hr monitor to obtain both resting and exercising hr s a more meticulous approach to the collection of data could potentially indicate that hr can be accurately predicted using reserves . Furthermore, better collection of hr data could also indicate that a direct relationship between% vo2r and% hrr does exist . Such results would support the findings of brawner and associates, dalleck and kravitz, suain and leutholtz, and swain and colleagues (1,4,8,9). Lastly, it is possible that not all participants reached the desired exercise intensity, as the workload was a predictive calculation derived from their maximal test . In conclusion, this study indicated that vo2 could be more accurately predicted than hr during moderate intensity exercise . The weak correlation between vo2r and hrr indicates that caution should be used when relying on a hr to determine vo2.
Although fish farming in iraq started in 1955 with a small pond in al - zaafaraniya, south of baghdad city, an advance was achieved in fish farming industry in iraq during the seventies and early eighties of the last century when many fish farms were established especially in the middle of iraq . However, such achievement was hindered due to consequences of the war situations during 19801988 and 1991 as well as the economic sanction imposed by the un against iraq on august 6, 1990 . During the last few years, a great advance was achieved in fish farming in general and fish cages in particular due to the increasing demand on fish protein as well as the increasing investment in fish - culture industry in most provinces of iraq . According to the statistics, a total of 441 working fish farms are scattered in iraq . Of these farms, a total of 72 working fish farms are situated in babylon province alone with a water area of 44.5% of the total water area of fish farms in iraq . Under extensive fish culture and inadequate administrative and control measures, fish farms are vulnerable to great hazards due to the infection with parasites and other disease agents [2, 4, 5]. Many parasite species can easily spread among fishes suffering from crowd and bad managements, especially those parasites with direct life cycles . In connection with the parasites of cultured fishes of babylon province, mhaisen et al . Surveyed the literature on the parasitic fauna of fishes of al - furat fish farm (previously known as babylon fish farm), which is the biggest fish farm in babylon province, and showed that the parasitic fauna of fishes of that farm included 60 valid parasite species (10 protozoans, three myxozoans, one trematode, 29 monogeneans, five cestodes, three nematodes, two acanthocephalans, six crustaceans, and one mollusc larva). Such data are scattered in different local journals, unpublished theses, and few other sources . Therefore, the present paper was aimed at gathering data from the literature concerning all fish farms of babylon province and providing a list of parasite species according to their major groups as well as a host - parasite list for cultured fishes of these farms and some other fish species found in such farms . Such parasite list will help owners of fish farms and fish veterinarians to know what sort of parasites are found in their fish farms, which will help them later in taking appropriate measures for their control . A total of 50 references (33 published articles, 12 unpublished theses, two unpublished reports, one book, one conference abstract, and one review article) dealing with the parasites of farm fishes of babylon province were used to prepare the present paper . Data from such references was gathered to provide host - parasite and parasite - host lists . The systematic account of these parasites is based on some electronic sites [812] as well as some taxonomic references [1316]. The index - catalogue of parasites and disease agents of fishes of iraq was used to indicate the total number of fish hosts harbouring each parasite species in the whole waters of iraq . So far 25 chronologically arranged references [1842] were concerned with the parasitic fauna of al - furat fish farm . Only seven references [4349] were concerned with the parasitic fauna of al - shark al - awsat fish farm . The literature concerned with fish parasites of other farms in babylon province included those from al - latifiya fish farm [6, 5052]; al - bajaa fish farm; abdul - razzak al - janabi fish farm; fawzi al - janabi fish farm and ali al - hayali fish farm; three fish farms at al - iskandariya district: abdul - hadi al - matloob fish farm, hussain al - gaiem fish farm, and maki chinak fish farm; technical institute of al - musaib fish farm; and al - manahil (al - bilad) fish farm at al - iskandariya district . In addition, surveys were done from some unnamed fish ponds such as those at al - mahaweel district, al - musaib district, al - iskandariya district, and sadat al - hindiya district as well as some other unnamed farms in the province [6165]. Surveying the literature concerning the parasites so far recorded from fish farms of babylon province showed the presence of 92 parasite species . These parasites included one mastigophoran, three apicomplexans, 13 ciliophorans, five myxozoans, five trematodes, 45 monogeneans, five cestodes, three nematodes, two acanthocephalans, nine arthropods, and one mollusc . The common carp was found to harbour 81 species of parasites, the grass carp 30 species, the silver carp 28 species, l. abu 13 species, c. auratus one species, and h. fossilis one species . The layout and names of the major taxonomic groups (phyla and classes) followed a checklist of an fao fisheries technical paper . These major groups represent the concerned phyla of the parasites, but due to the great numbers of parasite species of the phylum platyhelminthes, its three classes (trematoda, monogenea, and cestoda) were applied in addition to their phylum . The parasite - host list is arranged in the major groups (phyla or classes) of parasitic fauna according to kirjuina and vismanis . For each major group, a list of species together with their hosts and concerned references is given . To economize space, names of fish farms also the systematic account of all major groups is given down to the specific name of all parasites . For each parasite species, all records in farm fishes in babylon province are given together with the first record of each concerned parasite in iraq as well as the present number of all hosts so far known in iraq for each concerned species based on the index - catalogue of parasites and disease agents of fishes of iraq . The phylum mastigophora is represented in farm fishes of babylon province with only one parasite species of the genus ichthyobodo . The systematic account of this parasite, followed by parasite - host list, is given here . Phylum mastigophora class kinetoplastidea order kinetoplastida family bodonidae ichthyobodo necator (henneguy, 1884) pinto, 1928 class kinetoplastidea order kinetoplastida family bodonidae ichthyobodo necator (henneguy, 1884) pinto, 1928 family bodonidae ichthyobodo necator (henneguy, 1884) pinto, 1928 ichthyobodo necator (henneguy, 1884) pinto, 1928 ichthyobodo necator (henneguy, 1884) pinto, 1928 ichthyobodo necator (henneguy, 1884) pinto, 1928, was erroneously reported as costia necatrix from the skin and gills of c. carpio . The first record of c. necatrix in iraq was from body surface of h. fossilis from al - ashar canal at basrah . Seven fish host species are so far known for this parasite (as c. necatrix) in iraq . The phylum apicomplexa, which is known as phylum myzozoa according to worms, is represented in farm fishes of babylon province with three species; two of them belonged to the genus eimeria and one unspecified species to the genus haemogregarina . Phylum apicomplexa class sporozoa order eucoccidiorida family eimeriidae eimeria dogieli (dogiel, 1948) pellerdy, 1963 eimeria mylopharyngodoni chen, 1956 family haemogregarinidae haemogregarina sp . Order eucoccidiorida family eimeriidae eimeria dogieli (dogiel, 1948) pellerdy, 1963 eimeria mylopharyngodoni chen, 1956 family haemogregarinidae haemogregarina sp . Family eimeriidae eimeria dogieli (dogiel, 1948) pellerdy, 1963 eimeria mylopharyngodoni chen, 1956 family haemogregarinidae haemogregarina sp . Eimeria dogieli (dogiel, 1948) pellerdy, 1963 eimeria mylopharyngodoni chen, 1956 eimeria dogieli (dogiel, 1948) pellerdy, 1963 eimeria mylopharyngodoni chen, 1956 family haemogregarinidae eimeria dogieli (dogiel, 1948) pellerdy, 1963, was recorded from the intestine of c. carpio . So far, this is the only record of e. dogieli from fishes of iraq . The specific name was misspelled as mylopharyngodon and no authority, site of infection, parasite description, and illustration were given for this parasite by hussain et al . . So this record is considered as questionable especially if we take in consideration that hussain et al . Examined c. carpio externally while e. mylopharyngodoni is known to infect intestine, kidneys, and liver of fishes . Three species of haemogregarina were so far recorded from blood of three fish species in basrah province only . So we think that the record of haemogregarina sp . From gills of c. carpio by hussain et al . With neither description nor a good illustration is considered as questionable . The phylum ciliophora is represented in farm fishes of babylon province with 13 species, three of which belonged to the genera chilodonella, ichthyophthirius, and tripartiella, five to the genus apiosoma, and four to the genus trichodina in addition to unspecified species of the genus trichodina . Phylum ciliophora class kinetophragminophorea order cyrtophorida family chilodonellidae chilodonella cyprini (moroff, 1902) strand, 1928 class oligohymenophorea order hymenostomatida family ichthyophthiriidae ichthyophthirius multifiliis fouquet, 1876 order petrichida family epistylididae apiosoma amoebae (grenfell, 1887) lom, 1966 apiosoma cylindriformis (chen, 1955) apiosoma minuta (chen, 1961) lom, 1966 apiosoma piscicola blanchard, 1885 apiosoma poteriformis (timofeev, 1962) lom, 1966 order mobilida family trichodinidae trichodina cottidarum dogiel, 1948 trichodina domerguei (wallengren, 1897) trichodina gracilis polyanskii, 1955 trichodina nigra lom, 1960 trichodina sp . Tripartiella amurensis (chan, 1961) class kinetophragminophorea order cyrtophorida family chilodonellidae chilodonella cyprini (moroff, 1902) strand, 1928 family chilodonellidae chilodonella cyprini (moroff, 1902) strand, 1928 family chilodonellidae chilodonella cyprini (moroff, 1902) strand, 1928 chilodonella cyprini (moroff, 1902) strand, 1928 class oligohymenophorea order hymenostomatida family ichthyophthiriidae ichthyophthirius multifiliis fouquet, 1876 order petrichida family epistylididae apiosoma amoebae (grenfell, 1887) lom, 1966 apiosoma cylindriformis (chen, 1955) apiosoma minuta (chen, 1961) lom, 1966 apiosoma piscicola blanchard, 1885 apiosoma poteriformis (timofeev, 1962) lom, 1966 order mobilida family trichodinidae trichodina cottidarum dogiel, 1948 trichodina domerguei (wallengren, 1897) trichodina gracilis polyanskii, 1955 trichodina nigra lom, 1960 trichodina sp . Tripartiella amurensis (chan, 1961) order hymenostomatida family ichthyophthiriidae ichthyophthirius multifiliis fouquet, 1876 family ichthyophthiriidae ichthyophthirius multifiliis fouquet, 1876 ichthyophthirius multifiliis fouquet, 1876 family epistylididae apiosoma amoebae (grenfell, 1887) lom, 1966 apiosoma cylindriformis (chen, 1955) apiosoma minuta (chen, 1961) lom, 1966 apiosoma piscicola blanchard, 1885 apiosoma poteriformis (timofeev, 1962) lom, 1966 apiosoma amoebae (grenfell, 1887) lom, 1966 apiosoma cylindriformis (chen, 1955) apiosoma minuta (chen, 1961) lom, 1966 apiosoma piscicola blanchard, 1885 apiosoma poteriformis (timofeev, 1962) lom, 1966 apiosoma amoebae (grenfell, 1887) lom, 1966 apiosoma cylindriformis (chen, 1955) apiosoma minuta (chen, 1961) lom, 1966 apiosoma piscicola blanchard, 1885 apiosoma poteriformis (timofeev, 1962) lom, 1966 family trichodinidae trichodina cottidarum dogiel, 1948 trichodina domerguei (wallengren, 1897) trichodina gracilis polyanskii, 1955 trichodina nigra lom, 1960 trichodina sp . Tripartiella amurensis (chan, 1961) trichodina cottidarum dogiel, 1948 trichodina domerguei (wallengren, 1897) trichodina gracilis polyanskii, 1955 trichodina nigra lom, 1960 trichodina sp . Tripartiella amurensis (chan, 1961) trichodina cottidarum dogiel, 1948 trichodina domerguei (wallengren, 1897) trichodina gracilis polyanskii, 1955 trichodina nigra lom, 1960 tripartiella amurensis (chan, 1961) chilodonella cyprini (moroff, 1902) strand, 1928, was recorded from skin, buccal cavity, and gills of c. idella [27, 29, 43, 51], skin, fins, buccal cavity, and gills of c. carpio [27, 30, 43, 51, 53], and skin and gills of h. molitrix [27, 43, 51]. The first record of c. cyprini in iraq was from skin, buccal cavity, and gills of mystus pelusius from tigris river at baghdad . Ichthyophthirius multifiliis fouquet, 1876, was recorded from skin and gills of c. idella [27, 29, 43, 51, 56], skin, fins, and gills of c. carpio [25, 27, 29, 30, 35, 43, 45, 47, 49, 51, 5356, 59, 60, 65], and skin and gills of h. molitrix [27, 29, 45, 47, 51]. The first record of i. multifiliis in iraq was from the skin and gills of chelon subviridis (reported as mugil dussumieri) from tigris river near baghdad . Apiosoma amoebae (grenfell, 1887) lom, 1966, was reported from skin, buccal cavity, and gills of c. idella [20, 24, 27], skin and buccal cavity of c. carpio [27, 55], and gills and buccal cavity of h. molitrix [20, 27]. It is appropriate to mention here that a. amoebae was reported as glossatella amoebae [27, 55]. The first record of a. amoebae in iraq was from the skin, buccal cavity, and gills of c. idella from babylon fish farm . Apiosoma cylindriformis (chen, 1955) was reported from gills of c. idella [20, 24, 27], buccal cavity and gills of c. carpio, and gills of h. molitrix [20, 21, 27]. The first record of a. cylindriformis in iraq was from gills of c. idella and h. molitrix from babylon fish farm . Apiosoma minuta (chen, 1961) lom, 1966, was recorded from skin of c. carpio [45, 47]. So far, a. minuta has two fish host species in iraq as it was recently recorded from luciobarbus xanthopterus by al - salmany . Apiosoma piscicola blanchard, 1885, was recorded from skin, buccal cavity, and gills of c. idella [27, 51], skin, buccal cavity, and gills of c. carpio [27, 29, 51, 55], and buccal cavity and gills of h. molitrix [20, 21, 27, 29, 51]. A. piscicola was recorded for the first time in iraq from skin, buccal cavity, and gills of c. idella, c. carpio and h. molitrix from al - suwaira and al - latifiya fish farms . Apiosoma poteriformis (timofeev, 1962) lom, 1966, was recorded from skin and gills of c. idella [20, 24, 27] and buccal cavity and gills of c. carpio . It is appropriate to mention here that a. poteriformis was reported as glossatella poteriformis by al - zubaidy . A. poteriformis was recorded for the first time in iraq from gills of c. idella from babylon fish farm . Trichodina cottidarum dogiel, 1948, was recorded from skin and gills of c. carpio [45, 47, 49, 54, 59, 60] and skin and gills of h. molitrix [45, 47]. T. cottidarum was recorded for the first time in iraq from gills of c. carpio from a manmade lake at baghdad city . Trichodina domerguei (wallengren, 1897) was recorded from skin, fins, buccal cavity, and gills of c. idella [24, 27, 29, 43, 51, 56], skin, fins, buccal cavity, and gills of c. carpio [25, 27, 29, 30, 35, 43, 51, 53, 55, 56, 59], skin, buccal cavity, and gills of h. molitrix [20, 27, 51], and skin and gills of l. abu [23, 56]. The first record of t. domerguei in iraq was from skin, fins, and gills of eight freshwater fish species from tigris river, al - tharthar lake, and fish markets in baghdad city . So far, t. domerguei has 39 host species in iraq and, therefore, it is the most prevalent ciliate species among fishes of iraq . Trichodina gracilis polyanskii, 1955, was recorded from skin of c. carpio [45, 47]. Trichodina nigra lom, 1960, was recorded from skin and gills of the three carp species: c. idella, c. carpio [27, 29, 35, 49, 55, 59, 60], and h. molitrix [27, 29]. The first record of t. nigra in iraq was from skin and gills of c. carpio and gills of h. molitrix from babylon fish farm . Unidentified specimen of trichodina was recorded from l. abu with no mention to site of infection . In addition to 24 recognized trichodina species so far recorded from fishes of iraq, some unidentified species of trichodina were so far recorded from six fish species . Tripartiella amurensis (chan, 1961) was recorded from skin of c. carpio [45, 47]. The phylum myxozoa is represented in farm fishes of babylon province with five species: one species belonged to the genus myxobilatus and four species to the genus myxobolus . Phylum myxozoa class myxosporea order bivalvulida family sphaerosporidae myxobilatus legeri (cpde, 1905) family myxobolidae myxobolus dogieli bykhovskaya - pavlovskaya & bykhovski, 1940 myxobolus muelleri btschli, 1882 myxobolus oviformis thlohan, 1892 myxobolus pfeifferi thlohan, 1895 order bivalvulida family sphaerosporidae myxobilatus legeri (cpde, 1905) family myxobolidae myxobolus dogieli bykhovskaya - pavlovskaya & bykhovski, 1940 myxobolus muelleri btschli, 1882 myxobolus oviformis thlohan, 1892 myxobolus pfeifferi thlohan, 1895 family sphaerosporidae myxobilatus legeri (cpde, 1905) family myxobolidae myxobolus dogieli bykhovskaya - pavlovskaya & bykhovski, 1940 myxobolus muelleri btschli, 1882 myxobolus oviformis thlohan, 1892 myxobolus pfeifferi thlohan, 1895 family sphaerosporidae myxobilatus legeri (cpde, 1905) myxobilatus legeri (cpde, 1905) myxobolus dogieli bykhovskaya - pavlovskaya & bykhovski, 1940 myxobolus muelleri btschli, 1882 myxobolus oviformis thlohan, 1892 myxobolus pfeifferi thlohan, 1895 myxobolus dogieli bykhovskaya - pavlovskaya & bykhovski, 1940 myxobolus muelleri btschli, 1882 myxobolus oviformis thlohan, 1892 myxobolus pfeifferi thlohan, 1895 myxobilatus legeri (cpde, 1905), erroneously reported as myxobllatus legerl, with no given authority, description, and illustration, was recorded from skin and gills of c. carpio . Myxobolus dogieli bykhovskaya - pavlovskaya & bykhovski, 1940, was recorded from kidneys and gallbladder of l. abu . The first record of m. dogieli in iraq was mainly from the external surface of heart, liver, and ovaries of l. abu from tigris river at baiji town . Myxobolus muelleri btschli, 1882, was recorded from intestine and liver of c. carpio with the specific name spelled as mlleri . The first record of m. muelleri in iraq was from gills of luciobarbus xanthopterus, reported as b. xanthopterus . Myxobolus oviformis thlohan, 1892, was recorded from skin, intestine, and kidneys of c. carpio [27, 54, 59]. The first report of m. oviformis in iraq was from gill arches and heart of four fish species . Myxobolus pfeifferi thlohan, 1895, was recorded from gills, intestine, liver, kidneys, and gallbladder of c. idella, gills, gallbladder, intestine, kidneys, and liver of c. carpio [25, 27, 29, 53, 59], gills, liver, and intestine of h. molitrix [27, 29], and gills, intestinal wall, and gonads of l. abu . The first report of m. pfeifferi in iraq was from gills of acanthobrama marmid from tigris river at mosul city . So far, m. pfeifferi is the prevalent myxozoan among fishes of iraq as it has 35 fish host species . The class trematoda of the phylum platyhelminthes is represented in farm fishes of babylon province with five species; two species belonged to the genera apharyngostrigea and ascocotyle and three species to the genus diplostomum . Phylum platyhelminthes class trematoda order diplostomida family strigeidae apharyngostrigea cornu (zeder, 1800) family diplostomidae diplostomum indistinctum (guberlet, 1923) hughes, 1929 diplostomum paraspathaceum schigin, 1965 diplostomum spathaceum (rudolphi, 1819) olsson, 1876 order plagiorchiida family heterophyidae ascocotyle coleostoma (looss, 1896) looss, 1899 order diplostomida family strigeidae apharyngostrigea cornu (zeder, 1800) family diplostomidae diplostomum indistinctum (guberlet, 1923) hughes, 1929 diplostomum paraspathaceum schigin, 1965 diplostomum spathaceum (rudolphi, 1819) olsson, 1876 order plagiorchiida family heterophyidae ascocotyle coleostoma (looss, 1896) looss, 1899 family strigeidae apharyngostrigea cornu (zeder, 1800) family diplostomidae diplostomum indistinctum (guberlet, 1923) hughes, 1929 diplostomum paraspathaceum schigin, 1965 diplostomum spathaceum (rudolphi, 1819) olsson, 1876 apharyngostrigea cornu (zeder, 1800) apharyngostrigea cornu (zeder, 1800) diplostomum indistinctum (guberlet, 1923) hughes, 1929 diplostomum paraspathaceum schigin, 1965 diplostomum spathaceum (rudolphi, 1819) olsson, 1876 diplostomum indistinctum (guberlet, 1923) hughes, 1929 diplostomum paraspathaceum schigin, 1965 diplostomum spathaceum (rudolphi, 1819) olsson, 1876 family heterophyidae ascocotyle coleostoma (looss, 1896) looss, 1899 ascocotyle coleostoma (looss, 1896) looss, 1899 ascocotyle coleostoma (looss, 1896) looss, 1899 apharyngostrigea cornu (zeder, 1800) was recorded as metacercaria from mesentery, coelom and liver of c. carpio . No more records are so far known on the occurrence of a. cornu from fishes of iraq . The adult worm of this parasite was detected from the intestine of the purple heron ardea purpurea in bahr al - najaf depression . Ascocotyle coleostoma (looss, 1896) looss, 1899, was recorded as metacercaria from gills of h. fossilis . This parasite was reported for the first time in iraq from gills of h. fossilis and l. abu from diyala river . The adult worm of a. coleostoma was detected from the grey heron a. cinerea in babylon (now al - furat) fish farm . Diplostomum indistinctum (guberlet, 1923) hughes, 1929, was recorded as metacercaria from eyes of h. molitrix . The first occurrence of metacercariae of d. indistinctum was from eyes of luciobarbus esocinus, reported as b. esocinus from fish market in mosul city . No more records are so far known on the occurrence of this parasite from fishes of iraq . Diplostomum paraspathaceum schigin, 1965, was recorded as metacercaria from eyes of both c. idella and c. carpio . Diplostomum spathaceum (rudolphi, 1819) olsson, 1876, was recorded as metacercaria from eyes of the three carp species: c. idella [43, 56], c. carpio [29, 43], and h. molitrix [43, 56]. The first occurrence of metacercariae of d. spathaceum was from eyes of c. luteus, reported as b. luteus, cyprinion macrostomum, and c. carpio from dokan lake . Thirty - four hosts are so far known for this parasite in fishes of iraq . Adult worms of this parasite were found in the intestine of some fish - eating birds such as the silver gull larus argentatus in bahr al - najaf depression . The class monogenea of the phylum platyhelminthes is represented in farm fishes of babylon province with 45 species: 12 species of the genus gyrodactylus, 26 species of dactylogyrus, and one species of each of the genera pseudacolpenteron, diplozoon, eudiplozoon, paradiplozoon, and microcotyle in addition to some unidentified species of dactylogyrus and diplozoon . It is appropriate to mention here that this group is considered as monogenea by some electronic sites [912] but as monogenoidea in some references [14, 66]. Phylum platyhelminthes class monogenea order gyrodactylidea family gyrodactylidae gyrodactylus baicalensis bogolepova, 1950 gyrodactylus ctenopharngodontis ling in gusev, 1952 gyrodactylus elegans von nordmann, 1832 gyrodactylus kherulensis ergens, 1974 gyrodactylus macracanthus hukuda, 1940 gyrodactylus malmbergi ergens, 1961 gyrodactylus markevitschi kulakovskaya, 1952 gyrodactylus medius kathariner, 1895 gyrodactylus menschikowi gvosdev, 1950 gyrodactylus salaris malmberg, 1957 gyrodactylus sprostonae ling, 1962 gyrodactylus vicinus bychowsky, 1957 order dactylogyridea family dactylogyridae dactylogyrus achmerowi gusev, 1955 dactylogyrus amurensis akhmerov, 1952 dactylogyrus anchoratus (dujardin, 1845) wagener, 1857 dactylogyrus arcuatus yamaguti, 1942 dactylogyrus barbioides gusev, ali, abdul - ameer, amin & molnr, 1993 dactylogyrus cornu linstow, 1878 dactylogyrus crassus kulwiec, 1927 dactylogyrus ctenopharyngodonis achmerow, 1952 dactylogyrus dogieli gusev, 1953 dactylogyrus ergensi molnr, 1964 dactylogyrus extensus mueller & van cleave, 1932 dactylogyrus gobii gvosdev, 1950 dactylogyrus hypophthalmichthys akhmerov, 1952 dactylogyrus inexpectatus izjumova, in gusev, 1955 dactylogyrus jamansajensis osmanov, 1958 dactylogyrus lamellatus akhmerov, 1952 dactylogyrus latituba gusev, 1955 dactylogyrus lopuchinae jukhimenko, 1981 dactylogyrus minutus kulwiec, 1927 dactylogyrus navicularis a. gusev, 1955 dactylogyrus phoxini malevitskaia, 1949 dactylogyrus propinquus bychowsky, 1931 dactylogyrus sahuensis ling in chen et al ., 1973 dactylogyrus simplex bychowsky, 1936 dactylogyrus skrjabini akhmerov, 1954 dactylogyrus vastator nybelin, 1924 dactylogyrus spp . Pseudacolpenteron pavlovskii bychowsky & gussev, 1955 order mazocraeidea family diplozoidae diplozoon paradoxum nordmann, 1832 diplozoon sp . Eudiplozoon nipponicum (goto, 1891) paradiplozoon barbi (reichenbach - klinke, 1951) family microcotylidae microcotyle donavini van beneden & hesse, 1863 order gyrodactylidea family gyrodactylidae gyrodactylus baicalensis bogolepova, 1950 gyrodactylus ctenopharngodontis ling in gusev, 1952 gyrodactylus elegans von nordmann, 1832 gyrodactylus kherulensis ergens, 1974 gyrodactylus macracanthus hukuda, 1940 gyrodactylus malmbergi ergens, 1961 gyrodactylus markevitschi kulakovskaya, 1952 gyrodactylus medius kathariner, 1895 gyrodactylus menschikowi gvosdev, 1950 gyrodactylus salaris malmberg, 1957 gyrodactylus sprostonae ling, 1962 gyrodactylus vicinus bychowsky, 1957 order dactylogyridea family dactylogyridae dactylogyrus achmerowi gusev, 1955 dactylogyrus amurensis akhmerov, 1952 dactylogyrus anchoratus (dujardin, 1845) wagener, 1857 dactylogyrus arcuatus yamaguti, 1942 dactylogyrus barbioides gusev, ali, abdul - ameer, amin & molnr, 1993 dactylogyrus cornu linstow, 1878 dactylogyrus crassus kulwiec, 1927 dactylogyrus ctenopharyngodonis achmerow, 1952 dactylogyrus dogieli gusev, 1953 dactylogyrus ergensi molnr, 1964 dactylogyrus extensus mueller & van cleave, 1932 dactylogyrus gobii gvosdev, 1950 dactylogyrus hypophthalmichthys akhmerov, 1952 dactylogyrus inexpectatus izjumova, in gusev, 1955 dactylogyrus jamansajensis osmanov, 1958 dactylogyrus lamellatus akhmerov, 1952 dactylogyrus latituba gusev, 1955 dactylogyrus lopuchinae jukhimenko, 1981 dactylogyrus minutus kulwiec, 1927 dactylogyrus navicularis a. gusev, 1955 dactylogyrus phoxini malevitskaia, 1949 dactylogyrus propinquus bychowsky, 1931 dactylogyrus sahuensis ling in chen et al ., 1973 dactylogyrus simplex bychowsky, 1936 dactylogyrus skrjabini akhmerov, 1954 dactylogyrus vastator nybelin, 1924 dactylogyrus spp . Pseudacolpenteron pavlovskii bychowsky & gussev, 1955 order mazocraeidea family diplozoidae diplozoon paradoxum nordmann, 1832 diplozoon sp . Eudiplozoon nipponicum (goto, 1891) paradiplozoon barbi (reichenbach - klinke, 1951) family microcotylidae microcotyle donavini van beneden & hesse, 1863 family gyrodactylidae gyrodactylus baicalensis bogolepova, 1950 gyrodactylus ctenopharngodontis ling in gusev, 1952 gyrodactylus elegans von nordmann, 1832 gyrodactylus kherulensis ergens, 1974 gyrodactylus macracanthus hukuda, 1940 gyrodactylus malmbergi ergens, 1961 gyrodactylus markevitschi kulakovskaya, 1952 gyrodactylus medius kathariner, 1895 gyrodactylus menschikowi gvosdev, 1950 gyrodactylus salaris malmberg, 1957 gyrodactylus sprostonae ling, 1962 gyrodactylus vicinus bychowsky, 1957 family gyrodactylidae gyrodactylus baicalensis bogolepova, 1950 gyrodactylus ctenopharngodontis ling in gusev, 1952 gyrodactylus elegans von nordmann, 1832 gyrodactylus kherulensis ergens, 1974 gyrodactylus macracanthus hukuda, 1940 gyrodactylus malmbergi ergens, 1961 gyrodactylus markevitschi kulakovskaya, 1952 gyrodactylus medius kathariner, 1895 gyrodactylus menschikowi gvosdev, 1950 gyrodactylus salaris malmberg, 1957 gyrodactylus sprostonae ling, 1962 gyrodactylus vicinus bychowsky, 1957 gyrodactylus baicalensis bogolepova, 1950 gyrodactylus ctenopharngodontis ling in gusev, 1952 gyrodactylus elegans von nordmann, 1832 gyrodactylus kherulensis ergens, 1974 gyrodactylus macracanthus hukuda, 1940 gyrodactylus malmbergi ergens, 1961 gyrodactylus markevitschi kulakovskaya, 1952 gyrodactylus medius kathariner, 1895 gyrodactylus menschikowi gvosdev, 1950 gyrodactylus salaris malmberg, 1957 gyrodactylus sprostonae ling, 1962 gyrodactylus vicinus bychowsky, 1957 family dactylogyridae dactylogyrus achmerowi gusev, 1955 dactylogyrus amurensis akhmerov, 1952 dactylogyrus anchoratus (dujardin, 1845) wagener, 1857 dactylogyrus arcuatus yamaguti, 1942 dactylogyrus barbioides gusev, ali, abdul - ameer, amin & molnr, 1993 dactylogyrus cornu linstow, 1878 dactylogyrus crassus kulwiec, 1927 dactylogyrus ctenopharyngodonis achmerow, 1952 dactylogyrus dogieli gusev, 1953 dactylogyrus ergensi molnr, 1964 dactylogyrus extensus mueller & van cleave, 1932 dactylogyrus gobii gvosdev, 1950 dactylogyrus hypophthalmichthys akhmerov, 1952 dactylogyrus inexpectatus izjumova, in gusev, 1955 dactylogyrus jamansajensis osmanov, 1958 dactylogyrus lamellatus akhmerov, 1952 dactylogyrus latituba gusev, 1955 dactylogyrus lopuchinae jukhimenko, 1981 dactylogyrus minutus kulwiec, 1927 dactylogyrus navicularis a. gusev, 1955 dactylogyrus phoxini malevitskaia, 1949 dactylogyrus propinquus bychowsky, 1931 dactylogyrus sahuensis ling in chen et al ., 1973 dactylogyrus simplex bychowsky, 1936 dactylogyrus skrjabini akhmerov, 1954 dactylogyrus vastator nybelin, 1924 dactylogyrus spp . Pseudacolpenteron pavlovskii bychowsky & gussev, 1955 family dactylogyridae dactylogyrus achmerowi gusev, 1955 dactylogyrus amurensis akhmerov, 1952 dactylogyrus anchoratus (dujardin, 1845) wagener, 1857 dactylogyrus arcuatus yamaguti, 1942 dactylogyrus barbioides gusev, ali, abdul - ameer, amin & molnr, 1993 dactylogyrus cornu linstow, 1878 dactylogyrus crassus kulwiec, 1927 dactylogyrus ctenopharyngodonis achmerow, 1952 dactylogyrus dogieli gusev, 1953 dactylogyrus ergensi molnr, 1964 dactylogyrus extensus mueller & van cleave, 1932 dactylogyrus gobii gvosdev, 1950 dactylogyrus hypophthalmichthys akhmerov, 1952 dactylogyrus inexpectatus izjumova, in gusev, 1955 dactylogyrus jamansajensis osmanov, 1958 dactylogyrus lamellatus akhmerov, 1952 dactylogyrus latituba gusev, 1955 dactylogyrus lopuchinae jukhimenko, 1981 dactylogyrus minutus kulwiec, 1927 dactylogyrus navicularis a. gusev, 1955 dactylogyrus phoxini malevitskaia, 1949 dactylogyrus propinquus bychowsky, 1931 dactylogyrus sahuensis ling in chen et al ., 1973 dactylogyrus simplex bychowsky, 1936 dactylogyrus skrjabini akhmerov, 1954 dactylogyrus vastator nybelin, 1924 dactylogyrus spp . Pseudacolpenteron pavlovskii bychowsky & gussev, 1955 dactylogyrus achmerowi gusev, 1955 dactylogyrus amurensis akhmerov, 1952 dactylogyrus anchoratus (dujardin, 1845) wagener, 1857 dactylogyrus arcuatus yamaguti, 1942 dactylogyrus barbioides gusev, ali, abdul - ameer, amin & molnr, 1993 dactylogyrus cornu linstow, 1878 dactylogyrus crassus kulwiec, 1927 dactylogyrus ctenopharyngodonis achmerow, 1952 dactylogyrus dogieli gusev, 1953 dactylogyrus ergensi molnr, 1964 dactylogyrus extensus mueller & van cleave, 1932 dactylogyrus gobii gvosdev, 1950 dactylogyrus hypophthalmichthys akhmerov, 1952 dactylogyrus inexpectatus izjumova, in gusev, 1955 dactylogyrus jamansajensis osmanov, 1958 dactylogyrus lamellatus akhmerov, 1952 dactylogyrus latituba gusev, 1955 dactylogyrus lopuchinae jukhimenko, 1981 dactylogyrus minutus kulwiec, 1927 dactylogyrus navicularis a. gusev, 1955 dactylogyrus phoxini malevitskaia, 1949 dactylogyrus propinquus bychowsky, 1931 dactylogyrus sahuensis ling in chen et al ., 1973 dactylogyrus simplex bychowsky, 1936 dactylogyrus skrjabini akhmerov, 1954 dactylogyrus vastator nybelin, 1924 pseudacolpenteron pavlovskii bychowsky & gussev, 1955 family diplozoidae diplozoon paradoxum nordmann, 1832 diplozoon sp . Eudiplozoon nipponicum (goto, 1891) paradiplozoon barbi (reichenbach - klinke, 1951) family microcotylidae microcotyle donavini van beneden & hesse, 1863 diplozoon paradoxum nordmann, 1832 diplozoon sp . Eudiplozoon nipponicum (goto, 1891) paradiplozoon barbi (reichenbach - klinke, 1951) diplozoon paradoxum nordmann, 1832 eudiplozoon nipponicum (goto, 1891) paradiplozoon barbi (reichenbach - klinke, 1951) family microcotylidae microcotyle donavini van beneden & hesse, 1863 microcotyle donavini van beneden & hesse, 1863 gyrodactylus baicalensis bogolepova, 1950, was recorded from skin, fins, and gills of c. carpio [25, 27, 29, 52]. The first report of g. baicalensis in iraq was from skin, buccal cavity, and gills of c. carpio from al - suwaira and al - latifiya fish farms . Gyrodactylus ctenopharngodontis ling in gusev, 1952, was recorded from skin, fins, buccal cavity, and gills of c. idella [24, 27]. The first report of g. ctenopharngodontis in iraq was from gills of c. idella from babylon fish farm . No more hosts are so far recorded for this parasite in iraq . Gyrodactylus elegans von nordmann, 1832, was recorded from skin, fins, buccal cavity, and gills of both c. idella [27, 29, 52] and c. carpio [2527, 29, 30, 32, 33, 43, 50, 52, 53, 55, 56, 60] as well as from gills of h. molitrix and from l. abu with no mention to site of infection . The first report of g. elegans in iraq was from c. carpio from al - zaafaraniya fish farm and l. abu from al - latifiya fish farm . Gyrodactylus kherulensis ergens, 1974, was recorded from skin and gills of c. idella and skin, fins, and gills of c. carpio [18, 27]. The first report of g. kherulensis in iraq was from gills of c. carpio from babylon fish farm . Gyrodactylus macracanthus hukuda, 1940 (reported as g. paralatus gusev, 1955), was recorded from skin and gills of c. carpio and skin, fins, and buccal cavity of h. molitrix . Later on, it was reported from both hosts as g. paralatus also . According to gussev and pugachev et al . No more hosts are so far known for g. macracanthus or its synonym g. paralatus from fishes of iraq . Gyrodactylus malmbergi ergens, 1961, was recorded from skin, fins, and gills of c. carpio and from skin and gills of h. molitrix . This was its first report in iraq and no more hosts are so far known for g. malmbergi from fishes of iraq . Gyrodactylus markevitschi kulakovskaya, 1952, was recorded from skin and gills of c. carpio [27, 45, 47, 60]. The first report of this parasite in iraq was from gills of capoeta trutta (reported as varicorhinus trutta) from tigris river at baiji town . Gyrodactylus medius kathariner, 1895, was recorded from skin and fins of c. carpio . The year of authority of this parasite was erroneously given as 1893 instead of 1895 by al - zubaidy as well as by three other references according to mhaisen and abdul - ameer . Also the authorship of this parasite was given as katheriner instead of kathariner according to monodb . Gyrodactylus menschikowi gvosdev, 1950, was recorded from skin and gills of c. carpio . The first report of this parasite in iraq was from gills and skin of c. carpio and skin, fins, and gills of l. abu both from hilla river . Gyrodactylus salaris malmberg, 1957, was recorded from skin and gills of c. carpio [27, 29]. The first report of this parasite in iraq was from gills and skin of c. carpio from al - furat fish farm . The year of authority of this parasite was reported as 1956 instead of 1957 by al - zubaidy and al - jadoaa . Gyrodactylus vicinus bychowsky, 1957, was recorded from skin, fins, and gills of c. carpio [27, 35]. The first report of this parasite in iraq was from skin, fins, and gills of c. carpio from al - furat fish farm . Now, it has three host species in iraq . Finally, the unidentified gyrodactylus species reported from c. carpio [34, 38] were the same 11 species which had been recorded in al - zubaidy . In iraq, so far 15 fish host species were reported for some unspecified gyrodactylus species . Dactylogyrus achmerowi gusev, 1955, was recorded from gills of c. carpio [19, 27, 29, 30, 32, 33, 36, 54, 55, 60]. The first report of d. achmerowi in iraq was from gills of c. carpio from al - wahda fish hatchery at al - suwaira and babylon fish farm . Dactylogyrus anchoratus (dujardin, 1845) wagener, 1857, was recorded from gills of c. carpio [45, 47]. The first report and description of d. anchoratus in iraq were from gills of c. carpio from tigris river at al - zaafaraniya [85, 86]. Dactylogyrus arcuatus yamaguti, 1942, was recorded from gills of c. idella and skin, buccal cavity, and gills of c. carpio [25, 27, 35, 36, 49, 52, 55, 59, 60]. The first report of d. arcuatus in iraq was from skin, buccal cavity, and gills of c. carpio from al - suwaira and al - latifiya fish farms . Dactylogyrus barbioides gusev, ali, abdul - ameer, amin & molnr, 1993, was recorded from gills of c. carpio . D. barbioides was described as a new species from gills of barbus grypus from tigris river near baiji town . Dactylogyrus cornu linstow, 1878, was recorded from gills of c. carpio [27, 59]. D. cornu was recorded for the first time in iraq from gills of five fish species from diyala river . Dactylogyrus crassus kulwiec, 1927, was recorded from gills of c. carpio [45, 47, 55, 60]. D. crassus was recorded for the first time in iraq from gills of c. carpio from al - shark al - awsat fish farm . It is appropriate to mention here that the specific name crassus was misspelled as carassus by al - rubaie et al . . Dactylogyrus ctenopharyngodonis achmerow, 1952, was recorded from gills of c. idella from al - shark al - awsat fish farm . This is the only report on the occurrence of d. ctenopharyngodonis in fishes of iraq . Dactylogyrus dogieli gusev, 1953, was recorded from gills of c. carpio in fish cages and an earthen pond in sadat al - hindiya . The first report of d. dogieli was from five fish species from euphrates river at al - musaib city and its full description and illustration were published later by al - sa'adi et al . . Dactylogyrus ergensi molnr, 1964, was recorded from gills of c. carpio from al - furat fish farm . Dactylogyrus extensus mueller & van cleave, 1932, was recorded from gills of c. idella, buccal cavity and gills of c. carpio [25, 27, 29, 30, 32, 33, 36, 43, 45, 47, 52, 54, 55, 59, 60], and gills of h. molitrix [29, 45, 47]. The first report of d. extensus in iraq was from the buccal cavity and gills of c. carpio from al - suwaira and al - latifiya fish farms . D. solidus which was also recorded from the same host by salih et al . As well as by mhaisen & abul - eis and al - rubaie et al . D. extensus and its synonym d. solidus have so far 17 fish host species in iraq . Dactylogyrus gobii gvosdev, 1950, was recorded from skin and gills of c. carpio [45, 47, 60]. D. gobii was recorded for the first time in iraq from gills of c. carpio from al - shark al - awsat fish farm . Dactylogyrus hypophthalmichthys akhmerov, 1952, was recorded from skin, buccal cavity, and gills of h. molitrix [21, 27, 43, 45, 47, 52, 56]. It is reliable to state here that d. hypophthalmichthys was reported as neodactylogyrus hypophthalmichthys by asmar et al . . The first report of d. hypophthalmichthys in iraq was from the buccal cavity and gills of h. molitrix from al - suwaira and al - latifiya fish farms . H. molitrix is the only host so far known for d. hypophthalmichthys in iraq . Dactylogyrus inexpectatus izjumova, in gusev, 1955, was recorded from skin and gills of c. idella [27, 29, 52], gills of c. carpio [27, 29], and gills of h. molitrix . The first report of d. inexpectatus in iraq was from skin and gills of c. idella from al - suwaira and al - latifiya fish farms . Dactylogyrus jamansajensis osmanov, 1958, was recorded from gills of c. carpio [45, 47]. The first report of d. jamansajensis in iraq was from gills of c. luteus from manmade lakes, north of baghdad . Dactylogyrus lamellatus akhmerov, 1952, was recorded from skin, fins, buccal cavity, and gills of c. idella [24, 27, 43, 52, 56] and gills of c. carpio . The first report of d. lamellatus in iraq was from the skin, buccal cavity, and gills of c. idella from al - suwaira and al - latifiya fish farms . Dactylogyrus latituba gusev, 1955, was recorded from gills of both c. idella and c. carpio [25, 27] as well as from the buccal cavity and gills of h. molitrix . The first report of d. latituba in iraq was from gills of c. luteus from manmade lakes, north of baghdad . Dactylogyrus lopuchinae jukhimenko, 1981, was recorded from gills of c. carpio [45, 47, 54]. D. lopuchinae was recorded for the first time in iraq from gills of c. carpio from al - shark al - awsat fish farm . Dactylogyrus minutus kulwiec, 1927, was recorded from skin, fins, and gills of c. carpio [27, 30, 32, 33, 45, 47, 55, 56, 59, 60]. The first report on this parasite in iraq was from gills of c. carpio from tigris river at al - zaafaraniya, south of baghdad, and al - qadisia dam lake, while its description and illustration were given later by mhaisen et al . . Dactylogyrus navicularis a. gusev, 1955, was recorded from fins, buccal cavity, and gills of c. carpio [27, 35, 60]. The first report of d. navicularis in iraq was from the buccal cavity, fins, and gills of c. carpio from al - furat fish farm . C. carpio is the only host so far known for d. navicularis in iraq . Dactylogyrus phoxini malevitskaia, 1949, was recorded from skin and gills of c. carpio [45, 47, 60]. The first record of d. phoxini in iraq was in june 1995 from gills of c. carpio from tigris river at al - zaafaraniya but the report was published later by balasem et al . . Dactylogyrus propinquus bychowsky, 1931, was recorded from gills of c. carpio [27, 35, 49]. The first report of d. propinquus in iraq was from gills of c. carpio from al - furat fish farm . C. carpio is the only host so far known for d. propinquus in iraq . The first report of d. sahuensis in iraq was from fins and gills of c. carpio from al - furat fish farm . C. carpio is the only host so far known for d. sahuensis in iraq . Dactylogyrus simplex bychowsky, 1936, was recorded from c. carpio with no mention to site of infection . The first report of d. simplex in iraq was from gills of c. carpio from the new fish farm of the fish research center at al - zaafaraniya . Dactylogyrus skrjabini akhmerov, 1954, was recorded from buccal cavity and gills of c. carpio [27, 45, 47] and buccal cavity and gills of h. molitrix [21, 27, 52]. The first report of d. skrjabini in iraq was from buccal cavity and gills of h. molitrix from al - suwaira and al - latifiya fish farms . Dactylogyrus vastator nybelin, 1924, was recorded from gills of c. idella and skin and gills of c. carpio [26, 27, 30, 32, 33, 35, 36, 43, 49, 52, 53, 56, 59, 60, 64]. The first report of d. vastator from iraq was from skin and gills of c. macrostomum from tigris river at baghdad . So far, d. vastator was reported from 33 fish host species from north, middle, and south of iraq . Unidentified dactylogyrus species were recorded from skin and gills of c. idella and from skin, buccal cavity, and gills of c. carpio [34, 38, 56]. Some of these specimens were larval stages, while the unidentified dactylogyrus species of al - zubaidy et al . [34, 38] were the same 15 species which were recorded in al - zubaidy . In iraq, so far nine fish host species were reported for some unspecified dactylogyrus species . Pseudacolpenteron pavlovskii bychowsky & gussev, 1955, was recorded from fins and gills of c. carpio [25, 27, 45, 47] and gills of h. molitrix . The first report of p. pavlovskii from iraq was from skin and gills of c. carpio from babylon fish farm . This parasite was reported for the first time in iraq from gills of carasobarbus luteus, reported as barbus luteus, from al - husainia creek, karbala province . Some other unidentified diplozoon species occurred as larvae in 12 fish host species in iraq . This parasite was recorded for the first time in iraq from gills of c. carpio from a manmade lake in baghdad as diplozoon nipponicum but then it was reported by its valid name e. nipponicum by all subsequent researchers . Paradiplozoon barbi (reichenbach - klinke, 1951) was recorded from gills of c. carpio as diplozoon barbi . This parasite was reported for the first time in iraq from gills of chondrostoma nasus, c. regium, and c. carpio from tigris river at baghdad as diplozoon barbi . Also, all the subsequent records in the iraqi literature, except the checklists of mhaisen and abdul - ameer, referred to this parasite as d. barbi . According to khotenovsky, d. barbi is a synonym of p. barbi . Microcotyle donavini van beneden & hesse, 1863, was recorded from gills of l. abu from babylon fish farm . The class cestoda of the phylum platyhelminthes is represented in farm fishes of babylon province with five species: one species of each of the genera bothriocephalus, ligula, and neogryporhynchus as well as two species of proteocephalus . Phylum platyhelminthes class cestoda order bothriocephalidea family bothriocephalidae bothriocephalus acheilognathi yamaguti, 1934 order diphyllobothriidea family diphyllobothriidae ligula intestinalis (l., 1758) bloch, 1782 order proteocephalidea family proteocephalidae proteocephalus osculatus (goeze, 1782) nybelin, 1942 proteocephalus torulosus (batsch, 1786) nufer, 1905 order cyclophyllidea family dilepididae neogryporhynchus cheilancristrotus (wedl, 1855) baer & bona, 1960 order bothriocephalidea family bothriocephalidae bothriocephalus acheilognathi yamaguti, 1934 order diphyllobothriidea family diphyllobothriidae ligula intestinalis (l., 1758) bloch, 1782 order proteocephalidea family proteocephalidae proteocephalus osculatus (goeze, 1782) nybelin, 1942 proteocephalus torulosus (batsch, 1786) nufer, 1905 order cyclophyllidea family dilepididae neogryporhynchus cheilancristrotus (wedl, 1855) baer & bona, 1960 order bothriocephalidea family bothriocephalidae bothriocephalus acheilognathi yamaguti, 1934 family bothriocephalidae bothriocephalus acheilognathi yamaguti, 1934 bothriocephalus acheilognathi yamaguti, 1934 order diphyllobothriidea family diphyllobothriidae ligula intestinalis (l., 1758) bloch, 1782 family diphyllobothriidae ligula intestinalis (l., 1758) bloch, 1782 ligula intestinalis (l., 1758) bloch, 1782 order proteocephalidea family proteocephalidae proteocephalus osculatus (goeze, 1782) nybelin, 1942 proteocephalus torulosus (batsch, 1786) nufer, 1905 family proteocephalidae proteocephalus osculatus (goeze, 1782) nybelin, 1942 proteocephalus torulosus (batsch, 1786) nufer, 1905 proteocephalus osculatus (goeze, 1782) nybelin, 1942 proteocephalus torulosus (batsch, 1786) nufer, 1905 family dilepididae neogryporhynchus cheilancristrotus (wedl, 1855) baer & bona, 1960 neogryporhynchus cheilancristrotus (wedl, 1855) baer & bona, 1960 neogryporhynchus cheilancristrotus (wedl, 1855) baer & bona, 1960 bothriocephalus acheilognathi yamaguti, 1934, was recorded from the intestine of both c. idella [24, 56] and c. carpio [2527, 52, 53, 62]. It is appropriate to mention here that this worm was reported by its synonym b. opsariichthydis by salih et al . And al - zubaidy . The first report of b. acheilognathi in iraq was from the intestine of c. carpio from different fish farms near baghdad . Two other species of bothriocephalus, b. gowkongensis yeh, 1955, and b. opsariichthydis yamaguti, 1934, were also reported from iraq . According to molnr, both, b. acheilognathi and both of its above - named synonyms has so far a total of 21 host species in iraq . Ligula intestinalis (l., 1758) bloch, 1782, was recorded from the body cavity of both c. idella [24, 27, 29] and c. carpio . L. intestinalis was reported for the first time in iraq as a plerocercoid from the body cavity of leuciscus vorax (reported as a. vorax) from shatt al - arab river ., the adult stage of l. intestinalis was reported from the intestine of the moorhen gallinula chloropus chloropus from around baghdad . Proteocephalus osculatus (goeze, 1782) nybelin, 1942, was recorded from the intestine of c. carpio . The first report of this parasite in iraq was from the intestine of l. vorax (reported as a. vorax) from al - tharthar lake . Proteocephalus torulosus (batsch, 1786) nufer, 1905, was recorded from intestine of c. carpio . The first report of this parasite in iraq was from the intestine of c. carpio from a fish farm near baghdad city . Neogryporhynchus cheilancristrotus (wedl, 1855) baer & bona, 1960, was recorded from the intestine of c. carpio by al - zubaidy as gryporhynchus cheilancristrotus . The first report of n. cheilancristrotus in iraq was from the intestine of l. abu from diyala river . The phylum nematoda is represented in farm fishes of babylon province with three species: unidentified larval species of the genus contracaecum as well as one species of each of the genera cucullanus and rhabdochona . Phylum nematoda class secernentea order ascaridida family anisakidae contracaecum spp . Family cucullanidae cucullanus cyprini yamaguti, 1941 order spirurida family rhabdochonidae rhabdochona hellichi (srmek, 1901) order ascaridida family anisakidae contracaecum spp . Family cucullanidae cucullanus cyprini yamaguti, 1941 order spirurida family rhabdochonidae rhabdochona hellichi (srmek, 1901) family anisakidae contracaecum spp . Family cucullanidae cucullanus cyprini yamaguti, 1941 cucullanus cyprini yamaguti, 1941 cucullanus cyprini yamaguti, 1941 family rhabdochonidae rhabdochona hellichi (srmek, 1901) family rhabdochonidae rhabdochona hellichi (srmek, 1901) rhabdochona hellichi (srmek, 1901) unidentified larval species of contracaecum was recorded from the intestinal wall, body cavity, liver, spleen, heart, and gonads of c. carpio [27, 29] and intestinal wall of l. abu [22, 37, 42]. Larvae in iraq was from the body cavity and different viscera of 10 fish species from different inland waters of iraq . Adult worms of contracaecum spp . Were detected from six species of aquatic birds in iraq, egretta alba, e. garzetta, ardeola ralloides, botaurus stellaris, ardea purpurea, and ceryle rudis, from bahr al - najaf depression . The first report of this parasite in iraq was from the intestine of alburnus caeruleus and luciobarbus xanthopterus (reported as b. xanthopterus) from al - tharthar lake . Rhabdochona hellichi (srmek, 1901), erroneously reported as r. bellichi, was recorded from intestine of the three carp species: c. idella, c. carpio, and h. molitrix . Ali et al . Reported this parasite (also erroneously as r. bellichi) from the intestine and coelom of l. xanthopterus (reported as b. xanthopterus), h. fossilis, and mystus pelusius (reported as m. halepensis). The phylum acanthocephala is represented in farm fishes of babylon province with two valid species of the genus neoechinorhynchus . Phylum acanthocephala class eoacanthocephala order neoechinorhynchida family neoechinorhynchidae neoechinorhynchus iraqensis amin, al - sady, mhaisen & bassat, 2001 neoechinorhynchus rutili (mller, 1780) hamann, 1892 class eoacanthocephala order neoechinorhynchida family neoechinorhynchidae neoechinorhynchus iraqensis amin, al - sady, mhaisen & bassat, 2001 neoechinorhynchus rutili (mller, 1780) hamann, 1892 order neoechinorhynchida family neoechinorhynchidae neoechinorhynchus iraqensis amin, al - sady, mhaisen & bassat, 2001 neoechinorhynchus rutili (mller, 1780) hamann, 1892 family neoechinorhynchidae neoechinorhynchus iraqensis amin, al - sady, mhaisen & bassat, 2001 neoechinorhynchus rutili (mller, 1780) hamann, 1892 neoechinorhynchus iraqensis amin, al - sady, mhaisen & bassat, 2001 neoechinorhynchus rutili (mller, 1780) hamann, 1892 neoechinorhynchus iraqensis amin, al - sady, mhaisen & bassat, 2001, was recorded from intestine of both c. carpio and l. abu [22, 50]. It is appropriate to mention here that this species was reported as n. agilis from c. carpio and l. abu by al - zubaidy and ali et al . The first report of n. iraqensis was as species de novo from the intestine of l. abu from the euphrates river at al - fallujah region, while its description was given later by amin et al . . N. iraqensis and the misidentified n. agilis have so far 24 fish host species in iraq . Neoechinorhynchus rutili (mller, 1780) hamann, 1892, was recorded from the intestine of the three carp species: c. idella, c. carpio [27, 29], and h. molitrix . N. rutili was firstly recorded by herzog from l. xanthopterus (reported as b. xanthopterus) from tigris and diyala rivers near baghdad and from l. abu (reported as mugil abu) from citscher oasis near al - fallujah . The subphylum crustacea of the phylum arthropoda is represented in farm fishes of babylon province with nine species: one species of each of the genera argulus, dermoergasilus, paraergasilus, lamproglena, and lernaea and three species of ergasilus in addition to unidentified species of ergasilus . Phylum arthropoda subphylum crustacea class maxillopoda order arguloida family argulidae argulus foliaceus (l., 1758) order cyclopoida family ergasilidae dermoergasilus varicoleus ho, jayarajan & radhakrishnan, 1992 ergasilus barbi rahemo, 1982 ergasilus mosulensis rahemo, 1982 ergasilus sieboldi von nordmann, 1832 ergasilus sp . Paraergasilus inflatus ho, khamees & mhaisen, 1996 family lernaeidae lamproglena pulchella von nordmann, 1832 lernaea cyprinacea l., 1758 class maxillopoda order arguloida family argulidae argulus foliaceus (l., 1758) order cyclopoida family ergasilidae dermoergasilus varicoleus ho, jayarajan & radhakrishnan, 1992 ergasilus barbi rahemo, 1982 ergasilus mosulensis rahemo, 1982 ergasilus sieboldi von nordmann, 1832 ergasilus sp . Paraergasilus inflatus ho, khamees & mhaisen, 1996 family lernaeidae lamproglena pulchella von nordmann, 1832 lernaea cyprinacea l., 1758 order arguloida family argulidae argulus foliaceus (l., 1758) order cyclopoida family ergasilidae dermoergasilus varicoleus ho, jayarajan & radhakrishnan, 1992 ergasilus barbi rahemo, 1982 ergasilus mosulensis rahemo, 1982 ergasilus sieboldi von nordmann, 1832 ergasilus sp . Paraergasilus inflatus ho, khamees & mhaisen, 1996 family lernaeidae lamproglena pulchella von nordmann, 1832 lernaea cyprinacea l., 1758 family argulidae argulus foliaceus (l., 1758) argulus foliaceus (l., 1758) family ergasilidae dermoergasilus varicoleus ho, jayarajan & radhakrishnan, 1992 ergasilus barbi rahemo, 1982 ergasilus mosulensis rahemo, 1982 ergasilus sieboldi von nordmann, 1832 ergasilus sp . Paraergasilus inflatus ho, khamees & mhaisen, 1996 family lernaeidae lamproglena pulchella von nordmann, 1832 lernaea cyprinacea l., 1758 dermoergasilus varicoleus ho, jayarajan & radhakrishnan, 1992 ergasilus barbi rahemo, 1982 ergasilus mosulensis rahemo, 1982 ergasilus sieboldi von nordmann, 1832 paraergasilus inflatus ho, khamees & mhaisen, 1996 lamproglena pulchella von nordmann, 1832 lernaea cyprinacea l., 1758 argulus foliaceus (l., 1758) was recorded from gills of the three carp species: c. idella, c. carpio [25, 27], and h. molitrix as well as from fins of c. auratus . A. foliaceus was reported for the first time in iraq from skin of c. carpio from al - zaafaraniya fish - culture station and c. luteus (reported as b. luteus) from al - habbaniyah lake . Dermoergasilus varicoleus ho, jayarajan & radhakrishnan, 1992, was recorded from gills of l. abu [31, 40]. This crustacean was reported for the first time in iraq from gills of l. abu from shatt al - arab river . This crustacean was firstly detected from gills of b. grypus from tigris river at mosul city by fattohy and its full description as a new species was achieved by rahemo . Ergasilus mosulensis rahemo, 1982, was recorded from gills of the three carp species, c. idella, c. carpio, and h. molitrix as well as gills of l. abu . This crustacean was firstly detected from gills of l. abu from tigris river at mosul city by fattohy and its full description as a new species was achieved by rahemo . Ergasilus sieboldi von nordmann, 1832, was recorded from gills, buccal cavity, and skin of the three carp species: c. idella [27, 29], c. carpio [25, 27, 29, 51, 55], and h. molitrix [27, 29, 56]. The first report of e. sieboldi in iraq was from gills of l. vorax (reported as a. vorax) from al - habbaniyah lake . Ergasilus sp . Was recorded from l. abu with no mention to site of infection . In addition to 11 species of ergasilus so far recorded from fishes of iraq, some specimens of unidentified ergasilus species were also reported from 12 fish species in iraq . Paraergasilus inflatus ho, khamees & mhaisen, 1996, was recorded from gills of h. molitrix . This crustacean was reported as a new species from gill rakers of l. abu from shatt al - arab river . The first report of l. pulchella in iraq was from gills of chondrostoma regium and capoeta trutta (reported as varicorhinus trutta) from tigris river at mosul city . Lernaea cyprinacea l., 1758, was recorded from skin, fins, and gills of the three carp species, c. idella [24, 2729, 43, 51], c. carpio [6, 2531, 39, 41, 43, 44, 48, 51, 5355, 57, 61, 63], and h. molitrix [21, 2729, 43], as well as from gills of l. abu . The first report of the anchor worm l. cyprinacea in iraq was from skin, fins, buccal cavity, pharyngeal cavity, gills, and anus of seven freshwater fish species from al - zaafaraniya fish - culture station . L. cyprinacea is the commonest crustacean among fishes of iraq as it has so far 30 fish host species in iraq . The phylum mollusca is represented in farm fishes of babylon province with only one parasite species of the genus unio . The systematic account of this parasite, followed by parasite - host list, is given here . Phylum mollusca class bivalvia order unionoida family unionidae unio pictorum (linnaeus, 1758) order unionoida family unionidae unio pictorum (linnaeus, 1758) family unionidae unio pictorum (linnaeus, 1758) unio pictorum (linnaeus, 1758) unio pictorum (linnaeus, 1758) unio pictorum (linnaeus, 1758) was recorded from gills of both c. carpio [27, 29] and h. molitrix . The first report of the glochidial larvae of u. pictorum in iraq was from gills of eight freshwater fish species from diyala river . It is appropriate to mention here that the authority of u. pictorum was erroneously stated as zhadin, 1938, in all the iraqi literature except al - salmany . The following host - parasite list for fish parasites in fish farms of babylon province is compiled . For each host, the scientific names of all recorded parasites are alphabetically enlisted under their major parasitic groups . To economize space, these can be obtained from the account of each concerned parasite species in the part of major groups of parasitic fauna within the results and discussion part . Ciliophora: apiosoma amoebae, a. cylindriformis, a. minuta, a. piscicola, a. poteriformis, chilodonella cyprini, ichthyophthirius multifiliis, trichodina cottidarum, t. domerguei, t. gracilis, t. nigra, and tripartiella amurensis . Myxozoa: myxobilatus legeri, myxobolus muelleri, m. oviformis, and m. pfeifferi . Monogenea: dactylogyrus achmerowi, d. amurensis, d. anchoratus, d. arcuatus, d. barbioides, d. cornu, d. crassus, d. dogieli, d. ergensi, d. extensus, d. gobii, d. inexpectatus, d. jamansajensis, d. lamellatus, d. latituba, d. lopuchinae, d. minutus, d. navicularis, d. phoxini, d. propinquus, d. sahuensis, d. simplex, d. skrjabini, d. vastator, dactylogyrus spp ., diplozoon paradoxum, diplozoon sp ., eudiplozoon nipponicum, gyrodactylus baicalensis, g. elegans, g. kherulensis, g. macracanthus, g. malmbergi, g. markewitschi, g. medius, g. menschikowi, g. salaris, g. sprostonae, g. vicinus, paradiplozoon barbi, and pseudacolpenteron pavlovskii . Cestoda: bothriocephalus acheilognathi, ligula intestinalis, neogryporhynchus cheilancristrotus, proteocephalus osculatus, and p. torulosus . Crustacea: argulus foliaceus, ergasilus mosulensis, e. sieboldi, lamproglena pulchella, and lernaea cyprinacea . Ciliophora: apiosoma amoebae, a. cylindriformis, a. minuta, a. piscicola, a. poteriformis, chilodonella cyprini, ichthyophthirius multifiliis, trichodina cottidarum, t. domerguei, t. gracilis, t. nigra, and tripartiella amurensis . Monogenea: dactylogyrus achmerowi, d. amurensis, d. anchoratus, d. arcuatus, d. barbioides, d. cornu, d. crassus, d. dogieli, d. ergensi, d. extensus, d. gobii, d. inexpectatus, d. jamansajensis, d. lamellatus, d. latituba, d. lopuchinae, d. minutus, d. navicularis, d. phoxini, d. propinquus, d. sahuensis, d. simplex, d. skrjabini, d. vastator, dactylogyrus spp ., eudiplozoon nipponicum, gyrodactylus baicalensis, g. elegans, g. kherulensis, g. macracanthus, g. malmbergi, g. markewitschi, g. medius, g. menschikowi, g. salaris, g. sprostonae, g. vicinus, paradiplozoon barbi, and pseudacolpenteron pavlovskii . Cestoda: bothriocephalus acheilognathi, ligula intestinalis, neogryporhynchus cheilancristrotus, proteocephalus osculatus, and p. torulosus . Crustacea: argulus foliaceus, ergasilus mosulensis, e. sieboldi, lamproglena pulchella, and lernaea cyprinacea . Ciliophora: apiosoma amoebae, a. cylindriformis, a. piscicola, a. poteriformis, chilodonella cyprini, ichthyophthirius multifiliis, trichodina domerguei, and t. nigra . Monogenea: dactylogyrus arcuatus, d. ctenopharyngodonis, d. extensus, d. inexpectatus, d. lamellatus, d. latituba, d. vastator, dactylogyrus sp . Crustacea: argulus foliaceus, ergasilus mosulensis, e. sieboldi, and lernaea cyprinacea . Ciliophora: apiosoma amoebae, a. cylindriformis, a. piscicola, a. poteriformis, chilodonella cyprini, ichthyophthirius multifiliis, trichodina domerguei, and t. nigra . Monogenea: dactylogyrus arcuatus, d. ctenopharyngodonis, d. extensus, d. inexpectatus, d. lamellatus, d. latituba, d. vastator, dactylogyrus sp . Crustacea: argulus foliaceus, ergasilus mosulensis, e. sieboldi, and lernaea cyprinacea . Ciliophora: apiosoma amoebae, a. cylindriformis, a. piscicola, chilodonella cyprini, ichthyophthirius multifiliis, trichodina cottidarum, t. domerguei, and t. nigra . Monogenea: dactylogyrus extensus, d. hypophthalmichthys, d. inexpectatus, d. latituba, d. skrjabini, gyrodactylus elegans, g. macracanthus, g. malmbergi, and pseudacolpenteron pavlovskii . Crustacea: argulus foliaceus, ergasilus mosulensis, e. sieboldi, lernaea cyprinacea, and paraergasilus inflatus . Ciliophora: apiosoma amoebae, a. cylindriformis, a. piscicola, chilodonella cyprini, ichthyophthirius multifiliis, trichodina cottidarum, t. domerguei, and t. nigra . Monogenea: dactylogyrus extensus, d. hypophthalmichthys, d. inexpectatus, d. latituba, d. skrjabini, gyrodactylus elegans, g. macracanthus, g. malmbergi, and pseudacolpenteron pavlovskii . Crustacea: argulus foliaceus, ergasilus mosulensis, e. sieboldi, lernaea cyprinacea, and paraergasilus inflatus . Crustacea: dermoergasilus varicoleus, ergasilus barbi, e. mosulensis, ergasilus sp ., and lernaea cyprinacea . Crustacea: dermoergasilus varicoleus, ergasilus barbi, e. mosulensis, ergasilus sp ., and lernaea cyprinacea.
Chronic pain affects from 25% to 60% of patients submitted to breast cancer treatment, and thus this is an important clinical problem which involves intra- and postoperative factors . Chemotherapy is associated with more severe and more persistent physical symptoms and thus patients should receive appropriate care to mitigate the effects . Fatigue is higher in patients with signs and symptoms such as neck pain, shoulder pain, reduced mobility, and bad body image, demonstrating a multidimensional character that affects psychological and physical aspects of the patient's life . Studies have shown that patients submitted to breast cancer surgery may have central hypersensitivity involving painful symptoms of the neck, axilla, and shoulder . The type of surgery, the lack of a spouse, postoperative infection, the number of lymph nodes resected, radiation therapy, high body mass index (bmi), low socioeconomic level, and advanced disease are associated with pain . Nerve injuries due to surgical trauma and the formation of neuromas are associated with postoperative pain after breast cancer . In relation to the forms of treatment, one study reported that 22.3% of 461 patients with shoulder bursitis were successfully treated with local therapy on the medial border of the scapula . However, a meta - analysis was unable to confirm that transcutaneous electrical stimulation of the nerve is an efficacious form of treatment for pain in these patients . Women have reported that yoga can improve several domains of their quality of life during chemotherapy . This mind - body intervention may help, but more randomized controlled trials about the effects of yoga on the cognition of women with breast cancer under adjuvant chemotherapy treatment should be carried out . The objective of this study was to evaluate pain in women with breast cancer treatment - related lymphedema and the characteristics of aggravating factors and coping mechanisms . Descriptive, qualitative study assessing pain reported after treatment of breast cancer surgery in a group of 46 women . All patients who had been subjected to breast cancer treatment and who were in rehabilitation due to lymphedema in the clinica godoy in so jose do rio preto, sp, in 2010 were considered for participation in this study . The following variables were investigated using a questionnaire: type and length of surgery, number of radiotherapy and chemotherapy sessions, infection, pain (at removed breast or at another site of the body), intensity of pain, and factors that improved and worsened the pain . The intensity of the pain was evaluated using a visual scale of 0 to 10 (no pain: 0; mild pain: 1 to 5; moderate pain: 6 to 9; severe pain 10). The responses to each item were analyzed as a frequency of events (%) and mean or median for age . All patients who accepted to participate in the study signed informed consent forms and the study was approved by the research ethics committee of famerp (protocol number 85923). About half (52.1%) of the participants performed modified radical surgery (91.3% unilateral and 8.6% bilateral) and only 17.37% underwent breast reconstruction surgery; the surgery was performed after more than 19 months in almost 74% of cases . Six to ten sessions of chemotherapy were used in 63.04% of the cases and more than 30 sessions of radiotherapy in 71.3% in a period from 1 to 120 months were used (table 1). Insignificant pain was reported by 32.61% of the women and 67.3% said they suffered pain; it was mild in 28.26% of the cases (scale 15), moderate in 34.78% (scale 69), and severe in 4.35% (table 2). The main coping mechanisms were painkillers for 41.30%, rest for 21.74%, participating in religious services for 17.39%, and chatting with friends for 8.70% (table 2). Another characteristic was that 39.1% of the women reported that they had pain outside the region of the surgery (such as pain in the body and neck, headaches, etc .) Within 6 to 12 months after the surgery which reduced after two years . At the site of the breast, 39.13% reported that the pain persisted for up to one year and 4.34% for up to two years (table 2). Worsening of the pain with physical effort was reported by 50% of the cases, with stress by 34.8%, heat by 13.0%, and cold by 2.2% (table 2). This study shows the prevalence of pain, the coping mechanisms, and the aggravation factors in women treated for breast cancer and subsequently for lymphedema . Pain was reported by 67% of the study sample, with varying intensities of pain and using different mechanisms for its control . However, unconventional options were also reported as a mechanism of coping with pain such as participating in religious services as described by 17.3% of the patients . Thus, a search for something higher and spiritual constituted a protective mechanism against physical pain . Another aspect that calls the attention is that more than 52% of these patients were college graduates and that the level of education did not interfere in their pursuit for faith as a mechanism of pain control . Several factors are cited that can affect pain such as psychological aspects, infection, immunotherapy, type of surgery, nerve injuries, and bursitis; [18] an association of these factors may aggravate the patient's status even further . Another way of controlling pain was chatting with friends as mentioned by 8.6% of the women; thus, a mechanism for pain control that involved spiritual or psychological acts was used by more than 25% of the cases . An important differential that caught the attention was in relation to pain and its location either at the site of surgery or in other parts of the body . The reports of pain in other locations were associated with emotional aspects associated with treatment and sometimes with the type of surgery used . However, the reduction of pain over time was important in the evolution of patients . Worsening of pain on physical exertion was reported by 50% of the patients, with stress being the second most common factor in 34.7% of patients . Thus, several factors that interfere in the pain were identified by participants as were the mechanisms (physical, psychological, religious, and environmental) they used to control it . In addition to the physical and psychological aspects, a broad approach to these patients involves an investigation of difficulties experienced in their day - to - day lives, coping mechanisms, nutritional aspects, and the adaption to their reality; this approach must be individualized [912]. In respect to the occupation of participants in this study, many reported that the surgery took place after they had retired; a second group requested retirement due to illness . The retired individuals had activities in their homes and remained with their families and those who had requested sick leave, even after two years, did not return to their normal activities as they reported feeling a loss of the physical functioning of the arm on the operated side . The majority of individuals who continued in their jobs were self - employed in informal activities and without the necessary documentation to claim sick allowance and thus could not stop working . The majority of participants had completed college education, but this did not change their psychological, social, and physical difficulties . Most did not receive psychological support or any type of physical therapy after surgery . These data highlight the multifactorial consequences of breast cancer treatment, which often are associated and are difficult to detect and so it is difficult to give the required support . In this study, it was found that most of the participants did not receive any kind of psychological and physical support from different health services during treatment . Treatment programs that involve a multidisciplinary team and that more broadly address the problems of these patients are thus recommended [1315] and pain should always be taken into account during treatment . The results of this study show the need for rehabilitation programs immediately after breast cancer surgery . It is being recognized by more and more health care professionals that morbidity after breast cancer treatment causes functional sequelae that interfere in the daily life of patients [13, 14]. In conclusion, many mastectomized patients with lymphedema complain of pain, but pain is often underrecognized and undertreated.
Clinical and radiologic findings of three patients are summarized in table 1 . A 32-year - old woman presented with chest discomfort . It had been diagnosed as alveolar soft - part sarcoma and the patient had undergone radiotheraphy . Chest radiography at that time had revealed a small, well - defined nodule in the left lower lung zone, but biopsy was not performed . Enhanced ct scan demonstrated a clearly enhanced nodule containing focal low attenuation, and clearly enhanced tubular structures, thought to be dilated vessels, were seen in the left lower lobe (fig . The attenuation of the focal low - attenuated area and the tubular structures was 1812 h and 928 h, respectively, while the attenuation of back muscle was 4213 h. wedge resection of the lung was performed, and gross examination showed that tumor size was 2.01.51.5 cm (fig . Microscopic examination revealed dilated vessels corresponding to the enhanced tubular structures demonstrated by ct scan (fig . The pathologic diagnosis was alveolar soft - part sarcoma . A 24-year - old man presented with neck stiffness and headache . Eight years previously a chest wall mass originating from the right second and third ribs had been removed . A chest radiograph obtained on admission showed a mass in the right paratracheal area and multiple nodules in both lungs . Ct scan demonstrated a 55-cm - sized, well - circumscribed, enhanced mass compressing the superior vena cava in the right paratracheal area . High - attenuated tubular structures within the mass, suggesting dilated vessels, were noted, and multiple enlarged hilar lymph nodes and nodules were observed in both lungs . Brain mr images showed clearly enhanced nodules in the right cerebellar hemisphere and at the right occipital pole . Resection of the cerebellar lesion and biopsy of the pulmonary nodule were performed, and pathologic examination revealed metastatic alveolar soft - part sarcoma of the brain and lung . A 31-year - old woman was admitted with left lower leg mass and pain, and she also complained of coughing and blood tinged sputum . Enhanced ct scan revealed multiple enhanced masses of various sizes and intratumoral dilated vessels (fig . It had been diagnosed as alveolar soft - part sarcoma and the patient had undergone radiotheraphy . Chest radiography at that time had revealed a small, well - defined nodule in the left lower lung zone, but biopsy was not performed . Enhanced ct scan demonstrated a clearly enhanced nodule containing focal low attenuation, and clearly enhanced tubular structures, thought to be dilated vessels, were seen in the left lower lobe (fig . The attenuation of the focal low - attenuated area and the tubular structures was 1812 h and 928 h, respectively, while the attenuation of back muscle was 4213 h. wedge resection of the lung was performed, and gross examination showed that tumor size was 2.01.51.5 cm (fig . Microscopic examination revealed dilated vessels corresponding to the enhanced tubular structures demonstrated by ct scan (fig . Eight years previously a chest wall mass originating from the right second and third ribs had been removed . A chest radiograph obtained on admission showed a mass in the right paratracheal area and multiple nodules in both lungs . Ct scan demonstrated a 55-cm - sized, well - circumscribed, enhanced mass compressing the superior vena cava in the right paratracheal area . High - attenuated tubular structures within the mass, suggesting dilated vessels, were noted, and multiple enlarged hilar lymph nodes and nodules were observed in both lungs . Brain mr images showed clearly enhanced nodules in the right cerebellar hemisphere and at the right occipital pole . Resection of the cerebellar lesion and biopsy of the pulmonary nodule were performed, and pathologic examination revealed metastatic alveolar soft - part sarcoma of the brain and lung . A 31-year - old woman was admitted with left lower leg mass and pain, and she also complained of coughing and blood tinged sputum . Enhanced ct scan revealed multiple enhanced masses of various sizes and intratumoral dilated vessels (fig . Alveolar soft - part sarcoma was initially defined in 1952 by christopherson et al . (6) and was described as' characterized by pseudoalveolar or organoid arrangement of cells in relation to numerous delicate endothelial - lined vascular sinuses and septa' . They described twelve cases of rare soft tissue tumor that showed remarkable uniformity and an alveolar pattern . Because of the uncertain origin of the cells, they were forced to use the descriptive term' alveolar soft - part sarcoma' . Gross pathology shows the tumors to be firm, often bulging slightly, variegated in color, and frequently hemorrhagic and necrotic; a tumor may clearly extend into blood vessels . The mass often appears to be well encapsulated but almost always there is histologically proven infiltration . Microscopically, the granular cells are arranged in nests (' alveoli') separated by connective septae that have a rich vascular networks (3). The tumor usually involves patients between the second and fourth decade of life, and involves the lower extremities, the head and neck, the upper extremities, and the trunk, in decreasing order of incidence . There is female predominance and the median age at diagnosis is 22 years for females and 27 years for males (3). Alveolar soft - part sarcoma is of particular clinical significance because of the high prevalence of metastases; at original diagnosis this involves 24 percent of patients . Of those without metastases at original diagnosis, 40% develop these within five years, 62% within 10 years, and 85% within 20 years (3). Pulmonary metastases are found in 42 - 65% of patients (2), and the lung is the most commonly involved site by metastatic tumor the brain and skeleton are the next most commonly invowed sites . The median survival term in patients in whom metastases do not develop is 11 years, but with metastases, this period is reduced to three years (7). In cases of extrapulmonary disease that can be controlled, complete resection of pulmonary metastases of soft tissue sarcoma, including alveolar soft - part sarcoma, can prolong survival even if multiple pulmonary metastases are present (8). Because of the high prevalence of pulmonary metastasis and good clinical results of pulmonary metastasectomy, annual screening of chest ct for an indefinite period might be beneficial for patients with alveolar soft - part sarcoma . The conventional radiographic and sonographic findings of this condition at the primary site are nonspecific (2). When unenhanced ct is used, the soft - tissue tumor is of lower attenuation than muscle (2). Enhanced ct scans show significant tumor enhancement and tortuous, dilated, and early draining veins (1, 2, 5) angiography of primary site lesions revealed hypervascularity of the tumor, with multiple enlarged tortuous vessels, prolonged tumor staining and early draining veins (1, 2, 5). On mr imaging, the mass showed high signal intensity on t1-weighted image and very high signal intensity on t2-weighted image . Tubular areas of signal void within the tumor, as seen on both t1-weighted image and t2-weighted image, are typical of rapid blood flow in enlarged vessels (2, 5). On ct, our cases typically showed dilated, tortuous and tubular structures within the pulmonary metastatic masses, and these represent engorged vessels . These ct findings are the same as those for primary masses of alveolar soft - part sarcoma and may reflect the characteristic pathologic findings of this rare malignant tumor . These findings are, therefore, even if not specific, helpful for differential diagnosis of pulmonary masses . They are, however, present only in cases involving masses larger than 2 cm . Because enhanced ct is not generally accepted in work up for pulmonary metastasis, our observation is in addition, limited . In conclusion, we have described the ct findings of pulmonary metastatic alveolar soft - part sarcoma of the lung which on enhanced scans manifested as clearly enhanced pulmonary nodules or masses with dilated and tortuous intratumoral vessels, similar to those of the primary lesion . Alveolar soft - part sarcoma is rare, but in cases involving a clearly enhanced pulmonary mass with tortuous and dilated vessels in young adult patients, the possibility of metastatic alveolar soft - part sarcoma should be included in the differential diagnosis.
A second primary tumor (spt) or secondary malignancy refers to the occurrence of a new cancer that follows a previously treated malignant neoplasm, but it is not considered a metastasis of the initial neoplasm . Second or multiple primary cancers represent 16% of all new primary cancers in cancer survivors in the united states . Overall, patients who survive certain types of cancer are at increased risk of a second primary cancer . Considering all cancers, after a first cancer diagnosis, males have a 9.7% chance of developing a spt within 10 years (about 1 in 10), and women have a 7.7% chance (about 1 in 13). The most common second primary cancers were found in lung, breast, colon, head and neck, uterine cervix, skin, and prostate . A male predominance was observed for each of the 4 major cancer types to which both sexes are susceptible (lung, colon, head and neck, and melanoma). The 10-year cumulative risk of a lung spt was 1.6% for males, and 0.7% for females . Environmental exposure and genetic factors play a role in the pathogenesis and natural history of a second cancer, as in the first cancer . A number of genetic factors (e.g., normal genetic variations in the genome) will interact with environmental and/or occupational factors to modulate lung cancer susceptibility, survival, and therapy . The chance of developing a second cancer depends on a number of factors, including the original type of cancer, age at diagnosis, sex, types of therapy received, effects of using therapeutic interventions, lifestyle choices, tobacco use, environmental exposures, genetic predisposition, the theory of a common clonal origin, and the screening effect . A second primary lung cancer after the complete resection of a primary lung cancer developed in 2.2% of patients during a 19-year follow - up period in a korean population . The overall 5-year survival rate from the time of detecting the second primary lung cancer was 47.8%, and the 5-year survival rate of patients who underwent resection of the spt was 77.0% . Surgical resection was shown to be feasible and effective in the management of a second primary lung cancer . Long - term surveillance for 5 years or more is essential for early detection, and it will increase the chance of resection of a second primary lung cancer . Several genes may contribute to inter - individual variations in dna repair capacity and cancer susceptibility . Thus, polymorphisms of genes involved in the major nucleotide excision repair pathway are of particular interest . Tumor necrosis factor - alpha (tnf-) is an important factor in cancer development and spread . It was shown that blood levels of tnf- were elevated in patients with solid tumors . Therefore, it is reasonable to hypothesize that the expression level of tnf- may be involved in cancer pathogenesis . Single - nucleotide polymorphisms within the tnf- gene promoter region have been reported to be associated with susceptibility to various types of cancers . Several cytokine promoter polymorphisms, including tnf--308 and tnf--238, were associated with altered protein levels and/or rates of transcription . Among the polymorphisms associated with an elevated production of tnf- were g / a transitions at positions -308 and -238; these polymorphisms have been the subject of many molecular epidemiological studies that investigated their significance in carcinogenesis . The association between some tnf- promoter polymorphisms and the risk of lung cancer remains controversial . The first lung cancer study that focused on the tnf--308 and tnf--238 polymorphisms investigated 202 chinese patients with non - small cell lung cancer (nsclc). They found that the -308 g / a and the -238 g / a polymorphisms in the tnf- promoter region were significantly associated with lung cancer susceptibility . Moreover, these 2 polymorphisms were shown to be related to the severity of disease . The -308a allele positively correlated with lung cancer development and progression; conversely, the -238a allele had a protective impact on the development of lung cancer . In the present study, we investigated whether the tnf--308 and tnf--238 polymorphisms were associated with the susceptibility and severity of lung cancer as a spt in a sample of croatian patients . This study included 104 patients with lung cancer (70 males and 34 females; mean age 68.25, range 5085 years) as second primary malignancies (group lc2). In addition, prior to the detection of lung cancer, 7 patients had previously had 2 malignant tumors, including cancers in the bladder and larynx, larynx and prostate, breast and thyroid, larynx and oral cavity, stomach and breast, lung and kidney, and skin and skin . The first control group (lc1) comprised 201 unrelated patients with lung cancer (174 with nsclc and 27 with sclc). The second control group (hc) comprised 230 healthy blood donors, from the clinical hospital blood donor registry, rijeka, matched for sex and age to the study group . All tested groups were from the croatian population living in primorsko - goranska county . In the study group (lc2), 7 patients had 3 different primary tumors and all other patients (n=97) had 2 primary malignant tumors . Of the 104 patients, 70 (67.3%) were male and 34 (32.7%) were female (ratio, 2.1:1). The average ages at the time of tumor diagnosis and the length of the disease - free interval between treatment of the first primary tumor and the detection of lung cancer are presented in table 1 . The anatomical locations of primary tumors were in the larynx in 22 patients (21.1%); skin in 14 patients (13.4%); breast in 13 patients (12.5%); uterus in 12 patients (11.5%); oral cavity, prostate, and bladder in 8 patients (7.7%); colon and pharynx in 5 patients (4.8%); kidney in 3 patients (2.8%); and lung, parotid, penis, small bowel, liver, and stomach in 1 patient (1.0%). Among skin tumors, there were 8 melanomas, 3 squamous carcinomas, and 2 basal cell carcinomas . Among the 7 patients (6.7%) with 3 primary tumors, the secondary malignancy locations were equally represented among the patients: 1 patient each had a tumor in the larynx, skin (basalioma), breast, oral cavity, prostate, kidney, and thyroid . When classified according to sex, the most frequent sites for primary tumors in males were the larynx (n=21; 30.0%), skin (n=13; 18.6%), and prostate (n=9; 12.9%). In females, the leading primary tumor sites were the breast (n=14; 41.2%) and uterus (n=12; 35.3%). Among all patients, 85 (81.8%) were smokers and 19 (18.2%) were non - smokers . The proportion of male smokers was significantly higher than females (male: female ratio, 4.5: 1; p<0.001). Among patients with lung cancer, 49 (47.1%) had squamous carcinomas and 27 (26.0%) had adenocarcinomas . At the time of diagnosis, 34 patients (32.7%) were in tumor - node - metastasis (tnm) stages i and ii, and 70 patients (67.3%) were in tnm stages iii and iv . Multiple tumors in a patient were classified as either synchronous or metachronous, based on moertel s definition: synchronous tumors occurring within 6 months of detecting the first primary tumor, and metachronous tumors occurring more than 6 months after detecting the first primary tumor . This study was carried out in accordance with the code of ethics of the world medical association (declaration of helsinki). For each of patient, we prospectively collected data on the tumor site, patient age at diagnosis, sex, smoking status, stage of lung cancer, and histological differentiation . Time elapsed between the appearance of primary and secondary tumors, therapies for primary tumors, and some data about the lc1 group were collected retrospectively . The analysis of tnf- gene polymorphisms was performed by polymerase chain reaction / restriction fragment length polymorphism (pcr - rflp) analysis under conditions described by shih et al . . Group differences in patient genotype and allele distributions were analyzed for statistical significance with the test or fisher s exact test, as appropriate . Odds ratios (or) and 95% confidence intervals (ci) were calculated to evaluate the effects of different genotypes / alleles . This study included 104 patients with lung cancer (70 males and 34 females; mean age 68.25, range 5085 years) as second primary malignancies (group lc2). In addition, prior to the detection of lung cancer, 7 patients had previously had 2 malignant tumors, including cancers in the bladder and larynx, larynx and prostate, breast and thyroid, larynx and oral cavity, stomach and breast, lung and kidney, and skin and skin . The first control group (lc1) comprised 201 unrelated patients with lung cancer (174 with nsclc and 27 with sclc). The second control group (hc) comprised 230 healthy blood donors, from the clinical hospital blood donor registry, rijeka, matched for sex and age to the study group . All tested groups were from the croatian population living in primorsko - goranska county . In the study group (lc2), 7 patients had 3 different primary tumors and all other patients (n=97) had 2 primary malignant tumors . Of the 104 patients, 70 (67.3%) were male and 34 (32.7%) were female (ratio, 2.1:1). The average ages at the time of tumor diagnosis and the length of the disease - free interval between treatment of the first primary tumor and the detection of lung cancer are presented in table 1 . The anatomical locations of primary tumors were in the larynx in 22 patients (21.1%); skin in 14 patients (13.4%); breast in 13 patients (12.5%); uterus in 12 patients (11.5%); oral cavity, prostate, and bladder in 8 patients (7.7%); colon and pharynx in 5 patients (4.8%); kidney in 3 patients (2.8%); and lung, parotid, penis, small bowel, liver, and stomach in 1 patient (1.0%). Among skin tumors, there were 8 melanomas, 3 squamous carcinomas, and 2 basal cell carcinomas . Among the 7 patients (6.7%) with 3 primary tumors, the secondary malignancy locations were equally represented among the patients: 1 patient each had a tumor in the larynx, skin (basalioma), breast, oral cavity, prostate, kidney, and thyroid . When classified according to sex, the most frequent sites for primary tumors in males were the larynx (n=21; 30.0%), skin (n=13; 18.6%), and prostate (n=9; 12.9%). In females, the leading primary tumor sites were the breast (n=14; 41.2%) and uterus (n=12; 35.3%). Among all patients, 85 (81.8%) were smokers and 19 (18.2%) were non - smokers . The proportion of male smokers was significantly higher than females (male: female ratio, 4.5: 1; p<0.001). Among patients with lung cancer, 49 (47.1%) had squamous carcinomas and 27 (26.0%) had adenocarcinomas . At the time of diagnosis, 34 patients (32.7%) were in tumor - node - metastasis (tnm) stages i and ii, and 70 patients (67.3%) were in tnm stages iii and iv . Multiple tumors in a patient were classified as either synchronous or metachronous, based on moertel s definition: synchronous tumors occurring within 6 months of detecting the first primary tumor, and metachronous tumors occurring more than 6 months after detecting the first primary tumor . This study was carried out in accordance with the code of ethics of the world medical association (declaration of helsinki). For each of patient, we prospectively collected data on the tumor site, patient age at diagnosis, sex, smoking status, stage of lung cancer, and histological differentiation . Time elapsed between the appearance of primary and secondary tumors, therapies for primary tumors, and some data about the lc1 group were collected retrospectively . The analysis of tnf- gene polymorphisms was performed by polymerase chain reaction / restriction fragment length polymorphism (pcr - rflp) analysis under conditions described by shih et al . . Group differences in patient genotype and allele distributions were analyzed for statistical significance with the test or fisher s exact test, as appropriate . Odds ratios (or) and 95% confidence intervals (ci) were calculated to evaluate the effects of different genotypes / alleles . The distributions of tnf--308 and tnf--238 polymorphism genotypes are presented in table 2 for different histological types of lung cancer . We did not find any association between the particular genotypes and the length of the disease - free interval . Furthermore, we did not find an association between the genotypes and different clinical stages of lung cancer according to the tnm classification . The distributions of tnf--308 and tnf--238 polymorphism genotypes and alleles among lc1 and lc2 group patients are presented in tables 3 and 4 . There was no statistically significant difference in the distributions of the tnf--308 polymorphism genotype and alleles between the lc1 and lc2 groups or between the lc2 and hc groups . The frequencies of the tnf--238 polymorphism gg genotype and the g allele were higher in the lc2 group relative to the lc1 group, but the differences did not reach significance (p=0.054 and p=0.057, respectively). Similar differences were found in the tnf--238 polymorphism gg genotype and g allele between the lc2 group and the hc group (p=0.054 and p=0.057, respectively). In terms of the different types of lung cancer, patients with a second primary nsclc more frequently had tnf--238 polymorphism gg genotypes and g alleles relative to patients with a first primary nsclc (the differences approached statistical significance: p=0.060, p=0.064, respectively). However, patients with a second primary sclc and those with a first primary sclc were not significantly different in the distributions of tnf--238 polymorphism gg genotypes or g alleles . All (100%) patients of group lc2 (n=104) had the gg genotype (homozygous gg) and the g allele . We investigated the relationship between tnf--308 and tnf--238 promoter polymorphisms and the susceptibility to lung cancer as a spt . The results revealed that the tnf--238 polymorphism had a possible impact on the incidence of lung cancer and the incidence of nsclc as a group lc2 . We found that 100% of patients of a group lc2 had the tnf--238 polymorphism gg genotype and the g allele . This frequency was nearly significantly different from that observed in the 2 control groups (lc1 and hc). In patients of group lc2 that had an nsclc, the patients with sclc in group lc2 (only 6) also had 100% frequency of the gg genotype and the g allele, but this was not significantly different from the frequency observed in patients with sclc in group lc1 . This study is the first to show that 100% of patients in group lc2 had the tnf--238 polymorphism gg genotype and the g allele . This result provided evidence that the tnf--238 gg genotype was associated with a greater possibility of developing lc2 . Thus, this polymorphism could have a promotional effect on lung cancer and the development of nsclc in the group of patients with lc2 . In this study, the mean disease - free period in females was 13.52 years; this was significantly longer (p<0.001) than the mean disease - free period in males (6.73 years). We speculate that this finding may have been due to the fact that most of the young women had breast cancer and uterine cancer as a first cancer and that there were fewer smokers among females than among males; both factors may have influenced our results that suggested a genetic predisposition to lung cancer . Our results are inconsistent with those of a meta - analysis from 2011 that included 34 679 patients with cancer and found no significant association between the tnf--238 polymorphism and the risk for cancer (including lung cancer). However, because lung cancer is affected by inflammatory cytokines, our results are consistent with those from an interesting study in an italian population (163 cases), which included 66 patients with severe rheumatoid arthritis . Also, shih et al . Found that the gg genotype of tnf--238 tended to be associated with advanced lung cancer . Lung cancer is a multi - factorial disease; it results from complex interactions between environmental and genetic factors . It is possible that variants at some loci may confer a modest risk of cancer . However, some environmental factors may predominate in the development of lung cancer, like living habits and exposure to carcinogens . Without considering these factors, it may not be possible to determine the role of tnf--308 and tnf--238 polymorphisms in lung cancer development . Some short nucleotide polymorphisms of cytokines may exert complex and interacting functions with each other, which may alter the effects of tnf--308 and tnf--238 polymorphisms in the pathogenesis of lung cancer . Therefore, other polymorphisms should be taken into account to ascertain the true effects of cancer risk factors . Tnf--238 polymorphism gg genotype and the g allele could have a promotional effect on the development of nsclc as a second primary tumor . Our findings should be validated in larger, prospective studies or multicenter, case - control studies . Also, studies with patients with different ethnic backgrounds would allow further exploration of the genetic pathogenesis of second primary lung cancers.
It was defined as the urine albumin - to - creatinine ratio (uacr) of 30 to 300 mg / g and originally used to predict chronic kidney disease and diabetic nephropathy . Previous studies have confirmed that there is an increased risk of cardiovascular disease and death related to microalbuminuria . The association between microalbuminuria, progressive atherosclerotic vascular disease, and renal damage has been well established in patients with systemic diseases . Microalbuminuria is extensively believed to reflect a generalized impairment of the endothelium which is an important determinant of vascular disease . Thyroid hormones are also found to be associated with endothelial damage and cardiovascular disease [69]. Previous studies have shown that thyroid hormones, via dilation of blood vessel, production of vasodilator molecules, inhibition of angiotensin ii receptor, and its signal transduction, regulate endothelial function and vascular homeostasis [7, 8]. Moreover, in experimental models, triiodothyronine (t3) had important effects on the vascular system, such as inducing relaxation of vascular smooth muscle cells through a direct or indirect effect [8, 9]. The epidemiology study of microalbuminuria also revealed a close association between systemic endothelial dysfunction and vascular disease . However, evidence for the association between thyroid hormones and microalbuminuria in a community - based study, if any, is scarce . Just recently, mervat and colleagues reported that subclinical hypothyroidism is independently associated with microalbuminuria in 147 prediabetic adults . But the sample size was relatively small and it was not a community - based study . In this large community - based cross - sectional study, we aimed to investigate the relationship between thyroid hormones and microalbuminuria in middle - aged and elderly chinese population . A cross - sectional survey was performed during 2008 - 2009 in a population from songnan community located in baoshan district, shanghai . The selection criteria and design of the study briefly, in phase 1 (june and july 2008), we conducted the investigation on residents aged 40 years or older . All participants received a fasting blood test . Based on the results of fasting glucose level and history of diabetes, participants were categorized into three groups: normal glucose regulation (ngr), defined as a fasting plasma glucose level less than 5.6 mmol / l (<101 mg / dl) and no history of diabetes; impaired glucose regulation (igr), defined as a fasting plasma glucose level of 5.6 to less than 7.0 mmol / l (101 to <126 mg / dl) and no history of diabetes; and diabetes, defined as a fasting plasma glucose level of 7.0 mmol / l or greater (126 mg / dl) or a history of diabetes . Because subjects with lower glucose levels were expected to have a lower participation rate than those with higher glucose levels, we randomly selected participants from these groups according to a ratio of 1.44 (ngr): 1.2 (igr): 1 (dm) to receive a more comprehensive survey including thyroid function test, a standard 75-g ogtt, blood and urine collection, an anthropometric measurement, and a questionnaire survey in phase 2 (june through august 2009). A total of 3455 persons with blood and urine samples were included in phase 2 survey . Exclusion criteria were (i) participants having a personal history of overt hyperthyroidism, hypothyroidism, or thyroiditis and having been taking thyroxine or antithyroid drugs (n = 12); (ii) taking medications affecting thyroid function such as amiodarone, lithium, antipsychotic drugs, and antiepileptic drugs (n = 5); (iii) history of thyroidectomy (n = 2); (iv) participants with uacr 300 mg / g (n = 34). Moreover, 56 participants without complete thyroid function or uacr data were also excluded from the study . Finally, a total of 3346 subjects (1336 men and 2010 women) remained in the present analysis . The participants (3346 subjects) and the nonparticipants (6839 subjects) were similar in characteristics, such as sex and age distribution . This study was approved by the institutional review board of the rui - jin hospital, shanghai jiao tong university school of medicine, and was in accordance with the principle of the helsinki declaration ii . Sociodemographics, medical history, and lifestyles including smoking and drinking status were conducted by trained personnel . Current smoking was defined as yes if a subject smoked at least one cigarette per day or seven cigarettes per week in the past 6 months . Current alcohol drinking was defined as yes if a subject consumed alcohol at least once a week in the past 6 months . Body mass index (bmi) was calculated using the formula of weight / height (kilograms per squared meters). Three sitting blood pressure measurements taken consecutively at 1 min interval after at least 5 min rest using an automated electronic device (omron model hem-752 fuzzy; omron co., dalian, china) were averaged for analysis . Hypertension was defined when systolic blood pressure (sbp) was above 140 mmhg, when diastolic blood pressure (dbp) was above 90 mmhg, or when taking medication for blood pressure control . All participants were admitted after an overnight fast of> 10 h and two - point (0 and 2 h) ogtt with a 75 g glucose load was performed . The fasting and postloading venous blood samples were collected into a routine tube, respectively, and were immediately centrifuged on site at 4c . Sera for the measurement of thyroid function were aliquot and kept frozen at 80c until use . Blood glucose, serum creatinine, lipid profile including total cholesterol (tc), triglycerides, high - density lipoprotein cholesterol (hdl - c), low - density lipoprotein cholesterol (ldl - c), and high - sensitivity c - reactive protein (hs - crp) were measured within 1 h of collection with an automated biochemical instrument (bayer biochemical autoanalyzer advia 1650, bayer, leverkusen, germany). Hyperlipidemia was defined according to the national cholesterol education program adults treatment panel iii (ncep atpiii) criteria . The abbreviated modification of diet in renal disease (mdrd) study formula as recalibrated for chinese was used to calculate the estimated glomerular filtration rate (egfr) expressed in ml / min/1.73 m: 186 (serum creatinine value in mol / l 0.011) 1.154 (age) 0.203 (0.742 if female) 1.233 (the number 1.233 is the adjusting coefficient for chinese). A first - voided, early - morning spot urine sample was collected for assessing the uacr within 1 h after sample collection . Urinary albumin concentration was measured using immunoturbidimetry (beijia biochemistry, shanghai, china), and urinary creatinine concentration was determined by a modified jaffe method on an automatic analyzer (beckman lx/20, brea, ca). Uacr was calculated as urinary albumin concentration divided by creatinine concentration and expressed in mg / g . Microalbuminuria was defined as the uacr ranges within 30300 mg / g and macroalbuminuria as 300 mg / g [2, 15]. We measured thyroid stimulating hormone (tsh), free triiodothyronine (ft3), free thyroxine (ft4), thyroid peroxidase antibody (tpoab), and thyroglobulin antibody (tgab) levels . Thyroid function tests were analyzed in the clinical laboratory for endocrinology, shanghai institution of endocrine and metabolic diseases, which was certified by college of american pathologists . Serum ft3 (reference range: 2.62 to 6.49 pmol / l), ft4 (reference range: 9.01 to 19.04 pmol / l), and tsh (reference range: 0.35 to 4.94 iu / ml) were determined by chemiluminescent microparticle immunoassay method (architect system; abbott laboratories, abbott park, il). The coefficients of variations were 4.78.0% for ft3, 2.65.3% for ft4, and 3.13.4% for tsh . Serum anti - thyroid antibodies (tpoab and tgab) were measured by chemiluminescent microparticle immunoassay using architect anti - tpo and anti - tg on the architect i system (abbott laboratories). Reference ranges were tpoab <5.61 iu / ml (with an interassay cv of 4.36.8%) and tgab <4.11 iu / ml (with an interassay cv of 3.25.2%). All of the statistical analyses were performed using sas version 9.2 (sas institute, cary, nc), and a p value <0.05 (two - sided) indicated statistical significance . Continuous variables were summarized as means sd or medians (interquartile ranges) for skewed variables . Triglycerides, fasting plasma glucose (fpg), ogtt 2-hour plasma glucose (ogtt-2 h pg), hs - crp, ft3, ft4, tsh, tpoab and tgab, and uacr were normalized by logarithmically transformed before analyses because of nonnormal distribution . Demographic and metabolic features in each quartile were described and p for trend is measured using linear regression analysis across ft3 quartiles . To investigate the association of ft3 categories and microalbuminuria, univariate- and multivariate - adjusted logistic regression models were performed with microalbuminuria as the dependent variable and ft3 categories as the independent variable along with the potential confounding factors . In the multivariable logistic regression models, we adjusted for the potential confounding factors including age, sex, bmi, smoking and drinking status, sbp, and dbp, tc, triglycerides, fpg and ogtt-2 h pg, tpoab and tgab, hs - crp, use of angiotensin - converting enzyme inhibitor (acei) and/or angiotensin receptor blocker (arb) drugs, and egfr . Odds ratios (or) with the lowest quartile were computed as the reference group . To study if the association with ft3 and microalbuminuria was more prominent in some specific subgroups, we further performed stratified analyses on the associations between ft3 and the risk of microalbuminuria by the major risk factors including sex, age, bmi, hypertension, hyperlipidemia, diabetes according to ogtt diagnosis criteria, hs - crp, and egfr . Odds ratios were calculated for an increase of each 1 s.d . In log ft3 concentration in subgroups of the strata variables . Interactions of each above strata variable with the association of serum log ft3 and microalbuminuria risks were tested by introducing the variable log ft3 into the univariate model . Among the 3,346 participants, the prevalence of microalbuminuria was 9.9% and no difference was found between males and females (9.2 versus 10.4%, p> 0.05). Ft3 concentrations in male were significantly higher than that in female (p <0.0001). Then participants were divided into four groups according to quartiles of ft3 with the first quartile representing the lowest one and the fourth quartile representing the highest one (4.28, 4.294.60, 4.614.93, and> 4.94 metabolic risk factors including sex distribution, current smoking or drinking status, bmi, dbp, triglycerides, tc, ldl - c, ft4, and egfr increased significantly with the increment of ft3, whereas age, ogtt-2 h pg, hdl - c, tsh, and uacr decreased (all p for trend <0.05). Compared with the participants in the lowest quartile of ft3, those in the second, the third, and the highest quartiles have significantly lower levels of uacr as follows: 6.08 (2.8612.86) versus 5.93 (2.7012.39), 5.93 (2.7912.48), and 5.28 (2.6512.12) mg / g (p for trend <0.05, table 1). Nevertheless, no statistical difference was found for sbp, use of acei / arb drugs, fpg, tgab, tpoab, and hs - crp among the four groups . The prevalence of microalbuminuria differed in the four groups with different ft3 quartiles . From the lowest quartile across to the highest one of ft3 quartiles, the prevalence of microalbuminuria was 13.2, 9.5, 8.6, and 8.2%, respectively (p for trend = 0.0005, figure 1). Compared to the lowest ft3 quartile, the second, the third, and the highest quartiles showed a significant decrease in the prevalence of microalbuminuria (p = 0.02, 0.003, and 0.0009, resp . ). In the univariable logistic regression model, the odds ratio (or) for the highest ft3 quartile compared with the lowest quartile was 0.58, 95% confidence intervals (95% ci) 0.420.81, p = 0.001, and those in the second and third quartile were also less likely to have microalbuminuria (or: 0.69, 95% ci, 0.510.93; p = 0.02 and or: 0.62, 95% ci, 0.450.85; p = 0.003 resp . ). After adjusting for age, sex, bmi, and smoking and drinking status, participants in the second, third, and highest quartiles were still less likely to have microalbuminuria compared with those in the lowest quartile (adjusted or: 0.72, 95% ci, 0.520.99, p = 0.04; 0.66, 95% ci, 0.480.91, p = 0.01; and 0.58, 95% ci, 0.410.82, p = 0.002, resp . ). After further adjustments for other potential confounding factors including sbp, dbp, tc, triglycerides, fpg and ogtt-2 h pg, tpoab and tgab, hs - crp, use of acei / arb drugs, and egfr, a fully adjusted logistic regression (table 2) showed similar associations for the third and highest quartile but not for the second quartile (adjusted ors for the highest versus the lowest quartile: 0.61, 95% ci, 0.430.87, p = 0.007, and for the third versus the lowest quartile: 0.70, 95% ci, 0.500.98, p = 0.04). The tests for the trend in the multivariable analyses were all significant (p for trend 0.01) (table 2). We also conducted linear regression analyses for ft3 and uacr . In both simple and multivariate - adjusted linear regression analyses, ft3 levels were negatively and significantly associated with uacr (p values <0.05). Then we excluded the participants with abnormal ft3 levels (ft3 <2.62 or 6.49 pmol / l, n = 41). We found a similar inverse association between serum ft3 and microalbuminuria in unadjusted, adjusted, and fully adjusted model (table 2). Compared with those in the lowest quartile, highest quartiles were still less likely to have microalbuminuria (or 0.69; 95% ci, 0.490.98, p = 0.04) (table 2). Ft4 and tsh were not significantly associated with microalbuminuria in all the models generated (data not shown). Furthermore, stratified analyses for multivariate - adjusted or of microalbuminuria with each 1 s.d . Inverse associations between microalbuminuria and ft3 were significant in both age strata (<60 and 60 years), both bmi strata (<24 and 24 kg / m), both hypertension strata (yes and no), both diabetes strata according to the ogtt diagnosis criteria (yes and no), and both hs - crp strata (<3 and 3 mg inverse associations between microalbuminuria and ft3 were also persisted in male, those without hyperlipidemia and egfr 90 ml / min per 1.73 m. no significant associations were detected in female, those with hyperlipidemia and egfr <90 ml / min per 1.73 m (figure 2). Nevertheless, interactions between ft3 and these risk factors were not significant (p> 0.05) (figure 2). In this community - based cross - sectional study, we found serum ft3 levels were inversely associated with microalbuminuria which is a powerful predictor of generalized vascular endothelial impairment . Microalbuminuria is a marker of increased risk of cardiovascular (cv) and renal morbidity and mortality in diabetic subjects . Meanwhile, microalbuminuria is a marker of increased risk for diabetes mellitus, hypertension, deterioration of renal function, and cv morbidity and all - cause mortality in nondiabetic subjects . Jones et al . Reported the prevalence of microalbuminuria in 22,244 subjects aged from 6 to> 80 years to be 7.8% (6.1% in males and 9.7% in females) with progressively increasing prevalence in adults> 40 years of age from the third national health and nutritional examination survey (nhanes). Our data showed the prevalence of microalbuminuria was 9.9% (9.2% in males versus 10.4% in females) in participants aged 40 or above . Microalbuminuria is regarded as an integrated marker of systemic endothelial dysfunction and vascular disease via strong associations with blood pressure levels, metabolic status, lipids, and smoking habits . In our study, we confirmed participants with microalbuminuria were more likely to have higher levels of bmi, sbp and dbp, fpg, ogtt-2 h pg, tc, triglycerides and hs - crp (all p <0.05), and lower levels of hdl - c (all p <0.01) compared to those normoalbuminuria . Thyroid hormone has crucial effects on the vascular system and is also found to be associated with endothelial damage [20, 21]. Endothelial impairment has well - known effects on the development of hypertension, coronary artery disease, chronic heart failure, peripheral artery disease, diabetes, and chronic renal failure . Serum ft3 level has been shown to be associated with impaired endothelial function as assessed by flow - mediated dilation in patients with chronic kidney disease . By now no large - scale population - based studies have been published to focus on the association with thyroid function and microalbuminuria . Only one study found an association between subclinical hypothyroidism and microalbuminuria in 147 prediabetic subjects . However, their sample size was small and the study was not population - based . In our study, after adjustment for a wide spectrum of lifestyle and biochemical risk factors we found ft3 was inversely associated with microalbuminuria in a large population . These findings raised the possibility that normal thyroid function within the higher range was related to lower prevalence of microalbuminuria . However, the exact mechanisms for inverse associations between ft3 and microalbuminuria are not well defined . Patients with subclinical hypothyroidism have endothelial dysfunction characterized by reduced endothelium - dependent vasodilatation and impaired nitric oxide availability . Moreover, this abnormal vascular profile is partially independent of dyslipidemia and restored by levothyroxine treatment [7, 24]. Vascular function is impaired in patients with mild and subclinical hypothyroidism, as documented by the increase in systemic vascular resistance and arterial stiffness and by the impaired endothelial function . The previous study showed the t3-induced decrease in systemic vascular resistance may be explained by direct modulation of endothelium - dependent and -independent vasoregulation . In experimental models, it was also shown that ft3 had antiatherosclerotic effects on the vascular bed via its effect on mitochondrial oxidation systems, induction of vasodilatatory molecules, and inhibition of angiotensin ii receptor expression and downstream signal transduction [8, 25]. Thus, endothelial damage / dysfunction, closely associated with the pathogenesis of atherosclerosis, impairs endothelium - dependent vasodilatation which may mediate with microalbuminuria . It is highly possible that low ft3 could be a marker of endothelial dysfunction similar to presence of microalbuminuria and both might be mediated by unmeasured or unknown factor . In addition, thyroid hormone is closely associated with the metabolic syndrome (mets), a syndrome of insulin resistance, obesity, hypertension, and dislipidemia . The data from nhanes iii showed strong positive associations between microalbuminuria and mets . Meanwhile, insulin resistance is generally hypothesized to be the major underlying pathophysiological process of mets . Low thyroid function (even in the euthyroid state) was reported to be associated with increased insulin resistance, which might mediate the association with microalbuminuria . Reported endothelial dysfunction exists in euthyroidism patients with hashimoto's disease and autoimmune reactivity, indicating tpoab might be responsible for the endothelial dysfunction and the subsequent microalbuminuria . However, our data showed the link with microalbuminuria was roughly similar for ft3 with and without tpoab . This demonstrates that the effect of ft3 on microalbuminuria might be independent of thyroid autoimmunity . Additionally, increasing evidence shows that systemic and vascular low - grade inflammation, as reflected by increased serum concentration of hs - crp, may provide a mechanism for the pathogenesis of microalbuminuria . However, we found ft3 inversely associated with microalbuminuria either in subjects with hs - crp 3.0 mg / l or <3.0 mg / l, indicating that the association between ft3 and microalbuminuria cannot be explained by systemic inflammation . Our study was the first to report an independent association between ft3 concentration and microalbuminuria in a large population . Firstly, our study was cross - sectional designed, and no causal relationships can be established . Secondly, the urinary albumin excretion was measured on a morning spot - urine sample . We acknowledge that a 24 h urine sample would provide more stable measures for urinary albumin excretion . However, it was reported that the use of spot specimens for urinary albumin - to - creatinine ratio was more convenient for a large cohort survey and the results correlated well with 24 h urinary albumin excretion and could be used as a reliable alternative to 24 h urinary albumin excretion in large epidemiological research . Thirdly, our study included only middle - aged and elderly subjects, and the results may not be applied to a general population with different age composition . In summary, the present study shows inverse correlation between microalbuminuria, an established marker of generalized vascular endothelial impairment, and serum ft3 . Moreover, this inverse association also exists in subjects classified as being euthyroid, which implicates low normal thyroid function could adversely affect microalbuminuria even in euthyroidism . We therefore propose to consider ft3 levels in addition to mets and other risk factors that are traditionally associated with microalbuminuria . Further studies are needed to explore the mechanisms responsible for the correlation between ft3 and microalbuminuria.
Across many areas of science, technological and conceptual advances are resulting in the increasingly rapid generation of large amounts of data . While research has always involved the collection and organization of data, the volume, variety, and velocity of current big data production presents new opportunities and challenges in both scale and complexity . At the same time, there is a broader cultural shift underway from approaches that kept data mostly private with sharing of resultant knowledge in the form of publications to an information - based culture that dynamically engages the scientific community through the active sharing of both data and publications . Big data are not only a new reality for the biomedical scientist, but an imperative that must be understood and used effectively in the quest for new knowledge . Needed are new approaches for data management and analysis that allow scientists to better access and extract value from data so as to advance research and discovery.1 2 key stakeholders in the coming biomedical big data ecosystem include data providers and users (eg, biomedical researchers, clinicians, and citizens), data scientists, funders, publishers, and libraries . Implementation of such an envisioned biomedical big data ecosystem will depend upon cultural changes and require updated policies related to funding, data sharing, and data citation . Metrics on the efficacy of data sharing and re - use may help inform important decisions at funding agencies and research institutions about the support for and career advancement of biomedical scientists, and could be invaluable in incentivizing necessary cultural changes across the biomedical research enterprise . The aim of this perspective is to describe how the national institutes of health (nih) has started catalyzing such changes through its big data to knowledge (bd2k) initiative . A report to the nih advisory committee to the director from its data and informatics working group (diwg) in june 2012 (http://acd.od.nih.gov/diwg.htm) recommended the establishment of a broad and inclusive trans - nih program for addressing the opportunities and challenges presented by biomedical big data . Indeed, the spirit of the report suggested that failure of the nih to act at this crucial juncture would represent institutional malpractice (as stated by one diwg member). To emphasize the need for a coherent and forward - looking response to these opportunities, the position of associate director for data science (adds) at nih was created with the goal of placing big data and data science at the highest level of decision - making at the nih . The first adds, dr philip bourne, has begun his tenure at the nih, and among his responsibilities is oversight of the bd2k initiative . Bd2k has been formulated as a programmatic response to the diwg recommendations and consists of four focused areas: (1) improving the ability to locate, access, share, and use biomedical big data; (2) developing and disseminating data analysis methods and software; (3) enhancing training in biomedical big data and data science; and (4) establishing centers of excellence in data science . Taking into account the substantial current investment of individual nih institutes and centers in bioinformatics and computational biology within their own spheres of interest, bd2k has first focused on identifying the pressing general but unaddressed needs related to biomedical big data, assessing community expectations, and identifying current policies and practices related to the production, handling, and utilization of biomedical big data . Outreach to the diverse community of stakeholders mentioned above has been at the forefront of these efforts through targeted requests for information (rfi), workshops, consultation with leaders in various fields, as well as extensive discussions across agencies and within the nih about the relationship(s) between potential bd2k components and other ongoing activities (http://bd2k.nih.gov/workshops.html). The resulting input strongly ratified the diwg's recommendations that supporting the development of software and applications for analyzing biomedical big data was only part of the solution . Further support came from the holdren / ostp memo issued in february 2013 (http://www.whitehouse.gov/blog/2013/02/22/expanding-public-access-results-federally-funded-research) and directing all agencies of the government to ensure the results of federally funded scientific research are made broadly available . A first step in fostering the emergence of data science as a discipline relevant to biomedicine is the development of solutions to specific high - need challenges facing the research community . Thus, the first bd2k funding opportunity was for investigator - initiated centers of excellence in data science (rfa - hg-13 - 009); this called for proposals to test and validate novel ideas in data science that not only focused on particular challenges but also had the potential for broad impact . The second launched bd2k area aimed to enhance the training of methodologists and practitioners in data science . Umbrella include computer science, mathematics and statistics, biomedical informatics, biology and medicine, and others, all incorporated as data science. At the same time, the generation of large amounts of data together with the complex questions being posed, requires interdisciplinary teams to design the studies and perform the subsequent data analyses . The bd2k training initiatives seek to seed the development of investigators in all parts of the research enterprise who are well - trained in data science . Hand - in - hand with training is the need for cultural changes to assure that the contributions of scientists well - trained in data science are appreciated and rewarded, including the provision of appropriate career paths with commensurate incentives and rewards . A fundamental question for bd2k is how to enable the identification, access, and citation of (ie, credit for) biomedical data . The diwg proposal for federated data catalogs, as distinct from data repositories, requires descriptions of and pointers to the data . Inherent in data discovery is the need for a sustainable and scalable plan to create and maintain a discovery system that allows researchers to readily find and cite biomedical data . Indeed, sustainability and scalability are two intertwined issues that must be addressed in order for the advances made possible by bd2k to have a lasting effect . A necessary first step has been recognition of the need to assemble and validate ideas drawn from the broader scientific community in developing a data discovery index (ddi). The goal of the initial efforts in this area is to define an effective and efficient mechanism(s) for indexing that will enable the discovery of relevant, existing datasets through the use of metadata and index terms . The ddi will enable advanced approaches to search, integrate, and facilitate visualization of data . Stakeholders will be encouraged to learn from related efforts in other fields, and conduct short - term pilot studies to explore different ways in which a ddi might be developed and used . Central to development of the ddi will be the ability to link data to associated publications to enhance discovery and facilitate better understanding and interpretation of data and associated analyses . Again, cultural and policy changes will likely be needed to both enhance data - sharing policies and incentivize data sharing . Among the difficult challenges is how best to create greater value from the expanding availability and use of electronic health records (ehrs). Clinical data from ehrs, together with individual health data captured by various personal devices, offer considerable opportunities for advancing clinical and biomedical research . Data from clinical sources could very well provide a cost - effective means to study different health interventions, to conduct large - scale surveillance of disease incidence and progression in real time, to identify patient cohorts for recruitment into clinical trials, and more . However, unlike most other forms of biomedical research data, clinical data are typically captured outside of traditional research settings and must then be re - purposed for research use . Changes in policies and practices are needed to govern research access to clinical data sources and facilitate their use for evidence - based learning in healthcare . Improved approaches to patient consent and improved quality and quantity of clinical data available for research (eg, by creating shared libraries of phenotype elements and algorithms and by providing access to key sources of data), as well as new methodologies for analyzing clinical data, are all needed for ethical and informed use of these data . While a ddi will undoubtedly help to make data findable and citable, large datasets can also give value beyond their original purpose when the data are appropriately annotated so that they can be used in a meaningful way . Critical to all of these activities will be the need for formulating, conducting, and maintaining community - based data and metadata standards efforts for reproducibility that will: (1) be driven by the clearly identified needs of the community; (2) include a core group of developers with appropriate expertise; (3) engage stakeholders across the community from the outset and in an ongoing way; and (4) evolve based on broad input to become increasingly dynamic and responsive to community needs . Addressing the challenges associated with biomedical big data must of necessity engage all parts of the big data ecosystem . While these challenges are complex, they are also addressable . Bd2k is deploying an integrated plan of action that will tackle numerous aspects of the big data challenge, including multiple elements of data science, training, policy, and community behavior . By fostering open communication with the stakeholders, bd2k will contribute to the broader vision for the future of biomedical big data . While the nih is only one part of the biomedical big data ecosystem, it can provide leadership for convening stakeholders, providing seed funding, developing metrics for success, implementing a working process, establishing and modifying policies, and supporting basic infrastructure that can catalyze long - term solutions for the biomedical research community . Through a working partnership between and among the relevant stakeholders, useful solutions will emerge and lead to vibrant and sustainable models for capitalizing on biomedical big data and translating data into new knowledge.
Comparative modeling methods, also known as homology modeling or template - based modeling methods, are used widely to model protein structures . Significant improvement in the accuracy of homology models stemming from various advancements has been described in other publications . However, even when using multiple templates, the resulting homology models can show significant deviation from the crystal structures, especially in certain local areas of the protein structure, depending on the sequence alignments . In order to be useful for functional analysis and drug design one of the outstanding problems in protein structure prediction is the lack of a consistent and reliable method for refinement of low resolution protein structural models to atomic level accuracy . However, limitations in the conformational search occur in the energy driven torsional monte carlo . These limitations can possibly be overcome by using the force driven molecular dynamics (md) methods that enable going over energy barriers . Therefore, an md simulation offers an attractive force driven conformational search method that overcomes the pitfalls of monte carlo - based methods . All - atom md simulations, also known as cartesian md simulations, have shown limited success in protein structure refinement . However, in combination with knowledge based potentials and/or restraints using experimental data, structural refinement has been achieved using all - atom md simulations . Mirjalili and feig have shown that the use of restraints to the starting structure during md gives better refinement than without . This is in agreement with the findings from the work of shaw and co - workers . Feig et al also showed that the md ensemble averaged structures show better refinement than selecting one conformation from the ensemble . In this paper, we focus on the use of md methods for structure refinement without restraints . The generalized newton euler inverse mass operator (gneimo) method is an md simulation method based on the use of internal coordinates . The use of the gneimo torsional dynamics method combined with the temperature replica exchange (rexmd) method has been demonstrated for refinement of protein homology models without the use of knowledge based restraints . Treating the high frequency degrees of freedom as rigid using hard holonomic constraints, along with temperature based replica exchange, leads to efficient conformational sampling in the low frequency torsional degrees of freedom . In this paper, we have applied the gneimo - rexmd method for the refinement of 30 proteins from the list of target proteins released by the critical assessment of techniques for protein structure refinement and prediction (casp) casp8 and casp9 . These 30 target proteins consist of both structure prediction and structure refinement categories from casp8 and casp9 . Since the focus of this work was to examine the performance of gneimo - rexmd method for homology model refinement, we chose casp targets for which the crystal structures were available at the time of our study we have studied the extent of structure refinement that gneimo provides without using experimental data as restraints . Our ultimate goal is to examine if the gneimo torsional md, in combination with torsional monte carlo method with and without experimental data is capable of protein structure refinement . Briefly, gneimo is a constrained md method using internal coordinates, where the high frequency degrees of freedom are held rigid using holonomic constraints and the protein is modeled as a collection of user - defined rigid bodies known as clusters connected by flexible hinges . Clusters can range in scale from single atoms to helices to whole domains of proteins, as chosen by the user . The equations of motion in internal coordinates are coupled, and the computational cost of solving the coupled equations of motion in internal coordinates scales as cubic power of the number of degrees of freedom . Using the gneimo algorithm, however, the computational cost scales linearly with the number of degrees of freedom that enables the use of torsional md computationally feasible for protein simulations . Other groups have used the gneimo algorithm for torsional md simulations and for nmr structure refinement . We have incorporated various advanced internal coordinate dynamics techniques to make the current implementation of gneimo a robust md technique for long time scale simulations . We have also demonstrated the use of gneimo for structure refinement of small proteins and for mapping the domain motion in proteins . In this paper we have used the gneimo torsional md that restricts internal motion to torsional angles for refining protein homology models . The gneimo - based protocol for protein structure refinement combines the gneimo torsional md method with the rexmd method for extended conformational search in torsional space . The gneimo - rexmd protocol used for refinement is an adaptation of the protocol previously derived for protein structure refinement . Briefly, the gneimo constrained md simulations were carried out using the gneimo code using the amber99sb force field with the generalized born / surface area (gb / sa) obc implicit solvation model, an interior dielectric value of 1.5 for the solute, and exterior dielectric constant of 78.3 for the solvent . We used a solvent probe radius of 1.4 for the nonpolar solvation energy component of gb / sa . The nonbonded forces were switched off at a cutoff radius of 20 . Gneimo md simulations were performed using all torsional degrees of freedom and at constant temperature using the nose - hoover method, a lobatto integrator, and an integration time step size of 5 fs . We added the temperature replica exchange md (rexmd) algorithm to the gneimo md method to enhance conformational sampling . Gneimo all - torsion rexmd was then performed using 32 replicas over the 310415 k range of temperatures, and the temperature sorting was done based on the metropolis algorithm every 5 ps . We have studied refinement of casp targets from two categories: (1) the refinement category where a given decoy is to be refined and (2) the structure prediction category where only the sequence of amino acids in the target protein is provided . In this paper, we have studied 23 proteins of various sizes from the casp8 and casp9 refinement category and 7 proteins from the structure prediction category . The decoy structures for these 23 proteins were downloaded from the casp web site: www.predictioncenter.org . These structures were first subjected to all - atom conjugate gradient minimization using the sander program and the amber99sb force field . A total simulation time of 15100 ns (for each replica) for 32-replicas gneimo - rexmd simulations were performed for each target . The list of 23 target proteins from the structure refinement category (tr) and seven target proteins from structure prediction category (t0) selected from the casp8 and casp9 is shown in table i. the casp10 targets were not released when we started this work . In addition, we wanted to validate the use of gneimo method for known targets and derive a protocol before we applied it to unknown targets . The starting decoys for the structure refinement targets were taken from the casp web site . For the targets from the structure prediction category, we derived homology models using the modeler method . The template structures for the modeler program were chosen by using the pdb sequence query search for sequences of 3080% identity to the target . We removed the structure of the target protein and its close homologues (that were published after the respective casp competitions) from the template structure search, to avoid any bias . One hundred models for each target were generated using modeler, and these models were then clustered into five groups . The best representative structure out of the five groups, as scored by procheck g - factor, was chosen to be the starting decoy for refinement for that target . Minimization on each decoy was run using the sander utility in the amber suite and with the amber ff99sb force field, followed by gneimo - rexmd simulations . The third column in each block shows the scores for the best structure submitted to casp . We have calculated several metrics to assess the native likeness of the conformations sampled by gneimo - rexmd . We have calculated the root - mean - square deviation in coordinates (rmsd) of the backbone atoms to the x - ray and nmr structures . The rmsd was calculated using snapshots from the combined rexmd trajectory of all replicas . To determine whether the refinement in the model structure came from the secondary structure regions or the loop regions, we further calculated the rmsd of the backbone atoms in the secondary structure region (as defined by the set of residues which are in helix or sheet in the native conformation) and the rmsd of the backbone atoms for the whole structure including the loops and termini, both with respect to native structures . We used the package known as mdanalysis for the rmsd calculations . To measure the extent of refinement in the overall packing and fold of a protein, we compared the percent of native contacts made in the gneimo simulation trajectories with those in the native crystal and nmr structures . We calculated the n n matrices consisting of pairwise c(i)c(j) atom distances for the native structures, and for the whole trajectories of gneimo - rexmd, where n is the number of residues in the protein, and i and j are residue indices . A pairwise c(i)c(j) distance smaller than 8 was considered a contact and given an index value of 1 . Hence, the calculation of the contact map results in the construction of an n n contact matrix consisting of 0 s (atom pairs farther than the 8 distance cutoff or within the 4-residue neighbor cutoff in sequence) and 1 s (an atom pair within the 8 cutoff and more than 4 residues apart in sequence). We then considered each c pair in the simulation snapshots to be a contact, if the distance between both the atoms were within 0.5 of the same distance in the contact map of the native structure . We calculated the percentage native contacts as (number of identical contacts between native and decoy / total number of contacts seen in the native structure). Mdanalysis was used to calculate the pairwise distances needed for determination of percent native contacts . The gdt and tm scores are commonly used metrics in the casp assessments since they are more stringent than rmsd . Gdt is defined as the average number of aligned c atoms that fit under a distance - to - native cutoff for four different cutoff distances . The most often used set of cutoff values is {8, 4, 2, 1} () and is referred to as gdt_ts . The tm - score was developed to correlate well with human - expert assessment of protein model quality and to address the limitations of other scores such as rmsd and gdt . The program maxcluster (www.sbg.bio.ic.ac.uk/maxcluster) was used to calculate the gdt_ts and tm - scores . The crystal and/or nmr structures used as reference structures for the calculation of the above - described metrics were downloaded from the pdb web site (www.rcsb.org) corresponding to each target as indicated by casp . If the native structure was deposited as an nmr ensemble, we used the top ranked nmr structure as the reference . Table i lists the gdt, the tm score, and the rmsd for the c atoms of the best structures from the gneimo - rexmd trajectories for 23 different casp targets in the refinement category . The gdt and the tm scores for the best structure from gneimo improved in comparison to the starting decoy for 19 out of 23 proteins . The gdt scores showed an increase of up to 14.0 points while the increase in tm score is up to 0.13 . The extent of refinement by gneimo is comparable to the best structure submitted to casp for each target . The average increase in gdt score over all the 23 targets is 4.9, 0.04 in tm score, and 0.52 in rmsd . This was a modest improvement for 19 out of 23 targets and in 4 cases there was no refinement from the starting model . To understand if there is a correlation between the extent of structure refinement and the secondary structure content, we calculated the secondary structure content of all the tr targets . We observed more than a 5.0 point improvement in the gdt scores for the tr429, tr435, tr453, tr454, tr464, tr476, tr530, tr557, tr574, and tr624 targets, and these proteins showed at least 55% secondary structure content . Some of the refinement targets for which there was little to no refinement by gneimo had less than 40% secondary structure content . Tr462, a target that showed little improvement with gneimo, is a two - domain protein connected by a linker region . Although the overall refinement is 3.3 in gdt score, we observed refinement of 5.7 and 6.2 in the gdt scores for the individual domains . Other targets such as tr576, tr594, tr606, tr614, and tr622 showed no improvement in gdt scores . Tr614 has a loop that is 25 residues long, and tr606 has two loops that are 10 and 15 residues long that showed no improvement for gneimo simulations . Figure s1 of the supporting information shows the extent of the refinement in the secondary structure regions in the target protein structures . Apart from the refinement category, we also predicted the structures for the casp8 and casp9 targets in the structure prediction category denoted as the t0 targets . As described in the computational methods section, we first derived a homology model for each target using the modeler program starting from the amino acid sequence . We avoided using crystal structures that were published after the corresponding casp8 or casp9 assessment dates as templates for the homology models . Starting from the homology models, we performed the gneimo - rexmd simulations for refinement of the model . Table ii shows the extent of refinement (gdt and tm scores) yielded by just the refinement cycle using the gneimo simulations . All the structures predicted were within 4 rmsd from the respective crystal structures except for t0488 . The average improvements of 4.5, 0.04, and 0.7 in gdt, tm scores, and rmsd, respectively, were obtained using gneimo - rexmd refinement simulations . The rmsd of the structural models generated by modeler ranges from 1 to 9 . In every case, we observed substantial refinement in the structures compared to the starting decoy from modeler . Figure 1 shows the contact map for targets with substantial refinement (t0453, tr429, tr454, tr530) and one target that did not show any refinement (tr576). These figures show the absolute distance between each residue in the gneimo refined model and the same residue in the corresponding crystal structure for various targets . The deeper the red color, the farther it is from the crystal structure . The cartoon picture of the backbone of the initial decoy, the gneimo refined, and the experimental structures are also shown for these targets . The white regions in the contact map are closer to the native structure, and the dark red regions are farther from the corresponding native structures . Figure 1a for target t0453 shows substantial improvement in the packing of the loop structure with the rest of the protein structure upon gneimo refinement . The long - range inter - residue contacts for residues in the loop region between 30 and 40 with residues that are in the core of the protein between 40 and 60 (shown in dark red in contact map for the starting decoy) improve from 14 to 16 to 24 as seen in the contact map for the refined structure . This is an example where most of the refinement is in the packing of the loop with the secondary structure core of the rest of the protein . Figure 1b for tr429 shows that residue ranges 2531 and 3642 form a -sheet that is missing in the starting decoy structure (see dashed line rectangles in figure 1b). The three - strand -sheet motif that ranges from residues 2060 improves its packing to the rest of the protein, upon gneimo refinement . Figure 1c shows that the two helices in the target tr454 are already formed in the initial decoy . Refinement in this situation requires improved packing of these two helices shown in dashed rectangles in figure 1c . As seen in figure 1c, tr454 shows the long - range helix packing into a more nativelike structure, resulting in an rmsd of 2.47 in coordinates of the backbone atoms to the crystal structure . Gneimo - rexmd refinement of the tr530 target leads to the proper folding of the n - terminal region, as shown in figure 1d (dashed line rectangles). Refinement is also seen in other long - range contacts throughout the molecule, due to slight improvements in local packing . (a d) refinement of casp targets with different types of secondary structure as shown in the figures and corresponding distance - to - native map . The distance to native map shows how far each residue is corresponding to the native structure . (1a) example of refinement of loop structure; (1b) refinement of -sheet packing; (1c) packing of -helices; (1d) -sheet growth; (1e) example of a structure that was not refined by gneimo . Tr576 has a substantial -sheet content as well as long loop regions, and the carboxy terminus region of tr576 needed substantial refinement . This structure was deemed to be of high difficulty by the casp assessment team, due to crystal contacts . The starting decoy is misfolded into a partial antiparallel -sheet while it is a parallel -sheet in the native structure . This could not be refined by gneimo possibly due to a high energy barrier in refolding this region . We did not use explicit water in these gneimo simulations, which may play an important role in stabilizing the loop structures . Many of the targets studied here show better refinement with gneimo simulations than the best structure submitted for the casp assessment . It should be emphasized that we have compared the best structure by rmsd in coordinates generated by gneimo - rexmd simulations to the native structure and not utilized energy or scoring functions to pick the best structure . However, the gneimo method can be combined with any energy function or scoring function for picking the best structure . The chances of identifying the closest to native structure as the best scoring structure are improved if there is a substantial population of near - native conformations compared to the starting decoy generated during the gneimo - rexmd simulations . Therefore, we calculated the fraction of the population from gneimo - rexmd simulations that are closer to the native structure compared to the starting decoy to assess the enrichment of nativelike structures in the gneimo - rexmd trajectories . Figure 2 shows the population histogram with respect to gdt score and tm score for some targets, and figure s2 of the supporting information shows the same for all the targets . Population distribution of the ensemble generated from the gneimo simulations compared to the cartesian simulations for various refinement casp targets . It is seen that, for tr429 and tr454, a significant (over 50%) population shift occurs toward the native structure compared to the starting decoy (denoted by the dotted line in figure 2) in both gdt and tm scores . Structurally, both of these proteins are notable in that they have over 50% secondary structure content . Both tr568 and tr624 show a small percentage (about 1020%) of the population getting refined . The targets tr576, tr606, tr614, and tr622 show little to no population shift toward the native structure . These structures have less than 40% secondary structure content and have large loops, thus leading to poor refinement . Thus, gneimo shows effective refinement of secondary structure regions and their packing with the failures occurring in the refinement of the loop regions . We are exploring the use of gneimo simulations with side chain replacement methods to improve the refinement of loop regions . In this section we compare the effectiveness of the conformational sampling of gneimo method to cartesian md method . However, we are not examining the effectiveness of energy functions that are used for selecting the best structure for cases where the experimental structure is unknown . Our future goal is to combine gneimo with other refinement methods, namely those methods such as torsional monte carlo methods, that complement the torsional dynamics based conformational search of gneimo . Figure 2 shows the comparison of the population of structures in the gneimo ensemble that get refined with respect to the starting decoy structures as measured by the gdt scores, compared to the corresponding populations in the cartesian md . The md simulations for both gneimo and cartesian md were done with the identical forcefield and gbsa solvation . The dotted line in the figure shows the position of the starting decoy . Figure 2 shows that the relative proportion of gneimo ensemble that gets refined is more than the population of refined structures from cartesian simulations . This shows that the conformational sampling afforded by sampling torsional angles using the same forcefield is more effective in getting closer to the native structures than the cartesian all - atom simulations . Shaw and co - workers have tested the ability of several microseconds of cartesian md simulations to refine homology models for 25 casp refinement targets, 21 of which are common to our study . They observed that the long cartesian md simulations lead to unraveling of the homology model away from the crystal or nmr structures . Using the same forcefield we have shown in this paper that gneimo torsional dynamics method leads to more refinement than the cartesian md simulations . Using an energy function that would preserve and funnel toward the native structure is a critical component for structure refinement . In the next section, we compare the all - atom amber energy function that we have used with gneimo md in this study to the knowledge based energy function in rosetta . Scoring functions are important in selecting the most refined structure from the ensemble of conformations generated during the gneimo - rexmd simulations . In this paper, we have not addressed this issue . Briefly, we have calculated the all - atom amber energies and energies from the rosetta energy function for all the conformations generated in the gneimo - rexmd trajectories of three target proteins . The rosetta energy function is based largely on the charmm energy function with additional knowledge based hydrogen bond terms . As shown in figure 3, the rosetta energy function showed a more funnel like character for some of the targets, i.e., the near - native structures showed the lowest energy . Thus, use of the rosetta energy function could improve the selection of the best refined structure . However, for many other targets, both the amber and rosetta energy functions did not show a funnel like behavior . In the next stage of our study, we will examine many other knowledge based energy functions for rescoring the conformations generated by gneimo - rexmd simulations . Also, we will explore the use of a force field derived from the rosetta potential energy function for driving the gneimo dynamics . Potential energies of the conformations generated in the gneimo - rexmd simulation trajectories calculated using (a) amber99sb all atom forcefield and (b) rosetta energy function . An advantage of using the gneimo method is the time required to perform each sampling . By taking stable time steps of 10 fs, gneimo combined with rexmd is able to explore more regions of conformational space in the same number of processor cycles compared to cartesian simulations . While the targets in this study were run for up to 100 ns (for all replicas combined), existing casp teams which replace cartesian md with gneimo could simulate about 1 order of magnitude longer in the same clock time . Unlike torsional monte carlo method where the moves are random and scored by energy, the forces govern the moves in gneimo torsional md . Performing conformational search in the torsional degrees of freedom appears to focus the search in the low frequency degrees of freedom . Coupling gneimo torsional md with rexmd provides enough thermal energy to overcome barriers that can arise from the stiffness in the dynamic model from freezing high frequency degrees of freedom . Giving such high thermal energy to all atoms in the cartesian all - atom dynamics can however result in an unraveling of structured regions in the starting decoy . The gneimo approach is also highly extensible . Some monte carlo based methods that were used in casp restrict sampling of certain regions or attempt to rigidly dock individual domains of the same protein . The ability to perform dynamics of coarsened bodies is inherent within gneimo, and the generalized coordinate system can naturally incorporate constraints into the equations of motion to rigidify or free any desired degree of freedom . Further, gneimo is not restricted to any specific force field but rather has a modular design with an extensible interface class for any force field that can be wrapped to fit a template . Numerical integration methods for time propagation are also optional modules in gneimo, and methods, which rely on torsional monte carlo sampling (like rosetta), can use gneimo to directly sample coordinates in phase space based on any definable coordinate system . In this paper, we report some of the promising developments on the application of torsional molecular dynamics method (gneimo) to structure refinement of casp target proteins . We have applied brute force torsional md without any restraints on any part of the structure from known structural information to refining homology models . The torsional md refinement yields results that are better compared to the all - atom md simulations performed under the same conditions . There is still much progress and issues that need to be addressed to improve gneimo as a refinement tool . As observed in table i, the extent of refinement by gneimo is the same whether the starting decoy is of low resolution (greater than 5) or of high resolution (less than 3). One possibility is to test the extent of refinement using different clustering schemes in gneimo in combination with side chain refinement methods . The use of a force field tailored for protein structure prediction can also improve refinement with gneimo . If distance restraints are available from known experimental data, refinement can be improved at low resolution . Presently, we are working on all these aspects to make gneimo a robust and generic refinement tool . We have shown that the gneimo - rexmd simulation technique leads to refinement of up to 1.3 for 30 casp target proteins starting from their homology models of variable resolution . Gneimo method leads to refinement for 21 out of 23 refinement targets although the average refinement for 23 targets is 4.0 in gdt score, 0.04 in tm score, and 0.5 in rmsd in coordinates . Significantly, we did observe that gneimo with rexmd simulations enable focused conformational sampling in the low frequency torsional space that is essential for structure refinement . Further testing of this method, applying residue - based distance restraints obtained from experiments and testing a suitable energy function that provides identification of the native structure, is ongoing . Additionally feig and co - workers have demonstrated that an ensemble average shows better refinement than rather than a single structure from md simulations . Further testing of gneimo ensembles using an ensemble average could provide better refinement than seen in this study . Enhancement in side chain sampling can be obtained by combining the gneimo simulations with side chain reassignment from a rotamer library . Our ultimate goal is to combine gneimo torsional dynamics with a torsional monte carlo method and test the method in a future casp in the refinement category.
Antiphospholipd syndrome is a disorder of recurrent vascular thrombosis, pregnancy loss, and thrombocytopenia associated with persistently raised levels of antiphospholipid antibodies . It may be primary, occurs alone or secondary, associated with other autoimmune or rheumatic diseases, also known as hughes syndrome, as described by dr . It was diagnosed at first as hemolytic uremic syndrome and passed through different other presentations which fulfilled the diagnostic criteria of antiphospholipid syndrome . The case was a female female patient 13 years old, 1st kid of a low socioeconomic state family, from zagazig . The condition started (may 2007) by acute onset of vomiting not responding to any medication followed on 2nd day by vaginal bleeding for 13 days . She was referred to insurance hospital where they discovered on clinical examination, jaundice and pallor too . The patient was then referred to our hematology unit where complaining of dark colored urine, puffy eyelid, and on examination there was purpura on abdominal, and investigation at that time revealed the following . Cbc: hb 5.7 gm\dl, rc 21%, platelet 28000, serum creatinine 2 mg\dl, blood urea 113 mg\dl, coombs test both direct and indirect were negative, total billruibin 2.2 mg\dl, direct 0.2 mg\dl, esr 75, urine analysis showed rbc's over 100 . Our differential diagnosis at that level was as follows: hemolytic uremic syndrome, autoimmune hemolytic anemia, immunological disease, for example, sle, ttp (thrombotic thrombocytopenic purpura), chronic hemolytic anemia on exacerbation, post infection, for example, hbv . So we performed next investigations: blood film showed fragmented red cells, coombs test again direct and indirect were negative, osmotic fragility was negative, hemoglobin electrophoresis was normal, ana negative, anti - double - strand dna negative, abdominal us enlarged kidneys with mild splenomegaly, hepatitis markers hbv weak positive (hbs ag) by pcr negative, c3 normal . So diagnosis of hemolytic uremic syndrome was the most likely diagnosis at that level with raising creatinine to 10.5 mg\dl, dialysis was initiated (via subclavian catheter), plus supportive blood and platelet transfusion and electrolytes support . Actually our patient showed slow response to dialysis that she was dialysis to the extend dependent for 7 weeks; gfr by dtpa at that time was 15 ml\mint\1.73 m. so avf was done . Apart from 3 admissions to icu with malignant hypertension, no other systems were affected in the next 7 weeks . We gave plasma transfusion repeatedly to cut off the circuit of pathogenesis; actually it was a great surprise that dina began to be off dialysis, with normal cbc . The patient was discharged on conservative management of crf and a close followup twice weekly was done . Investigations on follow up after 2 months of admission (july 2007) showed cbc rc 3%, hb 9 gm\dl, platelet 224,000, urea 42 mg\dl, and creatinine 1.3 mg\dl . The patient readmitted 3 weeks later with acute pallor and jaundice again with purpura and hypertension with palpitation . Investigations at that time (august 2007) showed the following results: rc 7.8%, hb 7.6 gm\dl, platelet 94,000, coombs negative, creatinine 4.9 mg\dl, urea 167 mg\dl, urine rbcs over 100, c3 consumed (0.5), ana and anti - double - strand dna were negative, anca negative, esr 85 1st hour, 126 2nd hour . Bone marrow biopsy was done and revealed hyperactive bone marrow with no abnormal or megakaryocytic changes . Echocardiography showed grade 3\4 miteral regurgitation, grade 1\4 aortic regurgitation, pulmonary hypertension, and moderate pericardial effusion . Plasmapheresis was initiated 4 sessions at one week with no improvement so redialysis was repeated . One week later the patient complained of abdominal pain, abdominal us, right kidney enlargement for doppler study, and right renal artery blunted systolic filling denote obstruction at subsegmental branching . Our differential diagnosis at this level was: other types of vasculitis, 2ry antiphospholipid syndrome, other cause (protein s.c deficiency), 1ry antiphospholipid syndrome . So diagnosis of antiphospholipid syndrome was done and treatment was initiated as follows: steroid was initiated in september 2007 . Anticoagulation heparin followed by low molecular weight heparin was given as well as antiplatelet, cytotoxic in the form of cyclophosphamide 2 pulses i.v . After two weeks hemolysis regressed, but renal function was the same, gfr 30 ml\mint\1.73 m (improved). But the patient developed new signs of vascular affection in the form of lived reticularis on abdominal wall and leg with repeated chest pain . Note that the patient was on regular dialysis through avf, blood pressure fluctuations were on the high level always (so stoppage of anticoagulant was done), and antidepressant was added; blood pressure was controlled by it plus using one antihypertensive only but the patient lost 9 kg in the next 6 months . She complained of recurrent massive pericardial effusions and responded to daily dialysis for 15 days each time for 4 times with stoppage of anticoagulant . Intravenous immunoglobulin was given 5 days a week for three weeks, but unfortunately with no response as regards hemolysis or c3 level . Reinvestigating for acl, ana, anti - douple - strand dna, anca, and all four tests were negative . And two weeks later repeated acl was done and was negative . The patient was on the same management with gradual decrease of steroid because the only response was raising c 3 level without clinical response for 3 months; on the other hand, hypocalcaemia and other signs of side effects began . In november 2007, echocardiography was done for repeated chest pain with normal ecg showing unequal septal movement evident of myocardial infarction . Unfortunately, cardiac enzymes were not available at that time . In january 2008, sever chest infection followed by hemoptysis occurred . Tuberculin test done with double dose was negative and ct chest revealed wedge pulmonary infarction . In february 2008, head nodding and static tremors, possibility of parkinson's (basal ganglia vascular affection) occured, but the result of mri came to be frontal lobe atrophy from repeated infarctions mostly . In march 2008, avf rethrombosis occured and it was documented by doppler but after 24 hours of detection of thrombosis, fistula leakage and emergency closure were done . In april 2008, the patient developed sever chest pain diagnosed as pulmonary infarction for which she was admitted to intensive care unit and unfortunately died . The description of antiphospholipid syndrome has been one of the most striking developments in the field of immunology in the last two decades the earliest descriptions of the association between a circulating anticoagulant and vascular thrombosis in pediatric population are those of olive et al . Our case had the following positive clinical data: hemolytic anemia, thrombocytopenia, hematuria with impaired kidney function, interarenal vascular occlusion, pulmonary infarction, cardiac infarction, valvular lesion, recurrent pericardial effusion, avf thrombosis, and neurological insult . And the case had the positive laboratory data as follows: acl positive once, ana, anti - double - stranded dna negative, anca negative, c3 consumed, documented thrombosis (interarenal, intraglomular, pulmonary, avf, cardiac, cns), and normal pt, ptt . For diagnosis of antiphospholipid syndrome, the patient should fulfill the diagnostic preliminary criteria by sapporo which was proposed in 1998 one clinical plus one lab [1, 4]. Clinical criteria are as follows: (1)vascular occlusion artery, vein or minute vessels in tissue or organ with no inflammatory reaction around,(2)pregnancy related . Vascular occlusion artery, vein or minute vessels in tissue or organ with no inflammatory reaction around, laboratory criteria were as follows: (1)acl ab igg or igm positive in two separate samples 6 weeks apart,(2)lupus anticoagulant ab positive in two separate samples 6 weeks apart . Acl ab igg or igm positive in two separate samples 6 weeks apart, lupus anticoagulant ab positive in two separate samples 6 weeks apart . Catastrophic antiphospholipids syndrome (caps) (accelerated form of aps with consequent multiorgan failure, disturbance at small vessel level) hasinternational criteria for classification such as clinical evidence of vessel occlusions affecting 3 or more organs or systems, development of the manifestations simultaneously or in less than a week, confirmation by histopathology of small vessel occlusion in at least one organ, serological confirmation of the presence of apl (la and/or acl). The patient fulfilled criteria of a catastrophic antiphospholipid syndrome as the repeated acl was negative, but our patient showed multiorgan failure, and there is small vessel affection as those appearing in renal biopsy (microangiopathic glemuleropathy). The main organ affected and one of the earliest organ affections was the kidney, and our case died after 11 months of diagnosis although we start all kinds of therapy of apl . Our case in clinical presentation showed veins affection (lived reticularis), arterial affection (renal thrombotic microangiopathic, myocardial infarction, pulmonary infarction, and brain ischemic manifestation), and also showed cardiac valve affection which was also described in apl . Our patient 1st presentation was hemolytic anemia and thrombocytopenia which made us think that it is a case of hemolytic uremic syndrome, but it was actually a spectrum of clinical presentation of apl . As regards differential diagnosis of our case, the main differential diagnosis was to exclude sle as recently the presence of apl in lupus patients has been associated with development of occurrence of neuropsychiatry manifestation and ischemic microangiopathic nephropathy, as many studies reported prevalence of acl and la in juvenile sle ranges from 19%87% and from 10%62%, also the possibility of transformation of 1ry apl to sle after 6 months . So we repeatedly sample our patient for ana anti - double - stranded dna and it was negative all through till her death raising the possibility of 1ry apl rather than 2ry apl of sle but this didnnot exclude the possibility of lupus - like syndrome . As regards management of apl, being symptomatic apl, that is, apl positive patient with thrombosis, it needs the following: anticoagulant like other cases with thrombosis, although the duration and intensity of antithrombotic therapy are not yet clearly established . We started low molecular weight heparin followed by warfarin, but we needed to stop this treatment for many times because of uncontrolled hypertension or pericardial effusion; high doses of corticosteroid, cyclophosphamide, intravinous immunoglobulin, and plasmapheresis as well as hemodialysis all were tried as all are recommended in life - threatening apl or catastrophic antiphospholipid; although we gave many modalities of treatment, we lost our patient in a severe attach of pulmonary infarction and this showed us the important and the aggressive form of this disease, as high mortality up to 48% despite antithrombotic therapy had been reported in many studies [1, 5, 9]. Apl is recognized increasingly as a leading cause of vascular thrombosis in pediatric population; also catastrophic antiphospholipid syndrome should take much care and recognition by pediatricians being one of severe and rabidly fatal disease; we saw to share this case and we suggest further studies about diagnosis, presentation, and management of this serious disease.
A 42-year - old indian male had multiple small circumscribed wrinkled sacs like lesions on the shoulders and upper back of 4 months duration (fig . Skin biopsy was taken for histopathological examination and revealed minimal dermal perivascular chronic inflammatory cells infiltrate (fig . Elastic stain (verhoef - van gieson) showed loss of elastic fibers in the superficial dermis (fig . Direct immunofluorescence (dif) was negative for iga, igg, igm, c1q, c3 and fibrinogen . The patient did not have any symptoms or show any sings of antiphospholipid syndrome (aps), and screening for antiphospholipid antibodies (anticardiolipin profile, anti--2-glycoprotein, igg and igm, and lupus anticoagulant) were all negative . Antinuclear antibody (ana), ena screen (smb, smd, rnp-70, and rnp - a, rnp - c, ssa / ro52, ssa / ro60, ssb / la, cenp - b, scl-70, jo-1, ribosomal p and histones) were negative . Thyroid panel test showed normal free thyroxine and thyroid stimulating hormone, but the patient had a positive high titer of thyroid peroxidase antibody (anti - tpo antibody)> 116 iu/ ml . There are numerous reports and studies that link primary anetoderma (pa) to lupus erythematosus, but the relation has not been clearly established [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14]. Moreover, there are isolated repots of pa and autoimmune diseases like primary hypothyroidism, grave's disease, addison's disease, sjogren's syndrome, alopecia areata, vitiligo [14, 18, 19] and multiple sclerosis . Now, there is a growing body of evidence to consider pa as a cutaneous sign of positive antiphospholipid antibodies with or without fulfilling the criteria of aps [16, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34]. In our case, there is a high titer of antithyroid peroxidase antibodies, which is consistent with autoimmune thyroiditis . This is in accordance with the work of hodak et al . Who described a case of pa with grave's disease, positive lupus anticoagulant and autoimmune hemolysis . It is important to mention that grave's disease in the study of hodak et al . Had started 5 years after the onset of the pa . In our study, there were no clinical signs or symptoms of thyroid disease at the time of diagnosis; also free thyroxine and thyroid stimulating hormone were in a normal range, but we have to take the short duration of onset of pa in our case into consideration . Also, bergman et al . Described a case of primary hypothyroidism that developed 3 years after the onset of anetoderma . From our point of view and the aforementioned literature we have to think of pa as a cutaneous sign of autoimmunity and patients should be examined and carefully tested for autoimmune diseases, especially for antiphospholipid antibodies, lupus erythematosus and also thyroid antibodies . Patients should also be followed up because associated autoimmune diseases may develop later in the course of the disease, maybe years after the onset of anetoderma.
Predictions on the influence of climate change indicate that the number of intense tropical cyclones (known as typhoons or hurricanes) will increase . Indeed, the united nations intergovernmental panel on climate change reports that the average maximum wind speed of tropical cyclones will likely increase throughout the coming century (http://www.ipcc.ch). Reporting the details of case studies following contemporary tropical cyclones may improve our ability to predict how these changes will influence ecosystem recovery following tropical cyclone damage . Toward that end, we recently compared long - term resilience of guam s dominant arborescent species cycas micronesica following the 1997 supertyphoon paka and the 2004 typhoon chaba . Results indicated that invasions of the armored scale aulacaspis yasumatsui and the specialist butterfly chilades pandava to guam after the 1997 typhoon compromised the resistance to and resilience following the 2004 typhoon . The outcomes signified that studying the interactive aspects of the broad disciplines of invasion biology and climate change may inform future predictions and management decisions . Understanding biogeochemical responses to disturbances such as tropical cyclones is viewed as a major theme that would benefit from international coordination among researchers . Pursuit of a useful understanding of long - term ecosystem responses to large - scale disturbances requires an understanding of short - term mechanistic processes during recovery . In order to learn from ecosystem responses following the 8 nov . 2013 damage of typhoon haiyan, a tropical cyclone reported to exhibit some of the highest tropical cyclone wind speeds on record, we followed its path across several eastern philippine islands . A widespread phenomenon became apparent in most of the habitats that were damaged by this severe tropical cyclone . Numerous examples of partial leaf necrosis on intact leaves of trees in the cycadaceae and arecaceae families resulted during the desiccating damage imposed by the typhoon (fig . The result was a copious amount of arboreal dead leaf material attached to live leaves . Arecaceae and cycadaceae trees were uniquely damaged by typhoon haiyan in november 2013, leaving copious amounts of dead leaf material attached to partially live leaves . (b) group of cycas trees in barrier island of leyte exhibiting dead leaf material . However, the manner in which arboreal litter influences biogeochemical cycling has received much less attention . One of the most important effects of tropical cyclones is defoliation and the spatiotemporal influence of the resulting litter on subsequent geochemical processes . Litterfall associated with tropical cyclones is so voluminous that it often explains the majority of variation in annual litterfall among years . Litter associated with tropical cyclones and other disturbances such as severe freeze events is comprised of high quality green tissues that were detached before the typical nutrient resorption associated with the organ senescing processes . The combined effects of greater quantity and quality of litter indicate tropical cyclones exert a widespread and long - term influence on subsequent ecosystem processes . The role of suspended or arboreal litter in ecosystem processes has not been well - studied, and the literature is comprised of eclectic publications . Some species have been shown to self - retain dead leaves and branches, and other understory species have been shown to intercept a disproportionately high amount of falling litter . General quantity and traits of arboreal litter indicate the canopy location of suspended litter can be ephemeral as wind and branch movement repeatedly dislodge the litter, and decomposition of suspended litter that is artificially tethered in place is slow compared with litter on soil surfaces . The composition and abundance of arthropods in arboreal litter may be highly contrasting to that of litter deposited on the forest floor . Availability of arboreal litter is critically important for these specialist arthropods and insectivorous bird species that specialize on dead - leaf inhabiting arthropods . The dead leaflet material that was generated by the passage of typhoon haiyan (fig . 1) may influence biogeochemical processes in a manner that is distinct from any previously published information concerning arboreal litter . (1) the petiole, rachis, and many leaflets of the leaves on these damaged palm and cycad trees remained green and will likely retain the leaves on the trees for extended periods . The dead sections of these large leaves were not detached, they remained affixed to live leaves . Therefore, the routine dislodging by wind and branch movements that is characteristic of suspended, arboreal litter will not occur and the dead material will remain in place for extended periods of time . (2) initial quality of this form of litter will be greater than the other forms of suspended litter that are generated by interception of naturally abscised leaves . The leaf sections killed by the forces of typhoon haiyan did not proceed through the typical resorption of nutrients associated with natural organ senescence . (3) this dead leaflet material will be more exposed to solar radiation than forest floor litter or suspended abscised litter that is intercepted by the sub - canopy . As a result, the abiotic catabolism imposed by photooxidation of this unique form of canopy litter will exceed that of other forms of litter . (4) leaching of soluble materials will be greater in this typhoon - generated litter than in other forms of litter because precipitation is initially intercepted by the upper canopy and the dead material will remain exposed to leaching processes for much greater lengths of time than suspended detached litter or ground litter . When the dead leaflet material is ultimately detached, its quality will be disparate from that of other forms of litter due to preferential long - term leaching of soluble materials like potassium, phosphorus, sugars, and phenolics . (5) the proportion of decay attributed to catabolism of organic molecules by saprophytic organisms will be minimal in this form of litter, as the dry canopy environment is not conducive to mineralization by fungi and bacteria . (6) in contrast, the proportion of decay attributed to comminution by detritivorous insects and mites will be greater than in other forms of litter due to a greater length of time that these detritivores can feed on the dead materials . We believe this form of damage by tropical cyclones is commonplace, but has gone unnoticed until now . Its prevalence will be greater in regions like the philippines where a high proportion of the forest communities are comprised of arecaceae, cycadaceae, pandanaceae, and other families that contain species with large, tough leaves that are resistant to wind damage . Based on our field work, typhoon haiyan exerted direct damage in habitats containing five cycadaceae species and 70 species and infraspecific taxa of arecaceae . Future studies including this unique form of arboreal litter created by tropical cyclones would be of use for increasing our understanding of the mechanisms by which large scale disturbances influence biogeochemical processes.
Based on the report of world health organization (who), road traffic injuries (rtis) stand for 25% of injury - related mortalities . Traffic injuries constitute the largest proportion of unintentional injury deaths (33%) in the world . Road traffic injuries are the ninth leading cause of all deaths and leading contributor to the worldwide burden of disease . Yearly traffic collisions cause 1.2 million deaths and 20 to 50 million disabilities in the world . Who estimates that without proper measures, the incidence of traffic mortalities rise by 67% and in 2020 it will be the third cause of death among 5 to 45 year old people . Low and middle - income countries have 85 percent of traffic mortalities in the world . Only 14% of traffic mortalities occur in highly motorized countries (hmc) such as north america, australia, newzeland, japan and west europe although these countries have comprised about 60% of the world vehicles . Previous studies showed that death caused by traffic injuries has been increased in africa, eastern mediterranean and asia in recent years . In addition, the rate of traffic mortality to whole mortality is 2.5 in the world and 1.9 in the region (eastern mediterranean)while this ratio in iran is about 7.5 . While having one percent of the world population, one fortieth of road traffic accidents occur in iran . Studies have indicated that iran has too many road traffic injuries; for example, iran with rti fatality rate of 31 . 8 per 100 000 in the year 2007 had the highest rate in the region of eastern mediterranean . In iran, there are about one vehicle for every four individuals; in comparison, this ratio is higher than that of eastern mediterranean countries but lower than those of industrialized and developed countries . Moreover, rtis stand for the most prevalent cause of injury and the second leading cause of death in iran . A report by who showed that despite technological development, it is not clear why mortality rate in traffic injuries has a progressive trend in many countries . Although new traffic laws had effects on reduction of fatal and non - fatal rates of road traffic injuries in iran, injuries from road traffic incidents have been increased in recent years . Comparative studies show that an important portion of traffic mortalities occur in post - crash phase (after crash, until transferring the injured victim to hospital) in low and middle income countries . One noticeable part of these mortalities are preventable by proper pre - hospital interventions . One of the most important roles of pre - hospital services is life support and prevention of future disabilities in the post - crash phase . Triage of an injured patient to an appropriate trauma center can have an impact on morbidity and mortality . Studying the process and structure of pre - hospital service system provides knowledge and insight for assessment of these types of services . Despite the importance of pre - hospital service in preventing mortalities and disabilities caused by road traffic injuries, there is lack of knowledge in these fields, especially cultural and contextual aspects . Since the function of pre - hospital service is culture and context bound, qualitative approach was an appropriate method to design the study . Also, since process of pre - hospital services is a phenomenon that consists human relationships, action - interactions, and emotions that are usually hidden and could not be assessed by quantitative approaches, qualitative method was selected as an approach . By qualitative approaches, we can observe phenomenon from the eyes of the participants, and the hidden aspects of phenomenon can be explored . Determining the barriers of pre - hospital service in viewpoints of participants was another reason for selecting the qualitative approach . The aim of this study was to explore the barriers of pre - hospital service in traffic injuries in tehran, iran . Is suitable when the existing theories are inadequate to describe the phenomenon . In content analysis, concepts are gained directly and inductively from the raw data . It is a method with the purpose of providing knowledge, new understanding, and representation of realities . The aim of content analysis is to attain a summarized and comprehensive description of the phenomenon . In this method, another advantage of this method is the possibility of observation of the phenomenon, as viewed by the participants . First, interviewer proposed some general questions to start the interviews such as please explain one of your assignments . Then, based on the participants responses and trend of the interview, next questions were arranged . Each interview lasted about 45 - 60 minutes . The time and location of the interviews interviews were recorded in the form of audio files (mp3) and transcribed verbatim . In addition, researchers used other methods of data gathering such as field notes, focus groups and observation . For data gathering by observation, the researcher observed the scenes of crushing and recorded the observed events (field note). The se notes consisted of the type of crushing, the way first aids were provided to the injured victim, the person who provides aids to the injured person, the way the crowded people in scene of crush interfere with care giving process, and the way the action of other organizations interferes with interventions of pre - hospital service providers . In addition, one session of focus group for enrichment of data was performed with pre - hospital managers and paramedics . Eighteen participants were selected by purposive sampling and that those with at least 2 years experience in the field of pre - hospital services were considered to be enrolled in the study . The participants consisted of 14 paramedics, 1 dispatch operator, 2 pre - hospital service managers and 1 police officer . Each interview was recorded and typed verbatim by the first researcher . For analyzing the data at first, the researcher read the texts several times (intensive reading) for getting a complete understanding of the respondents accounts; then, the meaning units of phrases was identified and summarized . Finally, after coding, based on common properties or dimensions, similar codes were classified in higher - level categories with a new and high abstract label (table 1). Data collection continued until data saturation, meaning that adding further data does not offer any new information and new properties for each category . Example of meaning units, condensed meaning units, codes, subcategories and categories for examining the rigor of data and results, guba and lincoln strategies were used . To insure the credibility of data, several strategies were used including: a) prolonged engagement (data gathering lasted about one year and during this period, researchers were oriented to culture, language and environment of the participants and atmosphere of the field), b) peer review (data and interpretation of data were checked by other researchers), and c) member checking (data rechecked by participants and our interpretations from data were reviewed and confirmed by them). In addition, to insure the dependability of data, triangulation strategy for data collection such as interview, observation, focus group and field notes were applied . By this strategy, they were also informed about their authority for withdrawal from the study at any stage of study . In addition, their anonymity as well as confidentiality was insured and the participants signed the informed consent form . Ethics committee of tehran university and the department of social welfare and rehabilitation sciences approved the study . In addition, one abstract of the results of the study was mailed for participants interested in the findings . Data was gathered through semi - structured interviews . First, interviewer proposed some general questions to start the interviews such as please explain one of your assignments . Then, based on the participants responses and trend of the interview, next questions were arranged . Each interview lasted about 45 - 60 minutes . The time and location of the interviews interviews were recorded in the form of audio files (mp3) and transcribed verbatim . In addition, researchers used other methods of data gathering such as field notes, focus groups and observation . For data gathering by observation, the researcher observed the scenes of crushing and recorded the observed events (field note). The se notes consisted of the type of crushing, the way first aids were provided to the injured victim, the person who provides aids to the injured person, the way the crowded people in scene of crush interfere with care giving process, and the way the action of other organizations interferes with interventions of pre - hospital service providers . In addition, one session of focus group for enrichment of data was performed with pre - hospital managers and paramedics . Eighteen participants were selected by purposive sampling and that those with at least 2 years experience in the field of pre - hospital services were considered to be enrolled in the study . The participants consisted of 14 paramedics, 1 dispatch operator, 2 pre - hospital service managers and 1 police officer . Each interview was recorded and typed verbatim by the first researcher . For analyzing the data at first, the researcher read the texts several times (intensive reading) for getting a complete understanding of the respondents accounts; then, the meaning units of phrases was identified and summarized . Finally, after coding, based on common properties or dimensions, similar codes were classified in higher - level categories with a new and high abstract label (table 1). Data collection continued until data saturation, meaning that adding further data does not offer any new information and new properties for each category . For examining the rigor of data and results, guba and lincoln strategies were used . To insure the credibility of data, several strategies were used including: a) prolonged engagement (data gathering lasted about one year and during this period, researchers were oriented to culture, language and environment of the participants and atmosphere of the field), b) peer review (data and interpretation of data were checked by other researchers), and c) member checking (data rechecked by participants and our interpretations from data were reviewed and confirmed by them). In addition, to insure the dependability of data, triangulation strategy for data collection such as interview, observation, focus group and field notes were applied . By this strategy, data can be collected from many aspects and dependability of data will be increased . They were also informed about their authority for withdrawal from the study at any stage of study . In addition, their anonymity as well as confidentiality was insured and the participants signed the informed consent form . Ethics committee of tehran university and the department of social welfare and rehabilitation sciences approved the study . In addition, one abstract of the results of the study was mailed for participants interested in the findings . Based on the data, 13 categories were developed and classified into four main categories (table 2). Main categories were classified into four branches: categories and subcategories of the barriers to pre - hospital services in road trafficinjuries a.barriers related to people consisting of concepts (subcategory) that have at least one common property . This property included barriers that were in a way related to people such as laypeople involvement, inadequate knowledge about first aids and mistake calls.b.barriers related to metropolitan infrastructures . This category consisted of concepts whose common properties were problems related to characteristics and structure of the city such as traffic, accessibility to streets and naming of the alleys.c.barriers related to profession . In this main category, there were concepts that explain barriers related to pre - hospital service providing profession . In this category, there are three subcategories: professional autonomy, workload and work related injuries.d.the fourth main category named barriers related to managerial issues consisting of four concepts that were common in property of managerial problems in the field of pre - hospital service . These concepts were named inadequate telecommunication technology, human resources, inadequate workload related privilege and lack of organizational coordination . In the next section, you can see categorization of main categories and related quotations: barriers related to people consisting of concepts (subcategory) that have at least one common property . This property included barriers that were in a way related to people such as laypeople involvement, inadequate knowledge about first aids and mistake calls . This category consisted of concepts whose common properties were problems related to characteristics and structure of the city such as traffic, accessibility to streets and naming of the alleys . Barriers related to profession . In this main category, there were concepts that explain barriers related to pre - hospital service providing profession . In this category, there are three subcategories: professional autonomy, workload and work related injuries . The fourth main category named barriers related to managerial issues consisting of four concepts that were common in property of managerial problems in the field of pre - hospital service . These concepts were named inadequate telecommunication technology, human resources, inadequate workload related privilege and lack of organizational coordination . In the next section, you can see categorization of main categories and related quotations: a. barriers related to people: a. 1 laypeople -involvement: crowded area in the crash scenes is a problem that was brought up by the participants . Overcrowding around the crash scenes may act as a barrier for proper and on - time pre - hospital interventions . One of the participants (p2) stated: in many situations, when we arrive in the crash location, people crowding around the scene area barrier for timely interventions . A. 2- inadequate knowledge on first aids: usually, people directly perform interventions that may worsen the problems of the injured person . The cause of these inappropriate actions is usually inadequate knowledge about first aids interventions . As an example always people do not have enough skills for proper interventions in the accident scene and this problem may worsen the severity of injuries . A. 3- mistake calls: in many situations, people call the number of emergency medical system (ems), instead of other organizations such as firefighter and the other one of the participants (p3) noted: we have about 10000 calls, daily, in tehran that one half of them are mistake or irrelevant . B. barriers related to metropolitan infrastructure: b. 1- traffic: traffic jam is a serious problem and barrier for prompt interventions by pre - hospital service providers . One of the participants (p11) stated: many of our people have not acquired acceptable traffic culture and knowledge and this problem is a barrier for on time arrival to the scene and transporting the injured victim to hospital . B. 2- accessibility to streets and alleys: problem with accessibility is one of the barriers mentioned by the participants . One (p6) stated: in many situations, because the streets and alleys are too narrow or too crowded, we have to stop and leave the ambulance faraway from the crush location . This acts as one cause of delay and this is especially noticeable and problematic during transport of the injured victim to ambulance . B. 3- naming of alleys: in many locations, there is not a logical and systematic approach for naming the pathways . One participant (p1) expressed that: non - systematic approach for naming of alleys causes problems and makes it complicated for pre - hospital service providers to find a location . For example, many alleys are named using numbers followed by an alley named using a special noun . C. barriers related to profession: c. 1- professional autonomy: participants frequently mentioned inadequate professional autonomy . One participant (p5) announced that: the pre - hospital system does not consider our professional capacities, knowledge and experiences . When i consult with the physician in our center, the only order is to transport the patient to the hospital . C. 2- workload: based on the participants accounts, workload may act as a barrier for service providing . Specially, this is considerable about payment system . According to participants, salaries do not suit their workload . One said (p13): in this part of the city, we have a high number of accidents and traffic injuries, but this is not considered by administrators of pre - hospital system . In fact, there is no difference between a high workload center and other centers . C. 3- work - related injuries: many injuries may occur during care giving by pre - hospital service providers . When we transport an injured victim to ambulance, injuries such as low back injuries and other problems may occur . These problems may affect the paramedics quality of life and even at the end, lead to losing the job . D. barriers related to managerial issues: d. 1- inadequate telecommunication technology: improper and inadequate telecommunication technology was one of the problems often announced by the participants . One (p2) stated: in many situations, when our ambulance cannot go so closed to collision scene, we have to communicate to our dispatch center using mobile phones or landline telephone . In these situations, d. 2- inadequate human resources: one of the barriers of delivering proper pre - hospital services in viewpoints of care providers was inadequate number of care providers . In addition, the absence of physician in ambulances was another problem implied by many participants . One of the participants (p18) said: when i am bedside an injured woman, because of cultural and religious considerations, there are many limitations to give care to her . In these situations, having a woman technician in the ambulance may decrease such problems and promote the quality of care providing . D. 3- inappropriate workload related privilege: this problem was another issue noticed, by the participants . A participant (p12) announced: this problem may decrease our motivation and the quality of pre - hospital care . In fact, the level of rewards in high and low workload centers is equal and this is one of the weaknesses of our pre - hospital care system . D. 4- lack of organizational coordination: problems on inter- and intra - organizational coordination are are issue mentioned by the participants . One participant (p4) said: involvement of other service providers such as firefighters or police in the accident location can be disturbing as they may sometimes interfere with our activities . I think this problem is related to the lack of coordination between the involved organizations . This study was an attempt to identify the barriers of pre - hospital caregiving classified into four main categories: barriers related to people, to metropolitan infrastructure, to the profession and to managerial problems . They argued that administrational and organizational factors, also staff qualifications and competences, availability and distribution of resources, communication and transport, involved organizations and laypeople could act as barriers in the chain of care from crush incident to the hospital . Causes such as, administration, communication and transportation, involved organizations (named as lack of organizational coordination), and laypeople involvement, that have been brought up by haghparast et al . Are confirmed in the current study . Moreover, findings of the present study are in the same line with khorasani et al . Laypeople involvement, lack of coordination, inadequate pre - hospital services, and shortcomings in infrastructure are found as barriers of pre - hospital service in road traffic injuries both by the current study and in that of khorasani et al . In the study of big deli et al . Also traffic, crowding of laypeople in the scene of the crush and involvement of other organizations have been mentioned as factors that interfere with interventions of pre - hospital care providers in traffic accidents . One category of barriers identified by the present study is barriers related to people classified into three subcategories: laypeople involvement, inadequate knowledge about first aids, and mistake calls . Based on this study findings, one aspect that differentiates pre - hospital care in road traffic injuries from other fields of pre - hospital care giving is crowding of people in the scene of the crush . Based on the findings of the current study, this problem not only prevents prompt care provision to the injured victims, but also it may worsen the condition of the injured victim because of inappropriate interventions delivered by people in the scene of accident . Another result of our study was that although laypeople involvement has been considered as a barrier, according to many participants, if care providers manage the scene, laypeople could be turned into a capacity to accelerate and facilitate pre - hospital interventions . Findings presented by a few studies such as that of tannvik et al . Have emphasized this matter . Inadequate information and knowledge about emergency agencies and wrong calls were also combined in one category named the barrier related to people . Knowledge providing and distribution of information on first aids and pre - hospital service processes among people may act as a factor to reduce their improper interventions in the scene of the crush . Another category of barriers identified by this study is those of metropolitan infrastructure including traffic, accessibility of alleys, and naming of alleys and streets . In fact, this category of barriers, especially traffic, has a remarkable confounding role in pre - hospital service delivery . Traffic is a barrier that has been highlighted in both this study and several other studies . To resolve these problems, there is a need for comprehensive assessment of the pre - hospital system and the ways other organizations (for example firefighters, police, and so on) may involve and collaborate in this system . Our results also show that there are some profession - related issues that may act as barriers for pre - hospital care delivery . Several previous studies have considered workload and work related injuries as barriers for care providing in the pre - hospital situations . However, professional autonomy is a concept that in the current study was emphasized by many of care providers (paramedics) as a factor that can decrease their motivation . In addition, use of woman paramedics in ambulance crew is an aspect of barriers that has not been mentioned by other similar studies . Based on our research findings, it can be inferred that issues such as workload and work related injuries might strongly affect the circumstances and the manner of actions done by care providers . Further, these factors can diminish the staff s motivation, as mentioned by most participants . Lack of motivation, in turn, may act as a reason for worsening the quality of services . Some issues that my decrease the motivation of care providers and quality of care and noted by participants were payments that do not correspond to workload, inadequate number of colleagues, and the rigidity of the physician orders . Results of the present study showed that there are problems in the field of management . These factors were classified as barriers related to managerial problems, including inadequate telecommunication technology, inadequate human resources, inadequate workload based privilege, and lack of organizational coordination . Study of nielsen et al . Also showed that one of the problems in low and middle income countries was managerial issues . One of the aspects that can be mentioned as a barrier and was emphasized by participants was lack of women in ambulance crew . Studies show in advanced countries, women are in arrangement of paramedics . Another issue mentioned as barrier by participants was lack of physician in ambulance assignments . Studies show that pre - hospital systems in high - income countries use physicians in ambulance crew . For example, when interviewer was in the pre - hospital context to interview with paramedics, they had to go for emergency assignment and this was a factor that interrupted the interview process . In these conditions interviewer had to continue the interview in the ambulance during the assignment or after returning the participants from their assignment . Based on conditions, many interviews performed in ambulance crew and traffic and noises were factors that interfered with recording of interviews . This caused the interviewer to plan for another session of interview with the participant or recheck the previous interview with them . This study found that not only laypeople s involvement could not be seen merely as barrier but also it can be apotential in some situations and contexts, if care providers manage the scene of the crash . Based on these findings, a fundamental change in management system may lessen the problems and solve the barriers like metropolitan infrastructure and managerial issues . According to our knowledge, some barriers such as laypeople involvement, inadequate knowledge about first aids and wrong calls can be reduced through public education, information campaigns and changing attitudes . Because of socioeconomic and cultural context, future studies are suggested to explore issues related to laypeople s involvement and their interventions in the scene of collision, because based on the study findings, laypeople s involvement cannot be seen merely as a barrier . In addition, future studies are suggested to assess the effect of women (as care providers)and physicians in ambulance crew on quality of pre - hospital service provided specially for injured victims . Considering the fact that the process of providing pre - hospital service in traffic accidents is not well established other qualitative approaches such as grounded theory method is recommended for further studies . Findings of this study contribute to improvement of the pre - hospital service system and can be used by health policy makers . For example, laypeople education, improvement of organizational coordination, use of strategies to expand the professional autonomy of paramedics, and application of practicable strategies can increase the motivation of pre - hospital care providers . In addition, use of new and appropriate telecommunication technologies and recruitment of more women and physicians as the ambulance crew may be a part of their future programs to promote and improve the quality of pre - hospital care.
Cryoglobulinemia (cg) occurs when the serum contains cryoglobulins, either single or mixed immunoglobulins, which precipitate at low temperatures . This can result in an immune - complex mediated, small - to - medium vasculitis that most commonly affects the kidneys and the skin . Ie also has the ability to manifest clinically through immune - mediated phenomena and thus can cause overlapping symptoms, namely positive rheumatoid factor (rf) and glomerulonephritis (gn). A 42-year - old man with known history of ivdu and hcv infection presented with complaints of bilateral lower extremity edema, rash, increasing abdominal girth, diffuse abdominal pain, and generalized fatigue . His lower extremity edema had been present for 6 months and he had been evaluated for it on 2 previous occasions with negative lower extremity ultrasound examinations and had taken a 30-day course of furosemide with some improvement; all other symptoms were new to this presentation . Initial laboratory values were significant for creatinine 1.7 mg / dl (reference range 0.81.4 mg / dl), elevated white blood cell count of 18, 000/mcl (450011,000/mcl) and hiv screening test that was non - reactive . A percutaneous kidney biopsy was subsequently performed revealing igm dominant mesangioproliferative gn with features suggestive of cg (fig . 1, fig . 2, fig . 3, fig . 4), likely related to chronic infection including chronic hepatitis c or chronic bacterial infection . The patient was treated with corticosteroid therapy for presumed mixed cg, however on hospital day 3 his renal function continued to deteriorate and the patient was transferred to our tertiary care center for further evaluation for possible rituximab and/or plasmapheresis therapy . Upon arrival, the patient s physical exam was notable for anasarca, a grade 2 diastolic murmur and several painless, small, erythematous macules on bilateral lower extremities, not extending to the soles of his feet . Initial laboratory results were significant for creatinine 3 mg / dl (0.81.4 mg / dl), total protein 8.4 g / dl (6.38.2 g / dl), albumin 2.4 g / dl (3.45.0 g / dl), hemoglobin 8 g / dl (13.517.5 g / dl), white blood cell count 35,000/mcl (450011,000/mcl) and urinalysis with 30 mg / dl of protein; platelet count and the remainder of the comprehensive metabolic panel were within normal limits . Other pertinent laboratory studies revealed low c3 and c4 complement levels (c3 level 88252 mg / dl, c4 88206 mg / dl), negative rf, and negative cryoglobulins . Blood and urine cultures obtained prior to transfer both grew enterococcus faecalis, leading the care team to ascribe the bacteremia to a urinary source and the patient was started on daptomycin and ceftriaxone (due to a previous adverse reaction to ampicillin). Repeat blood cultures were again positive for e. faecalis . Due to the persistent bacteremia and the patient s high - risk behavior, a transthoracic echocardiogram (tte) demonstrated severe aortic insufficiency, severe left atrial enlargement, and thickening of the mitral valve leaflets with trace regurgitation . Subsequent transesophagealechocardiogram confirmed these results and revealed mitral and aortic valve vegetations (fig . 5, fig . 6, the antibiotic regimen was changed to vancomycin and gentamicin, with subsequent improvement in abdominal pain, lower extremity edema, and renal function (creatinine 1.8 mg / dl (0.81.4 mg / dl)). The cardiothoracic surgery team evaluated the patient and recommended valve replacement following completion of the antibiotic course and scheduled the patient for a clinic visit in six weeks . The delay was due to concern for continued ivdu after the procedure and subsequent reinfection . The patient was discharged to a skilled nursing facility to receive a total six - week course of antibiotics for infective endocarditis . He recovered and returned home without issues, but failed to follow up with his clinic appointment for primary care or for surgical evaluation.fig . The glomerular basement membranes are unremarkable without spikes, holes, splitting, or corrugation.fig . 2immunofluorescence.on a scale of 03 +, there is 2 + diffuse global granular mesangial with very segmental capillary loop staining for igm (leh) and 12 + c3 staining (right) in a similar pattern.fig . 2fig . 3kappa and lambda immunofluorescence.on a scale of 03 +, there is equal 12 + diffuse segmental to global granular mesangial staining for kappa and lambda.fig . 4electron microscopy.there are scattered mesangial immune complex deposits (leh) with vague annular substructure (right).fig . The glomerular basement membranes are unremarkable without spikes, holes, splitting, or corrugation . On a scale of 03 +, there is 2 + diffuse global granular mesangial with very segmental capillary loop staining for igm (leh) and 12 + c3 staining (right) in a similar pattern . There is equal 12 + diffuse segmental to global granular mesangial staining for kappa and lambda . There are scattered mesangial immune complex deposits (leh) with vague annular substructure (right). Type i is defined by the presence of a monoclonal immunoglobulin (ig), predominantly resulting from hematologic diagnoses such as waldenstrom s macroglobulinemia, chronic lymphocytic leukemia, and multiple myeloma . Type ii is a mixture of polyclonal ig and monoclonal ig, predominantly resulting from infection such as hcv or human immunodeficiency virus (hiv). Type iii consists solely of polyclonal ig, often associated with autoimmune disorders such as henoch - schonlein purpura, systemic lupus erythematosus, sjogren syndrome, connective tissue disease, sarcoidosis and others, but also has been described as resulting from hcv infection . Risk factors include: age greater than 60 years old, male gender, history of ivdu, poor dentition, structural heart disease, intracardiac or intravascular access devices, chronic hemodialysis, diabetes mellitus, and hiv infection . Duke criteria comprise a collection of major and minor criteria to establish a diagnosis of ie (outlined in table 1). Some clinical findings of ie overlap with cg including arthralgias, abdominal pain, petechiae, renal dysfunction, positive rf, elevated esr and crp . While c4 levels are typically low in both processes, reflecting ongoing consumption by immune complexes, c3 is also low in ie but is generally unaffected or only mildly diminished in cg .table 1endocarditis diagnostic criteria modified duke criteria.table 1major criteriapositive blood culturestypical pathogens from at least two separate cultures (staphylococcus aureus, streptococcus bovis or viridans, hacek * organisms, enterococcus)evidence of endocardial involvement by echocardiographyendocardial vegetation, perivalvular abscess, new partial dehiscence of prosthetics valve, new valvular regurgitationcoxiella burnetti blood culturesingle positive culture or anti - phase 1 igg antibody titer> 1:800minor criteriapredispositionheart condition, intravenous drug use, indwelling catheters, poor dentition, diabetes mellitus, hemodialysisfevergreater than or equal to thirty - eight degrees celsiusmicrobiologic evidencepositive blood culture not meeting major criteriavascular phenomenaseptic arterial or pulmonary emboli, mycotic aneurysms, intracranial or conjunctival hemorrhages, janeway lesionsimmune phenomenarheumatoid factor, glomerulonephritis, osler s nodes, roth s spotshighly probable: 2 major or 1 major and 3 minor or 5 minor criteria.possible: 1 major and 1 minor or 3 minor. *haemophilus, aggregatibacter, cardiobacterium, eikenella, kingella . Highly probable: 2 major or 1 major and 3 minor or 5 minor criteria . In cg, the renal disease is most commonly attributed to immune complex deposition, but may be a result of thrombotic disease . Microscopy of kidney tissue samples most often yields results consistent with mesangioproliferative gn with hypercellularity from an influx of inflammatory cells, immune deposition, and thickening of glomerular elements . In ie, renal disease is thought to be sequela of vascular occlusion by microthrombi that lead to local immune - mediated vasculitis . . As with cg, a membranoproliferative - pattern gn with igg and c3 deposition is the traditional pattern of injury for ie . However, a recent study of clinical specimens demonstrated that ie presented most commonly with crescentic gn, followed by diffuse proliferation, and finally mesangial proliferation without endocapillary proliferation or crescent formation, the latter of which was seen in this case . Confounding this case if the presence of dominant igm staining in addition to c3 . Thus, no definitive etiology could be identified by renal biopsy alone in this case . Early recognition of ie is important as it is reported that in - hospital mortality is 1823% and 6-month mortality is 2227% . However, in one case series, ie was unrecognized in almost 1/5 of cases at the time of nephrology consult . If a patient is clinically stable, treatment can wait until blood culture results are available and targeted therapy can be delivered . When patients are acutely ill, empiric treatment should be given after two or three sets of blood cultures are drawn and vancomycin is an appropriate choice for most patients . A cardiac surgery consultation is recommended for cases where complications arise or are suspected such as infection of prosthetic valves, heart block, systemic emboli, or new moderate to severe heart failure . Assessment of response is based on clinical observation; fever should resolve between 37 days, and repeat cultures should demonstrate clearance of bacteremia . Duration of therapy can vary based on organism, but is typically six weeks starting with the first day of negative blood cultures . This case highlights the similarity in risk factors, clinical findings, and renal complications of ie and mixed cg . It also provides an opportunity for reflection on the use of heuristics and how bias can affect diagnosis and treatment . Healthcare providers must maintain a broad differential and continue to re - evaluate the patient as additional information arises . Due to hcv infection and acute kidney injury, it was reasonable to suspect cg in this patient . Kidney biopsy results added support for this diagnosis, but mesangioproliferative gn is not specific for cg and chronic infection can yield similar results . In addition, it is very common to see a urinary tract infection (uti) in hospitalized patients . When blood cultures returned positive, they were believed to be from a urinary source, however ivdu should significantly elevate the suspicion for endocarditis . Anchoring to the original admit diagnoses, uti and cg, led to the belief that two distinct processes were occurring . However in this particular case, occam s razor appears to prevail and all findings could be attributed to ie . This research did not receive any specific grant from funding agencies in the public, commercial, or not - for - profit sectors.
Guanidine, first isolated in 1861 by oxidizing guanine, is found in a wide variety of natural products, including the amino acid arginine . Although known for more than 150 years, the first solid - state structure was solved in 2007 by co - crystallization with 2-amino-4,6-dimethyl-1,3,5-triazine . Two years later, the structure of the free base was reported, and more recently, neutron diffraction studies accurately positioned the hydrogen atoms (figure 1a). As a strong base (pkb 0.5), at physiological ph s guanidine exists in its protonated form, the highly stabilized guanidinium cation . The y - shaped guanidinium group is a highly symmetric planar functionality that can form two strong parallel hydrogen bonds with biologically relevant counterparts . Its geometry generates a more favorable hydrogen bond alignment compared to ammonium groups, which are also widely found in biomolecules . Additionally, binding can occur through both charge pairing and hydrogen bonding as the group maintains its protonated state over a wide range of ph . Moreover, unlike for the ammonium cations where the charge is localized (hard), the interaction with softer ions such as phosphates and sulfates is facilitated by delocalization of the positive charge in the guanidinium group (figure 1). Crystal structures and 2d representations of (a) free base guanidine, (b) a guanidinium carboxylate salt, (c) a propylguanidinium phosphate salt, and (d) a sulfate salt of a synthetic bisguanidinium receptor . Comparing the binding energies of ammonium and guanidinium groups indicates that both have high affinities and selectivities for phosphates and arsenates over other anions, while higher binding constants are found for the guanidinium groups . Furthermore, while the formation of ammonium phosphate complexes was found to be primarily entropy driven, favorable enthalpy and entropy changes were reported for the guanidinium - phosphate complexation . This distinct thermodynamic behavior was suggested to derive from differences in the solvation shell of the two groups . Isothermal titration calorimetry (itc) measurements with a series of substituted bicyclic guanidiniums and different counterions assigned a decisive role to solvation in this enthalpy entropy compensation and highlighted the significance of the coordinating ability of the counterion . Endocytosis, the energy - dependent vesicular uptake of extracellular substrates, has been established as the main mechanism for cellular uptake of nonviral vectors . Briefly, endocytosis pathways can be divided into clathrin - mediated endocytosis, the best characterized endocytic pathway; caveolin - mediated endocytosis; phagocytosis, typically restricted to specialized mammalian cells; and macropinocytosis, which refers to the formation of large endocytic vesicles . They differ in the composition of the vesicle coat (if any) and in the fate of the internalized particles . Although most receptors are internalized by clathrin - mediated endocytosis, other endocytic pathways are capable of selective receptor - mediated endocytosis events . After endocytosis, internalized cargo is trafficked into endosomes, from where it can either escape or be sorted back to the surface of the cell or into other compartments such as lysosomes for degradation . Nonendocytic delivery was initially suggested for the direct translocation of cationic peptides such as tat across cell membranes . Potential mechanisms for cellular uptake including clathrin- and caveolin - mediated endocytosis, macropinocytosis, phagocytosis, and direct translocation across the plasma membrane . Following the discovery of hiv - tat s basic sequence (tat4957, rkkrrqrrr) as the module responsible for cellular entry, alanine scan indicated that the arginine residues are critical for cellular uptake and that the analogous (arg9) displayed increased translocation efficiency . The nature of the counterion was also shown to play an important role in the translocation ability of oligo / polyarginines in vitro, with lipophilic anions altering the highly hydrophilic characteristic of the guanidinium - containing entities into lipophilic complexes, therefore facilitating the translocation through lipophilic membranes . The opposite effect is observed for hydrophilic anions, and while some amphiphilic counterparts were not shown to mediate such phase transfer, others could solubilize polyarginine (but not polylysine) in chloroform . In a similar context, wender and co - workers investigated the importance of hydrogen bonding to membrane embedded constituents and correlated it with cellular uptake . They compared the uptake of arg8 with that of octamers of monomethylated and asymmetrically dimethylated arginine . This observation correlates with the ability of the octamers to form bidentate hydrogen bonds, further supported by molecular modeling of the possible isomers of the methylated guanidiniums and estimation of their energies . Given the ability of the guanidinium group to bind biologically abundant counterions and its involvement in translocation through mammalian cell membranes, researchers have focused on the design and development of guanidinium - containing oligomeric transporters, with the aim of delivering diverse cargo into mammalian cells . Wender and co - workers have recently reviewed oligomeric and polymeric guanidinium - rich molecular transporters where the guanidinium groups are linked to either peptidic or peptoidic backbones as well as to longer oligocarbamates and oligocarbonates . The use of guanidinylated dendritic molecular transporters in cell transfection, as reported by goodman and others, has been presented by gillies and co - workers . Moreover, the utility of cationic lipids for gene delivery has recently been discussed by zhao and co - workers . This synopsis, therefore, focuses on the development of nonoligomeric nonpept(o)idic guanidinylated scaffolds designed and used to deliver low and high molecular weight cargo, from small drugs to quantum dots, across cellular membranes . Guanidinoglycosides are guanidinylated aminoglycoside antibiotics where all the ammonium groups are converted to guanidinium groups . Bodipy - tagged guanidinotobramycin and guanidinoneomycin were shown to translocate across the cellular membrane with considerably improved efficiency compared to their parent aminoglycosides (figure 3). Furthermore, significantly higher translocation efficiency was observed for guanidinoneomycin (gneo), containing six guanidinium moieties, compared to oligo - arg peptide arg9, suggesting that the semirigid and perhaps more globular organization of the guanidinium groups might play an important role in facilitating cellular uptake . The cellular binding and uptake of gneo at nanomolar concentrations exclusively depends on cell surface heparan sulfate (hs) proteoglycans . The number of guanidinium groups and, to a lesser extent, their spatial distribution on the guanidinoglycoside core, significantly impact cellular uptake . Comparing the uptake of monomeric and dimeric guanidinoglycosides derived from tobramycin, paromomycin, and neomycin b containing a different number and 3d arrangement of guanidinium groups established a correlation between valency and uptake efficiency (figure 4). Specific mutant cell lines derived from chinese hamster ovary (cho) cells, differing in their expression of hs, were used to investigate the effect of sulfation patterns on the cellular recognition and uptake of guanidinoglycosides . Hs - deficient cells showed very poor uptake (<5%, compared to wild type). Uptake of monomeric guanidinoglycoside constructs was reduced to <20% in undersulfated cell lines, when compared to wild - type cells . Unlike the monomeric carriers, the multivalent constructs were able to overcome lower sulfation levels and displayed higher uptake levels in such cell lines (between 50 and 75% compared to that observed in wild - type cells). These results identify the nature of cell surface hs as a key parameter affecting the cellular uptake and recognition of guanidinoglycosides, suggesting an additional variable to consider when evaluating the behavior of molecular transporters . Monomeric and dimeric structures of (a) guanidinoneomycin, (b) guanidinoparomomycin, and (c) guanidinotobramycin . When biotinylated gneo is conjugated to streptavidin - coated quantum dots (qd525), approximately 90% of internalized nanoparticles colocalize with lysosomes after 3 h, suggesting that gneo can deliver very high molecular weight cargo (> 10 da) to these organelles (figure 5). To facilitate conjugation of diverse biomolecules, an n - hydroxysuccinimide activated ester of guanidinoneomycin was prepared (gneo - nhs, scheme 1). Two lysosomal enzymes, -d - glucuronidase and -l - iduronidase, were conjugated to gneo without interfering with their enzymatic activity and delivered to patient cells lacking the corresponding lysosomal enzyme in sufficient amounts to restore normal turnover of glycosaminoglycans . Gneo - qd525 conjugate colocalizes with lysosomes . Wild - type chinese hamster ovary cells were incubated with 5 nmol after the cells were rinsed three times, fresh medium was added, and 2.5 h later, they were rinsed with hank s balanced salt solution and labeled with hoechst dye and lysotracker red . Images were captured with a deltavision restoration microscope system and were deconvolved to show the localization of (a) gneo - qd525 and (b) lysosomes in a single z - stack plane . The merged images from (a) and (b) are shown in (c) with the outline of cells (hatched line) drawn based on a phase contrast micrograph . Copyright 2010 nature publishing group . To shed light on the uptake mechanism of guanidinoglycosides, fluorescently tagged streptavidins [streptavidin pycoerythrin - cy5 (st pecy5), streptavidin phycoerythrin (st pe), and streptavidin cy5 (st cy5)] were used as model cargos . Evaluating the uptake and binding of streptavidin gneo conjugates by flow cytometry and cell - surface fret analysis suggested that heparan sulfate proteoglycan aggregation is a pivotal step for endocytic translocation of guanidinoglycosides . This pathway can be altered by selective acylation of guanidinoneomycin - based transporters with long alkyl chains, which enhances the macromolecular cellular uptake with little or no heparan sulfate aggregation (figure 6). These findings suggest an alternative and distinct pathway involving hydrophobic interactions impacting membrane curvature while assisting the uptake . It is worth noting that hs proteoglycans were also identified as cell surface receptors for tat internalization . The ability of a carrier to bind to hs is not always sufficient, however, for efficient uptake, as suggested by the differences observed for arginine - rich cell penetrating peptides (cpps) featuring d - amino acids compared to those with l - amino acids, both having comparable hs binding constants . The ability to cluster hs has been shown to contribute to the efficiency of endocytosis of cpps, typically initiated through electrostatic interactions with cell - surface hs . The endocytic mechanism of internalization has been proposed to relate to the nature and distribution of proteoglycans expressed on the cell surface, and a recent report established structure activity relationships for hs binding and uptake for a set of cpps where stoichiometry was the decisive factor . Searching for naturally occurring scaffolds, chung and co - workers focused on carbohydrates as polyfunctional cores for molecular transporters . The intracellular localization of myo- and scyllo - inositol dimers bearing eight guanidinium groups differed from that of the tat and (arg)8 peptides, suggesting a distinct clathrin - independent internalization pathway (figure 7). Unlike tat peptides, these compounds were found mainly along the heart, lung and brain tissues, displaying unique distribution both in vitro and in vivo . In addition, conjugation of doxorubicin to one of the transporters significantly increased drug uptake and its intracellular permeation in brain tissues . Representative structures of dimeric (a) myo - inositol and (b) scyllo - inositol . Despite of the promising features of these dimeric inositol based transporters, chung and co - workers explored a different scaffold, based on sorbitol, keeping the eight guanidinium units (figure 8a, b). Similarly to the di - inositol transporters, the internalization mechanism was observed to be different from that of the tat peptide . More significant was their high selectivity for mitochondria and higher distribution in the heart muscle and brain sections . Taking advantage of their ability to cross the blood brain barrier (bbb), these octa - guanidinylated sorbitol - based molecular transporters were covalently linked to paclitaxel (figure 8c). Furthermore, a conjugate of 3-azido-3-deoxythymidine (azt) showed effective cellular uptake in hela cells with preferred localization in mitochondria and nucleoli (figure 8d). 5-fluorouridine (5-fu) was covalently attached to the sorbitol - based molecular transporter through a succinate ester linker at position 5 of the ribose ring (figure 8e). Like the azt derivative, 5-fu conjugates displayed good cellular uptake and mitochondrial localization . Although they showed different biodistribution in mouse tissue, these 5-fu conjugates crossed the bbb and showed more potent in vitro cytotoxicity than unconjugated 5-fu . Internalization of quantum dots (qd) into hela cells was facilitated using a biotinylated version of the sorbitol - based transporter bound to streptavidin qd conjugates (figure 8f). After long incubation times, these conjugates appeared mostly in the perinuclear region but did not enter the nucleus . Moreover, qd conjugates were observed to efficiently cross the bbb in mice when administered via tail vein . Finally, lipidated derivatives of the sorbitol - based transporters were evaluated for their ability to condense either dna or sirna and their applicability in nonviral gene delivery systems (figure 8 g). Compounds with a short lipid chain (c12) were proven particularly useful for nucleic acid condensation, whereas those with a long lipid chain (c30) were optimal for surface modification of nucleic acid containing lipid vesicles . Sorbitol - based transporters: (a) general structure of the sorbitol scaffold, conjugated with (b) fitc, (c) paclitaxel, (d) azt, (e) 5-fu, (f) biotin, and (g) fatty acids . Other scaffolds investigated by chung and co - workers include the disaccharides lactose, sucrose, and trehalose (figure 9), the monosaccharides glucose, mannose, allose, and galactose (figure 10), and monomeric myo- and scyllo - inositols (figure 11). Both the lactose- and sucrose - based transporters feature seven guanidinium groups tethered to the sugar through linkers of different lengths (figure 9a, b). For both, the intracellular localization was influenced by the length, or lipophilicity, of the linker and in the case of sucrose scaffold also by the nature of the fluorescent dye attached to the transporter . Representative structures of disaccharide - based transporters: (a) lactose, (b) sucrose, and (c) trehalose . Trehalose, a neuroprotective disaccharide with poor cellular uptake, was decorated with either six (td - g6) or eight (td - g8) guanidinium moieties and administered to huntington disease model mice (figure 9c). Td - g8 resulted to be more toxic than td - g6, and the latter showed enhanced neuroprotective activity compared to trehalose itself . While all monosaccharide - based molecular transporters shown in figure 10 displayed good permeability to brain tissues, the intracellular localization, particularly their mitochondrial affinity, was found to be related to their stereochemistry and to a lesser extent to the lipophilicity of the linker between the guanidinium moiety and the sugar scaffold . Representative structures of monosaccharide - based transporters: (a) glucose, (b) mannose, (c) allose, and (d) galactose . To better understand the correlation between stereochemistry and mitochondrial localization, a series of octa - guanidinylated molecular transporters based on two inositol stereoisomers, myo- and scyllo - inositol, was explored (figure 11). Derivatives of myo - inositol were found to target the mitochondria, whereas the more symmetric scyllo - inositol derivatives did not show significant mitochondrial colocalization . In addition, while all the transporters showed good affinity for brain tissues, scyllo - inositol - based transporters, unlike their myo - inositol stereoisomers, were widely distributed in all organs . It seems that although the structures of the saccharide scaffolds are closely related, several parameters need to be tuned for optimal organellar selectivity and tissue distribution . Representative structures of monomeric inositol - based transporters: (a) myo - inositol and (b) scyllo - inositol . As mentioned above, the use of high - order guanidinylated dendrimers was reviewed by gillies et al . Related reports, include different platforms such as dendronized nanoparticles, vivo - morpholinos, and guanidinium dendron carbon nanotubes (figure 12). Jeong and co - workers encapsulated a hydrophobic peptide model drug into dendritic amine and guanidinium group - modified nanoparticles . In this system, up to four amine or guanidinium groups are connected to a hydrophobic stearyl tail through a short oligophenylalanine linker introduced to provide structural rigidity (figure 12a). It was shown that the uptake efficiency increased with the number of the positively charged groups and that guanidinium - functionalized nanoparticles had better ability to cross membranes than the amino - functionalized ones . Moreover, this transporter structure was conjugated to a morpholino oligomer yielding a product referred to as a vivo - morpholino (figure 12b). This structure also proved to effectively deliver morpholino antisense oligomers into a wide variety of tissues in living mice . Also aimed to deliver antisense oligonucleotides, chi et al . Designed and prepared multiwalled carbon nanotubes conjugated to positively charged dendrons bearing either ammonium or guanidinium groups (figure 12c). Interestingly, ammonium - decorated nanotubes displayed notably better sirna complexation, cellular uptake and gene silencing activity than the guanidinium - decorated counterparts . Dendrimer - like transporters: (a) dendronized nanoparticles, (b) vivo morpholinos, and (c) a representative structure of guanidinium carbon nanotubes . Ungaro and co - workers reported the synthesis of calix[n]arenes (n = 4, 6, and 8) bearing guanidinium groups in the aromatic rings (upper rim) showing good water solubility and proven ability to bind linear and plasmid dna (figure 13a). Their findings indicate that cell transfection, promoted by guanidinylated calix[n]arenes, is highly influenced by small changes in conformation, ring size, and the nature of alkyl substitutions in the lower rim . Subtle changes in these constituents notably affect their ability to bind to dna and condensate it, which correlates with their ability to transfect cells and deliver dna . These upper rim guanidinylated calix[n]arenes, however, showed low overall transfection efficiency and relatively high cytotoxicity . These drawbacks were overcome by attaching the guanidinium groups to the lower rim, through the phenolic moieties (figure 13b). Calixarenes, containing four guanidinium groups, showed significantly enhanced cell transfection efficiency and reduced cytotoxicity compared to the upper ring analogues . Moreover, one of the reported structures, linking the guanidinium moiety and the phenolic oxygen through a propylene bridge and lacking substitutions in the upper rim, had higher transfection efficiency than lipofectamine when formulated with 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine (dope). Again, like the upper rim modified platforms, subtle structural variations in these vectors can cause drastic changes in their biological properties . More recently, calixarenes bearing four lysine or arginine units on either the upper or lower rim were disclosed (figure 13c). These tetraargininocalixarene constructs displayed higher efficiency in dna transfection when compared to their lysine counterparts . Representative structures of (a) upper rim guanidinocalixarenes, (b) lower rim guanidinocalixarenes, and (c) tetraargininocalixarene . A family of tetraguanidinium vectors that efficiently internalized in human tumor cells was reported by giralt and co - workers . These vectors consist of chiral bicyclic guanidinium subunits linked together through short thioether spacers (figure 14). It was shown that these compounds translocated through hela membranes more efficiently than antp or tat peptides at very low concentrations . These compounds appear to specifically accumulate in mitochondria and showed no cytotoxicity at relevant concentrations . Following kinetic and temperature - dependent experiments showed that the internalization pathway involved both active energy - dependent transport and passive internalization . As apparent from the above discussion, guanidinium groups confer unique translocation features upon polyfunctional scaffolds . Their introduction into diverse cores frequently involves modification of the corresponding polyamines . Numerous strategies to convert a primary amine into a guanidinium group, we briefly describe the synthetic approaches used in the reports mentioned above (figure 15). For the conversion of aminoglycosides into guanidinoglycosides, we typically use boc - protected triflylguanidine, a reagent developed by goodman and co - workers . Ungaro and co - workers used either boc - protected triflylguanidine or n, n-di(tert - butoxycarbonyl)thiourea in the presence of mercuric chloride to guanidinylate the calix[n]arenes derivatives . The use of di - boc-4-pyrazole-1-carboximidamide, described by drake et al ., was the choice of battigelli et al . To guanidinylate the carbon nanotube transporters, and morcos and co - workers used o - methylisourea for the vivo - morpholinos conjugates . Reaction conditions: (a) (i) dcm, tea, 78 c; (ii) tfa, rt; (b) (i) dimethylformamide (dmf), tea, rt; (ii) tfa, rt; (c) (i) dmf, diea, rt; (ii) tfa, rt; (d) 1,3-dimethyl-2-imidazolidinone, 1-hydroxybenzotriazole, tea, ammonium hydroxide, 50 c . Although high yields were reported for the guanidinylation of primary and secondary amines using protected thioureas, the need for mercury salts makes this procedure somewhat less atractive . The use of di - boc-4-pyrazole-1-carboximidamide is often associated with moderate yields, while high to quantitative yields have been reported for the use of triflylguanidine . The inexpensive o - methylisourea is useful for guanidinylation in aqueous solutions without the need for a deprotection step, and high yields were also reported for its reactions . When it comes to the perguanidinylation of multifunctional scaffolds, triflylguanidine remains our guanidinylating agent of choice . The introduction of guanidinium groups into multifunctional scaffolds has proven to be a very efficient strategy to generate cellular delivery vehicles . Such molecular transporters have been shown to facilitate the delivery of diverse cargos, ranging from low molecular weight small molecules to extraordinary high molecular weight quantum dots . Certain guanidinylated scaffolds have also been shown to overcome important biological barriers such as the bbb, allowing for the delivery of therapeutic agents to the brain . Although extensive efforts have advanced the utility of diverse guanidinium - based molecular transporters, the fundamental understanding of their cell entry processes and the role of guanidinium groups in cellular delivery remain elusive . Subtle structural changes in the carriers and linkers often result in unexpectedly distinct celullar delivery profiles or in completely different biodistribution patterns and intracellular localization . Systematic correlations between structural features of the molecular transporters and both their cellular delivery efficiency and biodistribution at the organelle / organ level are therefore needed . A deeper molecular level understanding of these processes will likely further advance such guanidinium - rich molecular transporters as therapeutic and diagnostic tools.
Health is a state of complete physical, mental, and social well - being and not merely an absence of a disease or infirmity.1 health is not something that one possesses as commodity but connotes rather a way of functioning within one s environment.2 oral health is an integral part of general health.3 loss of tooth reduces the quality of life . Prevalence of dental caries and periodontal disease determines the oral health, the former being one of the most widespread chronic diseases in the world . Dental caries is modern civilization scourge, and no nation or race has escaped the ill effects.4 oral health consists of two dimensions . First, the physical, oral health status in terms of number of teeth, mouth opening, periodontal status etc . These dimensions are needed for characterization of oral health.5 the essential part of achieving and maintaining readiness to deploy and fight is oral health . The dental health of the central reserve police personnel has a significant impact on their operations since the untreated oral conditions can result in increased rates of disease and non - battle injury for deployed police personnels.6 central reserve police force (crpf) is the largest of india s central armed police forces . It functions under the aegis of ministry of home affairs, government of india.7 no data on the oral health status among the central police personnel s of mathura city is available . Hence, this study was undertaken to assess oral health status and treatment need of central reserve police personnels of mathura city . The police personnels who were absent on the day of examination were examined on the next scheduled date . The average time for the examination and data collection were 10 min for each subject . The daily and weekly schedules were prepared and in a single day maximum of 20 - 25 subjects were examined . The examinations were carried out on all the week days excluding the second saturdays, sundays, and holidays . Clinical examinations were done using who standard criteria as mentioned in who oral health proforma, 2013 to assess the oral health status . The clinical examination was carried out by a trained examiner who was initially trained and supervised by an expert clinician in the department of public health dentistry . The ethical clearance was approved from the ethical committee of kd dental college and hospital, mathura prior to the start of the study and consent was obtained from crpf authorities . The collected data were entered in the microsoft excel sheet and analyzed using the spss, version 22.0 statistical package (ibm corporation). A t - test was used to compare the distribution of oral health status variables, according to age groups . Four hundred and seventy - five central reserve police personnels were selected from mathura city out of which 473 (99.6%) were males and 2 (0.4%) were females . Total 62.3% subjects belonged to 21 - 35 years age group (table 1). Among the 475 (100%) central reserve police personnels, 382 (80.4%) subjects had healthy teeth (table 2). Mean dentition status among study subjects was found to be 0.66 2.08 (table 3). Pockets were present in 28 (5.3%) study subjects, enamel fluorosis was present in 116 (24.4%) study subjects, and none of the study subjects had oral mucosal lesions . Total 9 (1.9%), and 4 (0.8%) study subjects needed partial denture in upper and lower arch, respectively . In the subjects above 35 years of age mean bleeding, pocket, loss of attachment, and enamel fluorosis were 0.24, 0.06, 0.17, and 0.34, respectively (table 4). Among the 475 (100%) central reserve police personnels, no treatment was needed for 24 (5.1%) subjects while 56 (11.8%) needed preventive or routine treatment, prompt treatment including scaling was needed for 382 (80.4%) subjects and, immediate treatment (urgent treatment needed due to pain or infection of dental and or oral origin) was needed for 13 (2.7%) subjects (table 5). No data on the oral health status among the central police personnel s of mathura city is available . Hence, this study was undertaken to assess oral health status and treatment need of police personnels in mathura city . In our study, a total of 475 (100%) central reserve police personnels were selected, out of which 297 (62.3%) were of the age group below 35 years, and 178 (37.7%) were of the age group above 35 years . Mean age of study subjects was 35.3 years, in the study conducted by spalj et al.8 in our study, mean dentition status among study subjects was found to be 0.66 2.08 . However, mean dentition status was 1.02 0.94, 2.98 3.12, and 2.69, in the studies conducted by sohi et al.,9 bhardwaj et al.,10 naveen and reddy,11 respectively . Among the 475 (100%) central reserve police personnels, bleeding was present in 122 (25.7%) study subjects . This was similar to study conducted by ahuja and darekar12 in which 74% subjects were healthy and 25% subjects presented with bleeding on probing . No treatment was needed for 24 (5.1%) subjects while 56 (11.8%) needed preventive or routine treatment, prompt treatment including scaling was needed for 382 (80.4%) subjects, and immediate treatment (urgent treatment needed due to pain or infection of dental and or oral origin) was needed in 13 (2.7%) subjects in the present study . The sample of police personnel provides a unique opportunity to study a large population from diverse geographic backgrounds . The adverse outlook of this occupation makes it necessary for the government to either build health care, clinics equipped with efficient manpower, especially for the police personnel or to regularly organize treatment camps . The study concludes that prompt treatment is required in large section of this study population.
Septic arthritis of the temporomandibular joint (tmj) is an uncommon condition in children and it is rarely reported in the literature . The infection carries the risk of severe long term morbidity and mortality if effective diagnosis and treatment are not instituted . The clinical presenting features in neonates and young infants are non - specific requiring a high degree of clinician suspicion to elucidate the diagnosis . The most common clinical clues at presentation include trismus, pain, tenderness, swelling and erythema . The signs and symptoms may be similar to other conditions including cellulitis, rheumatological disease, dental abscess, pharyngitis, retropharyngeal abscess, peritonsillar abscess, acute otitis media, mastoiditis, parotitis, submandibular sialadenitis, and lymphadenopathy . We report an eleven - month - old infant whose clinical presentation of fever, mild facial swelling and trismus lead to the elucidation of right sided tmj septic arthritis via magnetic resonance imaging (mri). An eleven - month - old boy presented with 24 h history of right facial swelling and 48 h history of persistent fever, drooling and poor oral solid intake . He had had one week history of upper respiratory tract infection including coughing, rhinorrhoea and intermittent fever and had been treated with antipyretic and oral fluid . He was not on regular medications and was up - to - date with his immunization . Physical examination revealed facial asymmetry due to mild - moderate pre - auricular swelling and tenderness including right zygomatic arch region and infratemporal fossa . He had a fever of 39.1c, a white cell count of 24.0x10/l, neutrophils 74% and a c - reactive protein concentration of 145 mg / l . He was admitted to the paediatric unit of the hospital and was commenced on intravenous antibiotics (ceftriaxone, gentamicin and metronidazole). Mri on day 3 of admission revealed an inflammatory and apparently infective process in the right temporal region and infratemporal fossa (figures 1 and 2). The primary focus of the inflammation / infection was at the right temporomandibular joint with effusion and subtle marrow change . There was evidence of the thickening of the meninges through the right squamous temporal fossa and mild oedema within the right mastoid air cells and the other surrounding soft tissues . The radiological images suggested septic arthritis of right tmj, supported by the clinical findings of guarding to contact, and apparent limitation of mandibular movement . Infective disease specialist, otorhinolaryngologist and maxillofacial surgeon were consulted and the recommendation was to continue conservative management with intravenous antibiotic treatment, which was switched to single agent of timentin (ticarcillin and clavulanate) from day 5 of admission . A central venous catheter was inserted to facilitate the administration of antibiotic . Blood culture collected before the commencement of antibiotic remained negative after five days . His oral solid intake progressively returned to normal, after a week of nasogastric feeding, with no evidence of impairment of jaw movement . An ultrasound study of his right face and neck on day 8 of admission reviewed no focal fluid collection in the region . C - reactive protein returned to normal on day 10 of admission . In view of the unusual nature of severe infection of this young patient, a repeated mri on day 13 of admission showed improvement in the extent of inflammatory response around the right tmj with small persistent joint effusion . The overall shape of the joint was preserved although the size appeared slightly smaller (figures 1 and 2). The patient was discharged home on day 16 of admission and continued on daily intravenous ceftriaxone for one more week as outpatient, followed by a further three months of oral cephalexin treatment . The patient received three months of oral antibiotic treatment and showed no signs of tmj dysfunction or deformity . Twelve months after the acute event, our patient continued to demonstrate normal oromotor function . Septic arthritis of the tmj is an uncommon condition in children and it is rarely reported in the literature . Investigation of children with febrile illness with facial swelling should consider tmj infection as potential diagnosis . The signs and symptoms may be similar to other conditions including cellulitis, rheumatological disease, dental abscess, pharyngitis, retropharyngeal abscess, peritonsillar abscess, acute otitis media, mastoiditis, parotitis, submandibular sialadenitis, and lymphadenopathy . A retrospective review shows septic arthritis of the tmj is most often found in male adult with an average age of 36 years . Patient with this condition may not have the classic presentation of septic arthritis, i.e. Febrile illness with unilateral swollen and tender tmj . Risk factors for septic arthritis of the tmj include head and neck infection, systemic and autoimmune disease, iatrogenic causes, burn wounds, blunt trauma and sexual contact . Staphylococcus aureus and staphylococcus saprophyticus, streptococcus species, neisseria and haemophilus influenzae are the most commonly isolated pathogen . The causative organism may infect the joint by hematogenous spread, from a contiguous infection, by penetration injury of the joint space or following a blunt trauma of the joint . Severe complications may arise from septic arthritis of the tmj including intracranial abscess, joint destruction, ankylosis and impaired growth of the condyle . A causative organism was not identified in our patient, we hypothesis that the preceding upper respiratory tract infection induced hematogenous spread of the offending organism . Surgical drainage of the joint was avoided in view of surgical induced joint ankylosis, and the patient was making an excellent clinical recovery . Laboratory investigations include neutrophilia and elevated c - reactive protein concentration is supportive for the diagnosis . Mri can demonstrate the joint effusion and allows the evaluation of the joint surfaces and adjacent soft tissues during the acute infection . The computed tomography is useful in the chronic stage of infection to demonstrate the bony changes related to cortical breakdown, osteomyelitis, and ankylosis of the tmj . With such an unusual infection it is always prudent to exclude a primary immune deficiency especially x - linked chronic granulomatous disease . Intravenous broad - spectrum antibiotics should be commenced empirically once the septic arthritis is suspected as delayed treatment could result in severe complications . Even a small detriment to joint growth has profound and long - term effects on mandibular function and facial appearance . The antibiotic treatment regime can be modified if the culture and the sensitivity results are available . Aggressive intravenous antibiotic therapy, and possibly surgical drainage of the tmj, are paramount in preventing both acute and chronic complications of the disease process . Surgical drainage of the infected tmj, such as needle aspiration, arthroscopy and arthrotomy, is indicated when abscess formation is developed, bony involvement is suspected, or poor response to antimicrobial treatment alone . The use of intra - articular antimicrobial irrigation is controversial and there is a concomitant risk of introducing new microbe into the infected joint . During the acute infection, these patients should be followed up to monitor the development of complication and the growth of the mandible . In conclusion, septic arthritis of the tmj is an uncommon condition in children, but one is a differential diagnosis for the child with facial swelling especially with concomitant trismus.
Esophageal cancer is one of the most common digestive malignancies with the 5-year relative survival rate less than 20%, ranking the fourth leading cause of cancer death among both men and women in china . Like most solid tumors, pathological tumor node metastasis (tnm) stage is still a main prognostic indicator of esophageal cancer patient survival . However, the molecular heterogeneity and complexity of esophageal cancer make clinical outcomes difficult to predict and even patients within the same stage present wide variations in survival . Therefore, it is urgent to develop novel biomarkers or models for survival risk prediction in esophageal cancer which would provide patients more effective therapies . Long non - coding rnas (lncrnas) are mostly defined as rna transcripts exceeding 200 nucleotides (nt) in length apparently without protein coding capacity . Accumulating evidence indicates that lncrnas play critical roles in a spectrum of biological processes via transcriptional, post - transcriptional and epigenetic mechanisms . Dysregulation of lncrnas has been observed in various cancers, including breast cancer, colorectal cancer, lung cancer, prostate cancer as well as esophageal cancer [610]. It has been reported that dysregulated lncrnas are associated with cancer pathogenesis and function as oncogenic or tumor suppressive regulators in cancer development . They have been shown to affect cell proliferation, migration, and invasion through regulating the expression of genes involved in various tumorigenetic pathways . One of the recent techniques used for transcriptome analyses is rna sequencing (rna - seq), a next - generation sequencing technique with high - sensitivity, high - throughput and the ability of detecting novel exons, splice sites, and transcripts . However, rna - seq based work focused on the prognostic power of lncrna signatures for survival risk of esophageal cancer patients is quite rare . In this study, we identified for the first time a rna - seq based lncrna signature as a predictor of survival risk of esophageal cancer patients using a cohort of more than 100 cases from the cancer genome atlas (tcga) database . Cox regression analysis and risk score model method were utilized to develop an 8-lncrna signature which could distinguish patients with good and poor survival . A higher area under curve (auc) of the receiver operating characteristic (roc) curve confirmed good sensitivity and specificity of the prognostic model while multivariate cox regression analysis and stratified analysis indicated the independence of predictive capacity of the 8-lncrna prognostic signature from other clinicopathological factors . Furthermore, our functional enrichment analysis suggested that the eight predictive lncrnas were probably involved in the progression of esophageal cancer through exerting their roles in esophageal cancer related biological processes and pathways, such as regulation of glucose metabolic process, positive regulation of mapk cascade, and amino acid and lipids metabolism . The preprocessed level 3 rna - seq data and corresponding clinical information of esophageal cancer patients were collected from the cancer genome atlas (tcga) database (http://cancergenome.nih.gov/) (as of september, 2016). The patients meeting the following criteria were included in the study: (1) patients with complete information of lncrna expression profiles and clinical characteristics (including age, gender, race, stage, survival status and survival time) (2) the overall survival time was more than one month . According to the inclusion criteria, a total of 122 esophageal cancer patients were enrolled in the study (table 1). The gene expression profiling data of the 122 esophageal cancer samples and 11 normal samples were downloaded from the tcga database . The package of edger in r language was employed to identify the differentially expressed lncrnas between esophageal cancer and normal tissues with the \|log2fc\|> 2 and fdr <0.01 set as the threshold . Then the unsupervised hierarchical clustering was performed based on the expression of these altered lncrnas by the pheatmap package in r (https://cran.r-project.org/web/packages/pheatmap/index.html, version 1.0.8). The association between the expression of differentially expressed lncrnas and patient overall survival was evaluated by univariate cox proportional hazards regression analysis using the survival r package . Only those lncrnas with p - value <0.05 were considered as candidate variables and entered into a stepwise multivariate cox regression analysis tested by aic (akaike information criterion, assessing the goodness of fit of a statistical model) to identify the predictive model with the best explanatory and informative efficacy . Then, a lncrna - related prognostic model was established to evaluate each patient s survival risk as follows: where k is the number of prognostic lncrnas, ci represents the coefficient of the ith lncrna in the multivariate cox regression analysis, vi is the expression value of the ith lncrna . The lncrnas with ci> 0 were defined as high - risk signatures while those with ci <0 were defined as protective lncrnas . According to the predictive lncrna signature model the patients were then classified into high - risk or low - risk group using the median risk score as the cutoff value . Overall survival curves were generated using the kaplan - meier method, and two - sided log - rank tests were employed to compare the differences in overall survival time between the high - risk and low - risk patient groups . The sensitivity and specificity of the lncrna prognostic model to predict clinical outcome were evaluated by calculating the area under curve (auc) of the receiver operating characteristic (roc) curve in the r package of survival roc . To determine whether predictive capacity of the lncrna signature was independent of other clinical factors (including race, gender, stage, and age) of esophageal cancer patients, multivariate cox regression analysis was carried out using overall survival as the dependent variable and lncrna signature and other conventional clinical factors as independent variables . For clinical features with p - value <0.01 in cox regression analysis, stratification analysis was further performed to determine whether the lncrna signature exhibit prognostic value within the same clinical factor . To identify potential biological processes and pathways which the predictive lncrnas were involved in, first, the pearson correlation coefficients between the expression profiles of the eight prognostic lncrnas and protein - coding genes (pcgs) were calculated to determine the co - expression relationships of the lncrnas and pcgs . The pcgs with \|pearson correlation coefficient\|> 0.40 were considered to be lncrnas - related pcgs . Gene ontology (go) biological process (bp) and kyoto encyclopedia of genes and genomes (kegg) pathway enrichment analysis were carried out for those pcgs using the database for annotation, visualization, and integrated discovery (david, https://david.ncifcrf.gov/, version 6.8). The p - value <0.05 was set as the cutoff criterion for both go and kegg functional analysis . The preprocessed level 3 rna - seq data and corresponding clinical information of esophageal cancer patients were collected from the cancer genome atlas (tcga) database (http://cancergenome.nih.gov/) (as of september, 2016). The patients meeting the following criteria were included in the study: (1) patients with complete information of lncrna expression profiles and clinical characteristics (including age, gender, race, stage, survival status and survival time) (2) the overall survival time was more than one month . According to the inclusion criteria, a total of 122 esophageal cancer patients were enrolled in the study (table 1). The gene expression profiling data of the 122 esophageal cancer samples and 11 normal samples were downloaded from the tcga database . The package of edger in r language was employed to identify the differentially expressed lncrnas between esophageal cancer and normal tissues with the \|log2fc\|> 2 and fdr <0.01 set as the threshold . Then the unsupervised hierarchical clustering was performed based on the expression of these altered lncrnas by the pheatmap package in r (https://cran.r-project.org/web/packages/pheatmap/index.html, version 1.0.8). The association between the expression of differentially expressed lncrnas and patient overall survival was evaluated by univariate cox proportional hazards regression analysis using the survival r package . Only those lncrnas with p - value <0.05 were considered as candidate variables and entered into a stepwise multivariate cox regression analysis tested by aic (akaike information criterion, assessing the goodness of fit of a statistical model) to identify the predictive model with the best explanatory and informative efficacy . Then, a lncrna - related prognostic model was established to evaluate each patient s survival risk as follows: where k is the number of prognostic lncrnas, ci represents the coefficient of the ith lncrna in the multivariate cox regression analysis, vi is the expression value of the ith lncrna . The lncrnas with ci> 0 were defined as high - risk signatures while those with ci <0 were defined as protective lncrnas . According to the predictive lncrna signature model, the risk score of each of the 122 patients was calculated . The patients were then classified into high - risk or low - risk group using the median risk score as the cutoff value . Overall survival curves were generated using the kaplan - meier method, and two - sided log - rank tests were employed to compare the differences in overall survival time between the high - risk and low - risk patient groups . The sensitivity and specificity of the lncrna prognostic model to predict clinical outcome were evaluated by calculating the area under curve (auc) of the receiver operating characteristic (roc) curve in the r package of survival roc . To determine whether predictive capacity of the lncrna signature was independent of other clinical factors (including race, gender, stage, and age) of esophageal cancer patients, multivariate cox regression analysis was carried out using overall survival as the dependent variable and lncrna signature and other conventional clinical factors as independent variables . For clinical features with p - value <0.01 in cox regression analysis, stratification analysis was further performed to determine whether the lncrna signature exhibit prognostic value within the same clinical factor . To identify potential biological processes and pathways which the predictive lncrnas were involved in, functional enrichment analysis was performed . First, the pearson correlation coefficients between the expression profiles of the eight prognostic lncrnas and protein - coding genes (pcgs) were calculated to determine the co - expression relationships of the lncrnas and pcgs . The pcgs with \|pearson correlation coefficient\|> 0.40 were considered to be lncrnas - related pcgs . Gene ontology (go) biological process (bp) and kyoto encyclopedia of genes and genomes (kegg) pathway enrichment analysis were carried out for those pcgs using the database for annotation, visualization, and integrated discovery (david, https://david.ncifcrf.gov/, version 6.8). The p - value <0.05 was set as the cutoff criterion for both go and kegg functional analysis . According to the cutoff criteria, a total of 265 differentially expressed (including 112 upregulated and 153 downregulated) lncrnas were identified between esophageal cancer tissues and normal tissues . The results of unsupervised hierarchical cluster analysis in figure 1 showed that the esophageal cancer samples could be clearly distinguished from the normal controls with the expression of differentially expressed lncrnas . To identify prognosis - related lncrnas, we first used univariate cox regression analysis to evaluate the associations between the expression level of each of the differentially expressed lncrnas and patients overall survival, and found that 13 lncrnas were significantly related to overall survival (p<0.05). Then a stepwise multivariate cox regression analysis was performed and eight lncrnas therein (as shown in table 2) were finally screened out to establish a predictive model . As previously described, the predictive model was defined as the linear combination of the expression levels of the eight lncrnas weighted by their relative coefficient in multivariate cox regression as follows: survival risk score = (0.0398 expression value of gs1 - 600g8.5) + (0.9990 expression value of linc00365) + (0.6216 expression value of ctd-2357a8.3) + (13.6225 expression value of rp11 - 705o24.1) + (1.7105 expression value of linc01554) + (0.8297 expression value of rp1 - 90j4.1) + (6.2336 expression value of rp11 - 327j17.1) + (1.2226 expression value of linc00176). Among these, gs1 - 600g8.5, linc00365, ctd-2357a8.3, rp11 - 705o24.1, linc01554, and rp1 - 90j4.1 showed positive coefficients in cox regression analysis, indicating high - risk signatures for these six lncrnas since their high expression signified a shorter overall survival of patients . For the remaining two lncrnas, we observed negative coefficients in cox regression analysis, implying that these lncrnas could be regarded as protective lncrnas since patients with higher expression levels of these lncrnas tended to have longer overall survival compared with those with lower expression levels of these lncrnas . For each of the 122 patients in our study, we were able to calculate an 8-lncrna expression - based survival risk score (referred to as srs) and assigned them into a high - risk group or a low - risk group using the median risk score of 1.058 as the cutoff point . As a result, 61 patients were classified into the high - risk group since their srss were greater than the cutoff value, whereas the other 61 patients were assigned to the low - risk group with their srss less than the cutoff point (figure 2a). The kaplan - meier overall survival curves of the two groups based on the eight lncrnas were notably different (log - rank p=1.72 e-05 <0.001), showing overall survival in 9.89% and 50.5% at five years for patients with high - risk and low - risk srs, respectively (figure 2b). The prognostic power of the 8-lncrna signature was evaluated by calculating the auc of roc curve . Higher auc indicates better model performance and auc more than 0.80 is considered good performance . In our study, the roc curve analysis achieved auc of 0.845, showing good sensitivity and specificity of the 8-lncrna signature model in predicting esophageal cancer patient survival risk (figure 2c). Multivariate cox regression analysis demonstrated that the 8-lncrna signature risk score maintained an independent predictive ability from other clinical factors (hr=5.951, 95% ci 2.57713.741, p=2.95 e-05, shown in table 3). Meanwhile, we also found that tnm stage was an independent predictor for overall survival of esophageal cancer patients . Therefore, stratification analysis was further carried out to examine whether the 8-lncrna signature could provide predict value for patients within the same tnm stage . Because the sample sizes in stage i and iv were too small to draw any reliable conclusions (n=14 in stage i and n=6 in stage iv), stratification analysis was performed only in stage ii and iii patients . Log - rank test for patients in stage ii demonstrated that the 8-lncrna signature could distinguish patients with significantly different survival (p=0.015, figure 3a). Similar predictive value of the 8-lncrna signature was observed for stage iii patients (p=0.003, figure 3b). Altogether, these results indicated that the prognostic capability of the 8-lncrna signature is independent of conventional clinical factors for survival prediction of esophageal cancer patients . We performed go and kegg functional enrichment analysis for the pcgs co - expressed with the lncrnas in the predictive signature to reveal the potential functions of the eight prognostic lncrnas . The results showed that co - expressed pcgs were enriched in 73 go bp terms, which mainly clustered in regulation of diverse biological processes (such as go: 0010906~regulation of glucose metabolic process, go: 0010628~positive regulation of gene expression, go: 0043410~positive regulation of mapk cascade), transport of various substances (including go: 0071805~potassium ion transmembrane transport, go: 0035879~plasma membrane lactate transport, go: 0098719~sodium ion import across plasma membrane) and response to different stimulants (such as go: 0042594~response to starvation, go: 0043627~response to estrogen, go: 0042493~response to drug) (the top 30 go bp terms were shown in table 4). Fourteen kegg pathways were enriched which mainly focused on digestive functions (including hsa04971: gastric acid secretion, hsa04974: protein digestion and absorption, hsa04972: pancreatic secretion) and basic substance metabolism (such as hsa01200: carbon metabolism, hsa00280: valine, leucine and isoleucine degradation, hsa01100: metabolic pathways and hsa00071: fatty acid degradation) (figure 4). According to the cutoff criteria, a total of 265 differentially expressed (including 112 upregulated and 153 downregulated) lncrnas were identified between esophageal cancer tissues and normal tissues . The results of unsupervised hierarchical cluster analysis in figure 1 showed that the esophageal cancer samples could be clearly distinguished from the normal controls with the expression of differentially expressed lncrnas . To identify prognosis - related lncrnas, we first used univariate cox regression analysis to evaluate the associations between the expression level of each of the differentially expressed lncrnas and patients overall survival, and found that 13 lncrnas were significantly related to overall survival (p<0.05). Then a stepwise multivariate cox regression analysis was performed and eight lncrnas therein (as shown in table 2) were finally screened out to establish a predictive model . As previously described, the predictive model was defined as the linear combination of the expression levels of the eight lncrnas weighted by their relative coefficient in multivariate cox regression as follows: survival risk score = (0.0398 expression value of gs1 - 600g8.5) + (0.9990 expression value of linc00365) + (0.6216 expression value of ctd-2357a8.3) + (13.6225 expression value of rp11 - 705o24.1) + (1.7105 expression value of linc01554) + (0.8297 expression value of rp1 - 90j4.1) + (6.2336 expression value of rp11 - 327j17.1) + (1.2226 expression value of linc00176). Among these, gs1 - 600g8.5, linc00365, ctd-2357a8.3, rp11 - 705o24.1, linc01554, and rp1 - 90j4.1 showed positive coefficients in cox regression analysis, indicating high - risk signatures for these six lncrnas since their high expression signified a shorter overall survival of patients . For the remaining two lncrnas, we observed negative coefficients in cox regression analysis, implying that these lncrnas could be regarded as protective lncrnas since patients with higher expression levels of these lncrnas tended to have longer overall survival compared with those with lower expression levels of these lncrnas . For each of the 122 patients in our study, we were able to calculate an 8-lncrna expression - based survival risk score (referred to as srs) and assigned them into a high - risk group or a low - risk group using the median risk score of 1.058 as the cutoff point . As a result, 61 patients were classified into the high - risk group since their srss were greater than the cutoff value, whereas the other 61 patients were assigned to the low - risk group with their srss less than the cutoff point (figure 2a). The kaplan - meier overall survival curves of the two groups based on the eight lncrnas were notably different (log - rank p=1.72 e-05 <0.001), showing overall survival in 9.89% and 50.5% at five years for patients with high - risk and low - risk srs, respectively (figure 2b). The prognostic power of the 8-lncrna signature was evaluated by calculating the auc of roc curve . Higher auc indicates better model performance and auc more than 0.80 is considered good performance . In our study, the roc curve analysis achieved auc of 0.845, showing good sensitivity and specificity of the 8-lncrna signature model in predicting esophageal cancer patient survival risk (figure 2c). Multivariate cox regression analysis demonstrated that the 8-lncrna signature risk score maintained an independent predictive ability from other clinical factors (hr=5.951, 95% ci 2.57713.741, p=2.95 e-05, shown in table 3). Meanwhile, we also found that tnm stage was an independent predictor for overall survival of esophageal cancer patients . Therefore, stratification analysis was further carried out to examine whether the 8-lncrna signature could provide predict value for patients within the same tnm stage . Because the sample sizes in stage i and iv were too small to draw any reliable conclusions (n=14 in stage i and n=6 in stage iv), stratification analysis was performed only in stage ii and iii patients . Log - rank test for patients in stage ii demonstrated that the 8-lncrna signature could distinguish patients with significantly different survival (p=0.015, figure 3a). Similar predictive value of the 8-lncrna signature was observed for stage iii patients (p=0.003, figure 3b). Altogether, these results indicated that the prognostic capability of the 8-lncrna signature is independent of conventional clinical factors for survival prediction of esophageal cancer patients . We performed go and kegg functional enrichment analysis for the pcgs co - expressed with the lncrnas in the predictive signature to reveal the potential functions of the eight prognostic lncrnas . The results showed that co - expressed pcgs were enriched in 73 go bp terms, which mainly clustered in regulation of diverse biological processes (such as go: 0010906~regulation of glucose metabolic process, go: 0010628~positive regulation of gene expression, go: 0043410~positive regulation of mapk cascade), transport of various substances (including go: 0071805~potassium ion transmembrane transport, go: 0035879~plasma membrane lactate transport, go: 0098719~sodium ion import across plasma membrane) and response to different stimulants (such as go: 0042594~response to starvation, go: 0043627~response to estrogen, go: 0042493~response to drug) (the top 30 go bp terms were shown in table 4). Fourteen kegg pathways were enriched which mainly focused on digestive functions (including hsa04971: gastric acid secretion, hsa04974: protein digestion and absorption, hsa04972: pancreatic secretion) and basic substance metabolism (such as hsa01200: carbon metabolism, hsa00280: valine, leucine and isoleucine degradation, hsa01100: metabolic pathways and hsa00071: fatty acid degradation) (figure 4). Esophageal cancer is a global health threat with high morbidity and mortality . Owing to the heterogeneity, conventional prognostic systems such as tnm staging system often show insufficient prediction for risk stratification and clinical outcome estimations . Therefore, considerable efforts have been made in recent decades to develop novel prognostic signatures to promote the prediction of esophageal cancer patient survival [2022]. As a new focused class of ncrnas, lncrnas were indicated to participate in multiple biological processes including x chromosome inactivation, genomic imprinting, and tumor related alterations . Accumulating reports have demonstrated the dysregulation of lncrnas and their potential as biomarkers in various cancers [2426]. Li et al . Have established a 3-lncrna signature associated with the patient survival in esophageal squamous cell carcinoma using microarray analysis . However, microarray technology has some undesired shortcomings, such as the bias due to the probe selection and the limitation of identifying only known transcripts . Compared with microarrays, rna - seq technique has been developed as an emerging sequencing technology with advantages reducing these defects and standing out especially with the ability of finding novel transcripts . Nevertheless, research investigating the impact of lncrnas in esophageal cancer patient survival, which were based on rna - seq technology, are quite deficient . In this study, we developed an 8-lncrna signature which was able to predict the clinical outcome of esophageal cancer . To the best of our knowledge, this is the first lncrna - related predictive model based on rna - seq technology using a cohort of more than 100 cases in esophageal cancer . The differentially expressed lncrnas were first screened out between esophageal cancer and normal tissues with the data downloaded from tcga database . Then the expression profiles of these lncrnas of 122 esophageal cancer patients were analyzed by univariate and stepwise multiple cox proportional hazards regression analysis . Then eight lncrnas were finally identified to establish a predictive model based on the linear combination of these lncrnas . A distinctive separation was observed in survival curves between patient groups with high - risk and low - risk scores using the predictive model . And roc analysis achieved an auc of 0.845 which demonstrated high sensitivity and specificity of the lncrna signature model . When taking other clinical factors together, multivariate cox regression analysis revealed that the 8-lncrna signature was independent of these conventional clinicopathological factors including race, gender, age, and tumor stage . Further stratification analysis indicated favorable discrimination of the 8-lncrna signature in predicting different survival of patients in the same tnm stage . This might provide more references for clinical doctors to select better individualized and effective treatment for patients with different survival risk . The carcinogenesis of esophageal cancer is a multi - step process hallmarked by a series of genetic alterations . Although growing attention has begun to focus on the study of lncrnas, the functions of most lncrnas are still unknown . Computational annotation of lncrna functions through their co - expressed mrnas has been proven to be effective . In the present study, we performed go and kegg enrichment analysis for the co - expressed mrnas of the eight lncrnas to explore the functions of the predictive lncrnas . The results showed that the prognostic lncrnas were involved in significant biological processes such as regulation of glucose metabolic process, positive regulation of gene expression, positive regulation of mapk cascade and enriched in kegg pathways including gastric acid secretion, amino acid metabolism (valine, leucine and isoleucine degradation), and lipids metabolism (fatty acid degradation). Studies have identified aberrant glucose metabolic processes in esophageal cancer, including high plasma and urine glucose levels, excessive glucose uptake and accumulation and alterations of glucose metabolism associated genes . Higher gastric acid secretion has been observed in gastroesophageal junction adenocarcinoma, including barrett esophageal cancer compared to normal subjects . Amino acid and lipids metabolism disorders, such as increasing leucine level and highly activated fatty acids metabolism, were also found to be responsible for the development of esophageal cancer . Therefore, it is plausible to infer that the eight prognostic lncrnas participate in the progression of esophageal cancer through interacting with pcgs in these esophageal cancer - related biological pathways . This study identified a rna - seq based 8-lncrna signature which could predict the survival risk of esophageal cancer patients . The signature displayed independent prognostic capacity of conventional clinicopathological factors and could robustly predict survival outcomes of esophageal cancer patients within the same tnm stage . It could be used to identify patients with high - risk scores who will benefit from more effective and individualized therapy . It could not only serve as a novel potential biomarker for esophageal cancer patient survival risk stratification, but also provide us a better understanding of molecular mechanisms involved in the development of esophageal cancer . However, further clinical studies validating the predictive efficacy of the signature and experimental research investigating the functions of the prognostic lncrnas need to be conducted.
Angioid streaks (as) are narrow, irregularly radiating lines emanating from the peripapillary area, resulting from pathological dehiscences in bruch's membrane . While they may be idiopathic, they are often associated with systemic conditions, most commonly pseudoxanthoma elasticum (pxe), paget disease, and sickle haemoglobinopathies . While generally asymptomatic, secondary complications may arise from choroidal neovascularisation (cnv), subretinal haemorrhage, and retinal pigment epithelial detachments (ped). Involvement of the macula may result in vision impairment and metamorphopsia [3, 4]. Polypoidal choroidal vasculopathy (pcv) is an exudative chorioretinopathy characterised by grape - like polypoidal subretinal vascular lesions associated with branching vascular networks (bvn). Controversy exists over whether pcv represents a subtype of neovascular age - related macular degeneration or a separate disease entity with different risk factors and outcomes . Typically, recurrent serous exudation and haemorrhage result in detachments of rpe which may cause fibrotic scarring, chorioretinal atrophy, and vision loss . Pcv can be difficult to distinguish from typical type 1 cnv in neovascular age - related macular degeneration, hence it is often underdiagnosed . Indocyanine green angiography (icga) is the gold standard for pcv diagnosis by highlighting typical polyp lesions and bvn within the choroidal vasculature . Previous studies have demonstrated that the anti - vegf agents ranibizumab and aflibercept are well - tolerated and efficacious treatments in the management of pcv . However, their use in patients with pcv associated with as has not been previously documented in the literature . We report the long - term follow - up of 3 clinical cases of pcv secondary to as in which the patients were managed with intravitreal anti - vegf injections with and without photodynamic therapy (pdt). A 52-year - old caucasian man presented with a 2-week history of central distortion in his right eye . He had a family history of advanced pcv secondary to as in his mother (case 2) (fig . Dilated fundus examination of both eyes showed dark brown irregularly radiating linear lines emanating from the peripapillary area including through the macula, corresponding to the characteristic clinical appearance of as (fig . Peds and subretinal haemorrhage adjacent to the area of as in the right macula were noted clinically and on spectral domain optical coherence tomography examination (sd - oct; topcon corporation, tokyo, japan). There were no other fundus features suggestive of pxe such as peau d'orange or comet lesions . There were no other related systemic clinical findings including skin lesions suggestive of any connective tissue disease . The patient was reviewed by a dermatologist and physician who did not find any obvious associated systemic disorders . Fluorescein angiography (fa) demonstrated an occult (type 1) choroidal neovascular membrane, and icga demonstrated grape - like choroidal vascular lesions consistent with a diagnosis of pcv . Intravitreal ranibizumab injections were commenced at 4 weekly intervals according to a treat - and - extend protocol . Following the stability of visual acuity and oct parameters, the interval between injections was increased by 2 weeks to a maximum of 12 weeks . Retreatment criteria and the next treatment interval were based on evidence of disease activity including: loss of visual acuity of more than 5 etdrs letters, presence of new haemorrhage, increased subretinal, intraretinal, or sub - rpe fluid . Subretinal haemorrhage was absent at 5 months; however, a small but persisting ped was observed, and the condition remained stable at 2 years with good vision . However, at 3 years following commencement of treatment, whilst the treatment interval was at 12 weeks, the patient became symptomatic of a paracentral scotoma . Clinical examination revealed a large subretinal haemorrhage, subretinal fluid (srf), and ped in the inferior macula (fig . 1e, f) demonstrated early filling of a grape - like polypoidal structure with leakage in the late phase suggestive of pcv secondary to as . Icga also revealed an associated bvn in both the early and late phase (fig . The patient received a combination of intravitreal ranibizumab with full fluence visudyne pdt, and the injection treatment interval was reduced to 4 weeks until the lesion stabilised . Following this, further ranibizumab injections were administered according to the indicated treat - and - extend protocol . At 56 months, visual acuity stabilised to 20/20 in the right eye . Figure 2 demonstrates an improved oct after the treatment and a repeat icga showing regressed pcv . In total, 19 ranibizumab injections were administered with a mean interval between consecutive injections of 89 days . Fifty - seven months following the initiation of this treatment, the patient was receiving injections every 6 weeks, then he switched from ranibizumab to intravitreal aflibercept on a similar treat - and - extend protocol according to the same retreatment criteria . Eight injections of aflibercept were subsequently applied during a 27-month follow - up period, with a mean of 85 consecutive days between aflibercept injections . At the final follow - up (84 months), visual acuity was 20/20 and oct revealed an absence of srf; however, a small but persisting ped (fig ., a total number of 27 injections (19 ranibizumab and 8 aflibercept) were given in the right eye with an average of 90 days between injections (both types). In the first year, the injection interval was 31 days on average with ranibizumab, and 62 days in the 4th year with aflibercept . An 84-year - old caucasian woman (mother of case 1) presented with bilateral, long - standing, poor vision since the age of 47 years . She had a history of controlled hypertension, atrial fibrillation, and hydrocephalus; however, no other known systemic disease associated with as . Her visual acuity was 20/400 in the right and 20/200 in the left eye, and she had bilateral, hypertrophic, disciform, macular scars and evidence of as . Fa and icga were performed and demonstrated bilateral pcv with polyp lesions and bvn (fig . 2). Due to her poor vision and established fibrotic macular scars, treatment was conservative . A 72-year - old caucasian woman with hypertension and hypercholesterolemia presented with a central scotoma in her left eye . Her best corrected visual acuity was 20/50 in the right eye and 20/50 in the left eye . Fundoscopy showed irregular lines with a brownish appearance emanating from the optic disc and spreading radially in various directions in both eyes, corresponding to the clinical appearance of as . There was no evidence of peau d'orange or other peripheral retinal abnormalities . In the left macula, oct, fa, and icga examination demonstrated srf, intraretinal fluid, and a type 1 choroidal neovascular membrane without any evidence of pcv at this stage . A diagnosis of cnv secondary to as in the left eye was made, and intravitreal ranibizumab injections were commenced according to a treat - and - inject protocol utilizing oct and visual acuity criteria as outlined above for the first 2 years . A total of 14 ranibizumab injections were given from baseline to 24 months, then no injections were given until 46 months . She was reviewed by a dermatologist and physician who found no other systemic illnesses associated with as . At 46 months follow - up of the left eye, the patient experienced new symptoms of distortion in the previously unaffected right eye . Clinical examination revealed a discreet orange - red nodule in the nasal right macula adjacent to the as (fig . Oct revealed typical features of pcv, including a ped with an underlying polypoidal lesion (fig . 3 g, h) demonstrated a polypoidal lesion with terminal aneurysmal dilatations at the edge of the as near the macula with an associated bvn . The patient was initiated on aflibercept injections in the right eye according to a treat - and - extend protocol . At this point, visual acuity was 20/40 in the right eye and 20/32 in the left eye . At 2 months follow - up, 3d) at 7 months showed an absence of srf and ped, with complete regression of the polypoidal lesion . A total number of 15 injections were given in the right eye over 33 months with an average of 68 days between injections . In the first year, injections were given on average every 46 days, and in the fourth year, on average every 66 days, demonstrating the ability to extend treatment intervals . At 53 months follow - up, the patient experienced worsening of vision in the left eye, 29 months after the last ranibizumab injection . 3o, p) revealed early filling of a grape - like structure suggestive of pcv with leakage in the late phase and an associated small bvn in both the early and late phase . Intravitreal aflibercept injections were commenced in the left eye according to a treat - and - extend protocol based on the same visual acuity and oct retreatment criteria detailed above . At 2 months after initiating aflibercept in the left eye, srf was absent (fig . 3k); however, the ped was still present . At the final follow - up, 26 months after aflibercept initiation (78 months following ranibizumab initiation), the ped was persisting; however, it had improved with the absence of srf and intraretinal fluid (fig . Final best corrected visual acuity was 20/20 in the right eye and 20/32 in the left eye . Subsequent icga demonstrated involution of the pcv lesions in the left eye . A total number of 18 injections were given in the left eye with an average of 65 days between injections . In the first year, injections were given on average every 33 days, and in the fourth year, on average every 57 days . A 52-year - old caucasian man presented with a 2-week history of central distortion in his right eye . He had a family history of advanced pcv secondary to as in his mother (case 2) (fig . Dilated fundus examination of both eyes showed dark brown irregularly radiating linear lines emanating from the peripapillary area including through the macula, corresponding to the characteristic clinical appearance of as (fig . Peds and subretinal haemorrhage adjacent to the area of as in the right macula were noted clinically and on spectral domain optical coherence tomography examination (sd - oct; topcon corporation, tokyo, japan). There were no other fundus features suggestive of pxe such as peau d'orange or comet lesions . There were no other related systemic clinical findings including skin lesions suggestive of any connective tissue disease . The patient was reviewed by a dermatologist and physician who did not find any obvious associated systemic disorders . Fluorescein angiography (fa) demonstrated an occult (type 1) choroidal neovascular membrane, and icga demonstrated grape - like choroidal vascular lesions consistent with a diagnosis of pcv . Intravitreal ranibizumab injections were commenced at 4 weekly intervals according to a treat - and - extend protocol . Following the stability of visual acuity and oct parameters, the interval between injections was increased by 2 weeks to a maximum of 12 weeks . Retreatment criteria and the next treatment interval were based on evidence of disease activity including: loss of visual acuity of more than 5 etdrs letters, presence of new haemorrhage, increased subretinal, intraretinal, or sub - rpe fluid . Subretinal haemorrhage was absent at 5 months; however, a small but persisting ped was observed, and the condition remained stable at 2 years with good vision . However, at 3 years following commencement of treatment, whilst the treatment interval was at 12 weeks, the patient became symptomatic of a paracentral scotoma . Clinical examination revealed a large subretinal haemorrhage, subretinal fluid (srf), and ped in the inferior macula (fig . 1e, f) demonstrated early filling of a grape - like polypoidal structure with leakage in the late phase suggestive of pcv secondary to as . Icga also revealed an associated bvn in both the early and late phase (fig . The patient received a combination of intravitreal ranibizumab with full fluence visudyne pdt, and the injection treatment interval was reduced to 4 weeks until the lesion stabilised . Following this, further ranibizumab injections were administered according to the indicated treat - and - extend protocol . At 56 months, visual acuity stabilised to 20/20 in the right eye . Figure 2 demonstrates an improved oct after the treatment and a repeat icga showing regressed pcv . In total, 19 ranibizumab injections were administered with a mean interval between consecutive injections of 89 days . Fifty - seven months following the initiation of this treatment, the patient was receiving injections every 6 weeks, then he switched from ranibizumab to intravitreal aflibercept on a similar treat - and - extend protocol according to the same retreatment criteria . Eight injections of aflibercept were subsequently applied during a 27-month follow - up period, with a mean of 85 consecutive days between aflibercept injections . At the final follow - up (84 months), visual acuity was 20/20 and oct revealed an absence of srf; however, a small but persisting ped (fig ., a total number of 27 injections (19 ranibizumab and 8 aflibercept) were given in the right eye with an average of 90 days between injections (both types). In the first year, the injection interval was 31 days on average with ranibizumab, and 62 days in the 4th year with aflibercept . An 84-year - old caucasian woman (mother of case 1) presented with bilateral, long - standing, poor vision since the age of 47 years . She had a history of controlled hypertension, atrial fibrillation, and hydrocephalus; however, no other known systemic disease associated with as . Her visual acuity was 20/400 in the right and 20/200 in the left eye, and she had bilateral, hypertrophic, disciform, macular scars and evidence of as . Fa and icga were performed and demonstrated bilateral pcv with polyp lesions and bvn (fig . 2). Due to her poor vision and established fibrotic macular scars, treatment was conservative . A 72-year - old caucasian woman with hypertension and hypercholesterolemia presented with a central scotoma in her left eye . Her best corrected visual acuity was 20/50 in the right eye and 20/50 in the left eye . Fundoscopy showed irregular lines with a brownish appearance emanating from the optic disc and spreading radially in various directions in both eyes, corresponding to the clinical appearance of as . There was no evidence of peau d'orange or other peripheral retinal abnormalities . In the left macula, oct, fa, and icga examination demonstrated srf, intraretinal fluid, and a type 1 choroidal neovascular membrane without any evidence of pcv at this stage . A diagnosis of cnv secondary to as in the left eye was made, and intravitreal ranibizumab injections were commenced according to a treat - and - inject protocol utilizing oct and visual acuity criteria as outlined above for the first 2 years . A total of 14 ranibizumab injections were given from baseline to 24 months, then no injections were given until 46 months . She was reviewed by a dermatologist and physician who found no other systemic illnesses associated with as . At 46 months follow - up of the left eye, the patient experienced new symptoms of distortion in the previously unaffected right eye . Clinical examination revealed a discreet orange - red nodule in the nasal right macula adjacent to the as (fig . Oct revealed typical features of pcv, including a ped with an underlying polypoidal lesion (fig . 3 g, h) demonstrated a polypoidal lesion with terminal aneurysmal dilatations at the edge of the as near the macula with an associated bvn . The patient was initiated on aflibercept injections in the right eye according to a treat - and - extend protocol . At this point, visual acuity was 20/40 in the right eye and 20/32 in the left eye . At 2 months follow - up, 3d) at 7 months showed an absence of srf and ped, with complete regression of the polypoidal lesion . A total number of 15 injections were given in the right eye over 33 months with an average of 68 days between injections . In the first year, injections were given on average every 46 days, and in the fourth year, on average every 66 days, demonstrating the ability to extend treatment intervals . At 53 months follow - up, the patient experienced worsening of vision in the left eye, 29 months after the last ranibizumab injection . 3o, p) revealed early filling of a grape - like structure suggestive of pcv with leakage in the late phase and an associated small bvn in both the early and late phase . Intravitreal aflibercept injections were commenced in the left eye according to a treat - and - extend protocol based on the same visual acuity and oct retreatment criteria detailed above . At 2 months after initiating aflibercept in the left eye, srf was absent (fig . 3k); however, the ped was still present . At the final follow - up, 26 months after aflibercept initiation (78 months following ranibizumab initiation), the ped was persisting; however, it had improved with the absence of srf and intraretinal fluid (fig . Final best corrected visual acuity was 20/20 in the right eye and 20/32 in the left eye . Subsequent icga demonstrated involution of the pcv lesions in the left eye . A total number of 18 injections were given in the left eye with an average of 65 days between injections . In the first year, injections were given on average every 33 days, and in the fourth year, on average every 57 days . Whilst cnv secondary to as is commonly reported, pcv secondary to as is often underdiagnosed for several reasons . Pcv is often mistaken for occult (type 1) cnv lesions, and its variable natural course further renders recognition difficult . Often icga, the gold standard for pcv diagnosis, is not performed or not available routinely in clinical practice despite being an important investigative tool . We hope to increase the awareness of pcv secondary to as and thus improve screening rates for these clinically important illnesses . It has previously been shown that in pcv patients who develop ranibizumab tachyphylaxis, switching therapy to aflibercept may maintain or improve visual acuity and regress polypoidal lesions without adverse events . However, the current literature does not document the use of these treatments in cases of pcv secondary to as . Here, we demonstrate that an inject - and - extend protocol with intravitreal aflibercept and ranibizumab is effective against pcv secondary to as . Promisingly, visual acuity improved in all 3 cases and polypoidal lesions were stabilised using both anti - vegf agents . In case 1, the injection frequency in the fourth year with aflibercept was less than in the first year with ranibizumab; however, this may be reflective of the stability of the lesion rather than a difference in the efficacy between the 2 agents . A comparison between aflibercept and ranibizumab for pcv secondary to as is beyond the scope of this report; however, it would represent an interesting study . Our cases were treated for 4 years; however, treatment may continue indefinitely in other patients reflecting the relapsing and remitting nature of pcv . While as is often asymptomatic, the presence of associated cnv or pcv may play a role in the choice of treatment for the patient . A previous study outlined a case of pcv secondary to as in a symptomatic patient with pxe treated with verteporfin pdt combined with triamcinolone and the anti - vegf agent bevacizumab . However, this management strategy was suboptimal, as visual acuity in the affected eye deteriorated from 20/125 to 20/200 . Pdt has been associated with retinal toxicity, scarring, and atrophy, thus caution must be taken in cases with as which suggest an inherently pathologically weak retina due to the structural dehiscences in bruch's membrane . However, it has been argued that a combination therapy using pdt and anti - vegf agents may be effective in the treatment of typical pcv by combining mechanisms of angio - occlusion and anti - angiogenesis . Indeed the multicentre, double - masked everest trial demonstrated a pdt - ranibizumab combination therapy to be more efficacious than an anti - vegf monotherapy . Thus, a pdt combination therapy was used in our case 1 when ranibizumab monotherapy maintenance treatment failed . Here, we describe the first cases reported of familial as with pcv in a mother and son . As are a hereditary condition (omim 607140) prompt recognition of as is important due to its common associations with various, life - threatening, systemic diseases . In particular, the connective tissue disorder pxe may affect the gastrointestinal tract, great vessels, and arteries which may result in sudden death due to haemorrhage and accelerated atherosclerosis . Similarly, the early diagnosis of pcv is critical for optimizing treatment outcomes and prognosis . One natural history study demonstrated that without treatment for over 2 years, pcv patients may experience persistent polypoidal lesions, repeated haemorrhage and leakage, and significant vision loss . In case 2, the definitive diagnosis of pcv occurred following the long - term recurrence of a lesion initially diagnosed as typical cnv secondary to as . Also, pcv is now recognised to be a variant of type 1 cnv lesions, with the bvn and pcv components growing below the rpe within bruch's membrane, often giving rise to the so - called double - layer sign on oct . We have previously described 2 cases of pcv secondary to choroidal nevus, where we hypothesised that possible chronic inflammatory changes may stimulate the growth of pcv . . It is possible that in our current study the dehiscences in bruch's membrane may give rise to a chronic inflammatory response also stimulating the development of pcv . A treat - and - extend protocol with intravitreal aflibercept and ranibizumab with or without pdt for the treatment of pcv secondary to as is safe and effective in the longer term . We recommend the use of icga to assist in the diagnosis of pcv secondary to as, since both conditions are underrecognised . Future studies are required to elucidate the underlying pathogenetic mechanisms in the development of pcv secondary to as as well as optimise treatment for improved patient outcomes.
The regulation of pancreas development from the gut endoderm is a complex process that involves a carefully balanced interplay between several common signaling pathways . Studies in several model organisms indicate conservation of the main mechanisms of pancreas development from lower to higher vertebrates (table 1). Gastrulation in the developing embryo (e7.5 in mice) results in the formation of three germ layers, namely the ectoderm, the endoderm, and the mesoderm . It appears that these germ layers do not further develop in isolation from each other, but signals emanate from one to pattern the other and vice versa (1). Several structures temporally participate in these early tissue interactions including the notochord (2), the cardiac mesoderm, the septum transversum mesenchyme and the lateral plate mesoderm (3), the aortic endothelial cells and the vitelline veins (4), and the developing pancreatic mesenchyme (5). Time scale of pancreas development in mouse and human the major sequences of developmental processes are similar in mouse and human embryonic pancreas . The striking difference resides in the duration of each stage, which can vary by a factor of 5 to 15 . As far as the pancreas is concerned, specific growth and differentiation factors released by adjacent tissues control a set of transcription factors expression, resulting in the patterning of the ventral and dorsal prepancreatic endoderm . The initial interactions usually confer competence to respond to additional inductive signals that establish organ determination and specification at particular time points referred to as competence windows . Although our present knowledge of extrinsic factors that control patterning, proliferation, and differentiation cues throughout the developing pancreas is not yet complete, there is strong evidence emanating from all vertebrate developmental models that complex spatio - temporal combinations of common signaling pathways are crucial for appropriate specification of pancreatic cell fates from the endoderm (69). These involve, for instance, the hedgehog, wnt, retinoid, and notch pathways, as well as activin / bone morphogenetic protein (bmp), fibroblast growth factor (fgf), vascular endothelial growth factor (vegf), epidermal growth factor (egf), and hepatocyte growth factor (hgf) signal transductions . An overview of the roles played in pancreas development by the most relevant growth and differentiation factors is summarized in table 2 . A detailed sequential presentation is difficult to address because many signaling events are not clearly dissociated from each other, while others function redundantly or intermittently at several developmental stages . It is also worth noting that the developmental effects elicited by these common signals can vary significantly according to the cell type being stimulated or according to the stage of development when the signal occurs . This level of complexity is further enhanced by the interplay between transcription factors that lie downstream of these extracellular stimuli and are responsible for observed developmental features . Overview of growth and differentiation factors that participate in vertebrate pancreas development cmes, cardiac mesoderm; dmes, dorsal mesenchyme; gcg, glucagon; gdf, growth differentiation factor; hb - egf, heparin - binding epidermal growth factor; ihh, indian hedgehog; lpm, lateral plate mesoderm; pepi, pancreatic epithelium; pmes, pancreatic mesenchyme; pnep, pancreatic ngn3 endocrine progenitor ., considerable progress has been achieved in understanding pancreas embryogenesis thanks to the numerous genetic studies involved in transcription factors . Most of them belong to the homeobox domain, the paired - box, or the basic helix - loop - helix families . The sequential expression of different transcription factors that control pancreas development has been extensively reviewed elsewhere (68,10). 1 with special focus on 1) the early pancreatic epithelial cells, 2) the pancreatic endocrine progenitors, and 3) the endocrine subtypes selection . Expression of different combinations of tfs determines the sequential lineage segregation in the developing pancreas . Not only are the combinations of tfs important, but the expression level of a particular tf is known to guide differentiation as well . The most relevant tfs that are landmarks for the selection of each stage / lineage are shown . It is presently well recognized that attempts to generate pancreatic cells from pluripotent cells should first establish a definitive endoderm (de) population as occurs in vivo . Wnt and nodal signals were identified as sufficient to induce de from mouse and human embryonic stem (he s) cells, but also from induced pluripotent stem (ips) cells . In this model, high concentrations of activin a (acta) are used to mimic the function of the endogenous endoderm inducer nodal (11). Beside the activation of this pathway, initial treatment with wnt3a or bmp4 was found to improve the efficiency of de induction by setting up a transient mesendoderm progenitor population, especially when cultures are performed in feeder cell - free conditions (12,13). Because active pi3k signaling is inhibitory for de differentiation from embryonic stem (es) cells, antagonists of this pathway (ly294002, wortmanin) were also included in some protocols, resulting in an increased efficiency (14,15). In addition, supplementation of sodium butyrate was found to support endoderm differentiation when combined with acta (16), though it remains unclear how this chemical participates in the signaling network required for the definitive endoderm . The standard and modified protocols for de induction from es cells allow for the derivation of at least 6080% cells that express the characteristic markers foxa2, sox17, gsc, and cxcr4 (table 3) but not the visceral endoderm marker sox7 (13,14,1620). Whether the quest for a universal -cell differentiation protocol is a desired myth or an achievable reality, it appears clear from the literature that combined wnt3a and acta treatments efficiently activate de phenotype in a vast majority of he s cell lines . In other words, the molecular events that lead de differentiation in vivo have been largely if not completely unraveled and implemented in vitro . Despite the recently described differences in the response of he s cell lines to differentiation cues (21), the variability and the low success rate in the outcome of initial pancreas differentiation protocols might first of all reflect the lack of such a universal developmentally based strategy that thoroughly reproduces in vivo events in the culture dish after the de stage . Derivation of pdx1 progenitors from he s cells recent models of pancreas differentiation from he s cells integrate bmp antagonism and retinoid signaling early after definitive endoderm induction . Cyclo, cyclopamine; dapt, n-[n-(3,5-difluorophenacetyl)-l - alanyl]-s - phenylglycine t - butyl ester (gamma secretase inhibitor); ebs, embryoid bodies; ex4, exendin-4; fbs, fetal bovine serum; its, insulin selenium transferring supplement; na, nicotinamide; nabut, sodium butyrate; ng, noggin; stz, streptozotocin; 3d, three dimensional . When maintained in culture without added growth factors or when transplanted under the kidney capsule of immunocompromised mice, hes - derived definitive endoderm cells could spontaneously generate hepatocyte - like cells characterized by the expression of afp, albumin (alb) (fig . This tendency for hepatic lineage commitment was also reported in several studies wherein hes - derived de cells were exposed to bmp and/or fgf, two growth factors implicated in embryonic liver induction and specification (13,20,22,23). The contribution of these pathways was further demonstrated by the treatment with bmp antagonist noggin and fgf receptor antagonist su5402, leading to the complete abrogation of hepatic gene expression from de progenies that otherwise was mainly composed of hepatocytes . As suggested from animal studies, these experiments also indicated that fgf did not function in the induction but essentially contributed to the amplification of liver progenitors initially induced by bmp signaling (20). Therefore, the molecular signals elicited by bmp and fgf are the main factors driving hepatocyte differentiation from hes - derived de cells . Expression of pancreas - related transcription factors in hes - derived de progenies treated with noggin, ra, and cyclopamine . Foxa2 and albumin expression in control cultures (a) and following treatment with noggin, ra, and cyclopamine (b). D: several nkx6.1 cells are also detectable, the majority of which express lower levels of pdx1 (not shown). (a high - quality digital representation of this figure is available in the online issue .) In vivo, bmp activity generated by the adjacent mesoderm derived structures and operating in the ventral endoderm is associated with hepatic induction during early embryonic development . On the contrary, ventral pancreas progenitors are located away from the midline endoderm with active bmp signaling, whereas bmp inhibition in the dorsal endoderm plays a crucial function in the acquisition of a pancreatic fate (3,24). The requirement for bmp antagonism is reverted after pancreas induction since this organ requires bmp signaling later on for the maintenance of pdx1 expression and further differentiation . This underscores the existence of a tight competence window and the versatile functions of growth factors during development, which complicates their in vitro implementation for directed differentiation of es cells (24). Retinoic acid (ra) plays a crucial role shortly after gastrulation in endoderm patterning and in the induction of pdx1 expression by pancreas progenitors in several organisms from xenopus to humans, which indicates the conservation of its activity during evolution (2527). This role for ra has already been evaluated during mouse and human es cell differentiation in vitro and has proven to be relevant for the induction of pancreatic gene expression, notably detectable levels of pdx1 (17,23,2832). In line with the in vivo recognized functions of these two signaling pathways on pancreas induction, recent studies have combined bmp inhibition (noggin supplementation) with retinoid signaling immediately after the de induction stage and succeeded in producing large amounts (up to 80%) of pdx1 pancreas progenitor cells (table 3), (13,14,20). Concomitantly, the proportion of hepatocytes that could be detected in these conditions was significantly reduced (fig . 2a and b), indicating an efficient blockade of their induction upon bmp antagonism (20,33). This combination was efficient not only in different he s cell lines, but also in ips cells (14), suggesting that the basic molecular events that should be monitored in order to design a universal pancreatic differentiation protocol are being progressively clarified . These minimally include the signals for the blockade of hepatic induction on the one hand and those required for pancreatic induction (namely pdx1 expression) on the other (fig . The necessity for such a combination was recently highlighted by treatment of hes - derived de cells with noggin in the absence of ra; this blocked the expression of liver markers, but did not generate pdx1 cells (20,23). This requirement for combining ra treatment with bmp antagonism could also be seen from another angle . Indeed, retinoid signaling was shown to activate bmp expression in several in vivo and in vitro systems including embryonal carcinoma cells and differentiating mouse embryoid bodies (3436). It is not yet clear whether this inductive effect on bmp also occurs during he s cell differentiation . Nevertheless, considering the in vivo function of bmp signaling in the midgut endoderm, it appears reasonable that both retinoid signaling and bmp antagonism were required in these studies for strong induction of a pancreatic fate from the de cells while preventing hepatic differentiation . However, it is likely that the effect of ra is context - dependent because when applied on hes - derived de cells cultured at low density, it resulted in the inhibition of smad 1/5/8 phosphorylation (23). It remains to be elucidated whether this later effect represents an artifact of in vitro culture or a specific retinoid effect at the single cell level . Four main pathways regulate the early stages of pancreas differentiation from definitive endoderm and control the acquisition of hepatic vs. pancreatic fate . Hedgehog signaling is well characterized as a potent inhibitor of pancreatic initiation (a) that is expressed in the hepatic domain (b) under the influence of fgf signaling . Retinoic acid is expressed in the developing pancreas and participates in the induction of pdx1 expression (c). It is also suggested to contribute to hepatic gene expression to a certain degree (d). An inhibitory effect of retinoic acid on smad1 - 5 - 8 phosphorylation (e) was demonstrated on hes - derived definitive endoderm seeded at low density, which contrasts with the early studies indicating activation of bmp signaling by retinoic acid . Bmp is well described as an inhibitor of early pancreas development (f) in contrast to its requirement for hepatic initiation (g). This potent inhibitory effect at early stages gets reverted afterward and pancreatic progenitors require bmp signaling (f). The inhibition of bmp signaling in the pancreatic domain is under the control of noggin (h). As for bmp pathway, fgf signaling via erk / mapk also controls early pancreas induction with high concentration being inhibitory (i) whereas low concentrations are required (j). In late stages, fgf plays in the proliferation of pancreas progenitors (j). On the contrary the markers displayed in the progenitors recall the current status of protein detection from differentiated he s cells . Other studies made use of ra combined with fgf ligands (fgf4, fgf7, fgf10) or of noggin combined with fgf2 and egf to induce pancreatic cells albeit at a lower efficiency than for combined noggin and ra (table 3) (13,14,16,17,19,23). Our unpublished observations suggest that fgf signals favor hepatic gene expression in the absence of bmp antagonism, but the addition of fgf ligands together with bmp antagonism (noggin) and ra enhances pancreatic gene expression . Furthermore, the induction of pdx1 cells by combined noggin and ra requires at least a basal level of fgf activity (via the extracellular signal regulated kinase / mitogen - activated protein kinase [erk / mapk] pathway [fig . 3]) given that the addition of the mapk - inhibitor u1026 prevented pancreatic cell differentiation (20). Because ra is known to act synergistically with fgf signaling during endoderm patterning, supplemented fgf can be seen as an enhancing factor in protocols that already integrate noggin and ra (13,14,19). This view might resolve the nature of factors x suggested as involved in the indirect effects of ra in xenopus dorsal endoderm (37). It was recently suggested that in the presence of fgf7, ra can efficiently induce pdx1 + progenitors from hes - derived de cells seeded at low density (550,000 cells / cm) and that this effect is paralleled by inhibition of smad1/5/8 phosphorylation, therefore recapitulating bmp antagonism described above (23). Recent analysis of the signaling network during early liver and pancreas development in 36 somites - stage mouse embryos further indicated that inhibition of the activin signaling significantly increases the proportion of cells fated toward pdx1 expression . These findings therefore suggest that the addition of activin inhibitors to the above - mentioned cocktails might still boost the occurrence of pdx1 cells in he s cell cultures (13,24). Despite the fact that combining bmp antagonism with retinoid signaling efficiently generates pdx1 pancreatic progenitors from hes - derived definitive endoderm, the requirement for hedgehog antagonism with cyclopamine during pancreas induction from these cells has not been clarified by these studies . The initial report by d'amour et al . (18) indicated the necessity to antagonize hedgehog signaling that is indeed activated during es cell differentiation, and it was shown to also play a negative role ex vivo by limiting endocrine and exocrine genes expression in embryonic (e12.5) mouse pancreatic explants (38,39). However, additional studies using noggin, fgf ligands, and/or ra after de induction succeeded in generating pdx1 cells without the use of hedgehog inhibitors (14,17,19,23). Owing to the previously described increase in the expression of hedgehog ligands and effectors upon de induction, we added cyclopamine to the combination of noggin and ra in our experimental setup, but it remains unclear whether this was mandatory for the efficient differentiation of pdx1 cells that we observed (20,38,39). While waiting for conclusive data on this issue, we speculate that supplementation of hedgehog antagonists would become obsolete in settings where combined noggin and ra treatment of de cells essentially induces a true pancreatic endoderm cell type, which is normally devoid of hedgehog activity . Gli1 transcripts profiling in cells treated with noggin and ra combination would help to clarify this issue . Pancreatic progenitor cells are characterized by their expression of a subset of transcription factors . For instance, the combined expression of pdx1, foxa2, sox9, hnf6, nkx6.1, and ptf1a (fig . The last two transcription factors are more specific to the pancreas, whereas pdx1, foxa2, sox9, and hnf6 are also detected in the adjacent duodenum, stomach, and liver . Following the induction of pancreas progenitor cells from hes - derived de cells, pdx1 co - expression with foxa2, sox9, and hnf6 was demonstrated in a context where hepatic (afp, alb) and intestinal (cdx1, cdx2) markers were not induced or only minimally induced (fig . However, none of the above - mentioned studies have assessed the expression of all the six transcription factors by the pancreatic progenitors obtained from he s cells . More specifically, demonstration of ptf1a expression has remained elusive until now, possibly owing to the lack of an optimal antibody against this protein . The same holds true for ngn3, which has been detected at the protein level in only few studies . In our experimental setup, ptf1a transcripts were detected only at low levels, whereas a significant emergence of nkx6.1-positive cells was denoted . Nevertheless, when combined with the demonstration of ins and pdx1 co - expression at later stages, these findings point toward the pancreatic nature of the differentiated pdx1 cells . Additional efforts are required to obtain their efficient differentiation toward pancreatic endocrine cells in vitro (table 3) (14,20). During pancreas development, the pdx1 progenitor cells are amplified thanks to signals emanating from the pancreatic mesenchyme . Fgf10 controls this mesenchymal - to - epithelial interaction, and its over - expression beyond the competence window (e11.5e13.5 in the mouse) significantly induces progenitor cell proliferation and arrests their terminal differentiation (40,41). This signaling operates via activation of the notch pathway, which temporally prevents progenitor differentiation toward the endocrine or exocrine fates (42). In agreement with these, fgf10 or egf supplementation induces the proliferation of hes - derived pdx1 cells, suggesting the contribution of erk / mapk and akt activation in this effect (14,16,20) furthermore, before the proliferation step, establishment of the pdx1 progenitors from hes - derived definitive endoderm was also shown to be highly dependent on erk / mapk signaling and possibly mediated by fgf2 (20,43). The initiation of endocrine differentiation from the pdx1 progenitors relies on the activation of ngn3 expression, which should be paralleled or preceded by downregulation of notch activity and further sustained by additional induction of pax6 (fig . Although the transcription factors cascade regulating pancreatic endocrine differentiation has been largely explored, to date the instructive extracellular signals that trigger their coordinated expression remain elusive . To this end, there is a growing list of proposed growth factors and chemicals whose effects will need to be extensively assessed in the endocrine commitment and maturation of pdx1 progenitors obtained in vitro from hes - derived de cells . These include but are not limited to wortmannin and ly294002 that are pi3k inhibitors (46), vegf (4,47), ra (26), conophylline (48), wnt, and hedgehog ligands (49,50), and antagonists of the notch pathway such as gamma secretase inhibitors, notch ligands, and soluble notch . Not only is the nature of these factors important, but their timely combination will also be of consideration given the demonstration of a competence window for endocrine patterning beyond which the endocrine differentiation arrest becomes irreversible (41). For instance in our experimental setup (table 3), fgf10 was supplemented for controlling pdx1 cell proliferation together with a gamma secretase inhibitor (compound e) for notch antagonism in view of endocrine induction (20). Considering the above - mentioned crosstalk between fgf10 and notch activity in the proliferation of the pdx1 progenitors, such a protocol therefore included two phenomenas that were actually antagonistic . This issue might explain the very limited progression toward endocrine cells that we obtained, a hypothesis that will need further evaluation by dissociating fgf10 treatment from notch inhibition in this protocol . The next step after endocrine commitment will be the further endocrine differentiation and maturation by use of old and new players such as betacellulin, activin, exendin-4, insulin - like growth factor, hgf, nicotinamide, bone morphogenetic protein, fgf, and ra (14,16,18,20,26,28). Similarly, three - dimensional cultures will merit further investigation in order to mimic the actual environment of the -cells in islets as was recently described in differentiating he s cells (16). Some of these factors have been used singly or in combination on pdx1 progenitors generated from he s cells . Because of the complex nature of pancreatic endocrine differentiation, which is presently not yet elucidated, the generation of mature and functional -cells has not been efficiently achieved in vitro . The demonstration of human - specific cell functions in animal models following transplantation of pancreatic progenitors derived from he s cells (19,32,51) underscores this gap in our current knowledge . Based on these data, it has been proposed that diabetic patients could be grafted with pancreatic progenitors derived from he s cells, eliminating the need for finding the efficient strategy for terminal differentiation in vitro . However, this approach would be hampered by teratoma formation from cells that were not fully differentiated at the time of transplantation (see below). Although the differentiation of specific cell types such as pancreatic cells can be established through their characterization with several markers, it remains obvious that no protocol currently offers a factual conversion of all cultured stem cells to the desired phenotype . Therefore, the possibility exists that other cell types or even multi- or pluripotent cells might be transplanted to the diabetic recipients, which raises safety issues . Indeed, despite the fact that functional -cells developed in vivo after transplantation of hes - derived pancreatic cell preparations, teratoma formation was also observed in at least 15% of grafts (19,51,52). Several tools have been investigated in order to select out the undifferentiated cells that express surface markers (ssea4, ssea3, tra-160, tra-181) or to specifically isolate the desired cell type (cell - trapping) that have been modified to express an antibiotic resistance gene, a fluorescent or a bioluminescent reporter under ck19, pdx1, or ins promoter (5355). Whereas these techniques would allow for the selection of differentiated -cells up to purity, transplantation of such cells is presently not approved given the genotoxic, the mutagenic, and the oncogenic risks associated with transgenic approaches . The in vitro differentiation of functional -cells from human pluripotent cells (he s and ips) represents a major challenge for diabetes cell therapy in the future . Progress toward this goal already encompasses the efficient generation of definitive endoderm and pdx1-expressing pancreas progenitors . The recent successful studies indicate that the differentiation of pdx1 progenitors concomitantly requires at least two events after de establishment in order to mimic the in vivo situation: blockade of hepatic induction by bmp antagonism and induction of pancreas progenitors by retinoid signaling (fig . Optimization of this step might still prove beneficial in order to unravel the role of fgf and hedgehog modulation and in order to definitely establish cells that undoubtedly show combined expression of the major transcription factors recognized for the multipotent pancreas progenitor, namely pdx1, ptf1a, and nkx6.1 . In addition, the recently described temporal shift in the requirement for bmp antagonism to a need for bmp signaling during mouse pancreas development will merit implementation in he s cell differentiation . Despite the latest data indicating differentiation of the pdx1 progenitors into insulin / c - peptide cells, we believe that additional work is needed for an optimal in vitro control of the progression toward ngn3-expressing endocrine progenitor cells and for the maturation of glucose - sensing and insulin - secretion functions in the -like cells . A step forward toward these goals will certainly shorten the time frame for achieving our objective of using he s cells as alternative sources of functioning -cells in diabetes cell therapy.
Celem niniejszej pracy jest przedstawienie obecnej taktyki postpowania zarwno diagnostycznego, jak i terapeutycznego u chorych z pseudotorbielami oraz torbielowatymi guzami trzustki . Praca zostaa napisana na podstawie materiau zebranego i przygotowanego w klinice autora oraz wspczesnych doniesie z zaprezentowanego pimiennictwa . W latach 20002012 w ii klinice chirurgii oglnej, gastroenterologicznej i nowotworw ukadu pokarmowego przebywao 179 chorych z torbielowatymi zmianami w obrbie trzustki, w tej grupie stwierdzono 12 przypadkw guzw torbielowatych oraz 167 zmian o charakterze rzekomych torbieli u 75 wykonano zabieg drenaowy na drodze operacyjnej, 48 chorych zakwalifikowano do endoskopowej cystogastrostomii lub cystoduodenostomii . W grupie pacjentw z guzem torbielowatym trzustki na 12 badanych u 6 (50%) wykonano lecznicz resekcj guza z odpowiedni czci narzdu . The aim of this paper was to present current management, both diagnostic and therapeutic, of patients with pancreatic pseudocysts and cystic tumors . The article has been written based on the material collected and prepared in the author's department taking into consideration contemporary approaches and opinions included in the references . In 20002012, the second department of general, gastrointestinal and oncological surgery of the alimentary tract treated 179 patients with cystic lesions in the region of the pancreas . The diagnostic procedures included: abdominal ultrasound and doppler examinations, ct and in certain cases, also eus and intraoperative ultrasound (ious). When a cystic tumor was suspected, contrast enhanced ct, mri or mrcp were conducted . Patients with suspected or confirmed neoplastic tumors were included in a separate group of cystic lesions . The majority of the lesions adhered to the pancreas but their outer outlines could border on various parts of the gastrointestinal tract and the mesentery or could be localized in the extraperitoneal space . The exact specification of adherence to, e.g. Head, body or tail of the pancreas, is quite troublesome . Therefore, in our department, the description usually specifies to which aspect of the pancreas, proximal or distal, the cyst adheres . In the material presented herein, the proximal localization (head, body) was found in 118 cases and distal (body, tail) in 61 cases . Most of the treated lesions ranged from 3 to over 25 cm . In 2000, we introduced endoscopic treatment method of pseudocysts . If in transabdominal ultrasound examination, cysts with defined walls are detected, located near the stomach or duodenum and indenting their lumina, eus and videoendoscopy are conducted to determine the indications for endoscopic cystogastrostomy or cystoduodenostomy . When the adequate puncture site in a non - vascular region has been selected in eus, it is marked with dye, spot coagulation or, currently, an appropriate drainage set is placed, directly guided by eus . With the use of the lateral - viewing duodenoscope and diathermy, the gastric wall and adjacent cyst are punctured . Subsequently, having conducted the aspiration attempt, the prosthesis is inserted which allows for free outflow of cystic contents (fig . The efficacy of the procedure is evaluated in sonography . Up to 2012, a total of 48 patients were qualified to endoscopic drainage procedures . Eus - guided pancreatic pseudocyst drainage . Puncturing the pseudocyst and inserting a guidewire eus - guided cystogastrostomy . The pancreatic juice flows through cystogastrostomy (prosthesis is inserted to the lumen of the pseudocyst) when the initial diagnosis cannot rule out a cystic neoplasm of the pancreas, the patient undergoes detailed imaging examinations, biochemical and serological tests as well as, in certain cases, fine - needle aspiration biopsy (fnab). In the subject group of 179 patients with cystic lesions in the region of the pancreas, 12 presented with cystic tumors and 167 with pseudocysts, both acute and chronic . From among all 167 cases with symptomatic cysts, 23 patients (13.8%) were monitored only (involution of the lesions during the observation period) and the remaining 144 patients were qualified to procedures . In 75 patients (44.9%), the resection of the cyst was possible in merely 4 cases, the remaining patients underwent drainage procedures . In 24 cases, cystogastrostomy (jurasz procedure) was conducted and in 39 cases, roux - en - y cystojejunostomy was performed . In the remaining patients, complex procedures took place, such as drainage of several cysts or total drainage of pseudocysts and pancreatic duct, also performed according to roux - en - y technique . During postoperative monitoring period, complications were observed in 16% of patients . Furthermore, 21 patients (12.6%) with thin - walled retention cysts secondary to acute pancreatitis (ap), were treated by external us - guided drainage . In 8 cases (38%), 48 patients (28.7%) were qualified to endoscopic cystogastrostomy or cystoduodenostomy if transabdominal ultrasound and subsequently, eus had confirmed such qualification . In all cases, it was attempted to introduce an appropriate stent (prosthesis) to the cysts through the wall of the gastrointestinal tract . The efficacy of the procedure was usually evaluated on the second day following the procedure by means of us examination . Drainage was successful in 37 patients, 12 of whom required correction of drain placement and a repeated procedure was necessary . Due to technical reasons, the placement of the drain was impossible in 11 patients (23%) they underwent surgical drainage instead . During examination, all patients underwent imaging examinations, such as: us examination with doppler mode, ct and in 4 patients, mri and mrcp . The analysis of the contents (cytology, level of markers, test for mucous content) was positive in only 2 patients . In the remaining cases the analysis was not reliable . Due to the results of imaging examinations, in 5 patients with slight lesions (mainly from 2 to 3 cm) in whom a benign serous cystic neoplasm was suspected (based on us / ct), observation was recommended . In this group, only one patient underwent fnab (unreliable attempt). Evident growth of the tumorous lesion was not observed in the monitored patients within the observation period of 23 years . In further 6 patients with over 4 cm lesions that showed features of neoplastic cystic tumors, resection of the tumor with an adequate part of the pancreas was performed . In 5 patients, fnab was conducted (the result was positive only in 2 cases, see above). In 1 patient, a malignant neoplasm was confirmed which infiltrated adjacent structures and gave metastases to the liver . This case was considered inoperative (as assessed in diagnostic laparoscopy with tissue sample collection). In patients diagnosed with and treated due to pancreatic pseudocysts, transabdominal ultrasound is the main diagnostic tool, followed by ct and eus, in qualifying patients to endoscopic treatment . In us thin - walled cisterns do not qualify to surgical treatment (anastomosis) due to the difficulties to perform anastomosis and the possibility of secondary leak . In such cases, external percutaneous drainage should be considered or, in certain favorable conditions internal endoscopic drainage . The treatment of pancreatic cysts should depend on their origin, character, size, location and individual clinical symptoms . Slight cysts measuring not more than 4 cm which appeared after acute pancreatitis receive conservative treatment . They are usually necrotic collections of aseptic contents which do not contain any large breakdown fragments . Slight lesions, from 3 to 4 cm, which may be effectively controlled by means of periodical us examinations, do not receive any treatment provided that their neoplastic character has been ruled out . Only large cysts are qualified to procedures usually those measuring more than 56 cm particularly when other characteristic clinical symptoms are present, such as pain, obstructive jaundice, obstruction in the upper gastrointestinal tract, decreased appetite or weight loss (fig . 2). A similar situation refers to cysts whose continuous growth is observed in us check - up examinations and complicated cysts . In accordance with current principles, uncomplicated cysts and fluid collections secondary to ap are not subject to treatment if the period of us observation and monitoring is shorter than 6 weeks . In the discussed period of time, from among 167 patients diagnosed with symptomatic cysts of the pancreas, 44.9% underwent surgeries, 12.6% were treated by means of external drainage and 28.7% by internal eus - guided endoscopic drainage . Still, the largest group underwent surgeries in spite of the fact that endoscopic procedures introduced in 2000 have a greater and greater therapeutic value . Such a procedure was performed in 48 patients, but in 11 of them (23%) endoscopic drainage was not successful mainly due to technical problems connected with prosthesis placement, difficult angular access of the endoscope to the cyst, bleeding and suspicion of perforation . In such cases, surgical drainage is the method of choice . In the remaining 37 patients with pancreatic pseudocysts, it allows for a rapid return to everyday activities, patients do not need to follow a rigorous diet (as it happens after a surgery) and their physical activity is not restricted due to the postoperative wound . At present, thanks to the advancement of diagnostic techniques, we may accurately assess and distinguish neoplastic cystic tumors of the pancreas from ordinary cystic lesions . It is currently believed that even 10% of all cases of cystic lesions are related to a neoplasm and that these lesions account for 15% of all pancreatic neoplasms . It is believed that asymptomatic lesions constitute approximately 3575% of cystic tumors of the pancreas . The appearance of certain symptoms may be associated with growth of the tumor, increased internal pressure or malignant transformation . The most common reported symptoms include: pain in the abdomen, nausea, vomiting and increased circumference of the abdomen, weight loss, obstructive jaundice, constipation or diarrhea and weakness . Patients with ipmn may be sometimes treated due to recurrent episodes of acute pancreatitis, particularly, in cases when the main pancreatic duct is involved . Large tumors are most frequently found in patients with mucinous cystic neoplasms (mcn) and spn these lesions usually give symptoms of a midabdominal tumor . Exacerbation of pain is associated with sudden hemorrhage to the tumor or results from its rupture (spn). In advanced malignant lesions, apart from the above - listed symptoms the following may occur: bleeding from the gastrointestinal tract in the case of gastric infiltration, portal hypertension and hemobilia as well as, eventually, diabetes when the pancreas is considerably damaged . In the analyzed time period, after thorough imaging analysis, including doppler ultrasound, contrast enhanced ct as well as in some cases, mri, mrcp and eus, 6 patients were qualified to fnab . Only in 2 cases did fnab and assessment of the aspirated fluid confirm the presence of a cystic neoplasm . Similarly in the quoted references imaging examinations are decisive when treatment - related decisions are made . The assessment of the aspirate is usually less specific and the final verification of the type of the tumor is acquired following the analysis of the postoperative specimen . In our material, the postoperative verification confirmed the presence of 3 serous cystadenomas and 3 mucinous cystadenomas in 2 cases, the lesions had already been identified as cystadenocarcinomas . In one patient, a malignant spn tumor was detected with slight metastases to the liver inoperative lesion . In further 5 patients, slight (23 cm) tumor - like lesions they were identified as serous tumors which do not require surgical treatment . In these patients, annual check - ups were recommended which in the period of analysis, did not show any growth or change of the inner structure . According to numerous authors, such lesions do not require immediate qualification to resection and in the majority of cases, they do not change their appearance during the monitoring period . In the next part, the material has been collected based on quoted recent publications concerning diagnosis, methods of differentiation and proposed types of treatment . They are usually solitary, with the size of 1.535 cm and constitute approximately 30% of all primary pancreatic cystic tumors . We mainly encounter two types of such lesions: microcystic cystadenomas and macrocystic or oligocystic cystadenomas . The microcystic form usually develops in women at the age of 5060 and its typical localization is the body and tail of the pancreas . It is usually asymptomatic and signals, such as pain in the abdomen, weight loss, feeling of distension in the epigastric region and peristalsis disorders appear when the lesion shows large growth . A typical form of the tumor is composed of a large number of small cysts lined with cuboidal epithelium producing glycogen . The size of the cysts ranges from 0.2 to 2.0 cm and the entire tumor measures 1.430 cm . The lesion may grow locally leading to almost complete damage of the healthy pancreatic tissue . In imaging examinations (us, ct), such a cystic tumor frequently has the appearance of a honeycomb which is related to a large number of small cysts (fig . The lesion is relatively well - circumscribed with a central scar or, sometimes, with a calcification . Serous cystadenoma (microcystic type). A typical serous tumor is composed of multiple small cysts and has a the cystic mass is situated in the pancreatic tail, about 5 cm in diameter ious . Well - defined microcystic tumor, poorly vascularized, adhering to the splenic vessels (arrow) (color doppler) doppler us . Serous cystadenoma in the head of the pancreas, poorly vascularized, 5.6 cm in diameter . Structure with oligocystic components and central scar (arrow) contrast - enhanced ct shows a classic serous cystadenoma in the head of the pancreas (4.1 2.9 cm). The lesion has the appearance of a solid mass with numerous small cysts and septations honeycomb structure . The calcified central scar (arrow) the other form of the tumor, i.e. Macrocystic or oligocystic cystadenoma, with its appearance resembles a mucinous cystic tumor and is difficult to differentiate based on imaging examinations . It develops equally frequently in men and women, usually, after the age of 40 . Therefore, the basic symptoms of its presence, next to the feeling of discomfort in the epigastric region and sometimes, pain when the lesion grows, is jaundice the tumor is composed of other well - circumscribed cystic lesions, with their diameters usually greater than 2 cm . They are generally single cysts or there are a few poorly circumscribed ones with some internal septations . In the present - day imaging diagnosis it is a modern us examination with the use of contrast media which considerably broadens diagnostic possibilities of doppler us examination . Ceus allows for a good visualization of the honeycomb appearance and of the septations inside a neoplastic cyst . When, in small cystic tumors, which do not qualify to surgical treatment, ceus is performed, there is no need for further examinations, such as mri, mrcp and even contrast enhanced ct . Owing to the high resolution, one may visualize lesions composed of very slight cysts (of 5 mm in diameter) in a more detailed manner as compared to conventional ultrasound . Moreover, eus examination enables precise visualization of the honeycomb structure, calcifications and internal septations . It appears that a confirmation of a microcystic structure is sufficient for differential diagnosis and there is no need for the analysis of the fluid collected by means of eus - guided fine - needle aspiration biopsy (eus - fna). What is more, the collection of adequate amount of fluid from such slight structures is frequently not feasible . Fluid aspiration and its examination seem necessary when a larger cyst with a suspected neoplastic character has been visualized ., the collected fluid does not show any traces of mucin or amylase, and the test for the contents of cell elements (cytological examination) is possible in less than 50% of cases . Contrary to patients with marked malignant transformation, the level of tumor markers (mainly cea) is low, usually below 5 ng / ml . In the material presented herein, in the case of serous tumors, aspiration demonstrated correct level of the markers (cea and ca 19 - 9) and cytological examination did not reveal any neoplastic cells . A great majority of pancreatic serous tumors are benign the possibility of their malignant transformation concerns less than 3% of cases . A surgical resection of such a tumor together with a fragment of the pancreas is indicated in symptomatic lesions, in tumors that grow during the period of monitoring and in cases in which malignant transformation cannot be ruled out . In patients qualified to surgical treatment, ious appears to be useful both in terms of final verification of the diagnosis and in the assessment of the range of resection . According to the quoted literature, the size of the tumor at which surgery should be considered is over 45 cm (fig . Up to 4 cm, tumors tend to grow slowly (on average, 0.12 cm / year). It was observed that tumors larger than 4 cm grow faster (on average, 1.6 cm / year), symptoms begin to appear and malignant transformation becomes more possible . It may happen that in imaging scans, a tumorous lesion shows local infiltration on its surrounding structures but the possibility to diagnose carcinoma based on imaging methods, endoscopic techniques and biopsy are limited . Serous cystadenoma situated in the pancreatic body . A symptomatic lesion, 5.5 cm in the diameter, qualified to surgical resection ious . Postoperative specimen the debate whether serous tumors should be only observed or at once qualified to surgical treatment is still open . There are mixed opinions since the cost of check - ups is considerable and the sole awareness of patients of a potential threat to develop a cancerous disease is of great significance . Thus, slight lesions with 23 cm in diameter should be observed (fig . 6). Check - up examinations, including us and ct, ought to be performed at least every 12 years . In the remaining cases, resection of the tumor it might be performed with the application of minimally invasive techniques when the lesion is relatively small and when surgical treatment should proceed with no complications . In our department, the aforementioned principles are observed . Contrast - enhanced ct a small hypodense cystic tumor in the pancreatic tail (2.5 2 cm). Poorly vascularized, multiple small cysts with polycyclic border of the tumor it is currently believed that mucinous cystic tumors constitute the most common form of pancreatic cystic neoplasms (1045%). Due to the fact that in numerous cases they resemble ordinary cysts, a proper differentiation between these two is essential . They are encountered almost only in women (95%), mainly middle - aged ones, and are usually localized in the body and tail of the pancreas . A mean size of the tumor during the first diagnosis equals> 5 cm and in certain cases, the lesion may grow even up to 2535 cm . Such lesions are usually solitary and do not maintain any connection with the main pancreatic duct . The most frequently encountered form of such a tumor is a single large lesion of cystic character with or without internal septations and with a well - defined, usually thickened outer wall (fig . 7). The cyst is lined with columnar epithelium which produces mucus that forms its inner layer . The outer layer is made up of stroma which resembles ovarian structure . These cysts usually contain a certain amount of mucous substance and one may also find sanguineous contents after hemorrhages or necrotic changes . Typical radiological features of the tumor include: septations, solid intramural nodules and peripheral calcifications . In ultrasound examination, a mucinous tumor is visualized as a hypoechoic, well - circumscribed mass with diverse, often irregular thickening of the wall and sometimes, with solid nodules or calcifications . Ceus examination allows for a better identification of the lesions within the wall (nodules) and thickened septations, which is caused by increased vascularity of the altered structures attesting to malignant transformation of the tumor . From the histological viewpoint the visualization of the symptom triad, i.e. Irregular cystic wall thickening, peripheral calcifications and intramural solid lesions, in us, ct or mri may correspond to malignant transformation even in 95% cases (fig . Only a slight per cent of mucinous cystic tumors present a microcystic form, usually a slight one, single with few septations . In such cases, the cystic lesion is situated in the head of the pancreas with low - grade dysplasia (arrows) us power doppler . Cystic neoplasm with reduced fluid capacity, mainly growth of solid tissue in order to learn the morphology of cystic tumors, eus examination is performed . This imaging modality provides reliable information allowing for the characterization of the cyst and determining the possibilities of resection when features of malignancy are present . Due to the extension of the method to include aspiration biopsy (eus - fna), it is possible to find extracellular mucus in the cystic fluid, conduct cytological or, more rarely, histological analyses, make biochemical assessment, determine the level of tumor markers and perform molecular analysis . As for cytological analysis, the reasons may be technical difficulties to collect fluid (too little amount) or low contents of cell elements in the aspirate . From among numerous tumor markers, the most specific is carcinoembryonic antigen cea . With the cut - off value of 200 ng / ml, higher values obtained in the analysis of cystic fluid indicate the presence of a mucinous tumor . In the statistical analysis for this level, a similar situation may be observed in the analysis of amylase concentration in the fluid . For the cut - off value of 250 iu / l, in the case of this level, the sensitivity and specificity constituted 45% and 89% respectively . Unfortunately, the papers published so far do not specify the level of cea marker above which the cancerous transformation might be confirmed . This is verified after thorough evaluation of all data, particularly, imaging examinations and possible findings of eus - fna . What is important is the fact that small mucinous tumors may manifest no symptoms in as many as 75% of patients . When symptoms appear, the tumor is usually large and potentially malignant . Therefore, in all such cases rapid diagnosis and surgical resection of the tumor are essential . The basis for the qualification to surgical treatment is the outcome of imaging examinations, clinical examination and, to a lower degree, also data obtained from cystic fluid aspiration . When a mucinous tumor of the pancreas is identified, one should remember that such lesions are frequently malignant or of highgrade dysplasia and they qualify to surgical treatment . A total evaluation of the tumor's character is possible only following its resection, after histopathological examination . In our material, the examination of a postoperative specimen verified the presence of malignant mucinous tumors in two patients . Aspiration biopsy performed prior to the procedure revealed elevated cea level and presence of mucus . Cytological examination gave positive outcomes in the analysis of fluid from the inside of the cystic tumor in only one patient . This type of tumor is usually encountered in elderly men, aged 6080 . In a large majority of patients, these lesions manifest no symptoms . The most common symptom is recurrent pain, similar to that in the course of chronic, exacerbating pancreatitis or episodes of acute pancreatitis, occurring mainly after meals . Such mucus travels to the pancreatic duct where it contributes to large pressure rise, which causes significant dilatation of the duct . The features of the tumor's image depend on its type and localization in the pancreas . We distinguish three types of ipmn: central type which encompasses the main pancreatic duct, peripheral type involving the branches of the ducts, and mixed - type . The central type is the most common (even up to 75% of cases). The lesion looks like a papilloma or polyp originating from the epithelium of the pancreatic duct . The changes are the effect of highgrade dysplasia of a borderline type or even a cancerous lesion (local or invasive). The peripheral type, which is encountered much more rarely, is mainly localized in the region of the uncinate process . One of the features of its morphological presentation is a set of cysts resembling a cluster of grapes . The cystic appearance of the lesion renders it similar not only to a mucinous tumor, but also to a pseudocyst . The grape - like presentation is caused by the dilatation of numerous peripheral pancreatic ducts filled with mucus . Sometimes, but rarely, instead of the lesion described above, a single, considerably dilated peripheral duct is visualized which forms the structure of the cyst . At present, imaging examinations, such as ultrasound, but ct and mri in particular, are capable of relatively accurate visualization of the dilatation of the pancreatic duct, secondary cystic lesions and connection between the duct and altered, dilated cystic structure . The decisive examinations in the case of ipmn are: mrcp, eus and ercp . Ercp allows for the identification of the intraductal lesions and collection of the pancreatic juice in order to determine the content of mucin, obtain a cytological picture as well as determine the level of tumor markers in the aspirate . This examination may simultaneously wash out and even temporarily decompress the duct from dense mucus . Mrcp, on the other hand, is the method of choice in the diagnosis and assessment of ipmn progression . It may prove more accurate in identification of the peripheral types as compared to ercp . The function of imaging examinations is also to determine a potential infiltration of adjacent tissues, enlargement of the lymph nodes or confirmation of existing metastases . Eus allows for visualizing the connection between the main pancreatic duct and dilated peripheral ducts . The examination is also performed to differentiate between hypoechoic mucus and internal growths of papillary or polypoid type . It accurately assesses the thickening of the ductal wall and in peripheral types the thickening of the cystic wall and presence of intramural nodules which are normally signs of malignant transformation . The risk of developing a malignancy rises with the increase in the diameter of the main pancreatic duct, in the case of visualizing a large amount of mucus crossing through the ampulla of vater and when obstructive jaundice and/or diabetes are clinically confirmed . Eus examination is extended by eus - fna thanks to which we may conduct cytological tests, determine the concentration of tumor markers and amylase and perform fnab of enlarged peripancreatic lymph nodes . The risk of malignant transformation of a central - type ipmn involving the main pancreatic duct is estimated at 70% . In the case of the peripheral type, it is 25% . Therefore, there are specific indications for a surgical resection of a central - type tumor, even for total pancreatectomy if the main duct is involved to a great extent . When the tumor is localized peripherally, the decision concerning surgical treatment should be made based on the general condition of the patient and on associated risks and benefits of such a procedure taking into account the presence of given symptoms . Furthermore, the progression of the local tumor (> 3 cm) and other patient's conditions should also be taken into consideration . In many cases intraoperative ultrasound examination helps to decide about the range of surgery and resection margin . It is also useful in differentiating between pancreatic cysts and cystic tumors if following the previous examinations, both imaging scans and others, there are still certain diagnostic doubts . So far, over 600 cases have been reported which constitutes approximately 1% of all cystic neoplasms of the pancreas . In over 90%, it develops in women in the 2 and 3 decades of life . Usually wellcircumscribed, the tumor is composed of solid part, cystic lesions, papillary structures, areas of necrosis and hemorrhagic components which are a result of numerous extravasations into the tumorous tissue . The tumors may develop both in the head and in the tail of the pancreas . It has been reported to grow to 135 cm but the average size is 6 cm . The recommended imaging examinations in the diagnosis of spn are: us, ct and mri . The scans reveal a well - encapsulated, heterogeneous tumor with areas of cystic degeneration and hemorrhagic components . Other examinations include tumor marker assessment, recommended markers are as follows: 1-antitrypsin, 1-antichymotrypsin, neuron - specific enolase (nse) and vimentin . Spn shows a relatively low malignancy potential and the risk is estimated at 15% . In 1015% of patients, the neoplasm metastasizes to the liver, infiltrates vascular structures and adjacent organs (fig . A surgical resection of the tumor with adequate portion of the pancreas (proximal or distal resection) is the treatment of choice . It is also recommended to surgically remove metastases and to perform lymphadenectomy . Even in such cases, the prognosis is good and longterm survival depends on the specific biology of the spn tumor . The remaining cystic neoplasms are encountered rarely and thus, their separate description is not needed . The tumor localized in the tail of the pancreas, approximately 10 cm in diameter . The content of the lesion is solid with signs of necrosis or hemorrhage which are responsible for the cystic capacity . The tumor with malignant potential malignant transformation of spn . Contrast - enhanced ct scans show a mixed solid and cystic mass in the pancreatic head (10 7.5 cm). Thick, well - circumscribed capsule, hyperdense areas due to hemorrhage, and areas of necrosis they are usually solitary, with the size of 1.535 cm and constitute approximately 30% of all primary pancreatic cystic tumors . We mainly encounter two types of such lesions: microcystic cystadenomas and macrocystic or oligocystic cystadenomas . The microcystic form usually develops in women at the age of 5060 and its typical localization is the body and tail of the pancreas . It is usually asymptomatic and signals, such as pain in the abdomen, weight loss, feeling of distension in the epigastric region and peristalsis disorders appear when the lesion shows large growth . A typical form of the tumor is composed of a large number of small cysts lined with cuboidal epithelium producing glycogen . The size of the cysts ranges from 0.2 to 2.0 cm and the entire tumor measures 1.430 cm . The lesion may grow locally leading to almost complete damage of the healthy pancreatic tissue . In imaging examinations (us, ct), such a cystic tumor frequently has the appearance of a honeycomb which is related to a large number of small cysts (fig . The lesion is relatively well - circumscribed with a central scar or, sometimes, with a calcification . The cystic mass is situated in the pancreatic tail, about 5 cm in diameter ious . Well - defined microcystic tumor, poorly vascularized, adhering to the splenic vessels (arrow) (color doppler) doppler us . Serous cystadenoma in the head of the pancreas, poorly vascularized, 5.6 cm in diameter . Structure with oligocystic components and central scar (arrow) contrast - enhanced ct shows a classic serous cystadenoma in the head of the pancreas (4.1 2.9 cm). The lesion has the appearance of a solid mass with numerous small cysts and septations honeycomb structure . The calcified central scar (arrow) the other form of the tumor, i.e. Macrocystic or oligocystic cystadenoma, with its appearance resembles a mucinous cystic tumor and is difficult to differentiate based on imaging examinations . It develops equally frequently in men and women, usually, after the age of 40 . Therefore, the basic symptoms of its presence, next to the feeling of discomfort in the epigastric region and sometimes, pain when the lesion grows, is jaundice the tumor is composed of other well - circumscribed cystic lesions, with their diameters usually greater than 2 cm . They are generally single cysts or there are a few poorly circumscribed ones with some internal septations . In the present - day imaging diagnosis it is a modern us examination with the use of contrast media which considerably broadens diagnostic possibilities of doppler us examination . Ceus allows for a good visualization of the honeycomb appearance and of the septations inside a neoplastic cyst . When, in small cystic tumors, which do not qualify to surgical treatment, ceus is performed, there is no need for further examinations, such as mri, mrcp and even contrast enhanced ct . Owing to the high resolution, one may visualize lesions composed of very slight cysts (of 5 mm in diameter) in a more detailed manner as compared to conventional ultrasound . Moreover, eus examination enables precise visualization of the honeycomb structure, calcifications and internal septations . It appears that a confirmation of a microcystic structure is sufficient for differential diagnosis and there is no need for the analysis of the fluid collected by means of eus - guided fine - needle aspiration biopsy (eus - fna). What is more, the collection of adequate amount of fluid from such slight structures is frequently not feasible . Fluid aspiration and its examination seem necessary when a larger cyst with a suspected neoplastic character has been visualized ., the collected fluid does not show any traces of mucin or amylase, and the test for the contents of cell elements (cytological examination) is possible in less than 50% of cases . Contrary to patients with marked malignant transformation, the level of tumor markers (mainly cea) is low, usually below 5 ng / ml . In the material presented herein, in the case of serous tumors, aspiration demonstrated correct level of the markers (cea and ca 19 - 9) and cytological examination did not reveal any neoplastic cells . A great majority of pancreatic serous tumors are benign the possibility of their malignant transformation concerns less than 3% of cases . A surgical resection of such a tumor together with a fragment of the pancreas is indicated in symptomatic lesions, in tumors that grow during the period of monitoring and in cases in which malignant transformation cannot be ruled out . In patients qualified to surgical treatment, ious appears to be useful both in terms of final verification of the diagnosis and in the assessment of the range of resection . According to the quoted literature, the size of the tumor at which surgery should be considered is over 45 cm (fig ., tumors tend to grow slowly (on average, 0.12 cm / year). It was observed that tumors larger than 4 cm grow faster (on average, 1.6 cm / year), symptoms begin to appear and malignant transformation becomes more possible . It may happen that in imaging scans, a tumorous lesion shows local infiltration on its surrounding structures but the possibility to diagnose carcinoma based on imaging methods, endoscopic techniques and biopsy are limited . Serous cystadenoma situated in the pancreatic body . A symptomatic lesion, 5.5 cm in the diameter, qualified to surgical resection ious . Postoperative specimen the debate whether serous tumors should be only observed or at once qualified to surgical treatment is still open . There are mixed opinions since the cost of check - ups is considerable and the sole awareness of patients of a potential threat to develop a cancerous disease is of great significance . Check - up examinations, including us and ct, ought to be performed at least every 12 years . In the remaining cases, resection of the tumor it might be performed with the application of minimally invasive techniques when the lesion is relatively small and when surgical treatment should proceed with no complications . In our department, the aforementioned principles are observed . Contrast - enhanced ct a small hypodense cystic tumor in the pancreatic tail (2.5 2 cm). It is currently believed that mucinous cystic tumors constitute the most common form of pancreatic cystic neoplasms (1045%). Due to the fact that in numerous cases they resemble ordinary cysts, a proper differentiation between these two is essential . They are encountered almost only in women (95%), mainly middle - aged ones, and are usually localized in the body and tail of the pancreas . A mean size of the tumor during the first diagnosis equals> 5 cm and in certain cases, the lesion may grow even up to 2535 cm . Such lesions are usually solitary and do not maintain any connection with the main pancreatic duct . The most frequently encountered form of such a tumor is a single large lesion of cystic character with or without internal septations and with a well - defined, usually thickened outer wall (fig . The cyst is lined with columnar epithelium which produces mucus that forms its inner layer . These cysts usually contain a certain amount of mucous substance and one may also find sanguineous contents after hemorrhages or necrotic changes . Typical radiological features of the tumor include: septations, solid intramural nodules and peripheral calcifications . In ultrasound examination, a mucinous tumor is visualized as a hypoechoic, well - circumscribed mass with diverse, often irregular thickening of the wall and sometimes, with solid nodules or calcifications . Ceus examination allows for a better identification of the lesions within the wall (nodules) and thickened septations, which is caused by increased vascularity of the altered structures attesting to malignant transformation of the tumor . From the histological viewpoint the visualization of the symptom triad, i.e. Irregular cystic wall thickening, peripheral calcifications and intramural solid lesions, in us, ct or mri may correspond to malignant transformation even in 95% cases (fig . Only a slight per cent of mucinous cystic tumors present a microcystic form, usually a slight one, single with few septations . In such cases, the cystic lesion is situated in the head of the pancreas with low - grade dysplasia (arrows) us power doppler . Cystic neoplasm with reduced fluid capacity, mainly growth of solid tissue in order to learn the morphology of cystic tumors, eus examination is performed . This imaging modality provides reliable information allowing for the characterization of the cyst and determining the possibilities of resection when features of malignancy are present . Due to the extension of the method to include aspiration biopsy (eus - fna), it is possible to find extracellular mucus in the cystic fluid, conduct cytological or, more rarely, histological analyses, make biochemical assessment, determine the level of tumor markers and perform molecular analysis . As for cytological analysis, the reasons may be technical difficulties to collect fluid (too little amount) or low contents of cell elements in the aspirate . From among numerous tumor markers, the cut - off value of 200 ng / ml, higher values obtained in the analysis of cystic fluid indicate the presence of a mucinous tumor . In the statistical analysis for this level, a similar situation may be observed in the analysis of amylase concentration in the fluid . For the cut - off value of 250 iu / l, in the case of this level, the sensitivity and specificity constituted 45% and 89% respectively . Unfortunately, the papers published so far do not specify the level of cea marker above which the cancerous transformation might be confirmed . This is verified after thorough evaluation of all data, particularly, imaging examinations and possible findings of eus - fna . What is important is the fact that small mucinous tumors may manifest no symptoms in as many as 75% of patients . When symptoms appear, the tumor is usually large and potentially malignant . Therefore, in all such cases rapid diagnosis and surgical resection of the tumor are essential . The basis for the qualification to surgical treatment is the outcome of imaging examinations, clinical examination and, to a lower degree, also data obtained from cystic fluid aspiration . When a mucinous tumor of the pancreas is identified, one should remember that such lesions are frequently malignant or of highgrade dysplasia and they qualify to surgical treatment . A total evaluation of the tumor's character is possible only following its resection, after histopathological examination . In our material, the examination of a postoperative specimen verified the presence of malignant mucinous tumors in two patients . Aspiration biopsy performed prior to the procedure revealed elevated cea level and presence of mucus . Cytological examination gave positive outcomes in the analysis of fluid from the inside of the cystic tumor in only one patient . This type of tumor is usually encountered in elderly men, aged 6080 . In a large majority of patients, the most common symptom is recurrent pain, similar to that in the course of chronic, exacerbating pancreatitis or episodes of acute pancreatitis, occurring mainly after meals . Such mucus travels to the pancreatic duct where it contributes to large pressure rise, which causes significant dilatation of the duct . The features of the tumor's image depend on its type and localization in the pancreas . We distinguish three types of ipmn: central type which encompasses the main pancreatic duct, peripheral type involving the branches of the ducts, and mixed - type . The central type is the most common (even up to 75% of cases). The lesion looks like a papilloma or polyp originating from the epithelium of the pancreatic duct . The changes are the effect of highgrade dysplasia of a borderline type or even a cancerous lesion (local or invasive). The peripheral type, which is encountered much more rarely, is mainly localized in the region of the uncinate process . One of the features of its morphological presentation is a set of cysts resembling a cluster of grapes . The cystic appearance of the lesion renders it similar not only to a mucinous tumor, but also to a pseudocyst . The grape - like presentation is caused by the dilatation of numerous peripheral pancreatic ducts filled with mucus . Sometimes, but rarely, instead of the lesion described above, a single, considerably dilated peripheral duct is visualized which forms the structure of the cyst . At present, imaging examinations, such as ultrasound, but ct and mri in particular, are capable of relatively accurate visualization of the dilatation of the pancreatic duct, secondary cystic lesions and connection between the duct and altered, dilated cystic structure . The decisive examinations in the case of ipmn are: mrcp, eus and ercp . Ercp allows for the identification of the intraductal lesions and collection of the pancreatic juice in order to determine the content of mucin, obtain a cytological picture as well as determine the level of tumor markers in the aspirate . This examination may simultaneously wash out and even temporarily decompress the duct from dense mucus . Mrcp, on the other hand, is the method of choice in the diagnosis and assessment of ipmn progression . It may prove more accurate in identification of the peripheral types as compared to ercp . The function of imaging examinations is also to determine a potential infiltration of adjacent tissues, enlargement of the lymph nodes or confirmation of existing metastases . Eus allows for visualizing the connection between the main pancreatic duct and dilated peripheral ducts . The examination is also performed to differentiate between hypoechoic mucus and internal growths of papillary or polypoid type . It accurately assesses the thickening of the ductal wall and in peripheral types the thickening of the cystic wall and presence of intramural nodules which are normally signs of malignant transformation . The risk of developing a malignancy rises with the increase in the diameter of the main pancreatic duct, in the case of visualizing a large amount of mucus crossing through the ampulla of vater and when obstructive jaundice and/or diabetes are clinically confirmed . Eus examination is extended by eus - fna thanks to which we may conduct cytological tests, determine the concentration of tumor markers and amylase and perform fnab of enlarged peripancreatic lymph nodes . The risk of malignant transformation of a central - type ipmn involving the main pancreatic duct is estimated at 70% . In the case of the peripheral type, it is 25% . Therefore, there are specific indications for a surgical resection of a central - type tumor, even for total pancreatectomy if the main duct is involved to a great extent . When the tumor is localized peripherally, the decision concerning surgical treatment should be made based on the general condition of the patient and on associated risks and benefits of such a procedure taking into account the presence of given symptoms . Furthermore, the progression of the local tumor (> 3 cm) and other patient's conditions should also be taken into consideration . In many cases intraoperative ultrasound examination it is also useful in differentiating between pancreatic cysts and cystic tumors if following the previous examinations, both imaging scans and others, there are still certain diagnostic doubts . Spn is a rare tumor . So far, over 600 cases have been reported which constitutes approximately 1% of all cystic neoplasms of the pancreas . In over 90%, it develops in women in the 2 and 3 decades of life . Usually wellcircumscribed, the tumor is composed of solid part, cystic lesions, papillary structures, areas of necrosis and hemorrhagic components which are a result of numerous extravasations into the tumorous tissue . The tumors may develop both in the head and in the tail of the pancreas . It has been reported to grow to 135 cm but the average size is 6 cm . The recommended imaging examinations in the diagnosis of spn are: us, ct and mri . The scans reveal a well - encapsulated, heterogeneous tumor with areas of cystic degeneration and hemorrhagic components . Other examinations include tumor marker assessment, recommended markers are as follows: 1-antitrypsin, 1-antichymotrypsin, neuron - specific enolase (nse) and vimentin . Spn shows a relatively low malignancy potential and the risk is estimated at 15% . In 1015% of patients, the neoplasm metastasizes to the liver, infiltrates vascular structures and adjacent organs (fig . A surgical resection of the tumor with adequate portion of the pancreas (proximal or distal resection) is the treatment of choice . It is also recommended to surgically remove metastases and to perform lymphadenectomy . Even in such cases, the prognosis is good and longterm survival depends on the specific biology of the spn tumor . The remaining cystic neoplasms are encountered rarely and thus, their separate description is not needed . Solid pseudopapillary neoplasm (spn). A large symptomatic lesion, well - circumscribed, surrounded by a thick capsule . The tumor localized in the tail of the pancreas, approximately 10 cm in diameter . The content of the lesion is solid with signs of necrosis or hemorrhage which are responsible for the cystic capacity . The tumor with malignant potential malignant transformation of spn . Contrast - enhanced ct scans show a mixed solid and cystic mass in the pancreatic head (10 7.5 cm). Thick, well - circumscribed capsule, hyperdense areas due to hemorrhage, and areas of necrosis the procedural treatment is applied in the case of large (> 4 cm), symptomatic cysts of the pancreas.endoscopic drainage constitutes an effective and safe method of minimally invasive treatment of pancreatic cysts and surgical procedures are an alternative when minimally invasive techniques are not possible.the differentiation of a neoplasm from a typical cyst is of fundamental significance for the selection of an adequate treatment method . The procedural treatment is applied in the case of large (> 4 cm), symptomatic cysts of the pancreas . Endoscopic drainage constitutes an effective and safe method of minimally invasive treatment of pancreatic cysts and surgical procedures are an alternative when minimally invasive techniques are not possible . The differentiation of a neoplasm from a typical cyst is of fundamental significance for the selection of an adequate treatment method . Authors do not report any financial or personal links with other persons or organizations, which might affect negatively the content of this publication and/or claim authorship rights to this publication.
A boy, 11 years old, was first seen for spinal deformity by a spinal surgeon in july 2011 . The child's medical history indicated that he had outpatient appointments with an orthopedic surgeon for limb deformities . Orthopedic status showed disproportionate stature; evident cachexia with a weight of 18 kg; decompensated, rigid, extended right - side cervicothoracic scoliosis with torso deviated to the right and head to the left up to 90 degrees of cobb's angle; gibbus on the right; hypertrophic muscles of the neck more severe on the right; clinodactyly and hypertrophied phalanges of the fingers; and grade photo of the patient at the intensive care unit of the regional children's hospital . Coordination tests were performed with intention; tendon reflexes of upper and lower limbs were brisk and symmetrical, and muscle strength of the upper limbs was graded as 5 and lower limbs as 3 according to american spinal injury association standard neurological classification of spinal cord injury . Emergency admission to the regional children's hospital was required on august 1, 2011, for a severe condition with complaints of spitting up blood, coughing, and frequent episodes of sleep apnea lasting to 1 minute . Mechanical lung ventilation and antibacterial and syndrome - based therapy were performed at the intensive care unit . Imaging findings: (a, b) radiographs of the spinal column: c4 lateral nonsegmented hemivertebra on the right (marked with arrow), scoliotic curve, and decrease in the volume of thoracic cage . An external fixation device was applied for halo - pelvic traction by a team of spinal surgeons of the kurgan ilizarov center on august 5, 2011 . The frame was set up in the distraction mode of 6 cm to correct spinal deformity and to create favorable conditions for lung excursion (fig . However, tracheostomy was performed due to progressive respiratory failure on august 1, 2011 . The patient's condition stabilized, and in 2 days he was transferred to autonomous breathing with tracheostomy and at 7 days he was transported to a somatic department (on august 25, 2011). Photo (a) and radiograph (b) of the patient following deformity correction with halo - pelvic system assembled of the ilizarov components . The patient was referred from the regional children's hospital to the neurosurgery department of the russian ilizarov scientific centre restorative traumatology and orthopaedics on september 29, 2011 . The diagnosis on admission indicated ollier disease with multiple deformities of axial skeleton, severe combined scoliosis with underlying systemic pathology and disturbed cervical spine formation, progressive muscular dystrophy, pneumofibrosis of the right lung, myocardiodystrophy, grade 3 cachexia, tracheostomy, and frame application for halo - pelvic traction . On admission to the center, the patient was able to stand independently for 10 minutes and walk with support for 6 minutes without respiratory distress or imbalance, but he needed help with his meals and getting up . His torso balance improved in the coronal plane with persistent decompensation, but sagittal balance was compensated (fig . 3a). Deformity correction with instrumentation was achieved up to 32 degrees (31 degrees) at the level of major cervicothoracic curve and up to 10 degrees (2 degrees) at the level of accompanying lumbar arch (fig . Operative intervention performed on september 29, 2011, included correction and multisupport instrumentation fixation of c2t9 segments with combined connector - type system (summit - expedium; depuy international ltd ., leeds, uk), posterior local spondylodesis, and dismantling of the halo - pelvic traction system (fig . The procedure involved incision at the level of c2c10 spinous processes after cleaning the operation field . Evident torsion to the apex of the arch was observed in the superior portion of the thoracic spine . Pairwise interlaminar screws were placed in c2; lateral mass screws (summit) introduced in c4, c5, c6; and transpedicular and intracorporeal screws inserted in t6, t7, t8, t9 (on the left) and t6, t7, t9 (on the right). Connecting rods 3.5/5.5 were laid over the fixation points and connected at c6t6 level bilaterally with four parallel connectors . Acute correction with tensioned rods was produced with distraction provided on the left and compression on the right under protection of methylprednisolone injection . The construct was reinforced by two transverse bars mounted to the cervical spine and one connection plate to the thoracic spine . Loss of blood volume was 200 ml (5% of the total body volume). Functional result assessed at 1 month after the surgery showed increased vital capacity of 56% according to spirometry findings and improved respiratory function with pulmonary stress test . The patient could walk with support for 30 minutes without respiratory imbalance, and he could eat food and stand up by himself, with torso balance in the coronal and sagittal planes being compensated (fig . Checkup radiographs showed adequate screw position; spinal deformity correction was achieved at the level of major cervicothoracic curve to 12 degrees (51 degrees) and at the level of accompanying lumbar arch to 10 degrees (2 degrees; fig . The child was discharged on november 4, 2011, and tracheostomy was removed on november 23, 2011 . Photo (a) and radiographs (b, c) of the patient prior to discharge . The objective of the demonstration was to emphasize medical considerations on system abnormalities of patients with severe progressive spinal deformity, so called high - risk deformities that entail a severely reduced quality of life due to static and dynamic imbalance and progressive life - threatening functional disorders.5 6 traditionally, such cases are considered incurable . It can be expected that the improved quality of peri- and postnatal care with persistent rate of worsening maternal health would inevitably lead to an increase in this patient cohort . The clinical example presented here demonstrates the modern possibilities of surgical treatment, which dramatically improved a child's condition and his quality of life . A strategy of surgical treatment involved application of an external stabilizing system in the position of moderate spinal correction and additional correction and stabilization with a posterior internal multisupport construct after improvement of his general condition . Tactical application included a halo - pelvic device assembled of ilizarov components and a hybrid system with transpedicular supporting points, connecting rods, and connectors that provided staged distraction during growth.7 8 9 legal consideration of surgical treatment with detailed substantiation of a lifesaving procedure is required.
Despite that the economy is developing steadily, quang tri province is still one of the poorest areas in vietnam which was affected by world war ii and the vietnam war . Its gross domestic product per person was around us$360 and the poor strata is consisted to take up 14% of the population, higher than 11% of total vietnam . Moreover, it is the most vulnerable provinces in the country for flood and drought . The provincial residents are still suffered from the land degradation and health disorders by damage agent orange . Nevertheless, the government support policy for financial and medical service is very insufficient1 . The 37,000 people with disabilities in quang tri including 15,000 damaged agent orange is treated by only 11 physical therapists2 . Thus, more health care personnel and network for rehabilitation are needed . To solve these social problems, the council of people s commissars of quang tri province established policies to increase up to 90% the approach for treatment for agent orange victims and 85% the early detection of children with congenital disabilities . According to these policies, the education for physical therapy has appeared to more important and urgent task . The educational institution for physical therapy assistant is quang tri medical college, which is the only place in quang tri province . In quang tri medical college, the curriculum of physical therapy for enrolled student in the department of medicine and nursing are 2 prerequisites such as introduction to physical therapy (theory 16 hours, practice 8 hours) and community - based rehabilitation (theory 15 hours, practice12 hours) (tables 1table 1.the curriculum of introduction for physical therapyno.contentscomplete timetheorypractice1introduction of rehabilitation, the role of nurse and maternity nurse for patient care2 - 2introduction of physical therapy and methods of therapy for rehabilitation2 - 3.1physical therapy and rehabilitation for joint disorders223.2physical therapy and rehabilitation for nervous disorders323.3physical therapy and rehabilitation for cerebral palsy disorders213.4physical therapy and rehabilitation for fracture disorders113.5physical therapy and rehabilitation for respiratory disorders213.6physical therapy and rehabilitation for patient of before and after operation113.7physical therapy and rehabilitation for pregnant woman of before and after childbirth1-total168 and 2table 2.the curriculum of community - based rehabilitationno.contentscomplete timetheorypractice1introduction of rehabilitation and community - based rehabilitation3 - 2general rehabilitation for patient with disabilities 113rehabilitation for patient with motor disability444rehabilitation for patient with visual disability115rehabilitation for patient with auditory - speech disability116rehabilitation for patient with sensory disability117rehabilitation for patient with epilepsy118rehabilitation for patient with abnormal behavior disability119rehabilitation for patient with learning disability1110methods for life adaptation11total1512). Moreover, the educational course for physical therapy assistant has been operated from 2012 and the recipients are the general public who work in local health center and clinic and which was established by government and volunteering group . It is consisted to 330 hours during 3 months period, presented as table 3table 3.the curriculum for physical therapy assistantno.contentscomplete timetheorypracticetotal1introduction of rehabilitation8082procedure and prevention of disability124163methods of physical therapy and rehabilitation164204rehabilitation by community814225rehabilitation for patient with motor disability and therapeutic instrument applied to everyday lives816246rehabilitation for patient with cerebral palsy832407rehabilitation for patient with palsy of upper and/or lower limb832408rehabilitation for patient with fracture832409rehabilitation for patient with visual disability4162010rehabilitation for patient with auditory - speech disability4162011rehabilitation for patient with learning disability4162012rehabilitation for patient with sensory disability4162013rehabilitation for patient with abnormal behavior disability4162014rehabilitation for patient with epilepsy41620total100230330 . The will of the government and quang tri medical college to educate for professional personnel of physical therapy is strong, however, due to financial deficits and other various limitations, there are difficulties to achieve this goal . For global approach and support to overcome these difficulties, we established the adaptive and reinforced curriculum for physical therapy assistant by korea international cooperation agency (koica). The curriculum includes the educational activities or its related plan and program according to educational purpose for the intent of educational institution . It includes the educational purpose and contents, teaching procedure, learning experience, and factors of education evaluation . In addition, the development of curriculum is focused on the analysis of demand and situation, plan about learning results, procedure organization, selection and preparation of teaching materials, provision of effective teaching, and evaluation . It is a comprehensive process and contains the gradual process for planning the educational procedure, enforcement, and evaluation, gradually . Thus, in procedure on development of curriculum, demand analysis should be performed preferentially in the process of plan to educational procedure and enforcement . After setting up the educational purpose and aim which are based on the demand, the educational contents are selected . Afterwards, the teaching materials is selected or adjusted, and the problems are assessed through the evaluation of teaching and learning . Vendrely and cater suggested that physical therapist education is made up of required subject including the liberal arts curriculum, basic medical subject, and physical therapy subject, and optional subject including clinical medical subject for acquiring the professional knowledge and therapy technique to establish the firm personality and work ethic5, 6 . It should be contained to learn the correct attitude as health and medical service personnel . Moreover, physical therapist education need to include the attitude of lifelong learning for new approach in the rapidly changing medical circumstances7, 8 . Therefore, it is very important to develop the proper curriculum for quang tri province of vietnam . In the present study, we planned to develop the education program for physical therapy assistant to provide high quality physical therapy service and professionalism . Thus, through the scientific and practical education for physical therapy assistant, it would be reinforced their capability as well as improved the quality of life of provincial residents . Subjects are consisted of 9 professors in quang tri medical college and 1 physical therapist in quang tri general hospital which are located in dong ha city, quang tri province . This study was also approved by the institutional review board (irb) of eulji university of korea (eu16 - 21). The general characteristics of the subjects were presented to table 4table 4.the general characteristics of subjectscontentsfrequency (number)percents (%) gendermale220.0female880.0age2130110.03140330.04150330.05160330.0affiliationcollage990.0hospital110.0degreebachelor550.0master220.0doctor330.0occupationphysical therapist110.0professor770.0medical doctor220.0careerunder 10 years330.010 years or more770.0 . The survey research to lecturer for education of physical therapy assistant in quang tri medical college was conducted as pre - analysis of demand for the physical therapy assistant curriculum development . First of all, it was measured as 4 question to ask the expectation of education using likert 5-point scale which subject was education beneficiary . Additionally, the priority rank of expectation and consciousness was measured in 5 subjects including introduction of rehabilitation, basic kinesiology, physical therapy for neurology & pediatrics, physical therapy for international medicine & surgery, and basic measurement for joint and muscle . Cronbach s value to measure the reliability coefficient of question for the educational expectation was 0.801 . The statistical analysis of the present study was used spss 18.0 package for window version . First, the reliability analysis was conducted to measure the internal consistency in research question . Second, the frequency analysis was conducted to measure the background variable and characteristics of subjects . Third, the descriptive statistics was conducted to measure the pre - analysis of expectation for the physical therapy assistant curriculum development . In addition, independent t - test and anova were conducted to measure differences of the educational expectation according to background variable of subjects . The yielder ranking was conducted to measure the consciousness priority of the curriculum subject . The educational expectation was conducted to analyze the pre - analysis of expectation for the physical therapy assistant curriculum development (table 5table 5.the educational expectationquestionminimummaximummeanstandard deviationavailability of acquisitional knowledge through the education454.500.53necessity of education555.000.00help to improvement of educator capability454.900.32confidence about education participation354.500.85total454.720.42). In results of educational expectation about the curriculum total educational expectation this data shows that the educational expectation was high . In the result of differences of educational expectation according to background variable, the score differences of educational expectation according to gender, major, and degree were not significant, however, the educational experience was significant (table 6table 6.the difference of educational expectation according to background variablesbackground variablescontentsfrequencymeanstandard deviationp-valegendermale24.750.350.90female84.710.31majormedicine44.750.200.61nursing54.650.38others15.00.degreebachelor54.700.410.77master24.660.14doctor34.870.18educational experienceyes44.930.130.04no64.580.30p<0.05). Mean (standard deviation) of experienced education group about physical therapy was 4.93 (0.13) and experience less was 4.58 (0.30). As shown in table 7table 7.consciousness priority of the curriculum subjectcurriculum subjectrank 1frequencyrank 2frequencyrank 3frequencyconsciousnessrankintroduction of rehabilitation8112basic kinesiology911physical therapy for neurology & pediatrics643physical therapy for international medicine & surgery911basic measurement for joint and muscle822, among the consciousness priority of each curriculum subject, the priority rank of basic kinesiology and physical therapy for international medicine & surgery were 9, which was the highest first rank frequency . Second rank subjects were introduction of rehabilitation and basic measurement for joint and muscle which priority rank were 8 and appeared to high consciousness . Third rank subject was physical therapy for neurology & pediatrics which priority rank was 7 . Therefore, the consciousness priority rank was appeared by basic kinesiology and physical therapy for international medicine & surgery, introduction of rehabilitation and basic measurement for joint and muscle, and physical therapy for neurology & pediatrics . The curriculum subjects were made up based on these results in the present study . The educational curriculum was developed according as attention points which was suggested by tyler (table 8table 8.development of curriculum of physical therapy assistantsubject / contentscomplete timetotal (h)theory (h)practice (h)introduction of rehabilitation301515purpose: introduction of rehabilitation is for understanding about physical therapy and fundamentals of patient care skills . It includes simple nursing procedures necessary, charting of the care and comfort of patients, and communication with patient care.1introduction of physical therapy5322public health151053education for patient s family and community - based rehabilitation1028basic kinesiology502030purpose: basic kinesiology is the integration of facts and principles derived from the field of anatomy, physiology, and biomechanics with implications for normal physical activity, conditioning and therapeutic exercise.1basic anatomy10552physical biomechanics6333range of motion10464therapeutic exercise using range of motion6245activities for patient transfer10466proper patient posture 826basic measurement for joint and muscle301515purpose: basic measurement for joint and muscle is designed to assist students in acquiring an understanding and skills in the basic measurement procedure of physical therapy including gross and definitive measurement of muscle strength, joint motion, and posture.1performance and contents of patient counsel3122systemic observation, mental status, and functional evaluation3123understanding of vital sign3124musculoskeletal evaluation7435neurological evaluation7436assessment and evaluation for special needs child743physical therapy for international medicine & surgery1304090purpose: physical therapy for international medicine & surgery includes the motor relearning program, evaluation and treatment of patient demonstration, treatment of various orthopedic conditions, application of therapeutic exercise techniques to regions of the body (shoulder, elbow & forearm complex, wrist & hand, hip, knee, ankle & foot, spine & posture), and treatment of other musculoskeletal conditions.1ready for patient care2112infection management10373understanding and care for disability12484physical therapy for patient with disability by defoliant 8265application of walking aid6246physical therapy for joint disorder13497physical therapy for muscular disorder 13498physical therapy for cardiopulmonary disorder10379physical therapy for amputation103710physical therapy for non - progressive central nervous system disorder 93611physical therapy for progressive central nervous system disorder93612physical therapy for peripheral nervous system disorder 103713physical therapy and functional evaluation for geriatric cognition disorder103714accident and emergency measure826physical therapy for neurology & pediatrics602040purpose: physical therapy for neurology includes motor relearning program, evaluation and treatment of neurological injury with patient demonstration, treatment of various conditions and treatment of other neurological and pediatric conditions.1development of functional movement in children5322development and adaptation of musculoskeletal system3213physical therapy and management for spine in children3124physical therapy and management for amputation and disorder in extremities3125physical therapy for development coordination disorder7256physical therapy for children with disability of motor and/or cognition8267physical therapy for children with cerebral palsy8268physical therapy for brachial plexus injury 4139physical therapy for spinal cord injury52310physical therapy for brain injury (traumatic brain injury, hydrocephalus, brain tumor)82611physical therapy for spina bifida31212physical therapy for special environment and consideration in children312field education90090final examination301020total420120300). Introduction of rehabilitation was comprised of 30 hours totally including 15 hours for theory and 15 hours for practice . It contains introduction of physical therapy, public health, and education for patient s family and community - based rehabilitation . Basic kinesiology was comprised of 50 hours totally including 20 hours for theory and 30 hours for practice . It contains basic anatomy, physical biomechanics, therapeutic exercise using range of motion, and the others . Physical therapy for international medicine & surgery was comprised of 130 hours totally including 40 hours for theory and 90 hours for practice . It contains physical therapy for muscular disorder, physical therapy for progressive central nervous system disorder, physical therapy and functional evaluation for geriatric cognition disorder, and the others . Physical therapy for neurology & pediatrics was comprised of 60 hours totally including 20 hours for theory and 40 hours for practice . It contains development of functional movement in children, physical therapy for children with cerebral palsy, physical therapy for brain injury (traumatic brain injury, hydrocephalus, brain tumor) and the others . Except these subjects, the curriculum contains 90 hours of practice for the field education and 30 hours including 10 hours of theory and 20 hours of practice for final examination . Totally, the educational curriculum contains 420 hours including 120 hours of theory and 300 hours of practice . In the present study, we developed the educational curriculum for physical therapy assistant based on local physical therapy condition in vietnam . Survey results of the educational expectation and consciousness priority of the curriculum subject for providing the high quality medical service to provincial residents and establishing the professionality in physical therapy assistant . We developed the education program for physical therapy assistant according to tyler model which affects continuously and enormously in the late 20th century4 . Advantages of tyler model are summarized as follows: first, it has a wide availability which could be applied any subject and level . Second, it provides the logic and reasonable procedure, which is easy to follow to curriculum developer and course planer . Third, it provides broad guideline through the emphasis on the behavior and experience of leaner . Fourth, it has the totality including purpose, experience selection, experience organization, evaluation, without the discrimination of educational procedure and course . In addition, tyler suggested the continuance, sequence, and integration as criteria of organization on the educational experience . The sequence is to provide the chance for exercise and development of the curriculum factors repeatedly . It is related to the continuance and to organize the understanding, function, attitude, and interest to the individual level widely, deeply, and highly . While the continuance means repetition of same contents, the sequence means the arrangement in order of different contents . The integration is to concatenate the curriculum factors horizontally . First of all, the educational expectation was conducted to analyze the pre - analysis of expectation for the physical therapy assistant curriculum development . In results of educational expectation about the curriculum, especially, in the question of necessity of education, all educational recipients graded 5-points . These results mean that the educational expectation about the program for physical therapy assistant was very high . Moreover, in the result of differences of educational expectation according to background variable, the differences of educational expectation on scores according as the educational experience were significant . The consciousness priority rank appeared in the order of basic kinesiology and physical therapy for international medicine & surgery, introduction of rehabilitation and basic measurement for joint and muscle, and physical therapy for neurology & pediatrics . The curriculum subjects were made up based on these results in the present study . The curriculum for physical therapy assistant was developed to 5 main subjects including totally 420 hours (120 hours of theory and 300 hours of practice). Basic kinesiology, basic measurement for joint and muscle, physical therapy for international medicine & surgery, and physical therapy for neurology & pediatrics. Except these 5 main subjects, the curriculum contains 90 hours of practice for the field education and 30 hours including 10 hours of theory and 20 hours of practice for final examination . These contents were also considered the curriculum in quang tri medical college which was accredited to the ministry of labor society of vietnam . Introduction of rehabilitation was comprised of 30 hours totally including 15 hours for theory and 15 hours for practice . It contains introduction of physical therapy, public health, and education for patient s family and community - based rehabilitation . Introduction of rehabilitation is for understanding about physical therapy and the fundamentals of patient care skills . Additionally, it includes simple nursing procedures necessary, charting of the care and comfort of patients, and communication with patient care . Basic kinesiology was comprised of 50 hours totally including 20 hours for theory and 30 hours for practice . The purpose of this subject is the integration of facts and principles derived from the field of anatomy, physiology, and biomechanics with implications for normal physical activity, conditioning and therapeutic exercise . Thus, it contains basic anatomy, physical biomechanics, therapeutic exercise using range of motion, and the others . Basic measurement for joint and muscle is designed to assist students in acquiring an understanding and skills in the basic measurement procedure of physical therapy including gross and definitive measurement of muscle strength, joint motion, and posture . Basic measurement for joint and muscle was comprised of 30 hours totally including 15 hours for theory and 15 hours for practice . It contains performance and contents of patient counsel, systemic observation of mental status and functional evaluation, understanding of vital sign, and the others . Physical therapy for international medicine & surgery is designed to understand the motor relearning program, evaluation and treatment of patient demonstration, treatment of various orthopedic conditions, application of therapeutic exercise techniques to regions of the body (shoulder, elbow & forearm complex, wrist & hand, hip, knee, ankle & foot, spine & posture), and treatment of other musculoskeletal conditions . Thus, physical therapy for international medicine & surgery was comprised of 130 hours totally including 40 hours for theory and 90 hours for practice . It contains physical therapy for muscular disorder, physical therapy for progressive central nervous system disorder, physical therapy and functional evaluation for geriatric cognition disorder, and the others . Especially, it also includes the physical therapy for patient with disability by defoliant which was considered the present condition in quang tri province of vietnam . Physical therapy for neurology & pediatrics was comprised of 60 hours totally including 20 hours for theory and 40 hours for practice . It contains development of functional movement in children, physical therapy for children with cerebral palsy, physical therapy for brain injury (traumatic brain injury, hydrocephalus, brain tumor) and the others . The purpose of physical therapy for neurology is to study about motor relearning program, evaluation and treatment of neurological injury with patient demonstration, treatment of various conditions and treatment of other neurological and pediatric conditions . The detailed contents are based on the present condition of this area . On the basis of survey results of the educational expectation and consciousness priority of the curriculum subject and on the present local physical therapy condition it will be necessary to have an additional study about the usability and effectiveness of the developed curriculum via the comparison with the educational satisfaction.
We report here the first case of spontaneous rupture of kidney resulting from mixed cryoglobulinemia . A 44-year - old man presented with sudden onset of fever, acute pulmonary edema, left flank abdominal pain unassociated with trauma, and rapidly progressive renal failure requiring dialysis . Computed tomography of the abdomen revealed a large perirenal hematoma of the left kidney . During conservative surgery, the patient underwent renal biopsy that showed renal vasculitis and membranoproliferative glomerulonephritis with intracapillary microthrombi . The patient was better after immunosuppressive therapy, with the disappearance of clinical symptoms and the recovery of baseline renal function . We report on this case and discuss a possible link between spontaneous rupture of kidney and mixed cryoglobulinemia - associated sjgren s syndrome . Spontaneous rupture of kidney (srk) is uncommon . In most cases, it was a complication of benign or malignant kidney tumors . Only a few cases are a result of systemic vasculitis, particularly polyarteritis nodosa (pan), microscopic polyangiitis, and in sporadic reports, wegener s granulomatosis . To our knowledge a 44-year - old white man was admitted to our hospital because of acute pulmonary edema . His past medical history was significant for chronic tobacco use and recurrent episodes of arthralgias over the course of 2 years, which were treated symptomatically by the patient by self - medication with analgesics, without any specific medical management . The patient had been well until 2 months earlier, when he began to have worsening of his physical state, anorexia, weakness, weight loss, and diffuse myalgias . Three days before admission, he complained of acute left flank pain unassociated with trauma . In addition, the patient reported a significant decrease of urine volume (<0.5 l urine in 24 hours) and dyspnea . Physical examination showed high blood pressure (170/100 mmhg), his temperature was 38.7c, and his respiratory rate was 34 breaths per minute . The abdomen was tender, mainly over the left upper quadrant, and had no palpable hepatosplenomegaly . Exam of the lower limb found petechial purpura on his left leg, with livedo reticularis . Otherwise, the examination was normal . Laboratory findings revealed a rapidly progressive renal failure with a serum creatinine level of 1.71 mg / dl (normal value, 0.61.2 mg / dl), which increased to 4.02 mg / dl over the next 2 days; a blood urea nitrogen level of 57 mg / dl (normal value, 720 mg / dl), which also increased to 123 mg / dl; a hypervolemic hyponatremia of 124 meq / l (normal value, 135145 meq / l); and normal serum electrolytes otherwise . Twenty - four hour urinary protein levels were 0.6 g/24 hours (normal value, <0.03 g/24 hours), and urinary sediment was positive for microscopic hematuria . Hemoglobin was 9.7 g / dl (normal value, 1316 g / dl), white blood cell count was 18,300/mm (normal value, 4,0001,000/mm), and platelets were 626,000/mm (normal value, 150,000300,000/mm). Erythrocyte sedimentation rate was 80 mm / hour (normal value, <15 mm / hour), with a c - reactive protein level of 11.3 mg / l (normal value, 612 mg / l). Lactate dehydrogenase levels were elevated (1,243 u / l; normal value, <250 u / l). Ch50 was not checked, but complement component c3 was normal, at 80 mg / dl (normal, 80170 mg / dl), and c4 was strongly reduced, at 6 mg / dl (normal, 1640 mg / dl). There was no monoclonal paraprotein detected both on serum and urine protein immuno - electrophoresis . In addition, five blood samples for cultures (both aerobic and anaerobic) were sent to the laboratory in view of the patient s fever, leukocytosis, and high erythrocyte sedimentation rate, all of which were negative . Renal ultrasound showed normal - sized kidneys without evidence of hydronephrosis, but it allowed us to identify a subcapsular renal hematoma that measured 6352 mm in the upper pole of the left kidney, which was confirmed by abdominal computed tomography (ct) scan (figure 1a). Multislice computed angiotomography scanning identified the hematoma and ruled out microaneurysms of the main renal arteries, other vascular abnormalities, and renal tumors (figure 1b). Hemodialysis therapy was started because of oliguria, continuous decrease of renal function, and persisting pulmonary edema despite high doses of furosemide (500 mg / day), and then the patient was operated on with conservative surgery for renal hematoma . Renal biopsy was performed, during surgery, the third day after admission and was submitted for light microscopic and immunofluorescence examination (figure 2). The specimen consisted of renal medulla and cortex with 14 glomeruli . There was significant hypercellularity of glomeruli with numerous infiltrating neutrophils, minimal endocapillary proliferation, and one cellular crescent . There were thickening and reduplication of the glomerular basal membrane, suggesting membranoproliferative glomerulonephritis . In two glomeruli, we noted intracapillary amorphous deposits compatible with microthrombi . The laboratory tests were sent immediately after we received the kidney biopsy results, 72 hours after the biopsy had been performed . Serological studies for antineutrophil cytoplasmic antibodies, antiglomerular basement membrane antibodies, anti - double - stranded dna antibodies, and anti - smith antibodies were negative, excluding respectively pauci - immune vasculitis, goodpasture s syndrome, and lupus vasculitis . Skin - biopsy specimen of a purpuric lesion showed leukocytoclastic vasculitis without immunoglobulin a deposition, and therefore did not support the diagnosis of henoch - schnlein purpura . Tests for cryoglobulinemia were positive, including the presence of cryocrit (12%) with type ii mixed cryoglobulin (immunoglobulin g / immunoglobulin m kappa). We made additional investigations to determine the cause of mixed cryoglobulinemia, and assessment for hepatitis c virus (hcv) rna and hepatitis b virus dna by polymerase chain reaction was negative . Tests for sjgren s syndrome were positive with the presence of anti - ro (ss - a), anti - la (ss - b), lymphocytic infiltrate in minor salivary gland biopsy (grade 3 according to chisholm and mason grading system), and positive schirmer s test . Screening for lymphoma remained negative despite initial elevated lactate dehydrogenase levels . All these data confirmed that the patient had srk caused by type ii mixed cryoglobulinemia - associated sjgren s syndrome . The patient received 3 days of pulses of methylprednisolone (1 g / day), followed by oral prednisone (1 mg / kg / day) with a decreasing regimen and 6 monthly pulses of intravenous cyclophosphamide (1 g) as induction of remission . The patient remained febrile during hospitalization, with temperatures ranging between 38.5c and 38.8c, until he received the third pulse of methyl prednisolone, which was administered 12 days after admission . Arthralgias and the other symptoms disappeared, proteinuria fell to only trace amounts, and renal function returned to its baseline value (0.9 mg / dl) over the next 2 weeks, and c4 levels were again normal . Ethical approval was not needed in the present case report, because it did nt include research or experiments . There was no risk of causing harm or violating the patient s right, since it was presented in a way that respected the patient s privacy and confidentiality . It was first reported by bonet in 1700 and later described in more detail by wnderlich in 1856.1,2 to date, more than 200 cases have been reported in world literature . To the best of our knowledge, tumors, particularly renal cell carcinoma and angiomyolipoma, are the most common cause of srk, occurring in 57%73% of cases.3,4 vascular disease was reported in a meta - analysis of spontaneous renal hemorrhage as the next most common offender, with pan occurring most frequently.5 other vasculitis was also reported to be associated with srk, especially microscopic polyangiitis and wegener s granulomatosis.68 our patient had srk, probably as a result of vasculitis; however, he had neither pan nor pauci - immune vasculitis . Furthermore, the patient had also neither renal tumor, both in radiologic and histologic findings, nor renal infection . On the basis of clinical context and laboratory findings, we suggest that srk was a result of cryoglobulinemic vasculitis associated with sjgren s syndrome . Prevalence of hcv infection in patients with mixed cryoglobulinemia ranges from 30% to 96%.9,10 the etiologies of negative - hcv mixed cryoglobulinemia are essential mixed cryoglobulinemia (27%); lymphoproliferative disorders, especially b - non - hodgkin s lymphoma and myeloma (26%); autoimmune diseases, particularly sjgren s disease and lupus (34%); and infectious disease (13%), including hepatitis b virus and hiv.11 in the study of trejo et al,12 of 443 patients with cryoglobulinemia, 10% had sjgren s syndrome . Inversely, in large series of patients with sjgren s syndrome, mixed cryoglobulinemia, especially type ii, was present in 20% of cases and was characterized by a low ch50, cutaneous vasculitis, and poor prognosis resulting from more severe internal organ involvement and frequent evolution to malignant lymphomas.1315 vasculitis in sjgren s syndrome can occur either as a cutaneous localized form (leukocytoclastic vasculitis) or as systemic necrotizing vasculitis involving small - sized and medium - sized arteries of various organs related to the presence of cryoglobulinemia.16 it is well known that in sjgren s syndrome type b, there is aneurysmal dilatation of arteries ., there are only three reports: achkar et al described first a ruptured aneurysm of the inferior pancreaticoduodenal artery in the setting of rheumatoid vasculitis - associated sjgren s syndrome,17 curi et al reported a contained rupture of the visceral aorta in a patient with sjgren s syndrome associated with osteoarthritis,18 and klingler et al reported a rupture of a spinal artery aneurysm attributable to exacerbated sjgren syndrome - associated cryoglobulinemic vasculitis.19 in contrast, it is also known that cryoglobulinemic vasculitis may induce injuries in the vascular wall . Herold et al described cryoglobulinemic vasculitis associated with aortic aneurysms in pediatric cases of kawasaki disease,20 and au et al reported structural aortic abnormalities in four patients with negative - hcv cryoglobulinemia, including multiple dissections, aneurysms, and congenital aortic arch abnormalities.21 therefore, we presume that an exacerbation of the sjgren s syndrome - induced cryoglobulinemic vasculitis with necrotizing arteritis and fibrinoid necrosis of the vessel s media led to weakening of the arterial wall, formation of kidney aneurysm, and spontaneous rupture . This mechanism may also explain the occurence of microaneurysms and srk in pan and in rare cases of microscopic polyangiitis and wegener s granulomatosis.2226
5-ht3a receptor constructs and transfection of cdnas cdnas encoding human wild - type and mutant 5-ht3a receptor subunits were cloned into pgw1 (17). Point mutations were introduced using standard molecular biological techniques (31), and all cdnas were sequenced to confirm fidelity . Wild - type and mutant receptor subunit cdnas were co - transfected into tsa-201 or hek-293 cells, with a cdna encoding green fluorescent protein to identify transfected cells . Transfection was performed by electroporation (400 v, 125 microfarads, infinite resistance) using a bio - rad gene electropulser ii (biorad) or the calcium phosphate precipitation method . Transfected cells were routinely cultured in dulbecco's modified eagle's medium supplemented with 10% (v / v) fetal bovine serum, 2 mm glutamine, 1 mm sodium pyruvate, 100 g ml streptomycin, and 100 units ml penicillin and maintained at 37 c for 2448 h in 95% air, 5% co2 at 100% humidity before use . Electrophysiological recordings whole - cell and outside - out patch configurations were used to record macroscopic and single channel currents, respectively, from transfected cells . The recording chamber was routinely superfused (5 ml min) with an extracellular solution (e1) comprising (in mm): nacl 140, kcl 2.8, mgcl2 2.0, cacl2 1.0, glucose 10, hepes 10 (ph 7.2, adjusted with 1 m naoh; final [na]o = 146 mm). Patch electrodes (resistance = 28 megohms when measured in solution e1) were filled with an intracellular solution (i1) containing (in mm): cscl 140, cacl2 0.1, egta 1.1, hepes 10 (ph 7.2, adjusted by 1 m csoh, final [cs]i = 143 mm). In solution i2, cscl was totally replaced by nacl (ph 7.2, adjusted by 1 m naoh, final [na]i = 145 mm). The intracellular free calcium concentration ([ca]i) for i1 and i2 was estimated to be 10 nm (35). To determine the relative permeability of na versus cs (pna / pcs), in solution e2, additional nacl totally replaced kcl in the extracellular solution, and the concentrations of cacl2 and mgcl2 were each reduced to 0.1 mm to minimize the influence of divalent cations upon the reversal potential of the macroscopic current response to 5-ht (e5-ht). The permeability of ca relative to cs (pca / pcs) was determined using an extracellular solution (e3) containing (in mm): cacl2 100, l - histidine 5, glucose 10 (ph 7.2) (20). To prevent changes in reference electrode potential during the superfusion of media with altered ionic composition, a bridge containing 3 m kcl in agar (4% w / v) was employed . Liquid junction potentials arising at the tip of the patch pipette were measured as described by fenwick et al . The membrane potential was stepped from 60 mv to 100 mv for 100 ms and subsequently ramped to + 60 mv within 1 s at the peak of the macroscopic current response to pressure - applied 5-ht (1 m). Care was taken to ensure that the agonist - induced current recorded at 60 mv before and immediately following the voltage ramp did not change . Subtraction of the leakage current recorded in the absence of 5-ht from the current recorded in the presence of the agonist yielded the current - voltage (i - v) relationship attributable to the 5-ht - evoked conductance increase . In the second method, peak current responses to pressure - applied 5-ht (10 m) were recorded at steady holding potentials closely bracketing e5-ht, which was subsequently determined by interpolation . Concentration - response relationships to 5-ht (0.3100 m) were determined from the peak inward whole - cell current response recorded in the presence of solutions e1 and i1 at a holding potential of 60 mv ., san jose, ca) by an equation of the form (eq . 1)\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{i}{i_{{\mathrm{max}}}}=\left(\frac{[a]}{[a]+[{\mathrm{ec}}_{50}]}\right)^{n_{h}}\end{equation}\end{document} where i is the peak current elicited by 5-ht at concentration [a], imax is the peak current evoked by a saturating concentration of 5-ht, ec50 is the concentration of 5-ht that evokes a half - maximal response, and nh is the hill coefficient . To combine the concentration - response data obtained from several cells, agonist - evoked currents were normalized and expressed as a percentage of the maximal inward current response produced by a saturating concentration of 5-ht obtained for each cell . Single channel currents evoked by pressure application of 5-ht (10 m) were recorded from excised outside - out membrane patches clamped at steady holding potentials within the range of 100 to + 100 mv as specified under results . The solutions used in such experiments were i1 and e1, e2, and e3, as appropriate . Additionally, in experiments that determined e5-ht and single channel conductance in mixtures of extracellular na and ca, nacl was held at a constant concentration of 95 mm in the presence of variable concentrations of cacl2 (103 10 m), sucrose (090 mm, as appropriate to maintain constant osmolarity), glucose (10 mm), and l - histidine (5 mm; ph 7.2). Currents were recorded using an axopatch-1d amplifier (axon instruments, union city, ca), low pass - filtered (5 khz, bessel characteristic), and recorded onto magnetic tape using a bio - logic dat recorder (bio - logic, claix, france) for subsequent offline analysis . Data analysis permeability ratios (relative to cs) were determined from measurements of e5-ht and calculated ion activities (i.e. The ionic concentration multiplied by ion activity coefficient (ion)). The latter were estimated (following the guggenheim convention) to be: cs 0.72 (140 mm cs) na 0.76 (140 mm na), and ca 0.26 (100 mm ca). For varying concentrations of ca in the presence of 95 mm na, the mean molal activity coefficients for cacl2 in the presence of 100 mm nacl, as tabulated by butler (36), allowed calculation of ca . Sucrose had a negligible influence upon na over the range of sugar concentrations employed (37). The permeability ratio pna / pcs was calculated from the goldman - hodgkin - katz (voltage) equation, (eq . 2)\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}\;e_{5{\mathrm{-}}{\mathrm{ht}}}=\frac{rt}{f}{\mathrm{ln}}\frac{(p_{{\mathrm{na}}}/p_{{\mathrm{cs}}})[{\mathrm{na}}^{+}]_{o}}{[{\mathrm{cs}}^{+}]_{i}}\end{equation}\end{document} where r, t, and f have their usual meaning and [na]o and [cs]i are the calculated activities of extracellular na and internal cs ions, respectively . This equation ignores the very small error anticipated due to the presence of low concentrations of permeant divalent ions (e.g. Ca) within the extra- or intracellular solutions . The permeability ratio, pca / pcs, was calculated from a modified goldman - hodgkin - katz (voltage) equation (38) which, with both na and ca present as permeant species in the extracellular medium but only cs and negligible ca present in the pipette (` intracellular') solution, can be written as (20) (eq . 3)\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}\;e_{5{\mathrm{-}}{\mathrm{ht}}}=\frac{rt}{f}{\mathrm{ln}}\frac{p_{{\mathrm{na}}}/p_{{\mathrm{cs}}}[{\mathrm{na}}^{+}]_{o}+4(p^{^{\prime}}_{ca}/p_{{\mathrm{cs}}})[{\mathrm{ca}}^{2+}]_{o}}{[{\mathrm{cs}}^{+}]_{i}}\end{equation}\end{document} where [na]o and [ca]o are the external activities of na and ca, [cs]i is the internal activity of cs, and pca / pcs is a modified term relating the permeability of ca to cs . Substituting for pca / pcs, the above can also be written as (eq . 4)\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}{\mathrm{exp}}^{e_{5{\mathrm{-}}{\mathrm{ht}}}f / rt}=\frac{p_{{\mathrm{na}}}[{\mathrm{na}}^{+}]_{o}}{p_{cs}[{\mathrm{cs}}^{+}]_{i}}+4\frac{p_{{\mathrm{ca}}}[{\mathrm{ca}}^{2+}]_{o}}{{\mathrm{p}}_{{\mathrm{cs}}}(1+{\mathrm{exp}}^{e_{5{\mathrm{-}}{\mathrm{ht}}}f / rt})[{\mathrm{cs}}^{+}]_{i}}\end{equation}\end{document} when ca was the sole cation in the extracellular medium, the term pna / pcs was omitted . For simplicity, in the text we routinely refer to ion concentrations (in square brackets) rather than activities (in parentheses), although the latter were used in all calculations of relative permeability and are presented in the figures where relevant . Single channel currents were low pass - filtered offline at 1 khz, digitized at 10 khz via a digidata 1302a (axon instruments) interface . Using the winedr v2 7.6 electrophysiology data recorder (j. dempster, dept . Of physiology and pharmacology, university of strathclyde, uk), single channel current amplitude histograms were constructed from sections of single channel activity in which unitary events predominated . A transition detection threshold of 3040% of the predominant unitary event amplitude events less than 1 ms in duration (classified as incompletely resolved), obvious artifacts, and those corresponding to multiple openings were rejected . The resulting mean open state amplitude histogram was fitted using an iterative least squares algorithm with a single gaussian probability density function from which unitary event amplitude (i) was determined . The analysis was restricted to the dominant unitary current state, as events of differing amplitude were relatively rare . Single channel conductance () is routinely reported as the chord conductance, i.e. = i/(vm e5-ht), where vm is the holding potential (including liquid junction potential correction) and e5-ht is the reversal potential of the agonist - evoked macroscopic response determined as described above under the appropriate ionic conditions . Statistical analysis was conducted using one - way analysis of variance with the post hoc dunnett or tukey test as appropriate . Residues in the ma helix of the 5-ht3a receptor influence ca permeability we recently demonstrated that arginine residues 432, 436, and 440 within the ma helix motif of the tm34 loop at positions termed ma 4, 0, and 4, respectively, which line putative cytoplasmic portals (fig . 1c), are responsible for the sub - ps single channel conductance of the 5-ht3a receptor (2, 22, 31). We began this study by testing the hypothesis that the same 5-ht3a ma helix arginine residues also serve as determinants of ionic selectivity . Figure 1.domains of the 5-ht3 receptor that influence ion conduction and selectivity between mono- and divalent cations . A, homology model of the wild - type 5-ht3a receptor using the nicotinic ach receptor of torpedo marmorata as a template (32). The functional modules are denoted: extracellular domain (ec), poreforming transmembrane helices (tm), and the portal - framing cytoplasmic ma helices (ma). B, sequence alignment of the second transmembrane domain (tm2) and flanking sequences of the human 5-ht3a and 5-ht3b subunits . The residue mutated to alanine in the mutant 5-ht3a receptor constructs is boxed . C, an alignment of amino acids within the ma helices of wild - type 5-ht3a and 5-ht3b subunits and the mutant 5-ht3a(qda) construct used in this study, indicating the positions of the 4, 0, and 4 residues (boxed). Amino acids are numbered according to the human 5-ht3a subunit amino acid sequence (51). Domains of the 5-ht3 receptor that influence ion conduction and selectivity between mono- and divalent cations . A, homology model of the wild - type 5-ht3a receptor using the nicotinic ach receptor of torpedo marmorata as a template (32). The functional modules are denoted: extracellular domain (ec), poreforming transmembrane helices (tm), and the portal - framing cytoplasmic ma helices (ma). B, sequence alignment of the second transmembrane domain (tm2) and flanking sequences of the human 5-ht3a and 5-ht3b subunits . C, an alignment of amino acids within the ma helices of wild - type 5-ht3a and 5-ht3b subunits and the mutant 5-ht3a(qda) construct used in this study, indicating the positions of the 4, 0, and 4 residues (boxed). Amino acids are numbered according to the human 5-ht3a subunit amino acid sequence (51). The relative permeability ratio, pna / pcs, of wild - type and mutant 5-ht3a receptors was calculated by determining e5-ht from voltage ramps (100 to + 60 mv) that coincided with the peak of the macroscopic current response to pressure - applied 5-ht in the presence of extracellular and intracellular electrolytes e2 and i1, respectively (fig ramp currents generated in the absence of 5-ht were subtracted from ramp currents in the presence of 5-ht yielding an inwardly rectifying i - v relationship with an e5-ht of 2.1 0.7 mv, corresponding to a pna / pcs ratio of 1.02 0.03 (n = 15; figs . 2b and 3a, table 1) for the wild - type receptor . In six cells from this population, e5-ht was redetermined following the exchange of solution e2 with e3 (i.e. Complete replacement of na by 100 mm ca), resulting in a negative shift in e5-ht to 6.1 1.3 mv accompanied by a pronounced depression in the amplitude of the 5-ht - evoked current and the loss of inward rectification in the i - v relationship, most noticeable at positive potentials (fig . 2b). Despite substantial inhibition of current amplitude by extracellular ca, pca / pcs (1.42 0.11, n = 6; fig . 3a, table 1) was greater than pna / pcs (p = 0.021, paired t test). Table 1the influence of mutations within the intracellular ma helix upon the permeabilities of ca and na relative to csrelative permeabilities (i.e. Pca / pcs and pna / pcs) were calculated from determinations of e5-ht in extracellular solutions containing na (solution e2) or ca (solution e3) as the permeant cation . , indicates significantly different from the appropriate wild - type permeability ratio, p <0.01 . Wt, wild - type; qda, 5-ht3a(r432q, r436d, r440a); qea, 5-ht3a (r432q, r436e, r440a); qaa, 5-ht3a(r432q, r436a, r440a); qfa, (5-ht3a(r432q, r436f, r440a); qra, 5-ht3a(r432q, r440a).5-ht3a constructs na extracellular ca extracellular e5-htpna / pcsne5-htpca / pcsnmvmv wt 5-ht3a 2.1 0.7 1.03 0.03 15 -6.1 1.3 1.42 0.11 6 r432q 6.0 0.9 1.20 0.04 17 -3.4 2.6 1.70 0.26 7 r436d -0.4 0.7 0.93 0.03 13 7.1 1.4 3.13 0.31 7 r440a 4.8 1.1 1.15 0.05 5 2.3 1.7 2.33 0.23 5 qda -1.3 1.0 0.90 0.04 10 9.4 1.4 3.65 0.30 * 10 qea -3.6 0.6 0.81 0.02 4 7.6 0.9 3.15 0.17 * 4 qaa -6.8 0.9 0.72 0.02 * 8 3.5 0.9 2.46 0.14 6 qfa -5.2 0.5 0.77 0.01 * 8 2.1 0.4 2.25 0.06 5 qra -2.0 1.6 0.88 0.05 7 -2.1 1.4 1.80 0.15 7 the influence of mutations within the intracellular ma helix upon the permeabilities of ca and na relative to cs relative permeabilities (i.e. Pca / pcs and pna / pcs) were calculated from determinations of e5-ht in extracellular solutions containing na (solution e2) or ca (solution e3) as the permeant cation . Statistical significance was determined by analysis of variance with dunnett's post hoc test . , indicates significantly different from the appropriate wild - type permeability ratio, p <0.01 . Wt, wild - type; qda, 5-ht3a(r432q, r436d, r440a); qea, 5-ht3a (r432q, r436e, r440a); qaa, 5-ht3a(r432q, r436a, r440a); qfa, (5-ht3a(r432q, r436f, r440a); qra, 5-ht3a(r432q, r440a). Experiments performed on the 5-ht3a(qda) mutant using the voltage ramp protocol yielded a pna / pcs ratio of 0.9 0.04 (e5-ht = 1.3 1.0 mv, n = 13), which is not significantly different from that of the wild - type 5-ht3a receptor (fig . An essentially identical value for e5-ht (1.4 0.5 mv, n = 3) was found from measurements of 5-ht - induced peak current amplitudes at potentials closely bracketing that of current reversal . However, in contrast to the negative shift in e5-ht found for the wild - type 5-ht3a receptor upon replacement of solution e2 by e3, that of the mutant displayed a robust positive shift to 9.4 1.4 mv (n = 10), reflecting a significant increase in pca / pcs to 3.65 0.34 (n = 10, figs . 2c and 3a, table 1). Despite the enhancement of pca / pcs at the 5-ht3a(qda) receptor, inhibition of the macroscopic current responses by extracellular ca persisted, although the effect was less pronounced at positive potentials due to the outward, versus mild inward, rectification typically observed in the ca- and na - containing extracellular solutions, respectively (fig ., we evaluated the contributions of the individual mutations r432q (ma 4), r436d (ma 0), and r440a (ma 4) to the enhancement of pca/ pcs . The r436d charge reversal mutation produces an 6-fold increase in single channel conductance compared with the wild - type 5-ht3a receptor (22). The 5-ht3a(r436d) receptor also had a significantly enhanced relative permeability to ca (pca / pcs = 3.13 0.31, n = 7, fig . By contrast, the introduction of aspartate did not significantly affect pna / pcs compared with wild - type 5-ht3a receptors (fig . Notably, the pca / pcs of the 5-ht3a(qda) receptor was not significantly greater than that of the 5-ht3a(r436d) receptor . The individual replacement of arg-432 by the equivalent 5-ht3b subunit residue glutamine had no significant effect on single channel conductance (22). Likewise, the permeability of ca relative to cs of the 5-ht3a(r432q) mutant was similar to that of the wild - type 5-ht3a receptor, suggesting that this ma helix residue has little influence on ion selectivity (fig . Although the 5-ht3a(r440a) mutant exhibited a trend toward an increased pca / pcs ratio relative to the wild - type receptor, the effect failed to reach statistical significance (fig . The validity of the above estimates of pna / pcs and pca / pcs for the 5-ht3a(qda) receptor construct relies upon the mutant retaining the near perfect cation versus anion selectivity of the wild - type receptor (pna / pcl = 53) (30). We tested this assumption rigorously by dilution experiments in which the nacl content of the extracellular solution e2 was reduced to 95, 50, and 20 mm by replacement with sucrose . Under such conditions a plot of e5-ht as a function of the logarithm of extracellular na activity, (na)o, was linear and yielded a slope of 59 mv / decade change in (na)o (fig . A similar dependence upon (na)o was observed under simplified bi - ionic conditions in which na totally replaced cs within the intracellular solution . Because only a slope of less than 58 mv / decade change in (na)o is compatible with anion permeation (28), we concluded that for the 5-ht3a(qda) receptor construct pna / pcl = . The foregoing results indicate that the arginine residues present at positions 436 (ma 0) and to a lesser extent 440 (ma 4) of the human wild - type 5-ht3a receptor collectively suppress the permeability of ca, but not na, relative to cs, revealing that the large intracellular loop of a cys - loop receptor is an important determinant of divalent versus monovalent cation selectivity . Figure 2.the reversal potential of 5-ht - evoked macroscopic currents at wild - type 5-ht3a and 5-ht3a(qda) receptor constructs with na or ca as the charge carrier in the extracellular solution . A, voltage ramp (left) applied at the peak of the macroscopic inward current response to pressure - applied 5-ht (1 m)(right) to determine the reversal potential (e5-ht) of the agonist - induced response mediated by the wild - type 5-ht3a receptor . B, current - voltage relationships recorded from a representative hek-293 cell expressing the wild - type 5-ht3a receptor with an intracellular cscl - based solution (i1) as reference and nacl (146 mm)-based solution e2 or cacl2 (100 mm)-based solution e3 as the extracellular electrolyte . Note the negative shift in e5-ht (arrow) upon complete iso - osmotic replacement of extracellular na by ca . C, current - voltage relationships recorded from a representative hek-293 cell expressing the 5-ht3a(qda) receptor under the conditions described in b. note that complete iso - osmotic replacement of extracellular na by ca causes a positive shift in e5-ht (arrow). The reversal potential of 5-ht - evoked macroscopic currents at wild - type 5-ht3a and 5-ht3a(qda) receptor constructs with na or ca as the charge carrier in the extracellular solution . A, voltage ramp (left) applied at the peak of the macroscopic inward current response to pressure - applied 5-ht (1 m)(right) to determine the reversal potential (e5-ht) of the agonist - induced response mediated by the wild - type 5-ht3a receptor b, current - voltage relationships recorded from a representative hek-293 cell expressing the wild - type 5-ht3a receptor with an intracellular cscl - based solution (i1) as reference and nacl (146 mm)-based solution e2 or cacl2 (100 mm)-based solution e3 as the extracellular electrolyte . Note the negative shift in e5-ht (arrow) upon complete iso - osmotic replacement of extracellular na by ca . C, current - voltage relationships recorded from a representative hek-293 cell expressing the 5-ht3a(qda) receptor under the conditions described in b. note that complete iso - osmotic replacement of extracellular na by ca causes a positive shift in e5-ht (arrow). The charge of the ma stretch 0 residue is the principal determinant of ca permeability we further evaluated the influence of charge at the ma 0 position by determining pca / pcs for constructs in which the residue was either negative (5-ht3a(r432q, r436e, r440a); abbreviated as 5-ht3a(qea)), neutral (5-ht3a(r432q, r436a, r440a) and 5-ht3a(r432q, r436f, r440a); abbreviated as 5-ht3a(qaa) and 5-ht3a(qfa), respectively), or remained positive (5-ht3a(r432q, r440a); abbreviated as 5-ht3a(qra)). Pca / pcs was significantly enhanced compared with the wild - type 5-ht3a receptor for the 5-ht3a(qea) construct . The enhancement observed for the 5-ht3a(qea) construct was not significantly different from that found for the 5-ht3a(qda) receptor . Although pca / pcs was not significantly different from the wild - type 5-ht3a receptor for the 5-ht3a(qaa), 5-ht3a(qfa), and 5-ht3a(qra) constructs, the ratio tended to decrease progressively as the charge at position 436 changed from negative, through neutral, to positive (fig . Thus, charge inversion appears necessary to cause a shift in e5-ht sufficient to produce a statistically significant difference in pca / pcs values . Finally, measurements of e5-ht with na as the predominant extracellular cation revealed no significant change in pna / pcs relative to the wild - type receptor, apart from the 5-ht3a(qaa) and 5-ht3a(qfa) constructs in which the ratio was modestly reduced to 0.72 0.02 and 0.77 0.01, respectively (fig ., the sign of the charge at position 436 clearly had no consistent influence upon pna/ pcs (fig ., the results suggest that the reversal of charge at position 436 within the 5-ht3a(qda) construct was the predominant cause of a greatly increased pca / pcs relative to the wild - type receptor . Figure 3.comparison of the relative ion permeability of wild - type and mutant 5-ht3a receptor constructs . A, bar graph illustrating the relative permeability (px / pcs) of na (black bars), or ca (white bars) relative to cs for the indicated receptor constructs . Permeability ratios were determined with i1 as the intracellular reference solution and nacl (146 mm)-based solution e2 or cacl2 (100 mm)-based solution e3 as the extracellular electrolyte . Mutant constructs in which pna / pcs or pca / pcs was significantly different from the appropriate wild - type control are indicated with an asterisk (p <0.05; analysis of variance followed by dunnett's post hoc test). B, relationship between the reversal potential of the macroscopic current evoked by 5-ht (e5-ht) and the activity of na in the extracellular medium ((na)o) for the 5-ht3a(qda) receptor construct . Responses were recorded with extracellular electrolytes based on solution e2 wherein the concentration of nacl was reduced by substitution with sucrose . Intracellular solutions were either cscl - based i1 (filled circles) or a solution in which nacl totally replaced cscl (open circles) to produce bi - ionic recording conditions . The regression line fitted to the filled circles yielded a slope of 59 mv / decade change in (na)o . Data points are the mean of 36 independent determinations of e5-ht, and vertical bars indicate s.e . C, relationship between pna / pcs (filled circles) and pca / pcs (open circles) and the charge of the amino acid residue at position 436 of the ma helix . Comparison of the relative ion permeability of wild - type and mutant 5-ht3a receptor constructs . A, bar graph illustrating the relative permeability (px / pcs) of na (black bars), or ca (white bars) relative to cs for the indicated receptor constructs . Permeability ratios were determined with i1 as the intracellular reference solution and nacl (146 mm)-based solution e2 or cacl2 (100 mm)-based solution e3 as the extracellular electrolyte . Mutant constructs in which pna / pcs or pca / pcs was significantly different from the appropriate wild - type control are indicated with an asterisk (p <0.05; analysis of variance followed by dunnett's post hoc test). B, relationship between the reversal potential of the macroscopic current evoked by 5-ht (e5-ht) and the activity of na in the extracellular medium ((na)o) for the 5-ht3a(qda) receptor construct . Responses were recorded with extracellular electrolytes based on solution e2 wherein the concentration of nacl was reduced by substitution with sucrose . Intracellular solutions were either cscl - based i1 (filled circles) or a solution in which nacl totally replaced cscl (open circles) to produce bi - ionic recording conditions . The regression line fitted to the filled circles yielded a slope of 59 mv / decade change in (na)o . Data points are the mean of 36 independent determinations of e5-ht, and vertical bars indicate s.e . C, relationship between pna / pcs (filled circles) and pca / pcs (open circles) and the charge of the amino acid residue at position 436 of the ma helix . The 5-ht3a(qda) receptor conducts ca less efficiently than na determinations of relative permeability based on reversal potential measurements do not predict the relative ease with which ion channels conduct permeant ions (39). For example, a binding site for ca within the permeation pathway may confer selectivity toward this divalent, yet retard its flux in comparison to monovalent cations . Indeed, we previously demonstrated that extracellular divalent cations (mg and ca) reduce the single channel conductance of the wild - type 5-ht3a receptor as estimated using fluctuation analysis (20). However, the latter technique tends to underestimate single channel conductance, and it is preferable to measure single channel conductances directly using excised patch recording (22). This is impossible for the wild - type 5-ht3a receptor, with a conductance of <1 ps, which is below the resolution of the patch - clamp technique (20, 21, 40). In contrast, the 5-ht3a(qda) construct has a single channel conductance that enables the direct resolution of single channel currents in outside - out patch recordings (slope conductance of 37 ps at 74 mv (22)). With na as the predominant extracellular cation, the single channel current - voltage relationship (i - v) for the 5-ht3a (qda) receptor shows a very mild outward rectification over a wide range of holding potentials (100 to 100 mv) and yields chord conductance levels of 46 and 40 ps at holding potentials of + 100 and 100 mv, respectively (fig . The latter is slightly larger than previously reported by us (22); this minor difference is due to the reduced concentration of divalent cations in the extracellular medium e2 . Inclusion of 1 mm ca and 2 mm mg (medium e1) also yielded an essentially linear i - v relationship but with a slope conductance corresponding to our previous estimate (22). In contrast to the mild outward rectification of the i - v relationship observed with na, when ca was the sole extracellular cation, 5-ht - evoked single channel events mediated by the 5-ht3a(qda) receptor displayed marked outward rectification (fig . 4, a and b) similar to that seen in whole - cell current recordings (fig . Negative holding potentials, single channel current amplitudes were greatly reduced in comparison to when na was the permeant cation . At the most negative potential studied (100 mv), where it can be assumed that single channel currents reflect inward movement of ca with little opposing outwardly directed monovalent cation flux, a single channel chord conductance of 5.7 ps was calculated, assuming an e5-ht of 9.4 mv obtained from macroscopic currents recorded under identical ionic conditions . The disparity between single channel current amplitudes recorded in na- and ca - containing solutions decreased as the holding potential was progressively shifted to more positive values (fig . 4c, which plots unitary current amplitude against driving force (i.e. Holding potential minus e5-ht). Indeed, at the most positive potentials examined, where an outwardly directed flux of cs predominates, single channel current amplitudes were indistinguishable between na- and ca - based extracellular solutions (fig . The data indicate that the 5-ht3a(qda) receptor construct, despite selecting for ca over na, conducts the former less efficiently, thus predicting that extracellular ca will (at negative holding potentials) reduce single channel conductance in mixtures of na and ca as we previously inferred for the human wild - type 5-ht3a receptor by fluctuation analysis (20). Figure 4.single channel currents mediated by the 5-ht3a(qda) receptor construct with na or ca as the extracellular permeant ion species . Single channel currents (i) were recorded from outside - out membrane patches in response to pressure - applied 5-ht (10 m) at holding potentials (vh) ranging from 100 to + 100 mv . The patch pipette contained cscl - based solution i1, and the patch was superfused with nacl (146 mm)-based solution e2 or cacl2 (100 mm)-based solution e3 . A, exemplar single channel currents with either na (left) or ca (right) as the extracellular permeant cation . B, current - voltage (i - v) plot for single channel currents recorded with nacl - based solution e2 (filled circles) or cacl2-based solution e3 (open circles) as the extracellular electrolyte . Note the mild outward rectification with na as the permeant ion species in the extracellular medium (compare dashed line fitted to data obtained at positive potentials to curve fitted to all data points). C, i - v plot depicted in b but with driving force (i.e. Vh the plot reveals that outwardly directed single channel current amplitudes, at the most positive vh values examined, are essentially identical in na- and ca - containing extracellular solutions when equivalent driving forces are considered . Each data point in b and c is the mean of 310 determinations of single channel current amplitude derived from different outside - out membrane patches . The vertical bars, when exceeding the size of the symbols, indicate s.e . Single channel currents mediated by the 5-ht3a(qda) receptor construct with na or ca as the extracellular permeant ion species . Single channel currents (i) were recorded from outside - out membrane patches in response to pressure - applied 5-ht (10 m) at holding potentials (vh) ranging from 100 to + 100 mv . The patch pipette contained cscl - based solution i1, and the patch was superfused with nacl (146 mm)-based solution e2 or cacl2 (100 mm)-based solution e3 . A, exemplar single channel currents with either na (left) or ca (right) as the extracellular permeant cation . B, current - voltage (i - v) plot for single channel currents recorded with nacl - based solution e2 (filled circles) or cacl2-based solution e3 (open circles) as the extracellular electrolyte . Note the mild outward rectification with na as the permeant ion species in the extracellular medium (compare dashed line fitted to data obtained at positive potentials to curve fitted to all data points). C, i - v plot depicted in b but with driving force (i.e. Vh the plot reveals that outwardly directed single channel current amplitudes, at the most positive vh values examined, are essentially identical in na- and ca - containing extracellular solutions when equivalent driving forces are considered . Each data point in b and c is the mean of 310 determinations of single channel current amplitude derived from different outside - out membrane patches . The vertical bars, when exceeding the size of the symbols, indicate s.e . Extracellular ca reduces the single channel conductance of the 5-ht3a(qda) receptor in a concentration - dependent fashion we examined the influence of extracellular ca upon the single channel conductance of the 5-ht3a(qda) receptor construct with an extracellular solution containing 95 mm na and varying concentrations of ca (10 nm to 30 mm). Chord conductances were derived from single channel current amplitudes recorded at a holding potential of 80 mv, and measurements of e5-ht were made from macroscopic currents in the ionic mixtures . In this series of experiments, e5-ht was estimated by interpolation between the peak amplitude of inwardly and outwardly directed 5-ht - induced currents that closely bracketed the zero current potential . Consistent with the relative permeability measurements made with high ca solution (e3, table 1), the addition of increasing concentrations of ca was associated with a progressive shift in e5-ht to more positive values (from 15.0 0.04 mv (n = 4) with [ca]o = 0.1 mm to 1.6 0.7 mv single channel conductance was reduced by 65% over the range [ca]o = 0.1 mm (41.3 0.88 ps) to [ca]o = 30 mm (14.8 0.63 ps; fig . 5, b and c). However, it should be noted that ca will itself contribute to inward current amplitude, particularly at high [ca]o . For this reason the calculation of an ic50 value for the suppressant effect of ca upon single channel conductance was inappropriate . A simple interpretation of such data is that ca reduces single channel conductance by binding to a site(s) of comparatively low affinity within the permeation pathway and thereby occludes the flux of na . In agreement with such a violation of the independent movement of na and ca within the permeation pathway, [na]o fixed at 95 mm (and assuming pna / pcs = 0.9; see above), pca / pcs was calculated from determinations of e5-ht to be 0.5 and 1.8 in the presence of 10 and 30 mm extracellular ca, respectively, versus the value of 3.7 found with ca as the sole extracellular charge carrier . Extracellular ca reduces single channel conductance of the 5-ht3a(qda) receptor in a voltage - independent manner blockade by ca of macroscopic current responses mediated by the 5-ht3a receptor expressed in mammalian cell hosts is voltage - independent (20, 21, 34, 40, 41). The readily resolvable single channel conductance of the 5-ht3a(qda) receptor allowed direct measurements of the influence of extracellular ca upon single channel current amplitudes over a range of membrane potentials . 6 depicts the results of such studies where [ca]o was varied between 0.3 and 10 mm in the presence of 95 mm na and single channel events evoked by pressure - applied 5-ht (10 m) were recorded from outside - out membrane patches at holding potentials in the range of 40 to 100 mv . At all concentrations of extracellular ca studied the single channel i - v relationship was well fitted by a linear function and showed no evidence of a region of negative slope conductance or outward rectification (fig . In addition, the single channel conductances derived from the slope of the i - v relationships (i.e. 35.8, 29.4, 25.0, and 20.0 ps in the presence of 0.3, 1, 3, and 10 mm ca, respectively) were in excellent agreement with the chord conductances reported in fig . 5c . Thus, the reduction in single channel conductance by extracellular ca is voltage - independent over the range of negative potentials examined . A, reversal potential of the macroscopic current response to 5-ht (e5-ht) with [na]o set to 95 mm and [ca]o within the range of 0.1 to 30 mm . Note that ca ion activity, (ca)o, rather than concentration, [ca]o, is plotted in a and c. data are the mean of 34 observations, and vertical bars indicate s.e . , p <0.01 as determined by analysis of variance followed by dunnett's post hoc test . B, single channel currents in response to 5-ht (10 m) applied by pressure to outside - out membrane patches voltage - clamped at a holding potential (vh) of 80 mv . Increasing [ca]o depresses unitary current amplitude in a concentration - dependent manner . Note that an increased driving force (i.e. Vh e5-ht) when [ca]o is equal to 10 and 30 mm (corresponding to a (ca)o of 3.12 and 8.56 mm, respectively; see a) partially masks the magnitude of the suppression of single channel current amplitudes by ca . C, concentration - response relationship illustrating the depressant effect of extracellular ca upon single channel conductance . Data are the mean s.e . Of measurements performed on 45 outside - out membrane patches under the conditions described in b. extracellular ca depresses the single channel conductance of the 5-ht3a(qda) receptor construct . A, reversal potential of the macroscopic current response to 5-ht (e5-ht) with [na]o set to 95 mm and [ca]o within the range of 0.1 to 30 mm . Note that ca ion activity, (ca)o, rather than concentration, [ca]o, is plotted in a and c. data are the mean of 34 observations, and vertical bars indicate s.e . , p <0.01 as determined by analysis of variance followed by dunnett's post hoc test . B, single channel currents in response to 5-ht (10 m) applied by pressure to outside - out membrane patches voltage - clamped at a holding potential (vh) of 80 mv . Increasing [ca]o depresses unitary current amplitude in a concentration - dependent manner . Note that an increased driving force (i.e. Vh e5-ht) when [ca]o is equal to 10 and 30 mm (corresponding to a (ca)o of 3.12 and 8.56 mm, respectively; see a) partially masks the magnitude of the suppression of single channel current amplitudes by ca . C, concentration - response relationship illustrating the depressant effect of extracellular ca upon single channel conductance . Data are the mean s.e . Of measurements performed on 45 outside - out membrane patches under the conditions described in b. extracellular na influences reduction of the single channel conductance of the 5-ht3a(qda) receptor by extracellular ca and mg the single channel conductance of the 5-ht3a(qda) receptor decreased from 41.1 to 26.5 ps when [ca]o was elevated from 0.1 to 3 mm in an extracellular solution containing 95 mm na (fig . 5c). However, only a modest decrease in single channel conductance (i.e. From 40.8 to 35.7 ps) occurred when solution e1 (containing 1.0 mm ca and 2.0 mm mg in the presence of 140 mm na) replaced solution e2 (containing 0.1 mm ca and 0.1 mm mg in the presence of 140 mm na). The 5-ps decrease in conductance is far less than that predicted (11 ps) from fig . 5c when [ca]o was increased from 0.1 to 1.0 mm, even neglecting a likely contribution from mg (20, 40). The reason for this apparent discrepancy is an interaction between [na]o and [cation]o (fig . Where [na]o was set at 140, 95, and 50 mm na (osmolarity maintained with sucrose), single channel chord conductance recorded at a holding potential of 80 mv in the presence of 0.1 mm each ca and mg was unchanged between the 140 and 95 mm solutions but was significantly depressed in the 50 mm solution (range 40.8 to 34.4 ps; fig . 7). However, in similar solutions containing 1 mm ca and 2 mm mg, single channel conductance decreased markedly in parallel with diminished [na]o (range, 35.7 to 18.3 ps; fig . The data are consistent with competition between na and divalent cations for a binding site within the permeation pathway . Figure 6.extracellular ca reduces the single channel conductance of the 5-ht3a(qda) receptor construct in a manner independent of transmembrane potential . A, single channel currents recorded from outside - out membrane patches in response to pressure - applied 5-ht (10 m). The currents were recorded at holding potentials in the range of 100 to 40 mv in the presence of 0.3, 1, 3, or 10 mm extracellular ca and 95 mm na . B, i - v relationships for single channel events recorded with [na]o constant at 95 mm and [ca]o present at 0.3 mm (filled circles), 1 mm (open circles), 3 mm (filled inverted triangles), or 10 mm (open inverted triangles). The relationship between single channel current amplitude and vh at each concentration of ca was fitted by linear regression analysis to yield slope conductance values of 35.8, 29.4, 25.0, and 20.0 ps in the presence of 0.3, 1, 3, and 10 mm ca, respectively . Dashed lines represent an extrapolation of each i - v plot to the zero current level (e5-ht). Each data point is the mean of single channel current amplitudes derived from recordings performed on 35 outside - out membrane patches, and the vertical bars indicate s.e . Extracellular ca reduces the single channel conductance of the 5-ht3a(qda) receptor construct in a manner independent of transmembrane potential . A, single channel currents recorded from outside - out membrane patches in response to pressure - applied 5-ht (10 m). The currents were recorded at holding potentials in the range of 100 to 40 mv in the presence of 0.3, 1, 3, or 10 mm extracellular ca and 95 mm na . B, i - v relationships for single channel events recorded with [na]o constant at 95 mm and [ca]o present at 0.3 mm (filled circles), 1 mm (open circles), 3 mm (filled inverted triangles), or 10 mm (open inverted triangles). The relationship between single channel current amplitude and vh at each concentration of ca was fitted by linear regression analysis to yield slope conductance values of 35.8, 29.4, 25.0, and 20.0 ps in the presence of 0.3, 1, 3, and 10 mm ca, respectively . Dashed lines represent an extrapolation of each i - v plot to the zero current level (e5-ht). Each data point is the mean of single channel current amplitudes derived from recordings performed on 35 outside - out membrane patches, and the vertical bars indicate s.e . Figure 7.the extracellular concentration of na influences depression of single channel conductance by extracellular divalent cations . Bar graph depicting the single channel conductance of the 5-ht3a(qda) receptor construct in the presence of 0.1 mm ca and 0.1 mg (black bars) or 1 mm ca and 2 mm mg (white bars) with [na]o set at 140, 95, or 50 mm . Increasing the extracellular concentration of ca and mg causes a significant reduction of single channel conductance at all concentrations of na examined, but the magnitude of the depression (expressed as a percentage within the white bars) increases as [na]o is reduced . In the presence of 0.1 mm ca and 0.1 mm mg reducing [na]o from 140 to 50 mm, but not to 95 mm, significantly decreases single channel conductance . With ca and mg present at 1 and 2 mm, respectively, reducing [na]o to both 95 and 50 data are the mean s.e . Of observations made from 310 outside - out membrane patches . , p <0.05; * , p <0.01; * , p <0.001; as determined by analysis of variance followed by tukey's post hoc test . The extracellular concentration of na influences depression of single channel conductance by extracellular divalent cations . Bar graph depicting the single channel conductance of the 5-ht3a(qda) receptor construct in the presence of 0.1 mm ca and 0.1 mg (black bars) or 1 mm ca and 2 mm mg (white bars) with [na]o set at 140, 95, or 50 mm . Increasing the extracellular concentration of ca and mg causes a significant reduction of single channel conductance at all concentrations of na examined, but the magnitude of the depression (expressed as a percentage within the white bars) increases as [na]o is reduced . In the presence of 0.1 mm ca and 0.1 mm mg reducing [na]o from 140 to 50 mm, but not to 95 mm, significantly decreases single channel conductance . With ca and mg present at 1 and 2 mm, respectively, reducing [na]o to both 95 and 50 data are the mean s.e . Of observations made from 310 outside - out membrane patches . , p <0.05; * , p <0.01; * *, p <0.001; as determined by analysis of variance followed by tukey's post hoc test . Effect of neutralizing the extracellular ring of charge of the 5-ht3a and 5-ht3a(qda) receptor extracellular ca exerts multiple effects upon the wild - type 5-ht3a receptor including reductions in the apparent affinity of agonist binding (34, 41), depression of macroscopic and single channel current amplitudes (20, 34, 42), and an acceleration of the kinetics of activation, deactivation, and desensitization (34). Hu and lovinger (34) demonstrated that neutralization of the extracellular ring of negative charge formed by the 20 aspartate residue (fig . 1b) at the extracellular vestibule of the mouse 5-ht3a receptor channel by the mutation d298a prevents modulation of macroscopic current kinetics and amplitude by ca . We have examined directly whether the ca - induced decrease in single channel conductance involves the homologous residue (asp-293) of the human 5-ht3a(qda) receptor construct and also have evaluated its effect upon the relative permeability to ca . We additionally determined the pca / pcs ratio for 5-ht3a receptors in which the d293a substitution was the only mutation present (i.e. 5-ht3a(d293a)). Application of 1 m 5-ht to cells transfected with the 5-ht3a(qda d293a) receptor cdna elicited only small inward current responses . Hence, we constructed full concentration - response relationships to 5-ht for both the 5-ht3a(qda) and 5-ht3a(qda d293a) receptors to determine whether the d293a mutation was associated with a reduction in the agonist potency of 5-ht . Such experiments demonstrated that relative to the 5-ht3a(qda) receptor (ec50 1.1 m), the 5-ht3a(qda d293a) construct is less sensitive to 5-ht (ec50 3.6 m; supplemental fig . Similarly, a comparison of wild - type 5-ht3a and 5-ht3a(d293a) receptors revealed that the d293a mutation decreases the potency of 5-ht, as the ec50 value was shifted dextrally from 3 to 7 m (supplemental fig . In subsequent experiments, 2 m 5-ht was employed to elicit macroscopic currents from cells expressing the 5-ht3a(d293a) and 5-ht3a(qda d293a) receptor constructs . In common with the 5-ht3a(qda) construct, the 5-ht3a(qda d293a) mutant maintained exclusive selectivity for cations because the slope of the relationship between the logarithm of (na)o and e5-ht was determined to be 61 mv / decade change in (na)o (fig . 8a). Using solutions e3 and i2 and the voltage ramp protocol, macroscopic currents mediated by the 5-ht3a(qda d293a) construct in response to 5-ht demonstrated an e5-ht of 40.5 0.8 mv (n = 6), corresponding to a pca / pcs ratio of only 0.25 0.01 (n = 6) compared with that of 3.7 for the 5-ht3a(qda) receptor (fig . (n = 6) for the 5-ht3a(d293a) mutant indicates a pca / pcs of 0.44 0.08 (n = 6), which is also significantly reduced versus the wild - type receptor (1.4). The pca / pcs ratios of the 5-ht3a(d293a) and 5-ht3a(qda d293a) receptors were not significantly different . The pna / pcs ratio of either construct was unchanged by the d293a mutation (fig . Thus, for both the wild - type 5-ht3a and 5-ht3a(qda) constructs, neutralization of the negatively charged 20 residue greatly reduces the pca / pcs ratio but has no effect upon pna / pcs . In addition to reducing the relative permeability of ca, as evidenced by a decreased sensitivity of e5-ht to changes in [ca]o (fig . 8c), the introduction of the d293a mutation into the 5-ht3a(qda) receptor construct also decreased single channel conductance . In the presence of 0.1 mm ca and 95 mm na, the single channel chord conductance of the 5-ht3a(qda d293a) receptor was 25.8 1.9 ps (n = 4) at a holding potential of 80 mv in comparison with 41.3 ps for the 5-ht3a(qda) construct . Similarly, the single channel chord conductance (at 80 mv) of the 5-ht3a(qda d293a) receptor determined with solution e2 (29.8 0.5, n = 7) was significantly less than that of the 5-ht3a(qda) construct (40.8 ps). However, no difference in single channel conductance was observed between the 5-ht3a(qda) and 5-ht3a(qda d293a) constructs over a range of positive holding potentials (60 to 100 mv), indicating that the d293a mutation preferentially suppresses inwardly directed cation flux (fig . As found for the 5-ht3a(qda) receptor, elevated [ca]o (0.130 mm) also depressed the single channel conductance of the 5-ht3a(qda d293a) construct in a concentration - dependent manner (fig . At all values of [ca]o studied, the single channel chord conductance of the 5-ht3a(qda d293a) construct was significantly less than that of the 5-ht3a(qda) receptor . Clearly, the alleviation of macroscopic current block by the d293a mutation (34) cannot be attributed to a reduced influence of ca upon single channel conductance . Figure 8.comparison of the relative ion permeability of wild - type, 5-ht3a(d293a), 5-ht3a(qda), and 5-ht3a(qda d293a) receptor constructs with na and ca . A, the relationship between the reversal potential (e5-ht) of the macroscopic current response to pressure - applied 5-ht (10 m) recorded from the 5-ht3a(qda d293a) receptor construct and the activity of na in the extracellular medium ((na)o). Responses were recorded with extracellular electrolytes based on solution e2 wherein the concentration of nacl was reduced by substitution with sucrose . Intracellular solutions were either cscl - based i1 (filled circles) or a solution in which nacl totally replaced cscl (open circles). The regression line fitted to the filled circles yielded a slope of 61 mv / decade change in (na)o . Data points are the mean of 36 independent determinations of e5-ht, and vertical bars indicate mean s.e . In a, c, d, and e, the dashed gray lines summarize the corresponding data for the 5-ht3a(qda) receptor construct illustrated in detail in figs . 3, 4, and 5 . B, bar graph illustrating the permeability (px / pcs) of na (black bars) or ca (white bars) relative to cs for the indicated receptor constructs . E5-ht was determined with an intracellular cscl - based solution (i1) as reference and nacl (146 mm)-based solution e2 or with cacl2 (100 mm)-based solution e3 as the extracellular electrolyte . Mutant constructs in which pca / pcs was significantly different from the wild - type control are indicated with asterisks (p <0.01; analysis of variance followed by dunnett's post hoc test). No significant differences in pna / pcs were found between the wild - type and mutant receptors . C, e5-ht for the 5-ht3a(qda d293a) receptor construct with [na]o set to 95 mm and [ca]o within the range of 0.1 to 30 mm . Note that ca ion activity ((ca)o) rather than concentration ([ca]o) is plotted . D, current - voltage (i - v) plot for single channel currents recorded from the 5-ht3a(qda d293a) receptor construct with nacl - based solution e2 as the extracellular electrolyte and i1 as the intracellular solution . Note that the additional d293a mutation causes an intensification of outward rectification and a reduced single channel conductance at negative potentials in comparison with the 5-ht3a(qda) construct . E, concentration - response relationship and exemplar single channel currents illustrating the depressant effect of [ca]o upon the single channel conductance of the 5-ht3a(qda d293a) receptor construct . Note that ca ion activity, rather than concentration, is plotted . Data are the mean s.e . Of measurements performed on 34 outside - out membrane patches at a holding potential of 80 mv under the ionic conditions described in c. comparison of the relative ion permeability of wild - type, 5-ht3a(d293a), 5-ht3a(qda), and 5-ht3a(qda d293a) receptor constructs with na and ca . A, the relationship between the reversal potential (e5-ht) of the macroscopic current response to pressure - applied 5-ht (10 m) recorded from the 5-ht3a(qda d293a) receptor construct and the activity of na in the extracellular medium ((na)o). Responses were recorded with extracellular electrolytes based on solution e2 wherein the concentration of nacl was reduced by substitution with sucrose . Intracellular solutions were either cscl - based i1 (filled circles) or a solution in which nacl totally replaced cscl (open circles). The regression line fitted to the filled circles yielded a slope of 61 mv / decade change in (na)o . Data points are the mean of 36 independent determinations of e5-ht, and vertical bars indicate mean s.e . In a, c, d, and e, the dashed gray lines summarize the corresponding data for the 5-ht3a(qda) receptor construct illustrated in detail in figs . 3, 4, and 5 . B, bar graph illustrating the permeability (px / pcs) of na (black bars) or ca (white bars) relative to cs for the indicated receptor constructs . E5-ht was determined with an intracellular cscl - based solution (i1) as reference and nacl (146 mm)-based solution e2 or with cacl2 (100 mm)-based solution e3 as the extracellular electrolyte . Mutant constructs in which pca / pcs was significantly different from the wild - type control are indicated with asterisks (p <0.01; analysis of variance followed by dunnett's post hoc test). No significant differences in pna / pcs were found between the wild - type and mutant receptors . C, e5-ht for the 5-ht3a(qda d293a) receptor construct with [na]o set to 95 mm and [ca]o within the range of 0.1 to 30 mm . Note that ca ion activity ((ca)o) rather than concentration ([ca]o) is plotted . D, current - voltage (i - v) plot for single channel currents recorded from the 5-ht3a(qda d293a) receptor construct with nacl - based solution e2 as the extracellular electrolyte and i1 as the intracellular solution . Note that the additional d293a mutation causes an intensification of outward rectification and a reduced single channel conductance at negative potentials in comparison with the 5-ht3a(qda) construct . E, concentration - response relationship and exemplar single channel currents illustrating the depressant effect of [ca]o upon the single channel conductance of the 5-ht3a(qda d293a) receptor construct . Note that ca ion activity, rather than concentration, is plotted . Data are the mean s.e . Of measurements performed on 34 outside - out membrane patches at a holding potential of 80 mv under the ionic conditions described in c. two decades of research have focused upon tm2 and flanking sequences as the primary determinants of ion selectivity in cys - loop receptors (2, 28, 43). For the nicotinic ach receptor in particular, multiple sites within such domains (i.e. The 1, 13, 16, and 17 positions; fig . 1b) that affect the conduction of ca have been identified (28, 43). We have added to this information by demonstrating that residues within the ma helix of the cytoplasmic loop of the 5-ht3a receptor, a cousin of the nicotinic ach receptors, exert a strong influence on the permeability of ca relative to monovalent cations . The replacement of arginine residues 432, 436, and 440 of the human 5-ht3a receptor by those aligned in the human 5-ht3b subunit (i.e. Glutamine, aspartate, and alanine), generating the 5-ht3a(qda) receptor construct, increased pca / pcs from a wild - type value of 1.4 to 3.7 . Such an effect was selective because neither pna / pcs nor pna / pcl was perturbed by the mutation, the latter being inferred by comparison with values within the literature (20, 29, 30). Notably, the 5-ht3a(qda) construct retained perfect selectivity toward cations versus anions, a finding that accords with the results of a recent study in which deletion of the entire intracellular loop of the 5-ht3a receptor had no measurable influence upon pna / pcl (44). Indeed, residues within and adjacent to tm2 govern charge selectivity in the 5-ht3a receptor (29). In particular, the single amino acid substitution e1a at the intracellular border of tm2 (fig . 1b) is sufficient to generate a 5-ht3a receptor that does not discriminate between monovalent cations and anions (pna / pcl = 0.89) (30). In future studies, it would be of interest to generate the e1a mutation within the 5-ht3a(qda) background to assess whether a role for the ma helix emerges when a primary determinant of charge selectivity is silenced . Our results indicate that the single mutation r436d is sufficient to enhance pca / pcs to an extent comparable with that found for the 5-ht3a(qda) construct . Analysis of several amino acid substitutions involving neutralization, or inversion, of charge at this key locus suggests that an electrostatic repulsive effect contributes to a suppression of pca / pcs in the wild - type receptor . The latter is consistent with the results of modeling studies that place the highly basic (pka = 12.48) guanidinium group of arg-436 within the intracellular permeation pathways (portals) located between adjacent ma - stretch -helices (26, 32). Because of the small maximal width of the portals (26), interactions between partially hydrated permeating ions and arg-436 are likely . Previous studies of ca permeation through homomeric 5-ht3 receptor channels have, because of its very low single channel conductance, been restricted to the analysis of macroscopic currents . The latter are suppressed by extracellular divalent cations by mechanisms that potentially include a reduction in agonist binding affinity (34, 41), an acceleration of the kinetics of deactivation and desensitization (34), and depression of macroscopic and single channel current amplitudes (20, 34), the latter being inferred by fluctuation analysis (20). In the present study, we employed the 5-ht3a(qda) receptor construct to examine directly the influence of extracellular ca upon single channel conductance . A solution (e3), in which ca was the sole extracellular cation, supported outwardly rectifying single channel currents but with a chord conductance at negative potentials (5.7 ps), which was greatly reduced in comparison with the value of 40.8 ps obtained with solution e2, in which na was the permeant species . Thus, although the permeability of ca relative to cs is greatly increased in the 5-ht3a(qda) construct, the channel does not conduct ca efficiently . Moreover, the addition of ca to na - containing extracellular solutions caused a concentration - dependent inhibition of the inwardly directed single channel current amplitude, in a manner reminiscent of that observed for single channel currents mediated by the nicotinic ach receptor of skeletal muscle (45). Such results are most parsimoniously explained by hypothesizing that the permeation pathway presents a low affinity binding site (or sites) for ca, which confers a degree of selectivity for ca over monovalent cations, yet with an off - rate sufficient to allow significant flux of both na and ca . An interaction between ca and na within the channel is supported by our experimental observations indicating the pca / pna ratio for the 5-ht3a(qda) receptor construct varies with [ca]o (i.e. The principle of independence is violated (39)). Consistent with this notion, the reduction in single channel current amplitude by extracellular divalent cations was more pronounced when [na]o was reduced . We considered the d293a residue of the human 5-ht3a receptor subunit, which forms the 20 negatively charged ring within the extracellular vestibule of the channel (2, 28), to be a strong candidate for the putative ca binding site that limits the rate of charge transfer through the channel (fig . Firstly, mutation of the corresponding residue (asp-298) of the mouse 5-ht3a subunit to alanine alleviates the block of macroscopic current responses by extracellular ca (34). Secondly, ca reduced the amplitude of single channel currents mediated by the 5-ht3a(qda) receptor construct in a voltage - independent manner . The lack of observable voltage dependence suggests a binding site for ca away from the electrical field of the membrane . However, because ca is permeant, rather than a simple impermeant open channel blocker, it remains possible that ca does interact with a site(s) within the field but that the increased driving force provided by hyperpolarization facilitates its dissociation and inward conduction . Note that this scenario is quite different from that found for an impermeant cationic blocker, for which the dwell time within the channel would be increased by hyperpolarization because of a decreased probability of the molecule exiting the pore to the extracellular environment . The potency of 5-ht to activate the mouse 5-ht3a(d298a) construct is less than for the murine wild - type receptor (34), a result concordant with our observation that the d293a mutation introduced into either the human wild - type or 5-ht3a(qda) construct causes a dextral shift in the 5-ht concentration - response relationship . The d293a mutation within the 5-ht3a(qda) receptor background caused a reduction in the amplitude of inwardly, but not outwardly, directed single channel current events . Such an effect is consistent with a simple electrostatic mechanism in which the negative charge of the five asp-293 residues acts to increase the local concentration of permeant cations within the extracellular vestibule . Removal of such charges would thus reduce a focusing mechanism, consistent with the appearance of a enhanced outward rectification in the i - v relationship for the 5-ht3a(qda d293a) receptor construct versus the 5-ht3a(qda) receptor . Very similar effects of neutralizing the extracellular ring of charge upon single channel conductance and rectification have been reported for nicotinic ach and anion - selective -aminobutyric acid type a and glycine receptors (28). Because of the very low conductance of the wild - type 5-ht3a receptor (20, 21) and kinetic properties of the 5-ht3a(d293a) receptor, which are unfavorable to fluctuation analysis (34),5 we could not estimate the effect of the d293a mutation upon single channel conductance in this construct . However, it is notable that the pattern of inward rectification of macroscopic currents mediated by the mouse 5-ht3a(d298a) mutant resembles that of the murine wild - type receptor (34). The latter differs from the present observations comparing the human 5-ht3a(qda) and 5-ht3a(qda d293a) receptors at the single channel level . An important difference in our studies is the removal of positive electrostatic potential in the intracellular vestibule by the mutations r432q, r436a, and r440a . Such charges are present in the mouse 5-ht3a and 5-ht3a(d298a) receptors studied by hu and lovinger (34) and would reduce the local concentration of permeant ions within the intracellular vestibule . It is possible that such an influence is dominant, masking any tendency toward linearization that might be anticipated from the 5-ht3a(d293a) mutation . Indeed, in our studies, the macroscopic current i - v relationship shifts from the pattern of inward rectification characteristic of the wild - type 5-ht3a receptor toward linearity in the 5-ht3a(qda) construct . For both the wild - type 5-ht3a and the 5-ht3a(qda) constructs, the introduction of the d293a mutation drastically reduced the pca / pcs ratio . Once more, such an effect can probably be attributed to a reduction in a local negative electrostatic potential that particularly favors the accumulation of ca at the entrance to the pore, as suggested by molecular dynamics simulations performed on models of the 7 and skeletal muscle nicotinic ach receptors (46, 47). Such simulations indicate monovalent cations to be stabilized at the 20 position, where the ion dwells during its passage through the channel axis . Also, heteromeric 42 nicotinic ach receptors, harboring a larger complement of 2 subunits in which lysine occupies the 20 position, appear to have reduced selectivity toward ca (48). However, the d293a residue does not contribute crucially to cation versus anion selectivity because the pna / pcl ratio for the 5-ht3a(qda d293a) construct was best described as infinite . A previous study of mouse 5-ht3a receptors in which cysteine residues engineered into the tm2 domain were successfully probed with a negatively charged sulfydryl - modifying agent (methane - thiosulfonate ethylsulfonate (mtses)) applied extracellularly also indicates that the extracellular ring does not totally select against anions gaining access to deeper regions of the pore (49). In addition, neutralization of the 20 charge was not required to invert the ion selectivity of the mouse 5-ht3a receptor (29). Finally, the recent crystal structure of a prokaryotic pentameric ligand - gated channel from erwinia chrysanthemi reveals that phenylalanine occupies the 20 position, yet the channel is cation - selective (50). Heteromeric 5-ht3 receptors expressed in tsa-201 cells assemble with the stoichiometry (5-ht3a)2(5-ht3b)3 as a subunit rosette in the order b - b - a - b - a, yielding 2 a - b, 2 b - a, and 1 b - b subunit interfaces (16). Hence, compared with homomeric receptors, the influence of arg-432, arg-436, and arg-440 will be removed from three of the cytoplasmic portals, irrespective of whether a clockwise or anticlockwise arrangement is assumed . Yet, the inclusion of the 5-ht3b subunit within heteromeric 5-ht3 receptors reduces permeability to ca (pca / pcs = 0.50.6) in comparison with homomeric receptors (pca / pcs = 1.11.4) (ref . This apparent paradox might be explained by the presence of a polar asparagine, rather than an acidic aspartate, at the 20 locus and a non - polar alanine, rather than an acidic glutamate, at the 1 position of the human 5-ht3b versus 5-ht3a subunit (fig . The present work suggests that the 5-ht3b subunit 20 asparagine may not be optimal for a high pca / pcs, ratio and it is known that a e1a substitution in the 7 nicotinic ach receptor abolishes permeabilty to ca (28). Macroscopic current responses mediated by the mouse 5-ht3a(d298a) receptor are insensitive to [ca]o within the range of 0.1 to 10 mm (34), but we observed that single channel currents recorded from the human 5-ht3a(qda d293a) receptor were reduced in amplitude in the presence of extracellular ca . Such an effect was concentration - dependent and occurred within the range observed for the 5-ht3a(qda) receptor . Hence, the asp-293 residue cannot be a crucial site at which ca binds to reduce single channel conductance in the 5-ht3a(qda) receptor construct . It remains to be explained why increasing [ca]o does not affect the macroscopic current response of the mouse 5-ht3a(d298a) receptor, yet clearly depresses single channel conductance in the human 5-ht3a(qda d293a) construct . However, as clearly noted by hu and lovinger (34), depression of single channel conductance cannot be the sole mechanism by which ca depresses macroscopic currents, because the magnitude of the effect is dependent upon the concentration of 5-ht employed to activate the receptor . Indeed, at saturating concentrations of 5-ht (i.e. 30 m), increasing [ca]o from 1.8 to 10 mm exerts only a modest depressant effect upon 5-ht - evoked currents (34), which might indicate that the influence of ca derives, at least in part, via indirect, or direct, actions upon ligand binding (41). In addition, ca accelerates desensitization of the murine wild - type 5-ht3a receptor, and the d293a mutation suppresses this effect . It is possible that the reduced relative permeability of the 5-ht3a(d293a) mutant contributes to such resistance . In summary, we have demonstrated that structural elements in both the intracellular and extracellular regions of the human 5-ht3a control relative permeability to ca . Our report represents the first description of the influence of the ma helix upon ion selectivity, a process that has conventionally been linked to specific amino acid residues within tm2 and immediate flanking sequences (28). It is thus an oversimplification to refer to a single selectivity filter in the 5-ht3a receptor and, by inference, in other cys - loop receptors . Instead, the extracellular vestibule, tm2, and the ma helix act collectively to determine ion permeation.
The growing market of animal health products may account for the potential revenue source of companion animal care . The animal health care has resulted in more than one billion sales in 2007 for novartis, pfizer and heska (kling 2007). The opportunity to carry out preclinical trials based on spontaneous tumors has been considered suitable to develop new effective drugs that could be quickly translated into clinical practice (de vico et al . ; vail and macewen 2000). For instance, naturally occurring tumors in dogs share similarities with human cancer both in terms of histopathological features and biological behavior (hahn et al . 1994). In the second half of the 1800s, a substantial parallel between the animal and human oncopathology, evidence showed that spontaneous animal tumors were similar to their human counterpart and information retrieved from them might have clinical implications (dobberstein 1937). In the early 1970s, the first world health organization (who) international histological classification of tumors of domestic animals was published (beveridge and sobin 1974, 1976). It was followed by the tnm classification of tumors in domestic animals based on the guidelines of the tnm classification of malignant tumors in man (owen 1980) showing that spontaneous canine and feline tumors are relevant models to study human cancers . We summarized such models in table 1 according to the review from de vico et al . Conversely, in vivo and in vitro models, based on rodents and cell lines, have displayed intrinsic limits, such as complexity of management (schiffer 1997) and the experimental methods used to induce the pathology (vail and thamm 2004), which make them different from the human spontaneous diseases (de vico et al . 2005; moore and siopes 2004).table 1summary of tumor features shared between animals and humans (allen et al . 2002)modelkind of tumorfeaturescanine / felinenon - hodgkin s lymphomaequates to intermediate and high - grade non - hodgkin s lymphoma in humansimmune system alterations seem to be associated with a higher incidence of non - hodgkin s lymphomais very sensitive to chemotherapycurrently used as a model for testing new chemotherapeutics and new forms of immunotherapy, as well as for the study of multiple drug resistance; it has been used as a relevant model for (1) developing hypoxic cell markers, (2) studying the effect of whole - body hyperthermia on the pharmacokinetics of systemic chemotherapy and (3) studying autologous bone transplantationcaninesoft tissue sarcomassimilar to pathological appearance, clinical presentation and behavior of human soft tissue sarcomasrespond to radiation therapy and chemotherapy in a manner similar to human pathologyhemangiosarcomas are extremely similar to human hemangiosarcomas with respect to histological pattern and metastatic behaviorcanineosteosarcomassimilar to human osteosarcomas with respect to the histology, metastatic behavior and clinical evolution of the diseasecanineoral malignant melanomasthey are chemoresistant and share many immunological targets with human oral malignant melanomasconsidered a relevant model for developing new immunotherapeutic approaches for both dogs and humansboth in dogs and humans, human tyrosinase dna vaccination is safe and potentially effectivecaninetransitional cell carcinomasshare histological appearance, biological behavior and response to therapy with invasive human transitional cell carcinomasconsidered a useful model for testing new photodynamic therapy technologies and chemotherapeutic combinationscaninemast cell tumorsmutation of exon 11 of c - kit occurs in 3050% of advanced mast cell tumors and is similar to that usually occurring in 5090% of human gastrointestinal stromal tumorscanine / felinemammary carcinomasvery similar to human breast cancers because of their hormonal dependence, spontaneous development in middle - aged to older animals, metastatic behavior toward regional lymph nodes and lungs, as well as adhesion molecules and neoangiogenesis patternsabnormalities in the nuclear dna content have been documented in both malignant and benign canine mammary tumors, but are more frequent in human mammary carcinomasmammary neoplasia in dogs may be a good molecular model for developing new antineoplastic strategies involving cyclin d1 and cyclin - dependent kinasesfeline mammary carcinoma shows age incidence, histopathology and pattern of metastasis similar to human breast cancerfeline mammary carcinoma shares common features with human inflammatory mammary carcinoma and human mammary carcinoma with osteoclast - like giant cellsepidermal growth factor receptor-2 (her-2) and recepteur d'origine nantais (ron) overexpressions qualify feline mammary carcinoma as homologous to human breast carcinomas which have poor prognosiscanineseminomasmetastases occur only in a small percentage of cases, whereas human seminomas have a marked tendency to metastasizehave a histogenetic behavior similar to that of human seminomascaninetransmissible venereal tumoris considered a promising model to study human kaposi s sarcomapercutaneous inoculation and intraarterial transplantation of tumor fragments in the canine lung result in predictable patterns of tumor growth resembling the solitary pulmonary nodules and metastatic disease found in humans summary of tumor features shared between animals and humans (allen et al . 2002) we searched pubmed / medline using the keywords spontaneous, animal, tumor, oncology and comparative alone or in combination and cross - referencing among published papers . Selected papers from 1910 to 2013 were chosen on the basis of their content (evidence - based quality and reliability). Spontaneous canine tumors, such as prostate carcinoma, chondrosarcoma, which accounts for approximately 10% of all primary bone tumors in dogs (beveridge and sobin 1976), axial osteosarcoma, which accounts for 80% of all primary bone tumors in dogs (thompson and pool 2002) and synovial cell sarcoma, have been extensively described representing valuable models to study carcinogenesis (vail et al . 1994). Most canine prostate carcinomas affect both elderly sexually intact dogs and dogs which had undergone surgical castration after reaching sexual maturity . In 20052009, a pilot study evaluated the incidence of spontaneous tumors in dogs living in the neighborhood of venice and vicenza (vascellari et al . Two thousand five hundred and nine out of 296,318 canine cases of neoplasia were diagnosed during the first three years . The estimated annual tumor incidence rate per 100,000 dogs was 282, with an equal distribution of malignant and benign tumors between male and female dogs . Moreover, in pure breeds, a twofold higher incidence of malignant tumors, with respect to mixed breeds, was observed . The incidence increased with age in both genders . Due to the dissimilar methodologies and variable reference populations of both european and north american veterinary cancer registries, the occurrence of spontaneous tumors in pet animals has been underestimated (bonnett et al . 1997; dobson et al . The animal tumor registry of genoa (italy) retrospectively showed that, from 1985 to 2002, 70% of all cancer cases in female dogs were located in the breast (merlo et al . The overall incidence of cancer was 99.3 per 100,000 dogs per year in male dogs and 272.1 in female dogs . Among domestic animal tumors, spontaneous squamous cell carcinomas provided additional information about the pathology of oral cancer (gardner 1996). As far as oral tumors affecting dogs are concerned, malignant melanoma accounts for the 4% of all canine tumors (macewen et al . Canine malignant melanomas and human melanomas are treated similarly using surgery and/or fractionated radiation therapy and immunological therapies (vail and thamm 2004; bergman et al . Therefore, canine malignant melanomas are suitable models for new immunotherapeutic protocols in humans (vail and thamm 2004). Dogs and cats also frequently develop squamous cell carcinomas (dorn and priester 1976; strafuss et al . Skin and subcutaneous tissue neoplasms are the most frequently recognized neoplastic disorders in domestic animals and can be caused by prolonged and continuous exposure to sunlight (madewell 1981). Alimentary tract cancer in dogs has lower incidence, with ductal and acinar carcinomas occurring most frequently in females than in males while intranasal tumors account for 12% of all canine neoplasms (priester 1974). Primary bone cancer is the second most often detected in dogs, and its main risk factors are ionizing radiations, chemical carcinogens and viruses (madewell 1981). Moreover, preexisting bone defects and skeletal abnormalities increase the risk factors from 60 up to 185 times for large and giant breeds with respect to small dogs (tjalma 1966). As to primary brain tumors, they account for approximately 2% of all cancer in human adults (mckinney 2004) and for 0.01% in dogs (lecouteur 1999). Specifically, gliomas are more common among brachycephalic breeds of dogs, especially boxers, english bulldogs and boston terriers (hayes 1976). High incidence rates were also detected for non - hodgkin s lymphoma in bitches and for non - hodgkin s lymphoma and skin cancer in male dogs (merlo et al . 2008). Additionally, the incidence rate of cancer increased with age ranging between 23.7 and 763.2 in bitches and 16.5 and 237.6 in male dogs aged 3 and> 911 years . Hemangiosarcoma, which can affect both cats and dogs, involves the musculoskeletal system, and mean survival time in dogs affected by this condition ranges from 267 (ogilvie et al . The ovarian and epithelial tumors, although quite rare in domestic animals, have been reported mainly in cats, dogs and horses (maclachlan 1987). A single unique gonadoblastoma tumor affecting human males or females has been observed in the testicle of a dog (turk et al . Spontaneous testicular tumors, which are quite common in aged dogs, are mainly seminomas (solitary, unilateral, more frequent in the right testicle and with a reduced possibility to develop metastases, if compared with humans) (kennedy et al . 1998), sertoli cell tumors and leydig cell tumors, which can coexist together along with seminomas (nielsen and kennedy 1990) and occur with the same frequency (maiolino et al . 2004). Suggested a possible correspondence between human spermatocytic seminomas and most canine seminomas in order to justify their low metastatic behavior, showing a potential predictive model for the development of a treatment protocol (maiolino et al . Further, rare dog tumors are fibrosarcoma, rib, vertebral body and pelvis tumors (both in dogs and in cats) (dernell et al . 1998; pirkey - ehrhart et al . 1992) and multiple myeloma (rare in cats and uncommon in dogs), which accounts for 8% of all canine hematopoietic tumors and affects older dogs with no breed or sex predilection (matus et al . 1986) and intracranial central neurocytomas (russell and burch 1959). Canine mast cell tumors, which account for 1621% in this species (thamm and vail 2001), display a molecular alteration in the proto - oncogene c - kit, which is involved in mast cell differentiation, proliferation, survival and activation (london et al . 1999). Specifically, the mutation of exon 11 of c - kit occurs in 3050% of advanced mast cell tumors and is similar to that usually occurring in 5090% of human gastrointestinal stromal tumors (heinrich et al . 2002; london et al . This evidence emphasizes a parallel between human and canine mast cell tumors underlying the possibility of using the canine model to develop new beneficial therapeutics for both species (pryer et al . Non - hodgkin lymphoma, which accounts for 5% of all malignant tumors and 83% of all hematopoietic malignancies in dogs (vail and thamm 2004), affects mostly middle - aged, older dogs (german shepherds, boxers, basset hounds and saint bernards) (macewen and young 1996) and both genders equally and corresponds to intermediate and high - grade non - hodgkin lymphoma in humans . It is generally associated with a poor prognosis, and in 7080% of cases, it is b - cell mediated, whereas in 2030%, it is t - cell mediated (macewen 1995). Due to its sensitivity to chemotherapy, it is used as a model both for development of new chemotherapy drugs and multiple drug resistance studies (vail and macewen 2000). Moreover, non - hodgkin lymphoma has been used to develop hypoxic cell markers, study the whole - body hyperthermia effects and evaluate autologous bone transplantation (vail and thamm 2004). Spontaneous osteosarcomas account for more than 70% of malignant bone tumors in cats (thompson and pool 2002; brodey and riser 1969). These tumors are relevant to human cancer biology, due to their relative resistance to chemotherapy (he et al . Injection - site sarcomas in felines, usually fibrosarcomas, are caused by an intense inflammatory reaction or vaccination adjuvants (carwardine et al . They are highly locally invasive and metastasize in up to 23% of the affected cases (seguin 2002). Although aggressive surgical resection remains the gold standard therapy, chemotherapy and radiation can prolong the survival times (hershey et al . 2000; kobayashi et al . 2002). Uterine tumors, rare in cats, account for 0.29% of feline neoplasms (miller et al . 2003), and they include endometrial adenocarcinoma, mixed mullerian tumor, leiomyoma which occurs most frequently (maclachlan and kennedy 2002) and myxoid leiomyosarcoma (cooper et al . Even if the etiology of nervous system tumors is not well understood, cats are commonly affected by meningiomas (troxel et al . However, the biological behavior of such tumor, except for the anaplastic type, is generally considered benign in both humans (louis et al . A pilot study evaluated the incidence of spontaneous tumors in cats living in the neighborhood of venice and vicenza (vascellari et al . Four hundred and ninety - four out of 214,683 feline cases of neoplasia were diagnosed during the first three years . For all tumors, the estimated annual incidence rate in cats per 100,000 was 77 . Furthermore, a 4.6-fold higher incidence of malignant tumors was observed in cats, if compared to benign pathology . Moreover, in pure breed cats, a twofold higher incidence of malignant tumors was observed and it increased with age in both malignant and benign pathologies, if compared with mixed breeds . In 2009, wells introduced the potential role of pets in offering a therapeutic value to humans (wells 2009b, 2012). Previous reports emphasized the role of dogs and cats as preventers of ill - health showing that pet owners are healthier than non - owners (parslow et al . 2002; anderson et al . 1992; baun et al . 1984; dembicki and anderson 1996; friedmann et al . 1980; katcher 1981; katcher et al . 1983; larson et al . 2010; sebkova 1977; siegel 1993; vormbrock and grossberg 1988). Furthermore, the ability of pets to facilitate human recovery has been explored (larson et al . 2010). For instance, friedmann et al . Reported that pet owners were significantly more likely to be alive 1 year after a heart attack than non - owners (friedmann et al . 1980) and dogs were stronger facilitators to recovery from ill - health than cats (friedmann and thomas 1995). Therefore, dogs may contribute indirectly to long - standing human physical health, which is of great importance, especially in immunocompromised patients (hemsworth and pizer 2006), possibly due to the increased physical activity which typically characterizes the ownership of a dog (larson et al . Dogs have also been used as warning systems for cancer (williams and pembroke 1989), epilepsy (brown and strong 2001; dalziel et al . 2003; strong et al . 1999, 2002) and diabetes (chen et al . 2000; they have also been used as therapists in nursing homes (crowley - robinson et al . 2002), visitors in pediatric hospital wards (moody et al . 2002), assistants for the disabled (davis et al . 2004; fishman 2003; sanders 2000), enhancers of psychological welfare (raina et al . 1999) and rehabilitators for prisoners (merriam - arduini 2000; strimple 2003). Spontaneous canine tumors, such as prostate carcinoma, chondrosarcoma, which accounts for approximately 10% of all primary bone tumors in dogs (beveridge and sobin 1976), axial osteosarcoma, which accounts for 80% of all primary bone tumors in dogs (thompson and pool 2002) and synovial cell sarcoma, have been extensively described representing valuable models to study carcinogenesis (vail et al . 1994). Most canine prostate carcinomas affect both elderly sexually intact dogs and dogs which had undergone surgical castration after reaching sexual maturity . In 20052009, a pilot study evaluated the incidence of spontaneous tumors in dogs living in the neighborhood of venice and vicenza (vascellari et al . Two thousand five hundred and nine out of 296,318 canine cases of neoplasia were diagnosed during the first three years . The estimated annual tumor incidence rate per 100,000 dogs was 282, with an equal distribution of malignant and benign tumors between male and female dogs . Moreover, in pure breeds, a twofold higher incidence of malignant tumors, with respect to mixed breeds, was observed . The incidence increased with age in both genders . Due to the dissimilar methodologies and variable reference populations of both european and north american veterinary cancer registries, the occurrence of spontaneous tumors in pet animals has been underestimated (bonnett et al . 1997; dobson et al . The animal tumor registry of genoa (italy) retrospectively showed that, from 1985 to 2002, 70% of all cancer cases in female dogs were located in the breast (merlo et al . The overall incidence of cancer was 99.3 per 100,000 dogs per year in male dogs and 272.1 in female dogs . Among domestic animal tumors, spontaneous squamous cell carcinomas provided additional information about the pathology of oral cancer (gardner 1996). As far as oral tumors affecting dogs are concerned, malignant melanoma accounts for the 4% of all canine tumors (macewen et al . Canine malignant melanomas and human melanomas are treated similarly using surgery and/or fractionated radiation therapy and immunological therapies (vail and thamm 2004; bergman et al . Therefore, canine malignant melanomas are suitable models for new immunotherapeutic protocols in humans (vail and thamm 2004). Dogs and cats also frequently develop squamous cell carcinomas (dorn and priester 1976; strafuss et al . Skin and subcutaneous tissue neoplasms are the most frequently recognized neoplastic disorders in domestic animals and can be caused by prolonged and continuous exposure to sunlight (madewell 1981). Alimentary tract cancer in dogs has lower incidence, with ductal and acinar carcinomas occurring most frequently in females than in males while intranasal tumors account for 12% of all canine neoplasms (priester 1974). Primary bone cancer is the second most often detected in dogs, and its main risk factors are ionizing radiations, chemical carcinogens and viruses (madewell 1981). Moreover, preexisting bone defects and skeletal abnormalities increase the risk factors from 60 up to 185 times for large and giant breeds with respect to small dogs (tjalma 1966). As to primary brain tumors, they account for approximately 2% of all cancer in human adults (mckinney 2004) and for 0.01% in dogs (lecouteur 1999). Specifically, gliomas are more common among brachycephalic breeds of dogs, especially boxers, english bulldogs and boston terriers (hayes 1976). High incidence rates were also detected for non - hodgkin s lymphoma in bitches and for non - hodgkin s lymphoma and skin cancer in male dogs (merlo et al . 2008). Additionally, the incidence rate of cancer increased with age ranging between 23.7 and 763.2 in bitches and 16.5 and 237.6 in male dogs aged 3 and> 911 years . Hemangiosarcoma, which can affect both cats and dogs, involves the musculoskeletal system, and mean survival time in dogs affected by this condition ranges from 267 (ogilvie et al . Although quite rare in domestic animals, have been reported mainly in cats, dogs and horses (maclachlan 1987). A single unique gonadoblastoma tumor affecting human males or females has been observed in the testicle of a dog (turk et al . Spontaneous testicular tumors, which are quite common in aged dogs, are mainly seminomas (solitary, unilateral, more frequent in the right testicle and with a reduced possibility to develop metastases, if compared with humans) (kennedy et al . 1998), sertoli cell tumors and leydig cell tumors, which can coexist together along with seminomas (nielsen and kennedy 1990) and occur with the same frequency (maiolino et al . Suggested a possible correspondence between human spermatocytic seminomas and most canine seminomas in order to justify their low metastatic behavior, showing a potential predictive model for the development of a treatment protocol (maiolino et al . Further, rare dog tumors are fibrosarcoma, rib, vertebral body and pelvis tumors (both in dogs and in cats) (dernell et al . 1998; pirkey - ehrhart et al . 1995; straw et al . 1992) and multiple myeloma (rare in cats and uncommon in dogs), which accounts for 8% of all canine hematopoietic tumors and affects older dogs with no breed or sex predilection (matus et al . Canine mast cell tumors, which account for 1621% in this species (thamm and vail 2001), display a molecular alteration in the proto - oncogene c - kit, which is involved in mast cell differentiation, proliferation, survival and activation (london et al . Specifically, the mutation of exon 11 of c - kit occurs in 3050% of advanced mast cell tumors and is similar to that usually occurring in 5090% of human gastrointestinal stromal tumors (heinrich et al . 2002; london et al . This evidence emphasizes a parallel between human and canine mast cell tumors underlying the possibility of using the canine model to develop new beneficial therapeutics for both species (pryer et al . Non - hodgkin lymphoma, which accounts for 5% of all malignant tumors and 83% of all hematopoietic malignancies in dogs (vail and thamm 2004), affects mostly middle - aged, older dogs (german shepherds, boxers, basset hounds and saint bernards) (macewen and young 1996) and both genders equally and corresponds to intermediate and high - grade non - hodgkin lymphoma in humans . It is generally associated with a poor prognosis, and in 7080% of cases, it is b - cell mediated, whereas in 2030%, it is t - cell mediated (macewen 1995). Due to its sensitivity to chemotherapy, it is used as a model both for development of new chemotherapy drugs and multiple drug resistance studies (vail and macewen 2000). Moreover, non - hodgkin lymphoma has been used to develop hypoxic cell markers, study the whole - body hyperthermia effects and evaluate autologous bone transplantation (vail and thamm 2004). Spontaneous osteosarcomas account for more than 70% of malignant bone tumors in cats (thompson and pool 2002; brodey and riser 1969). These tumors are relevant to human cancer biology, due to their relative resistance to chemotherapy (he et al . Injection - site sarcomas in felines, usually fibrosarcomas, are caused by an intense inflammatory reaction or vaccination adjuvants (carwardine et al . They are highly locally invasive and metastasize in up to 23% of the affected cases (seguin 2002). Although aggressive surgical resection remains the gold standard therapy, chemotherapy and radiation can prolong the survival times (hershey et al . 2000; kobayashi et al . 2002). Uterine tumors, rare in cats, account for 0.29% of feline neoplasms (miller et al . 2003), and they include endometrial adenocarcinoma, mixed mullerian tumor, leiomyoma which occurs most frequently (maclachlan and kennedy 2002) and myxoid leiomyosarcoma (cooper et al . 2006). Even if the etiology of nervous system tumors is not well understood, cats are commonly affected by meningiomas (troxel et al . However, the biological behavior of such tumor, except for the anaplastic type, is generally considered benign in both humans (louis et al . 2007) and common pets like dogs and cats (motta et al . 2012). A pilot study evaluated the incidence of spontaneous tumors in cats living in the neighborhood of venice and vicenza (vascellari et al . Four hundred and ninety - four out of 214,683 feline cases of neoplasia were diagnosed during the first three years . For all tumors, the estimated annual incidence rate in cats per 100,000 was 77 . Furthermore, a 4.6-fold higher incidence of malignant tumors was observed in cats, if compared to benign pathology . Moreover, in pure breed cats, a twofold higher incidence of malignant tumors was observed and it increased with age in both malignant and benign pathologies, if compared with mixed breeds . In 2009, wells introduced the potential role of pets in offering a therapeutic value to humans (wells 2009b, 2012). Previous reports emphasized the role of dogs and cats as preventers of ill - health showing that pet owners are healthier than non - owners (parslow et al . 2002; anderson et al . 1992; baun et al . 1984; dembicki and anderson 1996; friedmann et al . 1980; katcher 1981; katcher et al . 1983; larson et al . 2010; sebkova 1977; siegel 1993; vormbrock and grossberg 1988). Furthermore, the ability of pets to facilitate human recovery has been explored (larson et al . 2010). For instance, friedmann et al . Reported that pet owners were significantly more likely to be alive 1 year after a heart attack than non - owners (friedmann et al . 1980) and dogs were stronger facilitators to recovery from ill - health than cats (friedmann and thomas 1995). Therefore, dogs may contribute indirectly to long - standing human physical health, which is of great importance, especially in immunocompromised patients (hemsworth and pizer 2006), possibly due to the increased physical activity which typically characterizes the ownership of a dog (larson et al . Dogs have also been used as warning systems for cancer (williams and pembroke 1989), epilepsy (brown and strong 2001; dalziel et al . 2003; strong et al . 1999, 2002) and diabetes (chen et al . They have also been used as therapists in nursing homes (crowley - robinson et al . 2002), visitors in pediatric hospital wards (moody et al . 2002), assistants for the disabled (davis et al . 2004; fishman 2003; sanders 2000), enhancers of psychological welfare (raina et al . 1999) and rehabilitators for prisoners (merriam - arduini 2000; strimple 2003). Comparative oncology research has extensively relied on domestic animals (antinoff and hahn 2004; hansen and khanna 2004; hershey et al . 2005; nasir and reid 1999; seltenhammer et al . 2004; withrow and vail 2007). The comparative oncology trials consortium program infrastructure, founded in 2009 to design and execute clinical trials involving dogs affected by cancer and in collaboration with the drug manufacturing industry and nongovernmental groups interested in cancer drug development, aimed to answer biological questions which could inform the development path of novel agents for future use in human cancer patients in a timely and integrated manner (gordon et al . The recent identification of the canine genome has provided evidence of strong similarities with humans (lindblad - toh et al . 2005; obrien and murphy 2003; ostrander et al . 2006) such as several cancer - associated gene families (hoffman and birney 2007), as well as the presence of the similar genetic cancer - associated molecular alterations (haga et al . 2001 tumor initiation and progression are also influenced by age, nutrition, sex, reproductive status and environmental exposures in both human and canine cancers (bukowski et al . 1998; hayes and fraumeni 1977; misdorp 1996; mukaratirwa 2005; olson 2007; patronek et al . 1997). The lack of gold standards for the management of cancer in dogs and cats, as well as the high motivation of pet owners to seek out new options for its management, underlines the increasing need to evaluate novel therapeutics in these populations . In this ways, it is possible to assess the effectiveness of new agents when given alone or in combination with other therapies prior to the clinical testing of the drug . The enrollment of pet dogs in preclinical and clinical trials is now focusing on new anti - neoplastic drug research and development . Although regulation of animal research possesses guidelines such as the three rs (replacement, reduction and refinement) (russell and burch 1959), legal implications regarding the use of animal models for research purposes are still debated (griffin 1998; porrello et al . Although the existence of spontaneous pet tumors could be a reliable model for human cancers, many open questions, related to the opportunities to select and recruit new types of animal models in oncology and to their legal and ethical implications, remain unanswered . Unfortunately, the use of spontaneous animal models implicates several professional and ethical warnings with conflictory debates . In the recent years, researchers have also focused their exploration on a possible role of some dog species as early warning systems for cancer (williams and pembroke 1989), epilepsy (brown and strong 2001; dalziel et al . 2003; strong et al . 1999, 2002) and diabetes (chen et al . 2000; lim et al . 1992; mcaulay et al . 2001). While some dogs have showed innate ability to recognize with smell the human cancers in vivo, others have been trained to perform this skill (willis et al . 2004). From this overview, there is evidence that companion animal health care is impressively growing in terms of development of new therapies and diagnostic tools, nutrition and disease prevention (kling 2007). Conclusively, we strongly encourage the animalists, veterinarians and clinical researchers to join us and support ethical observational studies on domestic and laboratory animal spontaneous cancer epidemiology, early prevention, diagnosis and treatment in order to detect and possibly eliminate alimentary and environmental factors which can also be cancerogenic for humans . In fact, the shorter life span of animals, compared with that of humans, can give a rapid overview of many clinical features which also characterize human cancer . Furthermore, the pooling of anecdotal veterinary reports on drugs or multimodal animal cancer treatments, protocols and outcomes, including surgical pathology, oncoimmunology and molecular biology investigations, will give outstanding contribution to the interspecies translational information with undoubted mutual interspecies benefit . The pet psychological and physical support to the fitness and wellness of cancer patients should be more deeply investigated and largely extended in the palliation practice.
Health - related quality of life (hrqol) is a subjective assessment of the patient1 that is related to the perception of their health,14 well - being, and functioning,1 and also to their worries about the future.4 although the term hrqol is not yet precisely defined,5 it is a useful measure to assess the overall impact of a particular nosological entity in patient,2 and it complements the clinical information.3,6 there are several generic tools that can be used to assess hrqol; they can be used not only in the healthy population but also in specific populations4 because they allow to assess a wide set of important dimensions that can be used in different diseases.4,68 wilson and cleary9 propose a conceptual model to explain the relationships between clinical variables and measures of hrqol in order to establish the connection between diagnosis and therapy and, thus, to increase patients hrqol . This classification is divided into five levels biological and physiological factors, symptoms, functioning, general health perceptions, and overall quality of life and proposes causal relationships between all of them . With regard to the relationship between hrqol, the presence of hypertension (htn), and diabetes - related chronic complications, the literature is not consistent in pointing out that their presence decreases hrqol and, hence, the importance of evaluating the relationship between these variables and hrqol . Moreover, as far as we know, the only two researches carried out in portugal5,10 that have assessed the relationship between the most frequent chronic complications of diabetes and hrqol did not make the adjustment of the hrqol scores for any demographic and/or clinical variables that can mediate some of these relationships . On the other hand, they evaluated simultaneously type 1 and type 2 diabetic patients and did not assess chronic complications according to diabetes type . Lastly, the investigations that evaluated the relationship between the number of diabetes - related chronic complications and hrqol did not make stratified analyses according to sex that can bring important supplemental information to the one achieved for the whole sample . Concerning microvascular complications of diabetes, while some studies1012 have found that the presence of diabetic retinopathy was related to worse hrqol, sakamaki et al13 found no relationship between the two variables . Results of research on the relationship between diabetic nephropathy and hrqol have found, on one hand, that its presence was related to lower hrqol10,11 and, on the other hand, no relationship between both variables.13 concerning the relationship between diabetic neuropathy and hrqol, while sakamaki et al13 found no relationship between the two variables, some studies10,1417 have found that its presence was related to worse hrqol . As far as diabetes - related macrovascular complications are concerned, although there is evidence that the presence of cardiovascular disease (cvd) in persons with diabetes was related to lower hrqol,5,10,12,1416,1822 wasem et al17 found no relationship between the two variables . Regarding the relationship between the presence of peripheral arterial disease (pad) and hrqol, silva et al,5 neves et al,10 javanbakht et al,11 and quah et al16 have shown that patients with this chronic complication had worse hrqol, whereas sakamaki et al13 and wasem et al17 found no relationship between the two variables, and venkataraman et al22 reported that its presence was related to better hrqol . Regarding the relationship between htn and diabetes, while some studies5,10,23,24 have found that the presence of htn was related to lower hrqol, wexler et al20 and hill - briggs et al25 reported no relationship between the two variables . Finally, some researchers, looking at the relationship between the number of diabetes - related chronic complications and hrqol, have found that an increased number of complications was related to worse hrqol.16,26 however, caldwell et al27 did not find a relationship between the two variables . The aim of this cross - sectional study was to assess the relationship between hrqol, the presence or absence of htn, and diabetes - related micro- and macrovascular complications in type 2 diabetes, as well as the association between hrqol and the number of chronic complications, for the whole sample and by sex . We focused on the assessment of htn and long - term complications on hrqol in type 2 diabetic patients for three reasons: first, because htn is a highly prevalent comorbidity in these individuals; second, because it is highly associated with diabetes - related chronic complications; and third, because it can have a negative association with hrqol . A consecutive sample of 100 type 2 diabetic patients from the outpatient department of endocrinology, diabetes and metabolism, centro hospitalar so joo, porto, portugal, was recruited . Patients were included in the study if they had a diagnosis of type 2 diabetes, were at least 18 years old, and were not currently pregnant . The protocol for the study was approved by the ethics committee for health of centro hospitalar so joo, and informed consent was obtained from all participants before participating in the study . General hrqol was assessed by the portuguese version of the medical outcomes study 36-item short - form health survey (sf-36),28,29 and it was administered by one of the researchers . We evaluated the presence of some clinical variables that are reported in the literature to be related to hrqol among type 2 diabetes;5,1012,1424,26 these factors included microvascular complications, macrovascular complications, and htn . Type 2 diabetic patients were classified as on oral hypoglycemic agents or insulin therapy (the latter including patients only on insulin therapy or on oral hypoglycemic agents plus insulin therapy). Microvascular complications considered were diabetic retinopathy, diabetic nephropathy, and diabetic peripheral neuropathy; macrovascular complications were cvd and pad . We considered that the patients had retinopathy if they had at least microaneurysms, nephropathy if they had at least albumin excretion rate higher than 30 mg / g creatinine, peripheral neuropathy if they had two or more insensate sites across two feet for the 10 g monofilament or one or more insensate sites for the 128 hz tuning fork . Cvd was defined by the presence of at least one of the following conditions: angina pectoris or acute myocardial infarction or transient ischemic attack or stroke; and pad was defined as an ankle - brachial index of 0.9 or a prior history of lower extremity revascularization . Htn was defined by one of the following: systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or current use of antihypertensive agents or documented diagnosis of htn . The sf-36 healthy survey comprises 36 items covering functional health status and general health, currently and over the past 4 weeks, and measures eight dimensions: physical functioning, role - physical, bodily pain, general health, vitality, social functioning, role - emotional, and mental health . Raw dimension scores were transformed to scales ranging from 0 to 100, in which higher scores indicate higher hrqol . Statistical package for the social sciences version 20.0 for windows software (ibm corporation, armonk, ny, usa) was used for the analysis . Descriptive statistical analysis included calculation of means and standard deviations for cardinal variables, and frequencies for ordinal and nominal variables . The degree of association between pairs of variables was measured by pearson s (r) or spearman s (rs) correlation coefficients . As there were age - discrepancies within the sample (minimum age of 44 years old, and maximum age of 80 years old), we adjusted sf-36 results to the age of the participants; therefore, all other variables were related to the residuals of simple linear regression models having each dimension as a dependent variable and age as the independent variable . A consecutive sample of 100 type 2 diabetic patients from the outpatient department of endocrinology, diabetes and metabolism, centro hospitalar so joo, porto, portugal, was recruited . Patients were included in the study if they had a diagnosis of type 2 diabetes, were at least 18 years old, and were not currently pregnant . The protocol for the study was approved by the ethics committee for health of centro hospitalar so joo, and informed consent was obtained from all participants before participating in the study . General hrqol was assessed by the portuguese version of the medical outcomes study 36-item short - form health survey (sf-36),28,29 and it was administered by one of the researchers . We evaluated the presence of some clinical variables that are reported in the literature to be related to hrqol among type 2 diabetes;5,1012,1424,26 these factors included microvascular complications, macrovascular complications, and htn . Type 2 diabetic patients were classified as on oral hypoglycemic agents or insulin therapy (the latter including patients only on insulin therapy or on oral hypoglycemic agents plus insulin therapy). Microvascular complications considered were diabetic retinopathy, diabetic nephropathy, and diabetic peripheral neuropathy; macrovascular complications were cvd and pad . We considered that the patients had retinopathy if they had at least microaneurysms, nephropathy if they had at least albumin excretion rate higher than 30 mg / g creatinine, peripheral neuropathy if they had two or more insensate sites across two feet for the 10 g monofilament or one or more insensate sites for the 128 hz tuning fork . Cvd was defined by the presence of at least one of the following conditions: angina pectoris or acute myocardial infarction or transient ischemic attack or stroke; and pad was defined as an ankle - brachial index of 0.9 or a prior history of lower extremity revascularization . Htn was defined by one of the following: systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or current use of antihypertensive agents or documented diagnosis of htn . The sf-36 healthy survey comprises 36 items covering functional health status and general health, currently and over the past 4 weeks, and measures eight dimensions: physical functioning, role - physical, bodily pain, general health, vitality, social functioning, role - emotional, and mental health . Raw dimension scores were transformed to scales ranging from 0 to 100, in which higher scores indicate higher hrqol . Statistical package for the social sciences version 20.0 for windows software (ibm corporation, armonk, ny, usa) was used for the analysis . Descriptive statistical analysis included calculation of means and standard deviations for cardinal variables, and frequencies for ordinal and nominal variables . The degree of association between pairs of variables was measured by pearson s (r) or spearman s (rs) correlation coefficients . As there were age - discrepancies within the sample (minimum age of 44 years old, and maximum age of 80 years old), we adjusted sf-36 results to the age of the participants; therefore, all other variables were related to the residuals of simple linear regression models having each dimension as a dependent variable and age as the independent variable . Most patients were male, were receiving only oral hypoglycemic agents, and had the disease for <10 years . Only 21.0% of the patients did not have chronic complications related to diabetes . Among the chronic complications considered, the most prevalent was diabetic retinopathy, followed by cvd . The average values obtained by patients in the eight dimensions of the sf-36, as well as the association between these scores and age are shown in table 2 . There was no association between age and any of the eight dimensions of the sf-36 . The age - adjusted scores of hrqol by the presence or absence of each of the microvascular complications of diabetes are shown in table 3, macrovascular complications of diabetes in table 4, and htn in table 5 . There was an overall tendency for the presence of each of the chronic complications and htn to be related to worse hrqol in all dimensions of sf-36, regardless of age; the only exception was the dimension role - physical in diabetic nephropathy . The complications related to larger number of dimensions were peripheral neuropathy and cvd (all dimensions except bodily pain). In type 2 diabetic patients with retinopathy, pad, and nephropathy, the hrqol only differed on four, three, and two dimensions, respectively retinopathy (physical functioning, general health, vitality, and mental health), pad (physical functioning, role - physical, and general health), and nephropathy (general health and vitality). The most related dimensions to worse hrqol were general health and vitality; the general health dimension was lower in patients with any of the long - term complications and htn, whereas the vitality dimension was lower in htn and all diabetes - related complications with the exception of pad . On the other hand, the bodily pain was the dimension least related to hrqol because it was not related to any of these variables . The association between hrqol and the number of chronic complications for the whole sample and by sex are shown in table 6 . An increased number of long - term complications tended to be associated with worse hrqol in all dimensions of sf-36, regardless of age; the only exceptions were the bodily pain dimension in males and the role - emotional dimension in females . This association reached statistical significance in all dimensions with the exception of bodily pain in the total sample, all except bodily pain and social functioning in men, and only in general health, vitality, and mental health dimensions in women . In men, the most associated dimension of hrqol with the number of chronic complications was physical functioning, followed by general health, and then by role - physical . In women, the most associated dimensions with the number of complications were general health, followed by vitality . The dimensions physical functioning, role - physical, and role - emotional were only associated with the number of complications in males . The presence of diabetes - related chronic complications and htn, as well as the number of complications were related to worse hrqol regardless of age, with the different conditions being related to different dimensions of hrqol . The adjustment of sf-36 scores for age may obscure relationships between diabetes - related complications, htn, and hrqol dimensions that strongly depend on a concomitant relationship with age . Thereby, absolute scores on the sf-36 and their relationship with long - term complications and htn may have clinical usefulness, but to analyze their association with hrqol, which was our intention, it is recommended that values are adjusted for age if there are significant age - discrepancies within the sample . Regarding the relationship between the presence of retinopathy and hrqol, we found that having retinopathy was related to lower hrqol in terms of physical functioning, general health, vitality, and mental health . Our results partially overlap with the findings of neves et al10 for three reasons: first, these portuguese researchers have found that the diabetic patients with retinopathy had worse hrqol than those without retinopathy in all sf-36 dimensions; second, because in that study the authors included in their analysis a quite heterogeneous sample (ie, type 1 and type 2 diabetic patients), whereas we included a less heterogeneous one; and third, because they did not make the adjustment of the hrqol scores for age . Conversely, a cross - sectional study13 and a longitudinal study30 have found no relationship between the two variables . Contrary to what we expected, which was a reduced hrqol in both physical and mental component scores of the sf-36, we found that diabetic retinopathy appears to have a greater relationship with the physical component than the mental one . Our results showed that the presence of nephropathy was related to worse hrqol in terms of general health and vitality . These results are partially in agreement with the findings of neves et al,10 because these authors reported that its presence was related to lower hrqol in all dimensions of sf-36 . However, as mentioned earlier, the study of neves et al10 has methodological limitations that might have compromised the results . In contrast, two studies13,30 reported no relationship between the presence of nephropathy and hrqol . Surprisingly, we did not find differences between the two groups in the other dimensions of the sf-36, particularly in terms of mental component . We found that the presence of peripheral neuropathy was related to worse hrqol in all dimensions of sf-36 with the exception of the bodily pain dimension . These results are different from those obtained in another study,13 which did not find a relationship between peripheral neuropathy and hrqol . Our results are consistent with the findings of neves et al,10 given that in that study it was found that the presence of neuropathy was related to lower hrqol in all dimensions of sf-36, and partially overlapping with the results of other investigations.15,16 in the first study it was reported that having neuropathy was related to worse hrqol in terms of physical functioning and bodily pain dimensions of sf-36, while in the latter one it was found that the presence of peripheral neuropathy was related to lower hrqol in terms of physical and mental components of the aforementioned tool . Concerning the relationship between the presence of cvd and hrqol, wasem et al17 reported no relationship between the two variables, whereas we found worse hrqol in patients with cvd in all dimensions of sf-36 with the exception of bodily pain dimension when comparing with those that did not have the disease . These findings are partially in agreement with the results of four cross - sectional studies5,10,15,22 because in those studies, the presence of cvd was related to lower hrqol in terms of physical functioning, general health,5,10,15,22 role - physical,5,10,22 bodily pain, vitality,5,10,15 social functioning,15 and physical component.15,22 on the other hand, our findings are also partially in accordance with the results of two longitudinal studies,30,31 which found that the presence of cvd was related to worse physical functioning, general health, vitality, mental component,30 and physical component.30,31 our results and those obtained in those studies suggest that cvd causes physical and emotional constraints in patients with diabetes . With regard to physical impairments, it appears that hrqol and life expectancy of these patients are more reduced, given that cvd is the major cause of death in both diabetic males and females.32 thereby, it is not surprising that these patients become physically more limited, and, consequently, end up becoming psychologically more vulnerable . Regarding pad and its relationship with hrqol, sakamaki et al13 and wasem et al17 found no relation between the two variables, and surprisingly, venkataraman et al22 reported that patients with pad had better hrqol in terms of physical functioning, role - physical, and physical component than those without pad . Conversely, we found that pad was related to worse hrqol in terms of physical functioning, role - physical, and general health . Our results are partially in agreement with the findings of silva et al5 and neves et al,10 because it was found that its presence was related to worse hrqol in terms of physical functioning, bodily pain, vitality,5,10 role - physical, general health, social functioning, role - emotional, and mental health.10 however, it is worth noting that these studies5,10 have methodological limitations that might have jeopardized the results, namely, the inclusion of type 1 and type 2 diabetes, and the non - adjustment of the hrqol scores for age . As with diabetic retinopathy and nephropathy, although wexler et al20 and hill - briggs et al25 have found no relationship between the presence of htn and hrqol, in this study htn was related to worse hrqol in terms of general health and vitality . These findings are partially in agreement with the results of prior studies5,10,23,24 the presence of htn was related to lower hrqol in terms of physical functioning, role - physical, bodily pain,5,10 general health,5,10,23 vitality, social functioning, mental health,10,24 role - emotional,10 and mental component.24 these differences between the two groups may possibly be explained by its frequent association with cardiac, cerebrovascular, renal, and ocular complications . Isolated htn (except hypertensive crisis) is usually asymptomatic, and it is through predisposition to associated diseases that eventually may help to explain the worse hrqol of the hypertensive patients . On the other hand, various items of the sf-36 are somewhat similar to some symptoms of hypotension and hypoglycemia and, thus, may have contributed to this worse hrqol in the hypertensive patients . With regard to the relationship between the number of diabetes - related chronic complications and hrqol, while caldwell et al27 reported no relationship between the two variables, we found that an increased number of complications was associated with worse hrqol in all dimensions of sf-36 with the exception of bodily pain dimension . These findings are partially in agreement with the results of two cross - sectional studies.16,26 in the first study, it was found that a higher number of chronic complications was related to worse hrqol in terms of physical and mental component, whereas in the latter investigation, an increased number of long - term complications was related to worse hrqol in terms of physical functioning, role - physical, role - emotional, social functioning, and mental health . On the other hand, our results are partially consistent with the findings of a longitudinal study33 that have found that an increased number of complications predicted lower hrqol in terms of physical and mental components . In the present study, the existence of higher number of diabetes - related complications suggests a set of physical, emotional, and social constraints, which result from the emergence of new symptoms, the intensification of the treatment regimen, and a feeling of frustration, anger, and failure after the worsening of diabetes . Different hrqol dimensions were more strongly associated with the number of chronic complications in male and female patients . Some differences in the results between this study and others may be explained by the different generic tools used to assess hrqol that do not measure exactly the same dimensions . This would also allow multivariate analysis, thus decreasing the confounder effect of the remaining variables . Multiple testing also must be considered, as it increases the occurrence of statistically significant results; however, the comparison of our results with prior studies enhances their adequate interpretation despite this limitation . Furthermore, most of the patients interviewed were of low sociocultural status, which may have limited their understanding of the questions in the instrument used to assess hrqol . Generalization of our findings may be limited because our sample was collected in a central public hospital and might not be representative of the population with type 2 diabetes in other care centers or in primary care . Further studies are warranted to adjust the dimensions of hrqol to other variables (eg, sex, body mass index, type of diabetes and treatment regimens, and duration of the disease) to explore the relationships between hrqol and the presence of diabetes - related chronic complications and htn, which we think may explain some of these relationships . Further investigations on stratifying the severity of diabetes - related chronic complications are required because they may bring important complementary information . More studies are needed to explore sex differences with regard to the association between the number of long - term complications and hrqol and considering different groups of complications . Lastly, the use of qualitative methodology could be useful to obtain information complementary to that achieved by quantitative studies, allowing more accurate interpretation of some results . First, we included a more homogeneous sample (ie, only type 2 diabetic patients) in comparison to the two previous researches carried out in portugal5,10 which have included both type 1 and type 2 diabetic patients when assessing the relationship between diabetes - related chronic complications, htn and hrqol without making stratified analyses according to type of diabetes . Second, the age - adjustment of the sf-36 values to evaluate the relationship between chronic complications, htn and hrqol, given the age - discrepancies within the sample, which was not made in the two mentioned portuguese investigations . Third, we evaluated the association between hrqol and number of chronic complications by sex, which was not made previously,16,26,27 and we think may provide important supplemental information . In summary, our findings suggest that the presence and increased number of diabetes - related chronic complications, as well as the presence of htn were related to worse age - adjusted hrqol . It is worth noting that among diabetes - related long - term complications, peripheral neuropathy and cvd were more strongly related to patients age - adjusted hrqol . In males, there was a strong association between the number of chronic complications and hrqol than in their counterparts.
, they can occur at any age, and the clinical symptoms vary, ranging from asymptomatic laboratory abnormalities to life - threatening bleeding . The presence of life - threatening factor v inhibitor cases underscores the need for prompt diagnosis and treatment (1). Various causes of factor v inhibitors have been reported, and most of the cases described in japan developed after exposure to bovine thrombin preparations, which have been used frequently as topical hemostatic agents in various types of surgery . However, due to the increasing surgical use of recombinant human or bovine forms of thrombin, at least in developed countries, the number of factor v inhibitor cases associated with these products appears to be in decline . In parallel, attention has lately been focused on other possible causes of factor v inhibitor development (2). We herein report the case of a patient who developed idiopathic acquired factor v inhibitor and died due to an intracerebral hemorrhage . A 79-year - old japanese woman presented herself to another hospital with left pedal edema . She also received oral cefcapene pivoxil hydrochloride hydrate to treat her lower urinary tract symptoms . She was transferred to our hospital because a routine coagulation panel revealed a markedly prolonged prothrombin time of 60 s and an activated partial thromboplastin time of 120 s. her medical history included hypertension, diabetes mellitus, and coronary artery disease (cad). The cad first manifested as angina in june 2001, at which time her coronary angiography demonstrated triple vessel disease . In july 2001, she underwent coronary artery bypass grafting . In december 2012, she was re - admitted to our hospital because of a worsening angina, and she underwent percutaneous coronary intervention . Subsequently, she underwent percutaneous coronary intervention with a drug - eluting stent in november 2013 . No previous bleeding tendency was noted, and she had no significant family history of bleeding disorder . Her medication included clopidogrel sulfate, aspirin, cilnidipine, lisinopril hydrate, pravastatin sodium, and nicorandil . On admission to our hospital in october 2014, her height was 143 cm and weight was 43.1 kg; her body temperature was 37.2, blood pressure 109/47 mmhg, pulse 83/min, clear consciousness, no conjunctival pallor, and no icterus . . Left pedal tender swelling was observed and it was thought to be a hematoma . The measurement of the coagulation factor profile revealed a marked decrease in factor v activity to 3% and somewhat reduced activities of factors ii, ix, x, xi, and xii . The test for coagulation factor v inhibitor was positive (18 bethesda u / ml). Ast: aspartate - aminotransferase, alt: alanine - aminotransferase, -gtp: -glutamyl transpeptidase, ldh: lactate dehydrogenase, bun: blood urea nitrogen, cr: creatinine, ua: uric acid, wbc: white blood cells, rbc: red blood cells, hb: hemoglobin, hct: hematocrit, plt: platelets, pivka - ii: protein induced by vitamin k absence - ii, pt: prothrombin time, pt (inr): prothrombin time international normalized ratio, aptt: activated partial thromboplastin time, fib: fibrin, atiii: antithrombin iii, fdp: fibrin / fibrinogen degradation products, tt: thrombotest, hpt: hepaplastin test, lupus ac: lupus anticoagulant she was given vitamin k and six units of fresh frozen plasma, while the administration of clopidogrel sulfate was stopped, but her coagulation panel did not improve . On day 3 after admission she slipped into a coma, and an emergent brain computed tomography (ct) scan demonstrated she had suffered an intracerebral hemorrhage (fig . Ct of the head of the patient, a 79-year - old woman, obtained on day 3 after admission . Because platelets contain factor v, we transfused 10 units of platelets . In addition, prednisone 1 mg / kg daily was initiated in an attempt to suppress possible autoantibody production against coagulation factor(s). Despite these treatments, the patient's plasma demonstrated prolonged phospholipid - dependent in - vitro clotting tests, such as aptt . Mixing studies with pooled normal plasma failed to correct the abnormal aptt, in which the incubation time was two hours (fig . The levels of phospholipid - dependent clotting factors (factors ii, v, ix, x, xi and xii) were low, and we evaluated the possibility of lupus anticoagulant hypoprothrombinemia syndrome, but the dilute russell viper venom time was not measurable in this case . A laboratory diagnosis of la requires careful adherence to the following criteria: (i) the prolongation of at least one phospholipid - dependent clotting test, (ii) a failure to correct prolonged screening coagulation test upon mixing with pooled normal plasma, (iii) evidence that an inhibitory activity is dependent on phospholipids, and (iv) ruling out any other coagulopathies (such as factor viii inhibitor, heparin) (3). Our patient's laboratory data were consistent with criteria (i), (ii) and (iii), but the level of factor v activity was remarkably low, which was inconsistent with criterion (iv). In addition, the positivity of factor ii inhibitor might be a false - positive result, due to cross - reactivity with factor v inhibitor . Cross mixing test of the patient, a 79-year - old woman . According to the review by franchini and lippi (2), among the conditions associated with the development of factor v inhibitors, autoimmune disorders were present in 13% of the cases examined, whereas 22% were associated with cancer . The most common associated condition was the use of antibiotics, such as -lactams, aminoglycosides (especially streptomycin), cephalosporins, tetracyclines and fluoroquinolones (especially ciprofloxacin), which accounted for 42% of the reported cases . However, it is difficult to establish whether these latter conditions play a causative role in the development of the inhibitor because concomitant antibiotic therapy was also present in almost all cases (2). Cases of an acquired factor v inhibitor due to medications, such as rifaximin, dabigatran etexilate methanesulfonate and warfarin have been described (1,4,5). However, our patient's medications, except for furosemide and cefcapene pivoxil hydrochloride hydrate, had been continually taken for many years without developing any inhibitors . Furthermore, furosemide and cefcapene pivoxil hydrochloride hydrate were initiated after the development of symptoms . Bovine factor v acts as a potent immunological stimulus for the development of anti - bovine factor v inhibitors, which can then cross - react with human factor v. most patients with bovine thrombin - induced autoantibodies showed only coagulation laboratory abnormalities without any hemorrhagic complications, and the antibodies were frequently transient (2). Our patient underwent coronary artery bypass grafting about 10 years earlier, and it is unlikely that the surgery had been the cause of the later factor v inhibitor . Ongoing dapt at the time of development of afvi might have contributed the catastrophic outcome in this patient . According to the aforementioned systematic review, the treatment of acquired factor v inhibitors is based on two steps: the control of the bleeding and the eradication of the antibody . Although treatment is usually unnecessary for asymptomatic patients, a number of therapeutic options have been used in bleeding patients . In a systematic review, 71% had a satisfactory clinical response to the transfusion of platelet concentrates (2). In the same review, immunosuppressive regimens with corticosteroids alone or in association with cyclophosphamide or other immunosuppressants had been used successfully to suppress autoantibody production in 63%, with remission in 76% . The median time to remission was 6 weeks . In our patient, it is possible that the coagulation profile was not corrected because she had only been treated for 5 days (2). Recent studies have demonstrated the effectiveness of rituximab in a number of autoantibody - mediated diseases, including thrombotic thrombocytopenic purpura, autoimmune hemolytic anemia, immune thrombocytopenia and acquired hemophilia due to factor viii inhibitors . Rituximab has been used in three patients with severe pulmonary hemorrhage due to acquired factor v inhibitor (6). The early recognition and initiation of treatment for factor we herein presented a case of factor v inhibitor that resulted in fatal intracranial bleeding . The earlier recognition of this condition and timely initiation of treatment therefore appear to be of critical importance.
Survival data was obtained from records of the ministry of public administration and security as well as from medical records . All slide glasses and specimen photos were reviewed and the histopathologic type, histologic grade, lauren classification, depth of invasion, regional lymph node metastasis, and lymphatic invasion were evaluated . The reviewed cases were reclassified according to the seventh american joint committee on cancer / international union against cancer classification (ajcc / uicc) tumor - node - metastasis (tnm) cancer classification system.20 the world health organization classification system, which describes three main histological grades, including well, moderately, and poorly differentiated, was applied for histological typing and grading of adenocarcinoma.21 the mean follow - up period was 50.9 months (median, 53.3 months; range, 3 to 83 months). The median age was 60.5 years (range, 23 to 84 years) and the male to female ratio was 2.1 . Forty percent (n=183) of patients were diagnosed with early gastric carcinoma and 59.5 percent (n=269) with advanced gastric carcinoma . This study was approved by the institutional review board of korea university guro hospital (kuggr-2010 - 033). Representative portions from the leading edge of tumor infiltration were marked and used for tissue microarray construction . Tumor tissues of 2.0 mm core diameter were taken from donor ffpe blocks and arranged in empty recipient paraffin blocks to generate a tma . The tma blocks were cut into 4 m slices for immunohistochemical staining . A standard streptavidin - biotin peroxidase complex method was used . After deparaffinization and rehydration, slides were heated in a microwave oven for 15 minutes in 10 mm citrate buffer (ph 6.0) and treated with 3% hydrogen peroxide for 20 minutes . Antibodies used in this study were dbc1 (1:200, polyclonal, abcam, cambridge, ma, usa), sirt1 (1:200, clone h-300, santa cruz biotechnology, santa cruz, ca, usa), -catenin (1:500, clone -catenin-1, dako, carpinteria, ca, usa), and p53 (1:500, clone do-7, novocastra, newcastle upon tyne, england). Immunohistochemical analysis by optical microscope was performed by two pathologist (y.r.kang and b.h.kim). 1). The nuclear staining intensity (0, no staining; 1, weak; 2, moderate; 3, strong) and percent areas of positive tumor cell nuclei were graded . When more than 50% of tumor cells showed a moderate or strong staining intensity, we considered it to be positive staining . To examine the correlation between the expression of p53 and other proteins, a cut - off value of 50% was applied to the p53 . To analyze the -catenin expression in tumor cells, the positivity of nuclear, cytoplasmic, and membranous staining was also scored . For nuclear and cytoplasmic staining, cases with tumor cells expressing more than 30% -catenin were considered to be positive, and for membranous staining, cases with a pattern of loss of membranous -catenin staining of more than 30% of tumor cells were regarded as negative (fig . 1). Expression of dbc1 and sirt1 were found mainly in the nuclei . To decrease the effects of observer biases, dbc1 and sirt1 tma slides were analyzed by an image analyzer program (ia). A scanscope cs (aperio, vista, ca, usa) digital scanner was used for scanning of tma slides . A combination of the genie histology pattern recognition tool (aperio) and nuclear quantification cellular analysis tool (aperio) was used to select tumor cells . The algorithm was adjusted to match the requirements of our particular marker and staining set . These adjustments include segmentation type, nuclear curvature threshold, size, roundness, compactness, and elongation . The output data contained the nuclear staining intensity (1 +, 2 +, 3 +, and negative), positive tumor cell percentage, and cell count . The cut - off value of 30% was also applied to dbc1 and sirt1 staining . When the sum of 2 + and 3 + cells exceeded 30% of the total tumor cells, the case was considered to be positive . Kaplan - meier plots and the log - rank test were used to estimate survival rates . Overall survival was calculated as the time from diagnosis to the date of death or the last follow - up date . Correlations between the immunohistochemical expression of proteins and the clinicopathological characteristics were analyzed by pearson's tests, but histologic grade and pathologic stage were analyzed by a spearman's rank tests . Multivariate analysis was performed using the cox proportional hazard model . In all statistical analyses, archived formalin - fixed paraffin - embedded (ffpe) samples of gastric adenocarcinomas were obtained from 452 patients who underwent radical gastrectomy at korea university guro hospital from january 2002 to december 2005 . Clinicopathologic data, including age, sex, and distant metastasis were obtained from medical records . Survival data was obtained from records of the ministry of public administration and security as well as from medical records . All slide glasses and specimen photos were reviewed and the histopathologic type, histologic grade, lauren classification, depth of invasion, regional lymph node metastasis, and lymphatic invasion were evaluated . The reviewed cases were reclassified according to the seventh american joint committee on cancer / international union against cancer classification (ajcc / uicc) tumor - node - metastasis (tnm) cancer classification system.20 the world health organization classification system, which describes three main histological grades, including well, moderately, and poorly differentiated, was applied for histological typing and grading of adenocarcinoma.21 the mean follow - up period was 50.9 months (median, 53.3 months; range, 3 to 83 months). The median age was 60.5 years (range, 23 to 84 years) and the male to female ratio was 2.1 . Forty percent (n=183) of patients were diagnosed with early gastric carcinoma and 59.5 percent (n=269) with advanced gastric carcinoma . This study was approved by the institutional review board of korea university guro hospital (kuggr-2010 - 033). Representative portions from the leading edge of tumor infiltration were marked and used for tissue microarray construction . Tumor tissues of 2.0 mm core diameter were taken from donor ffpe blocks and arranged in empty recipient paraffin blocks to generate a tma . The tma blocks were cut into 4 m slices for immunohistochemical staining . A standard streptavidin - biotin peroxidase complex method was used . After deparaffinization and rehydration, slides were heated in a microwave oven for 15 minutes in 10 mm citrate buffer (ph 6.0) and treated with 3% hydrogen peroxide for 20 minutes . Antibodies used in this study were dbc1 (1:200, polyclonal, abcam, cambridge, ma, usa), sirt1 (1:200, clone h-300, santa cruz biotechnology, santa cruz, ca, usa), -catenin (1:500, clone -catenin-1, dako, carpinteria, ca, usa), and p53 (1:500, clone do-7, novocastra, newcastle upon tyne, england). Immunohistochemical analysis by optical microscope was performed by two pathologist (y.r.kang and b.h.kim). 1). The nuclear staining intensity (0, no staining; 1, weak; 2, moderate; 3, strong) and percent areas of positive tumor cell nuclei were graded . When more than 50% of tumor cells showed a moderate or strong staining intensity, we considered it to be positive staining . To examine the correlation between the expression of p53 and other proteins, a cut - off value of 50% was applied to the p53 . To analyze the -catenin expression in tumor cells, the positivity of nuclear, cytoplasmic, and membranous staining was also scored . For nuclear and cytoplasmic staining, cases with tumor cells expressing more than 30% -catenin were considered to be positive, and for membranous staining, cases with a pattern of loss of membranous -catenin staining of more than 30% of tumor cells were regarded as negative (fig . Expression of dbc1 and sirt1 were found mainly in the nuclei . To decrease the effects of observer biases, dbc1 and sirt1 tma slides were analyzed by an image analyzer program (ia). A scanscope cs (aperio, vista, ca, usa) digital scanner was used for scanning of tma slides . A combination of the genie histology pattern recognition tool (aperio) and nuclear quantification cellular analysis tool (aperio) was used to select tumor cells . The algorithm was adjusted to match the requirements of our particular marker and staining set . These adjustments include segmentation type, nuclear curvature threshold, size, roundness, compactness, and elongation . The output data contained the nuclear staining intensity (1 +, 2 +, 3 +, and negative), positive tumor cell percentage, and cell count . The cut - off value of 30% was also applied to dbc1 and sirt1 staining . When the sum of 2 + and 3 + cells exceeded 30% of the total tumor cells, the case was considered to be positive . Kaplan - meier plots and the log - rank test were used to estimate survival rates . The overall survival period was applied to this study . Overall survival was calculated as the time from diagnosis to the date of death or the last follow - up date . Correlations between the immunohistochemical expression of proteins and the clinicopathological characteristics were analyzed by pearson's tests, but histologic grade and pathologic stage were analyzed by a spearman's rank tests . Multivariate analysis was performed using the cox proportional hazard model . In all statistical analyses, dbc1 and sirt1 expression in gastric adenocarcinoma were observed in 271 (60%) and 255 (56%) patients . Expression of dbc1 and sirt1 were significantly associated with lower histologic grade, intestinal type of lauren classification, lower pathologic t stage (dbc1 and sirt1, p<0.001), and lower pathologic n stage (dbc1, p=0.002; sirt1, p<0.001). The lower tnm stage showed a significant association with sirt1 (p<0.001), but not with dbc1 (p=0.112). A statistically significant association between the absence of lymphatic invasion and both sirt1 (p=0.001) and dbc1 (p=0.004) was observed . Statistical associations between younger age (less than 60) and sirt1 (p=0.023) and dbc1 (p=0.001) were also observed . However, neither sex was associated with sirt1 (p=0.611) or dbc1 (p=0.080) expression (table 1). Sirt1 (p=0.011) and dbc1 (p=0.001) showed significant association with better patient survival (fig . A cytoplasmic, membranous, and nuclear pattern of -catenin expression was observed in 262 (58%), 367 (81%), and 28 (6%) cases, respectively . The cytoplasmic expression of -catenin was significantly associated with lower histologic grade (p<0.001), lower pt stage (p<0.001), lower pn stage (p<0.001), lower tnm stage (p<0.001), sirt1 (p<0.001), and dbc1 (p<0.001) (table 1). The membranous pattern of -catenin was also associated with lower histologic grade (p<0.001), lower pt stage (p<0.001), lower pn stage (p=0.003), lower tnm stage (p=0.001), sirt1 (p=0.002), and dbc1 (p=0.013). Cytoplasmic -catenin expression also showed a significant association with better patient survival (p=0.004) (fig . No association between p53 expression and sirt1 (p=0.063) or dbc1 (p=0.060) expression was observed (table 1). No pattern of -catenin showed any correlation with p53 expression (p=0.508, p=0.768, p=0.196, or membranous, nuclear, or cytoplasmic expression of -catenin, respectively). Simultaneous expression of both dbc1 and sirt1 was significantly correlated with high expression of cytoplasmic -catenin (p<0.001). However, the cases of concurrent negative expression of both dbc1 and sirt1 showed no association with cytoplasmic -catenin expression . Also, when dbc1 and sirt1 were expressed as concurrent negative, it was significantly associated with poor patient survival (p<0.001) (fig . 3). In contrast, there was no association between simultaneous expression of both dbc1 and sirt1 and expression of p53 (p=0.089). This means that expression of dbc1 was an independent good prognostic factor in gastric adenocarcinoma . Dbc1 and sirt1 expression in gastric adenocarcinoma were observed in 271 (60%) and 255 (56%) patients . Expression of dbc1 and sirt1 were significantly associated with lower histologic grade, intestinal type of lauren classification, lower pathologic t stage (dbc1 and sirt1, p<0.001), and lower pathologic n stage (dbc1, p=0.002; sirt1, p<0.001). The lower tnm stage showed a significant association with sirt1 (p<0.001), but not with dbc1 (p=0.112). A statistically significant association between the absence of lymphatic invasion and both sirt1 (p=0.001) and dbc1 (p=0.004) was observed . Statistical associations between younger age (less than 60) and sirt1 (p=0.023) and dbc1 (p=0.001) were also observed . However, neither sex was associated with sirt1 (p=0.611) or dbc1 (p=0.080) expression (table 1). Sirt1 (p=0.011) and dbc1 (p=0.001) showed significant association with better patient survival (fig . A cytoplasmic, membranous, and nuclear pattern of -catenin expression was observed in 262 (58%), 367 (81%), and 28 (6%) cases, respectively . The cytoplasmic expression of -catenin was significantly associated with lower histologic grade (p<0.001), lower pt stage (p<0.001), lower pn stage (p<0.001), lower tnm stage (p<0.001), sirt1 (p<0.001), and dbc1 (p<0.001) (table 1). The membranous pattern of -catenin was also associated with lower histologic grade (p<0.001), lower pt stage (p<0.001), lower pn stage (p=0.003), lower tnm stage (p=0.001), sirt1 (p=0.002), and dbc1 (p=0.013). Cytoplasmic -catenin expression also showed a significant association with better patient survival (p=0.004) (fig . No association between p53 expression and sirt1 (p=0.063) or dbc1 (p=0.060) expression was observed (table 1). No pattern of -catenin showed any correlation with p53 expression (p=0.508, p=0.768, p=0.196, or membranous, nuclear, or cytoplasmic expression of -catenin, respectively). Simultaneous expression of both dbc1 and sirt1 was significantly correlated with high expression of cytoplasmic -catenin (p<0.001). However, the cases of concurrent negative expression of both dbc1 and sirt1 showed no association with cytoplasmic -catenin expression . Also, when dbc1 and sirt1 were expressed as concurrent negative, it was significantly associated with poor patient survival (p<0.001) (fig ., there was no association between simultaneous expression of both dbc1 and sirt1 and expression of p53 (p=0.089). This means that expression of dbc1 was an independent good prognostic factor in gastric adenocarcinoma . Sirt1 is a nad - dependent protein deacetylase that deacetylates diverse proteins as mentioned above.22 - 24 the first identified target of sirt1 is the p53 tumor suppressor gene . Sirt1 deacetylates p53 and attenuates its ability for cell - cycle arrest and apoptosis, so the role of sirt1 is considered to be tumor promotion in carcinogenesis.7,22,23,25 another protein, dbc1, which is a sirt1-associated protein, negatively regulates sirt1 activity on p53 and inhibits tumor promoting activity of sirt1.2,15,26 these two proteins, sirt1 and dbc1, are thought to play a critical role in carcinogenesis . However, in gastric adenocarcinomas, only a few studies have been conducted with human tissue samples and their results are contrary to the previously known relationship between sirt1 and dbc1.10,11 therefore, in this study, we examined the expression of sirt1 and dbc1 in human gastric adenocarcinomas and analyzed the association with sirt1, dbc1, p53, -catenin, and clinicopathologic variables . Sirt1 and dbc1 positive expression showed a direct correlation with each other, a result that is in concordance with previous study data.11 one study reported that expression of dbc1 and sirt1 was significantly associated with shorter overall survival and greater risk of death, while expression of dbc1 is an independent prognostic factor for disease recurrence and poor survival outcome . However, in this analysis, conflicting results were produced . Dbc1 and sirt1 expression were correlated with better survival and good prognostic factors including lower histologic grade, intestinal type of lauren classification, lower pathologic t (dbc1 and sirt1, p<0.001) and n stage (dbc1, p=0.002; sirt1, p<0.001), and absence of lymphatic invasion (sirt1, p=0.001; dbc1, p=0.004). Notably, simultaneous expression of both dbc1 and sirt1 was significantly correlated with high expression of cytoplasmic -catenin (p<0.001) and better patient survival (p<0.001). We analyzed sirt1 and dbc1 expression by an image analyzer that was optimized for the counting of nuclear protein expression to decrease intraobserver or interobserver bias in positive cell counting . The resultant data showed mostly similar survival data and a similar relationship between the expression of these two proteins and clinicopathologic variables . We concluded that there was no critical error in interpreting the immunoexpression of sirt1 and dbc1 in tma slides . Of course, other biases including selection bias, preservation of tissues, conditions of tissue staining, and so on may lead to an interpretation error, but this is an ineluctable consequence of using both an image analyzer and optical microscope . We also analyzed p53 expression of gastric adenocarcinomas in association with sirt1, dbc1, and -catenin expression . Some recent study results indicate that sirt1 could serve as a tumor suppressor by interaction with other proteins such as -catenin, brca1, histone protein, and nf-b.3,24,27 - 29 therefore, we also examined the expression of -catenin in gastric adenocarcinomas and analyzed its association with sirt1 and dbc1 expression . Cytoplasmic and membranous -catenin expression showed significant associations with sirt1 and dbc1 expression, lower histologic grade, lower tumor stage, and prolonged patient survival . Correlations between nuclear sirt1 expression and -catenin expression in the cytoplasm and cell membrane in concordance with a previous study that investigated colon cancer tumorigenesis.3 on the other hand, nuclear expression of -catenin in this study was not correlated with sirt1 expression, which may be due to the difficulty in counting positive nuclei in the presence of cytoplasmic staining . Therefore, we evaluated the relationship between the expression of cytoplasmic -catenin and the others . Significant associations between sirt1 and -catenin in the cytoplasm and cell membrane suggest that sirt1 expression suppresses the localization of -catenin to the nucleus, and significantly attenuates its ability to activate transcription . This hypothesis could explain how sirt1 and cytoplasmic -catenin expression is correlated in gastric adenocarcinoma patients and how sirt1 and -catenin positive patients show better survival results . Similar to this hypothesis in this study, simultaneous expression of both dbc1 and sirt1 showed significant associations with a high expression of cytoplasmic -catenin (p<0.001) and better patient survival (p<0.001). Lymph node metastasis (p<0.001), depth of invasion (p<0.001), and lymphatic invasion (p=0.041) were consistent independent prognostic factors . However, in a previous study, expression of dbc1 was an independent prognostic factor for disease recurrence and poor survival outcome.11 this discordant result may be due to observation error in interpretation of immunohistochemical staining slides . Recent studies indicate that sirt1 can serve as both a tumor promoter and a tumor suppressor.3 some malignant tumors such as breast cancer, colon cancer, and prostate cancer are known to have increased nuclear expression of sirt1.4,6,7 however, in some tumors including glioblastoma, bladder cancer, and ovarian cancers, reduced expression of sirt1 was also found.30 these contradictory findings may be attributable to the diverse functions of sirt1, which can interact with both tumor suppressors and tumor promoters . In this study, we analyzed sirt1, -catenin, and p53 together . It appears that expression of sirt1 is closely associated with the cytoplasmic expression of -catenin in gastric adenocarcinoma . The relationship between sirt1 and p53 in gastric adenocarcinoma is unclear in these study results . We could not find a relationship between sirt1 and p53 (p=0.063) therefore, we believe this weak correlation may be due to a partial relationship between sirt1 and p53 . In another study, expression of p53 showed no significant correlation between sirt1 expression cases and negative cases.10 in spite of the interaction between sirt1 and p53, immunohistochemical correlation may or may not exist . More intense research dealing with sirt1 and sirt1-related protein is necessary to understand the diverse roles of sirt1 in gastric carcinogenesis . In some studies, first of all, dbc1 was found to augment signals of apoptosis via relocalization of the dbc1 c - terminal fragment into the mitochondria.26 dbc1 is also able to regulate the transcriptional activity of some nuclear hormone receptors including retinoic acid receptor and ar.26 dbc1 is known to inhibit sirt1 through a direct protein to protein interaction and thus promotes p53 mediated apoptosis.2 however, mutation of dbc1 does not direct binding to sirt1, so it should not affect enzymatic activity of sirt1 and the acetylation state of sirt1 substrates including p53.2 in this regard, a negative correlation is expected between sirt1 and dbc1 expressions . However, in this study, expression of dbc1 was significantly correlated with sirt1 expression (p<0.001). In agreement with our data, some study results revealed upregulation of dbc1 in various types of malignancies . Moreover, recent studies have reported a positive correlation of sirt1 and dbc1 expression in human cancer tissues.8,11 these results may be due to the recently identified diverse functions of dbc1 other than inhibition of sirt1.16,26 though dbc1 expression of gastric adenocarcinoma is associated with sirt1 expression, the role of dbc1 in gastric carcinogenesis may be other than inhibition of sirt1 . In summary, expression of dbc1, sirt1, and cytoplasmic and membranous -catenin in gastric adenocarcinoma showed significant correlations with each other . Furthermore, expressions of these three proteins were associated with lower histologic grade, lower tumor stage, and better patient survival . The role of sirt1 and dbc1 in gastric carcinogenesis should be considered carefully in correlation with other proteins that interact with sirt1 and dbc1.
Spinal injuries in small children are rare with a reported incidence of 0.2 to 0.5% of all fractures or dislocations and 1.5 to 3% of all lesions in the spine.1 it has been shown that the younger the child at the time of injury, the more likely the upper cervical spine is affected, with over 50% of spinal injuries in small children affecting this region . This relates to the child's greater head - to - body ratio, where more forces are centered around the junction between the large head and the smaller body.2 a subset of cervical spine fractures is the odontoid process fracture . In small children, this fracture typically involves the cartilaginous plate that separates the odontoid process from the body of the axis . The most common mechanism of such an injury is a head - on motor vehicle accident with a toddler restrained in a backseat by a four - point children's seat harness . Previous biomechanical investigations using simulation have shown vehicle speed greater than 40 km / h is sufficient to create the shearing forces required to cause such injuries in children under the age of 3.3 however, despite the increasing numbers of motor vehicle accidents occurring annually, resulting in increasing numbers of pediatric fractures, the optimal management of odontoid process fractures in small children remains undetermined . The current literature reports these fractures can be successfully treated with nonoperative management including cervical spine immobilization as well as operative interventions ranging from closed reduction and external fixation to posterior fixation or fusion of c1/c2.4 we present a case of a conservatively managed type 2 displaced odontoid process fracture in a 2-year - old girl . A 2-year - old girl who was previously healthy re - presented to the emergency department following a motor vehicle accident 6 days earlier . The parents describe a head - on motor vehicle accident at 90 km / h, with the toddler restrained in the backseat by a four - point child seat harness . The fracture was missed on initial presentation to the emergency department, which included a cervical spine x - ray; however, she remained reluctant to move her head and became distressed when not lying flat . On repeat presentation, she was comfortable at rest but lacked head control . Her neurologic examination was unremarkable, but a cervical spine computed tomography (ct) revealed a 45-degree flexed type 2 odontoid process fracture . She remained neurologically intact throughout and by 12 weeks had regained head control and a full range of movement without pain . Repeat ct scans were performed at 6 weeks, 10 weeks, and 6 months postinjury . These revealed callus formation at 6 weeks, followed by remodeling toward normal alignment by 6 months (fig . 1). Serial sagittal computed tomography images of a conservatively managed odontoid process fracture in a 2-year - old: (a) 45 degrees at initial presentation; (b) 45 degrees at 6 weeks postinjury with callus formation; (c) 40 degrees at 10 weeks postinjury; (d) 25 degrees at 6 months postinjury . This case represents the common mechanism of injury for this fracture in the pediatric population . Furthermore, it illustrates the difficulties in diagnosis, where patients may present without neck pain but rather a feeling of instability . In the pediatric population, reports of these symptoms are often very difficult to attain from the patient and therefore clinicians should maintain a high index of suspicion in a child who becomes distressed with movement or the requirement of head control and who has a significant mechanism of injury . If plain film x - rays are difficult to discern on initial presentation, further imaging is required . We utilized a ct scan to further delineate the bony injury, and due to the difficulty in interpreting the plain film x - rays in this child, we used limited upper cervical ct scans to monitor fracture healing . However, such scans should be used with caution in children due to the concerns of the ionizing radiation dose delivered.5 6 although limited scans deliver a low dose, they should nonetheless be avoided where possible.5 6 within the adult population, displaced type 2 odontoid process fractures can be treated operatively or nonoperatively, depending on the patient age, comorbidities, fracture pattern, and displacement.7 8 however, the management of such fractures in the pediatric population remains unclear . To our knowledge, there has only been one other case describing the management of a displaced type 2 odontoid process fracture in a small child . In this publication, bhagat and coauthors described operative intervention of a displaced type 2 odontoid process fracture in a 2-year - old girl.9 although they did not describe when union occurred, they noted marked remodeling of the fracture over a 30-month period . With such paucity of literature on this topic, it is unknown whether operative intervention aids fracture union and functional outcome in the small child . However, our case illustrates that odontoid process fractures in small children can be successfully managed without operative intervention despite significant angulation . In this case, developing union, with callus formation, was noted at 6 weeks postinjury, and progressive remodeling was seen to occur by the 6-month follow - up . We utilized a semirigid aspen collar rather than a rigid cervical spine orthosis, such as a halo vest traction, because of the higher rate of complications, such as pin site infections, pin loosening, brain abscesses, skin breakdown, and stiffness, associated with halo traction.10 11 furthermore, the bulk and size of halo traction in small children limits their mobilization and, due to their poor head control and crawling rather than upright gait, risks progressive loss of spinal alignment if the brace is not tightly fitted . The concern, however, with nonrigid immobilization is that of poor compliance, with the brace able to be removed by the patient or parents . In addition, within the adult population, higher union rates are reported with rigid halo vest immobilization compared with nonrigid immobilization, although this is debated and has not been analyzed in the pediatric population.12 13 in conclusion, this case illustrates that displaced type 2 odontoid process fractures in small children can be successfully treated conservatively in a semirigid cervical orthosis.
The term gossypiboma, derived from the latin word gossypium (cotton) and the swahilli boma the condition may manifest as an exudative inflammatory reaction with formation of abscess or aseptic fibrotic reaction with formation of a mass (2). The patient may present early with features of sepsis or may remain asymptomatic or may present with non - specific symptoms months or years after the operation . Although early recognition of this problem will ensure institution of appropriate treatment, every measure should be taken to prevent this complication . Herein, we report a case of gossypiboma presented as an abdominal lump 7 years after open cholecystectomy . A 54-year - old woman presented with a 2-years history of dull aching pain in the upper abdomen and 1 year history of an abdominal swelling . She developed fever and purulent discharge from the wound in the immediate postoperative period which was treated conservatively . On examination a large non - tender mass with restricted side to side mobility was found in the right hypochondrium extending up - to the right lumber region . Blood investigations revealed hemoglobin of 8.5gm / dl, leukocyte count of 9600/mm, serum bilirubin of 2.9 mg / dl and alkaline phosphatase of 507 u / l . The plain abdominal radiography did not show any sign of a radio - opaque marker in the abdomen . However, contrast enhanced abdominal computerized tomography revealed a 1512x10 cm round well - defined soft - tissue mass with multiple septae . Ct scan abdomen (both oral and intravenous contrast) showed a mass in right upper quadrant with displacement of gut loops towards the left . The mass showed contrast enhanced internal septa (white arrow) a radio - opaque material was seen in the lesion raising the suspicion of a foreign body . Common bile duct was dilated and contained a stone which was removed endoscopically . At exploration, a large globular lesion was found which was densely adhered to the adjacent structures (anterior abdominal wall, omentum, transverse colon, and antrum of the stomach). En - bloc resection of the mass along with the adhered transverse colon was done (figure-2). Cut section of the mass showed a large surgical sponge and a foreign body within it (figure-3).the patient had an uneventful recovery and she was well at 19-month follow - up . Operative photograph showing a large mass (white arrow) with adherent transverse colon (black arrow). Cut section of the mass showing a large sponge (white arrow) and a radio - opaque marker (black arrow) within it . Gossypiboma or a mass of cotton that is retained in the body following surgery is rarely seen in daily clinical practice . Although the real incidence is unknown, it has been reported as 1 in 100 to 3000 for all surgical interventions and 1 in 1000 to 1500 for intraabdominal operations (3,4) the clinical presentation of gossypiboma varies and depends on the location of the sponge and the type of reaction . There are 2 types of foreign body reaction in pathology: an exudates reaction leading to abscess formation or chronic internal or external fistula formation, and an aseptic fibrinous reaction resulting in adhesion, encapsulation, and eventual formation of granuloma of various size . They usually remain asymptomatic or present with pseudotumor syndrome, as in our case . Because, the symptoms of gossypiboma are usually nonspecific and may appear years after surgery, the diagnosis of gossypiboma usually comes from imaging studies and a high index of suspicion . The most impressive imaging finding of gossypiboma is the curved or banded radio - opaque lines on plain radiograph . The ultrasound feature is usually a well - defined mass containing wavy internal echogenic focus with a hypoechoic rim and a strong posterior shadow . On ct scan, a gossypiboma may manifest as a cystic lesion with internal spongiform appearance with mottled shadows as bubbles, hyperdense capsule, concentric layering, or mottled mural calcifications (6). When no radio - opaque marker is seen on x - ray or ct, the characteristic internal structure of the gauze granuloma is best visualized on magnetic resonance imaging . It may appear as a low - signal - intensity lesion on t2-weighted images with wavy, striped or spotted appearance (7). Nosher and siegel (8) described six patients in whom percutaneous extraction was successfully performed for removal of foreign bodies . Besides diagnostic and therapeutic difficulties the presence of a foreign body inside the patient can be easily proved and the patient may litigate the responsible surgeon because this is an avoidable problem . Moreover, gossypiboma may be misdiagnosed as a malignant tumor and lead to unnecessary invasive diagnostic procedures or extensive extirpative surgery (as in our case) which may result in further complications (9). Avoidance of leaving foreign bodies inside the patients could be possible by implementation of three measures: meticulous count of all surgical materials, thorough exploration of the surgical site at the conclusion of the procedures and routine use of surgical textile materials impregnated with a radio - opaque marker . Gawande et al (3) published an article about risk factors of retained foreign bodies (rfbs). Of the 8 risk factors, the authors identified [emergency operation, unexpected change in operation, more than one surgical team involved, change in nursing staff during procedure, body mass index (bmi), volume of blood loss, female sex, and surgical counts] only 3 were found to be statistically significant by multivariate logistic regression . The 3 significant risk factors were emergency surgery, unplanned change in the operation, and bmi . The counting of sponges and instruments was not a significant predictor in the multivariate model . Although all 3 factors were significant, the 9-fold increase in risk associated with emergency surgery was impressive . In addition, in 88% of the cases where there was a rfb and counts were performed, the counts were falsely called correct . The authors recommended radiographic screening at the end of high risk cases as a possible adjunct to improve detection of rfb . Although human errors cannot be completely avoided, continuous medical training and strict adherence to rules of the operation room should reduce the incidence of gossypiboma to a minimum.
Venous cannulation is the most common invasive procedures in several patients in hospital, including anesthesiologists daily . It leads to unwanted effects like pain, especially among patients who fear of needles . Fear of needles and pain associated with vein puncture is a common phobia in the general population . Fear of pain and anxiety associated with venous cannulation may contribute to the overall stress experienced by patients in the preoperative setting . There is a lack of cooperation, low rate of the successful procedure attempts, repeated attempts, additional pain, and a prolonged total procedure time . Injection - injury consequently leads to avoidance among patients from medical care and blood donating or precipitating fainting episodes . In addition, patients are often more satisfied with their doctors when they are treated by methods having minimal or no pain . Considering premedication relieves the anxiety and fear associated with pain as well as the pain itself . Choosing the more effective analgesia the effect of various pharmacological and nonpharmacological measures has been explored in several studies in order to minimize venous cannulation pain . However, there are a few cost - effective, simple and effective methods associated with pain reduction during venous cannulation . These methods commonly comprised local skin infiltration, topical application of eutectic mixture of local anesthetics (emla) or nonsteroid anti - inflammatory drugs (nsaids), topical alkaline vapocoolant spray, ibuprofen, etc . Injections in general and especially the placement of intravenous cannula are often the cause of pain and anxiety for many patients . However, there is no gold standard and there is a need for the development of innovative analgesic methods that are simple to use, painless, effective with a minimal delay, without adverse effects, and cost - effective . Thus, clinical practice has recently interested to incorporate various techniques for decreasing the discomfort and pain of procedures involving routine needle punctures, such as vein puncture, intravenous catheter placement, and lumbar puncture . Emla, a local anesthetic cream composed of a eutectic mixture of lidocaine and prilocaine provides . Hence, a topical formulation may offer substantial benefits for prevention of needle and procedural pain . A eutectic mixture of lidocaine and prilocaine (emla) is the first commercially developed topical transdermal anesthetic agent that provides adequate effective analgesic properties . Therefore, we aimed to compare the effect of elma cream and diclofenac gel in pain reduction in patients undergoing caesarian section surgery . This controlled, randomized clinical trial study has assessed the effect of diclofenac gel, emla and placebo on vein puncture pain . This study approved by the ethical committee of islamic azad university of tehran . The registration i d of study is registration i d in irct irct201008234618n1 . Ninety women aged 18 - 40 years old who were referred to imam ali hospital for elective cesarean section during 2010, were selected randomly through random scheduling . Exclusion criteria include addiction to opium or analgesics, mental, visual and verbal inability, use of analgesic in last 24 h, anaphylactic response to nsaid . Considering inclusion and exclusion criteria and homogeneity of samples, the researchers selected patients in cesarean section . Patients were allocated into three groups, diclofenac (n = 30), emla (n = 30) and placebo (n = 30). A sample size of 30 patients was calculated considering statistical power of 0.8 (1) and average effect of d = 0.7 . The questionnaire includes socio demographic characters, i.e. Age and weights were collected and visual analog scale (vas) was used to find the pain severity induced by venous puncture . One hour before puncture, 2 g emla (compound of 2.5% prilocaine, and 2.5% lidocaine tehran chemistry, iran) covered the skin (2.5 cm 4 cm) in the second group, diclofenac gel (1%) (behvasan, rasht) (1.9 cm 3.8 cm) was covered for patients left hand, and in the third group, placebo (vaseline, pars hayan, iran) include liquid and glycerine without lanolin and essence) was covered on patient's left hand . After 60 min ointments and gel were cleaned from the hand and side - effects were recorded . Then, venous puncture was done by angiocath number 3 - 5 min after puncture, pain was recorded by vas . The pain level categorization applied according polit and beck manual . The nurses who assessed the patients were blind to the design of the study . Beside descriptive method to calculate mean and standard deviation, we used anova and k to analysis the sample demographic characters . Ninety women aged 18 - 40 years old who were referred to imam ali hospital for elective cesarean section during 2010, were selected randomly through random scheduling . Exclusion criteria include addiction to opium or analgesics, mental, visual and verbal inability, use of analgesic in last 24 h, anaphylactic response to nsaid . Considering inclusion and exclusion criteria and homogeneity of samples, the researchers selected patients in cesarean section . Patients were allocated into three groups, diclofenac (n = 30), emla (n = 30) and placebo (n = 30). A sample size of 30 patients was calculated considering statistical power of 0.8 (1) and average effect of d = 0.7 . The questionnaire includes socio demographic characters, i.e. Age and weights were collected and visual analog scale (vas) was used to find the pain severity induced by venous puncture . One hour before puncture, 2 g emla (compound of 2.5% prilocaine, and 2.5% lidocaine tehran chemistry, iran) covered the skin (2.5 cm 4 cm) in the second group, diclofenac gel (1%) (behvasan, rasht) (1.9 cm 3.8 cm) was covered for patients left hand, and in the third group, placebo (vaseline, pars hayan, iran) include liquid and glycerine without lanolin and essence) was covered on patient's left hand . After 60 min ointments and gel were cleaned from the hand and side - effects were recorded . Then, venous puncture was done by angiocath number 3 - 5 min after puncture, pain was recorded by vas . The pain level categorization applied according polit and beck manual . Beside descriptive method to calculate mean and standard deviation, we used anova and k to analysis the sample demographic characters . A total of 90 cases allocated in three groups, i.e. Diclofenac (n = 30), emla cream (n = 30) and placebo (n = 30). Because of short term intervention there was no drop - out during interventions . The findings of the current study showed that there was no significant difference in demographic variables between the three groups at baseline in respect of age, weight and height [table 1]. In addition, there was no significant difference in education (p = 0.957) and place of residence (p = 0.861). Demographic data presented either as number of patients or meansd the distribution of pain severity during vein puncture in three groups is shown in table 2 . Our results showed that mild pain was more frequent in the diclofenac group versus two other groups [figure 1]. The mean and standard deviation of pain severity in the placebo group was 4.66 1.02, emla group 2.3 0.9 and 1.76 0.91 in diclofenac group . Hence, it is clear that the least pain was observed in diclofenac group and placebo has the most pain severity during vein puncture . Frequency of pain severity induced by vein puncture in 3 groups by visual analog scale in patients undergoing cesarean section effectiveness of diclofenac and emla on pain comparing pain severity induced by vein puncture using in pair groups, we found that the placebo group had significantly more pain severity than diclofenac and emla groups (p = 0.001). Furthermore, mean of pain severity in diclofenac and emla groups was 1.76 and 2.3, respectively (p = 0.006). Whitening, redness, urticarial, and itching were the short side - effects of local drugs and were appearing in 1 h after drug prescription . Our results showed that in about 20% of emla ointment whitening was appeared, and 80% of patients, no side - effect was appeared . In two other groups, our results showed that there was a significant difference between group in relation with side - effects (p = 0.0001). The results of our study showed that 93.33% of patients in diclofenac group and 63.33% in emla group had mild pain and 93.33% of patients in the placebo group had moderate pain during vein puncture . Mean score of pain severity were 4.66, 2.3, and 1.76 in the placebo, emla and diclofenac groups, respectively . There was significant difference between the three groups in pain severity (p = 0.001). Evaluated pain severity in three group, i.e. Sucrose 25% basement cream, emla cream and vaseline . They reported that there was a significant difference between the three groups (p = 0.001). The results comparing vein puncture pain between groups in current study indicated that pain severity in diclofenac group was less than emla group and the difference was significant (p = 0.006). Also compared the effect of diclofenac patch, emla cream and vaseline patch in pain reduction of 450 patients during vein puncture and indicated that all patients in the control group experienced vein puncture pain, but there was 37% pain reduction in emla group and 48% pain reduction in diclofenac patch (p <0.001). Our results also showed that placebo group experience more pain compared with diclofenac and emla group (p = 0.001). Similar to our findings the results of saxena et al . Showed that the mean pain severity in emla group was less than vaseline group (p <0.001). Also reported that there is a significant difference in pain reduction between diclofenac patch and placebo patch (p <0.005), which is in harmony with our findings of current study . In last decades, several researches have been conducted aimed to evaluate effectiveness of medicines . However, rare studies exist targeted to examine the effectiveness of topical nonsteroidal anti - inflammatory drugs . Totally, the findings of the current study revealed that pain severity in diclofenac group was lower than placebo and emla cream . This finding is in harmony with a study done by deshpande et al ., who reported higher effectiveness of diclofenac patch in comparing to emla among adult patients underwent surgery . In addition, we explored the probable side - effects of drugs (diclofenac gel, emla and baseline) which indicated the side - effects, mostly was blanching, redness, urticarial, and pruritus mostly occurred our hour after vein puncture, the most common side - effect was blanching in the emla group which was present in 20% of cases and in 80% of cases there were no side - effects . Showed that blanching is a common side - effect of emla in the hour of use . According to our findings emla and diclofenac gel reduce vein puncture pain significantly compared with placebo . However, we suggest using diclofenac gel rather than emla because it is more cost - effective and effective in the reduction of pain with less side - effects . Hence, we suggest that there should be some policies in health care centers and treatment clinics of our country in a different ward to use diclofenac gel as a choice to vein puncture pain we did not measure the anxiety level of samples, which is extremely related to pain severity . The samples selected only from one center . We could not measure the effect of pain severity on birth related outcomes.
Ultrapure paraffin oil is used for covering culture solution during artificial fertilization in vitro and micromanipulation of reproduction technique in vitro . This product has been classified as a class iii medical device according to the chinese classification rules of medical devices . According to the quality specification of this product the reaction between the tachypleus amebocyte lysate (tal) and the bacterial endotoxin normally occurs in water solution; that is why water insoluble oily medical devices cannot be analysed directly . There is no reference to this situation in the united states pharmacopeia [1, 2]. The major purpose of this report is to introduce a new method to analyse bacterial endotoxins present in ultrapure paraffin oil and other water insoluble oily medical devices . In this study, the agglutination interference test and the recovery test of soluble extraction of ultrapure paraffin oil have been studied in detail . This is a fundamental and methodological research of bacterial endotoxin analysis for water insoluble oily medical devices with gel - clot test . The ultrapure paraffin oil (lot number: 503687) was obtained from vitrolife sweden ab . The tachypleus amebocyte lysate with a sensitivity of 0.125 eu / ml (tal-01, lot number: 1301172) or 0.01~10 eu / ml (lot number: 1203020) and endotoxin - free water (ef - water, lot number: 1207040) were obtained from zhanjiang endosafe biological ltd . The tachypleus amebocyte lysate with a sensitivity of 0.125 eu / ml (tal-02, lot number: 130102) was obtained from zhanjiang bokang ocean biological co. ltd . Control standard endotoxins (100 eu per pack, lot number: 150601201176) were provided by the national institute for the control of food and pharmaceutical products, beijing, china . All equipment and material used in this study were treated at 250c for more than 1 hour in order to destroy exogenous bacterial endotoxins . The confirmation was performed according to the requirement of the chinese pharmacopoeia 2010, appendix ii, xie bacterial endotoxin test regulation [3, 4]. In order to determine the concentration of possible bacterial endotoxins in the ultrapure paraffin oil the latter was mixed with water and shaken to extract the endotoxin into the aqueous phase . The limit value in the bottom aqueous phase (l) and the endotoxin in the ultrapure paraffin oil have the following relationship: formula (1): lultrapure paraffin oil vultrapure paraffin oil recovery of the endotoxin (%) = lwater vwater formula (2): lwater = (l vultrapure paraffin oil recovery of the endotoxin)/vwater formula (1): lultrapure paraffin oil vultrapure paraffin oil recovery of the endotoxin (%) = lwater vwater formula (2): lwater = (l vultrapure paraffin oil recovery of the endotoxin)/vwater according to the regulation of the gel - clot method of the chinese pharmacopoeia, the recovery does not need to agree with the calculation of the limit value if the recovery is within 50200% . Otherwise, the limit value has to be calculated based on the recovery . The limit of ultrapure paraffin oil is 0.25 eu / ml according to the company standard . In the test, 10 ml ultrapure paraffin oil was taken and added to 10 ml endotoxin - free water and shaken for 15 min at 2000 rpm . The limit value in the top aqueous phase is 0.25 eu / ml if the recovery is within 50%200% . The sensitivity of the marketed tachypleus amebocyte lysate is usually within 0.50.03 so the maximum valid dilution ratio of the sample which is corresponding to different sensitivities of tachypleus amebocyte lysate is (1)mvd0.25=0.250.25=1; mvd0.125=0.250.125=2;mvd0.06=0.250.06=4; mvd0.03=0.250.03=8 . As the ultrapure paraffin oil is water insoluble oily liquid, its endotoxin must be water - extracted before analysis can be made . In order to determine possible interference factors, 10 ml ultrapure paraffin oil was mixed with 10 ml endotoxin - free water and shaken for 15 min at 2000 rpm . The bottom aqueous phase was then diluted twice with endotoxin - free water followed by endotoxin analysis according to the regulation of the chinese pharmacopoeia 2010, appendix ii, the bacterial endotoxin test sample for the interference test . Control standard endotoxins were prepared at different concentrations, 0.25, 0.125, 0.06, and 0.03 eu / ml, with endotoxin - free water . The endotoxin - free water and the twice diluted water phase were used as negative controls . Tachypleus amebocyte lysate preparations with a sensitivity of 0.125 eu / ml from two different suppliers were applied for comparison . The extraction rate of endotoxin from the ultrapure paraffin oil to the aqueous phase is defined by the absolute recovery, which must be within 50%200% in accordance with the requirements of the chinese pharmacopoeia . Otherwise the l = (l vultrapure paraffin oil)/(vwater recovery) value has to be calculated based on the recovery, to ensure the accuracy of the analysis . The determination is based on three endotoxin standards at 5, 10, and 20 eu / ml and one ultrapure paraffin oil water extract batch, respectively . 10 ml endotoxin - free water was added to 10 ml of each sample followed by shaking for 15 min at 2000 r / min . The bottom aqueous phase was then diluted 25, 50, 100, and 200 times with ef - water and the recovery was measured using the dynamic turbidimetric analysis method . 10 ml endotoxin - free water was added to 10 ml sample followed by shaking for 15 min at 2000 rpm . The bottom aqueous phase was then analysed for endotoxin according to the gel - clot method of the chinese pharmacopoeia 2010, appendix xie . Bacterial endotoxin analysis of twice diluted extracts was performed using a tachypleus amebocyte lysate with a sensitivity of 0.125 eu / ml [6, 7]. Table 1 shows the results of the confirmation of the sensitivity of two batches of tachypleus amebocyte lysate . Tal-02 (= 0.125) was obtained from zhanjiang bokang ocean biological co. ltd . Plus the results indicate a es within 0.52.0 and a et within 0.5es2.0es (es: bacterial endotoxin of standard, et: bacterial endotoxin of samples), and it confirms that twice dilution or more will not interfere with the bacterial endotoxin test . Plus the results of bacterial endotoxin recovery in aqueous extracts of the ultrapure paraffin oil are shown in table 3 . The data indicate that the recovery of the endotoxin from the ultrapure paraffin oil extract meets the requirement of the gel - clot method of the chinese pharmacopoeia which is in the range of 50%200% . Because of the water insolubility of the ultrapure paraffin oil, extraction with water is an indispensable part of the bacterial endotoxin test . Although ultrapure paraffin oil is water insoluble, there may still be some residues in the extracted aqueous phase, so it is important to confirm that the extraction solution will not interfere with the clot between the tachypleus amebocyte lysate and the endotoxin . Also, since the limit value of the endotoxin in the aqueous phase has the following relationship with the limit value of the endotoxin in the ultrapure paraffin oil, lwater = (lultrapure paraffin oil vultrapure paraffin oil percent recovery of the endotoxin)/vwater, the recovery of the endotoxin of the ultrapure paraffin oil must also be considered; otherwise the result of the test will not correlate with the endotoxin in the ultrapure paraffin oil . If the recovery of endotoxin in the ultrapure paraffin oil is within 50%200% after extraction, then it does not need to be recovered based on the method in chinese pharmacopoeia (converted at 100%). If not within 50%200%, the limit value of the endotoxin in the aqueous phase should be calculated based on the recovery . Due to the different concentrations of endotoxin, their proportion in the ultrapure paraffin oil phase and the aqueous phase and the recovery will be different . This is why the recovery test should be done with different concentrations of endotoxin . Till present, there are few studies of test methods for bacterial endotoxins in water insoluble oily medical devices . In this study, an interference test was applied to the aqueous phase extracted from the ultrapure paraffin oil to confirm that it did not interfere with the clot formation between the tachypleus amebocyte lysate and the endotoxin . To this end, ultrapure paraffin oil with different endotoxin concentrations was prepared and the recovery was tested using a dynamic turbidimetric method . All results were within 86.8%96.8% and thus met the requirement of 50%200% in the chinese pharmacopeia . This also indicates that the test method in this study is able to accurately determine the endotoxin concentration in ultrapure paraffin oil by analysis of the aqueous phase extract . In this study, we have shown that it is possible to analyse the concentration level of bacterial endotoxin in water extracts of ultrapure paraffin oil using the gel - clot method and verified the limit value of <0.25 eu / ml . At the same time, this study can be regarded as a supplement analysis method for water insoluble oily medical devices to the united states pharmacopeia chapter number 85 analysis of bacterial endotoxin and chapter number 161 blood and other liquid transfusion device and similar medical device.
Osteoarthritis (oa) is a major cause of disability affecting millions of people worldwide . In the united states alone, to date there are no proven therapies for the prevention or treatment of oa . Pain relief and visco - supplementation are prescribed to attenuate the symptoms of oa until disease progression significantly impairs joint function and joint replacements are required . The lack of disease modifying oa drugs (dmoads) may be a function of incongruence between in vitro models of oa and the pathogenesis in vivo, and between disease mechanisms in humans and model animals . To overcome these issues, there is increasing momentum to develop human cell - based organotypic models in vitro that functionally represent the osteochondral tissue directly affected by oa . The development of physiologically relevant models requires an understanding of the tissue architecture, physiology, and pathophysiological responses to biochemical (or biophysical) insults . This is especially the case for the osteochondral complex, where the main tissues, cartilage and bone, differ so substantially . Cartilage is composed of a collagen type ii / aggrecan - rich, highly hydrated, viscoelastic, anisotropic matrix that encapsulates chondrocytes within biochemically distinct chondrons . In contrast, bone is composed of a collagen type i - rich, laminated or woven calcified structure that is much stiffer and encapsulates osteocytes, osteoblasts, and osteoclasts, blood vessels, and nerves . The cartilage and bone are intimately connected at the osteochondral junction (ocj), a highly organized structure that represents a significant challenge to mimic in vitro by tissue engineering . It is composed of distinct, interacting layers that include (epi - to - diaphyseally) deep zone cartilage, a basophilic tidemark, calcified cartilage, the cement line, and the subchondral bone plate . Interestingly, there is growing evidence of significant biochemical communication between cartilage and bone across the ocj . In the pathogenesis of oa, changes in the physical linkage between cartilage and bone at the ocj are critical components of disease progression . These include remodeling of the tidemark, microcracks, and fissures in both tissues and ingrowth from the underlying bone of blood vessels and nerves, all of which may enhance the cartilage - bone crosstalk allowing a better passage of growth factors, cytokines, and signaling molecules . These ocj changes accelerate cartilage degeneration and are associated with joint pain and disease morbidity, pointing to the need of a better understanding of the complex network of interactions between bone and cartilage in oa . Cartilage and bone exist not only in a different matrix but also in very different biophysical environments . In vivo, there is a steep oxygen gradient from bone (essentially normoxic) to cartilage (extremely hypoxic). These differences are reflected in the in vitro culture systems often used to maintain chondrocytes and osteoblasts . Starved environment: low glucose, serum - free medium supplemented with pyruvate and abundant matrix precursors or -enhancing molecules (proline and ascorbate) in hypoxic conditions . However, osteoblasts are maintained in high glucose, serum - containing medium supplemented with -glycerol phosphate and vitamin d3 in normoxic conditions . With these fundamental environmental differences between chondrocytes and osteoblasts, it is not surprising that oa elicits specific responses from each tissue . Oa disease progression is most frequently characterized by a net loss of cartilage matrix that results from an imbalance between cartilage matrix degradation and synthesis by chondrocytes in the cartilage . Progressive chronic destruction of articular cartilage is the most obvious characteristic of oa, and the etiology of the disease is believed to be at the intersection of genetics and abnormal mechanical forces . Therefore, the primary locus of the disease is traditionally presumed to be the cartilage, and as a result, most in vitro oa models focus exclusively on cartilage to study oa disease mechanisms and therapeutic intervention . However, there is increasing evidence from in vivo and clinical studies that subchondral bone lesions may precede cartilage degeneration, implying that oa is an osteochondral disease and possibly bone dependent . In addition, it has been often reported that the health of mature articular cartilage in vitro is positively impacted by the presence of subchondral bone . Despite these observations, most in vitro oa research has not taken into account the effects of bone - cartilage interactions, focusing primarily on cartilage alone . This may account for the dearth of new therapeutics for the prevention and treatment of oa . We theorize that the development of a model system of osteochondral tissue using human cells in a physiologically relevant environment that can accurately replicate in vivo osteochondral tissue homeostasis and pathophysiology will lead to greater predictive power in the development of dmoads . The challenges in developing such a system include: (1) mimicking or inducing production of appropriate extracellular matrix critical to the function of cartilage and bone, (2) replicating the tissue architecture, (3) reconciling the different growth and maintenance conditions of bone and cartilage while promoting their interaction with each other, and (4) replicating the biomechanical environment known to be essential to cartilage and bone health . Current in vitro models to investigate bone - cartilage interactions are mostly limited to cell co - culture systems in which bone and cartilage cells are both exposed to the same medium, arguably a very distant condition from the in vivo environment . Here, we report the development of a bioreactor designed to accommodate the biphasic nature of an osteochondral plug by creating two separate compartments for the chondral and osseous microenvironments . These are separated only by the tissue itself and are supplied by a microfluidic system . The two microenvironments can be independently controlled and regulated via introductions of bioactive agents or candidate effecter cells, and the medium can be individually sampled for compositional assays . The central hypothesis of the study is that a gradient of tissue specific nutrients and conditions is required for the formation and maintenance of the osteochondral tissue . Furthermore, we hypothesize that induction of an oa - like condition in the engineered osseous or the engineered chondral component alone will induce a corresponding oa - like response in the other component . To test these hypotheses, we have generated distinct chondral and osseous zones within the same construct by controlling the different media exposures within the bioreactor . Then, we induced an oa - like response by exposing the osseous or chondral compartments to the pro - inflammatory cytokine (il-1) and assayed the intervening changes in expression and secretion from both the engineered chondral and osseous components . All chemicals used in this study were purchased from sigma - aldrich (st . Louis, mo) unless stated otherwise . The 3d structures of the bioreactor (figure 1) were modeled using magics 14 (materialise, belgium). The chamber and insert were fabricated using a stereolithography apparatus (envisiontec, germany) employing e - shell 300 as the resin . (a) an individual bioreactor composed of the removable insert (dark gray) within a chamber (light gray) of the microfluidic plate (b) and fixed in place with two o - rings . The osteochondral construct within the insert creates the final separation between the upper and lower medium conduits . Opposing gradients of chondrogenic and osteogenic factors and stimulants will aid in forming an interface . (b) a single bioreactor formed by the inset and lid in the context of a 24-well plate . Red circles indicate the o - rings that seal the joint space between lid / insert and chamber . Hbmscs were isolated from the femoral heads of patients undergoing total joint arthroplasty with irb approval (university of pittsburgh), cultured and expanded as previously described (caterson, 2002; song, 2004). Briefly, bone marrow was flushed out from the trabecular bone of the femoral neck and head using an 18-gauge needle and resuspend in dulbecco s minimal essential medium (dmem). The suspension was filtered through a 40 m strainer and the flow - through was centrifuged at 300 g for 5 min . After the supernatant was discarded, the pellets were suspended using growth medium (gm, -mem containing 10% fetal bovine serum (fbs, invitrogen), 1% antibiotics - antimycotic, and 1.5 ng / ml fgf-2 (raybiotech, norcross, ga)), and then plated into 150 cm tissue culture flasks at a density of 20,00040,000 nucleated cells / cm, and medium was changed every 3 to 4 days . The colony formation and trilineage mesenchymal differentiation capacity of hbmscs was validated before use (data not shown). All experiments were performed with passage 3 (p3) hbmscs from 3 patients (3 female patients 44, 52, and 72 years old), which were pooled for use in this study . The photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (lap) was synthesized as described by fairbanks et al . Mgl was synthesized by reacting gelatin with methacrylic anhydride (ma) in water according to a procedure previously described . Mha was prepared as previously reported using sodium hyaluronate powder (research grade, mw 66 kda, lifecore). Both mgl and mha were lyophilized and stored in a desiccator for future use . To test medium leakage between (1) the chamber wall and the insert and (2) the insert and scaffold material, the insert was filled with 10% mgl/0.15% lap in hbss and alexa fluor 488-conjugated soybean trypsin inhibitor (ti488, 21kd, molecular probes, ca) and alexa fluor 555-conjugated albumin from bovine serum (bsa) (bsa555, 65kd, molecular probes) were diluted in hbss individually at 10 g / ml and then perfused through the top and bottom of bioreactor, respectively, at 1 l / min . At different time points, effluent from the upper and lower medium conduits was collected and the fluorescence intensity at both wavelengths measured using a microplate reader (synergy ht, biotek, winooski, vt). Leaking between top and bottom conduits was estimated by the ratio of ti488 (bottom)/ti488 (top) and bsa555(top)/bsa555 (bottom). Because of the permeable nature of gelatin scaffold used as the scaffold model, leaking was assayed for 24 h only . P3 hbmscs were pelleted and drained completely in order to prevent the unwanted dilution of polymers . Chondrogenic cell suspension: hbmscs were resuspended in 10% mgl/1%mha/0.15% lap (w / v) hbss solution (ph adjusted to 7.4) at a final density of 20 10/ml (chondrogenic suspension). Hbmscs were resuspended in the 10% mgl/1% hydroxyapatite/0.15% lap (w / v) hbss solution (ph was adjusted to 7.4) at a final density of 20 10/ml . Osteochondral construct preparation: first, the insert was placed within a hollow cylindrical well to prevent suspension leaking from the pores in the insert . Second, 60 l of osteogenic suspension was pipetted into the insert and cross - linked using the uv light source . After 2 min of uv light exposure, the inset with photopolymerized osseous construct was removed from the chamber . Third, 30 l of chondral suspension was added on the top of osseous construct within the same insert and cured for another 2 min . Previous studies have shown cell viability within the scaffold> 90% after photo - cross - linking (data not shown). The second round of cross - linking had the added benefit of bonding the osseous and chondral layers together as well, and the fabrication of the osteochondral construct within the insert created a tight seal . The inserts with nave osteochondral constructs were placed into the microfluidic plate as shown in figure 1 . Chondrogenic medium (cm) was supplied through the upper conduit, while osteogenic medium (om) through the bottom conduit at a flow rate of 1 l / s . The following formulas were used for the differentiation media: om (gm supplemented with 10 ng / ml bmp-2 (peprotech, rocky hill, nj), 1% l - alanyl - l - glutamine (glutamax), 10 nm dexamethasone (dex), 0.1 mm l - ascorbic acid 2-phosphate (asa2-p), and 10 mm beta - glycerophosphate (-gp); cm (dmem supplemented with 10 ng / ml tgf-3 (peprotech), 1% its, 50 m asa2-p, 55 m sodium pyruvate, and 23 m l - proline). The perfusion rate was 1 l / min, and used syringes were replaced with syringes and new medium every 3 days . After 4 weeks of differentiation, engineered osteochondral tissues were collected for validation using real - time pcr and histological analysis, or treated with il1 - 1. chondral and osseous constructs were collected separately . To avoid the potential contamination, total rna was extracted using trizol (invitrogen) following the standard protocol and purified with the rneasy plus mini kit (qiagen, hilden, germany). Superscript iii kit (invitrogen) was utilized with random hexamer primers to complete the reverse transcription . Real - time rt - pcr was performed using the steponeplus thermocycler (applied biosystems, foster city, ca) and sybr green reaction mix (applied biosystems). Sox 9, aggrecan (acn), collagen type ii (col2a1), runx2, osteocalcin (ocn), and bone sialoprotein (bsp ii) expression were analyzed, and primer sequences are listed in table 1 . Monolayers of hbmscs cultured in gm on 2d tissue culture plastic were used as negative controls . Transcript level of 18s rrna was used as endogenous control, and gene expression folder changes were calculated using the comparative ct (ct) method . Aggrecan (acn), collagen type ii (col2), runt - related transcription factor 2 (runx2), osteocalcin (ocn), bone sialoprotein ii (bsp ii), and 18s rrna (18s). Intact engineered osteochondral tissues were fixed in 10% neutral buffered formalin (fisher scientific, pittsburgh, pa) for 7 days, dehydrated, embedded in paraffin with 10 m sections cut from each sample . Safranin o / fast green and alizarin red staining were used to detect the gag and calcium deposition, respectively . After 4 weeks of differentiation, engineered osteochondral constructs were treated with il-1 (10 ng / ml, r&d) on the chondral or osseous sides only to investigate the cell / neo - tissue response to pro - inflammatory cytokines and possible communication through the osteochondral construct . The media used in this test were cm without tgf-3 (chondral) and om without bmp-2 (osseous), both supplemented with 10 ng / ml il-1. There were three experimental groups: (1) cm / om, (2) cm + il1 - 1/om, and (3) cm / om + il1 - 1. the treatment lasted 7 days, with effluent medium collected and frozen at 1 and 7 days for elisa . After 7 days, the osteochondral constructs were bisected into the chondral and osseous halves and processed for gene expression analysis as described before . In addition to tissue specific gene expression, matrix metalloproteinase 1, 3, and 13 were also analyzed . Media was collected separately from chondral and osseous constructs, cleared of cell debris via centrifugation (1000 g), and analyzed via il-1 (abcam, cambridge, ma), mmp-1 (r&d), mmp-3 (abcam, cambridge, ma), and mmp-13 (abcam) elisas according to the manufacturers instructions . Significant differences were determined with anova followed by a bonferroni post hoc analysis for multiple group comparisons using spss statistics 21 (ibm, armonk, ny). Significance was determined at p <0.05 () and p <0.01 ( ). The robustness of the microbioreactor was tested by assessing the extent of leakage of two molecules perfused independently in the upper and lower medium conduits: (1) trypsin inhibitor (21 kda), with a molecular weight similar to the two commonly used osteoinductive (bmp-2, 26kd) and chondroinductive (tgf3, 25kd) factors, and (2) bsa (65kd), the most abundant protein in serum . As shown in figure 2a, after 24 h of perfusion, the extent of mixing between top and bottom was <1%, indicating there was minimal medium exchange through the interfaces between the chamber wall and the inset and between the inset and scaffold - only construct . These results were further confirmed by elisa assay for il-1 in both medium conduits during the il-1 test (figure 2b). (a) trypsin inhibitor-488 and bsa-555 were simultaneously perfused through the top and bottom space of bioreactor, respectively, and the percent leakage at different time was estimated based on the bottom / top ratio of 488 nm fluorescence readings (bottom / top 488) and top / bottom 555 nm fluorescence readings (top / bottom 555). (b) il-1 was included in top or bottom stream and perfused for 24 h. its concentration in top or bottom medium was then measured . The naive hbmscs seeded within tissue - specific scaffolds in the freshly fabricated osteochondral construct were induced to differentiate using cm in the top stream and om in the bottom stream . We anticipated chondrogenesis in the upper, chondral half of the construct and osteogenesis in the bottom, osseous half . After 4 weeks of differentiation, biphasic osteochondral constructs were produced (figure 4c). As shown in figure 3, cells in the chondral half showed enhanced expression of chondrogenic genes, including sox 9, aggrecan, and collagen type ii as compared to those in the osseous half, while the cells in the osseous half had higher expression of osteogenic genes, including runx2, osteocalcin, and bsp ii . Monolayers of hbmscs cultured in gm on 2d tissue culture plastic were used as negative controls . Histological staining with alcian blue / alizarin red revealed high matrix gag content in the upper chondral half than the osseous half (figure 4b); although the amount of calcium deposition in the osseous half was not detectable . Taken together, these results strongly indicate a spatially defined, biphasic differentiation of these engineered osteochondral constructs, with the chondral component undergoing more characteristic differentiation . In addition, h&e staining revealed a distinct, <100 m wide basophilic band in the interface between the chondral and osseous halves, potentially indicative of a developing tidemark (figure 4a). After 4 weeks of culture in the bioreactor, osteochondral constructs were separated into chondral (cartilage) and osseous (bone) components, and each were analyzed for expression of cartilage (sox9, col2, and aggrecan) or bone (runx2, osteocalcin, bspii) markers . Expression levels are normalized to 18s rrna and then to corresponding 2d control expression levels . Expression of cartilage markers was found only in the chondral component, and bone markers in the osseous compartment . p <0.05; * * p <0.01). Top, chondral component (cc); bottom, osseous component (oc). (a) alizarin red staining; (b) safranin o / fast green staining . Dashed lines indicate the border between cc and oc . As described above, the microtissue bioreactor presented here, with its two separate medium flow systems and biphasic construct compartments, has the capability for targeted treatment of one (or both) tissue construct(s) with soluble factors . Osseous and chondral components were separately treated with the pro - inflammatory cytokine il-1 (10 ng / ml) for 7 days (control conditions consisted of untreated osteochondral constructs), and the responses of each of the two components were separately analyzed . Media samples were collected from chondral and osseous components streams at days 1 and 7, and after day 7, the osteochondral constructs were separated into osseous and chondral components, and each was separately analyzed for gene expression of catabolic genes (mmp-1, mmp-3, and mmp-13) and either cartilage markers (sox9, col2, and aggrecan) (figure 5) or bone markers (runx2, osteocalcin, and bspii) (figure 6). Media samples from the chondral and osseous components stream collected at days 1 and 7 were analyzed via mmp-1, mmp-3, and mmp-13 elisas (figure 7). Effects of il-1 treatment on cartilage gene expression in the engineered osteochondral microtissue . After treatment of either osseous (bone) or chondral (cartilage) component with 10 ng / ml il-1 for 7 days, cartilage components were analyzed for the expression of cartilage markers and mmps . Expression levels were normalized to 18s rrna expression and then to corresponding gene expression under control conditions . * p <0.05; * * p <0.01 . Effects of il-1 treatment on bone gene expression in the engineered osteochondral microtissue . After treatment of either osseous (bone) or chondral (cartilage) component with 10 ng / ml il-1 for 7 days, bone components were analyzed for expression of bone markers and mmps . Expression levels were normalized to 18s rrna expression and then to corresponding gene expression under control conditions . Effects of il-1 treatment on osseous and chondral mmp secretion in the engineered osteochondral microtissue . After treatment of either osseous (bone) or chondral (cartilage) component with 10 ng / ml il-1 for 1 or 7 days, medium samples collected from the bone or cartilage medium compartment were analyzed by elisa for the levels of secreted mmp-1, mmp-3, and mmp-13 . Values were normalized to those measured under control conditions, which involved untreated osteochondral constructs . Treatment of chondral constructs with il-1 caused decreases in expression of cartilage genes sox9, col2, and aggrecan, consistent with physiological outcomes of damaged or stressed cartilage (figure 5). Chondral construct expression of these genes also decreased in response to il-1 treatment of osseous constructs, suggesting signaling between the osseous and chondral components . Evidence of this osseous - to - chondral communication was even more apparent in results concerning expression of catabolic genes; expression of mmp-1, mmp-3, and mmp-13 of the chondral constructs increased substantially in response to il-1 treatment of the osseous component . Crosstalk between the two components was also detected in the case of chondral - to - osseous communication (figure 6). Il-1 treatment of the chondral construct caused decreases in expression of the bone genes osteocalcin and bspii and increases in mmps production in the osseous construct, particularly mmp-13, which is one of the most important mediators of oa cartilage degradation . Elisa analysis of mmps secreted by the chondral and osseous components at different time points allowed for observations on the rate of signal propagation between the two components (figure 7). For example, the chondral construct responded to il-1 treatment of the osseous component with increases in mmp-1, mmp-3, and mmp-13 secretion . The chondral mmp-13 response occurred quickly, within 1 day, while the chondral mmp-1 response took 7 days . The chondral mmp-3 response time was intermediate between those of mmp-13 and mmp-1 . Again, these results are interesting considering the central role mmp-13 plays in cartilage degeneration . The osseous construct response to treatment of chondral component with il-1, however, was quick yet increased further over time, and by day 7 was overall stronger than the chondral responses to the osseous component treatment . It is worth noting that gene expression and protein levels of mmps should not be expected to be necessarily consistent . This stems from differences in the ways in which elisa and real - time pcr samples were collected and measured . Elisa samples consisted of culture media conditioned by cells for 24 h and were collected at day 1 or day 7 for each experiment . The proteins contained in day 1 samples were secreted between days 0 and 1, and day 7 samples contained proteins secreted between days 6 and 7 . Pcr samples, however, were collected after 7 days of cultures and represent the expressional activities taking place at the moment of collection . In other words, the mrna levels analyzed by pcr at day 7 are not necessarily totally reflective of the protein levels analyzed by elisa in day 7 conditioned media samples . This disconnect between pcr and elisa measurements may be more pronounced in mmps, which need to be translated, secreted, and then diffuse out of the 3d construct before they are detected by elisa . Furthermore, differences between pcr and elisa values also arise from differences in normalization . Pcr results are normalized to 18s rrna expression, thereby taking into account cell number . Elisa results are instead a representation of the entire culture and normalized to control conditions . Thus, any experimental treatment that may affect cell number would have a larger impact on elisa results than pcr results . Since chondrocytes are particularly sensitive to il-1, this may explain why results concerning cells of the chondral component exhibit the greatest degree of inconsistency between pcr and elisa measurements when chondral constructs are directly stimulated by il-1. In this study, we have developed a novel bioreactor system for the engineering of osteochondral tissue . Rt - pcr and histological analyses showed that hbmscs - derived nave constructs have been successfully differentiated into cartilage - like tissue on the top and bone - like tissue on the bottom, using separated culture medium for 4 weeks . A transition layer between 2 tissues is also observed . Our results show that il-1 exposure decreases the ecm anabolic gene expression but greatly enhances the levels of mmp expression and secreted amount into the medium . Interestingly, the il-1 insulted osseous construct induces a catabolic gene expression response into the untreated chondral component, which is not due to leakage of il-1, suggesting active osseous chondral interaction and the likely importance of bone injury in oa development . In this study, a dual - chamber bioreactor has been developed to generate and maintain osteochondral constructs derived from human hbmscs . The design parameters included individual compartments to separate the chondral and osseous microenvironments that are individually accessible for the introduction of bioactive agents and/or candidate effector cells, tissue and medium sampling, and compositional assays, including noninvasive imaging techniques . Furthermore, the total dimension and geometry of the bioreactor matches that of a multiwell culture plate chamber for the development of medium- to high - throughput analysis . Validation of the system included successful, simultaneous differentiation of osseous and chondral constructs from hbmscs from the same source (pooling of three donors) and subsequent application of il-1, a potent inflammatory mediator implicated in oa pathophysiology, to test the physiological response of the osteochondral construct . We have shown that in the course of 6 weeks, hbmscs undergo tissue - specific differentiation in response to the tissue specific growth media and hydrogel composition provided . The differentiating bbmscs expressed tissue - specific transcription factors and ecm molecules, as shown by rt - pcr and histological staining . Most impressively, there was an indication of a basophilic, tidemark - like zone separating the chondral and osseous components . The recreation of the a tidemark - containing biphasic tissue is vital to drug testing using an osteochondral organotypic culture since changes in the ocj are mechanistically involved in oa progression and likely to be a target of toxicants and dmoads . We subsequently tested the response of the msc - based osteochondral tissue to il-1. The test served two purposes: (1) to validate the utility of the bioreactor in osteochondral studies and (2) to assess the physiological replication of the oc tissue by the mscs in this bioreactor . Il-1 is almost ubiquitous in inflammatory diseases, is prominent in advanced oa in both the cartilage and synovial lining, and is frequently employed as a pathogenic initiator in in vitro models of oa . Application of il-1 to both osseous and chondral components results in clear matrix degeneration and phenotypic changes in the resident cells, similar to what has been observed in monocultures of chondrocytes and osteoblasts . While chronic degeneration of the articular surface is most prevalent in oa, it is not clear whether alterations in the subchondral bone or articular cartilage is the primary trigger in oa . Using the bioreactor in this study, we are able to study interactions between cartilage and bone that may contribute to oa progression clear osseous and chondral tissue interactions are observed when il-1 is applied to the osseous component, which results in low levels of anabolic gene expression (sox9, col2, and aggrecan) but robust expression of mmps in the cartilage component . Conversely, application of il-1 to the cartilage induces in bone low levels of anabolic bone gene expression (runx2, opn, and bspii) but robust expression of mmps . This apparently contradictory simultaneous induction of anabolic and catabolic processes within a tissue is entirely in keeping with the hypothesis that oa begins initially with a shift in the balance between anabolic and catabolic activities, followed by phenotypic changes in the cells in response to the modified environment . To some degree, inflammation can have beneficial effects on tissues, but at higher concentrations, inflammatory mediators induce tissue remodeling / destruction . Focusing on the response of chondral component to il-1 treatment of the osseous component, it is interesting that direct application of il-1 to the chondral component has a less impressive catabolic response than indirect exposure via the osseous component . This result implies that the affected osteoblasts in the osseous component are producing bioactive factors, in addition to il-1, that are causing greater catabolic responses than il-1 itself and vice versa . The formation of an ocj between regions of engineered cartilage and bone is often reported, but generally is not analyzed beyond histological identification . In our study, we have employed opposing chondrogenic and osteogenic nutrient gradients to stimulate ocj formation by nave, differentiating mscs . It is thus difficult to relate the potential tidemark development in our construct with frequently reported constructs that combine solid, porous polymeric sponges and hydrogels for osteochondral engineering in vitro or in vivo because of the great disparity in tissue architecture and scaffold biochemistry . Tidemark development similar to what is seen here has been reported in studies employing microparticle - mediated spatially restricted growth factor release, microbead - encapsulated msc - derived chondrocytes and osteocytes, mscs encapsulated within scaffold material gradients, and mscs stimulated by growth factor gradients . In all cases, the basophilic tidemark is indistinct and broad, particularly in models without loading . We expect that appropriate mechanical loading and enhancement of cell differentiation, e.g., with an oxygen gradient, may enhance the collection of metabolites to form the tidemark at the deep zone / calcified cartilage interface, particularly if differentiation is enhanced with an oxygen gradient and/or the addition of hydroxyapatite . While formation of ocj has been reported in in vivo implanted cell - seeded scaffold, there have been relatively few studies using a controlled bioreactor as reported here . The features of our bioreactor design, including separate compartments for the chondral and osseous microenvironments supplied by independent tissue - specific media that can be controlled and regulated via introductions of bioactive agents or candidate effecter cells, and capability of individual sampling of the different compartments, are thus of potential value in allowing more individual manipulations . Specifically, we envision its application for the assessment of drug and environmental factor toxicity . We have postulated that catabolic insults to one tissue component comprising the osteochondral unit would influence the other in a manner reminiscent of tissue degeneration in oa . Of particular interest is to investigate the communication of biomechanical signals / forces . To our knowledge, we are the first to provide evidence of communication between different compartments of an osteochondral construct in response to catabolic cues (il-1). The tissue responses reported here reflect a subset of pathophysiological conditions reported in in vitro and in vivo models of oa . Il-1 is utilized in models of both rheumatoid and oa, with the effect of causing cartilage matrix breakdown and down regulation of cartilage matrix gene expression as shown here . In contrast, il-1 has been reported to induce increased bone matrix deposition, although it is a matrix of inferior quality, which may explain the response of the osseous component to direct exposure to il-1 in our model . The fact that catabolic gene expression in the osseous component is more enhanced by exposure of the overlying chondral component to il-1 suggests that the chondral construct is producing additional signals and catabolic factors that travel to and affect the osseous component below, possibly constituting a form of intercellular communication not previously reported . In summary, we have fabricated a new microfluidic - based, multichamber bioreactor for osteochondral differentiation and toxicity testing . We demonstrated clear biphasic tissue differentiation in response to opposing chondrogenic and osteogenic gradients produced by tissue - specific differentiation factors supplied by independent medium streams to the chondral and osseous components of the construct . Finally we have shown that msc - based chondral and osseous tissues are capable of responding to il-1 in a relevant manner and that changes in one tissue compartment are communicated, and perhaps amplified, to the other along the osteochondral axis . This bioreactor / organotypic osteochondral culture combination will enable us to focus on the relationship of cartilage and bone in growth and degeneration and perhaps help to elucidate the roles of each tissue in oa . Finally, with minimal modification and appropriate coupling, the bioreactor reported here can be adapted as a tissue - specific component of an interacting multitissue bioreactor platform to study systemic multitissue interactions.
In recent years, we have witnessed a growing interest in the involvement of rna molecules in controlling gene expression . Numerous studies demonstrated that regulatory noncoding or non - protein - coding rnas (ncrnas and npcrnas) play equally important role as protein transcription factors in determining the repertoire of expressed genes virtually in all living organisms . The significance of ncrnas is evident from the results of analyses of the protein - coding capacity of sequenced genomes . The contribution of protein - coding regions decreases with the increase of complexity of organisms . In bacteria, unicellular eukaryotes and invertebrates, the coding sequences constitute 95, 30 and 20% of the genomic dna, respectively . In mammals, similar proportions of the coding and noncoding regions are observed within the sequences which are actually transcribed (3). There are also very little differences between the mammalian proteomes which suggests that the diversity observed on the phenotypic level is not determined by different sets of proteins, but rather by programs which govern their expression (4). A support for this view came recently from the discovery of the brain - specific har1f rna expressed during cerebral cortex development . Accelerated evolution of har1f - encoding region regulatory ncrnas have been identified in all domains of life and they have been shown to be involved in numerous mechanisms controlling expression of genes at all levels of transmission of genetic information from dna to proteins (6). They include epigenetic modification of chromatin structure (methylation and modification of histones), regulation of transcription by modulation of activity of rna polymerase and transcription factors, rna modification, mrna stability and translation . Unlike the infrastructural ncrnas (e.g. Trnas, rrnas, snrna and snornas), regulatory rnas are not transcribed constitutively in all cells . In many cases, their expression depends on the cell or tissue type, developmental stage or is controlled by epigenetic factors or environmental conditions (e.g. Hormones and stress). Specific changes in the expression of particular ncrnas in humans have been also linked to human neurobehavioral and developmental disorders and cancer (7). Initially, virtually all ncrnas were discovered by chance and there were no systematic approaches to identify the whole contents of the transcriptome . At the time of the publication of the first ncrna database in 1999 (8), since then, there has been a steady growth of reports on identification of new noncoding transcripts . In recent years, a large number of ncrnas sequences have been determined in large scale sequencing projects of cdnas (9,10), and small cytoplasmic rnas (11). There has also been a growing number of reports describing novel methods for computational identification of ncrna - encoding genes (12,13). Despite the advances in identification of new ncrnas both in prokaryotes and eukaryotes, our knowledge of the spectrum of their activities there are relatively few noncoding transcripts which have been at least partially characterized in terms of function or expression . This growth of interest in rna biology was a motivation for several databases published in recent years (e.g. 14,15). The purpose of the database is to serve and organize information concerning regulatory noncoding transcripts from all groups of organisms . In addition to the rnas for which regulatory activities have been documented, the database also contains sequences of ncrnas which are known to be expressed, but their role in a cell is still unknown . In comparison with other ncrna databases, which appeared in recent years, ncrnadb does not focus on any particular class of transcripts or taxonomic groups . The rnas included in the database have been demonstrated or are suspected to function without being translated into proteins and they are not constitutively expressed housekeeping transcripts (e.g. Trnas, rrnas and snrnas). As of august 2006, a significant growth of the amount of data, compared with previous edition published in 2003 which contained 300 sequences (16), is primarily due to systematic identification of noncoding transcripts in mammals by means of large scale cdna sequencing (8,9). These sequences account for over 90% of the data . On the other hand, certain groups of rnas (micrornas and snornas), which were present in previous editions, the primary source of sequences included in the database were the genbank (17) records . Human and mouse ncrna sequences are in part derived from h - invitational (18) and fantom3 (8) full length cdna databases, respectively . Computationally identified small cytoplasmic, bacterial rnas which are not annotated in the genomic sequences were derived from the rfam the database of rna families (15). Since many of the primary transcripts of the eukaryotic ncrnas are subject to alternative splicing, various splicing variants derived from the same gene are presented as separate entries . For ncrnas, for which there are no individual genbank records, predicted transcripts were extracted from genomic sequences using information provided in the feature tables or based on the multiple sequence alignments . Apart from the sequences, the entries contain supplementary information (when available) on experimentally verified activities, expression patterns and chromosomal localization . The annotations and genome mapping information for the sequences rely on data provided in original genbank records, h - invitational database of annotated human genes (release 3.4, july 2006), fantom3 database (march 2006 release) and ucsc genome browser database (19). Most of the entries for eukaryotic transcripts are linked to the ucsc genome browser (20). Individual nucleotide sequences can be retrieved in fasta format as separate entries or downloaded as batch files . In addition to the access to the database records, the search results also linked to full genbank entries . In the current version of the database we also included the blast server which allows to perform sequence similarity searches using the full ncrna database (64 mb). The browser section of the database is intended as a source of basic information on noncoding transcripts which have been at least partially characterized in terms of function or expression patterns . For such rnas we provide short descriptions of known activities described in the literature and relevant citations linked to the medline database . The browser entries also give access to the nucleotide sequences from the database (fasta) or the entire genbank records.
Enantioseparation (es) and enantiodiscrimination (ed) are, therefore, mandatory steps in the elucidation of enantiospecific biological functions on the molecular level . Interestingly, ed with isotropic solution nmr was first demonstrated by pirkle,1 who also developed enantioseparating chromatographic phases.2 however, since these times, the two areas have evolved increasingly independently . Although nmr studies are used to elucidate structural aspects of es,3a the role of enantioselective chromatography as a versatile tool in the understanding of ed obtained in nmr has been largely neglected . Since the early report of pirkle and pochapsky in 1986,3b isotropic solutionnmr studies have been used to investigate structural aspects of chromatographic enantiorecognition processes.3c,3d for this purpose, chemical shifts and nuclear overhauser effects (noes) were largely used . One recent approach for ed in nmr is the use of chiral anisotropic media.4 in general, anisotropic achiral media permit the measurement of nmr parameters that require a partial alignment of the solute (e.g. Residual dipolar couplings or chemicalshift anisotropies). Currently, a number of new alignment media that have been developed are becoming very important in the structural analysis of smalltolarge molecules.5 crosslinked acrylamidebased copolymers introduced in 20006a have been further developed into an alignment medium compatible with a wide range of solvents including dmso.6b recently, ed was reached by introducing a chiral, negatively charged monomer: (r)aphes [(2acrylamide)2phenylethanesulfonic acid, scheme s1 a].7 as expected from work with chiral ionexchange phases,8 ed was only reached for cationic (protonated basic) solutes (erythromefloquine, menthylamine, and strychnine). No ed was observed for the neutral compound menthol.7 this approach had been inspired by previous work in the field of enantioselective (chiral) liquidphase chromatography . Enantioselective chromatographic separation is based on the difference in the retention factor for a pair of enantiomers induced by a small difference in the phasetransfer equilibrium constants, owing to the involvement of diastereomeric associations (between the chiral solute and the chiral selector) in the phasetransfer process.8a a special class of stationary phases developed for enantioselective liquidphase chromatography (chiral ionexchange phases) employs positively and/or negatively charged immobilized chiral selectors, which can undergo the formation of diastereomeric associates (ion pairs) with protonated or deprotonated (ionisable) chiral solutes.8b, 8c it is now of interest to investigate whether or not the successful ed observed with a chiral alignment gel used for anisonmr (nmr spectroscopy under anisotropic conditions) can be further elucidated by using chromatographic studies employing the same alignment gel as a chiral separation medium in capillary electrochromatography (cec) (figure 1, and explanations given therein). For clarity, this approach should be clearly distinguished from techniques such as nmrce, capillary nmr, and chromatographic nmr spectroscopy.9 left side: schematic capillary / nmr tube cross section used for isotropic gelbased capillary electrochromatography (cec; upper part with isotropic mesh openings), and nmr under anisotropic conditions (anisonmr; lower part with anisotropic mesh openings). Right side: electrochromatogram obtained by cec (upper part), and twodimensional nmr spectrum with split signals attribtued to anisotropic alignment of the analyte (lower part). Ideally, for thermodynamic studies, the separation medium investigated through chromatographic studies should be identical to the alignment medium used for anisonmr . Many alignment media (e.g. Bicelles, phages), however, are incompatible with cec instrumentation, whereas homogeneous gels are separation media in plate gel electrophoresis (pge), in capillary gel electrophoresis (cge), and in gelbased capillary electrochromatography (gbcec).10 separation in pge and cge is based on differences in the migration velocity of the solute, induced by differences in the effective electrophoretic mobility of the solutes and the sieving effect . Separation in gbcec (in analogy to chromatography), however, is based on differences in the equilibrium constant characterizing the transfer of the solute from the electrokinetic transport zone (etz, where the velocity of the solute is given by the electrophoretic velocity of this solute and the electroosmotic velocity) into the interaction zone (iz, where the velocity of the solute is zero). We describe this equilibrium as a distribution process, although the highly swollen gel employed in gbcec is a homogenous phase (in contrast to the system of two interpenetrating continuous phases characteristic for cec with a monolith). Gbcec was introduced by fujimoto,10a using charged amphiphilic polyacrylamide gels for the separation of uncharged lowmolecularweight compounds . Gbcec with hydrophilic or amphiphilic gels with embedded or immobilized chiral selectors was also successfully applied to the separation of different enantiomers.10c10 g returning to previous results,7 we selected the synthetic antimalaria drug mefloquine (figure 2) with two stereocenters as the test solute.11a in recent years, the correct absolute configurations of the enantiomers of the erythro form, which is administered as the racemate, and of the enantiomers of the threo form, have been subject to debate . They were not reliably established until 20122013.11b11f in contrast to these difficulties, es of the four stereoisomers had been successfully achieved much earlier by using capillary electrophoresis (ce) with cyclodextrin derivatives or other native cyclodextrins as chiral selectors,12 and by using highperformance liquid chromatography (hplc) either with amylose tris3,5dimethylphenyl carbamatecoated silica gel13 or with a quinidinebased zwitterionic chiral stationary phase (chiral selector covalently anchored to the chemically modified silica gel).14 structural formulae of a) (+) erythromefloquine [(+) emq], b) ()erythromefloquine [()emq], c) (+) threomefloquine [(+) tmq], and d) ()threomefloquine [()tmq]. Against this background, in the present work, we study the chromatographic separation of the four stereoisomers of mefloquine [()threomefloquine (tmq) and ()erythromefloquine (emq)] by using gbcec, employing the same polymeric gel as the chiral separation medium as that already successfully taken for the ed of emq through anisonmr . The retention data gained by using gbcec will permit the determination of the ratio of (distribution) equilibrium constants . With this ratio, we confirm those parameters that have been calculated in the interpretation of previously reported anisotropic nmr data.7 with all types of gels obtained by varying the solvent composition, a good diastereoselective and enantioselective separation of the four mefloquine stereoisomers was achieved (figure 3). Although the peak of propranolol, taken as a marker of the holdup time (see the supporting information), can be characterized to be symmetric, the peaks for the more retarded stereoisomers of mefloquine show a characteristic (rightskewed) triangular broadened shape, which can be attributed to electromigration dispersion resulting from local electric field strength inhomogeneities . Separation of the four stereoisomers of mefloquine by gbcec with alignment gel as the separation medium with superimposed scaled trace for ()propranolol . Peak assignment: 1) ()propranolol; 2, 3) ()threomefloquine; 4) ()erythromefloquine; 5) (+) erythromefloquine; mobile phase = methanol / water (50:50, v / v) buffered with triethylamine / acetic acid, ph=6.66, electric conductivity=125 s cm, capillary dimensions=173 mm (102 mm)100 m, photometric ingel detection=283 nm, electrokinetic injection=2 kv2 s, separation voltage=2.0 kv, c (analyte in sample)=0.25 g l (analyte dissolved in mobile phase). We confirmed the suitability of propranolol as a marker of the holdup time by application of a second independent method, which is based on the comparison of retention times obtained for the two enantiomers of emq with two capillaries filled with alignment gel having different effective lengths (see the supporting information). It should be noted that, in gbcec, the holdup time can only be measured correctly if a marker is available that is not significantly retarded and has exactly the same effective electrophoretic mobility as the effective electrophoretic mobility of the analyte in the electrokinetic transport zone . The depicted peak distortions (figure 3) are typical in cec for charged analytes showing a strong interaction with the oppositely charged stationary phase (ionexchange cec).15 with analogously synthesized monolithic separation capillaries [employing (r)aphes as negatively charged comonomer] synthesized for comparison purposes under phaseseparation conditions (see the supporting information), leftskewed and rightskewed peaks can be obtained, dependent on the separation conditions (figures s1 a and s1 b). Under the conditions selected for recording the chromatogram depicted in figure 4 (see legend), peak broadening by electromigration dispersion is absent and a higher peak efficiency is obtained . Under these conditions, the method enabled baseline separation of the enantiomers of emq (figure 4). Spiking the sample with (+) emq hcl revealed a higher retention factor for (+) emq than for ()emq (figure s2). Repeatability of the data was confirmed by sequential runs (figure s3s5). For five repeated runs, the relative standard deviations of the observed mobilities are 2.02.3% (table s1). This corresponds to a relative confidence range of the mean of 2.52.9% (p=0.95, twotailed test). Separation of the enantiomers of erythromefloquine by gbcec with alignment gel as the separation medium . Peak assignment: 1) ()erythromefloquine; 2) (+) erythromefloquine; mobile phase = methanol buffered with 25 mm acetic acid and 2.66 mm triethylamine, ph=4.46, electric conductivity=55 s cm, capillary dimensions=234 mm (167 mm)100 m, photometric ingel detection=283 nm, electrokinetic injection=2.5 kv2 s, separation voltage=6.1 kv, c (analyte in sample)=0.25 g l (analyte dissolved in mobile phase). Following two different approaches (see the supporting information), we estimated the chromatographic separation factor (= ratio of the two retention factors) for tmq to be 1.08 and for emq to be 1.10 . These two factors correspond to the ratio of the two equilibrium constants related to the transfer from the electrokinetic transport zone to the interaction zone . Hence, the energetic differences g for the involved (distribution) equilibria are 190 and 230 j mol for tmq and emq, respectively . Taking propranolol as a nonretarded marker with an effective electrophoretic mobility similar to that of tmq and emq (figure 3 and scheme s1 b), we obtained the following retention factors: 0.41 and 0.44 for the two enantiomers of tmq, 0.73 for ()emq, and 0.80 for (+) emq . A larger difference in retention factors was obtained for the separation of tmq and emq, which corresponds to g=1.7 kj mol . Under isotropic conditions, anisotropic parameters such as dipolar couplings between two nmrsensitive nuclei are averaged to zero.16a socalled alignment media lead to a low degree of orientation and reintroduce observable couplings . By using only a weak alignment, the large dipolar couplings that are in the range of khz are scaled down to values in the range of hz (residual dipolar couplings).16b with a set of experimentally determined rdcs, an alignment tensor (a matrix with five independent elements) can be calculated, which describes the degree and orientation of the alignment . In 2001, the concept of the generalized degree of order (gdo) was introduced to describe the dynamics of protein fragments.17 gdo is a scalar quantity that is calculated from the alignment tensor (or the order tensor) through the determination of the euclidian norm of this matrix.16a the gdo can be employed to characterize differences in the strength of two alignments, as it is a quantity that is only dependent on the extent of dynamic averaging, arising from both overall alignment effects and internal motional effects (the latter being neglected with rigid molecules).16a, 17 for ()emq [in the gel prepared with (r)aphes] gdo=5.1910, which is smaller than for (+) emq [gdo=5.8510; absolute difference: 0.6610].7 under the assumption that a weaker alignment correlates with a weaker interaction of the solute with the polymer chains of the gel (being identical with a less stable diastereomeric association), the determined gdos correctly predict that ()emq elutes first in a chromatographic separation . The significance of this result was confirmed by a duplicate determination of gdo for ()emq.7 gdo1 = 5.2510 and gdo2 = 5.1310, resulting in a difference of 0.1210 . The confidence range of the arithmetic mean (p=0.95, onetailed test) is 0.3810, which is smaller than the difference between the values for the two isomers . With the aim to identify fragments of a protein, where motion has an effect on the accuracy of structure determination, tolman et al.17 defined a fragmentspecific internal gdo as the ratio of the observed fragment gdo to the alignment tensor gdo . We propose here the enantioselectivity parameter (= gdo ratio), referring to a nonracemic chiral alignment medium and a specific enantiomer pair . This parameter is quantified by determining the ratio of the (higher) gdo for enantiomer 1 to the (smaller) gdo for enantiomer 2 (with 1). In the present case, (emq)=gdo / gdo=1.13 . It is interesting to compare this value to the selectivity factor (= k / k) obtained for ()emq by gbcec, which is 1.10 . Both methods agree in terms of the sign [(+)> ()] and magnitude of the quantity describing ed / es, which should hold for cases in which the degree of alignment prevails the enantiospecific difference of the measured anisotropic parameter (here: rdc). Based on the retention factors determined by gbcec, it can be now concluded that the ed observed in the present case in anisonmr is directly related to the different degrees of transfer of the solute from the etz into the iz . This degree of transfer is quantified by the molar fraction x iz = n iz/(n it is directly accessible from chromatographic data through x iz = k/(1+k). For the different stereoisomers of mefloquine, it represents the fraction of those molecules being associated with the charged moieties of the crosslinked polymer acting as interaction sites . Although n total (= n etz+n iz) is kept constant, the values for x iz are 0.29 and 0.31 for the two enantiomers of tmq, 0.42 for ()emq, and 0.44 for (+) emq . With all types of gels obtained by varying the solvent composition, a good diastereoselective and enantioselective separation of the four mefloquine stereoisomers was achieved (figure 3). Although the peak of propranolol, taken as a marker of the holdup time (see the supporting information), can be characterized to be symmetric, the peaks for the more retarded stereoisomers of mefloquine show a characteristic (rightskewed) triangular broadened shape, which can be attributed to electromigration dispersion resulting from local electric field strength inhomogeneities . Separation of the four stereoisomers of mefloquine by gbcec with alignment gel as the separation medium with superimposed scaled trace for ()propranolol . Peak assignment: 1) ()propranolol; 2, 3) ()threomefloquine; 4) ()erythromefloquine; 5) (+) erythromefloquine; mobile phase = methanol / water (50:50, v / v) buffered with triethylamine / acetic acid, ph=6.66, electric conductivity=125 s cm, capillary dimensions=173 mm (102 mm)100 m, photometric ingel detection=283 nm, electrokinetic injection=2 kv2 s, separation voltage=2.0 kv, c (analyte in sample)=0.25 g l (analyte dissolved in mobile phase). We confirmed the suitability of propranolol as a marker of the holdup time by application of a second independent method, which is based on the comparison of retention times obtained for the two enantiomers of emq with two capillaries filled with alignment gel having different effective lengths (see the supporting information). It should be noted that, in gbcec, the holdup time can only be measured correctly if a marker is available that is not significantly retarded and has exactly the same effective electrophoretic mobility as the effective electrophoretic mobility of the analyte in the electrokinetic transport zone . The depicted peak distortions (figure 3) are typical in cec for charged analytes showing a strong interaction with the oppositely charged stationary phase (ionexchange cec).15 with analogously synthesized monolithic separation capillaries [employing (r)aphes as negatively charged comonomer] synthesized for comparison purposes under phaseseparation conditions (see the supporting information), leftskewed and rightskewed peaks can be obtained, dependent on the separation conditions (figures s1 a and s1 b). Under the conditions selected for recording the chromatogram depicted in figure 4 (see legend), peak broadening by electromigration dispersion is absent and a higher peak efficiency is obtained . Under these conditions, the method enabled baseline separation of the enantiomers of emq (figure 4). Spiking the sample with (+) emq hcl revealed a higher retention factor for (+) emq than for ()emq (figure s2). Repeatability of the data was confirmed by sequential runs (figure s3s5). For five repeated runs, the relative standard deviations of the observed mobilities are 2.02.3% (table s1). This corresponds to a relative confidence range of the mean of 2.52.9% (p=0.95, twotailed test). Separation of the enantiomers of erythromefloquine by gbcec with alignment gel as the separation medium . Peak assignment: 1) ()erythromefloquine; 2) (+) erythromefloquine; mobile phase = methanol buffered with 25 mm acetic acid and 2.66 mm triethylamine, ph=4.46, electric conductivity=55 s cm, capillary dimensions=234 mm (167 mm)100 m, photometric ingel detection=283 nm, electrokinetic injection=2.5 kv2 s, separation voltage=6.1 kv, c (analyte in sample)=0.25 g l (analyte dissolved in mobile phase). Following two different approaches (see the supporting information), we estimated the chromatographic separation factor (= ratio of the two retention factors) for tmq to be 1.08 and for emq to be 1.10 . These two factors correspond to the ratio of the two equilibrium constants related to the transfer from the electrokinetic transport zone to the interaction zone . Hence, the energetic differences g for the involved (distribution) equilibria are 190 and 230 j mol for tmq and emq, respectively . Taking propranolol as a nonretarded marker with an effective electrophoretic mobility similar to that of tmq and emq (figure 3 and scheme s1 b), we obtained the following retention factors: 0.41 and 0.44 for the two enantiomers of tmq, 0.73 for ()emq, and 0.80 for (+) emq . A larger difference in retention factors was obtained for the separation of tmq and emq, which corresponds to g=1.7 kj mol . Under isotropic conditions, anisotropic parameters such as dipolar couplings between two nmrsensitive nuclei are averaged to zero.16a socalled alignment media lead to a low degree of orientation and reintroduce observable couplings . By using only a weak alignment, the large dipolar couplings that are in the range of khz are scaled down to values in the range of hz (residual dipolar couplings).16b with a set of experimentally determined rdcs, an alignment tensor (a matrix with five independent elements) can be calculated, which describes the degree and orientation of the alignment . In 2001, the concept of the generalized degree of order (gdo) was introduced to describe the dynamics of protein fragments.17 gdo is a scalar quantity that is calculated from the alignment tensor (or the order tensor) through the determination of the euclidian norm of this matrix.16a the gdo can be employed to characterize differences in the strength of two alignments, as it is a quantity that is only dependent on the extent of dynamic averaging, arising from both overall alignment effects and internal motional effects (the latter being neglected with rigid molecules).16a, 17 for ()emq [in the gel prepared with (r)aphes] gdo=5.1910, which is smaller than for (+) emq [gdo=5.8510; absolute difference: 0.6610].7 under the assumption that a weaker alignment correlates with a weaker interaction of the solute with the polymer chains of the gel (being identical with a less stable diastereomeric association), the determined gdos correctly predict that ()emq elutes first in a chromatographic separation . The significance of this result was confirmed by a duplicate determination of gdo for ()emq.7 gdo1 = 5.2510 and gdo2 = 5.1310, resulting in a difference of 0.1210 . The confidence range of the arithmetic mean (p=0.95, onetailed test) is 0.3810, which is smaller than the difference between the values for the two isomers . With the aim to identify fragments of a protein, where motion has an effect on the accuracy of structure determination, tolman et al.17 defined a fragmentspecific internal gdo as the ratio of the observed fragment gdo to the alignment tensor gdo . We propose here the enantioselectivity parameter (= gdo ratio), referring to a nonracemic chiral alignment medium and a specific enantiomer pair . This parameter is quantified by determining the ratio of the (higher) gdo for enantiomer 1 to the (smaller) gdo for enantiomer 2 (with 1). In the present case, (emq)=gdo / gdo=1.13 . It is interesting to compare this value to the selectivity factor (= k / k) obtained for ()emq by gbcec, which is 1.10 . Both methods agree in terms of the sign [(+)> ()] and magnitude of the quantity describing ed / es, which should hold for cases in which the degree of alignment prevails the enantiospecific difference of the measured anisotropic parameter (here: rdc). Based on the retention factors determined by gbcec, it can be now concluded that the ed observed in the present case in anisonmr is directly related to the different degrees of transfer of the solute from the etz into the iz . This degree of transfer is quantified by the molar fraction x iz = n iz/(n etz+n iz). It is directly accessible from chromatographic data through x iz = k/(1+k). For the different stereoisomers of mefloquine, it represents the fraction of those molecules being associated with the charged moieties of the crosslinked polymer acting as interaction sites . Although n total (= n etz+n iz) is kept constant, the values for x iz are 0.29 and 0.31 for the two enantiomers of tmq, 0.42 for ()emq, and 0.44 for (+) emq . Taking a chiral alignment medium as the chiral separation medium, gbcec separates the four stereoisomers of mefloquine . This separation confirms our previous hypothesis that ed with this gel is based on the formation of localized diastereomeric associates between the charged solute and the oppositely charged moiety of the anisotropically deformed gel.7 consequently, ed is attributed to differences in the equilibrium constants related to associate formation . Through gbcec, retention data are obtained that permit the calculation of the degrees of transfer of the solute from the electrokinetic transport zone into the interaction zone and the precise quantification of the gibbs energy difference regarding the two diastereomeric associates involved in the enantioselective separation process . Hence, our approach provides physicochemical data that are important in the understanding and optimization of alignment processes.18 the capillary pretreatment procedure and the subsequent in situ synthesis of the polymeric gel are described in detail in the supporting information . Briefly, the separation capillaries are treated with a 30% (v / v) solution of 3(trimethoxysilyl) propylmethacrylate (bind silane) in acetone to introduce vinylic anchoring groups to the inner wall of the fused silica capillary . These vinylic anchoring groups enable the covalent attachment of the synthesized gel to the inner capillary wall, which is a prerequisite to obtain homogeneously filled capillaries . Subsequently, the chiral monomer (2acrylamide)2phenylethanesulfonic acid [(r)aphes], is copolymerized with n, ndimethylacrylamide (dmaa) and n, nmethylenebisacrylamide (bis) through a freesolution radical copolymerization (15 min at 70 c). The preparation procedure of the gel within the capillary is identical (with respect to concentration of monomers, reaction time, reaction temperature) to the one used for the measurement of anisonmr parameters with the exception of the absence or presence of a washing step (removal of nonreacted monomers), an additional drying and swelling step (in the case of anisonmr), and the solvent composition.7 although dmso was employed for anisonmr, the solvent is either methanol or methanol / water [(50:50, v / v) or (25:75, v / v)] in gbcec (after equilibration). Previous studies, however, with either methanol or dmso as the solvent demonstrated that the type of solvent has only a minor influence on the determined anisonmr parameters.19 in gbcec, the separation gel is buffered with triethylamine / acetic acid, ph*=4.56.7, whereas in anisonmr all experiments were performed in unbuffered gels . The influence of the electrolyte concentration on the alignment properties of a very similar achiral polyacrylamide gel [aphes replaced by 2(acrylamide)2methylpropanesulfonic acid (amps)] was determined by trigomourio et al.20 according to these results, we can assume a negligible influence of the selected buffer concentration (2.7 mm) on alignment properties and retention behavior . (for details refer to the supporting information).21 a scheme of the apparatus allowing ingel detection at 283 nm is shown in scheme s2 . As a service to our authors and readers, this journal provides supporting information supplied by the authors . Such materials are peer reviewed and may be reorganized for online delivery, but are not copyedited or typeset . Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
Bacterial strains and culture conditions: various oral bacteria were isolated from gingival plaque taken from maxillary premolars of the dogs with periodontal disease (table 1table 1.minimum inhibitory concentration (mic) of extracted polyphenolic compounds from japanese green tea and egcg against various isolates from oral cavity of dogscanine isolatesmic (mg / ml)polyphenolic compoundsfrom japanese green teaegcgporphyromonas endodontalis inu-10.80.1porphyromonas salivosa festa - s0.40.1porphyromonas gulae festa - g0.40.05prevotella intermedia inu - b40.10.025prevotella melaninogenica inu - bl10.20.05fusobacterium nucleatum inu - f20.20.05lactobacillus acidophilus inu - l30.20.05streptococcus spp . Inu-7a30.10.0125streptococcus spp . Inu - ps0.40.025a) polyphenolic compounds were using 95% ethanol at 80c for 4 hr to extract . It is a mixer that includes various components, such as tannins (egcg and other catechins), minerals, nitrogenous components, caffeine and lipids, etc . B) egcg is one of major polyphenolic compounds, which had been purified .) As previously described . The bacteria were grown in gam broth (nissui co., tokyo, japan) for 24 to 48 hr at 37c anaerobically . S. mutans was isolated by hirose et al . As previously described and was grown in brain heart infusion (bhi) broth (merck kgaa, darmstadt, germany) for 24 hr at 37c . A) polyphenolic compounds were using 95% ethanol at 80c for 4 hr to extract . It is a mixer that includes various components, such as tannins (egcg and other catechins), minerals, nitrogenous components, caffeine and lipids, etc . B) egcg is one of major polyphenolic compounds, which had been purified . Catechins: polyphenolic compounds were isolated from the leaf of c. sinensis by extraction using 95% ethanol (80c for 4 hr) as previously described . Five major catechins, epigallocatechin gallate (egcg), epicatechin gallate (ecg), epigallocatechin (egc), epicatechin (ec) and catechin (c), were detected at amounts of 17.8, 11.8, 4.2, 2.8 and 0.4%, respectively . Purified forms of these five major catechins were also purchased (nagara science co., ltd ., gifu, japan). Growth inhibition test: canine oral bacteria were used for the growth inhibition test . The pre - cultured bacteria were diluted to 10 cfu (colony forming units) per ml, and then, a mixture of polyphenolic compounds containing either the five major catechins and other components or only purified egcg was added to 1 ml of bacterial suspension . S. mutans (model bacterium) was added to each of the catechin (egcg, ecg, egc, ec or c) solutions, which had final concentrations of 0.2, 0.1, 0.05, 0.025 or 0.0125 mg / ml, and were mixed and incubated at 37c . One - hundred microliters of cell suspension, which had been treated with catechins for 3 or 5 hr, was used for a short - time killing assay and seeded on plates to incubate for 48 hr at 37c . Furthermore, cells were incubated with egcg for 24 hr to measure the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc). Controls were prepared by mixing 1 ml of bacterial suspension, 0.9 ml of bhi broth and 0.1 ml of hanks balanced salt solution (hbss, ph 7.4; gibco, grand island, ny, u.s.a . ). The mics of the polyphenolic compound mix and egcg are defined as the lowest concentrations that inhibited visible growth after overnight incubation . The mbc is defined as the lowest concentration of egcg that killed 99.9% of the initial inoculum in a given time using a plate count assay of viable cells . Biofilm formation test: the effect of egcg on biofilm formation of s. mutans was measured by using the minimum biofilm eradication concentration - high throughput plate (mbec - htp, innovotech, inc ., edmonton, ab, canada). The pre - cultured s. mutans was diluted to a final concentration of 10 cfu / ml . One - hundred microliters of the bacteria dilution was mixed with 100 l of 0.4 mg / ml egcg and was added into the 96-well microtiter plate . The plate was covered with a lid equipped with 96 pegs, the surface of which was covered with hydroxyapatite, with each peg dipping into the bacterial suspension . The plates were incubated at 37c with shaking for 12, 24 and 36 hr . After different periods of incubation, the pegs were broken off, and the biofilms were disrupted from the surface of the pegs in 200 l of physiological saline solution (pss) with a sonicator . Twenty - microliter aliquots of the cell suspensions were then inoculated on bhi agar plates . Biofilm susceptibility assay: biofilms of peg surfaces were formed from 24 hr cell suspensions of s. mutans containing 10 cfus / ml . The biofilms formed on the pegs were inserted into 0.2 mg / ml of egcg solution for 6 hr and 8 hr at 37c . The survival of the bacteria was assessed as described above using the colony count method . Observation of egcg - treated bacteria surface using field emission - scanning electron microscope (fe - sem): a 10 l cell suspension of s. mutans was treated with egcg at 37c for 24 hr, mixed with 10 l distilled water on a micro - glass and then dried . The dried cells were treated with saturated 70% ethanol for 5 min and saturated 100% ethanol for another 5 min . After air - drying, the sample was examined using a fe - sem (su8000; hitachi high - technologies corporation, tokyo, japan). The pegs treated with egcg were removed from the plate, on which biofilms had formed, and were rinsed in 0.9% physiological saline for 1 min to remove planktonic culture . The samples were fixed with 2.5% glutaraldehyde (kanto chemical co., inc ., osaka, tokyo, japan) in 0.1 m cacodylic acid (wako pure chemical industries, ltd ., osaka, japan) at 4c for 16 hr . The pegs were washed with 0.1 m cacodylic acid and distilled water for approximately 10 min each . Saturated 70% ethanol was applied for 15 to 20 min and then air - dried at least for 24 hr . Egcg interaction with streptococcal lta: the quartz crystal microbalance (qcm) is a very sensitive mass measuring device, which measures changes in resonant frequency upon a weight increase on the surface of sensor crystal oscillator . The use of qcm transducers offers sensitive, in situ detection of hybridization events, without the need for optical or redox indicators . Osaka, japan) has an automatic injection mechanism, mixer, sensor crystal oscillator and low capacity reaction vessel . The vibration frequency changes has 200 hz was defined as substances have attached to sensor crystal oscillator . Firstly, 500 l of hbss was added to the low - capacity reaction vessel and mixed at 6,000 revolutions per minute (rpm) to stabilize at 37c . Secondly, 5 l of egcg (16 mg / ml) was injected into the low capacity reaction vessel to stabilize . Subsequently, 5 l of block agent that did not react with the substances was injected to fix the rest of sensor where it was not bound with egcg . Finally, 5 l of streptococcal lta (1 mg / ml) from s. mutans (sigma - aldrich japan corporation, tokyo, japan) was injected to measure the frequency change . Statistical analysis: the function program in microsoft excel (microsoft corporation) was used to conduct f - tests and t - tests for our results (estimation of bacterial cell numbers). The bacteria cultures added different concentrations of catechins to incubate for 5 hr at 37c . The viable colonies were calculated in colony forming units and analyzed by t - test . Standard deviations indicated by error bars were calculated from 3 independent experiments . * significantly different from the untreated control (* p<0.05). The bacteria culture that added different concentrations of egcg was incubated for 3 hr and 5 hr at 37c . The viability (%) is represented as a percentage of the colonies that egcg cell culture is compared with control . Standard deviations indicated by error bars were calculated from 3 independent experiments . * significantly different from the untreated control (* p<0.05). The pegs inserted into 10 cfu / ml planktonic cultures that had added final concentration of 0.2 mg / ml of egcg to incubate for 12, 24 and 36 hr to form biofilm, respectively . The bacteria were moved from biofilm of pegs and seeded on agar plate to count and analyzed by t - test . Standard deviations indicated by error bars were calculated from 3 independent experiments . * significantly different from the untreated controls (* p<0.05).. after statistical analysis, p - values of less than 0.05 were considered statistically significant . The bacteria cultures added different concentrations of catechins to incubate for 5 hr at 37c . The viable colonies were calculated in colony forming units and analyzed by t - test . Standard deviations indicated by error bars were calculated from 3 independent experiments . * significantly different from the untreated control (* p<0.05). The bacteria culture that added different concentrations of egcg was incubated for 3 hr and 5 hr at 37c . The viability (%) is represented as a percentage of the colonies that egcg cell culture is compared with control . Standard deviations indicated by error bars were calculated from 3 independent experiments . * significantly different from the untreated control (* p<0.05). The pegs inserted into 10 cfu / ml planktonic cultures that had added final concentration of 0.2 mg / ml of egcg to incubate for 12, 24 and 36 hr to form biofilm, respectively . The bacteria were moved from biofilm of pegs and seeded on agar plate to count and analyzed by t - test . Standard deviations indicated by error bars were calculated from 3 independent experiments . * significantly different from the untreated controls (* p<0.05). Growth inhibition of canine oral bacteria: various bacteria isolated from oral cavity were sensitive to the polyphenolic compounds mix and egcg (table 1). Mic ranges were 0.10.8 mg / ml for polyphenolic compounds and 0.01250.1 mg / ml for egcg . Oral streptococci showed significant growth inhibition compared to controls which were in the absence of the polyphenolic compounds mix and egcg . 1, the growth of s. mutans was inhibited by three kinds of catechins (egcg, ecg and egc), and the order of their inhibitory effect is egcg> ecg> egc . The mic and mbc values of egcg against s. mutans were 0.125 and 0.1 mg / ml, respectively . The viabilities of s. mutans bacterial cells treated with 0.0125 to 0.1 mg / ml of egcg clearly decreased in the short - time killing assay (fig . The percentage of viable bacterial cells, which were incubated at concentrations of 0.0125 to 0.1 mg / ml of egcg for 3 hr and 5 hr comparing with controls, had decreased to 64.74.0% and 45.30.0%, respectively . Inhibitory effect of egcg on biofilm formation: the inhibitory effect of egcg on biofilm formation of s. mutans was measured by counting colonies that were recovered from biofilms formed on the surface of pegs, as described in materials and methods . The number of cfus obtained from biofilm, which had formed in the presence of egcg, was less than that formed in the control culture (t - test, p<0.05) (fig . 3), indicating that egcg had an inhibitory effect on s. mutans biofilm formation . Thus, this result suggests that egcg can interfere with some components of bacterial cells to inhibit biofilm formation . Biofilm susceptibility to egcg: the effect of egcg on the s. mutans biofilm was measured using a biofilm susceptibility assay as described above . For this purpose, biofilms were first established on pegs, and the biofilm bacteria were statistically significantly eradicated in 6 hr of incubation at a concentration of 0.2 mg / ml of egcg, and bacteria from the treated biofilm were seeded on a plate to count colonies within 10 mm of agarose (fig . Biofilm pegs treated different concentrations of egcg for 6 hr or 8 hr at 37c, and then, bacteria were moved from biofilm of pegs to count living cell within per 10 square millimeter where seeded on ager plate . ). The anti - biofilm effect of egcg was further enhanced after 8 hr incubation, when no colonies were recovered from biofilms treated with 0.2 mg / ml egcg . Biofilm pegs treated different concentrations of egcg for 6 hr or 8 hr at 37c, and then, bacteria were moved from biofilm of pegs to count living cell within per 10 square millimeter where seeded on ager plate . Bacterial cell surface damage after treatment with egcg: the effect of egcg on s. mutans cell morphology was observed using an fe - sem . As shown in fig . 5fig . (a) untreated control . (b) treated with 0.2 mg / ml of egcg for 24 hr . (d) biofilm bacteria were treated with 0.2 mg / ml of egcg for 24 hr . Cell surfaces treated with 0.2 mg / ml of egcg became muddled, and some cell membranes broke, leading to leakage of the cytoplasm (fig . These results demonstrate that the morphological changes to the cell surface were induced by egcg . The damage effect of egcg on s. mutans cell morphology and biofilm . (b) treated with 0.2 mg / ml of egcg for 24 hr . (d) biofilm bacteria were treated with 0.2 mg / ml of egcg for 24 hr . A microscopy image of the s. mutans biofilm attached on the peg is shown in fig . 5c, a regular form of the bacteria can be observed on the peg, showing the biofilm as a pellicle . The effect of egcg on s. mutans biofilm damage was similar to that in cell suspensions (fig . Cells of the biofilm became muddled, and some cell membranes broke, leading to leakage of cytoplasm . Intermolecular interaction between streptococcal lta and egcg: in the qcm test, when 5 l of egcg was injected, the vibration frequency decreased by 440 hz, and the amount of streptococcal lta solution injected each time was 5 l, the vibration frequency only reduced by 50 hz (fig . This is one of the most typical data, in that the same experiment done three times . The numbers in the figure represent injecting times . 1, 5 l of egcg . 2, 5 l of block agent . The numbers of 3 to 11 represent injected each 5 l of streptococcal lta . ). This is one of the most typical data, in that the same experiment done three times . The numbers in the figure represent injecting times . 1, 5 l of egcg . 2, 5 l of block agent . The numbers of 3 to 11 represent injected each 5 l of streptococcal lta . In this study, oral bacteria isolated from dogs were found to be sensitive to polyphenolic compounds and egcg, with streptococci being highly sensitive . In our previous study, we demonstrated that polyphenolic compounds inhibited plaque deposition, gingivitis and other porphyromonas in dogs when the compounds were supplemented in dog food (0.8 mg / g). We suspected that egcg was the most effective catechin, because mic increased when using purified egcg . One of the model bacteria of oral cavities in humans, s. mutans, adheres to the surface of the tooth indicating an area of demineralization of enamel [2, 27]. Besides, it has been reported that carotid injection of s. mutans and other related bacteria in a dog model leads to multifocal choroiditis with retinal detachment . In recent reports catechins of green tea have a broad spectrum of antimicrobial activity against both of gram - positive and gram - negative bacteria . In particular, galloylated derivatives, such as egcg, have been documented to possess antimicrobial effects against oral streptococci . Our study showed that one component of catechins, egcg, demonstrates the highest antibacterial activity against the growth of s. mutans . The mbc value was higher than mic . In the comparison of biofilm colonies formed in egcg - treated and untreated culture, the cfus of biofilm bacteria formed in the egcg - treated culture were obviously less than that formed in the untreated culture . It was suggested that egcg not only inhibits the growth of s. mutans, but also damages surface adsorption ability on the tooth in vitro . The antimicrobial mechanism of egcg is mainly attributable to irreversible damage of the microbial cytoplasmic membrane . Electron microscopic analysis showed that egcg induced cell membrane lysis and cytoplasm leakage as shown in fig . 5 . Microbial biofilms commonly exhibit increasing levels of resistance to most antibiotics or therapeutic agents . Biofilm cells have shown to be more tolerant of antibiotics comparing with planktonic bacteria, and this makes it hard to treat s. mutans with modern medicine [32, 37]. Our data certified further that bacteria in biofilms display lower susceptibility to egcg than those in suspension . As shown in fig . 5d, egcg has a striking effect on s. mutans cells, which establish the biofilm . Gram - positive bacteria develop a profound cell - envelope structure; they lack the normal outer membrane, and the cell wall is usually much thicker than that of gram - negative species, with multiple peptidoglycan layers . The lta is anchored to the plasma membrane and extends from the cell surface to the peptidoglycan layer . Lta and wall - teichoic acid create what has been aptly been described as a continuum of negative charge, which extends from the bacterial cell surface beyond the outermost layers of peptidoglycan . We performed a test on the intermolecular interaction with streptococcal lta and egcg, and the results indicated that egcg did not bind to streptococcal lta . Catechins are known to bind to various proteins (e.g., albumin, casein) to form macromolecular complexes in vitro [18, 25]. All results demonstrated that egcg interacts with other component (s) of the bacterial membrane, which could be some proteins, to inhibit biofilm formation and damage bacterial cells and biofilms, not through streptococcal lta . These findings highlight that the egcg of green tea may be an attractive candidate for the prevention and treatment of oral caries . Further work to understand the relation between egcg and components of the cell membrane is needed for the development of new means to fight the infections caused by canine oral bacteria in the future.
Endometriosis is a tricky albeit common disease whose management still largely relies on laparoscopic surgery . Surgical excision of ovarian endometrioma has positive effects on pain and on the chances of spontaneous conception in subfertile women . However, endometrioma surgery is complicated by concerns about recurrence and ovarian reserve as well as by a relative lack of knowledge on the pathophysiology of the disease [3, 4]. Surgery also represents a major cost for public healthcare because of the valuable human, technical, and logistic resources needed to operate on any single patient . Frequently, the cost profile of surgery in public funded healthcare is also worsened by cascading factors such as delays, cancellations, and long waiting lists . Therefore, the usage of the operating theatre (ot) is often under the spotlight of decision - makers . In spite of evidence that a great proportion of the ot time is lost on nonsurgical activities [7, 8], most surgeons have probably felt the pressure to finish a procedure at the planned time, victims of the belief that operative times are predictable . Our need for certainty makes it difficult to question the often wrong but somehow necessary prediction of surgical procedures duration, which is the basis for ot daily planning . Surgeons are naturally major determinants of surgical times, but no perfect predictive tool exists . In gynecological surgery, different operations require different times, but variability in the duration of the same intervention is also common . Laparoscopic surgery for endometriosis, apart from being often advanced, is peculiar because of a double nature, diagnostic and operative . In fact, see - and - treat interventions can be considered the norm rather than an exception . Ovarian endometriomas, for instance, are typically diagnosed at ultrasound but they often coexist with adhesions and peritoneal implants which can only be seen at laparoscopy . Technical and anatomical factors are usually considered responsible for the duration of surgery, but it would clearly be interesting to know if and how the duration of endometriosis surgery is predictable . In this study, we have analyzed the distribution of operative times from a series of laparoscopic removals of ovarian endometrioma, with a focus on possible predictive factors . We performed an analysis of operative times from a database including 148 cases of laparoscopic removal of ovarian endometriomas at the department of obstetrics and gynecology of the catholic university of the sacred heart, rome, italy . Our database was created for a study on endometrioma recurrence published by our group in 2014 . Only elective cases of laparoscopic excision of histologically confirmed endometriomas 2 cm in diameter were included, while cases with deep endometriosis were excluded . The cystectomy was always performed by means of laparoscopic stripping of the cystic capsule after careful identification of the cleavage plane . Laparoscopy was performed under the care of one experienced laparoscopic surgeon (sc) and in a standardized fashion, as elsewhere described by campo et al . . All patients gave their informed consent in written form preoperatively . In order to identify factors affecting the length of surgery, skin - to - skin operative times were analysed statistically together with other anonymized data . Operative times were then evaluated in bivariate analysis together with several anamnestic and clinicosurgical variables . Correlation between surgical time and continuous variables such as age, bmi, cysts number, and largest diameter (cm) was studied by spearman's rho . Association between categorical variables and surgical time was assessed by mann - whitney u test or kruskal - wallis one - way analysis of variance . Variables of epidemiological and anamnestic interest included dysmenorrhea, parity (1 delivery), infertility, and family history of endometriosis . The following categorical variables of technical interest were also considered: cyst number (single / multiple); cyst size (largest diameter 5 cm/>5 cm); cyst location (right / left / bilateral); peritoneal implants and adhesions; and intraoperative spillage of cystic contents . Furthermore, we aimed at identifying factors that could be associated with procedures which are either shorter or longer than the expected time according to measures of central tendency, such as the median . The reference group consisted of cases with a surgical time comprised within the first and the third quartiles (q1q3), while the other two groups consisted of cases, respectively, below the first quartile (<q1) and above the third quartile (> q3). Separate comparisons between the reference group and the other two groups were carried out by mann - whitney u test for continuous variables and chi - square test or fisher's exact test for categorical variables . Variables showing a significant association with surgical times in the previous analyses (p <0.05) were considered for simple and multiple logistic regression analyses . Crude and adjusted odds ratios (or and aor), with 95% confidence intervals (ci), were calculated to express the strength of associations between selected variables and surgical times . The statistical analysis was performed with spss statistics (ibm) for mac osx and manually . One hundred forty - eight laparoscopic surgeries for ovarian endometrioma were included in analysis . No conversion to laparotomy the median operative time was 70 minutes (mean: 75.14; 95% ci: 70.0380.24). However, analysis of the distribution showed a high dispersion of the data . While 50% of the cases had a surgical time within 1520 minutes above or below the median (interquartile range, iqr: 5593.75), the whole dataset ranged from 20 to 180 minutes, and the standard deviation was relatively large (31.4). When analyzing the entire dataset (table 1), no significant correlation was found between surgical times and age, bmi, and cystic diameter . Instead, the surgical time was significantly and positively correlated with the number of cysts (r = 0.202; p = 0.014). The median surgical time was significantly longer when multiple rather than single cysts were removed (77.50 versus 70 minutes; p = 0.021). Anamnestic factors such as dysmenorrhea and nulliparity were also significantly associated with longer operative times . The operative time was not significantly affected by cyst location, adhesions, peritoneal implants, spillage, or infertility . The reference group, serving as control, included 78 cases with an operative time ranging from the first quartile to the third quartile of the series (median: 70 minutes; iqr: 6080). A short time group (<q1) included 33 cases with a median of 40 minutes, while the long time group (> q3) consisted of 37 cases with a median of 115 minutes . Compared to the reference group, women in the short time group were significantly older (median age: 34 versus 30; p = 0.018), and their cysts were more frequently single (87.9% versus 69.2%; p = 0.039) and smaller than 5 cm (87.9% versus 62.8%; p = 0.008). Significantly less women in the short time group were nulliparous but the number of infertile patients was similar in both groups . No differences were found in adhesions and peritoneal implants rate, but the absence of spillage was associated with shorter times (69.7% versus 44.9%; p = 0.017). No statistically significant differences were found between the reference group and the longer operative time group except for a positive family history of endometriosis . Twenty - seven percent of women in the long time group had a family history of endometriosis compared to 8.9% in the reference group (10/37 versus 7/78; p = 0.011). Crude odds ratios, with 95% confidence intervals and p values, describing the strength of association between selected variables and operative times, respectively, shorter and longer than the reference group (q1q3) are presented in table 3 . A multiple logistic regression analysis, adjusting for the number and size of the removed cysts, identified older age and parity as independent predictors of shorter operative times (table 4). Longer operations were instead significantly associated with a positive familial anamnesis (aor: 3.639; 95% ci: 1.24610.627; p = 0.018). Finally, while shorter operative times did not appear to improve short - term postoperative outcomes, belonging to the long operative time group was associated with a significantly longer postoperative hospital stay (> 1 day for 35.1% of patients versus 15.4% in the reference group; p = 0.016) and a nonsignificantly higher postoperative complication rate (5.4% versus 0% in the reference group). Surgery, as a tool to treat medical conditions and improve quality of life, is invaluable . At the same time, costly operating theatres are a natural target of efficiency improving efforts . In our study, we analysed the operative times of laparoscopic surgery for endometrioma in order to evaluate to what extent they are predictable . Since historical data and the surgeon's expert judgement are, in combination, the most common predictive approach, we focused on their respective assumptions: a limited variation of operative times and adequate knowledge of predicting factors . The first one is the fact that the time needed to perform a laparoscopic removal of ovarian endometrioma is highly variable and, as such, difficult to predict by simply looking at historical data . This might sound familiar to endometriosis surgeons, but similar findings have rarely been described by dedicated scientific studies . Although half of the cases will last something within 15 minutes above or below the median time, a large proportion of the patients will have surgical times which are 50% or more shorter or longer . Endometriosis is certainly a complex disease, and this may reflect on the variability of operative times . Nevertheless, the cases in our study were quite homogeneous, since we only included patients with ovarian endometriomas 2 cm but excluded cases with elsewhere located deep endometriosis . Moreover, all the cases were performed under the care of an experienced laparoscopic surgeon, with a standardized stripping technique, and at the same institution, which is a reference centre for the treatment of endometriosis . Those conditions reduce the risk of performance bias, which is not uncommon in surgical research [13, 14]. A peculiarity of our study lies in the attempt to identify predictors of surgical duration for laparoscopic endometrioma surgery . Operative times are correlated to some of the surgical factors that we have analyzed, such as number and size of the cysts . However, other factors that might be considered to increase the surgical difficulty and to require additional time, such as adhesions and peritoneal implants, were not associated with a longer duration of the procedures . On the contrary, nontechnical factors such as age, parity, and family history were significantly associated with operative times . This further confirms the peculiar nature of endometriosis whose clinical manifestations and behavior change from patient to patient depending on factors so far largely unknown . For instance, the association of shorter times with older age or parity could be linked to a milder disease . Similarly, a positive family history for endometriosis, which we found to be associated with higher endometrioma recurrence rates, could be linked to a more severe disease and hence longer operative times . Finally, shorter operative times do not seem to ensure short - term benefits in terms of postoperative stay or complications . This might depend on the fact that the overall operative times in this series were relatively low because of the team experience . On the other hand, patients whose surgical time was above the third quartile had a significantly longer postoperative stay, and the two postoperative complications of this series were found in this group . These findings cannot directly support a causal relationship and are limited by the sample size and a lack of controlling for possible confounders . However, they can be useful to formulate hypotheses on how to identify cases at risk at the time of surgery, allowing for tailored postoperative care planning . Overall, our study highlights a new facet of the complexity of endometriosis and partially explains why it is difficult to predict operative times . The duration of laparoscopy is known to be less predictable than laparotomy, and this applies to different laparoscopic procedures . Laparoscopy in endometriosis patients has a diagnostic value and often leads to see - and - treat management of unexpected findings . Hence, strict time schedules are not convenient for endometriosis since they can lead to suboptimal surgery . Besides, the heterogeneity of endometriosis seems to reflect also on the severity of the disease, for which we are still probably lacking important knowledge and a reliable staging system . Similar lesions, such as ovarian endometriomas, have variable pathological and clinical behaviors in different women, thus leading to surgical difficulty of unpredictable level or, as we have previously documented, different rates of recurrence . Should we give up the efforts of making the operating theatre a more efficient place? To believe that surgical times, for any given operation, are standard and predictable according to historical measurements of central tendency such as the median, although tempting, is fallacious . The idea of standard procedures with standard times might satisfy our innate need for certainty and is also functional to other aspects of surgical planning, such as allocating instruments and staff . However, because of the great dispersion of surgical times around the average values, scheduling according to historical averages is based on probability [9, 17]. The surgeons themselves, unfortunately, do not seem to be able to provide a more accurate estimate of operative times . In this context, it would be reasonable to integrate predictive methods, based on historical data and the surgeon's estimate, with knowledge of the inevitable variability in operative time . Measuring and adapting to the variability in surgical durations should be a key process of modern operating theatre management . At the same time, efforts should be made in order to minimize the consequences to the patients and optimize resources' usage in other ways . On one hand, patient turnover could be improved by monitoring the time needed by standard perioperative procedures and activities [7, 8, 20]. On the other hand, we should maybe rethink our context where productivity is increasingly measured in number of procedures rather than in health outcomes, particularly in the case of such complex disease . Probably, the care of endometriosis patients should be provided by centres where the underlying production philosophy is mature enough to shift from a simplistic focus on quantity to a more refined demanding, but certainly patient - centered, interest for quality.
Vertebrobasilar dolichoectasia (vbd) is a condition characterized by ectasia, elongation and tortuosity of the basilar artery . It may manifest clinically by compression of the cranial nerves, ischemic symptoms or intracranial bleeding . Rarely, the dilated and ecstatic basilar trunk may manifest as obstructive hydrocephalus . We present a 60-year - old male with markedly dilated and elongated basilar trunk compressing the third ventricular outflow and, thus, presenting with hydrocephalus . A 60-year - old male presented to our outpatient department with complaints of headache since 2 years . He also complained of imbalance while walking and episodes of urinary incontinence since the past 5 months . On examination, he had bilateral papilloedema . He underwent further evaluation with magnetic resonance imaging (mri) of the brain, computerized tomography (ct) of the brain and ct angiography . Dilated and tortuous basilar artery was noted extending into the suprasellar space compressing the third ventricular outflow . Ct angiography of the brain showed dilated and tortuous left vertebral artery curving in the premesencephalic cistern from left to right and again from right to left . The basilar artery was also tortuous, with a diameter of 7.0 mm and extending into the suprasellar cistern [figure 1]. The patient underwent right - sided ventriculoperitoneal shunt and the headache improved postoperatively . Left and middle pictures show dilated ventricles with dolichoectatic basilar artery compressing the midbrain and outflow of the third ventricle . The right picture shows the computerized tomography angiogram with the dilated and elongated basilar trunk extending into the suprasellar space vbd is an anatomic variant that consists of enlargement and dilatation, often associated with a tortuous and elongated vessel . It is known by various names like dolichoectasia, megadolichoectasia fusiform aneurysm of the vertebral and basilar arteries and tortuous vertebrobasilar system . The prevalence of vbd is 4.4%, and it is more commonly observed in women . The major location for vbd is the basilar artery alone (40%), followed by bilateral vertebral arteries, basilar artery (22%) and both vertebral arteries (16%). The diagnostic criteria for vbd is a basilar artery or vertebral artery diameter> 4.5 mm or deviation of any portion of them higher than 10 mm from the shortest expected course, or basilar length> 29.5 mm or intracranial vertebral artery length> 23.5 mm . The vertebrobasilar system may be considered elongated if the basilar artery lies lateral to the margin of the clivus or dorsum sellae, or if it bifurcates above the plane of the suprasellar cistern . Hypertension, commonly associated with vbd, may cause continued stress on the walls of the artery and degrade the vessel wall by damaging and loosening the collagen and elastin meshwork that comprises the intima . Traditionally, vbd has been regarded as atherosclerotic in nature, much like aneurysms of the peripheral vascular system . However, recently, mizutani and aruga suggested that some cases represent a dissecting process . It may also be a congenital vasculopathy of the elastic layer of the arterial wall . It may present with varied clinical syndromes like cerebellar dysfunction, ischemic stroke, transient or permanent motor deficits, central sleep apnea, trigeminal neuralgia, hydrocephalus as well as brain stem compression syndrome . Clinical expression of this condition may be due to compression of the cranial nerves or brainstem, ischemia in the vertebrobasilar arterial territory and intracranial bleeding . Ikeda et al . Studied 7345 adult subjects, and found that 96 of them had asymptomatic vbd . Among these 96 subjects, hydrocephalus in vbd can be due to compression of the third ventricle by the ectatic, elongated and tortuous basilar artery . Only few cases of hydrocephalus due to direct compression of the aqueduct, foramen of monro or third ventricle have been reported in the literature. [1014] most of these cases have been reported in elderly patients . A peculiar mechanism of hydrocephalus by water - hammering effect due to the pulsating blood in the ectatic vessel, which creates cerebrospinal fluid outflow impairment through the third ventricle, has also been described . Management of vbd depends on symptomatic manifestations . For asymptomatic patients with vbd, functional testing such as brainstem auditory - evoked potentials (baeps), blink reflex (br) and motor - evoked potentials may be useful for long - term monitoring and may help in the decision - making process prior to the surgical approach for relief of subjective symptoms . Endoscopic third ventriculostomy may be technically difficult in these cases due to the odd anatomy of the basilar trunk . Vbd as a rare cause of obstructive hydrocephalus needs to be kept in mind by neurosurgeons.
Functional dyspepsia (fd) is a very common cause of upper gastrointestinal symptoms and discomfort . Fd has been defined as a functional gastrointestinal disorder (fgid) characterized by persistent or recurrent pain or discomfort centered in the upper abdomen that is not relieved by defecation or associated with changes in stool characteristics occurring at least once a week for at least 2 months in the absence of organic diseases . A diagnosis of fd can be made in children mature enough to provide an accurate history of pain that is present for at least a 12-week duration, which need not be consecutive, in the preceding 12 months, the recurrent discomfort is typically centered in the upper abdomen (above the umbilicus), and there is no evidence of organic disease (including at upper endoscopy). In addition, there is no evidence that dyspepsia is exclusively relieved by defecation or is associated with the onset of a change in stool frequency or stool form . There are two presentations of functional dyspepsia, which are ulcer - like dyspepsia and dysmotility - like dyspepsia . The low prevalence of organic disease found in dyspepsia supports the use of reassurance and empiric therapy as initial treatment, so the rome committee recommends that an upper gastrointestinal endoscopy should be performed in the presence of dysphagia, persistence of symptoms despite the use of acid reducing medications, or patients with recurrent symptoms after discontinuing such medications . Thus, different approaches with antisecretory, spasmolytic, prokinetic, and anti - inflammatory effects, and, most preferably, reduction of visceral hypersensitivity seem logical . This could explain the variety of drugs which show a positive symptomatic response, currently there is no fda - approved drug for treatment of fd . Despite the scant evidence, anti - secretory agents are frequently recommended in the treatment of patients with a predominant complaint of pain while prokinetic agents are frequently used for bloating and early satiety . Patients' symptoms that are severe enough to disrupt daily activities will likely benefit from pharmacologic therapy . Such therapy should be individualized and directed toward the predominant symptom . For patients with predominant dyspepsia (discomfort centered in the epigastrium, nausea, early satiety, postprandial fullness, recurrent emesis), a short course of empiric therapy with an h2-histamine receptor antagonists or proton pump inhibitors is acceptable . There are currently no pediatric data to support the long - term benefit of anti - secretory therapy in patients with fgids . The aim of this study is to compare the effectiveness of the commonly four most used drugs for treatment of dyspepsia in children, including omeprazole, ranitidine, cimetidine, and famotidine in a period of 4 weeks of treatment . In this clinical trial study, 169 children between 2 and 16 years old were enrolled with the diagnosis of fd which was made by a history of recurrent or persistent abdominal pain and discomfort which was typically centered in the upper abdomen for at least a 12-week duration without any evidence of organic disorder . The other symptoms were early satiety, postprandial abdominal floating or distention, nausea and vomiting . Patients over 18 years old, and whom their medical therapy could not be completed, and those with symptoms including, fever over 38 centigrade degrees, night sweating, weight loss more than 3 kg during past month, frequent vomiting, hematochezia or hematemesis, severe localized pain, and dysphagia, were excluded from this study . Then, for each patient, one of the acid suppressant medications, omeprazole, famotidine, ranitidine, or cimetidine, was administered, for a period of 4 weeks . The drugs dosage used was as follows: cimetidine: 10 mg / kg / dose b.i.d . (max: 300 mg/24 hr), famotidine: 1 mg / kg / day b.i.d . (max: 40 mg/24 hr), and omeprazole: 1 - 2 mg / kg / day q.d (max: 40 mg/24 hr). Patients were followed after 2 and 6 weeks from the beginning of the treatment . All the results, consisting of any probable side effects during the therapy and the effectiveness of the medication being used in treatment of the patients, were collected and analyzed . Collected chi - square test and students t - test were also used to analyze the results . The significant p - value in this study was determined to be less than .05 . 80 patients (43.7%) were males and 89 patients (52.7%) were females . The mean age of the patients was 7.4 3.2 years (range, 216 years). The mean duration of the disease among the patients was 15.9 14.2 months (range, 360 months), and the mean of their weight was 25.6 11.7 kg (range, 1260 kg). Ninety - nine patients (58.6%) had a positive family history of fd, and 52 patients (30.8%) were passive smokers . In 108 patients (63.9%), symptoms were related to food consumption; occurrence of the symptoms in 35 patients (32.4%) was before, in 55 patients (50.9%) was after, and in 18 patients (16.7%) was both before and after food consumption . In 61 patients (36.1%), there was no relation between meal consumption and symptoms . Of these 169 children, 37 (21.9%) patients were treated with cimetidine, 36 (21.3%) with famotidine, 44 (26%) with ranitidine, and 52 (30.8%) with omeprazole . The most common symptoms relieved regardless of type of medication were nausea (86.2%), vomiting (80.8%), and heart burn (79.5%). The distribution and percentage of symptoms being relieved regardless of the specific medication being administered are shown in table 2 . When different medications were compared, abdominal pain was improved in 45.9%, 65.9%, 66.7%, and 73.1% of cimetidine, ranitidine, famotidine, and omeprazole groups, respectively, these differences were statistically significant (p <.05). The distribution and percentage of symptoms being relieved in relation to the specific medication being used are mentioned in table 3 . The most influenced symptoms followed by medical therapy in relation with specific medication were chest pain in famotidine group (100%), vomiting in ranitidine group (92.7%), nausea and vomiting in cimetidine group (90%), and nausea in omeprazole group (87.8%), respectively . The least influenced symptoms followed by medical therapy in relation with specific medication was halitosis in all groups, 25.9%, 44.8%, 51.5%, and 52.5% in cimetidine, famotidine, ranitidine, and omeprazole groups, respectively . In 71 out of 169 patients (42%), all of the symptoms were relieved, not considering the specific medication being taken by them . When different medications compared 8 of 37 patients who took cimetidine cured completely (21.6%), this cure rate in other groups were 43.2% (19 out of 44) in ranitidine, 44.4% (16 out of 36) in famotidine, and 53.8% (28 out of 52) in omeprazole group, differences were statistically significant (p = .024). In the followups during 2 and 6 weeks after medical therapy, no side effects due to medical therapy were seen . Fgids, including functional abdominal pain (fap), are among the most common conditions in children . A school - based study in the united states showed a 38% overall weekly prevalence of abdominal pain and persistence of symptoms for more than 8 weeks in 24% of them . Children with abdominal pain were found to miss more school than their peers, and their parents frequently missed work to take care of their children . Some studies in children have shown an association between chronic or recurrent abdominal pain and higher depression and anxiety scores and poor quality of life . Despite its high frequency and significant impact on quality of life of children, there is only limited evidence to support most treatments that are commonly used to treat childhood fap . Dietary recommendations may be helpful for some patients with functional recurrent abdominal pain of childhood . There are different medical therapies with different medications for treatment of this disorder in children . In fd, patients' symptoms that are severe enough to disrupt daily activities will likely benefit from pharmacologic therapy . Treatment modalities include medications, diet modification, herbal preparations, and behaviorally psychologic interventions . Enteric - coated peppermint - oil capsules, believed to exert calcium channel blockade in smooth muscle, were shown in a randomized, placebo - controlled study to decrease the severity of abdominal pain, but not other symptoms in pediatric patients with irritable bowel syndrome . Pharmacotherapy for treatment of fgids consists of anticholinergic agents, tricyclic antidepressants, serotonergic agents, selective serotonin reuptake inhibitors, 5-ht3 receptor antagonists, 5-ht4 receptor agonists, and acid suppressive therapy . For patients with predominant dyspepsia (discomfort centered in the epigastrium, nausea, early satiety, postprandial fullness, recurrent emesis), a short course of empiric therapy with h2-receptor antagonists or proton pump inhibitors is acceptable . Some meta - analysis studies showed that h2-receptor antagonists did or did not have a significant therapeutic effect in fd [10, 11]. A meta - analysis of randomized controlled clinical trials has shown that there may be a benefit in the use of h2-receptor antagonists in patients suffering from fd . In another study, it was found that famotidine was equally effective as placebo . In a meta - analysis, proton pump inhibitors were regarded as superior to h2-receptor antagonists and antacids in patients with noninvestigated dyspepsia, h2-receptor antagonists and antacids showed positive effects in approximately 40% of patients (which is in the range of the placebo response rate) whereas proton pump inhibitors response rates were significantly higher, adding an additional 20% . In two preliminary studies of omeprazole, a proton pump inhibitor, for the treatment of nonulcer dyspepsia, only 50% of the patients treated with omeprazole had a response, as compared with 25% of those receiving placebo . In a double - blind randomized placebo - controlled study of 4 weeks of lansoprazole (a proton pump inhibitor) for the treatment of fd in chinese patients, findings implicated that proton pump inhibitors treatment was not superior to placebo for the management of fd in chinese patients . Several studies in the primary care setting have concluded that proton pump inhibitors are more effective than h2-receptor antagonists or antacids in treating heart burn and dyspeptic symptoms . Therefore, empiric acid suppression would seem to be the favored management approach for the treatment of fd . Since the various proton pump inhibitors are of equivalent efficacy and safety, the cost and acceptability of a particular proton pump inhibitor preparation may be more important when selecting among them than comparable efficacy . In this study, we compared the effectiveness of four medications including cimetidine, famotidine, ranitidine (all of them h2-receptor antagonists), and omeprazole (a proton pump inhibitor), for treatment of children with dyspeptic symptoms, to find the best one for this reason . So if a cure is defined as all symptoms relief after a period of 4 weeks treatment, our analysis indicates that there is a significant difference between response rate and the specific medication being used (p = .024), and it reveals that the most effective medication, when considering cure as all symptoms being relieved, was omeprazole with response rate of 53.8% and then with famotidine (44.4%), ranitidine (43.2%), and, at last, with cimetidine (21.6%). Although no significant difference (p = .06) was found in abdominal pain relief in relation with specific medication consumption, but due to higher response to omeprazole, it seems that omeprazole was better than others, and cimetidine had the least effect . Also, there were no significant differences between other symptoms relief (p>.05) and the specific medication taken by the patients, except for epigastric pain which responds significantly (p = .018) to famotidine and ranitidine with response rate of 68.4% then omeprazole with 66.7% and at last cimetidine with 28.6% . According to our results and the fact that no significant side effects being detected, and also due to the fact that ranitidine and omeprazole were the most effective medications on only one of the symptoms (epigastric pain) comparing with omeprazole that had the best result on all symptoms being relieved, it cannot be concluded that ranitidine and famotidine have equal or better effect in treatment of fd, but, in fact, the best medical therapy for treatment of fd is omeprazole, or in another way omeprazole is superior to h2-receptor antagonists for treatment of fd . At the end it is important to note that since 3 of 4 medications that we used in our study had approximately an equivalent efficacy and safety, the cost of a particular medication may be more important, when selecting among them, than comparable efficacy.
A 58-year - old african american gentleman presented with a history of idiopathic bulbar urethral stricture disease . He had been managed with multiple transurethral dilation and incision procedures for over a decade . Urodynamics testing had been performed previously and demonstrated a high - pressure, low - flow pattern with normal bladder capacity . His additional medical history included sarcoidosis, anemia of chronic disease, gout, and hypertension . Urinalysis, prostate - specific antigen levels, and testosterone levels were within normal limits . After extensive counseling, the patient was taken to the operating room for definitive management of his stricture with substitution urethroplasty . Four 4 - 0 vicryl stay stitches were placed in the urethra to allow for 180 degrees of rotation, providing excellent access to the dorsal urethra . The dorsal aspect of the urethra was opened sharply at the 12 o'clock position between the stay sutures (fig . The length of the urethrotomy was extended until the distal and proximal urethral segments easily accommodated 26-fr and 30-fr calibration, respectively (fig . The stricture ultimately measured 7 cm in length, and the urethral plate measured 1.2 cm in width . A buccal mucosal graft measuring 2.2 cm in width was harvested from the inner left cheek in standard fashion ., the ventral urethral plate was sharply incised in the midline without significant violation of the spongiosum . The wings of the opened plate were mobilized with sharp dissection to allow for an elliptical defect . The graft was sewn into place by using two separate 5 - 0 pds sutures in running fashion (fig . A lubricated 16-fr silicone foley catheter was then placed under vision across the repaired area (fig . The patient's postoperative imaging 3 weeks after the procedure demonstrated a patent, wide - caliber urethra (fig . 3). After 3 years of follow - up, he was voiding with a strong stream and with minimal pvr (<10 ml) at every visit . Historically, numerous surgical approaches have been used to treat these lesions . Depending on the length and location of the stricture as well as the experience and preference of the surgeon, strictures in the bulbar urethra may be approached in an excisional manner or with tissue substitution . Although excision with primary anastomosis can be applied to extensive proximal strictures, this is controversial and challenging for most practicing urologists . For longer or more complicated strictures, substitution techniques utilizing grafts or flaps substituted tissue is transferred into the urethra as either an onlay or inlay . In onlay procedures, the urethra and its overlying spongiosium the graft material is then either substituted in place of both the mucosa and sponge together (dorsal onlay) or in place of the mucosa only with the transected sponge being closed over the repair (ventral onlay). In contrast, in inlay procedures, only the mucosa of interest is opened throughout the length of the stricture, with the corresponding sponge remaining largely undisturbed . The mucosal edges are then usually undermined to some degree, and the graft material is then sewn to the edges of the mucosa . In general, bulbar strictures in excess of 2 cm are most commonly treated with ventral onlay of buccal mucosa . Although this is a fairly straightforward procedure anatomically, it does require disruption of the ventral spongiosum . It is important to recognize that this vascular tissue bed becomes much more concentrated ventrally as one moves from the distal to the proximal urethra . Division of this network of vessels can contribute to intraoperative blood loss that may limit visualization . Additionally, there is a theoretical benefit in terms of graft support if more of this vascular tissue could be spared . Furthermore, suture reapproximation of the spongiosum after a ventral onlay could create a furled lie of the graft, although this has not been established to the author's knowledge . This work builds on the concept of a " stricturoplasty " that takes advantage of the heineke - mikulicz principle . This was previously described by james pierce in 1962 and was resurrected by lumen et al . In 2010 . Andrich and mundy wisely point out that some patients may already have impaired retrograde blood flow (e.g., prior hypospadias repair), and preservation of vascularity seems prudent . The ventral inlay technique that we have described here is designed to minimize disruption of the spongiosum while providing a well - vascularized bed for the graft . Whereas this may not be well suited to every patient with stricture disease (i.e., patients with full thickness spongiofibrosis), for well - selected patients additionally, patients presenting for definitive repair of urethral stricture may require further urethral procedures for other reasons later in life . For example, many patients will go on to be screened and possibly treated for prostate cancer . Despite improvements in delivery of radiation and technical performance of radical prostatectomy, a subset of these men may require placement of an artificial urinary sphincter for treatment of incontinence . It is logical to assume that maximizing the potential of the urethra to resist compression - induced erosion would, therefore, be beneficial . This concept has been reinforced by utilizing surgical maneuvers to preserve the bulbar arteries at the time of anastomotic urethroplasty . Additionally, andrich and mundy point out that minimizing spongiosal trauma may also be of benefit to patients who require a subsequent urethroplasty . Thus, in the era of minimally invasive surgery, it seems prudent to preserve as much blood flow as possible as long as this does not compromise the treatment of the patient . Since this is the first description of an approach to bulbar stricture disease and a solitary case, it does not serve to establish superiority over ventral onlay substitution . However, our report demonstrates the feasibility of substitution urethroplasty for bulbar strictures with minimal disruption to the ventral spongiosum . One may argue that ventral onlay is often done in the proximal bulb, because this technique obviates the disruption associated with circumferential urethral mobilization . Alternatively, dorsal onlay could be performed as suggested by andrich and mundy . However, a few key points deserve consideration . Reviewed ventral, dorsal, and lateral graft onlays for bulbar strictures and showed no difference in overall success . More recently, figler et al . Also reviewed the impact of graft position and once again showed no appreciable differences between dorsal and ventral placement . However, what if the ventral onlay technique only failed to show superiority owing to division of the spongiosum? Barbagli's group suggested that " ventral placement of the graft is more efficacious in the proximal part of the bulbar urethra, where the spongiosum tissue is thicker and better vascularized . " Additionally, they felt that graft failure may stem from poor inosculation from insufficient vascularity of the graft bed, which would seem more likely when the graft is placed on the thinner dorsal spongiosum or on a divided ventral spongiosum . The approach described here takes advantage of having a graft in direct contact with an undisturbed bed of spongiosum at its thickest location . To the authors' knowledge, this represents the first description of a ventral inlay in the treatment of urethral stricture disease . Although further study will be required to determine the role of this type of repair in the reconstructive urologist's armamentarium, feasibility has been established and a logical rationale exists for considering this type of approach.
Stepwise fibrillation of proteins to amyloid structures and amyloid - like protein aggregates occurs naturally or is disease associated . The hallmark of amyloid aggregation is conversion of otherwise soluble proteins into -sheet conformations that locate extracellularly, in the cytoplasm or in the cell nucleus . Formation of amyloid - like protein aggregates unifies superficially unrelated human conditions such as alzheimer, parkinson, or huntington disease that are therefore consolidated as neurodegenerative deposition diseases . In brains of patients with alzheimer disease amyloid peptides (as) deposit in extracellular plaques or fibrils, whereas tau protein is enriched in intraneuronal neurofibrillary tangles . Protein deposition in parkinson disease occurs as characteristic lewy body inclusions in the cytoplasm of residual substantia nigra and dopaminergic striatal neurons . Huntington disease constitutes one out of nine neurodegenerative disorders that are characterized by aberrant deposition of signature proteins containing unstable homopolymeric repeats of the amino acid glutamine (polyq). Neuronal cells of patients with polyq - repeat - length diseases are characterized by occurrence of microscopically discernible nuclear inclusions (nis) that contain proteins with expanded polyq stretches, components of the ubiquitin - proteasome system (ups), and other cellular proteins . Aggregates can likewise be observed in other neural compartments such as the cytoplasm, dendrites, and axon terminals . A common feature of such inclusions is the recruitment of mutated, misfolded, and waste proteins that leave solution because of the tendency of normally buried hydrophobic domains to associate with one another . The presence of proteasome - dependent proteolytic activity in nis indicates that misfolded or excess polypeptides form intracellular aggregates before their degradation . Yet, the role of nuclear protein aggregates in disease pathology is still unknown, and their assumed function ranges from being cytotoxic to benign or even neuroprotective . This review compiles current knowledge about nuclear protein aggregation, e.g., amyloid deposition with special emphasis on their protein composition mandating the idea that this composition reflects perturbed protein interaction networks and holds the key for a better understanding of underlying pathologic as well as physiologic events . The term amyloid was first used in the 19th century by german pathologist rudolph virchow to describe starch - like deposits within mammalian cells based on similar tinctorial properties with polysaccharides from plants . However, virchow in fact observed waxy protein structures and the term amyloid currently designates proteinaceous deposits that are defined by one or more histochemical and biophysical features such as (1) an ultrastructure of long, unbranched fibrils of approximately 10 nm in width, (2) a green birefringence under cross polarized light after staining with the azo dye congo red, (3) a shift of fluorescence wavelength after staining with the dye thioflavin t, (4) a peak at around 1620 by fourier transform infrared spectroscopy indicating the presence of -sheet, (5) a crossed -pleated sheet structure, and (6) binding to amyloid - specific antibodies, peptides or compounds . In vitro characterization of amyloid fiber structure and the actual processes of amyloid protein fibrillation has largely progressed by usage of high - resolution structural studies, i.e., solid - state nuclear magnetic resonance (nmr). The process constitutes a nucleated growth mechanism that initiates stepwise fibrillation of otherwise soluble proteins with a lag phase of nuclei formation and proceeds by further association of monomers or oligomers . In the in vitro scenario fibril growth either occurs by transition of oligomeric species to spherical, chain - like fibrils or by sequential fibrillation of soluble oligomers, unstructured aggregates, and short curly protofibrils that finally form mature amyloid fibrils . It is important to note that structural intermediates of this fibrillation process may yet have varying amyloid features such as -structure, thioflavin t - binding or congo red - binding . Formation of intracellular amyloid deposits, e.g., in vivo protein fibrillation, may be initiated by local unfolding of native proteins through physiological fluctuations and subsequent fibrillation . Examples are changes of thermal conditions as observed in heat shock responses or experimentally induced fluctuations . Consistently, several reports describe controlled nucleation of protein fibrillation by heat shock, metals, or nanoparticles in vitro, in mammalian cell culture as well as in whole organisms . The analysis of intracellular amyloid formation has advanced in recent years due to the diagnostic value of amyloid fibril localization within cells and tissues . In line with this conformation - specific antibodies, peptides and luminescent compounds have proven useful to monitor amyloid distribution in fixed and living specimens; however, methods for the discrimination of specific fibrillation intermediates in vivo are still in their infancy . Amyloid - like nis in neurodegenerative aggregation diseases are predominately characterized by histological methods and biochemical purification of sds - insoluble protein fractions . At least nine cag - repeat diseases including huntington disease (hd), the spinocerebellar ataxias sca1, sca2, sca3, sca6, sca7, sca17, and dentatorubral pallidoluysian atrophy (drpla), as well as spinal and bulbar muscular atrophy (sbma) show nis in specific sections of postmortem brains . In postmortem brains of patients with hd it was discovered that mutated huntingtin forms mostly spherical or ovoid nis in cortical neurons (table 1; fig . 1). These nis were positioned throughout the nucleoplasm with a subfraction located in proximity to nucleoli . In a third of the observed neurons nis covered nearly 50% of the cross - sectional nuclear area . Electron microscopy revealed that nis in cortical hd neurons are composed of granules, filaments, and randomly or parallel oriented fibrils . Huntingtin and ubiquitin also occupy spherical nis in the striatum of mice transgenic for the huntingtin mutation . Interestingly, striatal neurons of symptomatic transgenic mice showed significant alterations of the nuclear envelope structure, i.e., numerous indentations of the nuclear membrane and aberrant clustering of nuclear pore complexes . In contrast, cross - sectional size of nuclei, nucleoli, and cajal bodies were described as unchanged . Notably, nuclear huntingtin occurs as an early pathological hallmark 12 wk before any murine phenotypes such as brain or body weight loss and approximately 5 wk before the first disease - specific symptoms . Anti a, detection by amyloid specific antibodies; ap, amyloid peptide binding; cr bi, congo red birefringence; cr / tht, congo red and/or thioflavin t binding; ihc, nuclear inclusions defined by immunohistochemistry; ni, nuclear inclusion . Figure 1 . Schematic representation of proteins in nuclear inclusions (nis) from patients with neurodegenerative polyq (cag) expansion diseases and respective animal or mammalian cell culture models . 20s, 20s proteasome subunits; 19s, 19s proteasome regulator subunits; 11s, 11s proteasome regulator subunits; ar, androgen receptor; atxn-1, ataxin-1; atxn-3, ataxin-3; atxn-7, ataxin-7; cbp, creb binding protein; eya, eyes absent protein; hnrnps, heterogeneous nuclear ribonucleoproteins; hsdj, dnaj protein homolog; hsp-70, heat shock protein 70; htt, huntingtin; mdm-2, mouse double minute protein; np, nanoparticle; npc, nuclear pore complex; numa, nuclear mitotic apparatus protein; pml, promyelocytic leukemia protein; qn, different length polyq repeat; sumo, small ubiquitin - related modifier; ub, ubiquitinated; tbp, tata binding protein . In sca1 nis are observable in neurons of the cerebral cortex, striatum, substantia nigra, and pontine nuclei . They have a diameter of 1 to 3 m and contain ataxin-1 and ubiquitin as well as unspecified proteins with polyq repeats (table 1; figs . 1 and 2). By transmission electron microscopy inclusions appear as round, spherical bodies in the nucleoplasm consisting of mixtures of granular and filamentous structures that are either organized in random or parallel arrays . Postmortem brains of sca3 patients display a similar morphology of nis in the spinocerebellum, cerebellar dentate nucleus, cerebral cortex, and striatum . They are immunoreactive with ataxin-3, ubiquitin, polyq repeat domains, and a variety of transcription factors (table 1; fig . 1). Additionally, nis of sca3 patients and respective mouse models have been shown to associate with pml and cajal nuclear bodies in nucleoplasmic microdomains . The incidence of nis in the polyq repeat diseases sca2 and drpla is low with 13% of affected neurons; however these nis are widely distributed within different brain regions . In sbma, also termed kennedy disease, the androgen receptor (ar) is mutated concerning expansions of its instable polyq repeats and accumulates in nis that appear in motor neurons of the brainstem, the spinal cord, but also in skin, testis, and additional visceral tissues (table 1; fig . 1). Unstable polyq repeats vary considerably with respect to their location in the primary protein sequence and length . It has been acknowledged very early that the distribution of nis within specific brain regions is not exactly correlated with degeneration of the affected neurons . While the striatum represents a brain region that undergoes extensive neurodegeneration in hd, only 14% of striatal neurons contain nuclear aggregates . Long - term observation of live neurons by automated microscopy revealed a higher viability of neurons with mutated huntingtin accumulating in nis compared with neurons with diffuse nuclear localization of huntingtin . These observations suggested a protective role of nuclear protein aggregation, i.e., a coping response of the nucleus to excess fibrillation of misfolded proteins . Consistent with this and as already mentioned above, ubiquitin, a main player of proteasome - dependent protein degradation, is identified by histopathology as a frequent component of nis . Likewise are heat shock proteins, 20s proteasomes and the 19s proteasome regulator units (table 1). With all the players on the field and the demonstration of proteasome - dependent proteolytic activity in nis a picture is emerging where at least a subpopulation of intranuclear protein aggregates operate as proteolytic centers . Here, formation of nis may serve recruitment of excess, mutated, or misfolded nuclear proteins to their degradation . Consistent with this idea it was shown that neurons have different capacities to clear mutated huntingtin, and that those with greater clearance capacities live longer . In this respect nis may have a protective function by sequestering and clearing excess or misfolded nuclear proteins from the nucleoplasm and thus maintaining protein homeostasis within the cell nucleus . While it seems appropriate to boost this cellular defense mechanism, e.g., nuclear proteolysis, with therapeutic interventions it must also be considered that protein components of nis are part of a nuclear proteome network and respective maintenance of cellular functions such as gene expression or progression of the cell cycle . Intracellular inclusions that were (1) biochemically isolated from cells transfected with expanded huntingtin polyq tracts and (2) subjected to mass spectrometry, contained among other components ubiquitin, hsp-70, cell cycle regulators p53 and mdm-2 as well as the general transcription factor tata binding protein (tbp). The transcriptional coactivator and histone acetylase creb - binding protein (cbp) that carries a stable polyq repeat was similarly identified as a component of nis (table 1; fig . 1 and 2). Moreover, proteins that interact with intracellular amyloid seem to participate in an amyloid interactome (table 1). This amyloid interactome is characterized by large proteins rich in predicted unstructured regions that hence may be targeted to amyloidogenic aggregation and depleted from their cellular functions . Based on the identified amyloidogenic candidate proteins affected nuclear functions may include ribosome biogenesis, rna processing, chromatin organization, transcription, and nuclear structure . Evidence for interactions between gene expression, protein aggregation, and altered gene expression has been provided previously; however, a global perspective on the role of such interactions in physiologic settings as well as in disease progression is clearly missing . Systematic profiling of protein aggregate compositions with unbiased systems biology approaches may provide for a promising concept to identify and eventually modulate the intracellular amyloid landscape in health and disease . Besides neurodegenerative protein deposition diseases aberrant fibrillation of proteins to intranuclear inclusion bodies has likewise been observed as a pathological feature in certain systemic autoimmune disorders such as scleroderma, in rodent animal models of xenobiotic - induced autoimmunity and in breast cancer . While these observations currently are of anecdotal nature, they suggest that amyloid protein fibrillation may play a role in other cells than neurons and in more pathologic disease conditions . Besides its disease association mounting evidence suggests that amyloid protein fibrillation also occurs naturally, i.e., in a healthy cellular environment . An increasing number of proteins with no link to protein aggregation diseases have been found to form, under defined in vitro conditions, fibrillar aggregates that have structural properties of amyloid fibrils, including binding to reporter dyes such as congo red and thioflavin t. this is consistent with the notion that the ability to convert into an amyloid structure is a generic property of polypeptide chains and virtually every protein has the potential to undergo amyloid fibrillation . Moreover, due to the advance of respective techniques functional amyloid is being increasingly identified in both, bacteria and eukaryotes . Examples are the mammalian protein pmel17 that forms amyloid structures and thereby accelerates covalent polymerization of reactive small molecules into melanin or peptide hormones that are stored as amyloid - like cross--sheet - rich conformations in pituitary glands . Here, the amyloid fold is clearly used in a non - disease - associated manner, namely in normal mammalian cell physiology . Consistent with this, bioinformatics - based approaches demonstrate that proteins containing stable polyq stretches promote polymerization of functional macromolecular complexes . A global genome survey from whisstock and the bulk of proteins with homopolymeric amino acid sequences, including polyq, perform roles in processes that are characterized by assembly of multiprotein or nucleoprotein complexes and involve rna and dna . Nuclear processes that account for gene expression are driven by dynamic formation and disassembly of macromolecules at the promoters of active genes: proteins containing stable polyq repeats such as creb - binding protein (cbp) and transcription factor sp1, or unstable homopolymeric polyq repeats such as the androgen receptor (ar), glucocorticoid receptor (gr), ataxin-7, and tata - binding protein (tbp) enable assembly of transcriptional initiation complexes (table 1; fig . 2). Here, polyq stretches might act as adaptor motifs that facilitate the formation of the transcription initiation machinery . In line with this, it was shown that homopolymeric glutamine (q)- and proline (p)-stretches activate transcription in vitro when fused to the dna - binding domain of yeast activator protein gal4 . Transcriptional activity correlated with the repeat length and was determined as being maximal with stretches of 10 to 30 q or 10 p residues . Given that polyq stretches seem to be associated with both, amyloid fibrillation in neurodegenerative aggregation diseases, and functional organization of the cell nucleus the question arises which features of polyq repeats contribute to either pathology or biological function . Is there a critical threshold of homopolymeric amino acid stretch length that decides if polyq plays a role in nuclear self - organization or acts as a prion? Indeed, a critical threshold concerning the length of consequent q residues exists in polyq deposition disorders that correlates with disease onset, and severity of disease . This critical threshold has been reproduced in biophysical in silico experiments, saccharomyces cerevisiae and animal models including caenorhabditis elegans, drosophila melanogaster, mice, zebrafish, and non - human primates . Since the length threshold is not uniform and may vary between 3540 q - residues in huntington disease and 2030 q - residues in spinocerebellar ataxia type 6 it is anticipated that the critical length of a polyq repeat is defined by additional context (fig . 2). Context dependency may concern the whole protein context, as well as sequences directly neighboring the polyq stretch . Confirmedly, a polyp repeat flanking the polyq stretches has been identified in huntingtin that acts as a cis - inhibitor of polyq aggregation . In line with results that polyq stretches of 10 to 20 q - residues maximally promote transcriptional activity it is tempting to speculate that shorter homopolymeric q - repeats sustain nuclear processes such as gene expression by their propensity to induce mild protein scaffolding, whereas the intrinsic capacity to form organized fibrillar structures with polyq length above a certain threshold disturbs the functional organization of the cell nucleus by excess fibrillation that culminates in formation of insoluble, amyloid - like protein aggregates in the nucleoplasm (table 1; fig . 1). Such yin and yang of polyq fibrillation may have prohibited evolutionary eradication of unstable, homopolymeric polyq repeats from genomes . Consistent with these ideas is the notion that the nucleus represents a highly crowded environment which on one hand essentially enables the self - organization of macromolecular, multiprotein complexes, however, likewise potentially promotes aberrant amyloid protein fibrillation through inordinate volume exclusion . Along these lines it was shown that xenobiotics such as certain heavy metals and nanoparticles sustain amyloid protein fibrillation, accumulate in specific nuclear microenvironments and locally induce nuclear amyloid . Thus, a fine balance between nuclear crowding and overcrowding may be required to maintain functional protein aggregation . Nuclear protein aggregation and amyloid deposition are a prominent hallmark of neurodegenerative protein deposit diseases . While it was initially thought that nuclear amyloid is in any case responsible for neural cell death, time - resolved experiments that correlate nuclear amyloid and neurodegeneration on the single cell level rather suggest a cell protective role . Aggregation of misfolded or excess nuclear proteins in spherical nucleoplasmic microenvironments may segregate dysfunctional proteins and recruit them for degradation by the ubiquitin - proteasome system . The frequent presence of ubiquitin, proteasomes and heat shock proteins in nis and the discovery of proteasomal activity in amyloid - like nucleoplasmic protein aggregates is consistent with this idea . Confirmedly, the nucleus establishes itself as a major cellular compartment for protein degradation, as it was shown recently in yeast that misfolded proteins are transported from the cytoplasm to nuclear degradation by chaperone sis1p . In addition to a protective role of nis in neurodegeneration evidence accumulates for functional amyloid in the nucleus . Amyloid - indicating features such as congo red and thioflavin t - binding as well as reactivity with amyloid - specific antibodies and peptides can be located in distinct subnuclear microenvironments under physiological conditions . Moreover, 55% of eukaryotic proteins are predicted to contain unstructured protein regions that are intrinsically amyloidogenic . To a certain degree the fibrillation capacity of amyloidogenic proteins may be required for nuclear function that is characterized by dynamic assembly and disassembly of large multiprotein and ribonucleoprotein complexes . An example is provided by the role of homopolymeric polyq repeats in the initiation of transcription . A better understanding of amyloid fibrillation in the nucleus is a prerequisite for promising therapeutic interventions in neurodegenerative protein deposition diseases . Before boosting the protein degradation machinery the function and topology of nis this requires development of methods that allow for the identification of different amyloid fibrillation steps in living cells and whole organisms . Comparative definition of protein aggregation landscapes by systems biology in different disease models as well as under physiological conditions has the potential to provide for global aggregome networks as sources for informed, personalized therapeutic interventions.
Only a few years ago, a paper on mobile learning would have focussed on the uses of a laptop personal computer (1). However, the pace of change in this area of technology means that many students will now not know even what a personal digital assistant is (2). In the recent past, e - learning designers created e - learning content for use on a desktop first and foremost, and subsequently thought about whether or how best to redesign the content for a mobile device . Today, many e - learning designers have developed a mobile first strategy - whereby they create content with a mobile device in mind in the first instance . Indeed, the pace of change has been so transformational that there is now debate as to what constitutes a mobile device or indeed what constitutes mobile learning . If all e - learning can be done on a mobile device, then, perhaps the specific terminology mobile learning should be abandoned altogether . Certainly, mobile learning can range from a formal registered approach to self - learning guided via the free e - courses which are designed in an andragogic way . The purpose of this article is to cover the wide range of resources provided by mobile learning - both formal and informal, and free and paid - for (3). It is also important to remember that learning is fundamentally a process and that mobile technology simply facilitates that process . In this era of rapid change, it is worth pausing and taking stock of the various advantages and disadvantages that are associated with mobile learning . In this short article, i outline these advantages and disadvantages and suggest methods to fully exploit the advantages and minimize the disadvantages . Mobile devices can enable students to learn in any time or location - however and wherever it suits them (5). Often they can learn when they have nothing else to do (when they can dedicate their undivided attention to relevant educational outcomes). These are environments that many educationalists could not have dreamt of a number of years ago - a library of resources available at the learner's discretion and in a physical environment where often they have to be - for example when travelling to college . Software applications (apps) now enable the access of content when the user is off - line . Access to resources is a fundamental first step in learning, and mobile learning enables a step change in this access . A second advantage associated with mobile learning is cost . As with any form of e - learning, there is the cost of hardware and software, the cost of faculty and learner time, and the cost of the physical learning environment (for example the computer aided learning room) (6 7). There is also the cost of the connectivity charge . For many of these components of the overall costs, however, mobile learning enables the educator to strike through certain aspects of the budget . Many students already have mobile phones or tablets, and many of these are already equipped with the necessary software . For students who do not have mobile devices, these can be purchased at low cost and at especially low cost when the purchase is made in bulk - for example, for a class of say 200 students . Medical education is expensive and so savings that can be made by these means will be significant (8 9). Another added value associated with mobile learning is that it enables more situated and contextual learning . This is both integral to the learning and to the technology enabled by mobile devices . For example, a student or postgraduate trainee may have an unanswered questions when on the ward . Mobile access will enable them to answer that question there and then and indeed to put their learning into action there and then - and sometimes to the benefit of patients . Such learning may take seconds, but it is actionable learning and it may be better embedded than learning that takes place in the lecture hall or the library . A fourth advantage is that mobile learning can enable the learner to do other activities while learning . These in turn may be listened too by means of live streaming, or users can download them and listen on an unconnected device . This enables learners to do whatever they like and still listen; they can drive, go to the gym or do housework . Once again, it is an example of technology that puts the learner in control (10 11). Mobile devices can also be used to enable quick, easy and continuous communication and interaction between learner and tutor and between learner and other learners (12). This can happen inside the lecture hall with students answering questions online by means of google surveys or twittering the content of the lectures to those who did not attend . It can also happen outside of the lecture hall with tutors communicating tasks to small or large groups of learners by means of their mobile devices and then learners collaborating amongst themselves to complete the tasks as a team ., it seems that there is a good match between the educational format and the technology . In the format of closed response assessment, a small amount of information can fit on a small screen with a question at the end . The learners can click on the answer that they think is correct and arrive immediately at the correct answer on the next screen . Software applications (apps) can enable banks of questions to be made available when off line . The provision is also often a good fit with learner behaviours - learners do not often wish to spend hours doing thousands of assessment questions - rather they often like doing small chunks of learning by assessment at a time, being able to monitor their progress towards achieving competence (13). Of course, mobile learning is not a panacea; like any other form of medical pedagogy, it has its downsides . One disadvantage of mobile learning is that it is completely reliant on technology and sometimes that technology is inadequate or fails . This can be related to hardware - with insufficient battery life or inadequate screen size (14 15). Memory may be limited or the software on the device may only support a limited number of file formats . As with most technology barriers, all these are being slowly overcome . Batteries have longer lives, internet speeds get faster, and the capacity for memory storage ever larger . There is a risk of distraction from learning by using a device that can be used for multiple purposes, and there is a risk of distraction from the constantly changing environments in which mobile devices are used (16). The advantage of availability can become a disadvantage if pursued too far . Thus, the constant opportunity for learning may mean that some students will never take a break, or will take their learning home with them to the detriment of their personal and family lives . High users of online learning will often have a healthy approach to this use; however, some availability may generate overuse or unhealthy use (17). There is also the potential for breakdown in barriers between personal usage of the mobile device and professional or educational use . For example, what policies should medical schools have regarding allowing their students to use medical school tablets to access personal email or watch films or communicate with their peers by means of social networks (18)? Should they ban this and if so would such a ban be reasonable? Or should they have any policy at all? These potential dilemmas should be thought through before medical education institutions move too quickly into mobile accessible curricula . There is also a risk that educators will become seduced by the technology and forget about the learning . However, it must never be forgotten that the aims and objectives of the learning should guide the methods of learning . It should also not be underestimated that for mobile learning to become useful, there has to be a teacher, a content creator, a supervisor, an online facilitator, in other words, a human resource at the other end . Lastly, any new technology will only work if the end - user can access it . In this regard, there may be a role for simple devices that can run on limited networks . (19 20) despite these caveats, there is no question but that mobile learning offers much potential . People are always learning, whether formally or informally, and mobile technology helps this process . In the future, it is likely that the strategy of mobile first, whereby providers of e - learning think of the user experience on a mobile first, will result in learners who increasingly expect that all e - learning provision will work seamlessly on a mobile . The issue of what constitutes mobile learning will also likely become less controversial as e - learning providers work towards device - responsive design where any learning content will work on any device in any place and at any time.
Hemophilia is an x - linked disorder and rare chromosomal disease that leads to excessive bleeding in men . The existence of the inhibitor causes severe complications in patients with hemophilia a (2). To treat patients with hemophilia resistant against factor viii, recombinant activated factor vll (rfviia) or activated prothrombin aryoseven is a biogeneric of rfviia marketed in iran which has recently shown a comparable efficacy with that of novoseven (4) and is the available rfviia product used to manage bleeding in hemophilia patients with inhibitors in iran aryoseventm [package insert]. On the other hand, administration of bypassing agents in patients with high titre inhibitors for bleeding episodes is very costly (5). Therefore, economic analysis of available bypassing agents could provide useful data to select eligible alternative therapeutic strategies to treat patients with haemophilia (6, 7). Although most of the studies show that bypassing agents are effective, none of them is permanently effective (8). The current study aimed to evaluate the cost - effectiveness of bypassing agents to treat patients with haemophilia and high titre inhibitors through decision analytic model, according to the iranian health care system . Considering the nature of the disease and small size of the study population, an open - label, non - randomized, cross - over and multi - center study was designed and performed in five hemophilia centers in iran . The hemophilia a patients with high titer inhibitors (5 bethesda units) were selected for the study.each patient was treated and evaluated for two separate bleeding events: one with aryoseven based protocol (protocol a) and the other one with feiba based protocol (protocol f). As part of the study to detect and evaluate the influence of tendency of the patients toward different types of bypassing agents, patients were categorized into three groups: group a (patients tending to use aryoseven), group f (patients tending to use feiba), and group o (patients indifferent to receive either product). Both protocols a and f were evaluated in all of the patients . Given that the current study was done based on the study by fenoc (2); hence, the subjects were selected in accordance with the inclusion criteria for the patients in his study . Patients with other clotting problems, advanced liver problems for short life expectancy, those who received blood products within five days prior to the test, and the patients with joint bleeding in the joint under study within seven days prior to their admission for the treatment were excluded . After categorization the patients were evaluated according to two bleeding events . According to the scoring scale of the patients in the study by kavakli et al . (9), the therapeutic protocol was employed in the first two hours after the onset of the pain and the subjects ` clinical symptoms and the responses were recorded after 1, 3, 6, 9, 12, 18, and 24 h of the product infusion . Protocols a and f (below section) were respectively employed in all groups after a bleeding event . Case report form (crf) was completed by the specialists to compare efficacy of the procedures in each bleeding event . Therapeutic protocols according to the injection protocol of the two products, in protocol a (treatment with aryoseven), all subjects received 90 - 120 g / kg body weight on arrival (time 0); 100 g / kg was considered as the mean dosage (2). Based on the subjective responses a specific therapeutic decision was made every three hours . In case of two failures with 90 g / kg, a dose of 270 g / kg was used . If no improvement was observed three hours after the next 270 g / kg dosage, it was considered as treatment failure for non - responders . In protocol f (treatment with feiba), all subjects received 50 - 75 u / kg body weight on arrival (time 0). Next dosage (75 - 100 u / kg) was injected after nine hours only if no relieving sign was observed . When there was no response, it was considered treatment failure . Effectiveness of therapy in patients a scoring system was used to determine the effectiveness of the applied therapies (table 1). Similar scoring systems had been employed in other studies for clinical assessment of efficacy (9). Two outcomes of pain and the extent of the movement limitation were evaluated in all subjects . If pain and/or movement limitation improved significantly, the score + + was recorded . But if they were worse or even not better, the score was recorded as -, except for hours 1 and 3 of assessment when score+ was recorded if no changes were observed in the symptoms of the subject . Evaluation of the patients ` clinical condition therapy success and therapy failure scores 16 + out of 28 were considered as therapy success . Scores less than 16 + and/or clinical necessity to switch or add another bypassing agent was considered as therapy failure . Determination of treatment cost to collect data concerning cost, a table was developed based on the previous studies conducted in iran or other countries . All direct medical and non - medical costs were collected . Hence, direct non - medical costs such as transportation cost were included . Therefor indirect and intangible costs were ignored (table 2). Ethical considerations the ethical committee of shahid beheshti university of medical sciences approved the current study, based on helsinki declaration . The present clinical trial was registered in irct website, under i d no.2013020612380n1 . Decision analytic model to provide an optimized strategy to treat hemophilia patients with inhibitors, a decision analytic model was designed . Data regarding the effectiveness of therapeutic protocols and also the related costs were transferred to the decision tree model . The tree age pro 2011 software was used in the study for modelling (10). To calculate the cost of medicines, basic prices extracted from the iranian food and drug organization (fdo) were used, since it is an official drug pricing reference in iran . One - way and two - way sensitivity analyses were used to assess the robustness of the results . A frequency domain for sensitivity analysis was 25% increase / decrease of the baseline results . According to the central bank of iran the official exchange rate was us$1 = 25,430 rls . 16 out of 36 subjects were excluded due to refusing to stay in the protocol and lack of cooperation and protocol deviation during the treatment . Hence, clinical and cost data on 20 patients with 40 bleeding episodes were extracted . Effectiveness scores are shown in table 3, based on the type of the received protocol in each time point of assessment . Effectiveness scores of patients with haemophilia a for feiba and aryoseven statistical relationship between the variables to statistically analyze the therapy success correlation, the mean of dosage, number of patients with treatment success and also effectiveness scores were used based on the applied therapeutic protocol . To assess the normality of data the kolmogorov - smirnov and shapiro - wilk tests were used . Based on normal and non - normal data, statistical parametric and non - parametric tests were used when the distributions were normal and non - normal, respectively . To determine the relationship between treatment success of protocol a in the groups, chi - square and the fisher exact tests were used . The obtained results showed no statistically significant differences between the groups receiving protocol a (p = 0.53), however group f had statistically significant success with protocol f, compared to the other groups (p = 0.03). Besides, the number of successes with each protocol was statistically equal in all of the groups (table 4). Although mean dosage of product in protocol a seems to be more in group f and o compared with group a, there were no statistically significant differences between protocols a and f in the treatment groups (p - value in protocols a and f were 0.18 and 0.19 respectively table 4 . Due to abnormality of data, the statistical relationship between the effectiveness scores of protocol a was evaluated by non - parametric kruskal - wallis test (p = 0.31). Mean effectiveness score with protocol f was observed in group f (p = 0.01) but there was no difference between the effectiveness of the protocols in groups a and o (table 5). No statistically significant difference was observed between the effectiveness scores of the subjects receiving protocols a and f (table 5(.several tests showed no statistically significant difference between the carry - over effect on the variables . Treatment success and mean dosage of products with each protocol in the treatment groups effectiveness score of protocols in different groups of patients cost - effectiveness analysis results of the cost - effectiveness analysis showed that administration of 90 g kg- aryoseven as the first line treatment with the cost of us $2,912 and 72% therapy success in one bleeding episode was more cost - effective compared to administration of 50 - 75 g kg- feiba as the first line of treatment with the cost of us$3,785 and 89% therapy success (table 6 and figure 1). The incremental cost - effectiveness ratio analysis (icer) was us$5,146 to manage an episode of bleeding to get one more unit of effectiveness using feiba vs. aryoseven as the first line of treatment . Although the results of the study confirmed cost effectiveness of aryoseven compared to feiba, it should be mentioned that according to table 6 these strategies are undominated . Cost - effectiveness rankings between the two strategies cost - effectiveness analysis between aryoseven and feiba sensitivity analysis to evaluate the results of sensitivity, one - way and two - way sensitivity analyses were employed . Results showed that the level of icer was sensitive to aryoseven cost changes in different hours, with more sensitivity at the early hours of treatment . Also, a one - way sensitivity analysis indicated the sensitivity of icer to the changes in feiba cost, which was in maximum and minimum in the first and ninth hours of treatment, respectively (figure 2). One - way sensitivity analysis between cost of feiba (cfhemodrugt1ni) and icer at 1-hour time point two - way sensitivity analysis was also performed to evaluate the sensitivity to the changes of variables such as medicine cost and effectiveness (figure 3).the results showed that with increase of the effectiveness or decrease of feiba cost within the first hour of treatment, feiba can be the cost - effective strategy, but in the area with the increase of feiba cost, aryoseven was more cost - effective . Two - way sensitivity analysis of cost and effectiveness of feiba (p_ft1) at 1 h time point the current study was the first pharmacoeconomics study to compare the cost - effectiveness of bypassing agents in hemophilia a patients with inhibitors through clinical trial and decision analytic model in iran . It was previously reported that bypassing agents are effective to control bleeding episodes in patients with haemophilia and inhibitors (11 - 14). In recent years several biogeneric medicines received marketing authorization from iran national regulatory authority (15). However, it is important to find out their cost effectiveness compared to the other brands in the market . The current study aimed to evaluate the cost - effectiveness of aryoseven-a biogeneric rfviia in iran market- compared with that of feiba through clinical trial and decision analysis model . Cost of medicines, applied dosage, and also therapy success in the first line of the treatment were among the most important elements in the economic analysis, similar to other studies (16). In the study by odeyemi et al . On bypassing agents modelling, in addition to medicine costs, hospitalization and transportation costs were also considered; considering the short time to control bleeding with rfviia compared to apcc and less cost, it was generally less costly (17). However other studies such as the one by fenoc (2) were also performed as an open label study . It should be noted that the impact of patients ` prior tendency to the type of products on the final effect of drugs, helped us to partially overcome the lack of blinding . Accordingly the results showed that prior tendency had no influence when the patients were treated with aryoseven but it did with feiba, which may be due to their different attitudes towards original and generic products of rfviia . As mentioned in the previous sections and according to the review by golestani et al . (18) although both drugs were effective none of them was permanently effective to treat joint bleeds . According to the results of the current study, the effectiveness of aryoseven and feiba were 72% and 89%, respectively . However aryoseven was a more cost effective medicine compared to feiba to manage bleeding in patients, although both strategies were undominated . The results of the current study were in line with those of the studies in which rfviia was introduced as the most cost - effective option comparing medicinal costs regarding bleeding episodes in patients with haemophilia a with bypassing agents in the first line of the treatment (19,20). Although the results of the remodelling showed that if the price of feiba reduced by 25% it will be a more cost - effective strategy . To determine the average cost per bleeding, stephens et al . (1) expressed that rfviia with us$28,076 had the lowest cost in the third line treatment compared to apcc in the first line of treatment due to unnecessary use of rfviia in the second and third line treatments . However, there are other studies which showed cost - effectiveness of feiba (3, 6). Different results may be due to different local cost of products, different dosage, and protocol of treatments and difference in assessment methods . Although the obtained results showed the cost - effectiveness of aryoseven, both strategies were undominated . On hence, both medicines can be applied in the first line of the treatment if the cost of feiba was reduced . Results of the current study can be considered for healthcare policy makers to select the best therapeutic strategies to be applied in the first line of treatment of patients with haemophilia a and inhibitors . It is important to emphasize that based on the results of the current study, due to higher efficacy of feiba, policy makers could decide to reduce feiba price to the extent that both medicines would be equally considered cost - effective . In this case competing interests this study received a research grant from the aryogen biopharmaceutical company, without any role or influence on design, administration, analysis, writing and submission of this article . Some of the authors received travel grants from baxter, csl, octapharma, novo nordisk, and aryogen companies . Authors' contributions study design, data collection and design of decision analysis model: mg; clinical trial design and evaluation of treatment effectiveness: pe; analysis of the cost - effectiveness and icer ratio: amc; all data supervision and study design review: hrr; review of clinical trial and data: js; in clinical trial contribution: rh, bh; evaluation of the effectiveness of the treatment: mn, mrm, hh and ght, review of the model and data analysis: ai; statistical analysis conduction: mtk.
The doses to critical organs of known sensitivity to the stochastic effects of radiation must be measured . The highest risk in dental radiography are for leukemia and cancer of the thyroid, even in doses as low as 500 msv . The aim of this study was to calculate the average surface radiation dose to the thyroid gland and gonads during full - mouth intraoral periapical (iopa) and maxillary occlusal radiography . The various materials used were routine diagnostic instruments, namely rubber bands, pocket dosimeter, x ray machine explor model, ektaspeed plus iopa films, ectaspeed plus occlusal film, and thyroid collar [figures 1 and 2]. Pd4507 [table 2, figures 3 and 4] with a digital display manufactured by bhabha atomic research centre, mumbai, maharashtra, india was used to measure the surface radiation dose . It also had the facility of depicting cumulative doses which made calculations of doses less than 1mr (10 gy) possible . This pocket dosimeter had the capacity to measure the maximum level of radiation upto 999mr (9.99 mgy). Other technical specifications are shown in table 1 specifications for a pocket dosimeter pocket dosimeter pd - 4507 pocket dosimeter with a charger besides, this pocket dosimeter had an added advantage over other conventional dosimeters in that the measured radiation dose was expressed in digits, which ruled out chances of error in reading, unlike the pen dosimeter, where the reading was to be read against a meter which gave room for error in visual reading . However, like the pen dosimeter, the pocket dosimeter also made use of a geiger - muller counter . The subjects for the study were average adult patients of age group ranging from 16 years to 65 years . The x ray machine used was explor model from confident dental equipments, with standardized 65 kvp and 10 ma . The films used were kodak ektaspeed plus iopa films of size 31 41 mm (1 1 inches) and kodak ektaspeed plus occlusal films of size 57 76 mm (2 3 inches). The patient was made comfortable and asked to sit on the dental chair . To measure the surface radiation dose at the thyroid level during full - mouth iopa radiography, the patient was positioned such that the mid saggital plane was perpendicular to the floor . For the maxillary five film series, the head of the patient was adjusted such that the maxillary occlusal plane was parallel to the floor . The x ray cone angulation was set at + 40, + 45, + 30, + 20 degrees for maxillary iopa films for anterior teeth, canines, premolars, and molars, respectively . For the mandibular five- film series, the head of the patient was adjusted such that the mandibular occlusal plane was parallel to the floor, upon opening the mouth . The x ray cone angulation was set at -15, -20, -10, -5 degrees for mandibular iopa films for anterior teeth, canines, premolars, and molars, respectively . The dosimeter was placed at three levels, namely the isthmus, right lobe, and the left lobe [figures 57] and the average dose was calculated . The total number of subjects taken amounted to 60, 20 each, for the three levels, namely the right lobe, the left lobe, and the isthmus . Dosimeter at the isthmus level of thyroid gland during full - mouth iopa radiography dosimeter at the right lobe level of thyroid gland during full - mouth iopa radiography dosimeter at left lobe level of thyroid gland during full - mouth iopa radiography to measure the surface radiation dose at the thyroid level during maxillary occlusal radiography [figure 8], the head of the patient was adjusted such that the mid saggital plane was perpendicular to the floor . The head of the patient was positioned such that the maxillary occlusal plane was parallel to the floor . Dosimeter at the thyroid gland level in maxillary occlusal radiography the x ray cone was angulated at + 65 degrees and positioned such that the central rays passed between the eyes through the base of the nose . The dosimeter was fixed in place as mentioned previously and doses were recorded at the thyroid level on the same patient . This procedure was done on a total of 20 subjects . To measure the surface radiation dose at the gonadal region [figure 9], during a 10 film full - mouth iopa radiography the dosimeter was placed in the center between the thighs, as close to the gonads as possible . Dosimeter at the gonadal region in full - mouth iopa radiography to measure the surface radiation dose at the gonadal region during maxillary occlusal radiography [figure 10], the patient was positioned as mentioned before and the dosimeter was placed on the center of thighs, as close to the gonads as possible . Dosimeter at the gonadal region in maxillary occlusal radiography after each reading, the dosimeter was reset and used for another subject . The digital readings were recorded, tabulated, and the average dose for single patient was calculated . The surface radiation doses observed at the isthmus level, at the level of the right and left lobe of the thyroid gland during a 10 film full - mouth iopa radiography, were 6.3 mrads, 13.9 mrads, and 12.6 mrads, respectively, and cumulative dosage was 10.93 mrads . The surface radiation dose at the thyroid level during maxillary occlusal radiography was 0.4 mrads . [tables 35] total number of subjects average surface radiation dose in full - mouth iopa radiographs(10 films) average surface radiation dose in maxillary occlusal radiography the surface radiation dose at the gonadal region during 10 film full - mouth iopa radiography and maxillary occlusal radiography was 0.4 mrads and 0.15 mrads, respectively . Dental radiographs and chest radiographs are the most common radiographs taken for routine investigations . Radiation exposure to patients in dental radiographic examination has been investigated time and again . Many of the previous studies were based on based on methods or conditions that do not provide good bases for comparison . Most of the dose determinations have been done with phantoms, mannequins or, with cadavers . No real effort has been made to determine the actual tissue - equivalence of dental phantoms as compared to living subjects . In 2004, mortazavi et al estimated the entrance surface doses (esd) to skin at mandibular or maxillary arcs using tld badges to be 1.1730.606 mgy . The use of pocket dosimeter in our study makes possible precise measurements of surface radiation dose in living subjects and rules out the chances of error in visual reading . Until 1982, the exposure dose to the thyroid gland in full - mouth examination using iopa radiography still remained as high as 49 mrads (julian gibbs). Similarly in 1980, danforth and gibbs reported the radiation dose to the thyroid using 20 iopa films to be 400 gy to 920 gy, and to the parotid gland to be 130gy to 480gy . In our study, during full - mouth iopa radiographic survey using 10 films, radiation exposure dose to the thyroid gland was calculated as 10.93 mrads and the average radiation dose at the thyroid level during maxillary occlusal radiography was calculated as 1.5 mrads . In 1982, julian gibbs et al calculated the gonadal dose from a full - mouth iopa survey to be 2 mrads . Similarly in 1993, tarnjit saini et al found the radiation dose to the male gonadal region to be 0.8 mrads using 20 iopa films . In our study the surface radiation dose at the gonadal region during a 10 film intraoral full mouth radiographic survey and in maxillary occlusal radiography was found to be 0.4 mrads and 0.15 mrads, respectively . To conclude, the surface radiation dose to the thyroid gland and the gonads was evaluated during a 10 film full - mouth iopa and maxillary occlusal radiography using a digital pocket dosimeter pd4507 . This study may be beneficial for the dental professionals worldwide to undertake proper precautionary measures to limit the radiation dose and allay the fear among the patients regarding the dental radiography, if any.
When the live poultry market surveillance program began in september 1999, eight of a total of 80 live poultry markets were selected to represent the 3 major regions of hong kong: hong kong island, kowloon, and the new territories . Since then, the number of markets has declined, and by may 2011, only 5 of the 8 selected live poultry markets continued in operation (of a total of 39 operating live poultry markets). We previously published data on the effect of introducing various interventions in live poultry markets, which included the ban on the sales of live quail and the introduction of rest days (7). In addition to collecting fecal droppings from the cage floors for virus isolation, we collected data on the total sales of chickens and minor poultry, the proportion of chickens imported as a ratio of the whole, the temperature and relative humidity, and the type of ventilation used, as described (7). Samples collected in virus transport medium were inoculated into 911-day - old embryonated eggs, and allantoic fluid with positive hemagglutination was confirmed and subtyped using standard antiserum . Because hpai a virus (h5n1) is rarely detected in live poultry markets in hong kong, we used isolation rates of influenza a virus (h9n2) as an indicator of the effect of these interventions on avian influenza virus circulation . The median numbers of samples collected weekly from chickens and minor poultry were 107 (range 3722) and 23 (range 1397), respectively (see technical appendix for weekly numbers of samples). The poisson generalized model (8) with influenza virus (h9n2) weekly isolation counts as the outcome variable was fitted as described (7) and adjusted for proportion of chickens imported; total sales of chickens and minor poultry; ventilation system; weekly average temperature; relative humidity; seasonal variations; sample size; and periods corresponding to the respective interventions: period i (no rest day), ii (1 monthly rest day with quail being sold in the live poultry market), iii (1 monthly rest day with elimination of live quail from the live poultry market), iv (2 monthly rest days), and v (ban on holding poultry overnight in live poultry market). These variables were considered potentially important confounders related to transmission efficiency of avian influenza virus (9), source, type, and volume of poultry . Weekly virus isolation counts were analyzed from september 22, 1999, through may 31, 2011 . A separate model for poultry and minor poultry was fitted, and all analyses were implemented by using r version 2.12.1 software (r development core team, vienna, austria). The figure shows overall isolation rates by week for chicken and minor poultry from 1999 through 2011; the table gives the parameter estimates for the final fitted models, which were adjusted for the effect of covariables that could affect the isolation of influenza in the study . For chickens and minor poultry, compared with the reference category of 2 monthly rest days, the ban on keeping live poultry overnight in live poultry markets was associated with dramatic and significant reduction of influenza virus (h9n2) isolation . The isolation rate of influenza virus (h9n2) among chickens declined 84% (adjusted relative risk 0.16; p = 0.006), and no influenza subtype h9n2 viruses were isolated from minor poultry after the ban on holding poultry overnight in live poultry markets was implemented . Higher volume of minor poultry sales was also significantly associated with higher isolation rate of influenza virus (h9n2). Weekly influenza virus a (h9n2) isolation rates for chickens (a) and minor poultry (b) in live poultry markets, hong kong, september 1999may 2011 . Vertical lines denote periods for different interventions: i, no rest day; ii, 1 monthly rest day with quail sold in live poultry markets; iii, 1 monthly rest day with no sales of quail in live poultry markets; iv,: 2 monthly rest days; v, ban on keeping live poultry overnight in live poultry markets . * reliable confidence interval cannot be estimated because of zero isolation of influenza virus (h9n2) from minor poultry after introduction of a ban on keeping live poultry overnight in live poultry markets . The seasonality coefficients and contribute to the estimated isolation rate in week t via the terms cos(2t/52) + sin(2t/52). A previous study that used a stochastic metapopulation model showed that frequent rest days in live poultry markets were effective for reducing transmission of avian influenza (h5n1) (10). Our findings show a large additional decline in the influenza virus (h9n2) isolation rate after implementation of a ban on keeping live poultry overnight, which suggests that this intervention has an even greater effect on reducing viral load in live poultry markets than the previous intervention of 1 or 2 rest days per month . While low pathogenic influenza virus (h9n2) was the indicator virus in our study, it is likely that these interventions would have comparable effects on highly pathogenic viruses such as avian influenza (h5n1); this effect has been demonstrated by mathematical modeling (10). Studies by others on social network analysis have shown that daily cage cleaning and disinfection of live poultry markets in southern china (11), and protective factors including removal of waste in indonesia (12) contributed to a reduction of hpai (h5n1) in live poultry markets . Taken together, these studies show that eliminating the carryover of live poultry in markets from one day to the next, in the form of rest days or a total ban, is highly effective for reducing viral amplification and persistence in live poultry markets and consequently minimizes zoonotic risk . Weekly number of samples and influenza virus (h9n2) isolation rates for live poultry markets, by poultry type, hong kong, september 1999013;may 2011.
Endoscopic internal drainage (eid) has been recently introduced for treatment of leaks and fistulas following bariatric surgery . Eid may be applied both as first - line treatment and as rescue therapy 1 2 . Fistulas following gastrointestinal surgery both for benign and malign pathologies remain a major clinical problem . Such complications account for significant morbidity and a mortality rate that can reach 60% if treatment is delayed 3 . Here we report our experience with eid as first - line treatment of fistula following different types of gastrointestinal surgery . To our knowledge, this is the first case series in which eid has been used systematically for fistulas following both upper and lower gastrointestinal surgery . From march 2013 to november 2015, 11 patients were treated at our endoscopic tertiary center for fistula following gastrointestinal surgery . The conditions with which these patients were diagnosed and procedures that they had undergone are as follow: four duodenal fistulas (biliopancreatic surgery), two colic fistulas (colorectal surgery), and five esophagogastric - jejunal fistulas (foregut surgery). All patients were referred to our endoscopy department by the surgical team performing the surgical procedure . Delay between first surgery and eid averaged 14 days (range 3 60). All patients presented either clinical evidence of sepsis (leukocytosis, fever, elevation on polymerase chain reaction) or output of enteric fluid from the drainage or medium contrast extravasation at swallow study / pelvic computed tomography scans . Opacification of fistula was always performed during endoscopic examination in order to evaluate the fistula opening, the collection, its shape and size . Endoscopic evaluation was of paramount importance to correctly decide the appropriate diameter and length of pigtail stent and its number . As shown in video 1, whenever feasible, we explored the cavity with the endoscope to clean and to perform necrosectomy (as reported by lemmers et al.4) if necessary . A duodenoscope or colonoscope was used because of the larger operative channel if a 10 fr pigtail stent was needed . We believe the key to success for treatment of gastrointestinal fistulas is to accomplish complete internal drainage of any collection and to induce granulation tissue formation by reaction to pigtail, considered as a foreign body, in order to promote healing . Video 1 endoscopic inspection of an esophago - jejunal anastomotic dehiscence of half the circumference with a concomitant mediastinal collection . To simultaneously achieve both aforementioned goals, a tandem catheter (boston scientific, massachusetts, boston, usa) was used to cannulate the defect and after guidewire insertion, one or more double pigtail stents (advanix boston scientific, massachusetts - boston - usa or visio g - flex europe, nivelles - belgium) were deployed (video 2). As a general rule, if an external drainage is present (surgical or radiological) the goal of eid is to reverse the drainage direction in order to remove early external drain, so a short stent usually is sufficient if no external drainage is present; one or two longer stents must be deployed to achieve complete cavity drainage . Video 2 endoscopic internal drainage with deployment of two double pigtail stents to treat an anastomotic dehsicence following total gastrectomy . In five of 11 patients a jejunal feeding tube was left in place . Systematic control was performed at 4 to 6 weeks in order to evaluate the evolution of the collection and to modify stent size according to the reduction of the cavity . A check endoscopy is important not only to evaluate treatment progression in order to adapt internal drainage, but also to promote healing by inducing trauma in the pseudocavity with the exchange of the double pigtail stents . After the first check endoscopy, oral diet resumption was allowed in four of five patients . Technical success was defined by achievement of all the following steps: visualization of fistula orifice, opacification of fistula and collection, selective catheterization, and deployment of the double pigtail stent . Technical success was achieved in all patients (100%). However, the day after the procedure, one patient presented with a perforation . This complication was not detected during the procedure and emergency surgery was required for esophago - jejunal anastomosis fistula . Clinical success was defined as follows: no signs of sepsis after oral diet resumption, no extravasation of medium contrast at follow - up procedures (endoscopy, ct scan, barium enema) and absence of enteral liquid output from the external drainage / fistula tract . Seven patients were found to be healed at first check endoscopy after an average of 35.6 days of treatment (range 28 58) (fig . 1); one patient was healed at the second check (60 days) and the last patient was healed after three endoscopic sessions (90 days). We reported one clinical failure consisting of one patient still under treatment after 570 days . That patient underwent total gastrectomy for t3n + gastric cancer and developed an anastomotic dehiscence of half of the circumference with a concomitant mediastinal collection after 11 days (video 1 and fig . 2). After three endoscopic sessions and two stent replacements (90 days), there was no extravasation of contrast medium and the stents were removed . Ct scan again showed a 10-cm mediastinal collection due to an anastomotic fistula, and eid therefore was repeated (fig . The span of time between endoscopic sessions was extended to 8 weeks instead of 4 weeks and he was allowed an oral diet . Endoscopic view of esophago - jejunal anastomotic fistula with surgical drainage in place (red arrow). B radiological view of esophago - jejunal anastomotic fistula with surgical drainage in place (yellow arrow). F healed fistula with no extravasation of medium contrast (yellow arrow). Radiological visualization of perianastomotic collection . Table 2 lists the type of scope used, the number of pigtail stents deployed, the number of overall and average endoscopy sessions, feeding method, and outcomes p: positive; n: negative; ut: under treatment . If present, the external drainage tube was removed before the first check endoscopy (4 6 weeks after start of eid). Overall we achieved clinical success in nine patients after an average of 44 days (28 90) treatment with a median of 2.3 endoscopic sessions (2 4) and deployment of 1.8 pigtail stents per patient (1 4). The overall clinical success rate was 82%, with eid failure in two of 11 patients (18%). No stent - related bleeding or stent migrations inside the abdominal cavity were observed in the study . At a median follow up of 291 days (10 leak and fistula following gastrointestinal surgery are usually treated minimally invasively with drainage tubes, oral alimentary stream diversion, and/or plugging . The most common approaches are insertion of temporary covered self - expandable metal stents (csems); clipping; plugging; gluing; and vacuum therapy 5 6 7 . Overall clinical success rate varied greatly among these different techniques without assurance of optimal results . Moreover, the rate of success decreases with the interval between initial surgery and endoscopic treatment . The most common treatment is with deployment of csems, but they are burdened by a high rate of migration due to altered anatomy and absence of stenosis coupled with the physiologically large diameter of the gastrointestinal tract . These factors do not allow the csems to guarantee watertight exclusion of a leak . Even if the stent remains in place, fluids pass in the space between the external sheets of the stent and the wall of the gastrointestinal lumen . Endoscopic internal drainage of intraabdominal collection was first used in the 1980 s by biliopancreatic endoscopists to treat pancreatic pseudocyst . One or several pigtails were introduced under endoscopic vision, puncturing the bulging collection in order to perform an anastomosis between the cavity and the gastrointestinal tract . Later, thanks to the advent of interventional endoscopic ultrasound, we had the opportunity to drain and create anastomosis between an abdominal collection that was neither bulging nor endoscopically visible and the gastrointestinal tract . Today newly designed, lumen apposing, fully covered self - expandable metal stent (la - fcsems) may be used for this purpose 8, enabling the emptying of the collection with resolution of this virtual third space . The wall defect (fistula - leak orifice) is already present . In our previous studies we demonstrated that a pigtail stent acts as a foreign body on the edge of the leak and inside the cavity, promoting its reepithelialization 1 9 10 . The new concept in our series is that eid can be used to internally drain perianastomotic fluid collection all along the gastrointestinal tract (esophagus, duodenum, colon). Application of pigtail drainage is feasible even in the colon and rectum, as shown in an interesting article by calzolari et al . We decided to systematically evaluate each patient after 4 to 6 weeks because pigtail stents have a small caliber and can easily get blocked . More importantly we believe that the stent being a foreign body promotes granulation tissue formation, as demonstrated by spontaneous double pigtail migration (fig . 4). D endoscopic view of a double pigtail stent migrated in the duodenal lumen 28 days after eid treatment . It is well known that early management of fistula should always be advocated because it correlates with higher clinical success rates . Therefore, we advocate eid as an early first - line treatment . Although eid often requires multiple endoscopic sessions, it is cost - saving compared to sems . Eid should be considered as an effective first option for leaks throughout the gastrointestinal tract . Eid is a valuable and efficient technique not only for leaks following bariatric surgery but even for leaks and fistulas following other type of upper and lower gastrointestinal surgery . As long as eid can achieve access to the fluid collection, internal drainage can be achieved all along the gastrointestinal tract.
All patients are subjected to pre - operative evaluation before surgery . The importance of pre - operative assessment and laboratory tests based on clinical examination has been well reviewed . Patients are classified into one of the 5 grades according to the american society of anesthesiologists (asa) physical status . However, it has been realised that many of the investigations are of minimal benefit . Many studies have questioned the need for these investigations . All the available literature which has addressed the issue of pre - operative tests and its cost implications have come from the developed countries and there are few such studies in our population . With raising costs, the health - care industry is looking to minimise additional expenses incurred through these investigations, and thus, it becomes prudent to be judicious in requesting for laboratory investigations . We studied to evaluate the additional cost implications of pre - operative investigations in patients scheduled for elective surgery compared to the currently available guidelines . A prospective study was undertaken between june and august 2011 at a tertiary care hospital . One hundred and sixty - three patients of either gender scheduled for elective procedures in the study period were included . All laboratory investigations ordered and the instrumental tests (electrocardiogram [ecg], echocardiogram [echo], chest x - ray [cxr]) performed were also noted . All values were categorised as normal or abnormal based on the reference values of the laboratory . Based on the national institute of clinical excellence (nice) guidelines, surgical grades were recorded and also asa physical status defined as per guidelines . Based on the guidelines, the tests were considered as required or not required . Any specific tests done on instructions by the anaesthesiologist or patients comorbid conditions were categorised as indicative tests . Any abnormal ecg with clinical correlation of signs and symptoms were further subjected to echo to determine the severity of the cardiac disease . Cost analysis was done for every test which was considered not necessary based on nice guidelines . A total of 163 patients of either gender scheduled for elective surgical procedures were prospectively evaluated . The missing data were of ecg, complete blood count (cbc) in one patient each, cardiac echo in two patients and international normalised ratio and blood grouping tests in five patients . The majority of the patients were adults and in asa physical status 1 and 2 . The nature of surgical procedures performed were equally distributed between grades 1 and 2 (40%) and grades 3 and 4 (47%). A total of 720 laboratory tests and 264 instrumental tests were done . Out of the 163 patients only 43 patients were subjected to laboratory tests as per the guidelines which translate to 26% . Out of the 984 tests done 515 tests were not indicated . The total number of tests which were found to be abnormal when performed in unindicated cases were seven, amounting to only 1.1% . Out of these seven tests, cxr was found to be abnormal in two patients . Both these patients underwent major surgical procedure (surgical grades 3 and 4) without perioperative events . Both these patients were young, clinically fit in asa physical status 1 and were incidental findings . This was neither correlating with the underlying disease nor the clinical condition of the patient . The most common investigation considered as routine was cxr irrespective of the age and the nature of the procedure . Out of 88 (55%) patients who had cxr, 82 (93%) were not indicated . In 75 (45%) patients where cxr was not done grouping and cross matching was done in 46% (39/84) of patients where expected intraoperative blood loss was minimal . Cost analysis was performed for unindicated tests and the savings for the patient and resource for the hospital calculated . For the 515 (52%) unindicated tests, the total cost amounted to rs . 171,358 . The major cost was towards performing echo and cxr accounting to more than 56% of the total cost [table 3]. Laboratory tests and instrumental tests the cost analysis considered as indicated and unindicated tests we observed that many patients were referred to the cardiologists for cardiac evaluation in patients scheduled for surgical procedure based on the age and nature of the procedure . Echo was not indicated in 74 out of the 81 patients (92.5%) as per guidelines . The present study results indicate that unnecessary investigations could be reduced significantly by good pre - operative evaluation of patients, with no loss of clinically relevant information and patient care . It is necessary to identify the risk factors and request for investigations based on the patient comorbid conditions and nature of surgery . Guidelines help to have a structured, patient directed, evidence - based approach for workup of these patients scheduled for surgery . Only 26% of patients were subjected to laboratory tests as per the guidelines, and 52% of the tests were not indicated . The total number of tests which were found to be abnormal when performed in unindicated cases were only seven . A cost analysis study showed a reduction of 63% of cost per patient by applying their institute guidelines . Similarly, in another study, selective ordering of investigations by the anaesthesiologists significantly reduced the number of tests and the cost by almost 25% and reduced by 41% if consultant assessed and ordered for the tests . The present study had a similar estimation of 63% cost reduction by applying the nice guidelines . The estimated saving in costs that was calculated refers to the application of the nice guidelines in our patient population . However, this may not be an ideal way of interpreting our data and draw conclusions . Compared to the developed countries, developing countries have limitations with respect to the access to health care . Awareness about the health issues is limited to education and affordability with the urban gaining advantage over rural population . Thus, in most instances what has been termed as the inappropriate test may be an appropriate test in the given situation . It has been defined as not indicated for few tests based on the population studied and abnormal results reported . The incidence of diabetes is on the rise, and the clinical scenario is changing . Based on this, blood glucose estimation, what is termed as unnecessary in the guidelines in select patients may be appropriate in our group of patients wherein they are evaluated for the 1 time for any pre - existing disease . Hence, this becomes an appropriate test in our population . Thus, a 52% reduction in the number of tests would lead to optimal utilisation of the hospital resources . The indirect additional cost to the patient in the present analysis was accounted towards cxr, coagulation profile and cardiac echo . We looked into the cardiac risk and pre - existing comorbidities and the need for cardiac echo testing . Based on these, we found more than 90% of the time echo requested by the physicians or surgeons was inappropriate leading to unnecessary testing and additional cost . Studies have looked into the need for the routine ecgs and chest radiographs . In a large study involving 6111 patients undergoing elective surgery, they found 18.3% were abnormal out of which only 5.1% (313 patients) were considered as useful which altered anaesthetic management . The presence of respiratory disease and age> 60 years with asa class 3 or more contributed to the probability of usefulness of the test . Similarly, routine ecg screening for patients with no cardiovascular risk added very little to predict perioperative cardiac complications . Inappropriate pre - operative tests can lead to a false - positive results, causing unnecessary delay in surgery and also add to patient anxiety . In two of our patients where ecg and echo were found to be abnormal neither required intervention nor influenced on the anaesthetic management in the perioperative period . In a report on the need for pre - operative ecg and its usefulness, the author suggested that clinical indicators like age more than 45 years, cardiovascular and respiratory diseases and surgical severity, should guide for pre - operative screening . We observed that many patients were referred to the cardiologists for cardiac evaluation in patients scheduled for surgical procedure based on the age and nature of the procedure . The need for this instrumental investigation was determined by the nature of the clinical status of the patient and also the severity of the existing comorbidities . Of the 81 patients who had echo, 74 patients (92.5%) echo were neither clinically indicated nor had pre - existing comorbidities . Echo has not been included in the nice guidelines . With increasing consumer protection issues, anaesthesiologists fear of the legal implications of leaving out tests, and there are very few studies which looked at the issues in adopting the guidelines with respect to the indian scenario . The issues were fear of cancellation, the force of habit passed down from senior colleagues, and most importantly legal implications . In another data analysis, it was observed that nearly 63.3% of the tests were unindicated . This was evaluated using patient questionnaire, and tests need was determined by the rule - based algorithm that depended on the patient's response . Their data included only neurosurgical patients which form a different group with respect to surgical risks and nature of the disease . In a retrospective cost analysis study, it is necessary that the guidelines should be pertinent to the population at large addressed . The laboratory tests should be patient centered and need - based which should add to the pre - operative preparation, intraoperative modification of anaesthesia care and post - operative management . All the current guidelines reported are based on the meta - analysis studied in the developed nations . In india, where more than 70% is rural population with awareness and literacy rate in health care is low, presentation to the hospital will always be at the advanced stage of the disease . Thus, there is a need for developing guidelines to suit our population considering the socio - economic status, time of presentation and the nature of the disease being treated . Certain tests though not indicated need to be done till we have sufficient evidence to omit from routine tests in our population . To our knowledge, this is first prospective study looking at the cost implications of pre - operative laboratory testing both indicated and unindicated and costs savings in the indian population . Pre - operative guidelines of laboratory testing fully introduced in clinical practice could notably increase efficiency without affecting the quality of care . We need to move away from ordering routine tests, to patient and disease specific and need - based laboratory testing . Considering the poor compliance of our population towards regular health check - ups, it may be necessary to develop guidelines pertinent to our country and further review the process.
Bile acid - sensitive ion channel (basic) is a member of the deg / enac family of cation channels . Other mammalian members of this gene family are the epithelial na channel (enac) and acid - sensing ion channels (asics). While the function of enac in epithelial na reabsorption and na homeostasis has been known for some time and the role of asics in neuronal transmission and sensation of painful acidosis has been revealed over the last years, the physiological role of basic has remained unknown . Cloned more than a decade ago from rat and mouse, it was originally named blinac, according to its predominant sites of expression, namely the brain, the liver and the intestinal tract . The human homolog was cloned shortly after basic and originally named inac (intestine na channel), because its expression was mainly restricted to the intestinal tract . While basic from mouse (mbasic) is a constitutively open, na - selective channel, its ortholog from rat (rbasic) is almost completely blocked by physiological concentrations of extracellular ca . The residual rbasic current is unselective but removal of extracellular ca opens rbasic and renders the channel more selective for na over k. a hallmark of deg / enac channels is the block by the diuretic amiloride . While mbasic is inhibited by micromolar concentrations of amiloride, rbasic is only partially inhibited by millimolar concentrations of the drug . The anti - protozoal diarylamidines, in particular diminazene and the related compound nafamostat, inhibit basic at micromolar concentrations . Because diarylamidines do not inhibit enac, they are well suited to distinguish between enac and basic currents in tissues and cells . Since its cloning, it was hypothesized that basic might be a ligand - gated channel, like other members of the deg / enac family, for example the hydra na channel (hynac) from the freshwater polyp hydra magnapapillata and fanac, the fmrfamide - activated na channel from snails, which have neuropeptides as ligands . The ligand - hypothesis gained further support when flufenamic acid (ffa) was identified as an artificial agonist of rbasic . Micro- to millimolar concentrations of ffa rapidly activate the channel, inducing na - selective currents . Recently we identified cholangiocytes, the epithelial cells lining the bile ducts, as the main site of basic protein expression in the liver . This in turn led to the identification of bile acids as agonists of rbasic, confirming that rbasic requires a ligand for activation . Based on the sensitivity of the channel for bile acids we changed the original name blinac to basic . Various bile acids naturally occurring in mouse, rat and pig bile, in particular hyodeoxycholic acid (hdca) and chenodeoxycholic acid (cdca), activate rbasic when applied individually . They are synthesized by hepatocytes and then transported via the bile ducts to the gallbladder, where they are stored and from which they are released into the small intestine when required . Rats lack a gallbladder; in these animals the bile is directly released from the major extrahepatic bile duct into the duodenum . Hundreds of different bile acids are known to date, the structure of their side chain, their stereochemistry and the number and position of their hydroxylgroups varies and determines their chemical properties . Furthermore the bile acid composition is highly variable between different species . In humans for example, the major bile acids are cdca and cholic acid (ca) whereas in rodents, ca, -muricholic acid (-mca) and hdca are the major bile acids . Ursodeoxycholic acid (udca) is the major physiological constituent of bear bile but it is also present in trace amounts in human and rodent bile . In traditional chinese medicine, udca isolated from bear bile has been administered as a remedy for liver diseases for almost 3000 y. in the 20th century, udca was discovered by western medicine as a compound capable of dissolving gallstones and inducing choleresis, an increased bile flow . Today udca is used to treat various cholestatic liver diseases, for example primary biliary cirrhosis, primary sclerosing cholangitis or intrahepatic cholestasis of pregnancy . Udca exerts its beneficial anti - cholestatic effect by several different but possibly linked mechanisms in hepatocytes and cholangiocytes . In hepatocytes, udca increases secretion by stimulating the expression of transporter proteins required for secretory processes, for example the bile salt export pump (bsep) and the multidrug resistance - associated protein 2 (mrp2), and by increasing the insertion rate of these proteins into the apical membrane cholangiocytes are constantly exposed to high concentrations of hydrophobic bile acids, which can damage the plasma membrane leading to cholangiocyte malfunction . Udca, a relatively hydrophilic bile acid counteracts this membrane - damaging effect in vitro . In addition, udca can stimulate secretion of cholangiocytes indirectly via intracellular signaling cascades that affect apically located proteins involved in secretion, e.g., purinergic py receptors and the chloride channel cftr (cystic fibrosis transmembrane conductance regulator). In this addendum to our previous study we demonstrate that the removal of extracellular divalent cations potentiates the activation of basic by bile acids . We had previously shown that removal of extracellular ca opens rbasic, suggesting that ca strongly stabilizes the inactive, resting state of rbasic . Here we determined the current amplitude of rbasic with different concentrations of extracellular mg in the absence of ca, revealing that mg also strongly inhibited rbasic with an ic50 of 79 10 m (n = 8; fig . 1). Thus, apparent affinity of mg - inhibition was 6-fold lower than of ca - inhibition (13 2 m, n = 8, p <0.01; fig . 1), showing that both, ca and mg, tightly control rbasic activity and stabilize its resting state . (a) upper panel, representative current trace from rbasic - expressing oocytes recorded in the absence of mg and decreasing [ca]e . Lower panel, representative current trace recorded in the absence of ca and decreasing [mg]e . (b) concentration - dependent inhibition of rbasic by extracellular ca (closed circles) and mg (open circles). Currents were normalized to the current in the presence of 10 nm ca or mg, respectively . Next, we tested whether removal of extracellular divalent cations affects the activation of basic by hdca and whether it can further increase basic activity in the presence of a maximal concentration of hdca . We used mg - free solutions and determined the ec50 of basic for hdca both in the presence (1.8 mm) and the absence (10 nm) of extracellular ca (fig . 2). Similar to our previous study, we found that 2 mm hdca did not change the concentration of free ca compared with standard bath (not shown), ruling out an unspecific effect via chelation of ca . Ec50 for hdca was modestly increased from 2.1 0.04 mm in the presence of ca to 1.6 0.05 mm in its absence (n = 8, p <0.01; fig . Moreover, at any concentration of hdca, the current amplitudes induced by hdca were three- to four - fold higher in the absence of extracellular ca than in its presence (fig . 2). (a) representative current traces from rbasic - expressing oocytes showing the concentration - dependent activation of rbasic by hdca in the presence of 1.8 mm extracellular ca and 1.0 mm extracellular mg (upper panel) or of 10 nm ca and 0 mg (-ca) (lower panel). (b) concentration - response curves for hdca in the presence of 1.8 mm extracellular ca and 1.0 mm extracellular mg (closed circles) or of 10 nm ca and 0 mg (open circles). Therefore, we previously proposed that removal of divalent cations does not simply unblock the basic pore but induces a conformational change that is associated with open gating of the channel . Assuming a pure effect of divalent cations on gating, potentiation of the basic current by removal of ca at a maximal concentration of hdca suggests that hdca is a partial agonist that does not induce full basic activity . Likewise, the similar hdca concentration - response relationship in the presence and absence of ca suggests that hdca further increases basic activity even when ca was not bound to the channel . These results, thus, argue that removal of ca and hdca gate basic by an allosteric mechanism, meaning that removal of ca and bile acids use two independent molecular mechanisms to synergistically influence the activity of basic . At present we cannot exclude, however, that a block by ca of the open basic pore is the reason of the increased current amplitude after removal of ca . Future studies determining the dependence of the open channel amplitude on ca will show whether ca has a pure gating effect on basic or a combined effect on gating and single channel amplitude . Udca and hdca differ only in the position of one hydroxyl group: in udca, c-7 is hydroxylated, whereas in hdca, c-6 is hydroxylated . The only difference between udca and cdca is the steric orientation of the hydroxyl group at position c-7: in udca it is in the -position, whereas in cdca it is in the -position (fig . 3a). Because of this high similarity we reasoned that udca might also activate rbasic . Indeed, udca robustly activated rbasic when applied at a concentration of 2 mm . The current amplitude induced by udca was even significantly higher (3.0 0.5 a, n = 10) than the amplitude induced by the same concentration of hdca (1.9 0.3 a, n = 10, p <0.05; fig . Activation of rbasic by udca was concentration dependent, similar to activation by hdca (fig . The ec50 value for udca was 2.5 0.04 mm (n = 10), similar to the ec50 for hdca (2.1 0.04 mm, n = 8). We have shown previously that when hdca and cdca are applied together at a concentration of 1.0 mm and 0.5 mm, respectively, they induce a significantly larger current amplitude than when applied individually, suggesting that both bile acids activate the channel synergisitically . Udca at a concentration of 1 mm together with 0.5 mm cdca induced a 3-fold larger current (2.4 0.3 a, n = 10) than udca applied alone at a concentration of 1.5 mm (0.7 0.1 a, n = 10, p <0.01), suggesting that cdca and udca synergistically activate rbasic . Structures were drawn using cs chemdraw ultra (cambridgesoft) based on structures available from pubchem (http://pubchem.ncbi.nlm.nih.gov/). Upper panel, representative current trace from an rbasic - expressing oocyte showing the activation of rbasic by individual application of 2 mm hdca or udca . Lower panel, quantitative comparison of current amplitudes induced by hdca (gray bar) and udca (black bar). Currents were normalized to the current induced by the removal of extracellular divalent cations (10 nm ca, 0 mg; error bars = sem (c) upper panel, representative current trace from an rbasic - expressing oocyte showing the concentration - dependent activation of rbasic by udca . Currents were normalized to the maximum current in the presence of 5 mm udca, which was 13.2 1.1 a (n = 8). Error bars = sem . Upper panel, representative current trace showing the activation of rbasic by individual application of 1.5 mm udca and by co - application of 1.0 mm udca and 0.5 mm cdca, respectively . Lower panel, quantitative comparison of current amplitudes induced by udca (gray bar) and udca / cdca (black bar). Currents were normalized to the current induced by the removal of extracellular divalent cations (-ca) which was 2.9 0.4 a (n = 10). Whether the effect of udca on rbasic activity is of any physiological or clinical relevance remains to be elucidated but it is tempting to speculate that rbasic is involved in absorptive and/or secretory processes in cholangiocytes and that it might represent an hitherto unknown molecular target of udca . Basic is a cation channel, however, and cation reabsorption by cholangiocytes would lead to a net fluid absorption and thus reduced bile flow, which contradicts the anticholestatic effect of udca . Future results will show whether basic contributes to cation transport in bile ducts and whether it promotes secretion or absorption by the bile duct epithelium . We had previously shown that removal of extracellular ca opens rbasic, suggesting that ca strongly stabilizes the inactive, resting state of rbasic . Here we determined the current amplitude of rbasic with different concentrations of extracellular mg in the absence of ca, revealing that mg also strongly inhibited rbasic with an ic50 of 79 10 m (n = 8; fig . 1). Thus, apparent affinity of mg - inhibition was 6-fold lower than of ca - inhibition (13 2 m, n = 8, p <0.01; fig . 1), showing that both, ca and mg, tightly control rbasic activity and stabilize its resting state . (a) upper panel, representative current trace from rbasic - expressing oocytes recorded in the absence of mg and decreasing [ca]e . Lower panel, representative current trace recorded in the absence of ca and decreasing [mg]e . (b) concentration - dependent inhibition of rbasic by extracellular ca (closed circles) and mg (open circles). Currents were normalized to the current in the presence of 10 nm ca or mg, respectively . Next, we tested whether removal of extracellular divalent cations affects the activation of basic by hdca and whether it can further increase basic activity in the presence of a maximal concentration of hdca . We used mg - free solutions and determined the ec50 of basic for hdca both in the presence (1.8 mm) and the absence (10 nm) of extracellular ca (fig . 2). Similar to our previous study, we found that 2 mm hdca did not change the concentration of free ca compared with standard bath (not shown), ruling out an unspecific effect via chelation of ca . Ec50 for hdca was modestly increased from 2.1 0.04 mm in the presence of ca to 1.6 0.05 mm in its absence (n = 8, p <0.01; fig . Moreover, at any concentration of hdca, the current amplitudes induced by hdca were three- to four - fold higher in the absence of extracellular ca than in its presence (fig . 2). (a) representative current traces from rbasic - expressing oocytes showing the concentration - dependent activation of rbasic by hdca in the presence of 1.8 mm extracellular ca and 1.0 mm extracellular mg (upper panel) or of 10 nm ca and 0 mg (-ca) (lower panel). (b) concentration - response curves for hdca in the presence of 1.8 mm extracellular ca and 1.0 mm extracellular mg (closed circles) or of 10 nm ca and 0 mg (open circles). Therefore, we previously proposed that removal of divalent cations does not simply unblock the basic pore but induces a conformational change that is associated with open gating of the channel . Assuming a pure effect of divalent cations on gating, potentiation of the basic current by removal of ca at a maximal concentration of hdca suggests that hdca is a partial agonist that does not induce full basic activity . Likewise, the similar hdca concentration - response relationship in the presence and absence of ca suggests that hdca further increases basic activity even when ca was not bound to the channel . These results, thus, argue that removal of ca and hdca gate basic by an allosteric mechanism, meaning that removal of ca and bile acids use two independent molecular mechanisms to synergistically influence the activity of basic . At present we cannot exclude, however, that a block by ca of the open basic pore is the reason of the increased current amplitude after removal of ca . Future studies determining the dependence of the open channel amplitude on ca will show whether ca has a pure gating effect on basic or a combined effect on gating and single channel amplitude . Udca and hdca differ only in the position of one hydroxyl group: in udca, c-7 is hydroxylated, whereas in hdca, c-6 is hydroxylated . The only difference between udca and cdca is the steric orientation of the hydroxyl group at position c-7: in udca it is in the -position, whereas in cdca it is in the -position (fig . 3a). Because of this high similarity we reasoned that udca might also activate rbasic . Indeed, udca robustly activated rbasic when applied at a concentration of 2 mm . The current amplitude induced by udca was even significantly higher (3.0 0.5 a, n = 10) than the amplitude induced by the same concentration of hdca (1.9 0.3 a, n = 10, p <0.05; fig . 3b). Activation of rbasic by udca was concentration dependent, similar to activation by hdca (fig . The ec50 value for udca was 2.5 0.04 mm (n = 10), similar to the ec50 for hdca (2.1 0.04 mm, n = 8). We have shown previously that when hdca and cdca are applied together at a concentration of 1.0 mm and 0.5 mm, respectively, they induce a significantly larger current amplitude than when applied individually, suggesting that both bile acids activate the channel synergisitically . Udca at a concentration of 1 mm together with 0.5 mm cdca induced a 3-fold larger current (2.4 0.3 a, n = 10) than udca applied alone at a concentration of 1.5 mm (0.7 0.1 a, n = 10, p <0.01), suggesting that cdca and udca synergistically activate rbasic . Structures were drawn using cs chemdraw ultra (cambridgesoft) based on structures available from pubchem (http://pubchem.ncbi.nlm.nih.gov/). Upper panel, representative current trace from an rbasic - expressing oocyte showing the activation of rbasic by individual application of 2 mm hdca or udca . Lower panel, quantitative comparison of current amplitudes induced by hdca (gray bar) and udca (black bar). Currents were normalized to the current induced by the removal of extracellular divalent cations (10 nm ca, 0 mg; -ca error bars = sem (c) upper panel, representative current trace from an rbasic - expressing oocyte showing the concentration - dependent activation of rbasic by udca . Currents were normalized to the maximum current in the presence of 5 mm udca, which was 13.2 1.1 a (n = 8). Error bars = sem . Upper panel, representative current trace showing the activation of rbasic by individual application of 1.5 mm udca and by co - application of 1.0 mm udca and 0.5 mm cdca, respectively . Lower panel, quantitative comparison of current amplitudes induced by udca (gray bar) and udca / cdca (black bar). Currents were normalized to the current induced by the removal of extracellular divalent cations (-ca) which was 2.9 0.4 a (n = 10). Whether the effect of udca on rbasic activity is of any physiological or clinical relevance remains to be elucidated but it is tempting to speculate that rbasic is involved in absorptive and/or secretory processes in cholangiocytes and that it might represent an hitherto unknown molecular target of udca . Basic is a cation channel, however, and cation reabsorption by cholangiocytes would lead to a net fluid absorption and thus reduced bile flow, which contradicts the anticholestatic effect of udca . Future results will show whether basic contributes to cation transport in bile ducts and whether it promotes secretion or absorption by the bile duct epithelium . Despite our recent progress regarding the expression pattern of basic in cholangiocytes and regarding its activation by bile acids, we are still far away from understanding the physiological role of basic in these cells, let alone its role in other tissues such as the intestinal tract or the brain . In future studies it will therefore be important to confirm expression of basic in cholangiocytes and to discover its cellular and subcellular expression pattern in other tissues . Furthermore it will be necessary to study basic in a native cell or a native epithelium or both, to unravel its role in ion transport mediated by these cells and epithelia.
Atypically resistant or even multidrug resistant enterobacteriaceae show a world - wide distribution which does not spare tropical and subtropical settings . Studies with civilian returnees from journeys to the tropics identified frequent intestinal colonization with such bacteria . In particular, enterobacteriaceae with extended - spectrum -lactamases (esbls) quantitatively dominate [14]. Antibiotic therapy, e.g., for the treatment of travellers diarrhea, facilitates colonization with esbl - positive strains . Resistant bacteria are selected under antibiotic pressure while the natural gut flora is depleted and thus deprived from its protective potential . The european union training mission (eutm) in mali, providing military training for local soldiers, is just one example . During a recent surveillance of eutm soldiers with diarrhea in koulikoro, mali, from the 49th calendar week 2013 to the 34th calendar week 2014, a total of 16 esbl - positive enterobacteriaceae, comprising 15 escherichia coli and one klebsiella pneumoniae, had been isolated from the stool sample of 13 out of 48 patients (27.1%) as recently described . As further demonstrated during this surveillance, there was a high degree of clonal diversity of the e. coli strains, suggesting multiple transmission events rather than ongoing nosocomial transmission within the field camp . Considering this high colonization pressure with esbl - positive enterobacteriaceae in mali, a molecular assessment of the 16 isolates from mali was performed to find out whether the pattern of genetic resistance determinants is similarly diverse . Altogether, 15 e. coli isolates and one k. pneumoniae isolate from stool samples of eutm soldiers in koulikoro, mali, with diarrhea were included in the assessment . Details on the mode of isolation of the strains, the observed resistance patterns, and the clonal distribution have been reported elsewhere . A duplex pcr was performed targeting blatem and blashv -lactamases as described [6, 7], followed by four simplex pcrs targeting the blactx - m groups i iv (table 1), respectively, also exactly as described [7, 8]. Amplicons were visualized using a lonza flashgel system (lonza rockland, inc ., rockland, me, usa) according to the manufacturer s instructions . Because not all blatem and blashv -lactamases are associated with esbl phenotypes, respective amplicons were subjected to sequencing . Amplicons were purified using the nat clean - up / nucleospin extract ii kit (macherey & nagel, dren, germany) according to the manufacturer s instructions . After purification, sequencing in both directions sequence alignment was performed using the software bionumerics 7.1 software (applied maths, sint - martens - latem, belgium). The alignment settings were open gap penalty 100%, unit gap penalty 0%, match score 100%, and fast algorithm (= minimum match sequence: 2, maximum number of 98). The analyses included sequences of enterobacteria (taxid: 91347) only and excluded models as well as uncultured / environmental sample sequences . As the blactx - m -lactamase groups that are identified by the above mentioned pcrs are generally associated with esbl phenotypes, no sequence - based discrimination was aspired here for economic reasons . Instead, discrimination on group level as allowed by the used pcrs was considered as sufficient . Altogether, 15 e. coli isolates and one k. pneumoniae isolate from stool samples of eutm soldiers in koulikoro, mali, with diarrhea were included in the assessment . Details on the mode of isolation of the strains, the observed resistance patterns, and the clonal distribution have been reported elsewhere . A duplex pcr was performed targeting blatem and blashv -lactamases as described [6, 7], followed by four simplex pcrs targeting the blactx - m groups i iv (table 1), respectively, also exactly as described [7, 8]. Amplicons were visualized using a lonza flashgel system (lonza rockland, inc ., rockland, me, usa) according to the manufacturer s instructions . Because not all blatem and blashv -lactamases are associated with esbl phenotypes, respective amplicons were subjected to sequencing . Amplicons were purified using the nat clean - up / nucleospin extract ii kit (macherey & nagel, dren, germany) according to the manufacturer s instructions . Sequence alignment was performed using the software bionumerics 7.1 software (applied maths, sint - martens - latem, belgium). The alignment settings were open gap penalty 100%, unit gap penalty 0%, match score 100%, and fast algorithm (= minimum match sequence: 2, maximum number of 98). The analyses included sequences of enterobacteria (taxid: 91347) only and excluded models as well as uncultured / environmental sample sequences . As the blactx - m -lactamase groups that are identified by the above mentioned pcrs are generally associated with esbl phenotypes, no sequence - based discrimination was aspired here for economic reasons . Instead, discrimination on group level as allowed by the used pcrs was considered as sufficient . From the analyzed strains, six out of 15 e. coli (40%) were positive for blatem in pcr . Sequence analyses identified blatem-1, a -lactamase which is not associated with an esbl phenotype, in all instances . All strains including both the 15 e. coli isolates and the single k. pneumoniae strain were positive for -lactamases of the esbl - associated blactx - m group i (table 1). Considering the high clonal diversity of the strains as previously described, the close homogeneity with only one detected blactx - m group and a complete absence of all other analyzed genetic esbl mechanisms is surprising . Obviously, genetic exchange due to international travel from and to terror- and war - haunted mali is low . This situation may allow for a wide spread of a restricted spectrum of resistance mechanisms under the selective pressure of antibiotic use and misuse . However, a french study with samples from adopted children from an orphanage in bamako, mali, allowed for the isolation of 52 esbl - positive enterobacteriaceae during a three - year - interval from 2002 till 2005 from 24 out of 25 adoptees after their arrival in france . Esbl - positive e. coli dominated quantitatively; the most frequent resistance determinant was the -lactamase encoded by blactx - m-15, a member of the blactx - m group i, which was identified in 93% of the e. coli strains . After 10 years, again all 16 isolated esbl - positive enterobacteriaceae were positive for blactx - m of group i, suggesting a locally very stabile distribution (table 2). While data on esbl distribution in western african mali are virtually absent, more respective data are available for western african ghana . In large hospitals of the ghanaian towns accra and kumasi, the proportion of esbl - positive strains among isolated enterobacteriaceae was reported to be about 50% . Of note, a broader distribution of blatem, blashv, and blactx - m genes was reported for the strains from kumasi, suggesting free regional and international exchange of resistance determinants . The introduction of esbl - positive enterobacteriaceae by european soldiers returning from deployments in subsaharan africa to their home countries is bothersome, because it bears the risk of transmission to their families . This phenomenon is well - documented for adopted children from mali, who have spread their colonizing esbl - positive strains among their french adoptive families in a percentage as high as 23% . It seems advisable to offer screening options for gram - negative pathogens for european soldiers who return from deployments in tropical high - endemicity settings with consecutive counselling regarding hand and sanitary hygiene to reduce the risk of transmission . One reason is the fact that there are numerous different molecular mechanisms, exceeding the potentials even of multiplex pcr approaches . However, if only singular or few resistance determinants circulate within a local population as suggested for mali, pcr - based screening might be successfully applied as recently demonstrated for a setting in madagascar . However, such interpretations are limited by the fact that only small numbers of esbl - positive strains from mali were assessed in this study as well as in previous assessments . A broader surveillance seems useful to more reliably estimate the true distribution of esbl - associated genes in colonizing enterobacteriaceae in mali in line with previous results from 10 years ago, bla ctx - m group i was confirmed as the major resistance mechanism of esbl - positive enterobacteriaceae in mali . European soldiers deployed in mali are at considerable risk of introducing such resistant strains to their home countries and their families . The obviously lacking diversity of locally distributed esbl - associated resistance genes in mali suggests successful use of pcr - based esbl screening approaches for this region.
Using multiple terminology in defining atypical celiac disease (cd) has confused many of the clinicians to recognise atypical forms of this common disorder . Cd is not considered an uncommon disorder any longer and is not a disease of essentially european origin (1). Nevertheless, recognising the existence of atypical forms known under the old terminologies like latent, silent and potential cd has introduced a new insight on clinical behaviour of this condition . That way the age of presentation of the disease has changed dramatically (1, 2) and the factors responsible for this change are mainly attributed to advances in diagnostic tools in recognising atypical and subclinical forms of the disease . Population screening with serological tests, have shown a cd screening prevalence of the order of 1% in the western hemisphere (3). European and asian studies involving healthy blood donors found a prevalence rate of 1 in 166 - 330 (4, 5) subclinical cd . According to the previous studies, screening based on antibodies only would underestimate the prevalence of cd due to false - negative results caused by the low sensitivity of tests (610). However, some studies suggest that the overestimation of cd frequency could also result from antibody based screening programmes due to a high rate of false positives (11). Terminologies like latent, silent and potential celiac disease can be confusing for clinicians and patients . Silent cd is not absolutely silent after all; patients show signs of cd with no significant symptoms . T - cell - mediated autoimmune processes are initiated by gluten exposure, leading to both intestinal and atypical extraintestinal manifestations . More and more diseases are proven to be associated with cd . In these conditions, screening is strongly recommended . Malabsorption can be silent like a mild anaemia (better defined under subclinical), or there is usually only moderate malabsorption, if any at all . Since gluten sensitivity is no longer limited to overt villous atrophy, and given the results of many studies (1315), we believe the answer is yes . Subclinical or so called silent cd cases are being detected in increasing numbers because of raised awareness of the disease . Presentations with atypical symptoms are the dominant form of disease manifestation and these comprise the sole and main part of the celiac iceberg (16). Whether they have positive serology with negative biopsy or increased t cells receptors with symptoms compatible with cd they could be classified as atypical cd . We are moving towards a lower threshold in implementing a gluten free diet (gfd). Villous atrophy is not mandatory any longer to qualify a patient for gfd . In fact a large number of patients present with non - celiac gluten sensitivity with completely normal biopsy and negative serology . There is a need to re - define the terminologies according to the modified treatment strategy in gluten related disorders . The main strategy for treatment should target symptomatic typical or atypical patients, and not asymptomatic cases . We propose a simplified classification by dividing and replacing previous terminology to typical, atypical and subclinical instead of silent / latent and potential . Celiac and non - celiac gluten sensitivity * suclinical: previously known as silent and atypical as known under latent and potential cd the disorder is now considered the result of a complex interaction between genetic and environmental factors such as gluten that classified from subclinical to severe malabsorption . In contrast to gluten sensitivity celiac disease development has a strong genetic component with a sibling relative risk (lambda (s)) of 30 . Recent studies using the human genome screening technique in families with multiple siblings suffering from cd have suggested the presence of at least 4 different chromosomes in the predisposition to suffer from cd (17). One susceptibility locus is the mhc (major histocompatibility complex) region, with a particular association with the hla - dq alleles dqa1 * 0501 and dqb1 * 0201 . However, shared - haplotype studies suggest that genes within the mhc complex contribute no more than 40% to the sibling familial risk of disease . Early studies showed that gliadin elicits an inflammatory t - cell reaction when added to intestinal biopsy specimens of celiac patients in vitro and a link to the genetic predisposition was provided by the isolation of gliadin - specific hla - dq2-restricted t - cell clones from cd mucosa (18, 19). However, the prevalence of hla - dq2 is high in the normal population (25 - 45%), suggesting the involvement of additional, and probably non - hla - linked genes in cd pathogenesis . The pathologic spectrum of the mucosal abnormalities seen on small intestinal biopsies, range from microscopic enteritis (marsh 0-ii) to macroscopic forms (marsh iiia - c) (20). Not every gluten - sensitised individual inevitably develops cd and not every celiac patient develops the destructive lesions such as marsh iii . Celiac disease is not exclusively due to antibody production either . A large proportion of the patients present with microscopic enteritis (marsh i - ii) whose diagnoses these 6 distinct and sequential phases of the cd are microscopic enteritis (me) marsh (0-ii). Some patients could still have subtle abnormalities at this stage like increased t cell receptors or alteration of enterocytes and microvillis . I) recruitment of t - lymphocytes> 25/100 enterocytes (intestinal - intraepithelial lymphocyte or iel; marsh i), ii) lymphocyte infiltration and crypt hyperplasia (marsh ii), iii) macroscopic enteritis (marsh iiia - c) partial villous atrophy (marsh iiia), iv) subtotal villous atrophy (marsh iiib) and v) total atrophy (marsh iiib) and total villous atrophy (marsh iiic) (6, 8). This sequential cascade suggests that a t - cell response to gliadin precedes, and very likely produces, the complete pattern of cd . The statistical comparison between antibody - positive and antibody - negative cases shows that the appearance of antibodies was seen predominantly in cases with serious mucosal damage in which iels was highly increased . However, it is hard to rule out the contribution of antibodies in genesis of an autoimmune condition like cd . The screening value of autoantibodies has been too optimistically overestimated, especially those on tissue transglutaminase antibodies (ttga) (24, 25). However, comparing the ttga to ema and aga, the sensitivity of ttga does not offer any advantages over ema for screening of the populations at high risk of cd (2628). It is time to re - evaluate our perception of intestinal pathology (29) in such terms, rather than by continued use of subjective degrees of villous atrophy (va), since absence of va is not evidence of absence of cd . Such terminology obscures the recognition of fundamental changes occurring within small bowel mucosa . In simple words, increased density of iel's and crypt hyperplasia form an essential phase in the disease pathogenesis sequence of progression . As cd with milder enteropathy is the most common form, histology cannot be considered as the gold standard any longer . Gluten sensitivity (gs) is characterised by negative antibodies and normal histology; it is defined as a non - allergic and non - autoimmune condition in which the consumption of gluten can lead to symptoms similar to those seen in cd . Until recently the terms gs and cd were used synonymously in literature (33) and it is not clear yet whether patients affected by gs will have some subtle intestinal and mucosal changes consistent with microscopic enteritis . Some gs patients would tolerate even more than 5 g gluten / day and still remain symptom free with negative serology (34, 35). Gs patients are gluten intolerant and gluten consumption does not lead to small intestinal damage, so it is not accompanied by the concurrence of ttg autoantibodies or autoimmune disease . In the study by kaukinen et al . Out of 94 adults with gi symptoms, 63% were affected by gluten foods and neither classified as cd nor as allergic (36). On the other hand around 50% of the gs patients were dq2/dq8 positive, which is similar to that of the general population, while celiac patients carry more than 95% in most regions of the word . However, while the prevalence of cd is roughly 1% within the general population, gs is thought to affect 6 to 10% of the general population (37). In some cases gs can present with normal or milder enteropathy seen as increased intestinal permeability, ibs, abdominal discomfort, pancreatic disorders, pain or diarrhoea; or it may present with a variety of extraintestinal symptoms including lymphoma, attention deficit disorder and neuropathy, autism and schizophrenia, infertility, ibd, muscular disturbances as well as osteopenia and osteoprosis (3842). According to current literature, a gfd is recommended to gluten sensitive cases with / without enteropathy . This policy includes a range of symptomatic gluten sensitive cases with atypical presentation including those with small bowel microscopic changes (marsh 0-ii) who are antibody negative or show characteristic features of other conditions . This is because these common systemic disorders have multifactorial etiology with a multitude of symptoms and complications inside and outside the small bowel . We still don't know how seriously subclinical cd will be affected by the long term complications if they are not treated with gfd . A marked increase in the prevalence of cd, especially the subclinical cd forms and non - celiac gluten sensitivity, seem to become a major health problem (4346). The clinician may often face the variability of histological and clinical aspects of cd (46) with uncertainty, as they might not quite fit into the diagnostic models in the current guidelines . Accumulated evidence supports that decreasing the treatment threshold for cases with atypical cd and those with gluten sensitivity, as the life quality of these cases will improve with gfd and the long - term health benefit of this strategy, would perhaps be also cost effective.
Amalgam has been shown to be an acceptable material with proven longevity for large posterior restorations . The major disadvantage of amalgam, however, is its inability to bond to dental hard tissues preventing reinforcement or strengthening of prepared tooth structure . This lack of bonding necessitates the use of macromechanical retentive features which are inherently destructive and cause further weakening of the remaining tooth structure . Resin bonding agents have been used for a number of years as an adhesive liner between dental amalgam and tooth structure . In vitro tests have shown that these adhesive systems form retentive bonds with amalgam that increase the shear bond strengths of amalgam to tooth and reduce microleakage [2, 3]. Other studies claim an increase in the fracture resistance of prepared teeth when restored with bonded amalgam [4, 5]. More recently, glass ionomer formulations have been tested as an adhesive liner with dental amalgam for the restoration of prepared teeth [68]. Certain glass ionomer formulations have demonstrated increased shear bond strength measurements of amalgam to dental hard tissue comparable to that achieved with resin systems [6, 8]. The fracture resistance of teeth restored with the bonded amalgam technique using restorative glass ionomer as an adhesive has been shown to be greater than that of prepared teeth alone and teeth restored with amalgam using a copalite liner . The objective of this study was to assess the resistance of premolars with traditional mod preparations to cuspal fracture when restored with amalgam that has been adhesively bonded to cavity preparations using various glass ionomer formulations as the adhesive liner compared to cavities restored with amalgam bonded with a resin luting liner and cavities restored with amalgam alone . Three groups of teeth restored with the amalgam bonding technique, 1 group of intact teeth, and 1 group of teeth restored with amalgam only were included in this study . The luting cements used as amalgam bonding agents utilized in this study are shown in table 1 . A power calculation determined that a minimum of 10 teeth were needed in each group . Eighty - five human maxillary premolars extracted for orthodontic reasons were collected and immediately stored in distilled water . The teeth were rinsed and any debris removed with a sonic scaler (titan sw scaler, dental ez group, stardental division, lancaster, pa, usa). The teeth were embedded in individual cylinders of autopolymerizing polymethyl methacrylate (fas - tray, harry j bosworth company, stokie, ill, usa) 1.0 mm short of the cementoenamel junction so as to mimic alveolar support for the tooth . After complete setting of the tray acrylic, the base was trimmed to expose a cross - section of the root in its apical one third . This allowed transmission of applied force entirely through tooth structure by preventing settling of the tooth within the tray acrylic during testing . The mounted teeth were randomly divided into five groups and stored in distilled water at 37c . Mesio - occlusal - distal (mod) cavities were prepared using a 245 bur (ss white, lakewood, nj, usa) in a high - speed handpiece (bellatorque, kavo dental corporation, lake zurich, ill, usa) with water spray . The dimension of each preparation was made proportional to the dimensions of the tooth to minimize variations as a result of the size of individual teeth . The pulpal floor was 2/3 of the height of the cusps measured from the cementoenamel junction . The facial - lingual width of the approximal boxes was 1/3 the maximum facial - lingual width of the tooth . The gingival floor of the approximal box was established 1.0 mm above the cementoenamel junction . The depth of the approximal box (from gingival cavosurface to axial wall) was 1.0 mm . All internal line angles were rounded and no additional retentive features were cut . Prior to actual cavity preparation, the outline of each cavity was drawn on the surface of the tooth as a preparation guide . The control group, of unprepared teeth, was returned to water for storage until testing . Number 1 tofflemire band (sullivan - schein dental, melville, ny, usa) was placed in a universal tofflemire matrix retainer (teledyne getz, elk groove village, ill, 60007) and secured around the tooth . The cavity walls were dried using clean compressed air . High copper, platinum modified, precapsulated, spherical amalgam (logic, sdi ltd, australia) was mechanically triturated (wig - l - bug, crescent dental mfg co, lyons, ill, usa) following the manufacturer's instructions followed by hand condensation into the prepared cavity . The matrix band was removed and the amalgam was carved and finished following the normal anatomical contours of the tooth . The cavity walls of the remaining three groups were coated with one of three luting cements: relyx arc (3 m espe, st . Paul, minn, usa), relyx luting (3 m espe, st . Paul, minn, usa), or ketac - cem (3 m espe, st . Following cement application, each tooth was immediately restored in the same manner as the amalgam group, with the amalgam being condensed into the cavity while the adhesive liners were still unset . The matrix band was removed to allow carving of the amalgam to normal anatomical contours and removal of any adhesive flash at cavosurface margins . Relyx arcthe cavity was etched with 37% phosphoric acid for 20 seconds using the total etch technique . The acid was then thoroughly rinsed for 20 seconds and the cavity was dried, but not desiccated, with compressed air . Paul, minn, usa) was applied to the cavity using a microapplicator tip and dried for 2 seconds with compressed air, leaving glossy cavity surfaces . The dentine bonding agent was then light cured for 10 seconds using visible blue light (optilux, demetron / kerr 21 commerce drive, danbury, conn, usa) at 460 m wavelength . The relyx arc resin luting cement was dispensed in equal amounts and mixed for 10 seconds and applied to the cavity using a microapplicator brush . The cavity was etched with 37% phosphoric acid for 20 seconds using the total etch technique . The acid was then thoroughly rinsed for 20 seconds and the cavity was dried, but not desiccated, with compressed air . Paul, minn, usa) was applied to the cavity using a microapplicator tip and dried for 2 seconds with compressed air, leaving glossy cavity surfaces . The dentine bonding agent was then light cured for 10 seconds using visible blue light (optilux, demetron / kerr 21 commerce drive, danbury, conn, usa) at 460 m wavelength . The relyx arc resin luting cement was dispensed in equal amounts and mixed for 10 seconds and applied to the cavity using a microapplicator brush . Relyx lutingthe cavity was washed with distilled water and dried with compressed air from the air - water tip, preventing desiccation of tooth tissue . The powder was fluffed and the standard powder: liquid ratio of 1.6:1, by weight, was dispensed on a glass slab . Mixing was completed with the aid of a metal spatula for 30 seconds . The cavity was washed with distilled water and dried with compressed air from the air - water tip, preventing desiccation of tooth tissue . The powder was fluffed and the standard powder: liquid ratio of 1.6:1, by weight, was dispensed on a glass slab . Mixing was completed with the aid of a metal spatula for 30 seconds . Ketac - cem the cavity was washed with distilled water and dried with compressed air from the air - water tip, preventing desiccation of tooth tissue . The powder was fluffed and the standard powder: liquid ratio of 3.8:1, by weight, was dispensed on a glass slab . Mixing was completed with the aid of a metal spatula for 30 seconds . The cavity was washed with distilled water and dried with compressed air from the air - water tip, preventing desiccation of tooth tissue . The powder was fluffed and the standard powder: liquid ratio of 3.8:1, by weight, was dispensed on a glass slab . Mixing was completed with the aid of a metal spatula for 30 seconds . The mixed cement all specimens (control and restored teeth) were stored in 100% humidity at 37c for 14 days prior to testing . The teeth were placed in a stabilizing ring, to avoid movement, and tested in compression in a hounsfield h50ks tensometer (tinius olsen ltd, redhill, surrey, uk) using a rounded, stainless steel, testing probe 5 mm in cross - section . The profile of the testing probe ensured contact with buccal and lingual inclines of cusp triangular ridges and not the amalgam restoration (see figure 1). The teeth were preloaded to a maximum force of 250 n at a speed of 5 mm / min for 10 times (with the speed of return at 50 mm / min), prior to catastrophic testing . The preloading was performed to simulate clinical situations where the occlusal surface of the tooth is subjected to repeated chewing forces . Following this preloading procedure, the teeth were tested to failure at a speed of 0.5 mm / min until cusp fracture occurred . Group means were analyzed using a one - way analysis of variance (anova) followed by a post hoc bonferroni multiple comparison test . During pretesting, a number of specimens were lost in each group due to early fracture . Two teeth were lost in the sound group, 1 in the amalgam group, 1 in the relyx arc group, none in the relyx luting group, and 2 in the ketac - cem group . The analysis of variance revealed that there were no significant differences in fracture resistance of teeth restored with amalgam alone or amalgam bonded with various luting cements (p>.05). There was however a highly significant difference between the intact teeth (control group) and all the restored groups (p <.05). The means and standard deviations, together with statistical significance, for the remaining teeth are presented in table 1 . This study did not attempt to characterize the fracture patterns of the failed specimens . Various studies have shown that prepared teeth fracture more readily than sound intact teeth [9, 10]. Ideally any material that is used to restore missing tooth structure should reinforce the tooth and minimize risk of cuspal fracture . In this study, the difference in resistance to catastrophic fracture between the sound (unprepared) teeth and restored teeth was highly significant . This supports previous findings that demonstrate the deleterious effect that cavity preparation has on the fracture resistance of posterior teeth . The results of this study showed no statistically significant differences in cusp fracture resistance between teeth restored with the bonded amalgam technique that utilized either a resin - based adhesive (relyx arc), glass - ionomer adhesive (ketac - cem), or resin - modified - glass ionomer (relyx luting) and a conventional technique using amalgam alone . These findings are contrary to those of other investigators, who have shown an increase in fracture resistance of teeth restored with the bonded amalgam technique using both resin and glass - ionomer luting cements when compared to teeth restored with amalgam alone [7, 12]. This difference may be explained by the size of the cavity preparation utilized in this study . The isthmus preparation dimensions of 1/3 the intercuspal width and the facial - lingual width of the approximal boxes at 1/3 the maximum facial - lingual width of the tooth could be considered conservative . This resulted in a significant portion of coronal dentin remaining, the mechanical properties of which contributed to similar fracture resistance values within the tested groups . Indeed, researchers that use more conservative cavity preparations have found that fracture resistance is not improved regardless of the restorative material or technique used . In a similar study where amalgam was bonded into cavities of similar dimensions using filled and unfilled resin systems, it was noted that there was no increase in the fracture resistance of restored teeth . This may explain the unexpected results shown by the sole resin system used, relyx arc, to bond amalgam in this particular study . Investigators who use larger cavity preparations have consistently shown that the use of the amalgam - bonding technique significantly contributes to the fracture resistance of the teeth [5, 14]. This particular research produced large variations in the standard deviation values among the tested groups . This may be attributed to the normal mechanical and anatomical variations of natural teeth, including, the ratio of enamel to dentin, cusp position and angulation, cusp height, and undetectable flaws . Another explanation of the high standard deviations may be due to the catastrophic forces applied in vitro until failure occurred . Such in vitro forces are applied at a constant direction and speed until failure occurs, which rarely mimics failure intraorally . Although the glass ionomer and resin - modified glass - ionomer luting cements used as adhesives for amalgam alloy did not increase the fracture resistance of the prepared teeth in this particular study, it must be noted that both conventional glass ionomer and resin - modified materials have shown increased measurements of shear bond strengths at the dentin / amalgam interface which has the effect of increasing retention of amalgam to tooth structure, thus diminishing the reliance on macromechanical features which has the overall effect of conserving tooth tissue [68]. Within the limitations of this study, the conclusion may be drawn that maxillary premolars restored with the bonded amalgam technique using glass ionomer, resin - modified glass ionomer, and resin luting cements do not improve measurements of fracture resistance . The clinical relevance of this is that amalgam bonded into conservative mod preparations of premolars does not offer increased strength to the tooth and restoration but only unnecessarily increases the complexity of the clinical procedure.
Peripheral nerves, especially those at the elbow region, such as the median nerve and ulnar nerve, are prone to entrapment neuropathies and injuries, such as cubital tunnel syndrome (1, 2), pronator teres syndrome (3), and nerve laceration (4). The traditional method used for evaluating these diseases with respect to preoperative diagnosis and postoperative rehabilitation is electrophysiological nerve conduction and electromyography; however, accurate evaluations using these methods largely depend on the operator s experience, and false negative results are common (5). A magnetic resonance (mr) image can display the soft tissue, including nerves, in t1 or t2 images, and the most commonly used method of mr for the diagnosis of peripheral nerve entrapment is to measure the cross sectional area (csa) and t2 signal intensity, which cannot distinguish axonal injury from demyelination (6). Moreover, the continuity and integrity of a nerve can hardly be detected based on two - dimensional (2d) images . Therefore, obtaining a three - dimensional (3d) display of a nerve that can be observed at any angle is essential for clinicians to make accurate assessments . Currently, the methods and sequences of mr neurography are mostly represented through diffusion tensor imaging (dti). Dti with fiber tracking was the first method used to obtain a 3d view of the nerve, which was originally applied to image the central nervous system . Diffusion is less restricted in the direction parallel to the nerve fiber than that perpendicular to the fiber (possibly due to the layers of myelin). Using special fiber - tracking software to reconstruct the dti data, it is possible to visualize the complete neural structure . In june 2004, skorpil et al . (7) demonstrated that peripheral nerves (the sciatic nerve in three healthy volunteers) could be imaged in vivo by using dti with fiber tracking . This technique has now become the most widely used method for evaluating the status of the median nerves in carpal tunnel syndrome (8), providing non - invasive imaging of peripheral nerve regeneration (9 - 11), and characterizing the structure of the brachial plexus (12). However, studies reporting the use of dti with fiber tracking post - processing at the elbow region are scarce . The following drawbacks of using dti for the elbow region, especially for the ulnar nerve, have been discussed: (i) it is superficial; (ii) close to the bone surface; and (iii) shows large angulations in the course of the nerve (13). Besides, these limitations of dti with respect to specific anatomical characteristics at the elbow region, the technique itself has many limitations . First, the quality of tractography depends on the parameters used in the acquisition step, such as the coils used, positioning of the patient, and other parameters including the b - value and the number of diffusion directions . (14) suggested using joint - specific coils at the joint region . When using this type of hard coil, the elbow, the subject must raise one hand above his / her head while in prone or supine position, which can be very uncomfortable . Second, during the post - processing phase before fiber tracking, the operator must choose a series of regions of interest (roi), which depends on the operator s anatomical knowledge . In addition to the dti fiber tracking technique, a novel post - processing method to reconstruct dti data was developed by skorpil et al . They used the maximum intensity projection (mip) technique to reconstruct sciatic nerves based on dti images in a single direction and in all directions . Furthermore, based on preliminary experiments performed at the wrist region, we found that images of a single direction could also be reconstructed by using volume rendering (vr) and mip . These two post - processing methods (vr and mip) may offer a more convenient procedure for dti because they do not require prior choice of rois . We predicted that the peripheral nerve at the elbow region could be three - dimensionally displayed based on the mr dti data with multiple post - processing techniques . We aimed to determine whether the vr or mip reconstruction for single direction would be more convenient than fiber tracking post - processing while acquiring approximate qualities of nerve imaging, and the consistency of vr / mip and fiber tracking post - processing for the same sample . The study had prior approval by our institutional review board and ethics committee, and all subjects provided informed consent prior to the study . A total of 24 elbows of 12 healthy, young volunteers were studied (six men, six women; age range 22 - 32 years). Nerves at the elbow region were analyzed, including the median nerve, ulnar nerve, and radial nerve . The exclusion criteria included general contraindications for mr imaging (mri), a prior history of trauma or surgery of the elbow, presence of rheumatoid arthritis, and presence of space - occupying lesions at the elbow . The mr images were acquired with a siemens magnetom verio 3 t mri scanner (siemens healthcare; erlangen, germany) using a 4-channal flex coil (flex small 4; siemens). The mri system s maximum field gradient amplitude was 45 mt / m with a slew rate of 200 t m s. during the scan session, the soft coil was placed around the elbow, and then a polymer brace was placed on the whole upper limb in order to restrain movement . The subject s upper extremity with the coil was immobilized with cushions, sandbags, and bandages . The elbow was then positioned to the center of the magnet bore . For imaging of the elbow, dti was performed using an echo planar inversion recovery (epir) sequence (tr = 15,300 ms; te= 92 ms; ti = 200 ms; fa = 90; etl = 1; fov = 250 250 mm; scan matrix = 128 128; average = 2) with 38 transversal slices of 3.0-mm thickness with no gap between the slices . Diffusion weighting with a b value of 1200 s / mm was applied in 20 encoding diffusion directions . In addition to the diffusion - weighted images, a single reference image without diffusion weighting (b = 0 s / mm) was acquired . For anatomical reference, a t1-weighted image (tr = 700 ms; te = 11 ms; fa = 150; etl = 3; thickness = 3 mm; gap = 0 mm; fov = 140 140 mm; scan matrix = 320 240; average = 1) was acquired . The scanning times for dti and t1-weighted image were 11 min 29 s and 2 min 1 s, respectively, for a total scan time of approximately 15 min . The raw dti images were transferred automatically to the siemens mr workstation (siemens syngo 3d). The x - axis represents the anterior posterior direction, the y - axis represents the left - right direction, and the z - axis represents the superior inferior direction . Based on the gradient table of the dti scan, direction 1 with coordinates (1, 0, 0) represents the anterior posterior direction . (15), the most suitable choice for single direction vr post - processing is direction 1, which is perpendicular to the nerves . The original dti images usually contained a series of t2-weighted images (b = 0 s / mm), which were generated as the first series in the result chart . The optimal choice (direction 1) was the second series generated in the result chart . During vr / mip reconstruction of images of direction 1, any high - signal noises of the veins or skin present in the reconstructed stereoscopic view were cut off in order to make the nerves as conspicuous as possible . One of the authors (y.y) was responsible for this part of the work . X - axis represents the anterior - posterior direction . Y - axis represents the left - right direction . Direction 1 with the coordinates (1, 0, 0) represents the anterior - posterior direction . The raw dti images were transferred to the siemens mr workstation (siemens syngo neuro 3d). Fiber tractography was then performed using a multiple seed roi technique, which employed fiber assignment by continuous tracking . Seed rois were drawn freehand at three anatomical locations (level to the supracondylar humerus, olecranon, and proximal radioulnar joint) on the raw dti images and reference t1-weighted images . Rois were confined precisely to the nerve border to avoid partial volume artefacts and to exclude any surrounding structure from the seed rois . In this study, the fractional anisotropy (fa) threshold value was 0.2 and the angulation tolerance was 30. the software s option step length was set to 0.47 mm . Another author (d .- s.j) was responsible for this part of the work . In order to compare the imaging quality among vr / mip and fiber tractography, we created a custom evaluation scale to assess each nerve in vr / mip and fiber tractography images . We set the scale based on whether the target nerve could be shown and the length of the nerve was complete as follows: score of 2, the target nerve (the median, radial, or ulnar nerve) at the elbow region was clearly shown and the entire length of the nerve was visible throughout the entire scanning section; score of 1, the target nerve at the elbow region was clearly shown, but its length was incomplete; score of 0, the target nerve was invisible . Two authors (w .- q.d and j .- h.g) were responsible for independently evaluating the imaging qualities according to this scale . One author (w .- q.d) evaluated the imaging qualities again 4 weeks later . For each nerve, scores of vr / mip reconstruction of the single direction and fiber tracking were compared using friedman test . Intraclass correlation coefficient (icc) analysis was used to compare the correlation of the imaging quality scores for each nerve among vr / mip and fiber tractography . Kappa values of 0 - 0.20 were considered to indicate poor agreement; 0.21 - 0.40, fair agreement; 0.41 - 0.60, moderate agreement; 0.61 - 0.80, good agreement; and 0.81 - 1.00, excellent agreement . Statistical analysis was performed by one author (j .- h.g) using spss statistical software, ver . The study had prior approval by our institutional review board and ethics committee, and all subjects provided informed consent prior to the study . A total of 24 elbows of 12 healthy, young volunteers were studied (six men, six women; age range 22 - 32 years). Nerves at the elbow region were analyzed, including the median nerve, ulnar nerve, and radial nerve . The exclusion criteria included general contraindications for mr imaging (mri), a prior history of trauma or surgery of the elbow, presence of rheumatoid arthritis, and presence of space - occupying lesions at the elbow . The mr images were acquired with a siemens magnetom verio 3 t mri scanner (siemens healthcare; erlangen, germany) using a 4-channal flex coil (flex small 4; siemens). The mri system s maximum field gradient amplitude was 45 mt / m with a slew rate of 200 t m s. during the scan session, the soft coil was placed around the elbow, and then a polymer brace was placed on the whole upper limb in order to restrain movement . The subject s upper extremity with the coil was immobilized with cushions, sandbags, and bandages . The elbow was then positioned to the center of the magnet bore . For imaging of the elbow, dti was performed using an echo planar inversion recovery (epir) sequence (tr = 15,300 ms; te= 92 ms; ti = 200 ms; fa = 90; etl = 1; fov = 250 250 mm; scan matrix = 128 128; average = 2) with 38 transversal slices of 3.0-mm thickness with no gap between the slices . Diffusion weighting with a b value of 1200 s / mm was applied in 20 encoding diffusion directions . In addition to the diffusion - weighted images, a single reference image without diffusion weighting (b = 0 s / mm) was acquired . For anatomical reference, a t1-weighted image (tr = 700 ms; te = 11 ms; fa = 150; etl = 3; thickness = 3 mm; gap = 0 mm; fov = 140 140 mm; scan matrix = 320 240; average = 1) was acquired . The scanning times for dti and t1-weighted image were 11 min 29 s and 2 min 1 s, respectively, for a total scan time of approximately 15 min . The raw dti images were transferred automatically to the siemens mr workstation (siemens syngo 3d). The x - axis represents the anterior posterior direction, the y - axis represents the left - right direction, and the z - axis represents the superior inferior direction . Based on the gradient table of the dti scan, direction 1 with coordinates (1, 0, 0) represents the anterior posterior direction . Therefore, according to skorpil et al . (15), the most suitable choice for single direction vr post - processing is direction 1, which is perpendicular to the nerves . The original dti images usually contained a series of t2-weighted images (b = 0 s / mm), which were generated as the first series in the result chart . The optimal choice (direction 1) was the second series generated in the result chart . During vr / mip reconstruction of images of direction 1, any high - signal noises of the veins or skin present in the reconstructed stereoscopic view were cut off in order to make the nerves as conspicuous as possible . One of the authors (y.y) was responsible for this part of the work . X - axis represents the anterior - posterior direction . Y - axis represents the left - right direction . Direction 1 with the coordinates (1, 0, 0) represents the anterior - posterior direction . The raw dti images were transferred to the siemens mr workstation (siemens syngo neuro 3d). Fiber tractography was then performed using a multiple seed roi technique, which employed fiber assignment by continuous tracking . Seed rois were drawn freehand at three anatomical locations (level to the supracondylar humerus, olecranon, and proximal radioulnar joint) on the raw dti images and reference t1-weighted images . Rois were confined precisely to the nerve border to avoid partial volume artefacts and to exclude any surrounding structure from the seed rois . In this study, the fractional anisotropy (fa) threshold value was 0.2 and the angulation tolerance was 30. the software s option step length was set to 0.47 mm . Another author (d .- s.j) was responsible for this part of the work . In order to compare the imaging quality among vr / mip and fiber tractography, we created a custom evaluation scale to assess each nerve in vr / mip and fiber tractography images . We set the scale based on whether the target nerve could be shown and the length of the nerve was complete as follows: score of 2, the target nerve (the median, radial, or ulnar nerve) at the elbow region was clearly shown and the entire length of the nerve was visible throughout the entire scanning section; score of 1, the target nerve at the elbow region was clearly shown, but its length was incomplete; score of 0, the target nerve was invisible . Two authors (w .- q.d and j .- h.g) were responsible for independently evaluating the imaging qualities according to this scale . One author (w .- q.d) evaluated the imaging qualities again 4 weeks later . The imaging quality scores were expressed as medians with ranges and mean with sd . For each nerve, scores of vr / mip reconstruction of the single direction and fiber tracking were compared using friedman test . Intraclass correlation coefficient (icc) analysis was used to compare the correlation of the imaging quality scores for each nerve among vr / mip and fiber tractography . Kappa values of 0 - 0.20 were considered to indicate poor agreement; 0.21 - 0.40, fair agreement; 0.41 - 0.60, moderate agreement; 0.61 - 0.80, good agreement; and 0.81 - 1.00, excellent agreement . Statistical analysis was performed by one author (j .- h.g) using spss statistical software, ver . All 24 elbows of the 12 young volunteers were tested, and stereoscopic displays of the nerves were obtained by using vr, mip, and fiber tractography post - processing methods (figure 1). For most elbows of volunteers, three main nerves could be seen clearly (figure 1), whereas only one nerve could be detected in other individual cases (figure 2). Owing to the relatively simple and direct operations enabled without requiring rois selection, the vr and mip post - processing methods were easier to perform compared to fiber tractography . Vr / mip reconstructions for single direction images and fiber tracking evaluation scores for each target nerve are listed in table 2, which did not differ significantly (p> 0.05). The imaging quality scores of fiber tractography and vr / mip were significantly correlated based on intraclass correlation coefficient (icc) analysis (icc ranged 0.709 - 0.901), which suggested good consistency between the scores obtained based on the fiber tractography and vr / mip for the same sample . The values of inter- and intraobserver agreements ranged 0.674 - 0.909, which showed good to excellent agreements . Abbreviations: ft, fiber tractography; icc, intraclass correlation coefficient; 1, interobserver value; 2, intraobserver value; ob1, observer 1; ob2, observer 2; ob2 sec ., observer 2 second time . In this study, we found that some images belong to a single direction (perpendicular to the nerve) after dti scan could be used for vr or mip reconstruction to display the peripheral nerves clearly . This method is similar to unidirectional diffusion - weighted mr neurography (dw - mrn) (16, 17). The difference of applied diffusion directions between dti and dw - mrn scans is that dti scan can select many non - parallel diffusion directions, and the data can be used for fiber tracking and measuring the fa, apparent diffusion coefficient (adc) value, while dw - mrn can choose fewer number of diffusion directions, increase the excitation frequency so the scan time is shorter, and signal - to - noise ratio increased for better mip or vr display . (17) indicated when they applied diffusion direction which is perpendicular to the nerve axons, the water molecular diffusion in the nerve was restricted, so the nerve tissue could all be imaged and showed high signal . We speculated that in dti scan, the data collected from the perpendicular direction may play an important role in calculating fa value, which is the key parameter for fiber tracking . Thus, vr and mip reconstruction of a single direction (anterior - posterior direction, perpendicular to the nerve) could predict the imaging quality of fiber tractography . The results of this study raise an important and essential question: under the same experimental conditions, why do the results show variation among different healthy volunteers? For some elbows of volunteers, three main nerves could be seen clearly (figure 1), whereas only one nerve could be detected in other cases (figure 2). Besides the reasons of anatomical characteristics at the elbow region and the limitations of the technique, one possible explanation for this variation is that slight movement of the limb is the key factor affecting imaging quality . In the image shown in figure 2, the ulnar nerve is located outside of the outline of the elbow . The median nerve and radial nerve are covered by the outline of the elbow, which suggests that the signal values of the nerve fibers were calculated incorrectly, thereby affecting fiber tracking . Vr and mip post - processing are more convenient to obtain compared to fiber tracking . The selection of rois is not required, which prevents subjective factors from influencing the results . The icc analysis result suggested that scores based on fiber tractography and vr / mip for the same samples were highly consistent . Therefore, vr and mip reconstruction can be used as a simple preview tool before performing complicated fiber tracking . During the dti scanning process using siemens magenetom verio 3 t mri scanner, the dti original images are generated in the order of the gradient table (table 1) from direction 1 to direction 20, and from average 1 to average 2 . The images belonging to direction 1 of average 1 will be generated within the first few seconds, and then vr and mip post - processing can be immediately performed to observe whether or not the nerve is displayed . If the nerves display, then, a scan with more directions and more averages should be continued for fiber tracking . If the nerves do not display, the scan should be terminated, and more measures for restricting movement of the elbow should be adopted . Using this method could help save a lot of time to the benefit of both the clinician and the patient . Some potential applications of vr and mip reconstruction include evaluation of the continuity and integrity of the nerve as well as detection of abnormal morphology, whereas the application of fiber tracking is specific to assessing the status of axons . According to sunderland s nerve injury classification (18), grade i, grade ii, and some grade iii injuries (axons or endoneurial tube disruptions) do not require surgical exploration, whereas grade iv - vi injuries (perineurium or epineurium damages, defects, or mixed injuries) do require surgical exploration as soon as possible . Therefore, vr and mip reconstruction might be able to show more details of damaged nerves, which unlike fiber tracking, do not only focus on the axons . Background tissue also can be reconstructed as reference landmark to show the damage site of nerve more clearly . Cubital tunnel syndrome is the second - most common nerve compression syndrome after carpal tunnel syndrome . Injury of the ulnar nerve at the elbow region is also common, because it is superficial and close to the bone surface . Stereoscopic display of the nerves at the elbow region can help the clinician understand the morphology of the target nerve . For example, in nerve entrapment patients, nerves can be detected as the features of increasing csa and flatten ratio (fr), which can be observed more easily in stereoscopic displays compared with conventional 2d images . By using these methods with the addition of fa and adc value measurements, dti appears to be a promising tool for future evaluations of the severity and rehabilitation of ulnar neuropathy . First, due to the limited choice of equipment and coil, the parameters and coil of the dti scan at the elbow region did not reach adequate optimization . Second, the measures adopted for restricting slight movement of the limb were not highly efficient . Stereoscopic displays of the peripheral nerve at the elbow region were achieved by using vr, mip, and fiber tracking post - processing methods based on raw dti images . The scores based on vr / mip and fiber tracking post - processing had good consistency for the same samples indicating that vr and mip reconstruction for single direction could be used as preview tools before fiber tracking to determine whether the raw images are satisfactory.
Removal of the corneal epithelium is an essential component of riboflavin - ultraviolet - a (uva) corneal collagen cross - linking (cxl) therapy to permit the penetration of riboflavin into the corneal stroma . Failure to achieve adequate stromal absorption of riboflavin may impair the efficacy of the cross - linking process; until now epithelial removal is performed mechanically . Excimer laser phototherapeutic keratectomy (ptk) is a well - known surgical technique that has successfully been used in the management of superficial corneal pathology, such as anterior corneal dystrophies, degenerations, and the treatment of keratoconus nodules . Transepithelial ptk (t - ptk) uses an excimer laser ablation in order to remove the epithelium and smooth the anterior irregular cornea . In this paper, we present a case of a keratoconic patient who underwent epithelial removal with t - ptk using a solid - state laser system followed by cxl treatment with riboflavin and uva irradiation . A twenty - four - year - old male with progressive keratoconus presented to our institute for consultation . At the time of the examination, uncorrected visual acuity (ucva) was 20/25 in the right and 20/63 in the left eye and best spectacle - corrected visual acuity (bscva) was 20/20 (manifest refraction plano1.00 15) in the right eye and 20/40 (manifest refraction1.25 4.00 125) in the left eye . A trial with a rigid gas - permeable contact lens demonstrated improvement in visual acuity of 20/20 in his left eye . Keratometric readings were 42.60 24/43.52 114 at the right and 47.51 46/ 56.98 136 at the left eye . In order to assess corneal thickness, a pachymetric map measurement was performed in the right (481 m central, 639 nasal, 550 temporal, 587 superior, and 547 inferior) and left (462 m central, 581 nasal, 569 temporal, 611 superior, and 541 inferior) eye by ultrasound pachymetry (sonogage, cleveland, ohio, usa). Epithelial thickness at the apex of the cone was 41 m in the left eye . A solid - state laser system with a wavelength of 213 nm (pulzar z1, customvis, perth, wa) was used for the procedure . The wavelength is generated using a major nd: yag laser system of 1064 nm, and through special cultivated crystals the 213 nm is finally used . The t - ptk ablation was performed in an 8.0 mm zone in an intended depth of 50 m . There was a correlation between the localized ablated stroma and the preoperative topography (figure 1). After t - ptk, riboflavin 0.1% solution was instilled every 3 minutes for approximately 30 minutes . Penetration of the cornea and presence of riboflavin in the anterior chamber (riboflavin shielding) were monitored by slit - lamp examination . Uva irradiation was performed using a commercially available uva optical system (uv - x illumination system version 1000, zurich, switzerland) with a light source consisting of an array of uv diodes (365 nm) with a potentiometer in series to allow regulation of voltage . Before treatment, intended irradiance of 3.0 mw / cm (5.4 j / cm surface dose after 30 minutes) was calibrated using the uv - a light meter yk-34uv (lutron electronic) which is supplied with the uv - x device . Irradiance was performed for 30 minutes, corresponding to a dose of 5.4 j / cm . During treatment, riboflavin solution was applied every 5 minutes to saturate the cornea with riboflavin . At the end of the procedure, a silicon - hydrogel bandage contact lens (lotrafilcon b, air optix, ciba vision14.0 mm diameter, 8.6 base curvature, and dk = 140 barrers) was applied until full reepithelialization . Postoperative medication included diclofenac sodium 0.1% (denaclof, novartis) for 2 days as well as antibiotic / corticosteroid (tobramycin / dexamethasone) drops (tobradex, alcon laboratories, inc .) Contact lens was removed at the fifth postoperative day, and no signs of edema or inflammation were cited by slit - lamp biomicroscopy . After the removal of the contact lens, the patient received corticosteroid drops (fml, fluorometholone 0.1%, falcon pharmaceuticals), tapering for the next 15 days, and he was encouraged to use artificial tears at least six times per day for 3 months postoperatively . One month after the combined t - ptk and cxl procedure, both ucva and bscva were improved at 20/32 and 20/25, respectively (manifest refraction 0.50 2.00 135). Corneal topography revealed a significant improvement (figure 2) which remained stable during the six - month follow - up period . Excimer laser phototherapeutic keratectomy (ptk) is a well - known surgical technique that has successfully been used in the management of superficial corneal pathology, such as anterior corneal dystrophies and degenerations . Demonstrated the safety and efficacy of ptk in the treatment of keratoconus nodules and the improvement of contact lens tolerance in these patients . T - ptk uses an excimer laser ablation to remove the epithelium and smooth the anterior irregular cornea . In this paper, the patient underwent t - ptk using a solid - state laser system before cxl with riboflavin and uva irradiation . The aim of t - ptk was epithelial removal and anterior cornea smoothening in order to decrease the irregular astigmatism . Reinstein has reported that in a study of 38 keratoconic eyes, an inferotemporal region of thin epithelium surrounded by epithelial thickening was found; a pattern coincided with the cone on topography . According to this keratoconic pattern, an intended ablation depth of 50 m has been chosen in order to use the patient's own epithelium as a masking agent allowing laser ablation to take place on the apex of the cone to regularize the stromal surface (figure 1). The significant improvement in visual acuity, especially in ucva, from the first postoperative month should be the result of t - ptk which decreases corneal irregularities as shown at the topography one month postoperatively (figure 2). Cxl after mechanical epithelial removal could potentially improve patient's ucva but not at the first postoperative month and in this amplitude . A possible limitation of this approach could be the decrease of corneal tissue by t - ptk at the cone apex before cxl . In conclusion, epithelial removal with t - ptk before cxl could improve patient's visual outcomes . Longer follow - up is necessary in order to evaluate the outcomes of this combined procedure.
Although first described by starr in 1886, telescopic copings were initially introduced as retainers for removable partial dentures (rpd) at the beginning of the 20 century . Because of its resemblance to the collapsible optical telescope, this system of double crowns, which can be fitted into each other, became known as the telescopic denture . Telescoping refers to the use of a primary full - coverage casting (coping / male telescopic portion) luted to the prepared tooth with a secondary casting (superstructure / secondary crown / female telescopic portion), which is a part of the denture framework and is connected by means of interfacial surface tension over the primary casting . Alternate descriptive terms are double crown, crown and sleeve coping, or konuskrone, which is a german term for a cone - shaped design . They act by transferring forces along the direction of the long axis of the abutment teeth and provide guidance, support, and protection from movements that might dislodge the rpds . Telescopic crowns can also be used as indirect retainers to prevent dislodgement of the distal extension base away from the edentulous ridge . Telescopic copings have been used for several years in oral rehabilitation of patients with advanced periodontal disease . Patients with periodontal disease undergoing prosthetic reconstruction often present with teeth with minimal supportive tissue and increased tooth mobility . Therefore, it is extremely important for the prosthesis not to cause periodontal destruction or worsen an existing periodontal condition . They are distinguished from each other by their retention mechanisms: cylindrical crowns that exhibit retention through friction fit of parallel - milled surfacesconical crowns or tapered telescopic crowns that exhibit friction only when completely seated using a wedging effect . The magnitude of the wedging effect is mainly determined by the convergence angle of the inner crown; the smaller the convergence angle, the greater is the retentive forcedouble crown with clearance fit (hybrid telescope or hybrid double crown) exhibits no friction or wedging during insertion or removal . Cylindrical crowns that exhibit retention through friction fit of parallel - milled surfaces conical crowns or tapered telescopic crowns that exhibit friction only when completely seated using a wedging effect . The magnitude of the wedging effect is mainly determined by the convergence angle of the inner crown; the smaller the convergence angle, the greater is the retentive force double crown with clearance fit (hybrid telescope or hybrid double crown) exhibits no friction or wedging during insertion or removal . A 65-year - old male reported to the department of prosthodontics, subharti dental meerut, with a chief complaint of loose dentures and soreness of the mouth . Patient gave a medical history of diabetes mellitus since 15 years and hypertension since 23 years . He gave a dental history of wearing the same maxillary denture and mandibular rpd since 10 years, which gradually became loose . A preliminary examination revealed that the patient had missing 31, 32, 37, 41, 42, 47 and completely edentulous maxillary arch . Also there was grade ii furcation involvement of 36 and grade i furcation involvement of 46 . Diagnostic impressions were made using irreversible hydrocolloid impression and an inter - occlusal bite registration was taken . The impressions were poured and the diagnostic models were mounted on a semi adjustable articulator . The opg revealed generalized horizontal bone loss up to the middle 1/3 of the roots, and bone loss up to apical 1/3 was seen in 36 and 46 . Also, furcation involvement was seen in 36 and 46, thus indicating severe periodontitis . It was decided to extract both the mandibular molars due to advanced periodontitis followed by a thorough oral prophylaxis and a flap surgery in 35, 45 regions to decrease the pocket depth . After ascertaining the decrease in tooth mobility and pocket depth, prosthetic rehabilitation was carried out . During the definitive intra - oral examination the potential abutments were evaluated clinically to determine their periodontal condition, pockets, mobility, caries, old restorations, vitality, abrasions, and supra - eruption [figure 2]. Surveying of patient cast the diagnostic findings were as follows: a discrepancy in the occlusal plane was noted due to supra - eruption of 33, 45the potential abutments had varying paths of insertionthe mandibular teeth were lingually inclinedthe abutments had a large crown: root ratio . A discrepancy in the occlusal plane was noted due to supra - eruption of 33, 45 the potential abutments had varying paths of insertion the mandibular teeth were lingually inclined the abutments had a large crown: root ratio . It was decided to prosthetically rehabilitate this patient with a telescopic denture for the mandibular arch and to use a complete denture for the maxillary arch . Intentional rcts were performed on 33, 34, 35, 43, 44 and 45 . Tooth preparation was done by preparing a chamfer finish line of 0.7 mm and axial wall heights of 4 mm in 33, 43, and 6 mm in 34, 35, 44, and 45 with a taper of approximately 8 - 10. after the mouth preparation in the mandibular arch, gingival retraction was done and a final impression was made with addition silicone using the putty - wash technique . The first master model was prepared from the impression for fabrication of the primary copings . This was followed by making an interocclusal record using putty and a face bow transfer . In the laboratory, the wax patterns were prepared for the primary copings on 33, 34, 35, 43, 44, and 45 . The patterns were milled to obtain a frictional surface for retention and then cast in to nickel chrome alloy (high chrome soft). Once the primary copings were evaluated for fit [figure 3], the copings were luted with temporary cement (zinc oxide eugenol) and an overimpression was made using the medium viscosity addition silicone impression material and the second master model was made [figure 4]. Bite registration was repeated and the models with the copings were mounted on a semi - adjustable articulator using the same face bow record . In the laboratory, the copings on the second master model were milled with a parallelometer to obtain a milled surface of minimum 4 mm for friction . The second master model together with the primary copings was duplicated and the refractory model was prepared . The cast partial framework was waxed up, which was then cast using a base metal alloy (cobalt - chrome) with the secondary coping overlay of the primary coping . After evaluating the fit of the framework in the mouth [figure 5], it was used as a carrier for cementing the primary copings in place . The primary copings were luted with glass ionomer luting cement (type i; gc fuji). A wax rim was prepared on the framework and acrylic teeth were set with the same shade as were veneered over the secondary coping . After verification of esthetics, function, and phonetics, the mandibular denture was processed [figure 6]. The completed prostheses were evaluated for function, esthetics, and phonetics [figure 7]. Primary coping fabrication master cast after lutting of primary coping metal framework with secondary coping intraoral view of final prosthesis a telescopic overdenture was chosen for this patient because of its good retentive and stabilizing properties, rigid splinting action, and better distribution of stresses . Other treatment options included extraction of the remaining teeth, followed by a conventional complete denture . This was not selected because extraction would have decreased the available support and proprioception provided by the teeth and their periodontal ligaments . Implant supported prosthesis was not opted for as the patient was medically compromised and also because of the cost involved in the procedure . Clinical longevity of a telescopic overdenture is essentially influenced by the applied restorative concept of connecting the removable denture with the remaining teeth . With regard to the number, alignment, and periodontal status of the remaining teeth, the clinician needs to select the appropriate retainer for a long - term successful restoration . In this situation, a total of 6 abutments for telescopic copings were used to support the overdenture, thus creating a quadrilateral configuration . It has been reported that at least two abutment teeth should be splinted when attachment prostheses are used to make the stress patterns more favorable . The advantage of opting for this treatment plan was to distribute the load among the remaining periodontally weakened teeth, thus acting as a rigid splint . This option was thought to have a better prognosis for the remaining teeth as well as to have a more retentive prosthesis . The recommended alloys for fabrication of copings are the high noble (ada type iv). Ag - au - pd alloys have better precision and better retention, but are technique sensitive and costly . Base metal alloys (cr - co) can also be used because they have low thermal conductivity, thus the patient does not experience unpleasant thermal sensation caused by excessive tooth preparation . The advantages and disadvantages of telescopic overdentures are summarized as follows: creation of a common path of insertioneasy to perform routine oral hygienerigid splinting actiondistribution of stresses to the abutment teethprovision of suitable abutments for rpds even when the remaining teeth are periodontally compromisedmuch easier insertion and removal for the patientaccommodates future changes in the treatment planpsychologically well - tolerated by patients . Creation of a common path of insertion easy to perform routine oral hygiene rigid splinting action distribution of stresses to the abutment teeth provision of suitable abutments for rpds even when the remaining teeth are periodontally compromised much easier insertion and removal for the patient accommodates future changes in the treatment plan psychologically well - tolerated by patients . Increased costcomplex laboratory proceduresextensive tooth reduction requiredincreased number of dental appointmentsdifficulty in achieving estheticsretention diminishes after repeated insertion / separation cyclesreadjustment of retentive forces is difficult . Complex laboratory procedures extensive tooth reduction required increased number of dental appointments difficulty in achieving esthetics retention diminishes after repeated insertion / separation cycles readjustment of retentive forces is difficult . It was decided to prosthetically rehabilitate this patient with a telescopic denture for the mandibular arch and to use a complete denture for the maxillary arch . Intentional rcts were performed on 33, 34, 35, 43, 44 and 45 . Tooth preparation was done by preparing a chamfer finish line of 0.7 mm and axial wall heights of 4 mm in 33, 43, and 6 mm in 34, 35, 44, and 45 with a taper of approximately 8 - 10. after the mouth preparation in the mandibular arch, gingival retraction was done and a final impression was made with addition silicone using the putty - wash technique . The first master model was prepared from the impression for fabrication of the primary copings . This was followed by making an interocclusal record using putty and a face bow transfer . In the laboratory, the wax patterns were prepared for the primary copings on 33, 34, 35, 43, 44, and 45 . The patterns were milled to obtain a frictional surface for retention and then cast in to nickel chrome alloy (high chrome soft). Once the primary copings were evaluated for fit [figure 3], the copings were luted with temporary cement (zinc oxide eugenol) and an overimpression was made using the medium viscosity addition silicone impression material and the second master model was made [figure 4]. Bite registration was repeated and the models with the copings were mounted on a semi - adjustable articulator using the same face bow record . In the laboratory, the copings on the second master model were milled with a parallelometer to obtain a milled surface of minimum 4 mm for friction . The second master model together with the primary copings was duplicated and the refractory model was prepared . The cast partial framework was waxed up, which was then cast using a base metal alloy (cobalt - chrome) with the secondary coping overlay of the primary coping . After evaluating the fit of the framework in the mouth [figure 5], it was used as a carrier for cementing the primary copings in place . The primary copings were luted with glass ionomer luting cement (type i; gc fuji). A wax rim was prepared on the framework and acrylic teeth were set with the same shade as were veneered over the secondary coping . After verification of esthetics, function, and phonetics, the mandibular denture was processed [figure 6]. The completed prostheses were evaluated for function, esthetics, and phonetics [figure 7]. Primary coping fabrication master cast after lutting of primary coping metal framework with secondary coping intraoral view of final prosthesis a telescopic overdenture was chosen for this patient because of its good retentive and stabilizing properties, rigid splinting action, and better distribution of stresses . Other treatment options included extraction of the remaining teeth, followed by a conventional complete denture . This was not selected because extraction would have decreased the available support and proprioception provided by the teeth and their periodontal ligaments . Implant supported prosthesis was not opted for as the patient was medically compromised and also because of the cost involved in the procedure . Clinical longevity of a telescopic overdenture is essentially influenced by the applied restorative concept of connecting the removable denture with the remaining teeth . With regard to the number, alignment, and periodontal status of the remaining teeth, the clinician needs to select the appropriate retainer for a long - term successful restoration . In this situation, a total of 6 abutments for telescopic copings were used to support the overdenture, thus creating a quadrilateral configuration . It has been reported that at least two abutment teeth should be splinted when attachment prostheses are used to make the stress patterns more favorable . The advantage of opting for this treatment plan was to distribute the load among the remaining periodontally weakened teeth, thus acting as a rigid splint . This option was thought to have a better prognosis for the remaining teeth as well as to have a more retentive prosthesis . The recommended alloys for fabrication of copings are the high noble (ada type iv). Ag - au - pd alloys have better precision and better retention, but are technique sensitive and costly . Base metal alloys (cr - co) can also be used because they have low thermal conductivity, thus the patient does not experience unpleasant thermal sensation caused by excessive tooth preparation . Moreover, they are easy to fabricate and more economical . The advantages and disadvantages of telescopic overdentures are summarized as follows: creation of a common path of insertioneasy to perform routine oral hygienerigid splinting actiondistribution of stresses to the abutment teethprovision of suitable abutments for rpds even when the remaining teeth are periodontally compromisedmuch easier insertion and removal for the patientaccommodates future changes in the treatment planpsychologically well - tolerated by patients . Creation of a common path of insertion easy to perform routine oral hygiene rigid splinting action distribution of stresses to the abutment teeth provision of suitable abutments for rpds even when the remaining teeth are periodontally compromised much easier insertion and removal for the patient accommodates future changes in the treatment plan psychologically well - tolerated by patients . Increased costcomplex laboratory proceduresextensive tooth reduction requiredincreased number of dental appointmentsdifficulty in achieving estheticsretention diminishes after repeated insertion / separation cyclesreadjustment of retentive forces is difficult . Complex laboratory procedures extensive tooth reduction required increased number of dental appointments difficulty in achieving esthetics retention diminishes after repeated insertion / separation cycles readjustment of retentive forces is difficult . Although fixed restoration provides favorable conditions for preservation of oral function, telescopic overdenture may be considered as another option, combining good retentive and stabilizing properties with a splinting action . The telescopic system may therefore be seen as providing suitable abutments for overdenture even when the remaining teeth are compromised . For other prostheses, excellent oral hygiene maintenance is essential for an optimal prognosis . With telescopic construction, apart from the splinting of the abutment teeth with the telescopic system, the gingival tissues are easily accessible around the entire marginal circumference of the abutment, thus permitting easy home care and oral hygiene . However, correctly implemented plaque control is fundamental in the prevention of recurrence of gingivitis.
The unique ability of toxoplasma gondii to multiply in virtually all the nucleated cells of the host body and its subsequent encystment in the various tissues, coupled with its wide host range consisting of all the warm blooded animals including humans, makes the parasite a matter of serious concern for both animal and human health in the modern times . In humans, the parasite is known to cause births with congenital anomalies (1, 2) coupled with ocular involvement (3, 4), acute encephalitis (5) particularly in immunocompromised individuals suffering from aids, transplant recipients and those under cancer therapy (6) while in animals particularly small ruminants, the parasite accounts encephalitis (7), abortions (8), neonatal infections (1, 2) coupled with weight loss, hepatomegaly and enlarged kidneys (9). The human rh strain, originally isolated from a boy with his name initials rh (10), is considered as the referral virulent strain of the parasite . The inability of this strain to form either oocysts in definitive host or bradyzoites in intermediate hosts (11) due to its prolonged passage in the mice (12) further compels the researchers to opt for this as the referral strain for various immunological and molecular biological studies . Rats and mice are routinely used as biological models for isolation, passage and diagnosis of t. gondii across the globe (12). Therefore, an attempt was made to study the behavior, course of toxoplasmosis and its actual kinetics in mice using the standard rh strain . Ten inbred swiss albino mice of either sex, 6 - 12 weeks of age and weighing about 25 - 30 grams were obtained from the laboratory of animal resource section, indian veterinary research institute (ivri), india . Animals were kept in polypropylene cages and acclimatized for a period of 15 days prior to experimentation under standard temperature, humidity and light cycle conditions . Animals were fed on a balanced diet ad libitum, consisting of crushed 62% wheat, 30% maize, 7% wheat bran and 1% common salt . Tachyzoites of t. gondii (rh strain) were obtained from the cryopreserved stock maintained at the divisional laboratory, indian veterinary research institute . The parasite strain had been maintained in the divisional laboratory in the cryopreserved form for over a decade by continuous serial passage in murine models since time over and over again . The cryopreserved stock of the rh strain was firstly inoculated into the mice through intraperitoneal route . The infected mice were later euthanized by chloroform anaesthesia upon the development of symptoms of toxoplasmosis . After cutting the abdominal skin aseptically, 7 - 10 ml of phosphate buffered saline (ph 7.2) was inoculated slowly into the peritoneal cavity and the peritoneal lavage was aspirated without causing any damage to the other organs . Enumeration was carried out as per the standard protocol (13). After adjusting the live cell count, behavior of mice along with their feeding pattern and general body condition coupled with symptoms of acute toxoplasmosis were regularly monitored and recorded . Similarly, ten mice were intraperitoneally injected with sterile pbs and served as uninfected negative control . The experiments to the laboratory animal were done as per the approval of university ethical committee . Ten inbred swiss albino mice of either sex, 6 - 12 weeks of age and weighing about 25 - 30 grams were obtained from the laboratory of animal resource section, indian veterinary research institute (ivri), india . Animals were kept in polypropylene cages and acclimatized for a period of 15 days prior to experimentation under standard temperature, humidity and light cycle conditions . Animals were fed on a balanced diet ad libitum, consisting of crushed 62% wheat, 30% maize, 7% wheat bran and 1% common salt . Tachyzoites of t. gondii (rh strain) were obtained from the cryopreserved stock maintained at the divisional laboratory, indian veterinary research institute . The parasite strain had been maintained in the divisional laboratory in the cryopreserved form for over a decade by continuous serial passage in murine models since time over and over again . The cryopreserved stock of the rh strain was firstly inoculated into the mice through intraperitoneal route . The infected mice were later euthanized by chloroform anaesthesia upon the development of symptoms of toxoplasmosis . After cutting the abdominal skin aseptically, 7 - 10 ml of phosphate buffered saline (ph 7.2) was inoculated slowly into the peritoneal cavity and the peritoneal lavage was aspirated without causing any damage to the other organs . After adjusting the live cell count, ten healthy mice of were injected intrapertitoneally with 100 virulent tachyzoites . Behavior of mice along with their feeding pattern and general body condition coupled with symptoms of acute toxoplasmosis were regularly monitored and recorded . Similarly, ten mice were intraperitoneally injected with sterile pbs and served as uninfected negative control . The experiments to the laboratory animal were done as per the approval of university ethical committee . The infected mice appeared more or less same as before infection up to 36 - 48 hours . Thereafter, symptoms like dullness reduced feed and water intake suggestive of anorexia, disinclination to move on their own and moved only when excited through external stimuli and raising of hair coat started to appear by day 3 post infection (pi) (fig . They got hurdled at the centre of the cage with closed eyes, tucked - up abdomen and arched back . From day 3 pi ascities resulting from pendulous abdomen began to appear and by day 4 pi all the mice developed signs of ascities and were seen having large pendulous abdomen resulting into respiratory distress as suggested by tachypnoea and gasping movement was seen (fig . 1, 2). Some of the mice with pendulous abdomen were found resting or hanging by keeping one or both the forelegs on the walls of animal cage or on the nozzle of the water bottle or on other lying mouse . By the end of day 4 pi few of the mice begin to die and by day 6 pi all the mice were dead because of acute toxoplasmosis . The pertioneal lavage collected from mice revealed creamy, cloudy, watery liquid which upon giemsa staining revealed a large number of crescent shaped tachyzoites (fig . Few of the macrophages were also seen which were filled by tachyzoites from inside, in some of the peritoneal lavages . While the uninfected mice remained healthy and did not show any of these abnormal signs and remained fully alert and active throughout the experiment . Toxoplasmosis is one of the most common zoonotic parasitic invasions worldwide, caused by the intracellular protozoan parasite t. gondii . The acute invasion of the parasite is a transient stage which is usually followed by chronic invasion when the parasites resides within tissue cysts localized mainly in the central nervous system, muscle, and eye tissues (14). It has proven that t. gondii invasion may alter the indefinite host natural defensive behavior in order to increase the risk of rodent predation by cats, the definite parasite hosts (15, 16). This could be attributed to the presence of the parasite in the specified regions of the brain in rodents affects its physiological functions often results in such behavioral changes which are supposed to favor the spread of the parasite to the definite host (17). These behavioral changes are highly specific as parasite invasion blocks rodent aversion toward cat predator odor (18, 19) but do not affect recognition of the non - feline predator scent (20). Rh strain of was isolated in usa (21) and has since been passaged continuously in laboratories either in vitro or in vivo throughout the world . Researchers prefer to use it as a standard referral stain mainly due to its inability to form bradyzoite stage (11), lethal effects on mice and good yield of tachyzoites in short time intervals . In the present study as all the mice experimentally infected with acute toxoplasmosis showed increased body size and pendulous abdomen . This could be explained because of significant increase in brain, spleen and body weight gain during acute phase of toxoplasmosis (22). The increase in brain mass can further be explained on the grounds of induction of inflammatory processes, which is further driven by many pro - inflammatory cytokines like il-1 and il-6 produced in brain during acute infection (23) coupled with accumulation of inflammatory cells into the brain tissue (24). Spleen is involved with the development of specific immunological response against the parasite antigens owing to its role as the predominant secondary lymphoid organ . This leads to intense accumulation of inflammatory cells, which is further reflected by pronounced increase in spleen weight (22). Some of the mice, particularly those with huge pendulous abdomen, were found resting or hanging by keeping one or both the forelegs on the walls of animal cage or on the nozzle of the water bottle or on other lying mouse . Initially all the infected mice showed exploratory behavior but as the symptoms of acute toxoplasmosis developed infected animals exhibited decreased climbing and rearing especially in the central part of the arena and usually remained confined to the periphery of cage in the later stages . It is a matter of common observation that in unfamiliar surroundings mice first explore the peripheral parts and then they move to the central, open part of the arena (25) as this is the most basic means of gathering information about the environment used by nocturnal species with poor vision (26). The change in behavior might be attributed to t. gondii infection that inhibited the natural course of exploration (22, 27). This further strengthens the manipulation theory stating that the parasite alters the behavior of the intermediate host to increase a chance of predation by the definitive host . Noteworthy, most of these behavioral changes were mostly pronounced during acute invasion (22, 27). Nevertheless the exact mechanism involved in behavior changes is yet to be fully understand; nevertheless factors like disturbance in the level of neurotransmitters in the brain (28, 29) can prove to be vital plot in this regard . Likewise behavioral changes are also observed in humans like differences personality features between toxoplasma infected (30), associations with schizophrenia (31), parkinson disease (32), epilepsy (33), or psychosis (34). The study has generated some important data related to behavioral alterations because of the effect of acute infection of parasite on normal behavior of infected intermediate host . A lot of research is still warranted to be done regarding behavioral alterations and to investigate through well planned studies the extent and true nature of behavioral alterations in toxoplasmosis and hence fortifying the research to adopt suitable strategies for coping up these alterations in infected individual at the behavioral level for animals in general and humans in particular.
A 47-year - old female was referred to department of oral and maxillofacial surgery with a chief complaint of pain and swelling of the lower lip . Intra - oral clinical examination revealed a well circumscribed swelling measuring 6 cm 6 cm in dimension i.r.t 41, 42, 43 . It was covered with a thin normal looking mucosa, no inflammatory or erythematous areas seen around the swelling . Palpation showed a firm mass, bony hard in consistency, not fixed to the underlying tissue . After taking consent from the patient, surgical excision was planned . The patient underwent an uneventful excision under local anesthesia and the excised specimen was sent for a histopathological examination . Grossly, the specimen consisted of a gray - white mass measuring 1 cm 1 cm in dimension . Serial histological section at 6 each were made, and then, stained with hematoxylin and eosin technique . Microscopic observation revealed parakeratinized stratified squamous epithelium cells with an underlying stroma is composed of dense connective tissue with few blood capillaries [figures 1 and 2]. H and e, stained section shows mature lamella lined by stratified squamous epithelium h and e, stained section shows hematopoietic areas the lesion is composed of mature lamellar bone, osteocytes, and adipocytes . The specimen is also showing hematopoietic areas . With these histological findings along with clinical findings suggestive of the osseous choristoma of the labial mucosa a choristoma is defined as a histologically normal tissue proliferation or nodule of a soft tissue type not normally found in the anatomic site of proliferation . These include cartilage, bone, glial tissue, gastric mucosa, and tumor - like masses of sebaceous glands . Most frequently observed choristomas of the oral cavity are those that consist of bone . The age range for reported cases of intraoral choristomas is between 8 and 73 years . Most intraoral choristomas develop in dorsal surface of the tongue around the foramen caecum, but lesions have also been reported in the tongue, buccal mucosa, and the lingual alveolus of the mandible . Clinically, it grows as a pedunculated lesion or firm nodular with size of mass may range between 0.5 and 2.0 cm in size . Most patients are unaware of the lesion, but may feel the symptoms of pain, dysphagia, or sometimes may have choking sensation, and nausea also have been reported in some cases . In the literature (pubmed search revealed), 88 cases have been described regarding the osseous choristoma in regard to tongue and buccal mucosa in common, but not in relation to the labial mucosa . The osseous choristoma to arise in the age group of 1262 years of age with the lesion occurring more frequently in the fifth decade of life . It has a slightly predisposition to occur more commonly in females than males with a ratio of 1.5:1 . Although there are various theories regarding the etiology of ocs, their exact origins remain mysterious and both developmental and trauma origins have been reported . Histologically, these are choristoma consists of a well - circumscribed mass of viable lamellar bone with a well - developed haversian canal system, surrounded by dense, fibrous connective tissue, and covered with stratified squamous epithelium . The lesion reported here is a rarest type of osseous choristoma seen in the labial mucosa and is the first ever reported a case of osseous choristoma originating in the labial mucosa . The number of reported cases or literatures is insufficient, to review this particular type of osseous choristoma . Awareness of this entity is required so that an early and accurate diagnosis can be made.
To report on a case of keratoconjunctivitis with periorbital edema after accidental exposure to undiluted juice of ecballium elaterium fruit during the ripening season of this plant . Keratoconjunctivitis with descemet's membrane folds and superficial upper corneal well - defined edematous areas were noted after an ecballium elaterium fruit burst and its juice splashed into the patient's left eye . Prompt administration of antibiotic and steroid eye drops along with per os antihistamine therapy, resulted in quick regression of symptoms . This report demonstrates the toxic effects of this herb to the eye and also that prompt therapy is effective . A 70-year - old male farmer presented to the emergency department with periorbital pain, periorbital and conjunctival edema, epiphora, hyperemia, irritation, and blurred vision from his left eye after inadvertent exposure to undiluted juice of ecballium elaterium fruit some hours earlier . The symptoms began within minutes after the herb's fruit bursted and juice splashed into the eye (fig . Slit lamp examination revealed corneal epithelial micro - erosions, corneal superficial well - defined edematous areas mainly in the peripheral upper quadrants (fig . The patient was treated promptly with eye irrigation, topical steroids for periorbital edema, combination of antibiotic and steroid eye drops along with antihistamine tablets . Within four days from the initiation of treatment, exposure to ecballium elaterium fruit juice, mainly in its undiluted form, may cause irritation of the mucous membrane . Elaterium is the dried sediment that deposits in the juice of the fruits of ecballium elaterium . It is a powerful cathartic and contains up to 30% of elaterin, which is the active ingredient . The toxic dose is 5 mg and the fatal dose 0.6 g. published adverse reactions related to the toxic effect of this remedy include localized swelling of the uvula, the nasal mucosa and others sites of the upper respiratory tract of allergy - prone patients . Also, there is a report of fatal cardiac and renal failure probably related to ecballium elaterium juice intake . Regarding the eye, kocak et al . Reported a case of late - onset diffuse lamellar keratitis 11 months after lasik, when an ecballium elaterium herb fruit burst and splashed into the patient's eye . Also, raikhlin - eisenkraft and bentur report 6 cases with ocular exposure presented with conjunctivitis, corneal edema, and erosion . This report demonstrates the toxic effect of ecballium elaterium juice to the eye and also, that prompt therapy results in rapid regression of symptoms.
Since the initial studies by murugan et al . In 2007, li7la3zr2o12 (llzo) has received much scientific attention as a solid electrolyte for beyond li ion battery llzo provides a high li - ion conductivity (10 to 10 s cm at ambient temperature) and a li transference number approaching 1, superior chemical stability against high voltage cathodes, and electrochemical inertness in a wide potential window of up to 6 v. in particular, its stability against li - metal as well as its thermal and mechanical robustness makes llzo garnet exceptionally well - suited to be used as a protecting layer for li - metal - based batteries . For pure llzo, two structural polymorphs are described in the literature . A low - temperature tetragonal modification with a completely ordered arrangement of li crystallizes in space group (sg) i41/acd (no . 142). This structure type is described as garnet - related framework with two types of dodecahedral lao8 polyhedra (8b and 16e) and zro6 octahedra (16c). Li occupies three different sites: the tetrahedral 8a site as well as distorted octahedral 16f and 32 g sites . In contrast to the tetragonal modification, the cubic high - temperature modification exhibits a disorder in the li distribution . This cubic modification shows sg ia-3d (no . 230) and has the garnet structure composed of a framework of 8-fold coordinated lao8 dodecahedra (24c) and 6-fold coordinated zro6 octahedra (16a). Li is located at interstitial sites, showing tetrahedral (24d), octahedral (48 g), and distorted 4-fold (96h) coordination . For use as li - ion conductors, the cubic modification is much more desirable as its li - ion conductivity is 2 orders of magnitude higher than for the tetragonal modification . The cubic modification of pure llzo is not stable at room temperature (rt). However, it can be stabilized at rt by the introduction of supervalent cations, which cause a reduction of the li content that leads to the introduction of additional vacancies at the li sites . The stabilization of the cubic modification was originally achieved by the incorporation of al . Further research led to the discovery of other cations that are capable to stabilize the cubic modification . Among these, ga turned out to be a promising candidate which has been studied extensively . Ga is incorporated at the li sites according to 3 li ga + 2 li, i.e., creating two vacant sites, li, in the li - sublattice . Ga - stabilized llzo is characterized by a li conductivity of 1.3 ms cm at rt, which is twice as high as for llzo stabilized with al . The reason for the higher li - ion conductivity of ga - stabilized llzo is, however, not yet fully understood . Because of their blocking effect, some of these ions are suspected to hinder the long - range li - ion transport and, therefore, to reduce li - ion conductivity . (llzto) with those of li6.15la3zr1.75ta0.25m0.2o12 (m = al, ga); according to their study pure llzto shows the highest li - ion conductivity, followed by llzto: ga and llzto: al . On the basis of the crystal chemical considerations, they attributed this behavior to the different site preference of al and ga . In particular, the larger ga prefers the 96h site, which seems to be less hindering for long - range li - ion transport, compared to al, located at the 24d site, which is a junction for li - ion diffusion . However, the site preference of ga is still under discussion as ga nuclear magnetic resonance (nmr) spectroscopy studies revealed different results . (2015), using very high magnetic fields (21.1 t), showed that the site preference of ga and al to occupy 24d and 96h voids in samples crystallizing with ia-3d is practically the same . Thus, despite all efforts, there is, so far, no satisfying explanation why some of the ga - stabilized llzo samples presented in literature show higher ionic conductivities . Investigations on ga - stabilized llzo single crystals might help us to shed light on this question . Up to now, very little research has been performed on llzo single crystals . The synthesis of applicable llzo single crystals is delicate . To the best of our knowledge, no single - crystal x - ray diffraction (sc - xrd) study on ga - stabilized llzo has been published yet . In the present study, another cubic modification of llzo, showing the acentric sg i-43d, has been observed for ga - stabilized llzo for the first time . Coarse - grained ga - stabilized llzo samples were synthesized via solid - state reaction and characterized by a rich portfolio of techniques including x - ray powder diffraction (xrpd), single - crystal x - ray diffraction (sc - xrd), neutron powder diffraction (npd), scanning electron microscopy (sem)/ backscattered electron (bse) imaging, energy - dispersive x - ray spectroscopy (edx), and li nmr spectroscopy . A series of li73xgaxla3zr2o12 with intended ga contents xga = 0.10, 0.15, 0.20, 0.30, 0.40, 0.50, 0.60 pfu was synthesized by high - temperature sintering in air . For reason of comparison, also one sample of al - stabilized llzo with an intended al content xal of 0.20 al pfu (li6.4al0.2la3zr2o12) was prepared under the same conditions . Results obtained by cheng et al . Concerning the densification and coarsening of llzo samples were taken into consideration for this synthesis . Li2co3 (99%, merck), ga2o3 (99.0%, aldrich), al2o3 (99.5%, aldrich), la2o3 (99.99%, roth), and zro2 (99.0%, roth) were used as reagents . The starting materials were weighed out in their intended stoichiometric proportions with an excess of 10 wt% li2co3 to compensate the loss of li2o during sintering . The reagents were mixed in an agate mortar under addition of isopropyl alcohol and then cold - pressed into pellets with a uniaxial press . The resulting pellets were put in an alumina crucible . To avoid undesired contamination with al from the crucible, the samples were placed on a pellet of pure llzo . The samples were heated to 850 c with a rate of 5 c / min and calcinated for 4 h. the resulting pellets were then removed from the furnace, ground in an agate mortar, and ball - milled for 1 h under isopropyl alcohol (fritsch pulverisette 7, 800 rpm, 2 mm zro2 balls). After drying under air, to avoid incorporation of al from the crucible, the samples were again placed on a pellet of pure llzo . To suppress formation of extra phases due to li loss during sintering, the sample pellets were covered with a pellet of pure llzo . For the final sintering, the pellets were heated in a muffle furnace in air with a rate of 5 c / min to 1230 c and sintered for 6 h. for xrpd investigations, small fragments of the sintered pellets were ground using an agate mortar . Pellets used for npd were stored in a glovebox under ar atmosphere to avoid reaction with co2 and h2o; also the whole processing after sintering, including grinding using an agate mortar and filling of the sample container has been performed under ar atmosphere . For sem analysis, polycrystalline chips from the pellets were embedded in an epoxy holder . The surface was ground and then polished using diamond paste . In particular, we put emphasis on the investigation of the grain size, morphology, phase composition, and the chemical homogeneity, i.e., the distribution, of ga, la, zr, using a backscattered electrons detector (bse) and energy - dispersive x - ray spectroscopy (edx) measurements, respectively . Patterns were recorded using a bruker d8 advance davinci design diffractometer with a lynxeye solid - state detector using cu k radiation . The synthesis products were characterized regarding the presence of extra phases as well as to determine the symmetry and unit - cell dimension of the samples . Data were collected between 10 and 80 2. evaluation of xrpd data was performed by rietveld refinement using the program bruker diffrac topas (version 4.2). Single - crystal x - ray diffraction data were collected on a bruker smart apex ccd - diffractometer . Samples for single - crystal studies were selected readily after removing of samples from the furnace, and data collection was done within 24 h after synthesis . So we exclude that the change in sg symmetry is due to the incorporation of h in the structure as it was suggested in literature . Single crystals were carefully hand - picked under the binocular loupe from the gently crushed pellets . For each composition, more than 10 crystals were selected on the basis of their optical properties (regular shape, clearness, and homogeneity in color). Selected crystals were glued on top of glass capillaries (0.1 mm) and tested on the diffractometer . A full set of intensity data was collected on 24 crystals for each composition to obtain good statistics and data sets for crystal chemical interpretation, resulting in a total of 21 data sets . Intensity data were collected with graphite - monochromatized mo k x - radiation (50 kv, 30 ma); the crystal - to - detector distance was 30 mm, and the detector was positioned at 30 and 50 2 using an -scan mode strategy at four different positions (0, 90, 180, and 270) for each 2 position . In total, 630 frames with = 0.3 were acquired for each run . With this strategy, data in a large q - range up to minimum d - values d = 0.53 could be acquired . This is necessary for accurate determination of anisotropic displacement parameters and to reduce correlation effects between atomic displacement parameters and site occupation numbers . Three - dimensional data were integrated and corrected for lorentz-, polarization, and background effects using the apex2 software . Structure solution (using direct methods) and subsequent weighted full - matrix least - squares refinements on f were done with shelx-2012 as implemented in the program suite wingx 2014.1 . Neutron powder diffraction data for a sample with a nominal ga content xga = 0.20 pfu were collected at the maier - leibnitz zentrum (mlz), frm - ii, munich, germany . Powder diffraction data were acquired at 298 k in constant wavelength mode using the high - resolution powder diffractometer spodi with ge551 monochromatized neutron radiation (= 1.5482). Experiments were performed in a 2 range of 3 2 154, step width of 0.045. data treatment of powder diffraction data sets as well as a combined refinement of neutron powder diffraction data and single - crystal x - ray diffraction data (with special emphasis of li - cationic distribution) was performed using the fullprof - suite of programs . Variable - temperature li nmr spectra and spin lattice relaxation rates were recorded with a bruker avance iii solid - state spectrometer connected to a 7-t magnet (bruker). The resonance frequency was 116 mhz; 90 pulse lengths ranged from 2 to 3 s depending on temperature . We used single - pulse and solid - echo experiments to record nmr line shapes . Lattice relaxation rates in the laboratory frame were measured by means of the saturation recovery sequence; the rates in the rotating frame were acquired by using the spin - lock technique, see epp et al . For details . To protect the samples permanently from humidity during the nmr measurements small pieces of the sintered garnets were put into glass ampules that were sealed under vacuum . Polished samples and pellet fragments were examined by sem - bse . For all compositions, grain sizes of> minor amounts of extra phases were documented by sem - bse due to a contrast in brightness and a different morphology . As already observed visually, extra phases are commonly found in the peripheral part of the pellets . Single grains are commonly separated by gaps, and small amounts of extra phases are located in these gaps . Only small open voids with a size of <10 m were found as inclusions within llzo grains . A representative sem - bse image of a pellet fragment is shown in figure 1 . Standard - free sem - edx analysis of llzo grains showed the presence of la, zr, and ga . The la / zr ratio of llzo grains was slightly below the theoretical values of 3:2, which is in accordance with site occupation refinements from sc - xrd data and microprobe measurements of ga - stabilized llzo by rettenwander et al . Ga contents of llzo grains were partially lower than the target stoichiometry; additionally, a slight variation of the ga content within single samples was noted . Edx mapping of llzo grains confirmed their homogeneity with regards to the distribution of la and zr within single grains . Extra phases observed by sem - bse can be divided due to their chemical composition seen by edx into three different phases . An extra phase with a la / zr ratio of 1:1 and another extra phase showing a la signal only were mainly found in the peripheral part of the pellets . For samples with nominal xga = (0.50, 0.60) per formula unit (pfu), another extra phase that only showed a ga signal in edx was found . The xrpd patterns of li73xgaxla3zr2o12 (gax) with nominal ga concentration xga = 0.10, 0.15, 0.20, 0.30, 0.40, 0.50, 0.60 pfu are shown in figure 1 . All garnet compositions exhibit reflections indicating cubic symmetry . For sample with xga = 0.10, tetragonal llzo was still present with a share of 18 wt% as determined by quantitative phase analysis using the rietveld method . For samples with xga 0.15, no tetragonal llzo phase was observed . La2zr2o7 and la(oh)3 were identified as extra phases and confirmed results of sem - edx investigations . Considering the sem - bse results, these extra phases occur predominantly in the rim of the pellets and formed due to evaporation of li during high temperature sintering . The presence of extra phases was accepted as they facilitate the extraction of single crystals . For sample with xga = 0.60, it has to be noted that for most samples, an additional weak reflection at 2 = 21.65 (equal to d = 4.10) was observed . This peak cannot be explained with llzo showing sg ia-3d or any extra phase . Comparison of xrpd patterns for samples ga10 (lowermost pattern), ga15, ga20, ga30, ga40, ga50, ga60 (uppermost pattern). Pattern contributions from extra phases are marked with colors: la2zr2o7 (blue), la(oh)3 (green), ligao2 (violet). The peak at 2 = 21.65 (red) could neither be attributed to cubic llzo with sg ia-3d nor to any extra phase . The inset shows a representative sem - bse image of a pellet fragment of sample ga40 to demonstrate the grain size and morphology . The experimental data and results of structure refinement for selected samples are reported in table 1 and table 2, while the fractional atomic coordinates, occupation numbers and equivalent isotropic, and anisotropic atomic displacement parameters are given in table s1 . Crystallographic information files (cif) with full structural information are deposited as supporting information . Xal value instead of xga_ref; fixed to the data obtained from edx analysis for sample al20_7 . Single - crystal x - ray intensity data processing gave strong evidence for the cubic crystal system for all data collected . For the data of sample ga10, with nominal ga concentration xga = 0.10 pfu, sg determination yields ia-3d symmetry as is widely accepted for llzo garnets . However, for all samples with nominal ga concentrations xga 0.15 pfu, intensity statistics and systematic extinctions did not result in sg ia-3d but yielded an acentric sg i-43d (no . 220). This sg was consistently obtained using all the different sg determination tools as implemented in wingx . Subsequent structure solution tests for samples with xga 0.15 pfu using commonly occurring sg ia-3d or tetragonal sg i41/acd and sg i41/a failed . Simulated precession images of the 0kl layer of samples ga10_1 and ga40_2, displayed in figure 2, obviously show the presence of bragg peaks with k = odd and l = odd in sample ga40 . These peaks are forbidden in sg ia-3d (as is also the case for sample ga10_1) but allowed in sg i-43d . The presence of bragg peaks with k = odd and l = odd in (b) is evident . Structure solution with direct methods using i-43d symmetry resulted in a structure model which could be refined down to wr2 values for all data <4% . The li - positions were located from difference fourier map calculations, in the final structure all atoms could be treated with anisotropic atomic displacement parameters . Our structure model has been found consistent with the one given by lager et al . And galven et al . Li leaching and replacement by h and up to now, no doubt on the violation of ia-3d symmetry for ga - stabilized llzo was reported . Samples of al - stabilized llzo, which are produced with the very same synthesis strategies, show ia-3d symmetry up to nominal compositions of xal = 0.30 pfu in single - crystal structure refinements . Different to the ia-3d structure of sample ga10 with a nominal ga content xga = 0.10 pfu, which exhibits 5 different atomic positions in the asymmetric unit, the crystal structure of ga - stabilized llzo with a nominal ga content xga 0.15 pfu, showing sg i-43d, exhibits seven different atomic positions: la occupies the 8-fold coordinated 24d position (site symmetry 2 ..), and zr is located at the octahedrally coordinated 16c position (site symmetry .3 . ). Li is found distributed over three different positions in i-43d: two of them correspond to the regular tetrahedral coordinated site of the silicate garnets (24d in ia-3d), nevertheless, they split into two positions, namely li1 (12a) and li2 (12b), both with site symmetry 4 .., and differ in both bond lengths and polyhedral distortion . The third li position, namely li3, has been found located on general position 48e, site symmetry 1, which is only partly occupied as is also common in llzo compounds . In contrast to common silicate garnets, the acentric structure exhibits two independent o positions, both in general position 48e . Table 1 shows a summary of the sc - xrd results of selected samples . More detailed information is given in table 2, while a comparison of the two structural models ia-3d and i-43d is given in table s1 . A graphical representation of the ia-3d and i-43d structures, including the li - network, is displayed in figure 3 . (a) crystal structure of ga - stabilized llzo with xga = 0.10 and sg ia-3d . Blue dodecahedra represent 8-fold coordinated la (at the wyckoff position 24c); green octahedra 6-fold coordinated zr (16a). The red spheres correspond to tetrahedrally coordinated li at the 24d (li1) site, yellow spheres correspond to distorted 4-fold coordinated li at wyckoff position 96h (li2). (b) crystal structure of ga - stabilized llzo with xga = 0.30 and sg i-43d . Blue dodecahedra represent 8-fold coordinated la (at the wyckoff position 24d); green octahedra 6-fold coordinated zr (16c). The red spheres correspond to tetrahedrally coordinated li at the 12a site (li1), orange spheres represent tetrahedrally coordinated li at the 12b site (li2); yellow spheres correspond to distorted 6-fold coordinated li at wyckoff position 48e (li3). (c) projection of li - network for sg ia-3d (left) and sg i-43d (right). Both, for the ia-3d and the i-43d structure, site occupation refinements yielded the octahedral 16c sites to be fully occupied by zr, no evidence was found for a ga substitution onto this site, so during refinements, the occupation of zr was fixed to the ideal value . For both structures, however, there is a small deficiency of la, and thus, the site occupation factor was allowed to vary freely during the refinements . The finding of a small la deficit is consistent with results from edx analysis . The amount of vacancies varies between 0.08(2) in ga - poor samples to 0.04(2) in ga - rich samples, and there are some evidence that the amount of vacancies at the la site steadily decreases upon increasing of ga content, see figure 4a . (b) ga is almost exclusively located at the smaller li1 site (12a), only for samples with high ga contents, a small amount of ga is located at the li2 site (12b). (c) as a function of the decreased total li content for samples with higher ga contents, the occupation of the li3 site decreases . For the ia-3d structure for the i-43d structure, the refinement strategy was modified slightly . In a first refinement cycle, the site occupation numbers of the li1, li2, and li3 sites were allowed to refine freely without constraints, assuming only the scattering power of li . These preliminary refinements showed a surplus of electron density at the li1 site (occupation of li larger than 1.00, the value of full occupation), and thus, a larger scatterer in this case ga must be present, whereas at the li2 site, the refined occupation was close to 1.00 or even lower, indicating some vacancies at this site . Li2 site values> 1.00 were observed only for higher overall ga contents . For occupation numbers> 1.00, a mixed occupation with li and ga was refined, assuming full occupation of these sites . With the model of site occupation refinement, we have strong evidence that ga shows a strong preference for the li1 site, only very small amounts of ga are found at the li2 site (figure 4b). It should be noted that the li2 site, thereby, is fully occupied in most cases and the li - content is 1.441.48 pfu . We are aware that this model of full occupation of li1 and li2 sites may suffer small deficits . However, it yields total ga contents which are very close to or even higher than those determined from edx analysis of the samples studied by sc - xrd . Assuming vacancy concentrations of 20% on both sites, this does not change the site preference of ga for the li1 site but only increases the overall ga content by 15% . Additionally, there is good evidence from neutron diffraction data that the amount of vacancies indeed is below 20% in ga - stabilized llzo samples (see below). The occupation of the li3 site generally was refined freely and gave a partial occupation of this site . With increasing ga substitution, the occupation of the li3 site decreases; that is, the trivalent cationic substitution reduces the amount of interstitial li (see figure 4c). The decrease of li with increasing ga is even more evident and with smaller scatter in the data when the total li content of the samples is taken into account . Therefore, with our high - quality, high - resolution x - ray data, we are able to deduce smooth trends in li - occupation in the llzo: ga series . For one composition (xga = 0.20), the derived structural model as well as the cationic distribution were simultaneously refined against high - resolution neutron powder diffraction (npd) and sc - xrd data . The best fit to the neutron data was indeed obtained with the i-43d model of this study, and the cationic distribution of x - ray data was proven . Also, in this combined simultaneous refinements, the la site shows the presence of a small amount of vacancies . Again, there is no clear evidence for ga on the li2 position, while it is enriched on li1; in the combined refinement, the ga and li occupation was refined freely without constraints: this strategy gave evidence for a rather low concentration of vacancies, both on li1 (14%) and li2 (12%) sites . The occupation of the li3 site is somewhat higher than in the single - crystal study but still comparable within estimated standard deviations . The substitution of ga into the structure of pure llzo stabilizes the cubic structure . The lattice parameters thereby are reduced from 12.985 to 12.965, and the decrease is almost linear, see figure 5 . The phase transition from ia-3d to i-43d does not cause a significant change in the lattice parameter . For small ga concentrations, we find as mentioned the ia-3d symmetry, while for refined ga contents xga_ref> 0.07, a reduction in symmetry to sg i-43d takes place . The reduction in symmetry most probably is induced by the strong ordering of ga onto one of the two possible tetrahedrally coordinated li sites, namely, onto li1 . The li1 site in i-43d is smaller as compared to the corresponding li1 site in ia-3d, and it is also slightly smaller as compared to the li2 site in i-43d, expressed by the smaller li o bond lengths and smaller volume . With increasing ga substitution, the li1o bond lengths successively decrease due to the smaller cationic size of ga as compared to li in tetrahedral coordination (0.47 and 0.59, respectively). The li2o bond lengths remain constant, or increase slightly with increasing ga content (see figure s2a, supporting information). This is seen as a further evidence that ga preferentially eenters the li1 site (12a). Obviously, it is the smaller and more distorted character of the li1 tetrahedron, which favors the ga substitution . The li3 site is a large cavity which has four li o bonds within 1.9 and 2.25 and two more distant li o bonds at 2.65 and 2.75, and thus, one may consider the coordination polyhedron of the li3 site as a strongly distorted octahedron . Each li3 site shares two of its triangular faces with neighboring li1 and li2 sites, while both the li1 and the li2 site share all of their four triangular faces with li3 sites . Thus, a three - dimensional network of face sharing li - sites is present, which forms a diffusion pathway for li ions . Li distances within the li - network: li1li3 is the shorter one with distances of 1.6, whereas the li2li3 distance is around 2.3 . Further crystal chemical considerations are given as supporting information . In figure 6, the li nmr line shapes and line widths of the central transition of ga20 and al20 are shown . Considering the li nmr central lines shown in figure 6b it is seen that the lines are stepwise narrowed with increasing temperature . As an example, at 213 k (60 c) the nmr signal of ga20 with xga = 0.20 is composed of two components viz . A broad gaussian shaped line and a motionally narrowed lorentzian one . Those ions contributing to the latter are already exposed to sufficiently fast exchange processes with rates exceeding the rigid - lattice line, the latter is estimated to be in the order of 8 to 9 khz . At even higher temperature, see the spectrum recorded at 273 k (0 c), all of the li ions participate in sufficiently fast li diffusivity to average dipole dipole couplings . Above 273 k, the line widths (see figure 6a) reached the so - called extreme narrowing regime . (a) motional narrowing of the li nmr central line of ga20 and al20; the inset shows a magnification of the quadrupole intensities of the two nmr lines recorded at 223 k (50 c) by means of a nonselective solid - echo experiment . (b) li nmr lines recorded at 116 mhz via single - pulse excitation . Essentially the same behavior is found for the sample al20 stabilized with 0.20 al pfu . The corresponding motional narrowing curve, however, is slightly shifted toward higher temperatures indicating somewhat lower diffusivity in the sample containing al . Looking at the quadrupole intensities, visualized by the solid - echo technique (see the inset of figure 6a), the contribution to the nmr line of ga20 is slightly reduced . The satellite transitions reflect the interaction of the spin-3/2 nucleus with the electric field gradients at the li sites in garnet - type llzo . A reduction in intensity (note that the spectra shown are scaled such that they have the same height) might be explained by faster li exchange processes in ga - bearing llzo . At very high temperatures, full averaging of the satellite intensities is seen for the two samples . In order to quantify ionic motion, we recorded li nmr laboratory - frame (1/t1) and rotating - frame (1/t1) spin lattice relaxation rates . The rates obtained are shown as a function of the inverse temperature in a semilogarithmic plot in figure 7a; in figure 7b, selected magnetization transients of the experiments in the rotating frame are displayed . Solid lines represent fits with stretched exponentials to extract the rates 1/t1. The inset in figure 7a shows the temperature dependence of the stretching exponent . As temperature decreases, the transients become more stretched . 200 k. as has been observed for other li - ion conductors this feature might correspond to a (local) maximum in the 1/t1(1/t) plot . Indeed, this behavior is seen in figure 7b, see the vertically drawn arrow . It could be interpreted as an additional li ion diffusion process that is absent for the sample stabilized by al instead of ga . For ga20, an activation energy of 0.39 ev can be roughly estimated . (a) 1/t1 magnetization transients, ranging from 233 to 373 k in steps of 20 k, which were analyzed by stretched exponentials (solid lines). Inset: variation of the stretching exponent as a function of the inverse temperature . The arrow points to a local minimum in the case of the ga20 sample . Besides this slight difference observed by rotating - frame spin - lock nmr, the two samples show two marked similarities that were also observed in earlier studies focusing on garnets stabilized by m cations: (i) 1/t1 passes through an extremely broad rate peak from which the high - t flank, characterized by a mean activation energy of 0.41 ev, is only partly accessible . On the other hand, (ii) up to ca . 400 k, the rates 1/t1(1/t) follow linear behavior in the arrhenius plot pointing to activation energies of 0.13 to 0.14 ev . These values characterize local li ion jumps in the garnet structure, whereas those deduced from 1/t1 might already be influenced by long - range ion transport . It is generally accepted that the garnets and garnet - related (synthetic) materials can adopt both cubic and tetragonal symmetries . The inorganic crystal structure database reports almost 500 entries for garnets, with the vast majority of 95% showing the cubic space group ia-3d . Few natural garnets like henritermierite ca3(mn, al)2(sio4)2(oh)4 and synthetic materials such as llzo are known to show tetragonal i41/acd symmetry . It is accepted that symmetry breaking from cubic ia-3d to lower symmetry takes place among others as a consequence of jahn teller distortion for mn bearing garnets, strain, and growth effects, cation ordering and incorporation of hydrogen atoms . In pure llzo, the symmetry breaking is explained by the complete ordering of li onto tetrahedral site . In a recent paper, report on the symmetry change from ia-3d to i213 during li / h exchange in the li7xhxla3sn2o12 and li7xhxla3nb2o12 systems . For li6-xhxcala2nb2o12, a change from ia-3d to the acentric space group i-43d is described as a consequence of li / h exchange upon leaching in acetic acid for 4 days by galven et al . Were not the first to report onto the unusual acentric cubic space group: the structure was first reported and solved by lager et al . In the unusual mineral compound katoite ca3al2(o4h4)3 . Using single - crystal x - ray diffraction, the authors describe a phase transition from ia-3d to i-43d occurring at high pressures (above 5 gpa). In this study, we observed the acentric sg i-43d for ga - stabilized llzo samples with nominal ga contents xga 0.15 pfu . This deviates from al stabilized llzo prepared under the same conditions, which still shows sg ia-3d . The present study indicates that for li - oxide garnets, sg i-43d seems to be more common than expected . Ga - stabilized llzo gets ordered in this acentric sg even without explicit aging or protonation . Previous studies reporting sg i-43d for li - oxide garnets correlated this phase transformation with a protonation process . As our samples were characterized immediately after the final calcination step, we exclude a phase transformation due to hydration caused by humidity from exposure to air as supposed by larraz et al . For pure llzo from i41/acd to ia-3d . Recently, larraz et al . Documented some additional weak reflections for pure llzo that was stored for three years, washed, and then heated to 300 c that cannot be explained using sg ia-3d . In contrast to this, ma et al . Did not observe any phase transformation for cubic llzo exposed to aqueous solutions and suggested that llzo with sg ia-3d is relatively stable, even at very high li / h exchange rates of 63.6%, which affected almost solely 4-fold - coordinated li at the 96h position . A particular focus of their study was on the presence of any other space groups such as i213 or i-43d, which was clearly denied by this paper . As the phase transition seems to be triggered by a splitting of the 24d position of sg ia-3d, which is not affected by the li / h exchange, it is hardly imaginable that a phase transition can be caused by a li / h exchange at the 96h position only; so possibly other processes such as the heating to 300 c might have caused the phase transition to i-43d mentioned by larraz et al . The additional reflections observed by larraz et al . At 2 = 21.5, 40.3, and 53.4, respectively, can indeed be attributed to the (310), (530), and (710) reflections of sg i-43d, as these reflections are forbidden for sg ia-3d . In this study, the reflection at 2 = 21.65 (d = 4.101) can be attributed to the (310) reflection of sg i-43d, which is expected to show a relative intensity of 1.2% compared to the strongest reflection at 2 = 30.81 (d = 2.90). The other additional reflections show an even lower relative intensity of 0.4% and 0.3%, respectively . However, due to their low intensity, these peaks might have been overlooked by previous studies on ga - stabilized llzo or incorrectly attributed to ligao2, which is a common extra phase and shows a reflection at 2 = 21.52 (d = 4.126). For the detection of ligao2, the use of most intense reflection at 2 = 22.55 (d = 3.94) is more advantageous . For a definite determination of the sg of ga - stabilized llzo, much experimental as well as theoretical effort has been undertaken to collect information on the site preference of ga and its influence on li - ion dynamics and li - ion conductivity . The position of these cations might influence the mobility of li ions due to a possible blockage of the diffusion path . Several studies using ga nmr spectroscopy lead to different interpretations, indicating either one or two ga positions . In this study, the refinement of sc - xrd and npd data suggests that ga is preferentially located at a single position, namely the tetrahedral 12a position, and only minor amounts of ga occupy the 12b position . As the coarse - grained llzo samples in this study were prepared at high temperatures, we cannot assess whether ga - stabilized llzo prepared under different conditions (e.g., at lower temperatures) still orders in sg i-43d . Further research will be needed to clarify the influence of the preparation condition on the crystal structure of ga - stabilized llzo . Despite that ga - stabilized llzo shows a different sg than al - stabilized llzo, the li - ion mobility is still comparable or even better than for al - stabilized llzo with sg ia-3d . It stands to reason that the structural differences will have a significant impact on the li - ion dynamics . However, investigations on the influence of the new cubic modification on the li - ion mobility are beyond the scope of this study and will therefore be reported in a subsequent paper . The present study reveals that ga - stabilized llzo shows the acentric cubic sg i-43d (no . 220), which is different from other members of the li - oxide garnet group that show sg ia-3d (no . 230). In contrast to other studies which observed this sg due to li / h exchange, the structural changes might be caused by the site preference of ga . Li nmr relaxometry and line shape studies support the findings by impedance spectroscopy revealing enhanced ion dynamics in ga - stabilized llzo as compared to llzo stabilized by al . This study stimulates further research for an understanding of the structureproperty relationship as a basis to improve the electrochemical capabilities of these electrolyte materials.
Dynamic morphological changes are mediated by continual, highly- regulated fusion and fission events, collectively termed mitochondrial dynamics . For example, the evolutionarily conserved gtpase, dynamin related protein1 (drp1), is known to participate in the process of mitochondrial fission . When mitochondria undergo fission, cytosolic drp1 translocates to the outer mitochondrial membrane, oligomerizes around mitochondrial tubules, and in conjunction with fis1 promotes division or fission . The fusion machinery consists mainly of three additional large dynamin - like gtpases: both mitofusin1/2 (mfn1/2) and optic atrophy protein 1 (opa1). The strict regulation of the equilibrium between fusion and fission events governs mitochondrial dynamics in a healthy cell and is crucial to maintaining mitochondrial function affecting energy supply, response to apoptotic stimuli, mitochondrial inheritance via admixture of mitochondrial - dna (mtdna), and translocation of mitochondria to regions of high - energy demand . Although mitochondrial dynamics are crucial in all cell types, neurons are thought to be especially dependent on a dynamic mitochondrial network . With their often long processes, neurons require proper distribution of mitochondria across long distances to satisfy the energy requirements of electrical excitability and synaptic transmission (fig ., healthy neurons precisely control mitochondrial dynamics in order to efficiently transport mitochondria to distal locations, including dendrites, axons, and synapses . Thus, mitochondrial function is essential to many neuronal functions, including energy production, ca buffering, axonal and dendritic transport, and release and re - uptake of neurotransmitter . In order to maintain a proper distribution of functional mitochondria, basal activity of the fission machinery is required to divide the mitochondrial network into transportable units which can then be efficiently distributed to synapses . Fusion is necessary to maintain bioenergetic integrity of mitochondria . If the balance of fission and fusion becomes biased (e.g., with increased fission or impaired fusion) excessive mitochondrial fragmentation is observed, resulting in impaired mitochondrial translocation and energy production at synaptic sites (fig . 1). The synaptic dysfunction thus induced is typically followed by dendritic and axonal degeneration, leading to neurodegeneration . Indeed, excessive mitochondrial fragmentation with damaged cristae and resulting impaired bioenergetics are often associated with many neurological conditions, including stroke, alzheimer s disease (ad), parkinson s disease (pd), and huntington s disease (hd), as well as several metabolic diseases . Pathological disturbance of mitochondrial dynamics can result from either: 1) rare genetic mutations, such as those described for mfn1/2, opa1 and drp1, which underlie charcot - marie - tooth neuropathy type 2a (cmt2a), autosomal dominant optic atrophy (adoa), and a rare disorder of brain development, respectively; or 2) altered posttranslational modifications (ptms) of the proteins encoded by these genes that are responsible for the fission / fusion machinery . In this review, we discuss recent findings concerning aberrant ptms which regulate the mitochondrial fusion and fission machinery and thus contribute to the pathophysiology of neurodegenerative diseases . In particular, we focus on involvement of nitrosative stress - induced drp1 activation, which results in excessive mitochondrial fragmentation, impaired bioenergetics, and consequent synaptic damage in neurodegenerative disorders . As mentioned above, tight regulation of the mitochondrial fission / fusion machinery is essential for maintaining healthy neuronal physiology . However, dysfunctional mitochondrial dynamics caused by impaired mitochondrial fission or fusion is a hallmark of many neurodegenerative diseases and contributes to their pathophysiology . Mitochondrial fusion is the process by which the inner and outer mitochondrial membranes are separately fused by opa1 and mfn1/2, respectively . For example, loss of function mutations in the mfn2 gene disrupts mitochondrial fusion, resulting in distal muscle weakness and sensory loss in cmt2a . To date, more than 40 mutations have been identified, most of which are located in the conserved gtpase domain of mfn2 . In mouse models, deletion in the mouse mfn2 gene causes severe mtdna loss and the accumulation of mtdna mutations . In addition, mfn2 deficiency results in decreased axonal transport of mitochondria, causing a lack of functional mitochondria at pre- and postsynaptic sites . Collectively, these findings suggest that mfn2 mutations in cmt2a patients may trigger defects both in mtdna integrity and mitochondrial transport, contributing to disease pathogenesis . Another neurologic disorder caused by disruption in the fusion machinery is adoa, representing an hereditary optic nerve atrophy characterized by degeneration of retinal ganglion cells and progressive loss of vision . Mutations in the opa1 gene, and to a lesser extent in opa3, underlie adoa . Similar to mfn2 mutations, over 100 reported mutations in opa1 target predominantly the gtpase domain . Opa1 is located at the inner mitochondrial membrane, regulating fusion and modeling of the cristae structures . Interestingly, most opa1 mutations identified in the gtpase domain cause adoa via a dominant - negative effect . Furthermore, heterozygous opa1 knockout mice manifest severe degeneration of the optic nerves, similar to human adoa . Due to decreased mitochondrial fusion activity, disruption of opa1 gtpase activity results in increased mitochondrial fragmentation, possibly making the retinal ganglion cells more susceptible to apoptosis . However, further analyses are needed to determine whether mitochondrially - mediated apoptotic pathways are implicated in the pathogenesis of adoa . Increased mitochondrial fragmentation due to excessive fission is found in many neurodegenerative disorders, including ad, pd and hd, as well as in ischemic brain damage . Drp1 consists of an n - terminal gtpase domain, followed by a helical domain (also termed dynamin - like middle domain), an insert b domain, and a c - terminal gtpase effector domain (ged) (fig . 2). The helical domain allows drp1 to form ring - like oligomeric structures at the outer membrane of mitochondria to induce mitochondrial fission upon hydrolysis of gtp . A recent case study reported a rare dominant negative mutation in the human drp1 gene . This mutation inhibits drp1 oligomerization and thus results in the appearance of elongated mitochondrial morphologies . These abnormal mitochondria lose their respiratory function and can no longer be effectively distributed to regions of high - energy demand, such as neuronal synapses . The dominant negative drp1 that results from this mutation caused impaired brain development, microcephaly, optic atrophy, lactic academia, and eventual death by 37 days of age . Additionally, drp1 activity can be tightly regulated by a series of ptms including s - nitrosylation, phosphorylation, sumoylation and ubiquitination, as depicted in figure 2 . These ptms generally do not target the drp1 gtpase domain but instead are located within or in close proximity to the ged domain, thereby allosterically influencing gtpase activity . Recently, we discovered that drp1 is a target of s - nitrosylation, a covalent modification of free thiol by a nitric oxide (no) group, forming an s- nitrosothiol (r - sno). Specifically, we found that s - nitrosylation of cys644 within the ged domain of drp1 enhances its gtpase activity and results in excessive mitochondrial fragmentation . S - nitrosylation of drp1 and its involvement in neurologic disorders will be discussed in detail in later sections . In addition to s - nitrosylation, drp1 undergoes multiple other ptms including phosphorylation, sumoylation, and ubiquitination . Among these drp1, it is known that phosphorylation of drp1 at ser616 by cdk1/cyclin b can increase its gtpase activity . In addition, cyclic amp - dependent protein kinase (pka) phosphorylates drp1 at ser637 and decreases drp1 activity by inhibiting molecular interactions . Pka - mediated phosphorylation can be counteracted by calcineurin - induced dephosphorylation of ser637, resulting in translocation of drp1 to the mitochondrial membrane . In an apparently paradoxical manner, however, calcium / calmodulin - dependent pka 1 (camki) can phosphorylate the same cysteine (ser637) as pka but rather induces recruitment of drp1 to the mitochondrial membrane to form a complex with fis1, resulting in increased mitochondrial fission . Therefore, additional studies are needed to clarify the basis for the differential effects of pka and camki. Drp1 activity is also be affected by sumoylation, which occurs within the insert b domain (fig . The attachment of a small ubiquitin - related modifier 1 (sumo1) to the lysine residues of drp1 stabilizes the protein and thus increases mitochondrial fragmentation . Several proteins, including sumo1, ubc9, and mapl, have been shown to mediate drp1 sumoylation . This stabilizing modification can be reversed by sentrin / sumo - specific protease (senp5). Parkin and mitochondrial e3 ligase protein march5 can ubiquitinate drp1 on lysine residues located in the middle domain . Initial studies reported that march5 activity promoted mitochondrial fusion and elongation, suggesting that march - dependent ubiquitination of drp1 inhibited its activity . Although one could speculate that different expression levels of march5 might cause these opposite effects, the physiological action of march5-mediated ubiquitination on drp1 certainly warrants further investigation . Parkin is a ring - type ubiquitin e3 ligase expressed in many human tissues, including brain, heart, testis, and skeletal muscle . In the human brain, parkin is primarily expressed in neurons in various regions, including the substantia nigra in the midbrain as well as in the ca1/3 region of the hippocampus . Since mutations in the parkin gene cause a familial form of pd, it is tempting to speculate that abnormal regulation of the parkin - drp1 pathway, which would lead to impaired mitochondrial dynamics, might contribute to the pathogenesis of pd . Mitochondrial fusion is the process by which the inner and outer mitochondrial membranes are separately fused by opa1 and mfn1/2, respectively . For example, loss of function mutations in the mfn2 gene disrupts mitochondrial fusion, resulting in distal muscle weakness and sensory loss in cmt2a . To date, more than 40 mutations have been identified, most of which are located in the conserved gtpase domain of mfn2 . In mouse models, deletion in the mouse mfn2 gene causes severe mtdna loss and the accumulation of mtdna mutations . In addition, mfn2 deficiency results in decreased axonal transport of mitochondria, causing a lack of functional mitochondria at pre- and postsynaptic sites . Collectively, these findings suggest that mfn2 mutations in cmt2a patients may trigger defects both in mtdna integrity and mitochondrial transport, contributing to disease pathogenesis . Another neurologic disorder caused by disruption in the fusion machinery is adoa, representing an hereditary optic nerve atrophy characterized by degeneration of retinal ganglion cells and progressive loss of vision . Mutations in the opa1 gene, and to a lesser extent in opa3, underlie adoa . Similar to mfn2 mutations, over 100 reported mutations in opa1 target predominantly the gtpase domain . Opa1 is located at the inner mitochondrial membrane, regulating fusion and modeling of the cristae structures . Interestingly, most opa1 mutations identified in the gtpase domain cause adoa via a dominant - negative effect . Furthermore, heterozygous opa1 knockout mice manifest severe degeneration of the optic nerves, similar to human adoa . Due to decreased mitochondrial fusion activity, disruption of opa1 gtpase activity results in increased mitochondrial fragmentation, possibly making the retinal ganglion cells more susceptible to apoptosis . However, further analyses are needed to determine whether mitochondrially - mediated apoptotic pathways are implicated in the pathogenesis of adoa . Increased mitochondrial fragmentation due to excessive fission is found in many neurodegenerative disorders, including ad, pd and hd, as well as in ischemic brain damage . Drp1 consists of an n - terminal gtpase domain, followed by a helical domain (also termed dynamin - like middle domain), an insert b domain, and a c - terminal gtpase effector domain (ged) (fig . 2). The helical domain allows drp1 to form ring - like oligomeric structures at the outer membrane of mitochondria to induce mitochondrial fission upon hydrolysis of gtp . A recent case study reported a rare dominant negative mutation in the human drp1 gene . This mutation inhibits drp1 oligomerization and thus results in the appearance of elongated mitochondrial morphologies . These abnormal mitochondria lose their respiratory function and can no longer be effectively distributed to regions of high - energy demand, such as neuronal synapses . The dominant negative drp1 that results from this mutation caused impaired brain development, microcephaly, optic atrophy, lactic academia, and eventual death by 37 days of age . Additionally, drp1 activity can be tightly regulated by a series of ptms including s - nitrosylation, phosphorylation, sumoylation and ubiquitination, as depicted in figure 2 . These ptms generally do not target the drp1 gtpase domain but instead are located within or in close proximity to the ged domain, thereby allosterically influencing gtpase activity . Recently, we discovered that drp1 is a target of s - nitrosylation, a covalent modification of free thiol by a nitric oxide (no) group, forming an s- nitrosothiol (r - sno). Specifically, we found that s - nitrosylation of cys644 within the ged domain of drp1 enhances its gtpase activity and results in excessive mitochondrial fragmentation . S - nitrosylation of drp1 and its involvement in neurologic disorders will be discussed in detail in later sections . In addition to s - nitrosylation, drp1 undergoes multiple other ptms including phosphorylation, sumoylation, and ubiquitination . Among these drp1 for example, it is known that phosphorylation of drp1 at ser616 by cdk1/cyclin b can increase its gtpase activity . In addition, cyclic amp - dependent protein kinase (pka) phosphorylates drp1 at ser637 and decreases drp1 activity by inhibiting molecular interactions . Pka - mediated phosphorylation can be counteracted by calcineurin - induced dephosphorylation of ser637, resulting in translocation of drp1 to the mitochondrial membrane . In an apparently paradoxical manner, however, calcium / calmodulin - dependent pka 1 (camki) can phosphorylate the same cysteine (ser637) as pka but rather induces recruitment of drp1 to the mitochondrial membrane to form a complex with fis1, resulting in increased mitochondrial fission . Therefore, additional studies are needed to clarify the basis for the differential effects of pka and camki. Drp1 activity is also be affected by sumoylation, which occurs within the insert b domain (fig . 2). The attachment of a small ubiquitin - related modifier 1 (sumo1) to the lysine residues of drp1 stabilizes the protein and thus increases mitochondrial fragmentation . Several proteins, including sumo1, ubc9, and mapl, have been shown to mediate drp1 sumoylation . This stabilizing modification can be reversed by sentrin / sumo - specific protease (senp5). Parkin and mitochondrial e3 ligase protein march5 can ubiquitinate drp1 on lysine residues located in the middle domain . Initial studies reported that march5 activity promoted mitochondrial fusion and elongation, suggesting that march - dependent ubiquitination of drp1 inhibited its activity . However, a subsequent report by karbowski et al . Found that march5 activity is required for mitochondrial fission . Although one could speculate that different expression levels of march5 might cause these opposite effects, the physiological action of march5-mediated ubiquitination on drp1 certainly warrants further investigation . Parkin is a ring - type ubiquitin e3 ligase expressed in many human tissues, including brain, heart, testis, and skeletal muscle . In the human brain, parkin is primarily expressed in neurons in various regions, including the substantia nigra in the midbrain as well as in the ca1/3 region of the hippocampus . Since mutations in the parkin gene cause a familial form of pd, it is tempting to speculate that abnormal regulation of the parkin - drp1 pathway, which would lead to impaired mitochondrial dynamics, might contribute to the pathogenesis of pd . Over the past twenty years, numerous reports identified no as a crucial protein - modifying molecule in s - nitrosylation . Depending on the target protein, the effects of s - nitrosylation can be quite diverse . No is typically generated from no synthase (nos) during the conversion of l - arginine to l - citrulline . No production can be controlled by transcriptional activation of inducible nos (inos), or by ca / calmodulin dependent activation of neuronal or endothelial nos (nnos or enos). A well - characterized pathway for no production in the nervous system occurs via activation of the n - methyl - d - aspartate - type glutamate receptor (nmdar). Nmdar stimulation results in the influx of ca, which, together with calmodulin, stimulates nnos to generate no (fig . 3). Prior to the discovery of s - nitrosylation, initial studies of no signaling concentrated on its physiological and neuroprotective functions mediated by activation of soluble guanylate cyclase (sgc) and subsequent production of cgmp (fig . Subsequently, accumulating evidence has suggested that in a variety of diseases, including ad, hd and pd, nitrosative and oxidative stress appear to trigger aberrant s - nitrosylation events that contribute to neurodegeneration . For example, pathological s - nitrosylation reactions include no - mediated modifications to parkin and protein disulfide isomerase (pdi) (fig . 3). Additionally, we recently discovered that aberrant s - nitrosylation of drp1 hyperactivates its mitochondrial fission activity . In this case, no triggers excessive mitochondrial fission accompanied by increased autophagy (mitophagy) and decreased atp production, which contributes to synaptic damage conditions such as ad . In addition to sno signaling pathways, no can react with superoxide anion, generated from mitochondrial as well as non- mitochondrial sources (e.g., nadph oxidase), to form peroxynitrite (onoo-) (fig . Peroxynitrite can result in lipid peroxidation and tyrosine nitration, another no - mediated ptm that causes pathological alteration in target protein activity, as reviewed elsewhere . Ad is the most common neurodegenerative disorder with increasing prevalence because of the aging demographic of our society . Ad is clinically characterized by loss of cognitive functions accompanied by intracellular neurofibrillary tangles, composed of hyperphosphorylated tau, and extracellular amyloid plaques, comprised of amyloid- (a) peptide . Soluble oligomers of a arise from the proteolytic cleavage of amyloid precursor protein (app) and are thought to contribute to the pathogenesis of ad . Additional evidence accumulated over the past decade has suggested that altered mitochondrial function associated with dysregulated mitochondrial dynamics is another key feature of ad . Recently, the neurotoxic a oligomers were reported to cause excessive mitochondrial fragmentation in cell - based models of ad . Specifically, a oligomer - induced generation of no resulted in s - nitrosylation of drp1 (forming sno - drp1), which was found to hyperactivate its gtpase activity and induce excessive mitochondrial fragmentation . The resulting disruption of mitochondrial bioenergetics caused a drop in atp levels and consequent synaptic damage . Drp1 as shown to be s - nitrosylated at cysteine residue 644, located in the ged domain . Importantly, mutation of drp1 cys644 to an alanine abrogated a/no - induced mitochondrial fission, consistent with the notion that the mitochondrial fragmentation was at least in part mediated by formation of sno - drp1 . In ad, the only neuropathological correlation with cognitive decline is the degree of synaptic loss . Interestingly, drp1-c644a (representing a nitrosylation - resistant mutant) prevented a-induced synaptic damage, suggesting that s- nitrosylation of drp1 at cys644 contributes to a toxicity in ad . Moreover, the non - nitrosylatable drp1 mutant prevented nitrosative stress - induced neuronal cell death, lending support to the role of sno - drp1 in no - mediated neuronal damage . Although this pathway of aberrant sno - drp1 formation was initially discovered in human ad brains and animal models of ad, many other neurodegenerative conditions, including pd and hd, are characterized by increased oxidative and nitrosative stress . Consistent with this notion, sno - drp1 is present at very early stages in the substantia nigra of pd animal models induced by pesticide exposure . Additionally, sno - drp1 appears to contribute to mutant huntingtin (mthtt)-induced neurotoxicity in cell - based and animal models of hd . Hd is an adult onset, genetic neurodegenerative disorder caused by an aberrant expansion of a trinucleotide cag repeat in the htt gene . The cag expansion, translated into an expanded polyglutamine (polyq) repeat in the mthtt protein, accelerates toxic misfolding of mthtt, leading to interference with several critical molecular cascades in the cell, and resultant degeneration of synapses and neurons in the affected brain area (i.e., the striatum and cortex). Similar to the effects of a oligomers on sno - drp1, expression of mthtt significantly increased no production and subsequently up - regulated the levels of sno - drp1 in primary cultures of cortical neurons . Further along these lines, sno - drp1 formation was considerably elevated in the striatum of a transgenic mouse model of hd as well as in human postmortem brains from hd patients . In a cell culture model of hd, transfection of a non - nitrosylatable mutant form of drp1 abrogated the neurotoxic effects of mthtt on mitochondrial fragmentation and dendritic spine damage, suggesting that sno - drp1 is a key mediator of this form of mthtt toxicity . Taken together, these findings imply that s - nitrosylation of drp1 may occur in other neurodegenerative conditions, contributing to aberrant mitochondrial dynamics and downstream neuronal damage . Over the past twenty years, numerous reports identified no as a crucial protein - modifying molecule in s - nitrosylation . Depending on the target protein, the effects of s - nitrosylation can be quite diverse . No is typically generated from no synthase (nos) during the conversion of l - arginine to l - citrulline . No production can be controlled by transcriptional activation of inducible nos (inos), or by ca / calmodulin dependent activation of neuronal or endothelial nos (nnos or enos). A well - characterized pathway for no production in the nervous system occurs via activation of the n - methyl - d - aspartate - type glutamate receptor (nmdar). Nmdar stimulation results in the influx of ca, which, together with calmodulin, stimulates nnos to generate no (fig . 3). Prior to the discovery of s - nitrosylation, initial studies of no signaling concentrated on its physiological and neuroprotective functions mediated by activation of soluble guanylate cyclase (sgc) and subsequent production of cgmp (fig . Subsequently, accumulating evidence has suggested that in a variety of diseases, including ad, hd and pd, nitrosative and oxidative stress appear to trigger aberrant s - nitrosylation events that contribute to neurodegeneration . For example, pathological s - nitrosylation reactions include no - mediated modifications to parkin and protein disulfide isomerase (pdi) (fig . 3). Additionally, we recently discovered that aberrant s - nitrosylation of drp1 hyperactivates its mitochondrial fission activity . In this case, no triggers excessive mitochondrial fission accompanied by increased autophagy (mitophagy) and decreased atp production, which contributes to synaptic damage conditions such as ad . In addition to sno signaling pathways, no can react with superoxide anion, generated from mitochondrial as well as non- mitochondrial sources (e.g., nadph oxidase), to form peroxynitrite (onoo-) (fig . Peroxynitrite can result in lipid peroxidation and tyrosine nitration, another no - mediated ptm that causes pathological alteration in target protein activity, as reviewed elsewhere . Ad is the most common neurodegenerative disorder with increasing prevalence because of the aging demographic of our society . Ad is clinically characterized by loss of cognitive functions accompanied by intracellular neurofibrillary tangles, composed of hyperphosphorylated tau, and extracellular amyloid plaques, comprised of amyloid- (a) peptide . Soluble oligomers of a arise from the proteolytic cleavage of amyloid precursor protein (app) and are thought to contribute to the pathogenesis of ad . Additional evidence accumulated over the past decade has suggested that altered mitochondrial function associated with dysregulated mitochondrial dynamics is another key feature of ad . Recently, the neurotoxic a oligomers were reported to cause excessive mitochondrial fragmentation in cell - based models of ad . Specifically, a oligomer - induced generation of no resulted in s - nitrosylation of drp1 (forming sno - drp1), which was found to hyperactivate its gtpase activity and induce excessive mitochondrial fragmentation . The resulting disruption of mitochondrial bioenergetics caused a drop in atp levels and consequent synaptic damage . Drp1 as shown to be s - nitrosylated at cysteine residue 644, located in the ged domain . Importantly, mutation of drp1 cys644 to an alanine abrogated a/no - induced mitochondrial fission, consistent with the notion that the mitochondrial fragmentation was at least in part mediated by formation of sno - drp1 . In ad, interestingly, drp1-c644a (representing a nitrosylation - resistant mutant) prevented a-induced synaptic damage, suggesting that s- nitrosylation of drp1 at cys644 contributes to a toxicity in ad . Moreover, the non - nitrosylatable drp1 mutant prevented nitrosative stress - induced neuronal cell death, lending support to the role of sno - drp1 in no - mediated neuronal damage . Although this pathway of aberrant sno - drp1 formation was initially discovered in human ad brains and animal models of ad, many other neurodegenerative conditions, including pd and hd, are characterized by increased oxidative and nitrosative stress . Consistent with this notion, sno - drp1 is present at very early stages in the substantia nigra of pd animal models induced by pesticide exposure . Additionally, sno - drp1 appears to contribute to mutant huntingtin (mthtt)-induced neurotoxicity in cell - based and animal models of hd . Hd is an adult onset, genetic neurodegenerative disorder caused by an aberrant expansion of a trinucleotide cag repeat in the htt gene . The cag expansion, translated into an expanded polyglutamine (polyq) repeat in the mthtt protein, accelerates toxic misfolding of mthtt, leading to interference with several critical molecular cascades in the cell, and resultant degeneration of synapses and neurons in the affected brain area (i.e., the striatum and cortex). Similar to the effects of a oligomers on sno - drp1, expression of mthtt significantly increased no production and subsequently up - regulated the levels of sno - drp1 in primary cultures of cortical neurons . Further along these lines, sno - drp1 formation was considerably elevated in the striatum of a transgenic mouse model of hd as well as in human postmortem brains from hd patients . In a cell culture model of hd, transfection of a non - nitrosylatable mutant form of drp1 abrogated the neurotoxic effects of mthtt on mitochondrial fragmentation and dendritic spine damage, suggesting that sno - drp1 is a key mediator of this form of mthtt toxicity . Taken together, these findings imply that s - nitrosylation of drp1 may occur in other neurodegenerative conditions, contributing to aberrant mitochondrial dynamics and downstream neuronal damage . In the present study, we review how ptms of drp1, particularly s - nitrosylation, may contribute to excessive mitochondrial fragmentation in neurodegenerative diseases . These findings strengthen the hypothesis that aberrant protein s - nitrosylation may play a key role in the contribution of mitochondrial pathology to many neurodegenerative diseases . Importantly, the demonstration that preventing formation of sno - drp1 can ameliorate a- or mthtt - induced synaptic damage in models of ad and hd, respectively, suggests a new therapeutic approach based on developing drugs to affect disease - related protein s - nitrosylation . Additionally, we anticipate that additional examples of aberrant s - nitrosylation as well as other ptm events will be discovered which influence mitochondrial dynamics and function in a number of other neurodegenerative disorders . Another important area for future research into the effect of sno - drp1 concerns the discovery of endogenous compounds, including nitrosylase and denitrosylase enzymes which regulate its formation and destruction . Related to this question, we recently discovered that sno - cdk5 can act as an endogenous nitrosylase, enhancing sno - drp1 levels via transnitrosylation of drp1 (i.e., sno - cdk5 donates an no group to drp1 to form sno - drp1). Further efforts to identify additional molecular controls of aberrant s - nitrosylation of drp1 will be critical in determining not only the downstream signaling consequences but also for formulating new therapies for several neurological disorders.
Nonmelanoma skin carcinoma (nmsc) is the most frequent cancer in the usa and worldwide; it has been increasing in overall incidence since the 1960's at a rate of 38% per year . With over 1.3 million new diagnoses of nmsc a year in the united states, it is both the diversity of types, of which there are 82, and biologic variability in phenotype that make the analysis of nmsc even more challenging . Although the incidence of basal cell carcinoma (bcc) exceeds cscc by a 5: 1 ratio, cscc is associated with the burden of mortality with a disease - specific yearly mortality rate of 1% per year as reported in the early 1990's . Despite the fact that the majority of these tumors present at early stages, cscc accounts for the majority of nmsc deaths and 20% of all skin - cancer - related deaths . Multiple etiologies exist for cscc, including environmental, genetic, viral and altered host immunity and virally mediated . The high incidence of cscc and bcc is caused by the mutagenic effects of ultraviolet (uv) light which is intensified by geographic latitude [1, 7]. Cscc and bcc are more common in fair skinned and anatomic sites exposed to the sun, such as head, neck, and extremities: head and neck is the most common site . Other known risk factors are male sex, advanced age, immunosuppression (induced or acquired), human papilloma viruses (hpv), chronic inflammation, and genetic diseases manifested in the skin [79]. Immunocompromised states are associated with a marked escalation of cscc of up to 64250 times greater than that in the general population compared to the 10-fold increased risk in bcc, causing a reversal of the typical ratio in immunocompetent individuals from 5: 1 to a range between 1: 1.8 and 1: 15 [10, 11]. Cscc tumors tend to be numerous, exhibit a strong propensity to recur, and metastasize at a high rate regardless of lesional size . Malignant lesions develop within 10 years after organ transplantation and up to 80% of these lesions contain hpv dna . Hpv has also been associated with cscc with evidence of a higher viral load of hpv dna in immunosuppressed patients [13, 14]. Likewise cscc that arises in sites of chronic inflammation, such as scars, sinus tracts, and burns, can also demonstrate more aggressive clinical behavior and a greater propensity to metastasize with an overall metastatic rate of 40% [6, 15]. Cutaneous genetically inherited skin conditions that have a known propensity of risk for developing cscc are albinism, xeroderma pigmentosum, and epidermodysplasia verruciformis [5, 16, 17]. With a cure rate of greater than 90% for the routine lesion, and the large number of low risk lesions, the significance of the diverse spectrum and numerous subtypes of cscc has been underappreciated given the often quoted 5-year recurrence and metastatic rates of 8% and 5%, respectively [6, 18, 19]. However recent changes to the american joint committee on cancer staging (ajcc) presented in the 7th edition focus on identifying clinical parameters that portend a worse prognosis to identify and stage appropriately that subset of cscc that progresses to metastatic disease . The high risk characteristics determined by the 7th edition ajcc staging manual include lesional size (> 2 cm), and high risk features such as a depth of invasion (> 2 mm, clark level iv), perineural invasion, tumor grade (poorly differentiated or undifferentiated), as well as high - risk anatomic sites (see table 1). The paradigm shift in the 7th edition recognizes that the classification of lesions with similar histopathologic features is more likely to have similar aggressive behavior and the features chosen were based on that behavior . For instance, tumor grade alone is significantly associated with mortality given a 5-year cure after therapy of 61.5% for poorly differentiated cscc compared to 94.6% for well differentiated cscc . High risk histologic features were defined as showing poor differentiation, spindle cell characteristics, necrosis, high mitotic activity, and deep invasion . Both the depth of invasion and presence of perineural invasion significantly correlate with prognosis: it clearly has been shown that thicker lesions have a higher rate of nodal metastases and recurrence . In fact a depth of> 4 mm thickness or clark level iv is associated with a twofold increased rate of recurrence or 5-fold increase metastatic rate . Similarly, perineural invasion is associated with a 5-fold increase in both the recurrence rate and metastatic rate . Although not identified in the 7th edition, other histologic features that are important in prognosis include lymphovascular invasion and the presence of inflammatory features such as the presence of eosinophils and plasma cells . Cscc in immunocompromised patients or those that arise in scars, sinus tracts, or burns all demonstrate a more aggressive biologic phenotype with a greater metastatic rate of up to 40% [6, 15, 22]. So subsets with a worse prognosis are critical to correctly stage and classify together in order to appropriately reveal a previously unrecognized metastatic potential . Likewise recurrent or persistent disease portends a worse survival of 78% 5-year survival compared to 97% for a primary lesion [20, 23]. Since the incidence of metastatic cscc is low, the outcome of metastatic cscc is not universally recognized as dismal at a-5 year survival of less than 35% . Prognostic features have not been universally accepted in cscc and the histologic features delineated above are advised for uniformity in standardizing clinical staging guidelines . Since cscc is typically deemed to have a good prognosis, the variants with the worst biology, by not being grouped together, are underpowered numerically in an outcome analysis . Thus the variety of subtypes of cscc certainly necessitates a comprehensive clinicopathologic classification system to group variants of scc based on their biologic aggressiveness or indolence . The 7th edition introduces the histologic features that need to be tracked and classified; yet a clinicopathologic classification system would allow lesions to be stratified based on biologic behavior and thus patients accordingly to optimize therapy . Clinicopathologic classification of cscc has been characterized by cassarino et al . To stratify lesions based on their malignant potential (table 2). In the era of personalized medicine, molecular markers have been used in many tumors to prognosticate and risk - stratify patients . Given the relative lack of recognition of the growing incidence of cscc and the inability to track the worst subset of cscc given the abundance of low risk lesions and the practice of not banking or staging lesions, these molecular studies have been relatively limited compared to the field of melanoma . A summary of the current literature on the molecular markers in cscc highlights some promising areas of research . The increased incidence of cscc in immunocompromised patients compared to bcc suggests a mechanism of viral pathogenesis . Hpv is important in immunosuppressed patients, which are known to have higher levels of hpv dna in cscc lesions . Evidence of a higher viral load has been reported in cscc in organ transplant patients with up to 80% of lesions containing hpv dna . However the variable quantity of hpv in immunocompetent individuals can range between 2770% depending on detection techniques [13, 17]. Thus the type of hpv, -papillomavirus species 2, is more often associated with cscc as opposed to the total amount of hpv dna present . Three theories have been suggested for the mechanism of hpv carcinogenesis: (1) uv radiation - induced immunosuppression to explain enhanced interaction between hpv and uv radiation [25, 26], (2) e6/e7 oncoprotein - related changes in p53 and rb tumor suppressor gene, and (3) integration of hpv dna disrupting genomic stability [17, 25]. Viral expression of e6 and e7 oncoproteins can inactivate p53 and rb tumor suppressor genes, leading to an uncontrolled system of cell proliferation and apoptosis . Association of viral pathogens such as human papillomavirus (hpv) with head and neck squamous cell cancer (hnscc), especially oropharyngeal cancer has been recognized over the past two decades . Hpv16 is the most common genotype in these tumours, whereas hpv6 and hpv11 can also be found in a minority of these cancers, implying that these low - risk hpv types are not entirely benign in hnscc . Hpv dna is closely associated with poorly differentiated cancers, positive lymph nodes, and late - stage disease, which portend a worse diagnosis . Hpv status is also associated with p16 expression and hpv+ tumours are less likely to harbour p53 mutations . A subset of hnscc patients who had hpv 16 infection confers a better prognosis . On the other hand, papillomaviruses (-hpvs) also play a role in the tumorigenesis of cscc as shown by both european and us studies . However, no high - risk types have been identified although there is an association of species 1 in scc . Other viruses, such as polyomavirus (mcpyv) have been shown to be causative agent in merkel cell carcinoma . The genetic progression model for head and neck squamous cell carcinoma (hnscc) demonstrates that loss of heterozygosity (loh) is common during the progression from premalignant lesion to malignant tumors . Tumor suppressor genes (tsgs) are usually found in the area of loss rendering the cells more susceptible to tumorigenesis . One of the most common regions, 9p21, has been reported in both hnscc and cscc . This region contains several tsgs, including p16ink4a (cdkn2a), p15ink4b, and mtap . Allelic imbalances are also found in other regions of cscc, including loh on 3p, 2q, 8p, and 13 and allelic gain on 3q and 8q . Such studies indicate that allelic imbalance and loh are recognized and relevant events in cscc and can be used for early diagnosis and tumor surveillance . Epigenetics refers to the molecular mechanisms that regulate gene expression without changes in the dna sequence . Epigenetic alterations include dna methylation and histone modifications, which consist of methylation, acetylation, phosphorylation, ubiquitination, and sumoylation, chromatin remodeling and micrornas [3638]. Changes in genomic dna methylation associated with cancer include global dna hypomethylation and gene - specific hyper- or hypomethylation . Tumor progression involved chromatin - mediated changes such as dna methylation yet the role of histone variants in tumorogenesis is unclear . All of these modifications of gene expression have been associated with the development of various tumor types, including hnscc and cscc [39, 40]. A higher frequency of foxe1 promoter hypermethylation was found in cscc compared to normal skin, indicating that foxe1 may be a target for aberrant methylation in cscc . Epigenetic dysregulation is thought to be involved in tumor biology and progression and thus may be relevant biomarkers for clinical prognosis . Promoter dna methylation gene panels have been described for screening of primary hnscc [41, 42], for determination of tumor recurrence and assessment of margin status during surgery . Recent evidence does show that the loss of histone variants, such as macro - h2a, positively correlates with an increasing malignant phenotype of melanoma cells and that macro - h2a is a critical component of chromatin that suppresses malignant progression of melanoma . However, a determination of methylation gene panels relevant in cscc is yet to be established . To date there has been no correlation of the significance of global histone modifications to prognosis in cutaneous scc although studies have shown them to be applicable in esophageal scc . A combination of different genes from different pathways may allow for a better determination of the aggressiveness of cscc to better correlate to prognosis . Messenger rna (mrna) and microrna (mirna) profiles have been described in both hnscc and cscc . Several mrna biomarkers for cscc were identified, including ccr10, ccl27, muc4, p16, mmp2, and mmp9 . A recent study has demonstrated that a distinct microrna profile is modulated by uv radiation . Mitochondrial mutation in hnscc has been well reported; however, only a few studies show the association of mitochondrial dna mutation and cscc . Several regions of mitochondrial dna were reported, including displacement loop (d - loop) and other regions . Therefore, mitochondrial mutations may correlate in the future with the phenotypic behavior of cscc . Even the spectrum of biologic behavior has been slow to be characterized given the previously very generic clinical criteria used to distinguish low risk lesions from a more aggressive lesions . Recent changes in the classification of the staging paradigm has better captured the histologic features of this more aggressive subset to allow for a more precision in identifying the worst subset . Incorporating a clear clinicopathologic classification system into stratifying lesions is necessary to better cluster them by their biology besides just histologic features.thus molecular analysis can potentially profile that subset with biomarkers chosen to best correlate with the biologic phenotype.
Malignant peripheral nerve sheath tumors (mpnsts) are a rare variety of soft - tissue sarcoma of ectomesenchymal origin, with an estimated incidence of 0.1/100,000/year . Recently, fluorodeoxyglucose (fdg) positron emission tomography / computed tomography (pet / ct) has been employed in differentiating the malignant from the benign peripheral nerve sheath tumors . The current case highlights an unusual pet / ct presentation of mpnst masquerading clinically as infected dermatoses . A 64-year - old male, working as a gardener, presented with multiple rapidly progressive asymptomatic skin colored raised lesions 2 - 4 cm in diameter, over the medial border of the right foot and lower part of the right leg since 1 month . The lesion was subjected to punch biopsy, with clinical differential diagnosis of sporotrichosis, actinomycetoma, linear cutaneous leishmaniasis and atypical mycobacterial infection . Histopathology revealed a malignant mesenchymal tumor, which was confirmed to be a mpnst on immunohistochemistry . Soon thereafter, the lesions rapidly increased in size and number and began to ulcerate and become painful [figure 1a]. Magnetic resonance imaging (mri) of the right lower limb revealed multiple enhancing nodular lesions seen involving the skin and subcutaneous tissue . Subsequently, a whole body 18f - fdg pet / contrast - enhanced computed tomography (cect) scan was performed on a whole body full ring pet / ct camera (discovery ste 16, ge). 370 mbq of 18f - fdg was administered intravenously subsequent to a 6 h fast . Whole body cect scan was performed after intravenous instillation of non - ionic contrast medium . After the ct scan, an emission scan was performed from head to thigh for 2 min per frame . Images were reconstructed by 3d vue algorithm (ge) and viewed on a xeleris workstation (ge) using the volumetric protocol . The study revealed multiple fdg avid nodular lesions (maximum standardized uptake value 18.6) in the right lower limb [figure 1b], which were many more in number compared with those picked up on cutaneous examination . Multiple nodular lesions (arrows) with evidence of ulceration on the medial aspect of the right leg and foot (a), which show increased fluorodeoxyglucose avidity (b) mpnsts arise from a peripheral nerve or its branches or from the nerve sheath . Although they may arise spontaneously, up to 50% arise in patients of neurofibromatosis 1 (nf1). Mri is the imaging modality of choice because it can reveal the nerve of origin and its relationship to adjacent structures . Although it is well - known that these tumors can extend for considerable distances along nerves, it may not always be possible to delineate the origin from the nerve . A recent study by bilgic et al . Has shown that the nerve origin could be identified only in 45 - 56% cases . There have been recent reports of fdg pet / ct in mpnst, most of which have been performed in patients with nf1 . It has been documented as a useful tool in monitoring clinically stable nf1 patients with plexiform neurofibromas as it could predict which were more likely to subsequently grow rapidly . It has also been found that in patients with nf1 harboring mpnsts; higher fdg uptake is associated with poorer survival rates . In addition, it can assist in guiding targeted needle core biopsies and may provide critical information in tumors that are not amenable to biopsy . It thus plays an important role in the staging, restaging and post - therapy follow - up of mpnst in nf1 . A high fdg uptake has been documented in sporadic mpnsts also, as is well seen in the coronal maximum intensity projection image [figure 2] in the present case . Coronal maximum intensity projection image showing high fluorodeoxyglucose uptake in multiple nodular lesions on the right lower limb, predominantly on the medial aspect mpnsts usually present as discrete masses, which may be multifocal, especially in cases of nf1 . The present case was unusual in its presentation, owing to the linear arrangement of multiple nodules over the lower extremity in a gardener, raising the possibility of infectious dermatoses . These multiple hypermetabolic mildly enhancing nodular lesions were restricted to the skin and subcutaneous tissue with no extension into the underlying muscles [figure 3]. The additional lesions detected on pet / ct were also proven to be mpnst based on histopathology with immunocytochemistry [figure 4]. Though multimodality therapy, including surgical resection and adjuvant radiotherapy, is available, the prognosis remains dismal . Axial contrast - enhanced computed tomography (a) and fused positron emission tomography / computed tomography (b) images of the leg (upper row) and foot (lower row) demonstrate multiple hypermetabolic mildly enhancing nodular lesions (arrows) involving the skin and subcutaneous tissue with no extension into the underlying muscles (a) h and e stain (10 [inset 40]) shows a tumor in the dermis with multiple spindle - shaped cells in whorls with pleomorphic nuclei forming a storiform pattern . (b) vimentin stain (10 [inset 40]) for mesenchymal structures is diffusely positive throughout the dermis
The recruitment of a sufficient number of growing follicles following ovarian stimulation increases the oocyte yield and subsequently the success rate after in vitro fertilization (ivf) treatment . However, some women exhibit a scarce ovarian response following controlled ovarian stimulation, and this phenomenon is mainly observed in the so - called poor responder patients.1 poor ovarian response (por) to gonadotropins affects ~9%24% of ivf patients, and many other causes could be involved, such as woman s age, endometriosis, genetic disorders, ovarian surgery, or even iatrogenic factors.2,3 until recently, there was insufficient evidence to uniformly define poor responder patients, and the most common criteria used were based on the low number of recovered oocytes following an adequate ovarian stimulation.48 more recently, a first systematic and realistic attempt to identify women who respond poorly to stimulation has been reported by the european society of human reproduction and embryology . Bologna criteria,9,10 which include: 1) advanced women s age (> 40 years) or any other risk factor for por; 2) recovering a few number of oocytes (<3 oocytes) following previous ovarian stimulation; and 3) abnormal ovarian reserve test (antral follicle count <57 or antimullerian hormone <0.51.1 ng / ml). However, it has been established that the presence of two of these criteria after maximal stimulation are sufficient to define a patient as poor responder in the absence of advanced maternal age or abnormal ovarian reserve test.9,10 several ovarian stimulation regimens have been used to treat poor responder women, but the results achieved are still controversial . These protocols include: varying the dose of gonadotropins or the day of menstrual cycle to initiate the stimulation,11,12 high dose of gonadotropins in a gonadotropin - releasing hormone (gnrh) long protocol,13,14 administration of gnrh and gonadotropins together in the follicular phase (flare - up protocol),15,16 pre - cycle estrogen treatment,17 growth hormone co - treatment,18 gonadotropin suppression with oral contraception before ivf cycle,19 using clomiphene citrate for stimulation20 or clomiphene citrate with human menopausal gonadotropin in an antagonist gnrh protocol,21 and non - stimulated (nature cycle) ivf treatment.22 all these stimulation regimens have been used with a limited success, and adequate number and quality of retrieved oocytes in poor responder women still remain the most challenging items in assisted reproductive technologies.23,24 a novel recombinant follicle - stimulating hormone (rfsh) analog, known as corifollitropin alfa, has recently been introduced for ovarian stimulation in patients undergoing ivf treatment.25 it contains an alpha subunit, which is identical to that of fsh, coupled to a hybrid subunit of the beta subunit of human fsh and the carboxy - terminal peptide of the beta subunit of human chorionic gonadotropin.26,27 clinical data on corifollitropin alfa have shown that it has longer half - life and slower absorption rate compared to rfsh.28,29 thus, the administration of a single dose of corifollitropin alfa during the early follicular phase of the menstrual cycle is capable of sustaining follicular development for the first week of stimulation unlike rfsh, which must be injected daily.30 moreover, it has been demonstrated that corifollitropin alfa is well tolerated by patients and its use does not present any adverse events or complications.31 because of its bioactivity profile, some authors have suggested that corifollitropin alfa may have a positive effect on stimulation outcome of poor responder women undergoing ivf treatment.32,33 in the present study, we evaluated the effect of the long - acting fsh and corifollitropin alfa on stimulation and clinical outcomes, when used for ovarian stimulation of poor responder women . A total of 85 poor responder women entered our ivf program from july 2013 to december 2015 . Bologna criteria:9 1) advanced women s age (> 40 years); 2) few number of retrieved oocytes (<3 oocytes) following previous ovarian stimulation; and 3) abnormal ovarian reserve test (antral follicle count <57 or anti - mullerian hormone <0.51.1 ng / ml). The patients were included in the study if they fulfilled all criteria or at least two of them . The patients were randomized before gonadotropin stimulation, using a computer - generated random assignment . Sealed envelopes were used until starting the process of randomization in order to conceal treatment allocation . The patients received all the information regarding the nature of the study and gave their written informed consent . The primary parameters considered for statistical analysis were: the number of recovered oocytes, the number of mature metaphase ii oocytes, the number of cleaving embryos, pregnancy rate, and implantation rate . We analyzed also the stimulation outcome, ie, canceled cycles, duration of stimulation, serum estradiol level, endometrial thickness on the day of human chorionic gonadotropin administration, fertilization rate, delivery rate, and miscarriage rate . The patients were prospectively randomized into two groups: corifollitropin (group a), as the study group, and daily rfsh injection (group b) as the control group . In group a, 42 women aged 40.33.9 years were stimulated with 150 mg / day clomiphene citrate, starting from days 2 to 6 of the cycle . On day 2 of the cycle, a single dose of 150 g of corifollitropin alfa (elonva, merck sharp & dohme limited, rome, italy) was administered and thereafter a dose of 225 iu / day of fsh (gonal f, merk serono, rome, italy) was administered daily, starting from day 7 of the cycle onward . In group b, 43 women aged 412.6 years received 150 mg / day clomiphene citrate from days 2 to 5 of the cycle and a dose of 225 iu of rfsh was administered daily, starting from day 3 of the cycle onward . When the leading follicle reached 14 mm, all patients in both groups received 0.25 mg / day gnrh antagonist . A dose of 10,000 iu of human chorionic gonadotropin (gonasi hp, ibsa, lugano, switzerland) was administered in order to induce the final ovulation maturation and trigger when the leading follicle reached a diameter of 1820 mm and at least two follicles of 1718 mm . Ovum pick - up was performed 36 hours after human chorionic gonadotropin administration and the retrieved oocytes were assessed for their maturity . Mature metaphase ii oocytes were inseminated by intra - cytoplasmic sperm injection and the resultant viable embryos were transferred into the patient s uterus . The embryo transfer was performed on day 3 after insemination and a dose of 50 mg / day of progesterone was administered in order to support the luteal phase after embryo transfer . The data were statistically analyzed using the jmp software (version 4.0.4; sas corp ., cary, nc, usa). According to the two - tailed hypothesis, the minimum total sample size required was 84 patients, at least 42 patients per group, in order to reach a statistical power of 80% with confidence intervals of 95% and an level of 0.05 . The two - tailed student s t - test, fisher s exact test, and two - by - two table were used to analyze the parameters between groups where appropriate, and the significance level was set at p0.05 . A total of 85 poor responder women entered our ivf program from july 2013 to december 2015 . Bologna criteria:9 1) advanced women s age (> 40 years); 2) few number of retrieved oocytes (<3 oocytes) following previous ovarian stimulation; and 3) abnormal ovarian reserve test (antral follicle count <57 or anti - mullerian hormone <0.51.1 ng / ml). The patients were included in the study if they fulfilled all criteria or at least two of them . The patients were randomized before gonadotropin stimulation, using a computer - generated random assignment . Sealed envelopes were used until starting the process of randomization in order to conceal treatment allocation . The patients received all the information regarding the nature of the study and gave their written informed consent . The primary parameters considered for statistical analysis were: the number of recovered oocytes, the number of mature metaphase ii oocytes, the number of cleaving embryos, pregnancy rate, and implantation rate . We analyzed also the stimulation outcome, ie, canceled cycles, duration of stimulation, serum estradiol level, endometrial thickness on the day of human chorionic gonadotropin administration, fertilization rate, delivery rate, and miscarriage rate . The patients were prospectively randomized into two groups: corifollitropin (group a), as the study group, and daily rfsh injection (group b) as the control group . In group a, 42 women aged 40.33.9 years were stimulated with 150 mg / day clomiphene citrate, starting from days 2 to 6 of the cycle . On day 2 of the cycle, a single dose of 150 g of corifollitropin alfa (elonva, merck sharp & dohme limited, rome, italy) was administered and thereafter a dose of 225 iu / day of fsh (gonal f, merk serono, rome, italy) was administered daily, starting from day 7 of the cycle onward . In group b, 43 women aged 412.6 years received 150 mg / day clomiphene citrate from days 2 to 5 of the cycle and a dose of 225 iu of rfsh was administered daily, starting from day 3 of the cycle onward . When the leading follicle reached 14 mm, all patients in both groups received 0.25 mg / day gnrh antagonist . A dose of 10,000 iu of human chorionic gonadotropin (gonasi hp, ibsa, lugano, switzerland) was administered in order to induce the final ovulation maturation and trigger when the leading follicle reached a diameter of 1820 mm and at least two follicles of 1718 mm . Ovum pick - up was performed 36 hours after human chorionic gonadotropin administration and the retrieved oocytes were assessed for their maturity . Mature metaphase ii oocytes were inseminated by intra - cytoplasmic sperm injection and the resultant viable embryos were transferred into the patient s uterus . The embryo transfer was performed on day 3 after insemination and a dose of 50 mg / day of progesterone was administered in order to support the luteal phase after embryo transfer . The data were statistically analyzed using the jmp software (version 4.0.4; sas corp ., cary, nc, usa). According to the two - tailed hypothesis, the minimum total sample size required was 84 patients, at least 42 patients per group, in order to reach a statistical power of 80% with confidence intervals of 95% and an level of 0.05 . The two - tailed student s t - test, fisher s exact test, and two - by - two table were used to analyze the parameters between groups where appropriate, and the significance level was set at p0.05 . Seventy - six out of the 85 studied women underwent the oocyte retrieval procedure, 37 women in group a and 39 women in group b. nine patients were canceled because of ovarian response failure to stimulation: five in group a and four in group b. on comparing between the two groups, no statistically significant differences were observed regarding the primary end points (number of retrieved oocytes, number of cleaving embryos, pregnancy and implantation rates, and all studied secondary end points as well). As depicted in table 1, demographic data, stimulation outcome, and table 2 shows comparable embryological results, without any relevant significant differences observed between groups a and b in terms of the number of retrieved oocytes (30.8 and 2.70.7, respectively), the number of mature oocytes (250.8 and 2.40.8, respectively), and the number of cleaving transferred (1.80.6 and 1.70.7, respectively). Higher, though not statistically significant, difference in favor of group a compared to b in terms of pregnancy rate per embryo transfer (21.6% in group a and 17.9% in group b), pregnancy rate per cycle (19% in group a and 16.3% in group b), implantation rate (14.7 in group a and 13.4 in group b), and delivery rate per transfer (13.5 in group a and 10.3 in group b). Ongoing pregnancy rate and miscarriage rate were also similar between the two groups (table 3). It implements the administration of exogenous gonadotropins to induce the development of a number of follicles, thus increasing the oocyte yield and the availability of transferable embryos . In turn, adequate oocyte recruitment and recovery, following ovarian - controlled hyperstimulation, increases the likelihood of conception . Patients.1 obviously, poor response to gonadotropins stimulation still remains the most challenging problem in assisted reproduction field . Several ovarian stimulation approaches have been implemented with the aim to improve ovarian response and clinical outcome in poor responder women, but the results are still low and are often controversial.1121 the main reason for this controversy is due to the difficulties in establishing a uniform definition of poor responder patients, which can be universally applied.34 recently, the european society of human reproduction and embryology working group on por has published a consensus paper describing three criteria to be used to identify women with por, the so - called bologna criteria.9,10 although these criteria remain the most realistic attempt to identify women with a high probability of having reduced ovarian reserves, further research on the various mechanisms and risk factors, which may differently influence the ovarian reserve, are still urgently needed . Moreover, a recent cochrane review, analyzing 10 published trials on different stimulation regimens used for the treatment of poor responder patients, did not find sufficient evidence to indicate any stimulation approach for routine clinical use to treat poor responder women.24 with the advent of dna technology, a novel recombinant long - acting gonadotropin, corifollitropin alfa, has been recently produced and became available for clinical application . Its efficacy to stimulate multiple follicular growths has been proven in normal responder s women undergoing ivf treatment.2830 a growing body of evidence, as reported in the literature, has widely demonstrated that long - acting fsh (corifollitropin alfa) is noninferior to daily rfsh . A systematic review and meta - analysis included four randomized trials, with a total of 2,326 normal responder patients, on the role of corifollitropin alfa compared to rfsh in a gnrh antagonist downregulation protocol . The analyzed data showed no significant difference in ongoing pregnancy rate, while an increased risk of developing ovarian hyperstimulation syndrome was observed in patients treated with corifollitropin alfa.35 more recently, another meta - analysis, analyzing three randomized controlled trials, involving 3,392 normal responder s women, on corifollitropin alfa compared to rfsh did not evidence any significant differences in all studied parameters, including pregnancy and live birth rates . The authors concluded that a single dose of corifollitropin alfa for the first 7 days of stimulation is similarly effective as daily injected rfsh.36 additionally, a cochrane review including six randomized controlled trials, with a total of 3,753 patients, resulted in no significant differences between patients treated with the long - acting fsh and daily rfsh . From the analyzed data it was concluded that the long - acting fsh is a safe treatment and equally effective compared to daily fsh, but its efficacy in the treatment of hyper or poor responder women is still to be demonstrated.37 moreover, treatment of older women (aged 35 to 42 years) with corifollitropin alfa has proven to be as efficacious as daily rfsh in terms of the number of recovered oocytes, pregnancy rate, and live birth rate.38 nevertheless, in all preliminary studies on corifollitropin alfa, poor responder patients were excluded from studied population due to their reduced ovarian reserve,37 but in our study we used a single dose of corifollitropin alfa administered in the early follicular phase of the menstrual cycle to induce multiple follicular recruitment and growth in poor responder patients undergoing ivf treatment . Previous studies have suggested that corifollitropin alfa might have a valuable role during the early phase of follicular development and growth . Based on the peculiar pharmacokinetic profile and bioactivity of corifollitropin alfa, which is characterized by around two - fold longer half - life in comparison to rfsh, it allows a sustained fsh activity during early follicular recruitment and development . This is a critical step for ovarian stimulation, particularly in the treatment of poor responder women.28,39 the use of corifollitropin alfa to treat poor responder patients was first reported by polyzos et al in two retrospective published studies.32,40 in one study, they retrospectively compared poor responder patients stimulated with corifollitropin alfa plus rfsh in a gnrh antagonist regimen, and poor responder patients stimulated with the standard human menopausal gonadotropin in a gnrh agonist protocol . The obtained results were comparable and the authors concluded that there is no relevant difference between the two groups regarding all studied parameters.32 in another retrospective study, the same authors reported on ovarian stimulation of two groups of poor responder women, based on their age: 40 years and 40 years groups . The patients in both groups were stimulated using corifollitropin alfa plus highly purified human menopausal gonadotropin in a gnrh antagonist regimen . The author observed a statistically higher pregnancy rate in favor of patients aged 40 years compared to those aged 40 years (28% and 0%, respectively), whereas all other studied parameters were similar between the two groups.40 recently, a randomized controlled trial showed that there were no statistically significant differences in all studied parameters, including pregnancy and live birth rates, between poor responder patients treated with corifollitropin alfa and those treated with daily rfsh (follitropin beta).41 additionally, rinaldi and selman33 have retrospectively compared poor responder women treated with corifollitropin alfa plus rfsh in a gnrh antagonist regimen and those stimulated with daily rfsh injection in a gnrh antagonist regimen . The achieved results were similar between the two groups for all analyzed parameters, including pregnancy and implantation rates . As the study was analyzed retrospectively and a small number of patients were included, the authors have addressed further studies on the possible positive effect of corifollitropin alfa when used for ovarian stimulation in poor responder patients.33 in the present prospective randomized study, we attempt ovarian stimulation in poor responder patients using a single dose of corifollitropin alfa administered in early follicular phase followed by rfsh in comparison to rfsh daily administered to stimulate poor responder women . In both gonadotropin treatment protocols, clomiphene citrate was added because it has an antiestrogen activity and is capable of interfering with endogenous estrogen in the pituitary gland and hypothalamus by blocking the receptor - binding sites through a feedback - mediating mechanism, resulting in higher production of fsh and lh hormones by the pituitary gland . It also activates the production of a high level of gonadotropins, induces the ovarian follicle s development, maturation, and ovulation . However, although a general agreement on the specific role of clomiphene citrate in ovarian stimulation is still lacking,42 some authors have suggested that it may have a positive effect in the treatment of patients with poor ovarian reserve.20,4345 our results show that corifollitropin alfa exhibits similar results compared with daily injected rfsh when used to stimulate patients who respond poorly to ovarian stimulation with standard stimulation regimens . Nevertheless, no significant differences were observed between the two groups in terms of all studied parameters, including number of retrieved oocytes, mature oocytes, fertilization rate, pregnancy rate, and implantation rate; evidence of a trend was observed in favor of the patients stimulated with corifollitropin alfa plus rfsh compared to those treated with daily rfsh . Our study shows that corifollitropin alfa appears to be as efficacious and efficient as a daily rfsh stimulation regimen, and suggests that it can be a reliable alternative stimulation regimen to treat patients with poor ovarian reserve . However, an additional study on a large number of patients should be addressed in order to establish the effective role of the long - acting fsh, corifollitropin alfa, in patients who respond poorly to gonadotropin stimulation.
We came across 3 cases of sarcomas, other than leiomyosarcomas, reported to arise from the broad ligament . To the best of our knowledge, no case of an embryonal rhabdomyosarcoma (erms) of the broad ligament has been reported in literature . We report about a 9-month - old female child with an erms of the broad ligament . The case we present here is a 9-month - old female child, presented to us with the complaints of straining at micturition and defecation since the age of 5 months . The patient was diagnosed with benign partial epilepsy of infancy and was under treatment for the same . No other comorbidities were present . On examination, the patient had multiple caf au lait spots over her body (more than 5 in number). However, there were no other features to suggest neurofibromatosis, like, family history, lisch nodules, axillary or inguinal freckling and osseous lesions . Abdominal examination revealed an abdominopelvic mass, in the midline, extending superiorly to just above the umbilicus . A contrast enhanced computed tomography (cect) scan of the abdomen and pelvis confirmed the clinical findings of an abdominopelvic mass of about 9.8 cm 6 cm 5 cm extending from the level of acetabulum in the pelvis, to the level of l3 vertebra [figure 1]. The mass was isodense to hypodense on a plain scan with heterogenous enhancement on post - contrast scan . The urinary bladder was pushed to the right side due to mass effect but planes were maintained . The uterus and adnexa were not visualized separately . A percutaneous biopsy done from the mass revealed erms . The metastatic work - up, including a bone scan, cect thorax and bone marrow examination, was normal . Contrast enhanced computed tomography scan showing the heterogenously enhancing mass arising from pelvis, with contrast in urinary bladder (a and b). Intra - operatively (c), the mass seen arising from the broad ligament, the uterus (a) and urinary bladder (b) separated the patient received neoadjuvant chemotherapy (nact), as per intergroup rhabdomyosarcoma study group (irs) iv protocol (vincristine, actinomycin and cyclophosphamide). An exploratory laparotomy performed revealed a lobulated mass of 10 cm 5 cm 5 cm [figure 1]. The mass had a pedicle and was seen to be arising from the left broad ligament . The uterus and adnexa were separate from the mass, pushed to the right side . The mass was densely adhered to the urinary bladder but could be separated by dissection . As per the tnm and irs staging, our patient was staged as stage i, group iii, with embryonal histology and thus classified as low risk subgroup . Rhabdomyosarcoma is the most common soft - tissue sarcoma of children accounting for 3 - 4% of all the childhood cancers . Rhabdomyosarcoma is known to be associated with caf au lait spots and neurofibromatosis type - i . Our patient though had caf au lait spots, did not have other features suggestive of neurofibromatosis . On reviewing the literature on broad ligament sarcomas (excluding leiomyosarcomas), we came across only 3 such cases . Have reported the first of those in 1985 in their series of eight patients of female genital tract sarcomas . The patient was 16 years of age with an alveolar rhabdomyosarcoma of the broad ligament . The second case was a 55-year - old female diagnosed and treated for an undifferentiated pleomorphic sarcoma of the broad ligament reported by bouraoui et al . The third patient, reported by diaz - murillo et al . Was a 27-year female with an undifferentiated round cell sarcoma of the broad ligament . Our patient is a 9-month - old child with an erms of the broad ligament . The patient reported by diaz - murillo was followed - up for 12 years without any recurrence . The treatment is decided after a tissue diagnosis and metastatic work - up of the patient . Our patient after appropriate investigations was diagnosed as a non - metastatic pelvic erms . On the basis of cect scan findings of an abutting urinary bladder and non - visualization of the left ureter, it was decided to start nact (irs iv protocol) and reassess for the response . On repeating a cect scan after nact, it was a stable disease; hence, it was decided to go ahead with surgery for appropriate local control of the tumor . Adjuvant radiation therapy was considered in view of the mass adherent to the urinary bladder, but withheld considering the age and low risk group of the patient . Broad ligament rhabdomyosarcomas are an extremely rare entity . To the best of our knowledge, this is the first case of an erms arising from the broad ligament in an infant.
Hamartomatous polyps are uncommon and usually found as a part of peutz - jegher syndrome, a rare autosomal dominant disorder associated with mucocutaneous pigmentation and family history . Hamartomatous polyps are composed not only of epithelial elements, but also of bundles of smooth muscle cells . Such hamartomatous gastric polyps are almost always found in patients with peutz - jeghers syndrome, in association with intestinal polyps . In hamartomatous polyps, there is an increased risk of malignancy in both the gastrointestinal and extra - intestinal sites [2 - 4]. The risk increases with age (range, 1 to 33%) between 30 and 60 years . Fifty seven years - old man with a history of epigastric pain was transferred from an outside hospital with the diagnosis of early gastric cancer . Physical examination and laboratory no pigmentation was found on the lips and perioral region, hands, buccal mucosa, and feet . Endoscopy revealed two masses on the greater curvature side of the prepyloricantrum: a round, 1 cm polypoid lesion and an irregular 3 cm elevated mass with superficial ulcerations (fig . 1a) and another 6 cm pedunculated polypoid mass on the greater curvature side of prepyloricantrum (fig . Upper gastrointestinal series showed three polypoid lesions in the gastric antrum which was considered as an advanced, borrmann type i carcinoma preoperatively (fig . Subsequently, the patient underwent a radical subtotal gastrectomy, which revealed a 6 5 cm mass confined to the mucosa of the greater curvature of the lower body and another 5.5 2 cm mass invading into the submucosa of the greater curvature of the antrum (fig . The 5.5 2 cm sized mass looked like two masses, but was a fused mass intraoperatively . Microscopic analysis of the specimen revealed adenocarcinoma within the hamartomatous polyps demonstrated by proliferation of smooth muscle bundles within the lamina propria and between carcinomatous glands (fig . 4a) with evidence of epstein - barr virus (ebv) in both lesions (fig . The patient had an uneventful postoperative course and was discharged home on the 9th postoperative day . Reported four cases of patients with multiple benign gastric hamartomatous polyps that were not associated with polyposis coli . However, syndrome - associated hamartomatous polyps may have malignant potential as cases of malignant hamartoma have been described [8 - 10]. From our review of the english - language literature, this case report appears to be the first documented case of adenocarcinoma derived from gastric hamartomatous polyps which was not accompanied by a known syndrome . We present a case report of a patient with malignant gastric hamartomatous polyps in the absence of familial polyposis nor pigmentation . Furthermore, this case exhibited positive expression via in ebv - encoded rna (eber) in situ hybridization . To date, there is no report about incidence of adenocarcinoma in hamartomatous polyps in eber - positive in situ hybridization.
Cervical carcinoma is one of the most common gynecologic cancers diagnosed and represents a substantial threat to the wellbeing and survival of the world's adult female population . While human papillomavirus (hpv) infection (both chronic and persistent) of the cervical epithelium cells is recognized as the major risk factor of cervical cancer, it is not sufficient for cancer occurrence, since most women with hpv infection do not develop cervical cancer [1, 2]. The tumorigenic process of cervical cancer involves an array of immunomodulatory cytokines as well, including interleukins (il) 6, 10, and 12 and transforming growth factor- (tgf-) [3, 4], all of which are expressed by tumor cells . Cervical cancer cells have also been shown to secrete the granulocyte macrophage - colony stimulating factor (gm - csf) [5, 6], an important hematopoietic cytokine that is known to contribute to the survival, proliferation, and differentiation of bone marrow hematopoietic cells and to function as a cofactor to influence differentiated cells of other lineages and hematopoietic multipotent precursor cells . In the immune response, gm - csf helps to regulate the response to infection and inflammatory signaling cascades by recruiting dendritic cells and triggering their maturation as well as promoting cell - mediated immunity pathways . Nevertheless, the immunological effect of cervical cancer - derived gm - csf in the cervical cancer environment is still poorly understood . In addition, the gm - csf receptor (gm - csfr) is expressed by many of the mature hematopoietic cell types, including neutrophils, monocytes, and eosinophils, as well as some nonhematopoietic cells, such as the small cell carcinoma lines and human prostate cancer cells [9, 10]. However, whether cervical cancer cells express gm - csfr and whether the gm - csf / gm - csfr signaling pathway contributes to the pathogenic process of cervical cancer remain unknown . Human carcinomas are also associated with upregulation of the inducible nitric oxide synthase (inos) and cyclooxygenase-2 (cox-2), which are otherwise generally not expressed in normal (noncancerous) tissues, with the exception of placenta, kidney, and brain [11, 12]. Human carcinomas exhibiting high levels of cox-2 and inos expression include those in stomach, liver, lung, pancreas, colon, and cervix [1315]. These two synthases are involved in many physiological and pathological processes, and their combined expression is closely related to the tumor biological behaviors of growth, progression, metastasis, and prognosis . A key function of inos is the enzymatic conversion of arginine to generate a locally high concentration of nitric oxide (no); from a tumorigenic perspective, the inos - mediated increase in no supports cancer development . On the other hand, cox-2 acts as one of the rate - limiting enzymes in the metabolic pathway that produces eicosanoids; from a tumorigenic perspective, the ptgs2 gene encoding cox-2 acts as an early response gene inducible by carcinogens, tumor promoters, and oncogenes . The demonstrated correlation of cox-2 expression with several types of tumors has suggested potential roles for this enzyme in cancer progression, specifically by inducing proliferation, enhancing mitogenesis, reducing cellular adhesion, and aiding in tumor escape from immune surveillance . The above evidence indicates that cervical cancer cells express gm - csf and synthases cox-2/inos . Intriguingly, it has been reported that gm - csf can upregulate cox-2 and inos expression in skin dendritic cells . However, whether and how gm - csf regulates cox-2 and inos expression in human cervical cancer tissues remain to be clarified yet . To this end, this study was designed to determine whether gm - csf plays a role in human cervical cancer via influence on inos / cox-2 and to investigate the related underlying mechanism; the results from this study were expected to not only provide insight into the pathogenic processes of cervical cancer but identify molecular factors representing potential therapeutic targets . Eighty - seven specimens of clinical stage ib - iib squamous cell carcinoma and 24 specimens of cervical intraepithelial neoplasia (cin) were obtained from adult female patients (age range: 3168 years, median: 46.51 years) who had undergone radical hysterectomy with lymphadenectomy . Sixteen normal cervical tissues were obtained from women who had undergone hysterectomy for nonneoplastic indications and had normal findings from the last cervical smear . The tissue samples were fixed in 4% buffered paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin for histological analysis . The cin and primary cervical cancer specimens were graded according to the who criteria (cin1, cin2, and cin3 classifications) and the international federation of gynecology and obstetrics (figo) criteria (ib1, ib2, iia, or iib cervical carcinoma stages). The study was approved by the ethics committee of southwest hospital for clinical investigation (chongqing, china). The paraffin - embedded tissue samples were sectioned (5 m thickness) and mounted on glass slides for ihc analysis according to the published staining methods . Briefly, after blocking of nonspecific sites (5% bovine serum albumin, room temperature, 30 min), the slides were incubated (4c, overnight) with the primary antibodies to mouse monoclonal anti - human gm - csf (1: 30 dilution; r&d systems, wiesbaden, germany), mouse monoclonal anti - human gm - csf receptor or (1: 50; santa cruz biotechnology, dallas, tx, usa), rabbit anti - inos (1: 100; boster, wuhan, hubei, china), and rabbit anti - cox-2 (1: 100; boster), followed by incubation (4c, overnight) with the appropriate secondary antibodies . Staining with diaminobenzidine (dab) tetrahydrochloride was performed to localize antigens, and the sections were further counterstained with mayer's hematoxylin . A pathologist who was blinded to the clinical features of the study participants evaluated the processed samples . Gm - csf, inos, and cox-2 immunoreactivity was graded according to the german immunoreactive scoring system, which involves rating the staining intensity in the cytoplasm on a scale from 0 to 3 (0: none, 1: weak, 2: moderate, and 3: strong). The numbers of positive and negative cells were counted (with no less than 500 cells considered as the minimum acceptable count number) and used to calculate the percentage of positive cells for scoring as follows: 0, no staining; (1), 110%; (2), 1150%; (3), 5180%; and (4), 81100% . The final immunoreactivity score was calculated by multiplying the staining intensity score by the score for positive cells percentage, with a range from 0 to 12 whereby 0 indicated no immunoreactivity, 14 indicated weak immunoreactivity, 58 indicated moderate immunoreactivity, and 912 indicated strong immunoreactivity . For statistical purposes, the final immunoreactivity scores were grouped as negative / weak immunoreactivity and moderate / strong immunoreactivity . The hela (hpv-18-infected), siha, and ca ski (with both of the latter being hpv-16-infected) human cervical cancer cell lines were purchased from the china center for type culture collection (wuhan, hubei, china). All cell lines were grown in rpmi 1640 medium (hyclone, logan, ut, usa) supplemented with l0% heat - inactivated fetal calf serum (hyclone) and 1% penicillin and streptomycin (beyotime, shanghai, china) at 37c in a 5% co2 atmosphere . To establish overexpression of gm - csf in the cervical cancer cell lines, the hgm - csf - expressing plasmid pcdna3.1()/gm - csf (hereafter referred to as pgm - csf) and the vector pcdna3.1() were purchased from invitrogen (san diego, ca, usa) and lipid - mediated dna transfection (lipofectamine 2000; invitrogen) was performed . To silence the expression of hgm - csf, the cancer cell lines were seeded in 6-well tissue culture plates (10 cells / ml) and transfected using the lipofectamine 2000 reagent to introduce 5 nm of gm - csf sirna (number sc39391; santa cruz biotech) or scrambled control sirna (number sc-37007, santa cruz biotech). For both procedures, at 48 h postinoculation, the cells were harvested for western blot analysis to verify the expression of hgm - csf by using an anti - human gm - csf antibody (r&d systems) as the primary antibody . The levels of inos / cox-2 protein in the processed cancer cells were detected by western blot as described previously . Briefly, cells were washed twice with pbs and lysed in ripa lysis buffer (beyotime). After measurement of total protein by the bicinchoninic acid (bca) assay, equal sample amounts were loaded for separation through a 412% gradient sds - polyacrylamide gel (invitrogen) and electrotransfer onto a polyvinylidene fluoride membrane . The membrane was then blocked with 5% skim milk powder, followed by hybridization with the respective primary and secondary antibodies . Quantitative analysis was carried out with a 9-image scanner densitometer (alpha innotech, santa clara, ca, usa) and normalized to the actin control . In addition, the signals were detected by use of a luminol chemiluminescence detection kit (santa cruz biotechnology) and exposure to hyperfilm (amersham, piscataway, nj, usa). All statistical analyses were performed by using the spss statistical software for windows (version 17.0; spss inc ., the chi - square test or fisher's exact test was used for association analysis between immunostaining results and clinical characteristics . General linear regression model was used to determine possible correlations between gm - csf negative / weak or moderate / strong expression and cox-2/inos protein expression in cervical cancer tissues . Unpaired student's t - test was used to evaluate the differences in cox-2 and inos protein expression among gm - csf transfected cells (gt), vector control (vt), and blank control (nt). As shown in figure 1 and table 1, the ihc assay showed positive expression of cox-2 and inos in 49 of 87 (56.3%) and 46 of 87 (52.9%) of the cervical cancer tissue samples, respectively . While similar positive rates for cox-2 and inos expression were observed in the cin tissues, the normal cervical tissues showed no cox-2 and inos expression . Furthermore, the detected cox-2/inos expression in tumor cells was mainly localized to the cytoplasm, showing a diffuse pattern; a lower level of expression was also detected in the adjacent stromal cells of the cancer samples . Although moderate / strong expression of gm - csf was detected in 44 out of the 87 (50.6%) cervical cancer tissues examined, this rate was markedly lower than that in the normal cervical tissues (81.3%) (table 1 and figure 2). In addition, expression of gm - csfr was detected at similar levels in both cervical cancer tissues and normal cervical tissues (figure 2); however, the gm - csfr expression was not present in the cervical cancer nest and instead was mainly localized within the stromal cells (figure 2). Associations analysis showed that neither cox-2 nor inos expression was associated with patient age, tumor size, or hpv infection (all p values> 0.05); however, both cox-2 and inos expressions were positively correlated with lymph node metastasis and figo stage (table 2) (p <0.05). No significant associations were found to exist between gm - csf expression and any of the patients' clinical parameters, for either the squamous cell carcinoma or cin patient groups (table 2) (p> 0.05). Rank correlation analysis to assess whether the enhanced cox-2/inos expression in cervical cancer cells was associated with gm - csf unexpectedly showed that the expression of gm - csf in cancer tissues had a significantly negative correlation with cox-2 (r = 0.500, p <0.05) and inos (r = 0.473, p <0.01) (table 3). The ca ski cervical cancer cells were found to express a low level of endogenous gm - csf, but the siha and hela cells did not express any detectable endogenous gm - csf (figure 3(a)). When transfected with gm - csf expressing plasmids, all of the cell lines showed appreciable expression of gm - csf protein, while the cells transfected with control vectors and blank control cells did not express any detectable gm - csf (figure 3(a)). Notably, all of these cell lines were found to express high levels of endogenous inos and cox-2 protein, and this expression was found to be downregulated by about 1-fold upon pgm - csf transfection (figures 3(b) and 3(c)). Transfection of the cervical cancer cells lines with a specific sirna sequence targeting the gm - csf gene led to undetectable gm - csf protein in the ca ski cells, which otherwise expressed a low level of endogenous gm - csf protein . As expected, transfection of the vector control did not influence the gm - csf expression, as compared with that detected in the untreated ca ski cells (figure 4(a)). The siha and hela cells, which did not express any detectable endogenous gm - csf protein, showed no effect upon transfection of the sirna sequence targeting gm - csf, neither on gm - csf expression nor inos and cox-2 expression (figures 4(b) and 4(c)). Cervical cancer cells express synthases cox-2/inos and gm - csf [5, 6, 1315], and gm - csf has been shown to upregulate the expression of the synthases cox-2/inos in skin dendritic cells . However, whether and how gm - csf regulates cox-2/inos expression in human cervical cancer cells remain unclear . In this study, we found that cox-2 and inos expression is upregulated in cervical cancer tissues, compared to normal cervical tissues, and that this upregulation is positively correlated with cancer metastasis and stage . Although about one - half of the cervical cancer tissues examined in our study showed strong / moderate gm - csf expression, this rate was lower than that for the normal cervical tissues (which had> 80% positive rate); moreover, the cervical cancer cells showed no detectable gm - csfr expression . Our statistical analysis indicated that the gm - csf expression detected in cervical cancer cells was negatively correlated with the cox-2 and inos expression in the same tissues, suggesting that the tissues with moderate / strong gm - csf expression are prone to having less cox-2 and inos expression, and vice versa . The downregulating effect of gm - csf on cox-2/inos expression was further confirmed in studies of three cervical cancer cell lines . Collectively, the results presented herein suggest that gm - csf may play a protective role against cervical cancer by downregulating the protumor factors cox-2 and inos via a gm - csfr - independent mechanism . Several previous lines of evidence obtained from studies involving patients with cervical cancer have implied the clinical significance and biologic roles of both inos and cox-2 [18, 2225]. A study of cervical carcinoma patients who underwent radiotherapy treatment showed that overexpression of inos and cox-2 was associated with greater risks of metastasis and death . Another study using the hela cell line showed associations of inos and cox-2 with lymph node metastasis and angiogenesis and demonstrated that the coordinated activities of these two synthases contribute to the development and occurrence of cervical cancer through their activation of the nf-b pathway . Similarly, a study by bandyopadhyay et al . Showed a positive association between cox-2 expression and lymph node metastasis in cervical carcinoma . These reports collectively support the proposed protumor role of cox-2 and inos in cervical cancer, which agrees with our observations of these two proteins being significantly enhanced in clinical samples of cervical cancer tissues and with the presumed promotion of lymph node metastasis and higher stage (more advanced) cervical cancer . The relationship between gm - csf and inos and cox-2 expression in immune cells is well documented . For instance, incubating peripheral blood mononuclear cells from a healthy donor with gm - csf alone or in combination with the cytokines vegf, tnf-, il-6, or il-1 results in upregulation of inos . In addition, the expression of cox-2 in activated (lipopolysaccharide- (lps-) stimulated) monocytes may be regulated by the activities of gm - csf - stimulated stat5 acting in cooperation with other transcription factors such as ap-1, il-6, and nf-b, while in skin dendritic cells, gm - csf induces inos expression by activating the nf-b pathway . However, the findings from our current study showed that gm - csf expression is negatively correlated with the cox-2 and inos expression in cervical cancer tissues . Furthermore, when we established ectopic overexpression of gm - csf in the cervical cancer cell lines hela, siha, and ca ski, we found a marked downregulation of the expression of cox-2 and inos, with knockdown of gm - csf expression leading to enhanced expression of both factors . These findings suggest that gm - csf might exert different effects on different cell types . Specifically, the gm - csf protein may promote the proinflammatory response in immune cells, such as apcs, to clear a pathogenic infection and may be useful to enhance a vaccine's efficiency by upregulating cox-2 and inos expression . As observed in this study, gm - csf might suppress the expression of both protumor factors cox-2 and inos in tumor cells, thereby exerting an antitumor effect; therefore, the decreased gm - csf expression in clinical cervical cancer tissues might be responsible for the upregulated expression of both protumor factors . Although the exact mechanisms underlying these potentially beneficial effects remain to be fully elucidated, the suppressive effects of gm - csf on cox-2 and inos expression in cancer cells appear to occur via a gm - csfr - independent pathway, as suggested by the lack of gm - csfr expression in cervical cancer cells . The occurrence of cervical carcinoma is closely related with hpv infection, and hpv may play an important role in development of the disease . It has been reported that hpv18 infection is associated with poor prognosis for patient survival and accelerated tumor growth . Specific hpv proteins, such as e2, e6, and e7, have been reported as associated with certain cytokine dysfunctions, and in hpv - infected cervical epithelial cells, the hpv e2 protein has been shown to induce il-10 activity and to cause immune suppression and persistence of the infection . The downstream effects of hpv infection suggest that the presence and activity of this viral pathogen may correlate with many inflammation - related molecules, such as gm - csf, inos, and cox-2 . Indeed, a study has shown that inos expression is significantly higher in keratinocytes transduced with e6 and e7 from hpv16 than in untransduced keratinocytes, implying that hpv might affect expression of inos . However, that in vitro study may not fully reflect the in vivo effects of hpv infection on the expression of the related factors, which may explain why we did not observe any correlation between hpv infection and the expression of gm - csf, inos, or cox-2 in our clinical cervical cancer tissue samples . Moreover, our finding of no hpv correlation is supported by observations from sarian et al ., who demonstrated that the hpv detection rates do not differ significantly across cox-2 protein expression strata, ranging from negative to strong expression . This study shows that tumor - derived gm - csf might elicit an antitumor response in cervical cancer by inhibiting the inos and cox-2 expression in cervical cancer cells in a gm - csfr - independent manner . These results suggest that gm - csf might be a helpful adjuvant that will aid in the design of a novel cervical cancer vaccine . Further studies are necessary to explore the detailed mechanism by which gm - csf regulates inos and cox-2 expression in cervical cancer cells to support the potential applications of gm - csf in clinical cervical cancer patients.
Epilepsy is a common clinical neurological disease, with a prevalence of approximately 1%, that is highly associated with inflammation and oxidative stress [14]. Epilepsy is characterized by uncontrolled discharges caused by neuronal hyperactivity in the temporal lobe and the hippocampus . Glutamate is a major excitatory neurotransmitter in mammalian brains and an agonist of the ka subtype of glutamate receptors, which is typically used to induce epilepsy in both rats and mice [57]. Ka - induced epilepsy symptoms are extremely similar to temporal lobe seizures that occur in humans [810]. Glutamate receptor - dependent overactivation or the lack of -aminobutyric acid (gaba) receptor - dependent inhibition causes central nervous system discharge, which is principally responsible for epilepsy [11, 12]. Several antiepileptic drugs, such as topiramate and gabapentin, inhibit excitatory glutamate receptors and the activation of inhibitory gaba receptors . However, more than 30% of patients who use traditional antiepileptic drugs experience uncontrolled seizures and side effects in clinics . Accordingly recently, controlling body temperature, particularly by inducing hypothermia, has been suggested to reduce seizure activity [16, 17]. Reported that cooling body temperature can effectively inhibit synaptic transmission and reduce spontaneous epileptiform discharge . Hypothermia is frequently used as a standard treatment for stroke, brain injury, and uncontrolled epilepsy . However, the specific function of cooling the brain and the underlying mechanisms remain unclear . Transient receptor potential ankyrin 1 (trpa1), which belongs to the trpa (ankyrin) family, has been reported to detect noxious thermal pain [19, 20]. Trpa1 is a calcium - permeable ion channel expressed in peripheral nociceptive neurons [22, 23] and the brain . Trpa1 activity can be regulated by protein kinase a (pka), protein kinase c (pkc), and extracellular signal - regulated kinase (erk) in inflammatory and neuropathic pain models [25, 26]. Trpv4 is associated with osmotic pressure and mechanical and thermal sensitivity that is expressed in heterologous systems [27, 28]. Mice without trpv4 receptor cannot regulate the serum osmolarity and are insensitive to noxious stimuli [29, 30]. Trpv4 was involved in several kinds of pain mediation including pain from mechanical hyperalgesia, complications of vincristine chemotherapy, diabetes, alcoholism, and acquired immune deficiency syndrome therapy [31, 32]. Recently, several pkc isoforms (pkc, pkc, pkc, pkc, pkc, and pkc) were reported to participate in epilepsy [34, 35]. Liu et al . Reported that, in a pilocarpine epilepsy model, pkc, pkc, and pkc were decreased in epileptic hippocampi . In addition, tang et al . Suggested that pkc increased in the hippocampus in pilocarpine - induced epilepsy . However, pkc and pkc were attenuated in an epileptic hippocampus . After the epileptic time was extended, pkc1, pkc, pkc, and pkc were potentiated at 7 and 31 days after pilocarpine injection . Extracellular signal - regulated kinase 1/2 (erk1/2) has been reported to be activated in neurons, particularly in people with epilepsy . Indicated that pkc is involved in microglial activation process under pathological conditions including neuroinflammation and neurodegeneration . During ka - induced epileptiform seizures, perk1/2 activation acupuncture has been used for over 3000 years and has been based on the traditional chinese medicine theory . Auricular vagus nerve stimulation is also reported to serve as an alternative therapy for drug - resistant epilepsy . Acupuncture is highly regarded as a crucial therapy in inhibiting ka - induced hippocampal cell death and inflammatory events in mice [4042]. In this study, we investigated the crucial effects and detailed cellular mechanisms of acupuncture by using an electrophysiology technique . Epileptic discharge was increased in the hippocampal ca1 region of ka - injected rats and further reduced by administering auricular and somatic acupuncture . The ppkc and perk1/2 protein levels were increased; however, ppkc proteins were reduced in the ka group . A total of 30 male sprague - dawley (sd) rats weighing 200300 g were used in this study . The institute of animal care and use committee of china medical university approved the use of these animals (permit no . In addition, the guide for the use of laboratory animals (national academy press) was followed . Four days before the electroencephalogram (eeg) and electromyogram (emg) recordings were conducted, all of the rats underwent stereotactic surgery . Stainless steel screw electrodes were implanted on the dura above the bilateral sensorimotor cortices to function as recording electrodes . Electrodes were connected to an eeg- and emg - monitoring machine (mploowsw, biopac systems, inc ., the epileptic seizures were captured using a video recording epileptic behavioral analysis system (seizurescan, clever sys ., inc ., virgina, usa) that occurred in the rats were confirmed by observing behavior (e.g., wet - dog shakes, paw tremors, and facial myoclonia under a freely moving and conscious state) and epileptiform discharges on eeg recordings . The rats were randomly divided into 5 experimental groups on which electrophysiological studies (15 rats in total and 3 rats in each group) and western blot analysis of trpa1, trpv4, pkc, pkc, and perk1/2 (15 rats in total and 3 rats per group) were performed after the rats experienced ka - induced epileptic seizures . Each experiment was divided into 5 groups: (1) a control group, in which the rats were injected with phosphate buffer solution (pbs) ip only; (2) the ka group, in which the rats were injected with 12 mg / kg ip of ka only; (3) the auricular group, in which the rats received 2 hz ea (anion placed at the ear apex and cathode placed at the ear lobe by using a clip electrode at a frequency of 2 hz; the stimulus intensity, indicated by a visual ear twitch, was maintained for 20 min and 10 min for the left and right ears, resp .) For 3 days / week for 6 weeks beginning on the day following the ka injection; (4) the st36-st37 group, in which the rats received 2 hz ea at the zusanli and shangjuxu (st36-st37) acupoints (anode placed at st36 and cathode placed at st37 by inserting 2 stainless steel acupuncture needles into the muscle layer) for 3 days / week for 6 weeks beginning on the day following the ka injection; and (5) the sham group, for which the methods used were identical to those used for the st36-st37 group; however, the needles penetrated the st36-st37 acupoints cutaneously without electric discharge for 3 days / week for 6 weeks beginning on the next day following the ka injection . All of the rats were sacrificed 6 weeks after the ka injection, and the hippocampi were removed for electrophysiological and western blot studies . Transverse hippocampal slices (450 m thick) were cut with a vibrating tissue slicer (campden instruments, loughborough, uk) and transferred to an interface - type holding chamber at room temperature . The slices were recovered for at least 120 min and then were transferred to an immersion - type recording chamber, perfused at 2 ml / min . We use the artificial csf (acsf) containing the following (mm): 119 nacl, 2.5 kcl, 26.2 nahco3, 1 nah2po4, 1.3 mgso4, 2.5 cacl2, and 11 glucose (the ph was adjusted to 7.4 by gassing with 5% co2 - 95% o2). Regarding extracellular recording, a glass pipette filled with 3 m nacl was placed in the stratum radiatum of the cal area to record field excitatory postsynaptic potentials (fepsps). Bipolar stainless steel electrodes (frederick haer company, bowdoinham, me, usa) were placed in the stratum radiatum to stimulate the schaffer collateral input . Regarding whole - cell patch recording, a pipette was filled with the following chemicals (in mm): 116 potassium gluconate, 6 kcl, 2 nacl, 30 hepes, 0.5 egta, 4 mg - atp, and 0.3 na - gtp (the ph was adjusted to 7.2 by using koh, and the osmolarity was adjusted to 300 mosm). The whole - cell patch recordings were performed on the ca1 pyramidal cells by using a visualized patch technique . An axopatch-700b amplifier (axon instruments, inc ., foster city, ca, usa) was switched to current - clamp mode once the whole - cell recordings were obtained, and the membrane potential was maintained at 65 mv . Recording was terminated and the data were discarded if the serial resistance or input resistance varied by more than 30% . All of the signals were filtered at 2 khz by using a low - pass bessel filter included in the axopatch-700b amplifier and digitized at 5 khz by using a ced micro 1401 interface that ran signal software (cambridge electronic design, cambridge, uk). Total proteins were prepared by homogenizing neurons in a lysis buffer containing 50 mm tris - hcl with ph 7.4, 250 mm nacl, 1% np-40, 5 mm edta, 50 mm naf, 1 mm na3vo4, 0.02% nan3, and 1x protease inhibitor cocktail (amresco, solon, oh, usa). The extracted proteins (30 g / sample assessed using the bca protein assay) were subjected to 8% sds - tris glycine gel electrophoresis and transferred to a pvdf membrane . The membrane was blocked using 5% nonfat milk in a tbs - t buffer (10 mm tris, ph 7.5, 100 mm nacl, and 0.1% tween 20), incubated with anti - trpa1 (1: 1000, alomone labs, jerusalem, israel), trpv4 (1: 1000, alomone labs), pkc (pser657) (1: 1000, millipore, billerica, ma, usa), pkc (1: 500, novus biologicals, littleton, co, usa), and perk1/2 (pthr202, ptyr204) (1: 500, novus biologicals, littleton, co, usa) in tbs - t containing 1% bovine serum albumin, and incubated for 1 hour at room temperature . Peroxidase - conjugated anti - rabbit and anti - mouse antibodies (1: 5000, jackson immunoresearch laboratories, west grove, pa, usa) were used as secondary antibodies . The bands were visualized using an enhanced chemiluminescencent substrate kit (thermo scientific, waltham, ma, usa) with las-3000 fujifilm (fuji photo film co. ltd ., minato, tokyo, japan). Where applicable, the image intensities of specific bands were quantified using nih imagej software (bethesda, md, usa). All of the statistical data are presented as the mean standard error . Statistical significance between each group was tested using analysis of variance, followed by a post hoc tukey's test (p <.05 was considered statistically significant). Epilepsy is a major neurological disease caused by the hyperexcitation of neuronal networks in mammalian brains . In this study, we injected rats with ka ip to induce epileptic seizures (30 sd rats; 12 mg / kg per rat). We counted wet - dog shakes, paw tremors, and facial myoclonia to ensure the creation of a successfully induced seizure model . Wet - dog shakes were indicated by intermittent polyspike - like eeg activity (figure 1(b)). Paw tremors were indicated by continual spikes in eeg activity (figure 1(d)). Based on these parameters, we used the rats that exhibited seizures with similar degrees of severity for each experiment to investigate the effect of ea on epilepsy (figure 1(e)). We subsequently verified changes in the ca1 synaptic function in the hippocampal slices from each group . We first used field recordings to recognize fepsp to measure excitatory spikes . In the hippocampal ca1 areas of the rats in the pbs group, rare spikes after fepsp occurred (0.17 0.17, n = 6 from 3 rats) (figure 2(a)). Regarding ka - treated rats, several spikes appeared, suggesting hyperexcitability in the epileptic rats (3.17 0.31, p <.05 compared with pbs group, n = 6 from 3 rats) (figure 2(a)). The hyperactivity was further attenuated by administering 2 hz auricular and st36-st37 ea (0.67 0.33 and 1.17 0.31 compared with ka group, n = 6 from 3 rats, resp . ), but not in the sham - operated group (2.67 0.33, n = 6 from 3 rats) (figure 2(a)). Similar results were obtained from the whole - cell patch recordings, as shown in figure 2(b). We then examined synaptic transmission by using input - output ratio curves to identify the relationship between fepsp response and stimulation strength . No significant differences were observed among the groups, indicating similar synaptic transmission among the rats in each group (figure 2(d)). We further tested whether epileptic discharge could alter presynaptic activity by assessing paired - pulse facilitation . The results indicated that the presynaptic activity was similar among all of the groups (figure 2(e)). The aforementioned data revealed that neuronal hyperexcitability was altered during an epileptic seizure without changing the basal transmission and presynaptic properties . According to in vitro and in vivo epilepsy models the results indicated that trpa1 protein levels increased after the ka injection, suggesting that upregulation caused by epileptogenesis occurred in the hippocampus of the rats (137.9% 8.8%, p <.05 compared with pbs group, n = 6) (figure 3(a)). The potentiated trpa1 was decreased by administering auricular ea (107.7% 7.9%, p>.05 compared with ka group, n = 3) (figure 3(a)), but not by administering ea at the st36-st37 acupoints (120.8% 14.2%, p>.05 compared with ear group, n = 6) (figure 3(a)) or performing sham operation at the st36-st37 acupoints (116.9% 9.8%, p>.05 compared with ear group, n = 6) (figure 3(a)). Therefore, we subsequently verified whether the trpv4 protein levels changed after ka injection . The data used in this study revealed that the trpv4 protein levels were unaltered after the ka injection (97.6% 3.0%, p> additionally, trpv4 protein levels were unchanged after administering auricular and somatic ea at the st36-st37 acupoints (106.4% 5.2% and 98.0% 3.3%, resp . A similar result was obtained for the sham group (99.2% 5.3%, p>.05 compared with pbs group, n = 6) (figure 3(a)). The significant change in ppkc isoforms that occurs during pilocarpine - induced epilepsy has been established [21, 22]. Therefore, we verified whether ppkc and ppkc were involved in ka - induced epileptic seizures . The results indicated that ppkc increased after ka injection (230.7% 22.9%, p <.05 compared with pbs group, n = 6) (figure 4(a)) and could be attenuated by administering auricular ea (135.7% 12.6%, p <.05 compared with ka group, n = 6) (figure 4(a)), but not by administering somatic ea (251.2% 35.1%, p>.05, n = 6) (figure 4(a)). This phenomenon was not observed in the sham group (332.1% 47.2%, p>.05, n = 6) (figure 4(a)). By contrast, ppkc decreased in the hippocampi of the ka - induced rats (77.9% 6.0%, p <.05 compared with pbs group, n = 6) (figure 4(a)). Potentiation was significantly reversed in the auricular ea group (161.6% 25.3%, p <.05 compared with ka group, n = 6), but not in the somatic or sham groups (97.0% 8.1% and 106.1% 9.7%, resp . In addition, we determined that the expression of perk1/2 was similar to that of ppkc. The perk1/2 expression was augmented in the ka group (161.7% 11.8%, p <.05 compared with pbs group, n = 6) and was decreased by administering auricular (117.5% 3.6%, p <.05 compared with ka group, n = 6) and somatic ea (103.6% 4.9%, n = 6) (figure 4(a)). This phenomenon was not observed in the sham group (135.5% 18.2%, n = 6) (figure 4(a)). We first induced an epileptic seizure animal model by using a ka ip injection to investigate the effect of 2 hz ea at the auricular and at st36-st37 acupoints . The results indicated that administering 2 hz ea at the auricular and at st36-st37 can effectively reduce electrical discharge in the hippocampal ca1 areas of ka - treated rats . We also observed that the trpa1, but not trpv4, protein levels increased in the ka - treated rats . Furthermore, ppkc and perk1/2 increased . Ea administered at the auricular, but not at the st36-st37 acupoints, reversed the protein change in the hippocampus . Thus, we obtained novel evidence suggesting that ea administered at the ear can reduce epileptiform discharge accompanied by changes in the trpa1, ppkc, ppkc, and perk1/2 signaling pathways . A cooling temperature of less than 24c is required to decrease neuronal function . In addition, inhibiting experimental seizures requires a temperature of less than 24c because complete cessation occurs at a temperature from <20c to 22c . Under this condition, the results indicated that trpa1, but not trpv4, protein levels increased in the ka - induced epilepsy model . This phenomenon could be reversed in the ea at auricular group but not the st36-st37 or sham groups . During a seizure, a high temperature has frequently been reported to trigger seizures . Because gabaa inhibitory synaptic transmission is highly sensitive to temperature, decreasing the temperature serves as a convulsant drug . Several pkc isoforms have been suggested to be involved in epileptogenesis based on a seizure model . Tang et al . Reported that pkc decreased in the stratum granulosum during pilocarpine - induced epilepsy . After epileptic time was extended to 31 days, pkc expression disappeared in the inner molecular layer of the hippocampus during a seizure . The result of this study indicated that pkc protein levels were attenuated in ka - induced seizure rats and could be potentiated in the auricular group but not the st36-st37 or sham groups . Furthermore, pkc demonstrated a dramatically different pattern in which pkc increased in the ka - treated rats and could be reversed by administering auricular ea but not by st36-st37 ea or sham control . These results are similar to those described in previous publications, which have reported that pkc increased in the stratum pyramidale of the ca3 area during pilocarpine - induced epilepsy . In addition, we observed that perk1/2 expression was augmented in the ka - induced seizure rats and reduced by administering auricular and somatic ea . These phenomena indicated the possible mechanisms that may be involved in ea - mediated antiepileptic curative effects . In summary, we determined the effect of auricular and somatic ea on ka - induced seizure rats . The results suggest that the therapeutic effect was due to reduced hippocampal hyperactivity accompanied by the trpa1, pkc, pkc, and perk1/2 signaling pathways . The related mechanisms could be reversed by administering auricular ea and could be partially reversed by st36-st37 ea . These novel findings are remarkable and compelling and reveal the crucial curative effects of auricular ea.
Fabry disease (fd) is an x - linked recessive lysosomal storage disorder caused by a defect in the gene that encodes lysosomal -galactosidase a and is known to affect various tissues including the kidney and heart . Patients with fd manifest a variety of clinical symptoms, such as acroparesthesias, angiokeratoma, hypohidrosis, corneal opacities, stroke, renal disorder and cardiac abnormalities . The manifestations are thought to be due to progressive accumulation of globotriaosylceramide (gl-3) and other glycosphingolipids within various cells such as vascular endothelium, cardiomyocytes, glomerular cells and renal tubular cells . The precise pathophysiological mechanisms of fd remain unclear, and coronary microvascular dysfunction appears to be one of the causes of cardiac complications in fd [14]. In several studies, gl-3 accumulation within vascular endothelial cells in the kidney and heart many studies have demonstrated that nitric oxide (no) plays an important role in the progression of endothelial dysfunction, atherosclerosis and chronic kidney disease (ckd) [811]. No is synthesized by endothelial, neuronal and macrophage isoforms of the enzyme no synthase (nos). Asymmetric dimethylarginine (adma) is an endogenous competitive inhibitor of nos, and serum adma levels have been suggested to be a surrogate marker of endothelial dysfunction and/or angiosclerosis . Recently, we revealed a significant relationship between adma, glomerular filtration rate and endothelial dysfunction of the coronary and peripheral arteries [12, 13]. From the results of not only our studies but also various earlier studies, we thought that adma may be involved in the underlying mechanism connecting the two pathological conditions between the kidney and the heart and may be a useful biochemical marker even in fd . The aim of our study was to examine the effect of ert on cardiac abnormalities and to determine the relationship between adma, coronary microvascular function and left ventricular hypertrophy (lvh) in fd patients . For these patients, -galactosidase a (agalsidase, fabrazyme; genzyme corp ., cambridge, ma, or agalsidase, replagal; shire plc, dublin, ireland) was administered intravenously every 2 weeks for a period of 12 months (at a dosage of 1 or 0.2 mg / kg body weight, respectively). Ten hypertensive patients without diabetes mellitus, inflammatory disease, infection, ckd or any cardiovascular complications were also evaluated as controls . In addition, 20 healthy volunteers (mean age: 29 4 years) were also evaluated to determine serum adma levels . Patients were excluded only if they did not agree to participate in the study . For the fd patients, we prospectively performed blood examination, echocardiography, exercise stress myocardial scintigraphy and coronary flow reserve (cfr) measurements before starting ert and at 3, 6 and 12 months . The experimental protocols were approved by the appropriate institutional review committee and performed in accordance with the helsinki declaration of 1975, as revised in 2008 . Transthoracic doppler echocardiography, which has been described previously, was used to measure cfr . The ultrasound beam was transmitted toward the heart to visualize coronary blood flow in the distal portion of the left anterior descending (lad) coronary artery by color doppler flow mapping . First, the left ventricle was imaged in cross - section along the longitudinal axis, and then, the ultrasound beam was inclined laterally . Next, coronary blood flow in the distal lad was examined under the guidance of color doppler flow mapping . After positioning a sample volume on the color signal in the distal lad, doppler spectral tracings of flow velocity were recorded by fast fourier transformation analysis . Adenosine-5-triphosphate was administered (140 g / kg / min, i.v .) For 3 min to record spectral doppler signals during hyperemic conditions . All of the patients underwent continuous heart rate and blood pressure monitoring throughout the study period . An ultrasound system computer was used to trace the contour of the spectral doppler signal for off - line analysis of coronary flow velocity . Peak diastolic velocity (pdv) was measured at the baseline and peak hyperemic conditions . We adopted a cfr of <2.0 as the cut - off value for the presence of significant coronary microvascular disease, as used previously . Two - dimensional guided m - mode echocardiography was performed to measure left ventricular wall mass . Left ventricular diastolic diameter (lvdd), left ventricular systolic diameter, diastolic thickness of the left ventricular posterior wall (lvpwt) and interventricular septum (ivst) were assessed in m - mode images in the parasternal longitudinal axis view . The m - mode analysis was performed according to the guidelines of the american society of echocardiography . The following formula was used to calculate the left ventricular mass index (lvmi) (g / m): on the same day as the cfr measurement, venous blood was collected from the patients after a 20-min period of supine rest in the morning following overnight fasting . In two dialysis patients, according to japanese society of dialysis therapy (jsdt) guidelines, blood samples were collected before each dialysis session at 2-day intervals . Serum was separated by centrifugation at 2500 g for 10 min at 4c and stored at 20c until analysis . The ckd epidemiology collaboration (ckd - epi) equation was used to estimate the glomerular filtration rate for evaluation of renal function . We used the computer software application statview 5.0 (sas institute, cary, nc) for all statistical analyses . Whitney u - test was used to analyze the significance of differences between two groups . Pearson's correlation coefficient was used to analyze relationships between variables . Results with p - values for these patients, -galactosidase a (agalsidase, fabrazyme; genzyme corp ., cambridge, ma, or agalsidase, replagal; shire plc, dublin, ireland) was administered intravenously every 2 weeks for a period of 12 months (at a dosage of 1 or 0.2 mg / kg body weight, respectively). Ten hypertensive patients without diabetes mellitus, inflammatory disease, infection, ckd or any cardiovascular complications were also evaluated as controls . In addition, 20 healthy volunteers (mean age: 29 4 years) were also evaluated to determine serum adma levels . Patients were excluded only if they did not agree to participate in the study . For the fd patients, we prospectively performed blood examination, echocardiography, exercise stress myocardial scintigraphy and coronary flow reserve (cfr) measurements before starting ert and at 3, 6 and 12 months . The experimental protocols were approved by the appropriate institutional review committee and performed in accordance with the helsinki declaration of 1975, as revised in 2008 . Transthoracic doppler echocardiography, which has been described previously, was used to measure cfr . The ultrasound beam was transmitted toward the heart to visualize coronary blood flow in the distal portion of the left anterior descending (lad) coronary artery by color doppler flow mapping . First, the left ventricle was imaged in cross - section along the longitudinal axis, and then, the ultrasound beam was inclined laterally . Next, coronary blood flow in the distal lad was examined under the guidance of color doppler flow mapping . After positioning a sample volume on the color signal in the distal lad, adenosine-5-triphosphate was administered (140 g / kg / min, i.v .) For 3 min to record spectral doppler signals during hyperemic conditions . All of the patients underwent continuous heart rate and blood pressure monitoring throughout the study period . An ultrasound system computer was used to trace the contour of the spectral doppler signal for off - line analysis of coronary flow velocity . Peak diastolic velocity (pdv) was measured at the baseline and peak hyperemic conditions . We adopted a cfr of <2.0 as the cut - off value for the presence of significant coronary microvascular disease, as used previously . Two - dimensional guided m - mode echocardiography was performed to measure left ventricular wall mass . Left ventricular diastolic diameter (lvdd), left ventricular systolic diameter, diastolic thickness of the left ventricular posterior wall (lvpwt) and interventricular septum (ivst) were assessed in m - mode images in the parasternal longitudinal axis view . The m - mode analysis was performed according to the guidelines of the american society of echocardiography . The following formula was used to calculate the left ventricular mass index (lvmi) (g / m): on the same day as the cfr measurement, venous blood was collected from the patients after a 20-min period of supine rest in the morning following overnight fasting . In two dialysis patients, according to japanese society of dialysis therapy (jsdt) guidelines, blood samples were collected before each dialysis session at 2-day intervals . Serum was separated by centrifugation at 2500 g for 10 min at 4c and stored at 20c until analysis . The ckd epidemiology collaboration (ckd - epi) equation was used to estimate the glomerular filtration rate for evaluation of renal function . We used the computer software application statview 5.0 (sas institute, cary, nc) for all statistical analyses . Whitney u - test was used to analyze the significance of differences between two groups . The main characteristics of the subjects are shown in table 1 . Among the study patients, three had a history of stroke (cases 1, 3 and 4) and two had received hemodialysis therapy (cases 1 and 4). At the baseline, -galactosidase activity was low in all patients, as determined by the measurement of the dried blood spots in a fluorescence assay using 4-methylumbelliferyl . In contrast, -galactosidase activity measured in leukocytes was low in only one patient (case 4). Serum adma levels were significantly higher in the fd group than in the hypertensive control and healthy control groups (figure 1a). As shown in table 2, the lvmi increased in three patients (cases 1, 3 and 4), and systolic function decreased in two (cases 1 and 4). Although findings of ischemia were not observed using exercise stress myocardial scintigraphy in all the study patients, cfr decreased in three patients (cases 1, 3 and 4) and two had angina - like chest pain (cases 1 and 3). Needless to say, cfr was significantly lower in the fd group than in the hypertensive control group (figure 1b). Table 1.patient characteristicscase 1case 2case 3case 4normal valuesage (years old)45197151sexfmfmhd duration (years)1223hypertension(+)()(+)()diabetes mellitus(+)(+)()()hyperlipidemia()()()()stroke(+)()(+)(+)acroparesthesias()()(+)()hypohidrosis()()()()angiokeratoma()()()()corneal opacities(+)()()()chest pain(+)()(+)()cr (mol / l)872.451.165.6976.1egfr (ml / min/1.73 m)13968rbc (10/l)4.105.533.054.20hemoglobin (g / l)114161103121tp (g / l)686968796482alb (g / l)394539363552t - chol (mmol / l)3.236.214.864.093.366.21tg (mmol / l)2.019.043.921.260.342.03hdl (mmol / l)0.980.961.140.961.03hba1c (%) 6.112.65.04.64.35.8ca (mmol / l)2.222.422.222.592.122.55p (mmol / l)1.681.190.872.160.771.39bnp (ng / l)321.52.459.6272.218.4hscrp (nmol / l)28.645.712.421.9<28.6adma (ng / l)0.850.480.550.70-galactosidase activitydried blood spot (agal u)16.911.215.115.220leukocyte (nmol / mg protein / h)61.252.375.532.149.8116.4hd, hemodialysis; cr, creatinine; egfr, estimated glomerular filtration rate; rbc, red blood cell count; tp, total protein; alb, albumin; t - chol, total cholesterol; tg, triglyceride; hdl, high - density lipoprotein cholesterol; hba1c, hemoglobin a1c; bnp, brain natriuretic peptide; hscrp, high - sensitivity c - reactive protein; adma, asymmetric dimethylarginine . Table 2.echocardiographic parameters at baselinecase 1case 2case 3case 4lad (mm)41394144lvdd (mm)49454847lvds (mm)37313333ivst (mm)16101212pwt (mm)13101112fs (%) 24303129ef (%) 58606657lvmi (g / m)203.293.8158.2151.9e / a ratio0.772.260.620.65dct (ms)346146200212ivcd (mm)168.95.713cfr1.773.412.311.45lad, left atrial dimension; lvdd, left ventricular end - diastolic dimension; lvds, left ventricular end - systolic dimension; ivst, interventricular septum thickness; pwt, posterior wall thickness; fs, fractional shortening; ef, ejection fraction; lvmi, left ventricular mass index; dct, deceleration time; ivcd, inferior vena cava diameter; cfr, coronary flow reserve . Fig . (a) serum adma levels in the healthy subjects, hypertensive patients and fd patients . (b) cfr in the hypertensive patients and fd patients . * p <0.05 patient characteristics hd, hemodialysis; cr, creatinine; egfr, estimated glomerular filtration rate; rbc, red blood cell count; tp, total protein; alb, albumin; t - chol, total cholesterol; tg, triglyceride; hdl, high - density lipoprotein cholesterol; hba1c, hemoglobin a1c; bnp, brain natriuretic peptide; hscrp, high - sensitivity c - reactive protein; adma, asymmetric dimethylarginine . Echocardiographic parameters at baseline lad, left atrial dimension; lvdd, left ventricular end - diastolic dimension; lvds, left ventricular end - systolic dimension; ivst, interventricular septum thickness; pwt, posterior wall thickness; fs, fractional shortening; ef, ejection fraction; lvmi, left ventricular mass index; dct, deceleration time; ivcd, inferior vena cava diameter; cfr, coronary flow reserve . Serum adma levels and cfr in the control patients and fd patients . (a) serum adma levels in the healthy subjects, hypertensive patients and fd patients . P <0.05 we evaluated the changes in serum adma levels, cfr and lvmi after starting ert, and these parameters mostly improved 12 months after starting ert compared with the values at baseline (figure 2a c). Two study patients had angina - like chest pain before the treatment; their symptoms disappeared following ert . Unfortunately, because of a worldwide shortage of available recombinant -galactosidase, cases 2 and 4 did not receive sufficient ert for the last 2 months of treatment . Table 3.laboratory data during ertcase 1case 2case 3case 4months03612036120361203612cr (mol / l)872.4879.3854.1903.740.438.141.243.565.674.773.254.9976.1995.91060.0986.0egfr (ml / min/1.73 m)15315715214968586085rbc (10/l)4.103.393.614.055.535.675.715.643.053.143.323.404.203.803.783.75hemoglobin (g / l)11492103116161163167167103102105110121108114114tp (g / l)68697071696968706863646979727373alb (g / l)39414242454545483934363736333232t - chol (mmol / l)3.233.473.133.706.214.975.904.424.864.504.035.174.092.772.842.64tg (mmol / l)2.011.680.821.559.403.425.013.133.921.102.673.411.261.461.240.69hdl (mmol / l)0.981.241.321.320.960.960.960.911.141.531.241.810.960.800.720.88hba1c (%) 6.15.95.16.312.610.611.811.25.04.6ca (mmol / l)2.222.252.102.302.422.322.322.472.222.322.402.252.592.152.252.17p (mmol / l)1.681.941.522.231.191.191.231.130.871.001.190.902.161.781.841.84bnp (ng / l)321.5169.268.353.32.43.12.26.459.669.127.677.7272.2173.2183.1144.4cr, creatinine; egfr, estimated glomerular filtration rate; rbc, red blood cell count; tp, total protein; alb, albumin; t - chol, total cholesterol; tg, triglyceride; hdl, high - density lipoprotein cholesterol; hba1c, hemoglobin a1c; bnp, brain natriuretic peptide . Fig . Laboratory data during ert cr, creatinine; egfr, estimated glomerular filtration rate; rbc, red blood cell count; tp, total protein; alb, albumin; t - chol, total cholesterol; tg, triglyceride; hdl, high - density lipoprotein cholesterol; hba1c, hemoglobin a1c; bnp, brain natriuretic peptide . Change of cfr, lvmi and adma during ert . (a) serum adma levels in individual patients . We examined the correlation of serum adma levels with cfr and lvmi and found that serum adma levels were significantly correlated with both cfr (r = 0.576, p <0.05) and lvmi (r = 0.874, p <0.0001) (figure 3a and b). There was also a significant relationship between cfr and lvmi (r = 0.545, p <0.05) (figure 3c). In addition, serum adma levels were significantly correlated with plasma brain natriuretic peptide (bnp) levels (r = 0.770, p = 0.0005). The main characteristics of the subjects are shown in table 1 . Among the study patients, three had a history of stroke (cases 1, 3 and 4) and two had received hemodialysis therapy (cases 1 and 4). At the baseline, -galactosidase activity was low in all patients, as determined by the measurement of the dried blood spots in a fluorescence assay using 4-methylumbelliferyl . In contrast, -galactosidase activity measured in leukocytes was low in only one patient (case 4). Serum adma levels were significantly higher in the fd group than in the hypertensive control and healthy control groups (figure 1a). As shown in table 2, the lvmi increased in three patients (cases 1, 3 and 4), and systolic function decreased in two (cases 1 and 4). Although findings of ischemia were not observed using exercise stress myocardial scintigraphy in all the study patients, cfr decreased in three patients (cases 1, 3 and 4) and two had angina - like chest pain (cases 1 and 3). Needless to say, cfr was significantly lower in the fd group than in the hypertensive control group (figure 1b). Table 1.patient characteristicscase 1case 2case 3case 4normal valuesage (years old)45197151sexfmfmhd duration (years)1223hypertension(+)()(+)()diabetes mellitus(+)(+)()()hyperlipidemia()()()()stroke(+)()(+)(+)acroparesthesias()()(+)()hypohidrosis()()()()angiokeratoma()()()()corneal opacities(+)()()()chest pain(+)()(+)()cr (mol / l)872.451.165.6976.1egfr (ml / min/1.73 m)13968rbc (10/l)4.105.533.054.20hemoglobin (g / l)114161103121tp (g / l)686968796482alb (g / l)394539363552t - chol (mmol / l)3.236.214.864.093.366.21tg (mmol / l)2.019.043.921.260.342.03hdl (mmol / l)0.980.961.140.961.03hba1c (%) 6.112.65.04.64.35.8ca (mmol / l)2.222.422.222.592.122.55p (mmol / l)1.681.190.872.160.771.39bnp (ng / l)321.52.459.6272.218.4hscrp (nmol / l)28.645.712.421.9<28.6adma (ng / l)0.850.480.550.70-galactosidase activitydried blood spot (agal u)16.911.215.115.220leukocyte (nmol / mg protein / h)61.252.375.532.149.8116.4hd, hemodialysis; cr, creatinine; egfr, estimated glomerular filtration rate; rbc, red blood cell count; tp, total protein; alb, albumin; t - chol, total cholesterol; tg, triglyceride; hdl, high - density lipoprotein cholesterol; hba1c, hemoglobin a1c; bnp, brain natriuretic peptide; hscrp, high - sensitivity c - reactive protein; adma, asymmetric dimethylarginine . Table 2.echocardiographic parameters at baselinecase 1case 2case 3case 4lad (mm)41394144lvdd (mm)49454847lvds (mm)37313333ivst (mm)16101212pwt (mm)13101112fs (%) 24303129ef (%) 58606657lvmi (g / m)203.293.8158.2151.9e / a ratio0.772.260.620.65dct (ms)346146200212ivcd (mm)168.95.713cfr1.773.412.311.45lad, left atrial dimension; lvdd, left ventricular end - diastolic dimension; lvds, left ventricular end - systolic dimension; ivst, interventricular septum thickness; pwt, posterior wall thickness; fs, fractional shortening; ef, ejection fraction; lvmi, left ventricular mass index; dct, deceleration time; ivcd, inferior vena cava diameter; cfr, coronary flow reserve . Fig . (a) serum adma levels in the healthy subjects, hypertensive patients and fd patients . (b) cfr in the hypertensive patients and fd patients . * p <0.05 patient characteristics hd, hemodialysis; cr, creatinine; egfr, estimated glomerular filtration rate; rbc, red blood cell count; tp, total protein; alb, albumin; t - chol, total cholesterol; tg, triglyceride; hdl, high - density lipoprotein cholesterol; hba1c, hemoglobin a1c; bnp, brain natriuretic peptide; hscrp, high - sensitivity c - reactive protein; adma, asymmetric dimethylarginine . Echocardiographic parameters at baseline lad, left atrial dimension; lvdd, left ventricular end - diastolic dimension; lvds, left ventricular end - systolic dimension; ivst, interventricular septum thickness; pwt, posterior wall thickness; fs, fractional shortening; ef, ejection fraction; lvmi, left ventricular mass index; dct, deceleration time; ivcd, inferior vena cava diameter; cfr, coronary flow reserve . Serum adma levels and cfr in the control patients and fd patients . (a) serum adma levels in the healthy subjects, hypertensive patients and fd patients . We evaluated the changes in serum adma levels, cfr and lvmi after starting ert, and these parameters mostly improved 12 months after starting ert compared with the values at baseline (figure 2a c). Two study patients had angina - like chest pain before the treatment; their symptoms disappeared following ert . Unfortunately, because of a worldwide shortage of available recombinant -galactosidase, cases 2 and 4 did not receive sufficient ert for the last 2 months of treatment . Table 3.laboratory data during ertcase 1case 2case 3case 4months03612036120361203612cr (mol / l)872.4879.3854.1903.740.438.141.243.565.674.773.254.9976.1995.91060.0986.0egfr (ml / min/1.73 m)15315715214968586085rbc (10/l)4.103.393.614.055.535.675.715.643.053.143.323.404.203.803.783.75hemoglobin (g / l)11492103116161163167167103102105110121108114114tp (g / l)68697071696968706863646979727373alb (g / l)39414242454545483934363736333232t - chol (mmol / l)3.233.473.133.706.214.975.904.424.864.504.035.174.092.772.842.64tg (mmol / l)2.011.680.821.559.403.425.013.133.921.102.673.411.261.461.240.69hdl (mmol / l)0.981.241.321.320.960.960.960.911.141.531.241.810.960.800.720.88hba1c (%) 6.15.95.16.312.610.611.811.25.04.6ca (mmol / l)2.222.252.102.302.422.322.322.472.222.322.402.252.592.152.252.17p (mmol / l)1.681.941.522.231.191.191.231.130.871.001.190.902.161.781.841.84bnp (ng / l)321.5169.268.353.32.43.12.26.459.669.127.677.7272.2173.2183.1144.4cr, creatinine; egfr, estimated glomerular filtration rate; rbc, red blood cell count; tp, total protein; alb, albumin; t - chol, total cholesterol; tg, triglyceride; hdl, high - density lipoprotein cholesterol; hba1c, hemoglobin a1c; bnp, brain natriuretic peptide . Fig . Laboratory data during ert cr, creatinine; egfr, estimated glomerular filtration rate; rbc, red blood cell count; tp, total protein; alb, albumin; t - chol, total cholesterol; tg, triglyceride; hdl, high - density lipoprotein cholesterol; hba1c, hemoglobin a1c; bnp, brain natriuretic peptide . Change of cfr, lvmi and adma during ert . We examined the correlation of serum adma levels with cfr and lvmi and found that serum adma levels were significantly correlated with both cfr (r = 0.576, p <0.05) and lvmi (r = 0.874, p <0.0001) (figure 3a and b). There was also a significant relationship between cfr and lvmi (r = 0.545, p <0.05) (figure 3c). In addition, serum adma levels were significantly correlated with plasma brain natriuretic peptide (bnp) levels (r = 0.770, p = 0.0005). Our study demonstrated that (i) cfr was significantly lower and serum adma levels and lvmi were significantly higher in the fd group than in the control group; (ii) ert increased the cfr, decreased the elevated serum adma levels and increased lvmi; and (iii) serum adma levels showed a significant relationship with cfr and lvmi in the fd patients . It has been thought that fd patients show changes not only in the myocardium but also in coronary vascular function . In fact, some studies have demonstrated that cfr is markedly decreased in fd [4, 1618]. Previous studies have also reported that 50% of fd patients complain of angina - like chest pain [19, 20]. In our study, two patients had angina - like chest pain and three had markedly impaired cfr, despite having no manifest coronary artery disease . Thus, coronary microvascular dysfunction may play a key role in the development of these symptoms . In many cases with cardiac symptoms, gl-3 accumulates in the endothelial, myocardial and smooth muscle cells, which causes microvascular dysfunction in coronary arteries and lvh . It has been shown that ert reverses the accumulation of microvascular endothelial deposits of gl-3 in several organs, including the heart [13, 7]. Furthermore, it is thought that the increased oxygen demand of the hypertrophied myocardium is related to myocardial ischemia in fd . There are also some studies that have reported that ert was effective in decreasing lvh and improving regional myocardial function [5, 21, 22]. Therefore, we speculated that ert could ameliorate coronary microvascular dysfunction in fd through these two mechanisms . On the other hand, two previous studies reported that no improvements in hyperemic myocardial blood flow, cfr and lvmi were observed by ert [23, 24]. However, the result of one study showed that the study patients felt subjectively better and suffered less pain after starting ert . In addition, plasma gl-3 levels decreased significantly after ert in both studies . From the results of these studies, ert could prevent the progression of impaired cfr and lvh but could not improve cfr and lvh in their study patients . Therefore, it also has been speculated that ert is less effective in patients with severe lvh . In the present study, ert improved not only cfr but also lvmi in most patients . We believe that the reason is that our study patients did not have such severe lvh . Several studies have demonstrated that serum adma levels increased in ckd patients and correlated with the severity of atherosclerotic lesions [911]. Thus, adma is thought to be an available biochemical marker of endothelial dysfunction and/or vascular lesions in patients with ckd . Our previous studies demonstrated that adma was significantly associated with cfr, endothelium - mediated vasodilation of the brachial artery and renal function [12, 13]. Adma is also known to be related to lvh in patients with ckd [26, 27]. In the present study, to ascertain a possible role of adma in fd and determine whether adma is a useful marker of cardiac manifestation in fd, we assessed the relationship between serum adma levels, cfr and lvmi . We found that serum adma levels were significantly correlated with cfr and lvmi in patients with fd as well as in non - fd patients . Furthermore, the levels tended to decrease with improving cfr and lvmi after starting ert . In the present study, serum bnp levels were also significantly associated with cfr and lvmi . However, because many papers demonstrated that adma induces cardiovascular dysfunction, it is reasonable to suppose that adma plays a key role in kidney and heart disease . Moreover, it has been reported that adma is more sensitive than n - terminal pro - bnp to diagnose heart failure in patients with congenital heart disease . Considering these findings, there is a possibility that serum adma levels may reflect the degree of cardiac damage even in fd . Acroparesthesia is one of the major symptoms in fd and it is known that ert could reduce this symptom . As mentioned above, our results show that serum adma levels increased in fd . Taken together, we speculated that adma might be involved in the acroparesthesia of fd though we do not have objective data on polyneuropathy . The main limitation of our study was the small number of study patients and heterogeneous patient group . We believe this is important because there are a few prospective and longitudinal studies that evaluated cardiac parameters on this topic . Therefore, we consider the results of the present study to be valuable for the management of patients with fd . Another limitation of the present study was that two study patients did not get adequate therapy for the last 2 months of the study period because of a worldwide shortage of recombinant -galactosidase . There is a possibility that the insufficient therapy might have affected these patients' conditions and clinical data . Clearly, if we were to evaluate a larger number of appropriately treated patients, the efficacy of ert and the usefulness of serum adma measurement would be more evident . Our data suggest that ert prevents progression of cardiac abnormalities, possibly by improving coronary microvascular dysfunction . In addition, we believe that adma may be a useful surrogate marker for cardiac lesions in fd . Further study is required to elucidate the precise mechanisms underlying ert and the change in adma among patients with fd and to establish a useful strategy for the prevention of organ damage . This study was presented in part at the annual meeting of the american society of nephrology, 2011.
Intrauterine contraceptive devices (iucds) are one of the most widely chosen contraceptive options with over 99% effectiveness in preventing pregnancy, a low side effect profile, and low cost.1) however, these devices are associated with a multitude of complications including the serious risk of potential device migration into the intraperitoneal cavity and surrounding viscera.1) the rate of transuterine perforation and migration of iucds into the abdominal cavity has been estimated at less than 0.1%.2) we report a 30-year - old female who presented with iucd migration into the urinary bladder with secondary calculus formation . A 30-year - old female was admitted who had been experiencing suprapubic pain and dysuria since previous 23 months . There was no history of hematuria, pyuria, lithuria, burning micturition, or fever with chills . Her abdomen was soft and non - tender, and the urinary bladder was not palpable . Urine analysis and microscopy showed 68 pus cells / high power field, and there were no red blood cells . Kidney, ureter, and bladder radiography showed a thin, elongated, t - shaped, calculus - like foreign body (figure 1). There was a small calculus of 3 mm3 mm size in the mid - pole of the left kidney . The urinary bladder was well distended and thin - walled, and a t - shaped echogenic focus with a curvilinear tail was observed within the bladder, suggestive of an iucd - copper t device (figure 2). Computed tomography (ct) showed a distended urinary bladder with evidence of a copper t noted in the urinary bladder with its thread encrusted (figure 3). The long arm of the copper t appeared to be impacted in the right lateral wall of the bladder and was projecting slightly beyond the surface of the urinary bladder . Cystoscopy showed an encrusted copper t in the bladder with an impacted tread in the right lateral wall of the bladder . The copper t was removed with the help of double j stent removal forceps after incising the thread on the lateral wall with a collins knife . Iucds are the most popular method of reversible contraception in india because of their high efficacy for fertility regulation, low - risk, low - cost, and lack of required maintenance.34) they are ideal for lactating women because they do not affect the quality and composition of breast milk.4) the possible complications associated with iucd use are device failure to prevent contraception, painful abdominal cramps, expulsion, complete or partial uterine perforation, device migration, menstrual disturbances, increased risk of ectopic pregnancy, septic and spontaneous abortion in case of pregnancy with an iucd, and pelvic inflammatory disease.4) migration of iucds to the rectum, sigmoid, small bowel, and peritoneum has been reported in literature.3) the majority of the migrated and misplaced iucds perforate the uterus completely and remain in the pelvic cavity while some become embedded in the omentum.2) early closed loop devices like the birnberg bow were associated with small bowel strangulation and were, hence, discontinued.2) copper coils induce marked peritoneal reactions, causing adhesion formation and bowel injury.2) lippes loops and saf - t - coils are inert and non - medicated, hence, they cause little biological reaction and can be quiescent for long periods.2) modern intraperitoneal iucds have low morbidity and can be left in situ.2) there are many reports in literature that describe migration of an iucd into the urinary bladder.5) normally, the uterus is in an anteverted and anteflexed position and lies in close proximity to the urinary bladder; thus, there is a high propensity of migration of the iucd into the bladder.5) the process of transmigration of foreign bodies across organs is quite complex and difficult to understand.5) once the iucd migrates to the urinary bladder, the patient will experience urinary symptoms like frequency, dysuria, hematuria, retention of urine, and fever due to infection.5) calculus formation occurs in long - standing cases.5) dietrich reported eight cases of intravesical iucd migration in 1992 with development of urinary symptoms as early as 3 months to as late as 5 years after insertion.36) the index patient was also having similar urinary complaints for three months prior to her admittance . Patients are usually treated for urinary tract infections as the symptoms are non - specific and the diagnosis is usually delayed.5) hence, the importance of a good and proper history cannot be overemphasized . Simple investigations like plain radiography and ultrasound of the pelvis are enough to arrive at a diagnosis.5) a ct scan can precisely locate the migrating iucd in the pelvic / abdominal cavity and help in planning the appropriate management.4) laboratory investigations usually reveal leukocytosis in infected cases, and the urine examination will reveal either pus cells or microscopic or gross hematuria.5) surgery is the mainstay of treatment for migrated iucds with calculus formation.5) it has been suggested that misplaced and migrated iucds should be removed even in asymptomatic patients because of the risk of adhesion formation and damage to the surrounding structures.4) a preliminary cystoscopic examination is mandatory to chart the course of further intervention.57) for mobile stones, the endoscopic methods will suffice.5) however, if the stone is large and fixed, then open suprapubic cystolithotomy is indicated for safe and complete removal.5) perforation and migration into adjacent structures are known complications of iucds and should be kept in mind when a female with a history of iucd insertion presents with urinary symptoms . The importance of a good history, appropriate post - insertion follow - up, and counseling of the patient cannot be overemphasized.
Skeletal muscle exhibits a highly adaptable protein organization whose properties can be easily changed by aging, temperature, exercise, nutrition, and incretion, to name a few . Skeletal muscle adaptability results in a hypertrophied or atrophied state; these states are primarily associated with slow or fast fibers . Slow or fast fiber characteristics are attributed to changes in the population of intracellular proteins of the muscle cells . One study concerning the content of b - crystallin and tubulin in 10 days of atrophied soleus muscle was previously reported . B - crystallin and tubulin expression is dependent on muscle fiber type; however, no study has investigated the response of b - crystallin and -tubulin with transition of myosin heavy chain (mhc) isoforms in plantar flexor muscles followed by hindlimb unloading . In this study, we focused on b - crystallin and tubulin because these proteins exhibit functional properties associated with the adaptability of skeletal muscles . B - crystallin is localized in the z - line of the sarcomere in cardiac and slow - twitch muscle . It acts as a molecular chaperone during the folding process of other specific proteins to prevent protein degradation . B - crystallin is activated in a phosphorylation - dependent manner and also acts to stabilize intermediate filament and cytoskeletal proteins in vitro . Another study showed that disorganized cytoskeletal components trigger the phosphorylation of b - crystallin through activating the p38 mitogen - activated protein kinase (mapk) pathway . B - crystallin is also known to be upregulated in slow twitch muscles, which have high oxidative capability and higher protein turnover rates compared to fast twitch muscles, which are characterized by their high glycolytic capability . Microtubules function as an intracellular frame to shape the cell, maintain cellular morphology, and transport proteins throughout the cell [11 - 13]. Microtubules exhibit a morphological, structural, and dynamic property called dynamic instability, which is regulated by the concentration of free- tubulin dimers . Microtubules are directly associated with myosin, and the localization of myosin filaments during sarcomere formation and myogenic cell morphology is partly dependent on the microtubule networking organization . It can be inferred that different muscle types are due to the different myogenic cell morphologies and their localization is related to differences in the formation and content of microtubules . The mhc is a major component of muscle and can be transformed into various isoforms in muscle cells in response to various stimuli . The increase in directional shifts from the mhc i to the mhc iia, mhc iid / x, and mhc iib isoforms, augments the velocity of the shortening and power generation capacity of the mhc, but decreases its atpase activity . Reported that an atrophy response due to unloading induces the shift of mhc isoforms from slow - twitch to fast - twitch muscle types . It is thought that b - crystallin, tubulin, and myosin work closely with one another during the processes of myocyte formation . B - crystallin plays a critical role in chaeperoning for tubulin, which functions to maintain muscle cell morphology and transport organelles in the cell . B - crystallin also indirectly supports power transmission since it acts as a chaperone protein for myosin, one of the main components of muscle . Microtubules, which are composed of tubulin, also directly associate with myosin to ensure its proper localization during sarcomere formation . These data suggest that the functional relationship between b - crystallin, tubulin, and myosin are important for muscular function and adaptability . We hypothesized that hindlimb unloading induces muscular atrophy with mhc isoform transformation toward the mhc imhc furthermore, we evaluated whether b - crystallin and -tubulin would show different expression patterns in different types of atrophied muscles as atrophied slow muscle shows a more sensitive response to both proteins compared to atrophied fast muscle . We examined the triceps surae in the hindlimbs (soleus, plantaris, and gastrocnemius muscles) because these muscles might exhibit similar functions and endure similar mechanical loads compared to other muscles in the hindlimbs, such as the tibialis anterior . We employed the unloading model to induce muscle atrophy and to quantitatively determine the relationship between the proteins (b - crystallin and tubulin) with the transition of mhc isoforms, and its significance to the adaptability of skeletal muscles . In this study, we utilized 12 seven - week - old adult male wistar rats that weighed 238.2 9.1 g. after 2 - 3 days of adaptation, the rats were randomly divided, with half placed in a control group and the half placed in an unloading group . The unloading group was tested using the hindlimb unloading model based on modified methods described by morey and atomi . All animals were provided standard rat chow and water ad libitum, and were housed at 22 - 24 c with a 12:12 h light - dark cycle . After 15 days, all rats were euthanized by cervical dislocation under anesthesia induced by diethyl ether for relieving pain (50 ml / kg body mass). The right sides of the whole soleus muscle, plantaris muscle, and gastrocnemius muscle were isolated, and the muscles were immediately frozen in liquid nitrogen and stored at -80 c until use . Animal use and maintenance was approved by the university of tokyo animal care and use committee . The three isolated whole muscles used in this study were crushed in liquid nitrogen and solubilized in a low - salt buffer containing 20 mm potassium chloride (kcl), 2 mm sodium phosphate (ph 6.8), 2 mm ethylene glycol tetraacetic acid (egta), 5 mm ethylene diamine tetraacetic acid (edta), 20 mm sodium fluoride, 1 mm sodium orthovanadate, a protease inhibitor containing 1 mm phenylmethylsulfonyl fluoride, 2 g / ml leupeptin, 2 g / ml aprotinin, 10 g / ml soybean trypsin inhibitor, and a phosphatase inhibitor containing 100 nm okadaic acid and 10 mm sodium -glycerophosphate . The homogenate was solubilized with an equal volume of 2 sodium dodecyl sulfate (sds) sample buffer [2 mm sodium phosphate (ph 6.8), 2% sds, and 16% 2-mercaptoethanol] and boiled for 2 min . The supernatant was collected and the protein concentration was determined using a protein determination kit (bio - rad; richmond, va, usa). Polyacrylamide gels (8.5% to 12%) were used as running gels to detect b - crystallin, tubulin, and myosin proteins . As stacking gels 1.6 g of sample was electrophoresed over a 24-h period at a constant voltage of 150 v and 4 c . Gels were stained with coomasie brilliant blue r-250 dye (sigma - aldrich co.; st . The relative percentage of mhc isoform content was calculated using imagej (national institutes of health, rockville pike bethesda, md, usa). The extracted plantaris muscle was used as a reference marker for analyzing the extracted soleus muscle because not all mhc isoforms in the plantaris muscle overlap with the mhc isoforms in the soleus muscle, and vice versa . The markers used for the gastrocnemius muscle were a mixture of extracted soleus and plantaris muscles . The following primary antibodies were used for immunoblotting: anti--tubulin (1:1000; sigma - aldrich co.; st . Louis, mo, usa) and anti-b - crystallin (1:5000, raised in rabbit against the c - terminal 10 peptides [(sh) kpavtaapkk] of human b - crystallin). After incubation with secondary antibodies, the membrane was incubated with an enhanced chemiluminescence kit (amersham biosciences corp . ; buckinghamshire, uk). B - crystallin was expressed in escherichia coli and tubulin (phosphocellulose column - purified tubulin, pct) was purified from the porcine brain . To estimate the exact quantity of each protein in the extracted samples, we used the purified proteins (b - crystallin, 5, 10, 20, 30, and 40 ng; -tubulin, 1, 3, 5, 7, 9, and 11 ng) that were used as standards for each protein because there was a linear relationship between the gradual contents of each protein and the band intensity (fig . We subsequently loaded different quantities of proteins extracted from the soleus (0.59 g), plantaris (5 - 13 g), or gastrocnemius (2 - 35 g) muscles and their corresponding protein standards onto the same gel . The contents of b - crystallin and -tubulin estimated from the extracted soleus (0.5 - 9 g), plantaris (5 - 13 g), or gastrocnemius (2 - 35 g) muscles are presented as ng/g . All data are presented as the mean standard deviation (sd) and the differences between control and experiment groups were analyzed using independent t - tests (spss ver . 1) whole values were considered statistically significant when p <0.05 or p <0.01 . In this study, we utilized 12 seven - week - old adult male wistar rats that weighed 238.2 9.1 g. after 2 - 3 days of adaptation, the rats were randomly divided, with half placed in a control group and the half placed in an unloading group . The unloading group was tested using the hindlimb unloading model based on modified methods described by morey and atomi . All animals were provided standard rat chow and water ad libitum, and were housed at 22 - 24 c with a 12:12 h light - dark cycle . After 15 days, all rats were euthanized by cervical dislocation under anesthesia induced by diethyl ether for relieving pain (50 ml / kg body mass). The right sides of the whole soleus muscle, plantaris muscle, and gastrocnemius muscle were isolated, and the muscles were immediately frozen in liquid nitrogen and stored at -80 c until use . Animal use and maintenance was approved by the university of tokyo animal care and use committee . The three isolated whole muscles used in this study were crushed in liquid nitrogen and solubilized in a low - salt buffer containing 20 mm potassium chloride (kcl), 2 mm sodium phosphate (ph 6.8), 2 mm ethylene glycol tetraacetic acid (egta), 5 mm ethylene diamine tetraacetic acid (edta), 20 mm sodium fluoride, 1 mm sodium orthovanadate, a protease inhibitor containing 1 mm phenylmethylsulfonyl fluoride, 2 g / ml leupeptin, 2 g / ml aprotinin, 10 g / ml soybean trypsin inhibitor, and a phosphatase inhibitor containing 100 nm okadaic acid and 10 mm sodium -glycerophosphate . The homogenate was solubilized with an equal volume of 2 sodium dodecyl sulfate (sds) sample buffer [2 mm sodium phosphate (ph 6.8), 2% sds, and 16% 2-mercaptoethanol] and boiled for 2 min . The supernatant was collected and the protein concentration was determined using a protein determination kit (bio - rad; richmond, va, usa). Polyacrylamide gels (8.5% to 12%) were used as running gels to detect b - crystallin, tubulin, and myosin proteins . As stacking gels 1.6 g of sample was electrophoresed over a 24-h period at a constant voltage of 150 v and 4 c . Gels were stained with coomasie brilliant blue r-250 dye (sigma - aldrich co.; st . The relative percentage of mhc isoform content was calculated using imagej (national institutes of health, rockville pike bethesda, md, usa). The extracted plantaris muscle was used as a reference marker for analyzing the extracted soleus muscle because not all mhc isoforms in the plantaris muscle overlap with the mhc isoforms in the soleus muscle, and vice versa . The markers used for the gastrocnemius muscle were a mixture of extracted soleus and plantaris muscles . The following primary antibodies were used for immunoblotting: anti--tubulin (1:1000; sigma - aldrich co.; st . Louis, mo, usa) and anti-b - crystallin (1:5000, raised in rabbit against the c - terminal 10 peptides [(sh) kpavtaapkk] of human b - crystallin). After incubation with secondary antibodies, the membrane was incubated with an enhanced chemiluminescence kit (amersham biosciences corp . ; buckinghamshire, uk). B - crystallin was expressed in escherichia coli and tubulin (phosphocellulose column - purified tubulin, pct) was purified from the porcine brain . To estimate the exact quantity of each protein in the extracted samples, we used the purified proteins (b - crystallin, 5, 10, 20, 30, and 40 ng; -tubulin, 1, 3, 5, 7, 9, and 11 ng) that were used as standards for each protein because there was a linear relationship between the gradual contents of each protein and the band intensity (fig . We subsequently loaded different quantities of proteins extracted from the soleus (0.59 g), plantaris (5 - 13 g), or gastrocnemius (2 - 35 g) muscles and their corresponding protein standards onto the same gel . The contents of b - crystallin and -tubulin estimated from the extracted soleus (0.5 - 9 g), plantaris (5 - 13 g), or gastrocnemius (2 - 35 g) muscles are presented as ng/g . All data are presented as the mean standard deviation (sd) and the differences between control and experiment groups were analyzed using independent t - tests (spss ver . 1) whole values were considered statistically significant when p <0.05 or p <0.01 . The body and tissue weight of the control and unloading groups were measured after 15 days of hindlimb unloading (table 1). There was a significant reduction in body weight in the unloading group compared to the control group (p <0.05). In addition, the weights of the soleus, plantaris, and gastrocnemius muscles of the unloading groups were each significantly reduced compared to the corresponding muscle in the control group, such as 121.6 18.0 (mg) to 54.9 4.2 (mg), 299.3 37.5 (mg) to 218.5 22.9 (mg), and 1454.8 140.3 (mg) to 988.3 57.0 (mg), respectively (p <0.01). Based on the whole muscle to body weight ratios, the unloading soleus (p <0.01), plantaris (p <0.05), and gastrocnemius (p <0.01) muscles were significantly atrophied . B - crystallin levels in the soleus (p <0.01), plantaris (p <0.01), and gastrocnemius (p <0.01) muscles were significantly changed in the unloading group compared to the corresponding muscles of the control group (fig . B - crystallin levels in the unloading group were approximately 28% lower than those in the control group . The b - crystallin content in the unloading plantaris and gastrocnemius muscles was reduced by more than 37% and 42% compared to the control group, respectively . The soleus muscle contained approximately 7-fold as much b - crystallin as the plantaris or gastrocnemius muscles did . There was a significant decrease of -tubulin in the unloading soleus muscle compared to the control muscle (p <0.01) (fig . 2b); however, we did not observe significant differences in -tubulin levels in the plantaris and gastrocnemius muscles between the control group and unloading group . The mhc isoforms in the soleus, plantaris, and gastrocnemius muscles were quantified as comparative values (fig . Each muscle expressed a different primary mhc isoform as shown in figures 2c, 3c, and 4c . Mhc i in the soleus muscle was significantly reduced in the unloading group compared to the control group (p <0.05). However, mhc ii was significantly increased in the unloading soleus muscle compared to the control soleus muscle (fig . High levels of mhc iid / x and iib expression were found in the plantaris muscle . Mhc iid / x was significantly reduced in the unloading plantaris muscle compared to the control plantaris muscle (p <0.01). Mhc iia and iib were significantly increased in the unloading plantaris muscle compared to the control plantaris muscle (p <0.01). Mhc iib was predominantly expressed in the gastrocnemius muscle and was significantly increased after hindlimb unloading, whereas mhc i significantly decreased after hindlimb unloading (fig . The body and tissue weight of the control and unloading groups were measured after 15 days of hindlimb unloading (table 1). There was a significant reduction in body weight in the unloading group compared to the control group (p <0.05). In addition, the weights of the soleus, plantaris, and gastrocnemius muscles of the unloading groups were each significantly reduced compared to the corresponding muscle in the control group, such as 121.6 18.0 (mg) to 54.9 4.2 (mg), 299.3 37.5 (mg) to 218.5 22.9 (mg), and 1454.8 140.3 (mg) to 988.3 57.0 (mg), respectively (p <0.01). Based on the whole muscle to body weight ratios, the unloading soleus (p <0.01), plantaris (p <0.05), and gastrocnemius (p <0.01) muscles were significantly atrophied . B - crystallin levels in the soleus (p <0.01), plantaris (p <0.01), and gastrocnemius (p <0.01) muscles were significantly changed in the unloading group compared to the corresponding muscles of the control group (fig . B - crystallin levels in the unloading group were approximately 28% lower than those in the control group . The b - crystallin content in the unloading plantaris and gastrocnemius muscles was reduced by more than 37% and 42% compared to the control group, respectively . The soleus muscle contained approximately 7-fold as much b - crystallin as the plantaris or gastrocnemius muscles did . There was a significant decrease of -tubulin in the unloading soleus muscle compared to the control muscle (p <0.01) (fig . 2b); however, we did not observe significant differences in -tubulin levels in the plantaris and gastrocnemius muscles between the control group and unloading group . The mhc isoforms in the soleus, plantaris, and gastrocnemius muscles were quantified as comparative values (fig . 2c, 3c, and 4c). Each muscle expressed a different primary mhc isoform as shown in figures 2c, 3c, and 4c . Mhc i in the soleus muscle was significantly reduced in the unloading group compared to the control group (p <0.05). However, mhc ii was significantly increased in the unloading soleus muscle compared to the control soleus muscle (fig . High levels of mhc iid / x and iib expression were found in the plantaris muscle . Mhc iid / x was significantly reduced in the unloading plantaris muscle compared to the control plantaris muscle (p <0.01). Mhc iia and iib were significantly increased in the unloading plantaris muscle compared to the control plantaris muscle (p <0.01). Mhc iib was predominantly expressed in the gastrocnemius muscle and was significantly increased after hindlimb unloading, whereas mhc i significantly decreased after hindlimb unloading (fig . There were obvious correlations between the changes observed in b - crystallin, -tubulin, and dominant mhc isoforms in the muscles . Muscle atrophy induced a reduced expression pattern of b - crystallin and -tubulin in plantar flexor muscles when the muscles shifted to the fast muscle fiber in the unloading group . Both proteins, b - crystallin and -tubulin, were downregulated in slow muscle (p <0.01). B - crystallin was significantly decreased in the fast muscles of the unloading group compared to the control group (p <0.01); however, -tubulin was not reduced . Hindlimb unloading leads to proteolytic processes related muscle atrophy . The proteolytic process induced by hindlimb unloading for example, chaperone - mediated autophagy (cma) is believed to be related to muscle atrophy . Hindlimb unloading leads to the loss of muscle activity in the hindlimbs against the gravitational force and induces muscular atrophy . A reduced gravitational stimulus due to suspending hindlimbs was designed to investigate the transition and degradation of proteins that contribute to hindlimb muscle atrophy . We show that hindlimb unloading results in reduced -tubulin expression, followed by reduced ab - crystallin expression in atrophied triceps surae muscles . Furthermore, our results show the expression pattern of these proteins in fast triceps surae muscles, plantaris, and gastrocnemius muscles, in conjunction with alterations of mhc isoforms by hindlimb unloading . Skeletal muscles are categorized as slow - twitch or fast - twitch muscles based on properties related to adaptable characteristics such as, tension, contraction velocity, force, and glycolytic and oxidative metabolism . Among the triceps surae muscles, we selected the soleus muscle as representative of slow - twitch muscle and the plantaris and gastrocnemius muscles as representatives of fast - twitch muscles . We investigated the responses of b - crystallin and -tubulin in these muscles after hindlimb unloading . It is thought that significant changes in the protein population contribute to muscular function, generation of force, or sensitivity to gravity stimuli . The different expression levels of proteins in these muscles are caused by different recruitment and loading patterns in response to mechanical unloading . This suggests that the different tensile contractions of a muscle that causes different loading force also affects the quantity of tubulin . Chaperone proteins are also involved in the passive and active tensile stress to protect its substrates . In this study, we showed that b - crystallin and -tubulin respond to unloading in mhc i - dominant slow muscle but not in mhc ii - dominant muscles . In our study, the -tubulin and b - crystallin levels exhibited similar changes between control and unloading soleus muscle (fig . However, we did not observe significant changes in -tubulin levels between the control and unloading group in the plantaris and gastrocnemius muscles (fig . Sakurai et al . Reported that the -tubulin transcript level did not change after 10 d of tail suspension even though the b - crystallin transcript level was attenuated when compared to controls . Based on a previous report and our results, tubulin appears to have a less sensitive response than does b - crystallin . In atrophied muscles tubulin is a scaffold protein, and is a net component of the cytoskeleton that maintains the cellular structure . The hypothesis that the degradation of tubulin accelerates muscular atrophy is supported by the fact that the most significant decrease in tubulin levels (p <0.01) was observed in the atrophied soleus muscle, which also showed the greatest extent of atrophy among the three skeletal muscles (47% reduction). By contrast, relatively less prominent atrophy was observed in the gastrocnemius (21% atrophied) and plantaris (16% atrophied) muscles, in which tubulin was not significantly altered when compared to the controls . Mhc is also associated with b - crystallin . B - crystallin assists in preventing the aggregation of myosin and maintains atpase activation . It is thought that hindlimb unloading directly or indirectly affects the activation of quiescent satellite cells in the disruption of sarcolemma . These stimulated satellite cells activate hepatocyte growth factor (hgf) and the activated hgf initiates a signaling cascade, such as the 38p mapk pathway . This signaling pathway is likely related to the expression of ab - crystallin and the mhc isoforms in this study . We used 4 mhc isoforms to reliably identify the dominant mhc isoform in slow - twitch and fast - twitch muscles . We utilized these isoforms because the dominant mhc isoform is partly related to the morphological, contractile, metabolic, and fatigue - resistant properties of muscles [34 - 36]. We analyzed whole plantar flexor muscles because a whole muscle unit, such as the soleus, plantaris, and gastrocnemius muscles, can be linked to its explicit function and adaptability, which were the factors of interest for this study, and because at the molecular level, the border of the medial or lateral portion of the muscle is quite ambiguous, which could lead to inconclusive or erroneous results . Our results indicate that the atrophied plantar flexor muscles began to shift and resemble a fast twitch muscle . This is consistent with other studies that demonstrated a fiber type transition caused by atrophy mainly due to a reduction in the fiber size of the muscle . These results may be related to b - crystallin and tubulin expressions since the expressions of these proteins are dependent on fiber type . We could not clearly detect changes of tubulin because we used whole fast muscles in this study; therefore, in future studies, we will analyze changes of tubulin in muscle fibers . In addition, future studies will also focus on identifying changes in protein levels in hypertrophied slow and fast muscles . Hindlimb unloading leads to proteolytic processes related muscle atrophy . The proteolytic process induced by hindlimb unloading for example, chaperone - mediated autophagy (cma) is believed to be related to muscle atrophy . Hindlimb unloading leads to the loss of muscle activity in the hindlimbs against the gravitational force and induces muscular atrophy . A reduced gravitational stimulus due to suspending hindlimbs was designed to investigate the transition and degradation of proteins that contribute to hindlimb muscle atrophy . We show that hindlimb unloading results in reduced -tubulin expression, followed by reduced ab - crystallin expression in atrophied triceps surae muscles . Furthermore, our results show the expression pattern of these proteins in fast triceps surae muscles, plantaris, and gastrocnemius muscles, in conjunction with alterations of mhc isoforms by hindlimb unloading . Skeletal muscles are categorized as slow - twitch or fast - twitch muscles based on properties related to adaptable characteristics such as, tension, contraction velocity, force, and glycolytic and oxidative metabolism . Among the triceps surae muscles, we selected the soleus muscle as representative of slow - twitch muscle and the plantaris and gastrocnemius muscles as representatives of fast - twitch muscles . We investigated the responses of b - crystallin and -tubulin in these muscles after hindlimb unloading . It is thought that significant changes in the protein population contribute to muscular function, generation of force, or sensitivity to gravity stimuli . The different expression levels of proteins in these muscles are caused by different recruitment and loading patterns in response to mechanical unloading . This suggests that the different tensile contractions of a muscle that causes different loading force also affects the quantity of tubulin . Chaperone proteins are also involved in the passive and active tensile stress to protect its substrates . In this study, we showed that b - crystallin and -tubulin respond to unloading in mhc i - dominant slow muscle but not in mhc ii - dominant muscles . In our study, the -tubulin and b - crystallin levels exhibited similar changes between control and unloading soleus muscle (fig . However, we did not observe significant changes in -tubulin levels between the control and unloading group in the plantaris and gastrocnemius muscles (fig . Sakurai et al . Reported that the -tubulin transcript level did not change after 10 d of tail suspension even though the b - crystallin transcript level was attenuated when compared to controls . Based on a previous report and our results, tubulin appears to have a less sensitive response than does b - crystallin . In atrophied muscles, tubulin begins to degrade after b - crystallin has degraded . Tubulin is a scaffold protein, and is a net component of the cytoskeleton that maintains the cellular structure . The hypothesis that the degradation of tubulin accelerates muscular atrophy is supported by the fact that the most significant decrease in tubulin levels (p <0.01) was observed in the atrophied soleus muscle, which also showed the greatest extent of atrophy among the three skeletal muscles (47% reduction). By contrast, relatively less prominent atrophy was observed in the gastrocnemius (21% atrophied) and plantaris (16% atrophied) muscles, in which tubulin was not significantly altered when compared to the controls . Mhc is also associated with b - crystallin . B - crystallin assists in preventing the aggregation of myosin and maintains atpase activation . It is thought that hindlimb unloading directly or indirectly affects the activation of quiescent satellite cells in the disruption of sarcolemma . These stimulated satellite cells activate hepatocyte growth factor (hgf) and the activated hgf initiates a signaling cascade, such as the 38p mapk pathway . This signaling pathway is likely related to the expression of ab - crystallin and the mhc isoforms in this study . We used 4 mhc isoforms to reliably identify the dominant mhc isoform in slow - twitch and fast - twitch muscles . We utilized these isoforms because the dominant mhc isoform is partly related to the morphological, contractile, metabolic, and fatigue - resistant properties of muscles [34 - 36]. We analyzed whole plantar flexor muscles because a whole muscle unit, such as the soleus, plantaris, and gastrocnemius muscles, can be linked to its explicit function and adaptability, which were the factors of interest for this study, and because at the molecular level, the border of the medial or lateral portion of the muscle is quite ambiguous, which could lead to inconclusive or erroneous results . Our results indicate that the atrophied plantar flexor muscles began to shift and resemble a fast twitch muscle . This is consistent with other studies that demonstrated a fiber type transition caused by atrophy mainly due to a reduction in the fiber size of the muscle . These results may be related to b - crystallin and tubulin expressions since the expressions of these proteins are dependent on fiber type . We could not clearly detect changes of tubulin because we used whole fast muscles in this study; therefore, in future studies, we will analyze changes of tubulin in muscle fibers . In addition, future studies will also focus on identifying changes in protein levels in hypertrophied slow and fast muscles . In conclusion, we found that b - crystallin and tubulin related to the transition of mhc isoforms were downregulated more explicitly in the atrophied soleus muscle rather than in the atrophied plantaris and gastrocnemius muscles . This finding may explain the phenomenon that the target proteins in the fast muscles reacted less sensitive to microgravity than those in the slow muscle . Further studies are needed to provide more clear mechanism regarding the adaptability of skeletal muscles from disuse or immobilization.
The nodes of the graph correspond to metabolic genes, and edges correspond to connections established by metabolic reactions . Metabolic genes x and y are considered connected if and only if there exists a metabolite that is present among the list of either reactants or products of reactions catalyzed by enzymes encoded by both x and y. the metabolic connectivity graph is used to calculate network distance (or metabolic separation) between genes . We define a pair of directly connected metabolic genes as being separated by distance 1 . In general, we define network distance between the genes x and y as the length of the shortest path from x to y on the metabolic connectivity graph . A hand - curated metabolic network model of s. cerevisiae (forster et al, 2003) was used to construct a comprehensive metabolic connectivity graph . While any metabolite can be used to deduce gene connectivity, the relationships established by the common cofactors, such as atp, are not likely to connect genes with similar metabolic functions . In compiling a global metabolic connectivity graph, we consider a subset of metabolites, which excludes most highly connected metabolic species . An exclusion threshold was determined based on the connectivity of the resulting gene dependency graph (supplementary figure 1). A total of 14 most highly connected metabolites (atp, adp, amp, co2, coa, glutamate, h, nad, nadh, nadp, nadph, nh3, orthophosphate, pyrophosphate) and their mitochondrial and external analogs were excluded . The general trends described in the paper are not sensitive to the precise choice of the metabolite set; however, the actual values change when more or less metabolites are considered . For detailed analysis, genes encoding enzymes that are part of known complexes, according to mips complex database (http://mips.gsf.de) and sgd (http://www.yeastgenome.org/), were masked as unassigned enzymes, so that their expression profiles would not be included in any of the analysis (36 enzyme - encoding genes in total). The rosetta's compendium' data set (hughes et al, 2000) measures expression profiles of over 6200 s. cerevisiae open reading frames (orfs) across 287 deletion strains and 13 chemical conditions . In addition, the data set contains 63 negative control measurements comparing two independent cultures of the same strain . These were used to establish individual error models for each orf, providing not only the raw intensity and ratio measurement values for each experimental data point, but also a p - value gauging the significance of change in expression level . We also used a data set from brown's group, containing 173 environmental perturbations (gasch et al, 2000), and a data set from young's group with 34 conditions describing seven environmental perturbation time courses (causton et al, 2001). Log 10 intensity ratios of each data set were normalized to have a mean of 0 and a variance of 1 . Separate time courses contained in these data sets were first normalized individually and then combined . Supplementary table 8 shows the relative variability of metabolic enzyme - encoding genes in each data set . The expression distance measure between orfs x and y is then taken to be 1s(px, py), where px and py are expression profile vectors of x and y, and s corresponds to spearman rank correlation coefficient, calculated according to press et al (2002). Combined expression profile vectors are formed by concatenation of log 10 ratio values from three data sets . Conditions missing experimental value for at least one of the genes were omitted in calculating the expression distance of a given gene pair . Genes that were missing expression values for more than 25% of conditions were not considered in the analysis . Information on transcription factor binding to the metabolic gene promoter sites was taken from lee et al (2002). A p - value threshold of 0.001 was used to select transcription factor binding occurrences . To assess genome context association based on the physical clustering of genes on the chromosome, we relied on gene order statistics . Chromosome clustering association score between genes x and y was calculated as s(xy)=ggp(dg(x, y)), where g is a set of genomes and p(dg(x, y)) is the probability of observing gene order distance dg(x, y) between genes x and y in a genome g, calculated based on the chromosome sizes of organism under the null hypothesis that genes are randomly ordered across the chromosomes . Orthology mapping was established using best bidirectional hits from kegg ssdb (itoh et al, 2004). Phylogenetic profile cooccurrence association (pellegrini et al, 1999) was assessed using hypergeometric probability, as described by bowers et al (2004). The orthology data set was constructed based on best bidirectional blastp hits against ncbi nr protein data set . The calculation was limited to organisms containing orthologs for at least 1% of s. cerevisiae genes . The elementary topological motifs of the metabolite graph were classified in terms of the expression properties of the genes involved . The elementary node structures were enumerated, and sets of genes that were connected in the appropriate topology were extracted for each structure . It is possible for one gene to be included in multiple occurrences of the same motif; in other words, we counted any substructure with the correct topology as an occurrence . Motif instances formed around top 14 most connected metabolites, as well as their mitochondrial and external forms, were not included in the analysis . Mean expression distances of different types of gene pairs were compared using the wilcoxon rank test (wilcoxon, 1945). In generating homolog - filtered data, a pair of metabolic genes was excluded from the analysis if the blast score comparing the two nucleotide sequences was below an e - value of 10 . The nodes of the graph correspond to metabolic genes, and edges correspond to connections established by metabolic reactions . Metabolic genes x and y are considered connected if and only if there exists a metabolite that is present among the list of either reactants or products of reactions catalyzed by enzymes encoded by both x and y. the metabolic connectivity graph is used to calculate network distance (or metabolic separation) between genes . We define a pair of directly connected metabolic genes as being separated by distance 1 . In general, we define network distance between the genes x and y as the length of the shortest path from x to y on the metabolic connectivity graph . A hand - curated metabolic network model of s. cerevisiae (forster et al, 2003) was used to construct a comprehensive metabolic connectivity graph . While any metabolite can be used to deduce gene connectivity, the relationships established by the common cofactors, such as atp, are not likely to connect genes with similar metabolic functions . In compiling a global metabolic connectivity graph, we consider a subset of metabolites, which excludes most highly connected metabolic species . An exclusion threshold was determined based on the connectivity of the resulting gene dependency graph (supplementary figure 1). A total of 14 most highly connected metabolites (atp, adp, amp, co2, coa, glutamate, h, nad, nadh, nadp, nadph, nh3, orthophosphate, pyrophosphate) and their mitochondrial and external analogs were excluded . The general trends described in the paper are not sensitive to the precise choice of the metabolite set; however, the actual values change when more or less metabolites are considered . For detailed analysis, genes encoding enzymes that are part of known complexes, according to mips complex database (http://mips.gsf.de) and sgd (http://www.yeastgenome.org/), were masked as unassigned enzymes, so that their expression profiles would not be included in any of the analysis (36 enzyme - encoding genes in total). We used three data sets as sources of gene expression information . The rosetta's compendium' data set (hughes et al, 2000) measures expression profiles of over 6200 s. cerevisiae open reading frames (orfs) across 287 deletion strains and 13 chemical conditions . In addition, the data set contains 63 negative control measurements comparing two independent cultures of the same strain . These were used to establish individual error models for each orf, providing not only the raw intensity and ratio measurement values for each experimental data point, but also a p - value gauging the significance of change in expression level . We also used a data set from brown's group, containing 173 environmental perturbations (gasch et al, 2000), and a data set from young's group with 34 conditions describing seven environmental perturbation time courses (causton et al, 2001). Log 10 intensity ratios of each data set were normalized to have a mean of 0 and a variance of 1 . Separate time courses contained in these data sets were first normalized individually and then combined . Supplementary table 8 shows the relative variability of metabolic enzyme - encoding genes in each data set . The expression distance measure between orfs x and y is then taken to be 1s(px, py), where px and py are expression profile vectors of x and y, and s corresponds to spearman rank correlation coefficient, calculated according to press et al (2002). Combined expression profile vectors are formed by concatenation of log 10 ratio values from three data sets . Conditions missing experimental value for at least one of the genes were omitted in calculating the expression distance of a given gene pair . Genes that were missing expression values for more than 25% of conditions were not considered in the analysis . Information on transcription factor binding to the metabolic gene promoter sites was taken from lee et al (2002). A p - value threshold of 0.001 to assess genome context association based on the physical clustering of genes on the chromosome, we relied on gene order statistics . Chromosome clustering association score between genes x and y was calculated as s(xy)=ggp(dg(x, y)), where g is a set of genomes and p(dg(x, y)) is the probability of observing gene order distance dg(x, y) between genes x and y in a genome g, calculated based on the chromosome sizes of organism under the null hypothesis that genes are randomly ordered across the chromosomes . Orthology mapping was established using best bidirectional hits from kegg ssdb (itoh et al, 2004). Phylogenetic profile cooccurrence association (pellegrini et al, 1999) was assessed using hypergeometric probability, as described by bowers et al (2004). The orthology data set was constructed based on best bidirectional blastp hits against ncbi nr protein data set . The calculation was limited to organisms containing orthologs for at least 1% of s. cerevisiae genes . The elementary topological motifs of the metabolite graph were classified in terms of the expression properties of the genes involved . The elementary node structures were enumerated, and sets of genes that were connected in the appropriate topology were extracted for each structure . It is possible for one gene to be included in multiple occurrences of the same motif; in other words, we counted any substructure with the correct topology as an occurrence . Motif instances formed around top 14 most connected metabolites, as well as their mitochondrial and external forms, were not included in the analysis . Mean expression distances of different types of gene pairs were compared using the wilcoxon rank test (wilcoxon, 1945). In generating homolog - filtered data, a pair of metabolic genes was excluded from the analysis if the blast score comparing the two nucleotide sequences was below an e - value of 10 . Mean expression distance is plotted as a function of the metabolic network distance separating the metabolic gene pairs for (a) all metabolic gene pairs; (b) positively coexpressed pairs and (c) negatively coexpressed pairs . (d) fraction of metabolic gene pairs that share at least one transcription factor binding site in their promoter region as a function of metabolic network distance . (a) all irreversible metabolite graph motifs consisting of two genes (x and y) are analyzed using only positively coexpressed gene pairs . The motifs are ordered according to the mean expression distance of gene pairs belonging to each motif . The pair types are ordered (from left to right) in the order of increasing mean expression distance.
Sigmund freud is rarely mentioned in scientific discourse without also belittling the lack of quantitative statistical evidence for his elaborate models . At the same time, his qualitative case reports and the conclusions he drew from them by far belong to the most well - known research in psychosomatic medicine . Despite all valid critique, one reason, we argue, may very well be the superiority of the single case study in first observing, describing, capturing, evaluating, and creatively reflecting on an infinite set of parameters surrounding any chosen topic . Out of this primary assessment, novel hypotheses and it is our objective to reapply such primary assessment to the case of adolescent brittle diabetes (or more generally speaking, the psychosomatic underpinnings of diabetes type 1 in minors and young adults), while also trying to answer calls for more quantitative and statistically reliable approaches to doing so . This in mind, we have first selected a highly quantitative case study on family dynamics and brittle diabetes and reviewed and reanalyzed its raw data through implementation of a new statistical procedure increasing the coefficient of determination in the new model by factor ten (while also presenting new and clearer findings), in order to then, in a second step, discuss and compare our results to possibly the historically most well - known set of qualitative case studies on the topic . We will start by briefly revisiting the literature on the psychosomatics of adolescent instable diabetes type 1, present a case vignette and basic data collection method of the original case study we reexamine (which may be skipped by those familiar with the work published by), followed by a detailed description of our new statistical approach and its results, concluding with a clear clinically oriented graphical presentation of our findings and their discussion in light of minuchin et al . One out of 600 us or european school - age children suffers from insulin dependent diabetes mellitus [3, 4]. Just about 33 percent of diabetics between 13 and 19 years of age manage to maintain tolerable glycemic control and a hba1c below 8; 6.3 percent suffered at least one episode of major hypoglycemia within the last three months [5, 6]. The devastating immediate and long - term effects of poor diabetic control are widely known and feared . 44 percent of the variance in blood glucose control can be statistically explained by psychological variables in these patients and their parents . A randomized controlled study further demonstrates how an intensive inpatient treatment program including psychoanalytic psychotherapy could effectively improve diabetic control in children . These cases of glycemic instability with no somatic explanation have been termed brittle diabetes by some authors and there is no doubt concerning the importance of further exploration of the causes and remedies surrounding this truly psychosomatic disease . While various aspects of brittle diabetes have been explored in recent years, including its exact definition, there seems to be a gap in the literature in exploring how emotional variables of all individuals within the family system may interact to affect glycemic control of the diabetic adolescent, the index patient of a dysfunctional family system . The little research which has sought to fill this gap (i.e., [2, 10, 11]) is primarily qualitative in nature and must face similar critique as all such work, as will be discussed in the last section of this study . The case and its psychosomatic background (adopted and revised from). The adolescent index patient of this case study was diagnosed with diabetes type 1 at age of four (clinical clues were polyuria, polydipsia, loss of appetite, a fungal infection, hba1c of 9.1 per cent, antibodies against islet cells, and gad65). Family dynamics surrounding this classic family of three (biological parents, single child) appeared unsuspicious notwithstanding the girl's history of poorly controlled bronchial asthma and allergic diseases . Yet at age of six, nocturnal hypoglycemia with loss of consciousness led to readmission to the hospital, during which another episode of profound hypoglycemia, this time in conjunction with a tonic - clonic seizure, occurred, thus further consolidating her parents' distress concerning hypoglycemia and hospital treatment . Once all educational efforts concerning the diabetic management were exhausted (including individual and family - based counseling, detailed and repetitive disease - specific education, and information about glycemic control mechanisms including the influence of nutrition, sport, and other aspects of blood sugar regulation), but a hba1c below 7 percent was never achieved, the family finally sought for psychosomatic family treatment . Psychodynamically based therapeutic analysis of the family dynamic suggested a conflict between the adolescent and her mother about who had control of the blood sugar levels . The mother's dominance seemed to have negative effects on her daughter's glycemic control . Fears of hypoglycemia were somewhat irrational with all three family members, including the father, who, at first sight, seemed rather more distant to the matter (literature proposes parental hypoglycemia avoidance behaviours to adversely affect glycemic control). The family's shock in relation to the diagnosis and mistrust of hospital personnel was discussed . Finally, a therapeutic intervention confronted them with their specific type of collusion concerning (in-)dependence, in which both parents, in their manifest statements, advocated for more self - confidence and extended duties on the side of the daughter, but on a more latent level, gave hints to their beloved little girl not being ready to take control over the blood sugar monitoring by herself . This mostly unconscious conflict had culminated in cloudy paths of communication concerning glycemic control, in nebulous distributions of duties within the family members, and, as a result of the arrangement, in deep dissatisfaction over the failure of proper diabetic control . While traditional case studies would focus on the qualitative data outlined above, we sought to amend such observations by a highly quantitative approach in order to produce more evidence based and reproducible results . Therefore, we aimed to statistically explore how specific basic affect states of all three individual family members may impact each other and the success of the diabetic management over a period of 120 days . To operationalize this quest, we drew on the standardized self - assessment manikin (sam), as developed by bradley and lang (for details see [13, 14]), asking all three family members to individually record on a daily basis their valence (mood), arousal (high versus low), and dominance (a sense of presence in the current environment). In addition the index patient was asked to obtain at least three daily blood glucose measurements (or more if required by the disease) over the same period utilizing a common standardized technique . This form of diary based data collection is also referred to as ecological momentary assessment with many benefits in terms of accuracy and validity of measurements . Standard deviations of the daily blood glucose measurements served as an indicator for glycemic variability, a measure which recent research has identified as the most precise predictor of diabetic control, followed by the hba1c - value in second place [1619], due to it being the best known predictor for diabetic complications and microvascular derailments in particular . Resulting from this data collection and primary analysis are ten time series: three time series for each of the three family members from the sam, affective valence (happy, sad), arousal (excited, calm), and dominance (a sense of presence, distance to the current environment), as well as one time series recording glycemic variability (daily standard deviations of measurements)., these ten time series were further analyzed by a completely new statistical approach to vector autoregressive (var) modeling . While past analysis of this same set of data (see) has also relied on basic var analysis, there had been some common shortcomings to the validity and scope of results, which we were able to remedy here, thus solving statistical shortcomings while also presenting completely new results in a clearer more clinically oriented fashion . How we were able to achieve this, the presentation of a newly developed optimized multivariate lag selection process in var analysis, and a comprehensive review of the principles of vector autoregression will be presented next . The use of vector autoregressive models (var) for the analysis of time series data in psychosomatic medicine (also widely used in neuroscience) allows treating a set of variables as jointly driven by the lagged values of all variables in the system with no a priori assignment of dependent and independent status being necessary . This technique seems particularly apt for research in psychosomatic medicine, where, among others, has long called for a more integrated (monistic) view on the complexity of dynamic dependencies and intertemporal reciprocal cause and effect relationships among different psychic as well as somatic variables . Any var model requires the user to select a maximum number of lags, which, in more practical terms, refers to how far back in time the user wants to go in the search for past recordings of all variables to predict the present value of one variable . The farther back in time the user decides to go, the more explanatory variables (lags) need to be included in the model because it used to be improper to exclude past recordings of explanatory variables, which lay in - between the present value and the most historic one [22, 23]. Unfortunately including more explanatory variables (going back further in time) is a double edged sword, since this would provide a var model more representative of reality (goodness of fit), but would also endorse one with less explanatory power (lower adjusted r). The latter is due to the tremendous penalty inflicted by the large number of explanatory variables (lags) in the model resulting in high estimation variance [22, 23]. This substantial drawback weakened the substance of empirical findings derived from var models, because researchers would either present results through models with teeth chattering low r values (see previously published results from the same raw data as one example) or adopt models only incorporating the effects of events preceding the predicted value of a variable by one day / one unit of time in the var (e.g., see). In order to alleviate this shortcoming of low adjusted r values in the standard vector autoregressive modeling approach, we developed a computer code implementing a statistical procedure recently published in parts in savin and winker and winker [26, 27], referred to as the optimized multivariate lag selection process, which allows (contrary to previous practice) excluding such explanatory variables (lags) from the var model which add little to its goodness of fit (estimated representativeness of reality) while nonetheless reducing its explanatory power (adjusted r). This admittance of holes to the lag structure (equations organizing the explanatory variables) allows us to now present an entirely new model exhibiting more detailed dynamics with a smaller number of parameters, for the data in this case resulting in about tenfold increase of the adjusted r value . Mathematical details of applying the optimized multivariate lag selection process to this var analysis of the ten time series of the data set at hand will be presented next (and may be skipped by the more clinically focused researcher). A standard vector autoregressive (var) model was constructed, using eviews 7.1 (qms, quantitative micro software, irvine ca), based on the ten time series we mentioned above . In order to focus on the innovative aspects of our methodology we will not delve into the details of var model construction, which have been described at length in preceding publications (i.e., [1, 24]). Given the large number of explanatory variables (the more lags, the more variables) and the limited number of observations, only a very limited number of lags (past days) could be considered while adjusted r would still be low, if we were to follow the standard modeling approach [22, 23]. The novel contribution is to maximize the informational content of the model by minimizing an information criterion [2527]. In more concrete terms, if we assume that any one value within the ten time series may have effects on any of the other values of all - time series with a delay of up to one week, a total of 710 parameters would have to be estimated . Given 120 observations in each time series, this results in tremendous estimation variance (very low r). Model selection criteria suggest using only one lag (assuming effects will take place within a day instead of within a week, which seems highly unrealistic but is a common approach adopted by other researchers in the field, including wild et al ., 2010) resulting in a total of only 110 parameters to be estimated with a still low r value of 0.02 for the model explaining glycemic variance . To resolve this dilemma, we drew on winker [26, 27] and savin and winker engaging in optimized multivariate lag structure analysis . Given the huge discrete search space of all possible lag structures, for example, for a maximum lag length of seven, heuristic optimization algorithms are used to this end . For this process, a computer code was developed using matlab r2011b with an interface to eviews 7.1, which implements a genetic algorithm for the search of an optimized lag structure making use of information criteria (bic) as in the standard selection procedure (see for more details). By providing an approximation to the minimum of the information criterion, the resulting model exhibits an optimized tradeoff between a good fit to the multivariate dynamics of the data and model parsimony . As a result, we obtained a model with only 70 parameters, but still cover effect delays up to one week . Since the maintained lags are selected based on their joint informational content (as measured by the information criteria), the procedure results in a model with much higher explanatory power (for predicting glycemic variability adjusted r value of 0.20 as opposed to 0.02 for the standard model with only one lag) and a richer dynamic . Given the rich dynamics between all variables of the model, besides considering single equations, the calculation of impulse response functions as in would be of interest . However, the zero constraints of the var model with holes preclude the application of standard methods for the calculation of confidence bands . Similarly, poor glycemic control (high glycemic variability) will correlate with low glycemic variability four days earlier, a calm mother three days earlier, an excited mother seven days earlier, a dominating mother four days earlier, a nondominating mother seven days earlier (although statistically insignificant), a sad father both five and six days earlier, a calm father both three and seven days earlier, and a dominating father both two and five days earlier . High glycemic variability will also correlate with a sad child six days later, an excited mother three days later, and a dominating father one day later . For a graphical representation see figure 2 . The optimized multivariate lag structure selection process provides one equation of seemingly unrelated multiple regression for each of the ten time series to be presented next . Three of them directly involve glycemic variability in addition to the one for glycemic variability itself, which shall be presented last (lags in parentheses): affective valence of the adolescent = 1 glycemic variability (6) + 2 valence adolescent (1) (r = 0.25, adj . R = 0.24); affective valence of the mother = 3 dominance adolescent (7) + 4 valence mother (5) + 5 arousal mother (6) + 6 arousal father (4) + 7 arousal father (6) (r = 0.21, adj . R = 0.18); affective valence of the father = 8 valence adolescent (3) + 9 valence adolescent (5) + 10 arousal mother (5) + 11 dominance father (3) (r = 0.21, adj . R = 0.18); arousal of the adolescent = 12 arousal adolescent (1) + 13 arousal adolescent (3) + 14 arousal adolescent (7) + 15 valence mother (4) + 16 arousal mother (3) + 17 valence father (2) + 18 valence father (6) (r = 0.30, adj . R = 0.25); arousal of the mother = 19 glycemic variability (3) + 20 arousal adolescent (7) + 21 dominance adolescent (5) + 22 arousal mother (5) + 23 arousal mother (7) + 24 dominance mother (1) + 25 dominance father (6) (r = 0.29, adj . R = 0.24); arousal of the father = 26 valence mother (4) + 27 dominance mother (6) + 28 arousal father (1) + 29 arousal father (2) + 30 arousal father (6) + 31 dominance father (1) (r = 0.19, adj . R = 0.15); dominance of the adolescent = 32 valence adolescent (1) + 33 arousal adolescent (5) + 34 arousal father (1) + 35 dominance father (1) (r = 0.25, adj . R = 0.22); dominance of the mother = 36 valence mother (7) + 37 dominance mother (1) + 38 dominance mother (3) + 39 dominance father (5) (r = 0.65, adj . R = 0.64); dominance of the father = 40 glycemic variability (1) + 41 dominance child (6) + 42 valence mother (5) + 43 valence mother (7) + 44 dominance mother (4) + 45 dominance mother (6) + 46 valence father (1) + 47 valence father (3) + 48 arousal father (3) + 49 dominance father (2) (r = 0.34, adj . R = 0.27); glycemic variability = 1 glycemic variability (4) + 2 arousal mother (3) + 3 arousal mother (7) + 4 dominance mother (4) + 5 dominance mother (7) + 6 valence father (5) + 7 valence father (6) + 8 arousal father (3) + 9 arousal father (7) + 10 dominance father (2) + 11 dominance father (5) (r = 0.28, adj . Affective valence of the adolescent = 1 glycemic variability (6) + 2 valence adolescent (1) (r = 0.25, adj . R = 0.24); affective valence of the mother = 3 dominance adolescent (7) + 4 valence mother (5) + 5 arousal mother (6) + 6 arousal father (4) + 7 arousal father (6) (r = 0.21, adj . R = 0.18); affective valence of the father = 8 valence adolescent (3) + 9 valence adolescent (5) + 10 arousal mother (5) + 11 dominance father (3) (r = 0.21, adj . R = 0.18); arousal of the adolescent = 12 arousal adolescent (1) + 13 arousal adolescent (3) + 14 arousal adolescent (7) + 15 valence mother (4) + 16 arousal mother (3) + 17 valence father (2) + 18 valence father (6) (r = 0.30, adj . R = 0.25); arousal of the mother = 19 glycemic variability (3) + 20 arousal adolescent (7) + 21 dominance adolescent (5) + 22 arousal mother (5) + 23 arousal mother (7) + 24 dominance mother (1) + 25 dominance father (6) (r = 0.29, adj . R = 0.24); arousal of the father = 26 valence mother (4) + 27 dominance mother (6) + 28 arousal father (1) + 29 arousal father (2) + 30 arousal father (6) + 31 dominance father (1) (r = 0.19, adj . R = 0.15); dominance of the adolescent = 32 valence adolescent (1) + 33 arousal adolescent (5) + 34 arousal father (1) + 35 dominance father (1) (r = 0.25, adj . R = 0.22); dominance of the mother = 36 valence mother (7) + 37 dominance mother (1) + 38 dominance mother (3) + 39 dominance father (5) (r = 0.65, adj . R = 0.64); dominance of the father = 40 glycemic variability (1) + 41 dominance child (6) + 42 valence mother (5) + 43 valence mother (7) + 44 dominance mother (4) + 45 dominance mother (6) + 46 valence father (1) + 47 valence father (3) + 48 arousal father (3) + 49 dominance father (2) (r = 0.34, adj . R = 0.27); glycemic variability = 1 glycemic variability (4) + 2 arousal mother (3) + 3 arousal mother (7) + 4 dominance mother (4) + 5 dominance mother (7) + 6 valence father (5) + 7 valence father (6) + 8 arousal father (3) + 9 arousal father (7) + 10 dominance father (2) + 11 dominance father (5) (r = 0.28, adj . The coefficients, their standard error, t - statistic, and probability referred to above, can be reviewed in table 1 . The development of a novel statistical methodology allowed us to disentangle the data and generate statistically reliable results in the form of ten equations . The dynamic of the results pertaining to glycemic variability, (thereby, it has to be taken into account that additional dynamic interactions arise due to spillover between equations, which are not considered here), taking into account the direction of coefficients, can be summarized in the following words and graphical representations . Low glycemic variability and, therefore, good diabetic control will correlate with the following: high glycemic variability four days earlier, an excited mother three days earlier, a calm mother seven days earlier, a non - dominating mother four days earlier, a dominating mother seven days earlier (although statistically insignificant), a happy father both five and six days earlier, an excited father both three and seven days earlier, and a non - dominating father both two and five days earlier . Low glycemic variability will also correlate with a happy child six days later, a calm mother three days later, and a non - dominating father one day later . For a graphical representation of this paragraph refer to figure 1 . Similarly, poor glycemic control (high glycemic variability) will correlate with low glycemic variability four days earlier, a calm mother three days earlier, an excited mother seven days earlier, a dominating mother four days earlier, a non - dominating mother seven days earlier (although statistically insignificant), a sad father both five and six days earlier, a calm father both three and seven days earlier, and a dominating father both two and five days earlier . High glycemic variability will also correlate with a sad child six days later, an excited mother three days later, and a dominating father one day later . A graphical representation of this paragraph is presented in figure 2 in clinical terms, this means, good diabetic control was preceded by attentive and alert (high arousal, excited) parents with a positive attitude (happy father), at the same time refraining from too much overwhelming presence (low dominance). Likewise, phases of good diabetic management were followed by a continuously distant father (low dominance), unfortunately a less alert mother (low arousal), and a content (happy) adolescent index patient . Similarly, mostly self - explanatory, graphical representations were constructed for the effects surrounding the affective valence of all three family members (see figures 3, 4, and 5). We picked these three timelines for more detailed examination, because the appropriate measurement of depressive symptoms (which at least at a distance somewhat relates to affective valence) in diabetics in general, remains to be a topic of current debate in the literature . In comparison to the results derived from the same set of raw data with a different statistical approach in an earlier publication, there are several improvements we were able to achieve:(i)increasing the coefficient of determination r for the model prediction of glycemic variability by factor ten (adjusted r value of 0.20 as opposed to 0.02) while incorporating significant effects of explanatory variables (lags) stemming from a longer period of time preceding the predicted event;(ii)presenting a more precise timeline of effects of various variables on each other, including glycemic variability and vice versa (e.g., a nondominating mother four days prior to a set day will increase glycemic control instead of a nondominating mother somewhere up to four days prior to a set day will increase glycemic control);(iii)isolating additional relationships between variables which did not reach statistical significance earlier or took more time to take effect than the time frame of the earlier models allowed for . Increasing the coefficient of determination r for the model prediction of glycemic variability by factor ten (adjusted r value of 0.20 as opposed to 0.02) while incorporating significant effects of explanatory variables (lags) stemming from a longer period of time preceding the predicted event; presenting a more precise timeline of effects of various variables on each other, including glycemic variability and vice versa (e.g., a nondominating mother four days prior to a set day will increase glycemic control instead of a nondominating mother somewhere up to four days prior to a set day will increase glycemic control); isolating additional relationships between variables which did not reach statistical significance earlier or took more time to take effect than the time frame of the earlier models allowed for . A more substantial contribution of this paper is the demonstration and practical application of the multivariate lag selection process to var analysis, resolving an essential shortcoming in var analysis of (relatively) small samples . Hence, this contribution to literature will have relevance beyond the case study approach but also to var - based studies of larger cohorts of patients (as e.g.,), significantly increasing either the number of effects analyzed (as in) or the statistical reliability (i.e., the adjusted r) with which results are presented . All in all, however, mathematically refined quantitative methodological approaches relying on modern computational technology can generate more specific, reproducible, and thus trustworthy results than purely qualitative (narrative) accounts, while still honoring the benefits of the case study approach aiming to explore previously unforeseen avenues fit for further vested inquiry (often costly to perform). Yet, we have to ask ourselves critically if the added mathematical complexity honors the overall value of the results a case study approach can provide . Revisiting the opening comments of this report in the context of brittle diabetes, it seems interesting to note that particularly the most highly acclaimed and clinically widely trusted research on brittle diabetes has also been the most severely and broadly criticized . So, for instance, more than ten years after the initial publication of the pioneering work of minuchin et al . In 1978 (on what they called psychosomatic diabetes) entitled psychosomatic families, critics commented as follows: as we conducted research and therapy with the families of diabetic children, we were impressed with both the limit of the formulation of the family's role in diabetes offered in psychosomatic families' and the uncritical acceptance that the book continued to enjoy . In their rather pointed article entitled the psychosomatic family' reconsidered ii: recalling a defective model and looking ahead coyne and anderson criticize minuchin et al . Primarily for their bold, yet statistically (allegedly) poorly supported, statements on the typical psychosomatic family psychosomatic family as featuring enmeshment, rigidity, overprotectiveness, and lack of conflict resolution and the children affected by brittle diabetes as having difficulty in handling stress, showing a tendency to internalize anger and being somewhat immature in their ability to cope with challenging situations) and their overgeneralizations of these overall weak findings on familial situations in one psychosomatic illness to various psychosomatic illnesses . More specifically, small sample sizes and poor documentation of methodology (or lack thereof) are being highlighted . Reflecting on such valid criticism in light of our own extensive research both on the subject of brittle diabetes in adolescents and on the various shortcomings of contemporary statistical approaches to time series data in psychosomatic medicine, we believe there is a case for both sides . On the one hand, we must vigorously support critics (i.e.,) in their call for much more detailed and sophisticated reports on and publication of statistical methodology in such complex and intricate research situations as are present in multivariate time series analysis . The reason lies in the fact that there is vast room for pitfalls and error with this type of research, if left in the hands of the mathematically inexperienced . On the other hand, however, we found for fact, that with the change of statistical approach, the results drawn from a given set of data may change somewhat, despite both methodologies being perfectly valid and academically accepted . So one wonders how this (agreeably small) imprecision of highly quantitative research is any different from the (possibly but not necessarily larger) inaccuracy of qualitative research due to subjectivity . [2, 11], the one finding which we were able to observe clinically before conducting any statistical testing at all, namely, that of a dominating mother having a negative effect on glycemic control of her child, was also a finding that both of our methodologies were able to report at a high level of significance . Described as overprotectiveness in families with brittle diabetes is very similar, if not the same, to what we were able to pinpoint in terms of exaggerated control of a mother over her glycemically out of control child .) Additionally, we also fear that critics of primarily qualitative case research (i.e.,) may not have realized the vastness of data inherent even in a small sample in time series analysis, an apprehension possibly supported by the fait accompli of not too many critics providing any statistically evidenced findings on the subject of brittle diabetes themselves (i.e.,). So in conclusion, we believe the careful observation of the clinically experienced therapist to be almost as valuable as the most substantiated and savvy statistical approach.
Sepsis affects approximately 700,000 people annually and accounts for about 210,000 deaths per year in the us . Its incidence is rising at rates between 1.5% and 8% per year, despite continuous progress in the development of antimicrobial therapeutics and supportive cares . Most of the sepsis is caused by bacteria and bacteria of abdominal origin contribute to the second major reason for sepsis . The septic peritonitis is the host's systemic inflammatory response to the bacteria, initiated by the pathogen associated molecule patterns (pamps) including lipopolysaccharide and lipid a from gram - negative bacteria and lipoteichoic acid and peptidoglycan from gram - positive bacteria . The inflammatory initiation leads to the release of chemokines, such as interleukin-8 (il-8), and proinflammatory cytokines, such as tumor necrosis factor- (tnf-), il-1, il-6, and il-12, in a massive amount . The cytokines, if not appropriately controlled, may severely impair the functions of vital organs or systems, resulting in death . Data from the ciaow study (complicated intra - abdominal infections worldwide observational study) showed that the overall mortality rate was 10.5% in the patients with septic peritonitis in a worldwide context . Septic peritonitis is characterized by a massive infiltration of neutrophils into the peritoneum in response to bacterial invasion, where they are activated and act as a first line of defense against the bacteria . Morphologically, the mature neutrophils are unique among the other white blood cells by their lobulated nucleus, which inspired the renaming of them as polymorphonuclear neutrophils (pmns). Based on cytokine production, macrophage activation, expression of toll - like receptor (tlr), and surface antigen expression, murine pmns have been classified into three types: normal pmn (pmn - n), pmn - i, and pmn - ii . Pmn - i produces il-12/ccl3, activates macrophages in a classical way, expresses tlr2/tlr4/tlr5/tlr8, and has a cd49dcd11b phenotype . Pmn - ii produces il-10/ccl2, activates macrophages in an alternative manner, expresses tlr2/tlr4/tlr7/tlr9, and possesses a cd49dcd11b phenotype . Pmn - n rarely produces cytokine / chemokine, displays no effect on macrophage activation, and expresses tlr2/tlr4/tlr9, with cd49dcd11b phenotypes . The pmn - n may convert to pmn - i or pmn - ii in response to bacterial infections . Recently, macrophages have been reported to cooperate with neutrophils by promoting extravasation and activation of pmns, even their clearance of bacteria . Accumulated evidence revealed that toll - like receptors (tlrs) could play a fundamental role in induction of hyperinflammation and tissue damage in sepsis . Tlrs are pattern recognition receptors (prrs) that sense microbial invasion and initiate innate immune responses . Positioned at the cell surface, tlr4 is essential for sensing lipopolysaccharide of gram - negative bacteria and tlr2 is involved in the recognition of a large panel of microbial ligands, while tlr5 recognizes flagellin . Endosomal tlr3, tlr7, tlr8, and tlr9 are specialized in the sensing of nucleic acids produced notably during viral infections or during bacterial infections . Conventionally, tlr9 is thought to sense microbial dna in endolysosomes and not at the cell surface . Recently, it has been found that tlr9 is expressed on the surface of human and mouse pmns, that the surface tlr9 (stlr9) senses dna in pmns, and that stlr9 expressing pmns (stlr9 pmns) have roles in inducing rapid inflammation . Human and mouse pmns spontaneously express stlr9, and the stlr9 expression is upregulated in response to pmn stimulation . In front of tlr9 ligands, tlr9 mice exhibited lower serum inflammatory cytokine levels, higher bacterial clearance, and greater survival after experimental peritonitis induced by cecal ligation and puncture (clp). A single injection of tlr9 antagonist protected the wide type mice, even when administered as late as 12 hours after clp . It has been proposed that, during infection, stlr9 pmns could be involved in the detrimental effect by releasing massive proinflammatory cytokines, including il-6 and tnf-, and possibly other cytokines in response to pamps [12, 13]. Interleukin-17 (il-17) is a cytokine family that signatures t helper 17 (th17) cell subset and contains six members (il-17a to il-17f), among which il-17a is considered as one of the major proinflammatory cytokines mediating the innate and adaptive immune responses against bacterial infections . In addition to th17 cells, t cells, innate lymphoid cells (ilcs), mast cells, pmns, and macrophages are also il-17 producing cells . Notably, innate immune cell - derived il-17 constitutes a major element in the immune response against infectious agents by recruiting pmns to the sites of infections and by inducing the production of antimicrobial peptides, cxc chemokines, and granulocyte colony stimulating factor (g - csf) [15, 16]. In bacterial infection, pmns act to produce increased il-17, which in turn recruits more pmns to join the fighting against invaded bacteria, resulting in increased production of innate immune cell - derived il-17 [16, 17]. Il-17 was found to work in an il-23-il-17 axis which was critical for the survival of the host infected with bacteria . In the axis, tissue - infiltrating pmns could be the main source of il-23 which induces production of il-17 from macrophages . In the present study, we kinetically observed the infiltration of pmns and expression of stlr9 and il-17 on / in the pmns in the peritoneal lavage cells (plcs) of mice intraperitoneally infected with various doses of escherichia coli (e. coli), aiming to find the correlation of the expression of stlr9 and il-17 on / in the pmns during the development of bacterial septic peritonitis . The achieved results could provide a basis for further investigation on the roles of stlr9 expressing pmns and il-17 producing pmns in bacterium caused septic peritonitis . The monoclonal antibodies of apc - conjugated rat anti - mouse cd45 antibody (561689), pe - conjugated rat anti - mouse cd14 antibody (561711), pe - conjugated rat anti - mouse cd11b antibody (561689), apc - cy 7-cojugated rat anti - mouse cd3 antibody (560590), and apc - conjugated rat anti - mouse ly-6 g antibody (560599) were purchased from bd bioscience . The fitc - conjugated active anti - tlr9 monoclonal antibody (26c593.2) was from abcam; fitc - conjugated anti - mouse il-17a antibody (506908) and percp - cy 5.5-conjugated rat anti - mouse il-17a (3354955) antibody were from biolegend . Cdna synthesis kit (transgene biotech, i21021) and two - step sybr green qpcr assays (transgene biotech, g31227) were from biotech . E. coli strain of jm109 was recovered from lyophilized powder by being suspended into lb medium and then cultured on lb agar plate at 37c overnight . The single colonies of e. coli on the plate were used as a group of original seed e. coli . Female icr mice were purchased from the experimental animal center, medical college of norman bethune, jilin university, and maintained in microisolator cages under specific pathogen - free conditions . The experimental manipulation of mice was undertaken in accordance with the national institute of health guide for the care and use of laboratory animals, with the approval of the scientific investigation board of science & technology of jilin province . To prepare e. coli culture, each single colony of e. coli was picked up and cultured in 5 ml lb medium at 37c with shaking at 200 rpm . When the od value (a600) of the culture reached 1.4, e. coli was harvested from the culture by centrifugation and mixed with 20% glycerol solution . The working seed e. coli were seeded into lb medium and cultured at 37c till the od value reached 0.7 followed by harvesting the e. coli pellet after centrifugation at 4000 g for 5 min . The e. coli pellet was diluted in a serial tenfold, plated on an eosin methylene blue agar plate, and then cultured at 37c for 14 hours . The colony - forming units (cfus) of the e. coli culture were calculated and determined as approximately 0.8 10 cfus / ml . For animal experiment use, the e. coli pellet was washed twice using sterile 0.9% nacl (saline) and then resuspended in 1.0 ml saline containing 0.4, 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, and was ready to use for infecting mice . For inducing bacterial septic peritonitis, mice were injected intraperitoneally (i.p .) With 1.0 ml of e. coli preparations containing 0.4, 0.8, 1.6, and 2.4 10 cfus / ml in sterile saline, respectively . The survival of the mice was monitored every 4 hours for 4 days . To harvest the peritoneal lavage cells (plcs) from mice either infected with e. coli or injected with saline, the mice (n 3 per group) were euthanized at defined time points with 50 mg / kg pentobarbital sodium followed by washing peritoneal cavities using 6 ml per mouse of ice - cold phosphate - buffered saline (pbs). The peritoneal lavage fluid was centrifuged at 750 g for 5 minutes at 4c for harvesting the plcs . Plcs were harvested from the nave mice and maintained in rpmi 1640 supplemented with 10% (v / v) fetal bovine serum (fbs) (gibco) and antibiotics (100 iu of penicillin / ml and 100 iu of streptomycin / ml). In experiments using viable bacteria, antibiotics were not added to the culture medium during the isolation, washing, or subsequent culturing period . No bacterial contamination was observed in plcs cultured in the absence of antibiotics . Cells were counted and then plated in 24-well cell culture plates (costar, cambridge, ma) at an approximate density of 1 10 cells / well . The plcs were cocultured with e. coli at 1 10 or 1 10 cfus / well or saline as a vehicle control for 14 hours, and then they were cultured with bfa for another 4 hours, in a 5% co2 humidified incubator at 37c . The plcs were collected and stained with fitc - labeled anti - il-17 mab, followed by flow cytometry analysis to detect the expression of il-17 . To count the numbers of cells in each peritoneal lavage sample the cell pellets were smeared on slides and then stained using hematoxylin - eosin (he) followed by counting the cell numbers on hemocytometer (beckman coulter, fullerton, ca) and taking photos of the cells . For surface staining, the plcs were stained with fluorescence - conjugated mabs against cd45, cd11b, and stlr9, respectively, for 30 minutes at room temperature in the dark followed by washing twice with pbs . For il-17 intracellular staining, the plcs were surface stained as described above and then fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin followed by staining with fitc - conjugated anti - il17a monoclonal antibody . All stained cells were analyzed by flow cytometer facscalibur (bd) and cytoflex (beckman coulter). The plcs were harvested from mice (n 6 per group) either infected with e. coli or injected with saline . The plcs were stained with fluorescence - conjugated mabs against cd14, cd11b, and cd3 for 30 minutes at room temperature in the dark, followed by washing twice with pbs . The stained cells were sorted using bd facs aria ii by the methods described in figure 5(g). The neutrophils were sorted by selection for cd3cd14cd11b cells, and t cells were sorted by selection for cd3 cells, according to the manufacturer's recommendations . T cells (defined as cd3 cells with a purity> 83% of living cells) and pmns (defined as cd3cd14cd11b cells with a purity> 96.7% of living cells) were obtained . Total rna was isolated from the plcs with trizol reagent and reverse - transcribed using cdna synthesis kit . Quantitative real - time pcr (qrt - pcr) was performed using two - step sybr green qpcr assays and the target mrnas were identified by the specific primers as follows: il-17a, forward: 5-aaggcagcagcgatcatccct; reverse: 3-tcttcattgcggtggagagtcc; gapdh, forward: 5-atcaccatcttccaggagcga; reverse: 3-tctcgtggttcacacccatca . The data were acquired using the step one real - time pcr system (applied biosystems). The procedure of the target mrna amplification was as follows: 1 cycle at 95c (30 seconds) followed by 40 cycles at 95c (5 seconds) and 64c (31 seconds). The relative amount of gene expression was calculated according to the formula 2, in which ct = [ct(gene) ct(gapdh)] and ct is the crossing threshold value returned by the pcr instrument for every gene amplification . Survival curves of mice were estimated using the kaplan - meier method and compared using the log - rank test . We considered the resulting p values of less than 0.05 (95% ci) to be statistically significant . Statistics were analyzed using graphpad prism 5.0 for windows (san diego, ca). To understand how neutrophils contribute to the development of sepsis, we first established a murine model of septic peritonitis induced by escherichia coli (e. coli). The female icr mice, 10 in each group, were intraperitoneally injected with 2.4 10, 1.6 10, 0.8 10, and 0.4 10 colony forming units (cfus) of e. coli, respectively, and then monitored for their physical conditions and survival . Around 6 hours after the infection, all of the 10 mice received 2.4 10 cfus of e. coli, 8 out of the 10 mice received 1.6 10 cfus of e. coli, and 5 out of the 10 mice that received 0.8 10 cfus of e. coli began to exhibit multiple neurological symptoms, including dispirited behavior, staggered gait, and trembling, but these phenomena were not present in all of the 10 mice that received 0.4 10 cfus of e. coli or saline . Around 18 hours after the infection, the infected mice began to die . Around 48 hours, the infected mice underwent a massive death, especially in those that received high infectious dose of e. coli . By 72 hours after the infection, 80%, 60%, and 40% of the mice infected with 2.4, 1.6, and 0.8 10 cfus of e. coli, respectively, died and yet 100% of the mice which were infected with 0.4 10 cfus of e. coli or received saline survived (figure 1). The results indicated that the infection with e. coli at 1.6 10 cfus and 2.4 10 cfus could induce septic peritonitis in icr mice . Since the polymorphonuclear cells (pmns) are documented as the first innate immune cells recruited at inflammation sites and play a central role in host defense, we next collected the peritoneal lavage cells (plcs) of the mice at 18, 48, and 72 hours after infection, respectively, for observing the infiltration and morphology of the pmns in the plcs . The collected plcs were fixed on glass slides, stained with h&e dye, and photographed . A massive infiltration of pmns was observed in the plcs from the infected mice . At 18 hours after infection, in the plcs of the mice infected with 1.6 and 2.4 10 cfus of e. coli, the majority of the cells with the size of 0.2~0.4 micrometer were pmns with a typical lobulated nucleus . Interestingly, there occurred a portion of giant cells, with the size of nearly 1.0~1.6 micrometer, characterized by lobulated nucleus that was squeezed to the marginal zone of the inner cell membrane . However, at 48 or 72 hours, the giant cells disappeared in the plcs of the mice infected with 1.6 and 2.4 10 cfus of e. coli (figure 2(a)). Morphologically, there are four types of cells in the plcs, including pmns, macrophages, lymphocytes, and the giant cells (figure 2(b)). When counting the cells, we found that at 18, 48, and 72 hours, pmns constituted 50%80% and macrophages constituted 20%40% of the plcs from the mice infected with 4 doses of e. coli, respectively, compared with nearly 30% for pmns and 60% for macrophages in the plcs from the mice that received saline . At 18 hours, interestingly, there were about 20% of the giant cells in the plcs of the mice infected with 1.6 and 2.4 10 cfus of e. coli (figure 2(b), upper right). Noticeably, when the mice died of the infection, we collected the plcs immediately and observed and found that plenty of bacteria coexisted with the pmns in the plcs of the mouse infected with 1.6 10 cfus of e. coli for 31 hours (figure 2(c)). These results indicate that neutrophils can move to infected peritoneal cavity by infiltration and change their morphologies after engulfing bacteria possibly . During septic peritonitis, a large number of pmns were recruited to the peritoneal cavity of the infected mice . To find the correlation of the numbers of pmns at various time points with the severity of the disease in mice infected with e. coli, the plcs of the mice were collected at 18, 48, and 72 hours after infection, counted by hemocytometer or flow cytometry after staining with fluorescence - labeled mab of anti - cd45 and anti - cd11b . At 18 hours, the numbers of plcs were nearly 1 10 cells / ml of peritoneal lavage fluid (plf) in the mice received saline (control mice), significantly increased in the mice infected with 3 doses of e. coli in a dose dependent manner, and even reached nearly 3 10 cells / ml of plf in the mice infected with 2.4 10 cfus of e. coli (p <0.05). At 48 hours, the numbers of plcs were less than 1 10 cells / ml of plf in control mice and significantly increased to over 3 10 cells / ml of plf in the mice infected with three doses of e. coli and even to about 4 10 cells / ml of plf in the mice infected with 1.6 and 2.4 10 cfus of e. coli (p <0.05). At 72 hours, the numbers of plcs were decreased to 0.5 10 cells / ml of plf in control mice and decreased to nearly 0.5 10 cells / ml of plf in the mice infected with three doses of e. coli (figure 3(a)). In the plcs from the infected mice, most of the cells were cd45 nucleated leukocytes, representing pmns, macrophages, and lymphocytes, respectively . At 18 hours, the pmns, macrophages, and lymphocytes were displayed on the left, middle, and right of the histogram, respectively, sequentially based on their nuclear structure and fluorescence intensity . At 48 and 72 hours, the pmns were found to be shifted to the right upper quadrant, indicating that the pmns were with more ovulated nuclei and expressed elevated cd45 . As shown in figure 3(b), at 18 hours after infection, the cd45 pmns constituted 61.5%, 49.6%, and 56.4% of the plcs in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, compared with 32.5% in the control mice . At 48 hours, the cd45 pmns were increased to 59.5%, 63.7%, and 54.7% in the plcs from the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, compared with 41.2% from the control mice . At 72 hours, the cd45 pmns constituted 62.2%, 62.8%, and 63.5% of the plcs in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, compared with 39.5% from the control mice . The pmns were further detected as cd11bcd45 pmns and cd11bcd45 pmns, and their ratios in the pmns were calculated . As shown in figure 3(c), the cd11bcd45 pmns constituted 49%, 36%, and 13% of the pmns in the plcs from the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli for 18 hours, respectively . At 48 hours after the infection, the cd11bcd45 pmns constituted 91%, 88%, and 86% in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively . At 72 hours after the infection, the cd11b pmns reached around 96% in all infected mice . Oppositely, at 18 hours, the cd11b pmns constituted 51%, 64%, and 87% of the pmns in the plcs from the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively . At 48 hours after the infection, the cd11b pmns constituted 9%, 12%, and 14% in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively . At 72 hours after the infection, the cd11b pmns only accounted for around 4% in all infected mice (figure 3(c)). The results indicated that the peritoneal infection of e. coli induced massive infiltration of cd45 pmns . Among the cd45 pmns, cd11b pmns were increased with the progression of infection . In parallel, the cd11b pmns were decreased . At 18 and 48 hours after infection, the ratios of cd11b pmns or cd11b pmns were correlated with the doses of e. coli negatively or positively . Since primary human and mouse blood neutrophils were reported to be able to express a functional stlr9, we tried to detect whether the expression of stlr9 was different on cd11b pmns and cd11b pmns and correlates with development of septic peritonitis in mice . The plcs collected from the mice which were infected with e. coli or received saline were gated for detecting the expression of stlr9 on both cd11b and cd11b pmns (figure 4(a)). Results showed that, at 18 hours after infection, the ratios of stlr9cd11b pmns were increased to 57%, 51%, and 70% of the cd11b pmns in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively (p <0.05), compared to 12% in the mice that received saline control (control mice). At 48 hours, the ratios of stlr9cd11b pmns were decreased to 2.3%, 2.3%, (p <0.05) and 8% in the infected mice, respectively, and less than 13% as in control mice . At 72 hours, the ratios of stlr9cd11b pmns were 7%, 5.5%, and 9% in the infected mice, respectively, compared with 18% in control mice (figure 4(b)). The ratios of stlr9cd11b pmns constituted 52%, 53%, and 68.5% of the cd11b pmns in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, for 18 hours (p <0.05), compared with 3.5% in control mice, sharply decreased to 9%, 6%, and 9.5% in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, for 48 hours, compared with 8% in control mice, and reached 10%, 6%, and 10% in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, for 72 hours, compared with 8% in control mice (figure 4(c)). These results indicated that e. coli could significantly induce the occurrence of both of the stlr9cd11b pmns and stlr9cd11b pmns in the peritoneal cavity at early stage of infection . Considering the fact that macrophages are the inflammatory cells which overlapped with the pmns in the inflammatory sites, we also observed stlr9 expression on macrophages in the plcs . The results showed that the ratios of stlr9 macrophages constituted 5%, 6%, and 6.7% (p <0.05) of the macrophages in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, for 18 hours, compared with 3% in control mice, decreased to 0.9% (p <0.05), 0.45% (p <0.05), and 3% in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, for 48 hours, compared with 5% in control mice, and reached 4.8%, 2% (p <0.05), and 2.8% in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, for 72 hours, compared with 5.5% in control mice (figure 4(d)). These results indicated that, unlike pmns, the macrophages barely express increased stlr9 during septic peritonitis . In recent years, primary human and mouse neutrophils have been found to be able to display autocrine il-17 activity that probably contributes to the etiology of microbial and inflammatory diseases . To find whether the different subtype neutrophil derived il-17 was involved in the development of septic peritonitis, we detected il-17 expression in the cd11b pmns and cd11b pmns (figure 5(a)) of the plcs collected from the mice which were infected with e. coli or received saline (control mice). It was found that the ratios of il-17cd11b pmns constituted 7%, 8.5%, and 10% of the cd11b pmns in plcs of the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, for 18 hours (p <0.05), compared with 25% in control mice, was 2% (p <0.05), 7.5% (p <0.05), and 25.5% in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, for 48 hours, compared with 25% in control mice, and reached 6%, 10%, and 8% in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, for 72 hours (p <0.05), compared with 27% in control mice (figure 5(b)). Noticeably, the saline injection seemed to stimulate neutrophils to express il-17 . To confirm this, the plcs were collected from the mice injected with saline at 18, 48, or 72 hours after the injection, respectively, and stained with fluorescence - labeled mab against cd45, cd11b, and il-17, followed by detection using flow cytometry . We found that saline stimulation tended to induce il-17 expression (not statistically significant) with a big range of variation individually (data not shown). The ratios of the il-17cd11b pmns were 5%, 4%, and 3% of the cd11b pmns in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, for 18 hours, compared with 7% in control mice; 3%, 11%, and 21.5% in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, for 48 hours, compared with 7% in control mice; and 7%, 8%, and 13% in the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, for 72 hours, compared with 9% in control mice (figure 5(c)). The results indicated that both of the cd11b pmns and the cd11b pmns could not increase il-17 expression during the development of septic peritonitis in mice induced by e. coli . Also, as shown in figure 5(d), macrophages could not increase the il-17 expression in response to e. coli . Obviously, the results were in disagreement with the reports showing that pmns could increase the production of il-17 during bacterial infections . To validate the in vivo expression of il17 in the plcs, the plcs pooled from the nave mice were cocultured with e. coli at 1 10 cfus, 1 10 cfus or saline for 14 hours, and then with bfa for another 4 hours . The cultured cells were harvested, stained with fitc - labeled anti - il-17 mab, and detected by flow cytometry . As shown in figure 5(e), e. coli could significantly increase il-17 plcs of the nave mice, indicating that e. coli could stimulate the plcs to express il-17 . To clarify this, we set up an experiment to detect il-17 mrna expression in the plcs from the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli for 18 hours, respectively, or from the control mice and found that the il-17 mrna levels in the mice infected with 3 doses of e. coli were significantly elevated in a dose dependent manner (figure 5(f)). Next we sorted cd3 cells and cd3cd14cd11b cells from the plcs of the mice which were infected with e. coli or received saline for 18 hours, detected their il-17 mrna expression by qrt - pcr, and found that il-17 mrna levels were significantly increased in both of the cd3 t cells (p = 0.0006) and the cd3cd14cd11b cells (p = 0.0434) in the plcs from the e. coli infected mice . Comparatively, the il-17 mrna levels in the cd3 t cells were much higher than those in the cd3cd14cd11b neutrophils . The result indicated that both of t cells and pmns were the il-17 producers in the peritoneal cavity during the development of septic peritonitis (figure 5(g)). The result suggests that the plcs from the infected mice did express il-17 and il-17 in the pmns might be secreted out during the development of septic peritonitis . To find the possible correlation between the production of il-17 and the expression of stlr9 in / on the pmns and macrophages in peritoneal cavity of the mice during septic peritonitis, we harvested the plcs from the mice infected with 0.8, 1.6, and 2.4 10 cfus of e. coli, respectively, at early stage of 8 hours after infection and detected their expression of il-17 and stlr9 . The expression was represented by mean fluorescence intensity (mfi) to emphasize the increased expression of il-17 and stlr9 per cell . As shown in figure 6, il-17 expression was significantly upregulated in the cd11b pmns from the infected mice and their levels were positively correlated with the doses of e. coli (figure 6(a)), while the upregulated il-17 expression was only observed in the cd11b pmns or macrophages from the mice infected with 0.8 10 cfus and 1.6 10 cfus of e. coli . The highest dose of e. coli could not induce the highest levels of il-17 in the cd11b pmns or macrophages after infection for 8 hours (figures 6(b) and 6(c)). This result reveals that il-17 may be mainly derived from cd11b pmns and secrete out earlier during bacterial infection in mice infected with high dose of e. coli . In parallel, we also tested the expression of stlr9 on pmns and macrophages and found that stlr9 expression was significantly downregulated in the cd11b pmns from the infected mice and the lowest downregulation happened in the cd11b pmns from the mice infected with 2.4 10 cfus of e. coli (figure 6(d)). In contrast, stlr9 expression was significantly upregulated in the cd11b pmns from the infected mice (figure 6(e)). Interestingly, the significantly upregulated expression of stlr9 was observed also in the macrophages from the mice infected with 2.4 10 cfus of e. coli (figure 6(f)). To validate whether other types of cells in the plcs also expressed il-17 and stlr9, the plcs from mice infected with e. coli for 8 hours were stained with fluorescence - labeled mabs against ly6 g, cd11b, il-17, or tlr9 and then analyzed by flow cytometry . The results showed that e. coli infection significantly increased il-17 expressing cd11bly6 g neutrophils (figure 6(h)) as well as stlr9 expressing cd11bly6 g neutrophils (figure 6(g)) in a dose dependent manner but could not increase il-17 expression in the stlr9cd11bly6 g neutrophils (figure 6(i)) at the time point . In parallel, the plcs were stained with fluorescence - labeled mabs against cd3, cd11b, and il-17 and analyzed . It was found that both cd3 t cells (figure 6(j)) in the plcs and cd3cd11b pmns (figure 6(k)) increased their il-17 expression in response to the infection with e. coli for 8 hours . This result indicates that, at early stage, stlr9 may be mainly expressed on cd11b pmns in plcs of the mice infected with e. coli, and with the infection progression, macrophages may become successors to replace the cd11b pmns as the major stlr9 cells . In this study, we tried to investigate the correlation of tlr9 expression with il-17 production in pmns during septic peritonitis and found that both stlr9 and il-7 could be expressed in the pmns infiltrated into the peritoneal cavity of the mice infected with e. coli . At early stage of the infection, stlr9 was increasingly expressed in the infiltrated cd11b pmns, and il-17 was increasingly expressed in both of the cd11b pmns and cd11b pmns . Il-17 expression in cd11b pmns was positively correlated with the doses of e. coli . When infected with the highest dose of e. coli (2.4 10 cfus), il-17 was increasingly expressed and stlr9 was decreasingly expressed in / on the cd11b pmn . When infected with the lowest dose of e. coli (0.8 10 cfus), both il-17 and stlr9 furthermore, we stained the plcs with both cd11b mab and ly6 g mab and confirmed that the cd11bly6 g pmns in the plcs could increase their expression of both il-17 and stlr9 in response to e. coli infection . Taken together, with the fact that highest dose of e. coli (2.4 10 cfus) was the most deadly and the lowest dose of e. coli was the least deadly to the mice with septic peritonitis, we may deduce that increased expression of both stlr9 and il-17 in cd11b pmns might benefit the survival of the mice with septic peritonitis and that the decreased expression of stlr9 and increased expression of il-17 in cd11b pmns may be detrimental to the mice . In recent years, pmns have been found to be able to express stlr9 which engage pamps, such as bacterial dna generated during infection, and damage - associated molecular patterns (damps), such as mitochondrial dna released from necrotic cells during sterile inflammation . The engagement, if intense, can prime pmns to release massively produced cytokines, leading to sepsis or systemic inflammatory response syndrome (sirs). In the present work, we found that the ratios of the stlr9cd11b pmns and stlr9cd11b pmns were significantly increased in the infiltrated cd11b pmns and cd11b pmns of the mice infected with e. coli for 18 hours, respectively . At this stage, we observed that a plenty of e. coli coexisted with the pmns . When observed at 48 hours and 72 hours after infection, the ratios of both of the stlr9cd11b pmns and stlr9cd11b pmns were sharply decreased to the levels as those in saline control . Possibly, the increased tlr9cd11b pmns and stlr9cd11b pmns present a rapid innate immune response of the pmns to bacterial invasion at the early stage of infection . Pmns armed with stlr9 were demonstrated to be able to sense extracellular ligands and consequently initiate tlr9 mediated signaling in an intracellular tlr9 independent way . The response could offer a rescue mechanism for pmn activation when pathogen derived tlr9 ligands cannot reach the endosome in the early stage of infection . Practically, the increased expression of stlr9 on pmns may be a proinflammatory activation marker and stlr9 pmn should benefit antibacterial defense during infection, evidenced by the following: (1) tlr9 agonists, if they exist, could activate stlr9 pmn, not tlr9-deficient pmns, to upregulate cd11b and secrete mip-2 and il-8 (cxcl8); (2) stlr9 pmns are involved in inducing the rapid inflammation which is needed in the initial phase of bacterial infection by secreting antimicrobial peptides and elastases; (3) encountering microbial dna, stlr9 signaling is able to activate pmns, and the activated pmn increases stlr9 expression . However, it is worthy to note that stlr9 pmns could be culprit for causing more severe pathological consequences . For instance, bacterial dna or formulated peptides released following sepsis were reported to activate p38 map kinase through binding stlr9 on pmns, leading to acute lung injury which is characterized by protein - rich pulmonary oedema (swelling) and accumulation of large numbers of pmns in the lungs . It is well established that pmns, as the firstly recruited inflammatory cells in infection sites, are il-17 producing cells . Il-17 secreted from pmns induces the release of proinflammatory factors from mesenchymal and myeloid cells, recruiting additional pmns . In addition to pmns, macrophages located in the epithelial barriers are also important sources of il-17 . In this study, we initially found that the average ratios of the il17cd11b pmns and il17 macrophages were significantly decreased in the plcs at 18, 48, and 72 hours in the mice infected with 3 doses of e. coli . Seemingly, the cd11b pmns and macrophages, in response to the peritoneal infection, could not produce increased il-17 . However, when checking mrna expression, we found significantly upregulated il-17 mrna in the plcs from the mice infected with e. coli for 18 hours (figure 5(e)). The data imply that the infiltrated cells in peritoneal cavity of the mice infected with e. coli could produce il-17 and the produced il-17 may promptly secrete out the cells, therefore resulting in the decreased ratios of il17cd11b pmns and il17 macrophages . To clarify this, we harvested the plcs from the mice infected with e. coli at early stage of 8 hours after infection and detected their il-17 levels which were expressed as mean fluorescence intensity (mfi). By doing this, we found that both of the cd11b pmns and cd11b pmns obviously produced il-17 in the early stage of the peritoneal infection caused by e. coli . To confirm this, we set up another experiment in which the plcs were harvested at 12 and 16 hours after the infection, cultured with bfa, a protein transport inhibitor capable of retaining the produced cytokines inside the cells, for additional 4 hours, and then detected their il-17 expression . We found that the bfa in vitro treatment significantly increased the average ratios of the il17cd11b pmns in cd11b pmns and il17 macrophages in macrophages (data not shown). The results support the assumption that produced il-17 in cd11b pmns and macrophages could be secreted out promptly in response to e. coli at the early stage of septic peritonitis in mice . Theoretically, deducing based on the published data, il-17 derived from cd11b pmns and macrophages may initiate a response to induce chemokines which in turn recruit more pmns [23, 24] to the site of infection [18, 25]. The new comer pmns join the fighting against invaded bacteria, resulting in more il-17 [16, 17]. In addition to the pmns, macrophages could be recruited by il-17 [20, 26], specifically by cd11b pmns derived il-17 . The increased macrophages could produce more il-17 to join the cd11b pmns produced il-17, playing a defense role in fighting bacterial infection and reducing mortality of mice with septic peritonitis . In addition to the pmns and macrophages, t cells were found as a major producer of il-17 in the mice with experimental sepsis induced by clp; t cell - derived il-17 could promote production of proinflammatory mediators, resulting in enhanced lethality . Compatibly, t cells, in this study, were confirmed to be involved in the development of septic peritonitis in mice by the evidence that (1) the il-17 expression was increased in the cd3 cells of the plcs from the mice infected with e. coli and (2) il-17 mrna levels were significantly increased in the sorted cd3 cells in the plcs from the mice infected with e. coli . Furthermore, il-17 expression was obviously increased in the sorted cd3cd11b pmns in the plcs from the mice infected with e. coli . Together, these data suggest that the pmns, like t cells, do express il-17 as a response to bacterial infection during septic peritonitis . Although cd3 cells were one of the major producers of il-17 during the development of septic peritonitis, probably, neutrophils might produce more il-17 compared to cd3 cells because the neutrophils, as we found in this study, could constitute up to 70% of the plcs (figure 3(b)), whereas cd3 cells only constituted 1015% of the plcs (data not shown). Interestingly, stlr9cd11bly6 g pmns, not stlr9cd11bly6 g pmns, were found to be able to increase their il-17 expression during septic peritonitis . Interestingly, we found a type of giant cells with increasing numbers in the plcs from the mice infected with e. coli up to 1.6 10 cfus . Morphologically, the giant cells (figure 2(a)) could be the pmns with a changed lobulated nucleus located near to cell membrane, being at least 4 times larger than the regular pmns . Obviously, the giant cells are the newly described cells and have several facets worthy of note . (1) kinetically, the giant cells only appeared at the early stage of the infection and were not observed in the plcs of the mice infected for 48 and 72 hours . (2) the giant cells only occurred in the plcs of the severely infected mice, not in the plcs of the mice infected with less amount (0.40.8 10 cfus) of e. coli . Possibly, the giant cells were developed from the regular pmns after engulfing a large amount of bacterial pathogens . Biologically, the appearance of the giant cells could be used as a cell marker for signifying a real danger because the giant cells were intimately correlated with the severity of the infection and the death of the mice.
Vegf and bfgf contents in various subtypes of grade i meningiomas vegf (upper panel) and bfgf (lower panel) contents were determined by elisa . N denotes the number of patients . For transitional meningiomas: vegf range [0.259.85] and bfgf range [67.4576.9]; for microcystic meningiomas: vegf range [0.715.29] and bfgf range [164.3520.0]; for meningothelial meningiomas: vegf range [0.874.98] and bfgf range [183.6573.7]; for fibroblastic meningiomas: vegf range [0.101.69] and bfgf range [142.1377.3]. * no statistical differences were observed between groups (mann - whitney u test).
The study base was the participants of the ongoing tsuruoka metabolomic cohort study, initiated in april 2012 (yamagata prefecture, japan), comprising individuals aged 35 to 74 years who were recruited among attendees of annual municipal or worksite health check - up programs in the city . A total of 1,422 women consented to participate during the first few months of the baseline period, and plasma metabolomic profiling in these women was completed by the end of 2014 . Only postmenopausal female participants were ultimately included in the analysis due to the small number of premenopausal women . Because there are known metabolic differences compared with natural menopause, participants with surgical or medical menopause were excluded . In addition, women who had used hormone therapy within 1 year or had undergone oophorectomy after menopause were excluded to eliminate the possible effect on the metabolic profile of administered estrogen and hormones excreted from postmenopausal ovaries . The study was approved by the medical ethics committee of the school of medicine, keio university, tokyo, japan (approval no . All data and samples were obtained in the baseline study, including anthropometrics, clinical biochemistry, and blood specimens for metabolomic profiling . Detailed information was collected through a standardized self - administered questionnaire on medical history, including treatment of hypertension, dyslipidemia, and diabetes; gynecological and reproductive history, including menopause status (pre / peri / post), ages at menarche and menopause, reason for menopause (natural / medical / others), number of pregnancies and deliveries, age at first delivery, use of hormone therapy (if yes, number of years used and when it was used last), and whether oophorectomy was ever performed; and lifestyle parameters such as smoking habits, alcohol intake, diet, stress, and physical activity . Wc was measured to the nearest 0.1 cm at the umbilicus at the end of a normal breath . If the umbilicus drooped down, the measurement was made midway between the inferior margin of the last rib and the top of the iliac crest in a horizontal plane . Bp was measured twice on one occasion in the sitting position using an automated sphygmomanometer (omron hbp - t105s - n), and the mean of the two measurements was used for analysis . Blood samples were collected in the morning between 8:30 and 10:30 after overnight fasting to avoid variation due to fasting and circadian rhythm . Plasma samples were collected with ethylenediaminetetraacetic acid-2na as an anticoagulant and kept at 4c immediately after collection . The samples were centrifuged for 15 minutes (1,500 g at 4c) within 1 hour of collection, divided into aliquots, and kept for a maximum of 6 hours at 4c until extraction of metabolites . Serum samples were collected with serum - separating medium and kept at room temperature after collection . Serum levels of total cholesterol, tg, and fasting plasma glucose were analyzed using enzymatic methods, and glycated hemoglobin (hba1c) was determined by immunoassay . Mets was defined using the modified national cholesterol education program adult treatment panel iii definition with 100 mg / dl as the cutoff for the glucose level . Because lower overall adiposity has been associated with an increased risk of medical conditions such as type 2 diabetes in asian countries, the threshold for wc was set according to the recommendation for asians established by several organizations, including the national cholesterol education program . Specifically, central obesity was defined as wc at least 80 cm, high bp as mean systolic / diastolic bp at least 130/85 mm hg or currently on antihypertensive therapy, high serum tg as at least 1.7 mmol / l (150 mg / dl), low hdl - c as less than 1.3 mmol / l (50 mg / dl), and high fasting plasma glucose as at least 5.5 mmol / l (100 mg / dl) or current use of antidiabetic medication . Nontargeted mass spectrometry - based metabolomic profiling was performed with fasting plasma samples via capillary electrophoresis time - of - flight mass spectrometry . Metabolite extraction from plasma was completed within 6 hours after collection to minimize the effect of metabolic changes in plasma . Capillary electrophoresis time - of - flight mass spectrometry analysis of cationic and anionic metabolites was performed as described previously . Raw data were processed using our proprietary software (masterhands). As a preliminary study, we identified 290 metabolite peaks (131 cations and 159 anions) in plasma: 154 known with standard compounds and 136 unknown . We decided a priori to measure absolute concentrations of 115 metabolites (63 cations and 52 anions) that were expected to be observed stably in most human plasma samples and matched with standard compounds . To monitor the stability of metabolome analysis, quality control samples were injected every 10 samples and assessed at the start of the analytical run and at intervals throughout the analysis . The technical variability and between - subject variability for each metabolite assayed is shown in supplementary table 1 . Participants were randomly allocated into two groups (2:1) to create two independent original and replication populations . Analyses were first performed in the original population, and then the same analyses were performed in the replication population to confirm the robustness of the study outcome . The two groups were stratified for age and batch number to eliminate any confounding effects of age and analytical variability between multiple batches . Out of 115 metabolites, 37 had plasma levels below the assay limits of detection (lod) in more than 90% of the participants, and therefore were excluded from further analyses . The remaining 78 metabolites were analyzed . For samples with levels below lod, which comprised less than 1% of all samples, the distribution of each metabolite concentration was tested for normality using quantile - quantile plots . On the basis of the shape of the distribution, most of the metabolite concentrations were log - transformed . The characteristics of participants with and without mets were compared by t test and test for continuous and categorical variables, respectively . Age - adjusted spearman correlation coefficients were calculated for each metabolite pair in the original population . Linear regression analysis was performed between the mets and non - mets groups with each metabolite concentration used as the outcome to examine the association between plasma metabolite concentrations and mets . Analysis of covariance was performed to adjust for possible confounders using age, ldl - c levels, current smoking status, current alcohol consumption, physical activity, and dietary intake level . We calculated p values using the benjamini and hochberg false discovery rate method (= 0.05) for analysis of the original population to adjust for independent multiple comparisons . Sensitivity analyses were performed by excluding (1) women with bmi at least 25 kg / m and (2) those taking medications for hypertension, dyslipidemia, and diabetes . Both sensitivity and replication analyses were conducted using the metabolites shown to be significantly associated with mets in the original population (unadjusted false discovery rate p <0.05). Sas 9.3 (sas institute inc, cary, nc) was used for all analyses . The study base was the participants of the ongoing tsuruoka metabolomic cohort study, initiated in april 2012 (yamagata prefecture, japan), comprising individuals aged 35 to 74 years who were recruited among attendees of annual municipal or worksite health check - up programs in the city . A total of 1,422 women consented to participate during the first few months of the baseline period, and plasma metabolomic profiling in these women was completed by the end of 2014 . Only postmenopausal female participants were ultimately included in the analysis due to the small number of premenopausal women . Because there are known metabolic differences compared with natural menopause, participants with surgical or medical menopause were excluded . In addition, women who had used hormone therapy within 1 year or had undergone oophorectomy after menopause were excluded to eliminate the possible effect on the metabolic profile of administered estrogen and hormones excreted from postmenopausal ovaries . The study was approved by the medical ethics committee of the school of medicine, keio university, tokyo, japan (approval no . All data and samples were obtained in the baseline study, including anthropometrics, clinical biochemistry, and blood specimens for metabolomic profiling . Detailed information was collected through a standardized self - administered questionnaire on medical history, including treatment of hypertension, dyslipidemia, and diabetes; gynecological and reproductive history, including menopause status (pre / peri / post), ages at menarche and menopause, reason for menopause (natural / medical / others), number of pregnancies and deliveries, age at first delivery, use of hormone therapy (if yes, number of years used and when it was used last), and whether oophorectomy was ever performed; and lifestyle parameters such as smoking habits, alcohol intake, diet, stress, and physical activity . Wc was measured to the nearest 0.1 cm at the umbilicus at the end of a normal breath . If the umbilicus drooped down, the measurement was made midway between the inferior margin of the last rib and the top of the iliac crest in a horizontal plane . Bp was measured twice on one occasion in the sitting position using an automated sphygmomanometer (omron hbp - t105s - n), and the mean of the two measurements was used for analysis . Blood samples were collected in the morning between 8:30 and 10:30 after overnight fasting to avoid variation due to fasting and circadian rhythm . Plasma samples were collected with ethylenediaminetetraacetic acid-2na as an anticoagulant and kept at 4c immediately after collection . The samples were centrifuged for 15 minutes (1,500 g at 4c) within 1 hour of collection, divided into aliquots, and kept for a maximum of 6 hours at 4c until extraction of metabolites . Serum samples were collected with serum - separating medium and kept at room temperature after collection . Serum levels of total cholesterol, tg, and fasting plasma glucose were analyzed using enzymatic methods, and glycated hemoglobin (hba1c) was determined by immunoassay . Mets was defined using the modified national cholesterol education program adult treatment panel iii definition with 100 mg / dl as the cutoff for the glucose level . Because lower overall adiposity has been associated with an increased risk of medical conditions such as type 2 diabetes in asian countries, the threshold for wc was set according to the recommendation for asians established by several organizations, including the national cholesterol education program . Specifically, central obesity was defined as wc at least 80 cm, high bp as mean systolic / diastolic bp at least 130/85 mm hg or currently on antihypertensive therapy, high serum tg as at least 1.7 mmol / l (150 mg / dl), low hdl - c as less than 1.3 mmol / l (50 mg / dl), and high fasting plasma glucose as at least 5.5 mmol / l (100 mg / dl) or current use of antidiabetic medication . Nontargeted mass spectrometry - based metabolomic profiling was performed with fasting plasma samples via capillary electrophoresis time - of - flight mass spectrometry . Metabolite extraction from plasma was completed within 6 hours after collection to minimize the effect of metabolic changes in plasma . Capillary electrophoresis time - of - flight mass spectrometry analysis of cationic and anionic metabolites was performed as described previously . Raw data were processed using our proprietary software (masterhands). As a preliminary study, we identified 290 metabolite peaks (131 cations and 159 anions) in plasma: 154 known with standard compounds and 136 unknown . We decided a priori to measure absolute concentrations of 115 metabolites (63 cations and 52 anions) that were expected to be observed stably in most human plasma samples and matched with standard compounds . To monitor the stability of metabolome analysis, quality control samples were injected every 10 samples and assessed at the start of the analytical run and at intervals throughout the analysis . The technical variability and between - subject variability for each metabolite assayed is shown in supplementary table 1 . Participants were randomly allocated into two groups (2:1) to create two independent original and replication populations . Analyses were first performed in the original population, and then the same analyses were performed in the replication population to confirm the robustness of the study outcome . The two groups were stratified for age and batch number to eliminate any confounding effects of age and analytical variability between multiple batches . Out of 115 metabolites, 37 had plasma levels below the assay limits of detection (lod) in more than 90% of the participants, and therefore were excluded from further analyses . The remaining 78 metabolites were analyzed . For samples with levels below lod, which comprised less than 1% of all samples, the distribution of each metabolite concentration was tested for normality using quantile - quantile plots . On the basis of the shape of the distribution, most of the metabolite concentrations were log - transformed . The characteristics of participants with and without mets were compared by t test and test for continuous and categorical variables, respectively . Age - adjusted spearman correlation coefficients were calculated for each metabolite pair in the original population . Linear regression analysis was performed between the mets and non - mets groups with each metabolite concentration used as the outcome to examine the association between plasma metabolite concentrations and mets . Analysis of covariance was performed to adjust for possible confounders using age, ldl - c levels, current smoking status, current alcohol consumption, physical activity, and dietary intake level . We calculated p values using the benjamini and hochberg false discovery rate method (= 0.05) for analysis of the original population to adjust for independent multiple comparisons . Sensitivity analyses were performed by excluding (1) women with bmi at least 25 kg / m and (2) those taking medications for hypertension, dyslipidemia, and diabetes . Both sensitivity and replication analyses were conducted using the metabolites shown to be significantly associated with mets in the original population (unadjusted false discovery rate p <0.05). Sas 9.3 (sas institute inc, cary, nc) was used for all analyses . The final data set included 877 participants, with 594 women in the original population and 283 in the replication population . In the original population, 149 women were diagnosed with mets and 445 women did not meet the criteria (non - mets). In the replication population, 72 women were diagnosed with mets and 211 were free of mets . The ratio of mets to non - mets participants was similar in the two populations (1:3). Women with mets were older than those without mets (65.5 vs 64.1 y, p = 0.004), and had higher tg (111.0 vs 76.0 mg / dl) and lower hdl - c (64.8 vs 75.3 mg / dl) levels . There was no significant difference in serum ldl - c levels (126.1 vs 124.9 mg / dl) or in lifestyle characteristics (current smoking, current alcohol consumption, calorie intake, and physical activity) between the two groups . Most characteristics of the replication population were similar to those of the original population (data not shown). The only discrepancy between the two populations was for alcohol intake, with significantly fewer women with mets having current alcohol intake in the replication population (7.0% vs 51.0%, p = 0.01). High correlations within groups of related metabolites were observed for amino acids and their metabolites, and for intermediates of the tricarboxylic acid (tca) cycle, suggesting the importance of these metabolites in revealing the network of biochemical reactions in humans (fig . 2). We discovered a total of 19 metabolites with plasma concentrations that differed significantly between mets and non - mets in the original population, of which 11 remained significant after adjusting for possible confounders (table 2). Plasma levels of branched - chain amino acids (bcaas) and their derivatives were 7% to 10% higher in mets . Levels of phenylalanine and tyrosine tended to be slightly higher in mets, but significance disappeared after adjustments . Other metabolites that showed significantly higher concentrations in mets included alanine, glutamate, alpha - aminoadipate, cystine, and proline . A suggestive positive trend could also be seen for lactate and pyruvate (p = 0.06 and 0.07, respectively). 3-hydroxybutyrate levels were 18% lower in the mets group compared with the non - mets group . Correlation matrix for plasma metabolite concentrations in the original population (n = 594). Age - adjusted spearman coefficients were calculated for each pair of metabolite levels in the original population . Out of the 19 metabolites, 16 (84.2%) could be replicated (table 2), including those related to bcaa metabolism, aromatic amino acid metabolism, alanine, aspartate and glutamate metabolism, glucose - alanine cycle, lysine metabolism, proline and arginine metabolism, and the tca cycle . Among these comparisons of the adjusted means of the 13 metabolites between non - mets and mets in the original population are shown in figure 3 . Sensitivity analyses excluding (1) participants with bmi at least 25 kg / m and (2) those taking medications gave similar results (data not shown). The adjusted mean concentration of each metabolite was calculated using the fully adjusted model of the original population . The final data set included 877 participants, with 594 women in the original population and 283 in the replication population . In the original population, 149 women were diagnosed with mets and 445 women did not meet the criteria (non - mets). In the replication population, 72 women were diagnosed with mets and 211 were free of mets . The ratio of mets to non - mets participants was similar in the two populations (1:3). Women with mets were older than those without mets (65.5 vs 64.1 y, p = 0.004), and had higher tg (111.0 vs 76.0 mg / dl) and lower hdl - c (64.8 vs 75.3 mg / dl) levels . There was no significant difference in serum ldl - c levels (126.1 vs 124.9 mg / dl) or in lifestyle characteristics (current smoking, current alcohol consumption, calorie intake, and physical activity) between the two groups . Most characteristics of the replication population were similar to those of the original population (data not shown). The only discrepancy between the two populations was for alcohol intake, with significantly fewer women with mets having current alcohol intake in the replication population (7.0% vs 51.0%, p = 0.01). High correlations within groups of related metabolites were observed for amino acids and their metabolites, and for intermediates of the tricarboxylic acid (tca) cycle, suggesting the importance of these metabolites in revealing the network of biochemical reactions in humans (fig . 2). We discovered a total of 19 metabolites with plasma concentrations that differed significantly between mets and non - mets in the original population, of which 11 remained significant after adjusting for possible confounders (table 2). Plasma levels of branched - chain amino acids (bcaas) and their derivatives were 7% to 10% higher in mets . Levels of phenylalanine and tyrosine tended to be slightly higher in mets, but significance disappeared after adjustments . Other metabolites that showed significantly higher concentrations in mets included alanine, glutamate, alpha - aminoadipate, cystine, and proline . A suggestive positive trend could also be seen for lactate and pyruvate (p = 0.06 and 0.07, respectively). 3-hydroxybutyrate levels were 18% lower in the mets group compared with the non - mets group . Correlation matrix for plasma metabolite concentrations in the original population (n = 594). Age - adjusted spearman coefficients were calculated for each pair of metabolite levels in the original population . Bcaa, branched - chain amino acid; tca, tricarboxylic acid . Out of the 19 metabolites, 16 (84.2%) could be replicated (table 2), including those related to bcaa metabolism, aromatic amino acid metabolism, alanine, aspartate and glutamate metabolism, glucose - alanine cycle, lysine metabolism, proline and arginine metabolism, and the tca cycle . Among these comparisons of the adjusted means of the 13 metabolites between non - mets and mets in the original population are shown in figure 3 . Sensitivity analyses excluding (1) participants with bmi at least 25 kg / m and (2) those taking medications gave similar results (data not shown). The adjusted mean concentration of each metabolite was calculated using the fully adjusted model of the original population . To the best of our knowledge, this is the first population - based cross - sectional study to report associations between polar metabolites and mets in postmenopausal women in a lean asian population (average bmi 23 kg / m) with average hdl - c levels of 72.1 mg / dl . Our data indicate that alterations in amino acid metabolism are present in both obese individuals and in women with normal bmi with mets after menopause . 4-methyl-2-oxopentaonate and 2-oxoisopentanoate, which are degradation products of bcaas, were significantly elevated in mets, in addition to bcaas themselves . The significant increase of bcaas in plasma in mets is consistent with many prior experimental and physiological studies . In a study of lean and obese individuals, bcaas accounted for the largest amount of variance in principal components analysis of metabolite data, along with derivatives from oxidation products of bcaas . This increase in levels of downstream metabolites indicates that the associations with mets are related to an alteration in the flux of bcaas through this catabolic pathway . A leading theory of the mechanism behind this physiological change is suppression of bcaa catabolism under insulin resistance . Diabetic rats and humans have significantly lower levels of branched - chain keto acid dehydrogenase (bckdh) enzyme activity, which is the rate - limiting step in overall catabolism of bcaas . Several theories have been proposed for the mechanisms underlying suppressed bckdh activity, including increased activity of bckdh kinase that inactivates bckdh, and reduction of adiponectin signaling via an adenosine monophosphate - activated protein kinase 2-dependent pathway . The increase of bcaas has also been explained based on amino acid breakdown products being released into blood because insulin resistance causes excess protein breakdown in skeletal muscle . A causal role for bcaas in metabolic disease via the mammalian target of rapamycin pathway or inhibition of glucose transport / phosphorylation has also been suggested . Whether the increase in bcaas reflects the consequence of diabetes pathogenesis or has a causal role in disease development has yet to be proven because many large epidemiological studies have had a cross - sectional or case - control design . Further work is needed to determine the mechanisms through which increased amino acid levels might contribute to metabolic diseases . In our study, alpha - aminoadipate levels were 6.4% to 17.6% higher in plasma of women with mets compared with those without mets . Aminoadipate was found to be the strongest biomarker of diabetes risk out of 70 metabolites screened in the framingham heart study, in which participants with the highest quartile of plasma aminoadipate had fourfold higher odds of developing diabetes over a 12-year follow - up period compared with those in the lowest quartile . There were no correlations between aminoadipate and bcaas . Because aminoadipate is generated by lysine degradation and may also serve as a substrate for enzymes downstream of tryptophan metabolism, the current and previous findings collectively suggest that the mechanism behind mets and insulin resistance involves alterations in these metabolic pathways, distinct from pathways of bcaas . 3-hydroxybutyrate, a ketone body, was significantly lower in mets compared with non - mets participants (5.7%-28.4%). This is in contrast to previous studies showing that alpha - hydroxybutyrate is a positive predictor of type 2 diabetes . Alpha - hydroxybutyrate is generated in the liver under increased oxidative stress; therefore, the different results and the suggestive trends in lactate and pyruvate concentrations may be indicative of down- and up - regulation of ketogenesis and its relationship to the tca cycle . Another hypothesis is that the phenotype of mets in our population differs from those in previous studies, especially with respect to the very high levels of hdl - c . A recent review article on bcaas in insulin resistance described two types of obesity in rodents with regard to impairment of bcaa metabolism . Similarly, the alpha - hydroxybutyrate levels might be regulated in a more complex manner . One of the strengths of our study is the characteristics of the participants . To our knowledge, metabolomic profiling of mets on a population of lean, older females has not been performed previously . The only study in women with normal bmi (average 24.4 kg / m) was reported by wrtz et al; however, the average age of the participants was 32.1 years . Metabolism changes significantly with age and data should be carefully interpreted, even with adjustment of statistical models for age . In addition, the average hdl - c level of our participants was much higher than those in other populations . Hdl - c levels are indeed high among japanese people in general, and have increased in the past few decades . As one of the components of mets, decreased hdl - c is an independent risk factor for cvd, and the tg / hdl - c ratio has a positive correlation with the insulin resistance index . Our data suggest that alterations in plasma amino acids may precede the reduction of hdl - c levels in the development of insulin resistance . The study has several limitations . When interpreting metabolic profiling data in a cross - sectional study, the temporality of cause - effect relationships is not assured and possible confounding of unmeasured factors may exist, even after adjustments . Equally of concern is that our data were derived from a single site, lacking test marker performance in an independent cohort . Therefore, follow - up and intervention studies along with those at different facilities are needed . Comparisons between pre- and postmenopausal women could not be performed because of the few premenopausal women enrolled in the study . Completion of a separate ongoing cohort with a working age population is awaited to investigate the impact of menopause on these metabolic changes . Other common sites for wc measurements include the midpoint between the lowest rib and the iliac crest; narrowest or widest wc; just below the lowest rib; and just above the iliac crest . However, there is no consensus on the optimal protocol for measurement of wc . An effort was made to eliminate conditions that could affect the metabolic profile, such as history of cancer and nonfasting status, but multiple drugs for treated participants may have acted as confounders . Various factors might also have influenced measurement variability in the metabolomics analysis, potentially causing nondifferential misclassification of metabolomics data . To minimize these variabilities, we set a uniform fasting condition for participants and standardized the quality control procedures for metabolomics analysis . This study shows that japanese postmenopausal women who develop mets may have elevated concentrations of multiple amino acids, including bcaas, alanine, glutamate, lysine, proline, and other polar metabolites such as alpha - aminoadipate . These findings may not be translatable to populations of non - japanese women without very high hdl - c levels, but identifying these metabolic changes may be useful for detection of high - risk individuals, including those with normal bmi and relatively high hdl - c levels . A follow - up survey is needed to examine whether these candidate metabolites provide better prediction than standard measures for future development of mets.
From may 2007 to february 2009, all of 132 patients with refractory epilepsy who were referred to ayatollah kashani comprehensive epilepsy program, isfahan university of medical sciences (isfahan, iran), were evaluated to establish tle based on eeg and mri findings . The inclusion criteria were age between 15 and 40, full scale intelligence quotient (fsiq) more than 70, at least elementary school education and absence of major mental or neurological disorders except for epilepsy . The exclusion criteria were informed consent withdrawal and exacerbation of seizures that led to invalid neuropsychological test performance . Control subjects who were matched for age and education, were selected from the patients accompanying persons . Finally, 29 right tle, 31 left tle, and 32 control subjects were recruited in this study . This checklist included questions about age, education, seizure duration, handedness and marital status . The wms - iii was used to assess auditory and visual declarative memory and working abilities in adults and adolescents . Primary subtests must be given to obtain index scores and optional subtests can be given to obtain supplementary information . In this study, primary subtests were used including logical memory i and ii, face i and ii, verbal paired associates i and ii, family picture i and ii, letter - number sequencing and spatial span . The index scores are obtained by summing these primary subtests.33 one study in iran revealed internal consistency of 0.65 to 0.85 for wms - iii subtests and 0.76 to 0.83 for wms - iii indexes by cronbach's alpha coefficient.34 wms - iii was administered by a student of phd in psychology . Measures of full scale, verbal, and performance intelligence quotient (iq) were obtained using wechsler adult intelligence scale - r (wais - r). In one iranian study, reliability and validity of wais - r were studied.35 the wais - r subscales showed reliability from 0.69 to 0.87 on test retest stability and their internal consistency was 0.77 to 0.88 with split - half coefficient.36 wais - r was administered by a student of phd in psychology . All data were compared between patients (right tle and left tle) and controls . The multivariate analyses of variance (manova) was used to analyze the data of wms - iii and wais - r between groups . From may 2007 to february 2009, all of 132 patients with refractory epilepsy who were referred to ayatollah kashani comprehensive epilepsy program, isfahan university of medical sciences (isfahan, iran), were evaluated to establish tle based on eeg and mri findings . The inclusion criteria were age between 15 and 40, full scale intelligence quotient (fsiq) more than 70, at least elementary school education and absence of major mental or neurological disorders except for epilepsy . The exclusion criteria were informed consent withdrawal and exacerbation of seizures that led to invalid neuropsychological test performance . Control subjects who were matched for age and education, were selected from the patients accompanying persons . Finally, 29 right tle, 31 left tle, and 32 control subjects were recruited in this study . This checklist included questions about age, education, seizure duration, handedness and marital status . The wms - iii was used to assess auditory and visual declarative memory and working abilities in adults and adolescents . Primary subtests must be given to obtain index scores and optional subtests can be given to obtain supplementary information . In this study, primary subtests were used including logical memory i and ii, face i and ii, verbal paired associates i and ii, family picture i and ii, letter - number sequencing and spatial span . The index scores are obtained by summing these primary subtests.33 one study in iran revealed internal consistency of 0.65 to 0.85 for wms - iii subtests and 0.76 to 0.83 for wms - iii indexes by cronbach's alpha coefficient.34 wms - iii was administered by a student of phd in psychology . Measures of full scale, verbal, and performance intelligence quotient (iq) were obtained using wechsler adult intelligence scale - r (wais - r). In one iranian study, reliability and validity of wais - r were studied.35 the wais - r subscales showed reliability from 0.69 to 0.87 on test retest stability and their internal consistency was 0.77 to 0.88 with split - half coefficient.36 wais - r was administered by a student of phd in psychology . All data were compared between patients (right tle and left tle) and controls . The multivariate analyses of variance (manova) was used to analyze the data of wms - iii and wais - r between groups . From may 2007 to february 2009, all of 132 patients with refractory epilepsy who were referred to ayatollah kashani comprehensive epilepsy program, isfahan university of medical sciences (isfahan, iran), were evaluated to establish tle based on eeg and mri findings . The inclusion criteria were age between 15 and 40, full scale intelligence quotient (fsiq) more than 70, at least elementary school education and absence of major mental or neurological disorders except for epilepsy . The exclusion criteria were informed consent withdrawal and exacerbation of seizures that led to invalid neuropsychological test performance . Control subjects who were matched for age and education, were selected from the patients accompanying persons . Finally, 29 right tle, 31 left tle, and 32 control subjects were recruited in this study . This checklist included questions about age, education, seizure duration, handedness and marital status . The wms - iii was used to assess auditory and visual declarative memory and working abilities in adults and adolescents . Primary subtests must be given to obtain index scores and optional subtests can be given to obtain supplementary information . In this study, primary subtests were used including logical memory i and ii, face i and ii, verbal paired associates i and ii, family picture i and ii, letter - number sequencing and spatial span . The index scores are obtained by summing these primary subtests.33 one study in iran revealed internal consistency of 0.65 to 0.85 for wms - iii subtests and 0.76 to 0.83 for wms - iii indexes by cronbach's alpha coefficient.34 wms - iii was administered by a student of phd in psychology . Measures of full scale, verbal, and performance intelligence quotient (iq) were obtained using wechsler adult intelligence scale - r (wais - r). In one iranian study, reliability and validity of wais - r were studied.35 the wais - r subscales showed reliability from 0.69 to 0.87 on test retest stability and their internal consistency was 0.77 to 0.88 with split - half coefficient.36 wais - r was administered by a student of phd in psychology . All data were compared between patients (right tle and left tle) and controls . The multivariate analyses of variance (manova) was used to analyze the data of wms - iii and wais - r between groups . The demographic characteristics of right tle patients, left tle patients and the control subjects were summarized in table 1 . The results of manova revealed a significant difference between groups [pillai's trace f (12,170) = 6.57, p <0.001]. Demographic characteristics by group as shown in table 2, results of manova revealed significant differences between the groups for mean scores on piq subscale of wais - r (p <0.001), fsiq subscale of wais - r (p <0.002) and all primary indexes of wms - iii (p <0.001). There were no significant differences between right and left tle regarding mean scores of wais - r and wms - iii . Wechsler adult intelligence scale - r and wechsler memory scale - iii primary indexes and their subscale scores and comparisons by group although the verbal intelligence quotient (viq) mean scores were significantly different between the left tle and the control group (p = 0.037), there were not any significant differences between the right and the control group . Many investigators have reported specific cognitive deficits that differentiate tle from the other types of epilepsy . Problems in memory have been the most shared cognitive deficit in patients with tle.37 early neuropsychological studies indicated that resection of the left temporal lobe may impair the ability to learn verbal materials while right temporal resection can produce a deficit in learning of nonverbal and visuospatial information.2438 these phenomena were also found in patients with unilateral temporal lobe seizures with less power.3940 however, there were other studies that failed to show material specific of memory impairment to lateralize right or left tle.4142 in our study, patients with tle as a single group, had significant lower scores in all of the memory indexes and in the most subscales in comparison with control subject . In addition, a comparison of the iq scores between patients with epilepsy and subjects in control group showed significantly higher scores in fsiq and piq scores . This findings were consistent with those studies that showed cognitive dysfunction in temporal lobe epilepsy.3032 in patients with the right tle or the left tle as two different groups, there were no significant differences between the two groups regarding scores of wais - r and wms - iii indexes and subscales . These results were not consistent with findings of doss et al.23 and wilde et al.29 that showed material - specific dichotomized deficits in tle patients who were undergone anterior temporal lobectomy . However, some researchers have reported non - specific memory deficits in right or left tle . Baker et al.32 and vannucci31 found that there were no significant disparities between auditory and visual scores of patients with left temporal focal epilepsy group . Bachtler and dodrill showed that no significant group differences were found for visual immediate or delayed or auditory immediate indexes.43 these results were consistent with our finding . First, most of the findings that revealed material - specific memory problems were based on patients who were undergone anterior temporal lobectomy . Second, wilde et al . Reported that the ability of the wms - iii to predict lateralization was particularly weak for those with left temporal dysfunction.29 finally, although many of the neuropsychological tests possessed face validity, their genuine capabilities to assess what was prepared for them were in doubt . In wms - iii, the visual memory items invite verbal encoding during inspection, thus contralateral temporal lobe may have alternative or supplementary strategy for encoding of seemingly visual items in non - dominant hemisphere temporal lobe epilepsy . The results of this study showed ability of the wms - iii to find cognitive decline in patients with tle . Although the sample size had enough power to reveal difference between patients with tle and control group, it was not able to differentiate right versus left epileptogenic zone . The wms - iii has multiple subscales but it would have been better if more cognitive assessment tools had been used to raise validity of the findings . Mb was the main designer of this study and involved in all clinical and neuropsychological evaluations.
Although conservative treatment has its indications, surgical treatment for acl injuries is the ideal choice for young and active patients because it allows them to return to sports but prevents the onset of additional knee injuries.1 surgical techniques have evolved over recent decades, and the current gold standard treatment is arthroscopic intra - articular acl reconstruction with autogenous grafts . The main aspects of the surgical technique required to achieve favorable clinical results include graft quality, tunnel positioning and efficient graft fixation, with sufficient tension to provide joint stability and a physiological range of motion with normal flexion and extension.2, 3 along with the increasing number of indications for surgery, increases in the frequency of revision surgery have also been reported . Revision surgery is now performed in 1040% of cases.4 the main cause of surgical failure and acl revision is inadequate tunnel positioning.5, 6 anatomical studies have reported that the anatomical acl origin and insertion is the most isometric graft position710 . However, it is known that even senior surgeons using conventional guides frequently fail to place the tunnels in the desired positions.11, 12 a recent resource for improving orthopedic surgery precision is the computer assisted orthopedic surgery (caos) system.13 navigation systems for acl reconstruction have been developed,1418 but no comparative clinical studies have unequivocally demonstrated their superiority in relation to conventional guides . In addition to enhancement of precision, such navigation systems can also facilitate choosing the location for bony tunnels based on the new isometry criterion . Before the advent of such systems, these data could not be supplied to surgeons intraoperatively . The purpose of the present study was to compare isometry and tunnel positioning using the caos system and using conventional guides . The inclusion criteria for the knee specimens used were that the cause of death was not traumatic or due to infectious disease and that there were no scars, hematomas or deformities in the lower limbs . The exclusion criteria diagnosed during arthroscopy were the presence of any ligament lesions, meniscal lesions or degenerative joint disease . Thirty - six freshly frozen, undamaged human knees from cadavers (18 pairs from 18 cadavers) were used in this study . Four of the cadavers were female and 14 were male, ranging in age from 38 to 76 years (mean, 51.9; standard deviation (sd), 11.9). The femur was cut 20 cm and the tibia 30 cm from the joint line . The iliotibial tract up to mid - thigh, the popliteus musculotendinous unit and the joint capsule were left intact . The knees were stored at 20 c and thawed for 12 hours at room temperature before testing . Prior to the procedures, the knees were subjected to an initial arthroscopic inspection to rule out any previous intra - articular lesions . The specimens were divided in two groups, each comprising one knee of each pair (randomized side distribution by means of flipping a coin). These groups were named group 1, which consisted of 18 knees that underwent acl reconstruction using conventional guides, and group 2, which consisted of the 18 opposite - side knees that underwent acl reconstruction assisted by the caos system and navigation instruments . All procedures were performed by the same surgeon, who had prior experience in acl reconstruction and navigation . In group 1, an acufex tibial guide was used (director model; smith & nephew, inc ., u.s.a .) With 55 sagittal angulation and 15 lateral inclination and with the tip of the guide directed toward the intercondylar eminence of the tibia, halfway between the anterior horn of the lateral meniscus and the medial tibial intercondylar tubercle . In this position, a four - hole wire guide was drilled from the anteromedial surface of the tibia to the tip of the guide inside the joint . A polyester suture wire (ethibond 5) was then pulled by the metal guide wire from the extra - articular anteromedial cortical surface of the tibia to the inside of the joint, exiting from the central point of the tibial tunnel, which was not drilled (figure 1). For the femoral tunnel, a 7-mm offset acufex femoral guide was introduced through the anteromedial portal and positioned at the femoral intercondylar notch, with the tip of the guide on the posterior wall at eleven oclock for the right knee and one oclock for the left knee . In this position, the four - hole wire guide was drilled from the medial surface of the lateral femoral condyle to the anterolateral surface of the thigh of the specimen . The end of the suture wire that had been left inside the joint was then pulled through the anteromedial portal and was led by the four - hole wire guide through the femur (figure 2). In this way, the suture wire was positioned at the central point of the femoral tunnel, which was also not drilled . Orthopilot (aesculap, tuttlingen, germany) is a computer system that provides three - dimensional (3d) real - time tracking of specific surgical instruments in relation to anatomical reference points that are acquired during surgery . The 3d tracking is performed without the need for additional preoperative planning or imaging such as tomography . The anterior cruciate ligament replacement software, version 1.1 (2002) was used together with its specific surgical instruments . To provide tracking, two passive rigid bodies with four reflective spheres each the hardwired cameras detected the signals reflected by the spheres and determined their spatial position . A third passive rigid body was connected to the specific instruments for each step of the surgery . In the orthopilot setup, the anatomical reference points and kinematics of the knee were entered and registered by the orthopilot software, thus allowing it to calculate the relative positions between the fixed sensors and anatomical landmarks . The anatomical reference points input to the system were obtained using a pointer connected to the mobile and passive rigid bodies (figure 3). Superficial anatomical landmarks were registered in sequence, starting with the anterior tibial tuberosity and extending to the anterior, medial and lateral tibial plateau borders . The following intra - articular structures were viewed by means of arthroscopy and registered with the pointer through the anteromedial portal: posterior cruciate ligament (pcl), anterior tibial insertion, medial intercondylar tubercle, posteromedial margin of the anterior horn of the lateral meniscus, at least five points of the anterior border of the femoral intercondylar notch at its transition with the joint cartilage and at least five points on the medial surface of the femoral lateral condyle . Subsequent palpation of the posterior wall of the femoral intercondylar notch in two positions, eleven and twelve oclock for the right knee and twelve and one oclock for the left knee, was carried out with a navigation - linked pointer . The first step in the navigation was the positioning of the tibial tunnel; a specific tibial guide connected to the mobile and passive rigid bodies was tracked, and its position was monitored on the computer screen (figure 4). As soon as the desired position of the tibial guide was achieved, the guide wire was drilled through the guide from the tibial anteromedial surface to the guide tip inside the joint . A polyester suture wire (ethibond 5) was pulled through the four - hole wire guide along the drilled path . The wire remained free inside the joint at the center of what would be the tibial tunnel, which was not drilled as in group 1 . The data for the tibial tunnel were stored in the computer and used to calculate the optimum entry point for the femoral tunnel in relation to that specific tibial tunnel . A navigated femoral guide was introduced through the anteromedial portal and was tracked by the navigation system, allowing the guide position on the medial wall of the lateral femoral condyle to be followed on the computer screen . In addition, it permitted the calculation of the following parameters: coronal angulation, distance from the femur posterior wall and graft isometry . The point selected for the center of the femoral tunnel respected the previously defined parameters and provided the best isometry . In this position, the four - hole wire guide was drilled from the medial surface of the lateral femur condyle to the anterolateral surface of the thigh of the specimen . The same procedures as followed for group 1 were performed in relation to the suture wire, and the femoral tunnel was not drilled . Thus, no tunnel was drilled, but the suture wire was positioned inside the knees, through the centers of what would be the femoral and tibial tunnels . A knot was made at the end of the suture wire, proximally to the femur . This knot had a diameter larger than 2.5 mm, and therefore, when the opposite end emerging from the tibia was pulled, it could not migrate beyond the femoral cortex . Another knot was made at the extra - articular tibial end of the wire at an arbitrary distance from its tibial exit . The wire length between the tibial exit and the distal knot, with the knee at 90 of flexion and total extension (figure 5), was measured using a manual pachymeter . The difference between these measurements corresponded to the difference between the distances from the femoral tunnel to the tibial tunnel at these positions . The graft was considered isometric if the length variation was zero . Through arthrotomy, the distances to the pcl, medial intercondylar tubercle and margin of the anterior horn of the lateral meniscus relative to the guide wire exit on the tibial joint surface were collected using a manual pachymeter . A digital photograph of the frontal view of the femoral intercondylar notch was taken with 90 knee flexion . A line perpendicular to the tibial joint surface was drawn through the center of the intercondylar notch (twelve oclock). The intersection of this straight line with the image of the cranial edge of the tibia determined the point that was considered the angular vertex . From this point the angle formed by these two straight lines was measured by the software (figure 6). Thirty - six freshly frozen, undamaged human knees from cadavers (18 pairs from 18 cadavers) were used in this study . Four of the cadavers were female and 14 were male, ranging in age from 38 to 76 years (mean, 51.9; standard deviation (sd), 11.9). The femur was cut 20 cm and the tibia 30 cm from the joint line . The iliotibial tract up to mid - thigh, the popliteus musculotendinous unit and the joint capsule were left intact . The knees were stored at 20 c and thawed for 12 hours at room temperature before testing . Prior to the procedures, the knees were subjected to an initial arthroscopic inspection to rule out any previous intra - articular lesions . The specimens were divided in two groups, each comprising one knee of each pair (randomized side distribution by means of flipping a coin). These groups were named group 1, which consisted of 18 knees that underwent acl reconstruction using conventional guides, and group 2, which consisted of the 18 opposite - side knees that underwent acl reconstruction assisted by the caos system and navigation instruments . All procedures were performed by the same surgeon, who had prior experience in acl reconstruction and navigation . In group 1, guide wire positioning was performed using conventional surgical instruments . For the tibial tunnel, an acufex tibial guide was used (director model; smith & nephew, inc ., u.s.a .) With 55 sagittal angulation and 15 lateral inclination and with the tip of the guide directed toward the intercondylar eminence of the tibia, halfway between the anterior horn of the lateral meniscus and the medial tibial intercondylar tubercle . In this position, a four - hole wire guide was drilled from the anteromedial surface of the tibia to the tip of the guide inside the joint . A polyester suture wire (ethibond 5) was then pulled by the metal guide wire from the extra - articular anteromedial cortical surface of the tibia to the inside of the joint, exiting from the central point of the tibial tunnel, which was not drilled (figure 1). For the femoral tunnel, a 7-mm offset acufex femoral guide was introduced through the anteromedial portal and positioned at the femoral intercondylar notch, with the tip of the guide on the posterior wall at eleven oclock for the right knee and one oclock for the left knee . In this position, the four - hole wire guide was drilled from the medial surface of the lateral femoral condyle to the anterolateral surface of the thigh of the specimen . The end of the suture wire that had been left inside the joint was then pulled through the anteromedial portal and was led by the four - hole wire guide through the femur (figure 2). In this way, the suture wire was positioned at the central point of the femoral tunnel, which was also not drilled . Orthopilot (aesculap, tuttlingen, germany) is a computer system that provides three - dimensional (3d) real - time tracking of specific surgical instruments in relation to anatomical reference points that are acquired during surgery . The 3d tracking is performed without the need for additional preoperative planning or imaging such as tomography . The anterior cruciate ligament replacement software, version 1.1 (2002) was used together with its specific surgical instruments . To provide tracking, two passive rigid bodies with four reflective spheres each the hardwired cameras detected the signals reflected by the spheres and determined their spatial position . A third passive rigid body was connected to the specific instruments for each step of the surgery . In the orthopilot setup, the anatomical reference points and kinematics of the knee were entered and registered by the orthopilot software, thus allowing it to calculate the relative positions between the fixed sensors and anatomical landmarks . The anatomical reference points input to the system were obtained using a pointer connected to the mobile and passive rigid bodies (figure 3). Superficial anatomical landmarks were registered in sequence, starting with the anterior tibial tuberosity and extending to the anterior, medial and lateral tibial plateau borders . The following intra - articular structures were viewed by means of arthroscopy and registered with the pointer through the anteromedial portal: posterior cruciate ligament (pcl), anterior tibial insertion, medial intercondylar tubercle, posteromedial margin of the anterior horn of the lateral meniscus, at least five points of the anterior border of the femoral intercondylar notch at its transition with the joint cartilage and at least five points on the medial surface of the femoral lateral condyle . Subsequent palpation of the posterior wall of the femoral intercondylar notch in two positions, eleven and twelve oclock for the right knee and twelve and one oclock for the left knee, was carried out with a navigation - linked pointer . The first step in the navigation was the positioning of the tibial tunnel; a specific tibial guide connected to the mobile and passive rigid bodies was tracked, and its position was monitored on the computer screen (figure 4). As soon as the desired position of the tibial guide was achieved, the guide wire was drilled through the guide from the tibial anteromedial surface to the guide tip inside the joint . A polyester suture wire (ethibond 5) was pulled through the four - hole wire guide along the drilled path . The wire remained free inside the joint at the center of what would be the tibial tunnel, which was not drilled as in group 1 . The data for the tibial tunnel were stored in the computer and used to calculate the optimum entry point for the femoral tunnel in relation to that specific tibial tunnel . A navigated femoral guide was introduced through the anteromedial portal and was tracked by the navigation system, allowing the guide position on the medial wall of the lateral femoral condyle to be followed on the computer screen . In addition, it permitted the calculation of the following parameters: coronal angulation, distance from the femur posterior wall and graft isometry . The point selected for the center of the femoral tunnel respected the previously defined parameters and the four - hole wire guide was drilled from the medial surface of the lateral femur condyle to the anterolateral surface of the thigh of the specimen . The same procedures as followed for group 1 were performed in relation to the suture wire, and the femoral tunnel was not drilled . Thus, no tunnel was drilled, but the suture wire was positioned inside the knees, through the centers of what would be the femoral and tibial tunnels . A knot was made at the end of the suture wire, proximally to the femur . This knot had a diameter larger than 2.5 mm, and therefore, when the opposite end emerging from the tibia was pulled, it could not migrate beyond the femoral cortex . Another knot was made at the extra - articular tibial end of the wire at an arbitrary distance from its tibial exit . The wire length between the tibial exit and the distal knot, with the knee at 90 of flexion and total extension (figure 5), was measured using a manual pachymeter . The difference between these measurements corresponded to the difference between the distances from the femoral tunnel to the tibial tunnel at these positions . The graft was considered isometric if the length variation was zero . Through arthrotomy, the distances to the pcl, medial intercondylar tubercle and margin of the anterior horn of the lateral meniscus relative to the guide wire exit on the tibial joint surface a digital photograph of the frontal view of the femoral intercondylar notch was taken with 90 knee flexion . A line perpendicular to the tibial joint surface was drawn through the center of the intercondylar notch (twelve oclock). The intersection of this straight line with the image of the cranial edge of the tibia determined the point that was considered the angular vertex . From this point the angle formed by these two straight lines was measured by the software (figure 6). In group 1, guide wire positioning was performed using conventional surgical instruments . For the tibial tunnel, an acufex tibial guide was used (director model; smith & nephew, inc ., u.s.a .) With 55 sagittal angulation and 15 lateral inclination and with the tip of the guide directed toward the intercondylar eminence of the tibia, halfway between the anterior horn of the lateral meniscus and the medial tibial intercondylar tubercle . In this position, a four - hole wire guide was drilled from the anteromedial surface of the tibia to the tip of the guide inside the joint . A polyester suture wire (ethibond 5) was then pulled by the metal guide wire from the extra - articular anteromedial cortical surface of the tibia to the inside of the joint, exiting from the central point of the tibial tunnel, which was not drilled (figure 1). For the femoral tunnel, a 7-mm offset acufex femoral guide was introduced through the anteromedial portal and positioned at the femoral intercondylar notch, with the tip of the guide on the posterior wall at eleven oclock for the right knee and one oclock for the left knee . In this position, the four - hole wire guide was drilled from the medial surface of the lateral femoral condyle to the anterolateral surface of the thigh of the specimen . The end of the suture wire that had been left inside the joint was then pulled through the anteromedial portal and was led by the four - hole wire guide through the femur (figure 2). In this way, the suture wire was positioned at the central point of the femoral tunnel, which was also not drilled . Orthopilot (aesculap, tuttlingen, germany) is a computer system that provides three - dimensional (3d) real - time tracking of specific surgical instruments in relation to anatomical reference points that are acquired during surgery . The 3d tracking is performed without the need for additional preoperative planning or imaging such as tomography . The anterior cruciate ligament replacement software, version 1.1 (2002) was used together with its specific surgical instruments . To provide tracking, two passive rigid bodies with four reflective spheres each the hardwired cameras detected the signals reflected by the spheres and determined their spatial position . A third passive rigid body was connected to the specific instruments for each step of the surgery . In the orthopilot setup, the anatomical reference points and kinematics of the knee were entered and registered by the orthopilot software, thus allowing it to calculate the relative positions between the fixed sensors and anatomical landmarks . The anatomical reference points input to the system were obtained using a pointer connected to the mobile and passive rigid bodies (figure 3). Superficial anatomical landmarks were registered in sequence, starting with the anterior tibial tuberosity and extending to the anterior, medial and lateral tibial plateau borders . The following intra - articular structures were viewed by means of arthroscopy and registered with the pointer through the anteromedial portal: posterior cruciate ligament (pcl), anterior tibial insertion, medial intercondylar tubercle, posteromedial margin of the anterior horn of the lateral meniscus, at least five points of the anterior border of the femoral intercondylar notch at its transition with the joint cartilage and at least five points on the medial surface of the femoral lateral condyle . Subsequent palpation of the posterior wall of the femoral intercondylar notch in two positions, eleven and twelve oclock for the right knee and twelve and one oclock for the left knee, was carried out with a navigation - linked pointer . The first step in the navigation was the positioning of the tibial tunnel; a specific tibial guide connected to the mobile and passive rigid bodies was tracked, and its position was monitored on the computer screen (figure 4). As soon as the desired position of the tibial guide was achieved, the guide wire was drilled through the guide from the tibial anteromedial surface to the guide tip inside the joint . A polyester suture wire (ethibond 5) was pulled through the four - hole wire guide along the drilled path . The wire remained free inside the joint at the center of what would be the tibial tunnel, which was not drilled as in group 1 . The data for the tibial tunnel were stored in the computer and used to calculate the optimum entry point for the femoral tunnel in relation to that specific tibial tunnel . A navigated femoral guide was introduced through the anteromedial portal and was tracked by the navigation system, allowing the guide position on the medial wall of the lateral femoral condyle to be followed on the computer screen . In addition, it permitted the calculation of the following parameters: coronal angulation, distance from the femur posterior wall and graft isometry . The point selected for the center of the femoral tunnel respected the previously defined parameters and provided the best isometry . In this position, the four - hole wire guide was drilled from the medial surface of the lateral femur condyle to the anterolateral surface of the thigh of the specimen . The same procedures as followed for group 1 were performed in relation to the suture wire, and the femoral tunnel was not drilled . Thus, no tunnel was drilled, but the suture wire was positioned inside the knees, through the centers of what would be the femoral and tibial tunnels . A knot was made at the end of the suture wire, proximally to the femur . This knot had a diameter larger than 2.5 mm, and therefore, when the opposite end emerging from the tibia was pulled, it could not migrate beyond the femoral cortex . Another knot was made at the extra - articular tibial end of the wire at an arbitrary distance from its tibial exit . The wire length between the tibial exit and the distal knot, with the knee at 90 of flexion and total extension (figure 5), was measured using a manual pachymeter . The difference between these measurements corresponded to the difference between the distances from the femoral tunnel to the tibial tunnel at these positions . The graft was considered isometric if the length variation was zero . Through arthrotomy, the distances to the pcl, medial intercondylar tubercle and margin of the anterior horn of the lateral meniscus relative to the guide wire exit on the tibial joint surface a digital photograph of the frontal view of the femoral intercondylar notch was taken with 90 knee flexion . A line perpendicular to the tibial joint surface was drawn through the center of the intercondylar notch (twelve oclock). The intersection of this straight line with the image of the cranial edge of the tibia determined the point that was considered the angular vertex . From this point the angle formed by these two straight lines was measured by the software (figure 6). The variation of the distance from the femoral tunnel to the tibial tunnel between flexion and extension was smaller in the orthopilot group [better isometry; group 1, mean = 4.2 mm (range, 1.19.4 mm); group 2, mean = 2.8 mm (range, 0.36.7 mm); p <0.05]. None of the other parameters showed any significant difference between the groups, and all of the tunnels were considered to be in satisfactory positions (table 1). The design of this study aimed to make simple comparisons using direct measurements between the positions of the acl reconstruction tunnels achieved using the orthopilot navigation system and using conventional guides . Furthermore, and perhaps more importantly, this study aimed to determine which of these techniques provided better isometry . The knees used in this study did not present any acl injury, and the acls were arthroscopically removed . The resection invariably leaves clear signs of the anatomical origin and insertion of the acl, and this may have assisted in positioning the guides, thereby potentially improving the results in both groups . Direct measurements using a manual pachymeter have the advantage of being simple and reproducible, particularly when performed by a single surgeon . The measurements were based on absolute points that were covered by radiolucent structures, including cartilage and other tissues . Thus, our data cannot be compared with radiographic data.19, 20 the measurements of the distance from the tibial tunnel to the pcl showed averages close to 10.2 mm in both groups . This result was higher than the expected result of 7 mm, potentially indicating that this was not a reliable reference point for tunnel positioning . Measurement (in degrees) of the coronal angle of the femoral tunnel using digital photography has not been reported previously in the literature . We chose to use this measurement because of its reproducibility and because it is easy to perform . Therefore, it was surprising that the two groups, which demonstrated no significant differences in any anatomical parameters, had different isometric behaviors . The orthopilot group not only had a lower average but also a lower interval between the minimum and maximum values, and this result was very consistent . Our interpretation of this finding is that there is a relatively broad acceptable area for positioning the centers of the tibial and femoral tunnels . Positioning the center of the tunnel at any of the infinite number of points inside these areas would be appropriate . Therefore, an infinite number of possible combinations of appropriate tibial tunnels with appropriate femoral tunnels for each knee are possible . The navigation system seemed to provide us with a femoral tunnel location (chosen after positioning the tibial tunnel) in an acceptable area of the femur in relation to the selected tibial position, thereby providing decreased variation of the graft length or better isometry . However, the navigation system presents some potential inconveniences, such as the longer time required for the procedure21 and morbidity related to the fixation of rigid bodies to bones . The results of this study suggested that there was no difference in the anatomical tunnel position between the groups . However, further studies with a larger number of cases are needed to confirm these results . Finally, the results of our study encourage us to continue evaluating the use of navigation for the improvement of knee surgery . Better isometry was achieved using the navigation technique (group 2) compared to the conventional technique (group 1).
Prosthetic dentistry revolves around the restoration or replacement of lost or missing teeth using crowns, bridges, and other dentures . In fixed partial dentures (fpds), dental luting agents serve as the link between the prepared, supporting tooth, and the prosthetic tooth material . An ideal dental cement to be used as a luting agent should be biocompatible, inhibit caries or plaque formation, have low solubility, have correct film thickness and viscosity, have long working time and short setting time, have high strength and stiffness comparable to dentin, show no microleakage, permit easy removal of excess material, and exhibit high retention . A wide range of luting agents with varying chemistries and properties are available on the market and strive to meet these high requirements . However, fpds are prone to failure, with the number one cause of failure being secondary dental caries [2, 3]. Secondary caries is readily formed in restoration margins or gaps where plaque, a biofilm containing various bacteria, can adhere . Among the bacterial species present in these biofilms, streptococcus mutans is recognized as the one most frequently involved in caries formation . These bacteria produce acids when metabolizing fermentable carbohydrates, which can then dissolve the calcium phosphate mineral content in enamel and dentin, eventually leading to a cavity or failure of the fpd . Colonization of bacteria often occurs at secluded locations in shortage of oxygen and mechanical disturbance and consequently bactericidal properties of dental cements are of particular importance since it is one of the few means by which bacteria can be inhibited in these sites . Antibacterial properties of dental cements have been evaluated in the past [79], and the bactericidal effects are often attributed to their low ph and/or release of fluoride ions . There is, however, conflicting data as to whether the low - level fluoride release or acidity of currently used materials is sufficient for long - term bactericidal effect, and which class of cement performs the best [7, 911]. Two popular assays used to evaluate antibacterial properties of dental cements are the agar diffusion test (adt) and direct contact test (dct). While the adt has been used successfully [7, 12], it has certain limitations . Results are semiquantitative and depend on solubility and diffusion properties of both the material tested and the medium used . To avoid the limitations of adt, dct was developed by weiss et al . And further used in several other studies of similar character [8, 9, 14, 15]. In dct, outgrowth of bacteria after close contact with a nonsoluble material is quantified by continuous measurements of optical density (od), which is proportional to the concentration of bacteria present in solution . In this study, a modified dct was used to evaluate the antibacterial properties of four commercially available dental cements (three acid - base reacting and one self - etching) and two acid - base reacting reference materials . A metabolic assay containing resazurin was used to quantify the bacteria present in solution after direct contact with the cements . Resazurin is a common metabolic activity indicator that has been shown to be effective in assessing bacterial viability and in biofilm quantification . It is a water - soluble dye that can be reduced to highly fluorescent resorufin by metabolically active bacteria . In addition, the effects of ph and fluoride on bacteria proliferation were evaluated by means of od measurements . Compositions, powder to liquid (p / l) ratio and suppliers of the dental cements investigated in this study are shown in table 1 . Relyx unicem (relyx) and ketac cem (ketac) were prepared using the aplicap activator and applier and mixed using a capmix universal mixing unit (3 m espe). Harvard cement (zinc phosphate, zp), ceramir crown & bridge (ceramir), calcium aluminate cement (ca), and glass - ionomer cement (gic) were all mixed by hand using a stainless steel spatula on a clean surface . After preparation, the cements were transferred to rubber molds, 1.5 mm deep and 5 mm in diameter . Light curing was used for relyx, while the remaining cements were allowed to set for 710 min at 37c in 100% relative humidity . After setting, samples were removed from the molds and returned to the oven for aging at 37c in 100% relative humidity for 10 min, 1 day, or 7 days before testing for antibacterial effect . As a control, samples of poly(methyl methacrylate) (pmma), a biomaterial considered inert in its fully polymerized state, were cut in the same dimensions from a solid rod . Streptococcus mutans (strain nctc 10449) was used to determine growth inhibition activity of the investigated cements, the effects of varying ph, and the bactericidal effect of fluoride . S. mutans has frequently been used to test antimicrobial activity [12, 1821] and is considered a primary etiological agent of caries . S. mutans was inoculated in brain - heart infusion (bhi) broth (sigma - aldrich, steinheim, germany) and cultured anaerobically at 37c to exponential phase (od600 = 1.0). The culture was centrifuged at 4000 rpm for 5 min and the bacteria pellet was resuspended in sterile h2o . The concentration of bacteria was adjusted to od600 = 1.0, which corresponds to 10 cfu / ml . The direct contact test (dct) performed by weiss et al . Is based on turbidimetric determination of continuous bacterial outgrowth from the material under investigation . In the modified direct contact test performed in the current study, resazurin was used as metabolic activity indicator to quantify viable bacteria instead of od measurements . In the resazurin assay, blue, nonfluorescent resazurin is reduced by metabolic intermediates (e.g., napdh) to pink resorufin, which is fluorescent and therefore a sensitive indicator of the amount of viable bacteria in the assay . Six samples of each cement type and a control group of six pmma samples were tested for each aging time . Each sample was placed at the bottom of a well of a 96-well plate (sterile, transparent, flat bottom, cellstar, greiner bio - one gmbh, germany) and 5 l of the s. mutans suspension was added and evenly distributed on the sample surface . Samples were then incubated at 37c for 1 h, during which time the suspension liquid evaporated, ensuring direct contact between bacteria and testing material . Immediately following this step, 135 l of mueller hinton (mh) broth culture medium (fluka) and 15 l resazurin (1.25 g / ml) were added to each well and incubation was continued at 37c for 100 min, after which fluorescence measurements of each well were made using a multimode microplate reader (infinite 200 pro, tecan, mnnedorf, switzerland) set to 530 nm excitation and 590 nm emission wavelengths . Samples were removed from the wells prior to fluorescence measurement since the testing materials themselves may influence the measurements . In order to calibrate results from the sample viability assays, a standard curve was made from a set of resazurin assays containing a serial dilution of bacteria . The standard curve provides a quantitative measure of viable bacteria in the sample assays by comparing the fluorescent signals from the sample assays to that of the serial dilution . For measuring the effects of varying ph on bacterial proliferation, 10 different buffer solutions (sigma - aldrich) ranging from ph 1 to 11 test solutions comprised of a suspension of s. mutans in bhi broth and ph buffer were prepared as detailed in table 2 . A solution containing sterile deionized h2o (hereafter referred to as h2o) instead of ph buffer the ph of h2o is around 7 and is not expected to have negative effect on bacterial growth . The resulting ph of the control and test solutions was measured using a ph meter (hi 83141, hanna instruments). Solutions were then incubated at 37c and od measurements at 600 nm were made on 3 ml aliquots from each solution using a spectrophotometer (uv-1650, shimadzu, kyoto, japan) after 0 to 10 hours of incubation . For these tests, od measurements were used because it offered a simple and reliable means of measuring the concentration of bacteria as a function of time . Three different aliquots from each solution were measured at each time, after which they were returned to their respective solutions . A reference solution containing only bhi broth culture medium and h2o was used to calibrate the instrument at 600 nm and create a baseline . Sodium fluoride (naf) powder (sigma - aldrich), h2o, and bhi broth were mixed to obtain 10 ml solutions with varying fluoride strength, from 0 to 2000 ppm . To each solution, 50 l of s. mutans suspension (od600 = 1.0) was added . Three samples at each fluoride concentration were incubated at 37c for 8 h, after which od measurements were made at 600 nm in the spectrophotometer . Using the statistical software package spss v19 (spss inc ., il, usa), a univariate analysis of variance (anova) was carried out to identify statistical differences in amount of viable cells after dct on the different materials (cements and control) and after varying time . Multiple comparisons were made using the sheffe's method, and differences were considered significant at the 95% confidence level (p <0.05). The standard curve from the dilution series resulted in a linear relationship between the degree of fluorescence and the known amount of bacteria present . Figure 1 shows the corresponding relationship between the number of viable bacteria, labeled as colony forming units (cfus) and fluorescence, measured in relative fluorescence units (rfus). A linear fit to the data was used to calibrate fluorescence measurements from the test cements relative to the dilution series and was utilized in the results shown in figure 2 . Figure 2 displays the number of viable bacteria for each material after the different aging times, where each bar is the average from six different wells . The strongest antibacterial activity was demonstrated by ca, which showed significantly different values from the control (p <0.0005) and all other cements (p 0.014) at all aging times . It also showed a significant decrease in cfus with increased aging time (p 0.006). Ceramir showed antibacterial properties after 10 min and 1 day of aging (p 0.004) but was not significantly different from the control after 7 days of aging . Relyx also showed significant difference from the control after 10 min aging (p = 0.022), but not after 1 day or 7 days . The remaining cements (zp, ketac and gic) showed either higher number of viable bacteria or were not significantly different from the pmma control at all instances . S. mutans proliferated between ph 6.3 and 8.6 and showed strongest growth in the neutral ph buffer and in the h2o control . The curves are characterized by an initial lag phase (02 h), followed by a growth phase and a final stationary phase . However, a considerable gap exists between the ph buffer 7 and h2o control in the latter part of the growth . This is likely due to the fact that as bacteria grow, acid is produced that will negatively affect the bacterial proliferation after approximately 6 h. the buffer, however, has the ability to regulate for this and proliferation continues until the stationary phase is reached at a later point . Figure 5 displays the effect of fluoride on bacterial proliferation . A negative effect (i.e., reduction in the bacterial growth rate) was observed even at 200 ppm, with an increasing effect with increasing fluoride concentration until an approximately steady state was reached at roughly 1400 ppm . The longevity of dental restorations is often determined by their ability to resist plaque formation and consequently avoid secondary caries [2, 3, 20]. Although it has been suggested that early plaque formation is largely influenced by the intraoral positioning of restorations and general oral environment, cements having antibacterial properties could successfully prevent or delay caries formation and prolong the lifetime of restorations . In the current study, antibacterial properties of different classes of dental cements were investigated by means of a modified dct . Previous studies have shown various glass - ionomer cements to have initial bactericidal properties due to low ph during curing or slow release of fluoride [7, 8, 10, 23], whereas another study showed polycarboxylate cement and zinc phosphate cement to be more active both initially and over time, which was attributed to low ph and fluoride release . In this study, the bioceramics ca and ceramir exhibited the highest degree of antibacterial activity, followed by the relyx resin cement . Zp showed some initial bacterial inhibition but was not significantly different from the pmma control and displayed a large spread in data . The pure gic and ketac showed higher bacterial content than the pmma control for all aging times, and the differences were significant in all cases except for ketac after 7 days of aging . Conventional luting cements that set by acid - base interactions produce an environment that is initially acidic, but approach neutrality during the course of the reaction . Low initial ph is recognized as beneficial with regards to bactericidal properties; however, this can have negative consequences as acid diffusion through thin dentin sections which can cause pulpal irritation has been reported for glass - ionomer and zinc phosphate cements . The minimum ph for organisms such as s. mutans to grow is approximately 5, and although this study showed clear inhibition from ph 6.3, none of the cements maintaining a ph below neutral after setting (ketac, gic, and zp all end up around ph 6) displayed any clear bactericidal properties . Relyx showed some antibacterial properties after being aged for 10 min, but at that time should no longer be acidic . The light cured relyx resin cement reaches ph 5 within minutes after onset of the reaction and quickly continues to ph 8 before stabilizing at a neutral level after 24 h . This means that the initial bactericidal effect of relyx could potentially be attributed to fluoride release which is highest during the first 24 h . However, gic also releases fluoride and here no antibacterial effect was observed at any aging time, so it is not clear that fluoride gives an antibacterial effect . Ceramir is also acidic during the first stages of the setting reaction, but quickly surpasses neutrality to reach basic levels . The ph of ca can reach over 12 during the reaction, but settles around 11.5 after final setting, while ceramir stabilizes around 8.5 after roughly 12 hours . As shown in figure 4, inhibition was also observed for higher levels of ph, and therefore the bactericidal properties displayed by ceramir during initial setting times and especially by ca could be due to the basic environment they produce . Since the ph of ceramir lowers after 12 h, it is also likely that another antibacterial mechanism, such as fluoride release, is responsible for the observed antibacterial effect . Fluoride is not only widely used as an anticariogenic agent in common dental products such as toothpaste and mouthwash, it is also present in a wide range of restorative materials . Among the cements investigated in the current study, fluoride is released from relyx, ceramir, ketac, and gic . However, within the limitations of the experiments performed, no conclusive evidence of antibacterial activity directly attributable to fluoride release from these cements could be shown . The anticariogenic effects of fluoride involve a variety of mechanisms, including inhibition of demineralization and enhancement of remineralization at the crystal surface as well as inhibition of bacterial growth and metabolism . This discussion will, however, be limited to the action of fluoride on bacteria . The decreasing od with increasing fluoride concentration indicates that increasing fluoride concentration negatively affects the bacterial growth rate . Fluoride can act in various ways to stop or slow bacterial growth; f or hf can cause direct inhibition of enzymes such as enolase, urease, phosphatases, or heme catalase, which are all involved in microbial cell metabolism; metallic fluoride complexes such as alf and befh2o can effect enzymes and regulatory phosphates, with either positive or negative outcome for the cell; and hf can act as a transmembrane proton carrier to disturb the cell membrane through a ph discharge . Direct inhibition of enzymes through binding of f / hf is a ph - dependent process, where f in many cases binds to sites normally occupied by oh, followed by binding to a proton . F has been shown to be unable to cross the cell wall and membrane, whereas hf passes unhindered . Well inside the cell, hf dissociates to create an acid environment and releases f, which will accumulate and disturb the enzymes present . In this case, hf acts as a proton carrier across the membrane and overloads the proton - extruding atpases, as extruded protons will reenter as hf and eventually cause cell starvation and generally deenergize the membrane through the ph discharge . It has been suggested that the ideal release profile of fluoride from dental cements should be characterized by an initial burst, followed by a stable, lower release rate . It should be noted that the amount of fluoride released from dental restoratives is far less than the amounts shown in figure 5 . Accumulated release varies between material types and products, but resin cements have been shown to have similar release profiles as resin - modified and conventional glass - ionomer cements, which are able to reach 15 ppm in the first week [20, 28, 29]. However, with the occurrence of shrinkage during setting, which allows gaps or voids to form between the cement and tooth, a strong initial fluoride release could increase fluoride concentration in the area, reducing the proliferation of bacteria and helping prevent secondary caries . A subsequent constant, lower level release of fluoride could aid in maintaining the resistance to bacteria and buildup of plaque and subsequent caries in sensitive areas . Based on the results from the dct in the current study, fluoride released from the cements similar conclusions have been drawn for fluoride releasing restoratives by others [10, 20]. Nonetheless, it is possible that synergetic effects of fluoride with, for example, ph result in the observed bactericidal effects with luting agents such as ceramir . The antibacterial properties of four commercially available luting agents and two reference materials were tested in this study . Only calcium aluminate cement showed significant antibacterial properties compared to the pmma control after aging for 10 min, 1 day, and 7 days . An increase in antibacterial effect was found for calcium aluminate cement with increased aging time . Antibacterial activity was also shown for ceramir after 10 min and 1 day aging and for relyx after 10 min aging . Cements having high rather than low ph after setting were shown to be more antibacterial, despite the fact that clear negative effects on bacteria proliferation were seen at ph levels below 6.3 and above 9.0 . No correlation between either acidic materials or fluoride release and antibacterial properties could be seen; rather, basic materials showed the strongest antibacterial properties.
Actinomycosis is an infrequent invasive chronic inflammatory disease caused mainly by actinomyces israelii, gram - positive, filamentous, anaerobic bacteria . Actinomycosis occurring primarily in urachal remnants is rare and may mislead the clinicians to diagnose urachal carcinoma . We report one such case of urachal actinomycosis in a 50-year - old male with lower abdominal pain and mass palpable mimicking a urachal tumor . A 50-year - old man, hailing from south india, came to the urology outpatient department with complaints of lower abdominal pain, dysuria, and dribbling of urine for 2 months duration . Clinically, he was afebrile and per - abdomen examination revealed a vague mass of 7 cm palpable at the suprapubic region . Computed tomography scan of the abdomen revealed an irregular mass seen superior to fundus of urinary bladder appearing adherent to mesentery and bowel loops and suggested an urachal remnant associated lesion with infiltration figure 1 . Excision of the mass with partial cystectomy and resection of involved ileal segments was done figure 2 . The specimen was sent for histopathological examination . Computed tomography of the abdomen showing an irregular mass seen superior to fundus of urinary bladder appearing adherent to mesentery and bowel loops grossly, we received an irregular soft tissue mass measuring 12 cm 15 cm 4 cm with umbilicus on the one side and dome of the bladder on the other side . We also received two loops of intestine with attached serosal mass measuring 4 cm 3 cm 2 cm . After adequate sampling, sections from the urachal remnant revealed actinomycotic colonies surrounded by microabscesses and dense inflammatory fibrotic lesion figure 3 . Small intestinal segments showed a similar lesion in the serosa but not in the mucosa, confirming it to be a primary urachal actinomycosis . Photomicrograph showing high power view of h and e section of actinomycotic colonies surrounded by microabscesses the patient was started on intravenous penicillin and recovered without complications . The most common risk factor includes use of intrauterine contraceptive devices in female, and others include history of bowel surgery such as perforated acute appendicitis, perforated colonic diverticulitis, penetrating trauma to the abdomen, and persistent urachal remnant . Our patient showed two of the risk factors of persistent urachal remnant and an earlier appendicectomy . The infiltrative nature of the bacteria into the surrounding organs can be explained by production of proteolytic enzyme . There are no specific radiological signs to distinguish actinomycotic lesions from malignancy due to its infiltrative nature . Definite diagnosis before surgery is difficult unless patient had discharging sinuses in the lower abdomen . Demonstration of the colonies of actinomycosis by histopathological examination is the definitive diagnosis, and hence, adequate sampling and extensive scanning must be done . In a study of 33 patients with pelvic actinomycosis by marella et al ., 19 patients had intrauterine contraceptive devices and only four of them had urachal remnants . Exploratory laparotomy was done in most of them, and definite diagnosis was made by histopathological examination after excision of the mass . It has a good prognosis and is curable with antibiotics such as penicillin and amoxicillin . It is important to consider it in the differential diagnosis of patients presenting with lower abdominal pain and hard suprapubic mass with or without discharging sinuses . To the best of our knowledge,
The prevalence of gestational diabetes mellitus (gdm) among pregnant women in the united states ranges from 3% to 7%, depending on the population studied . However, up to 50% of women with a history of gdm will develop type 2 diabetes in the decade following their gdm diagnosis . Women with both pre - gdm and gdm are at an increased risk for hypertensive disorders of pregnancy and can lead to higher maternal and fetal morbidity . On the reverse, hypertensive disorders of pregnancy can lead to significant increases in the risk for future cardiovascular events in diabetic women and women with a history of gestational diabetes . Some studies have shown that elevated bp before or during early pregnancy are associated with the development of gestational diabetes . We planned to evaluate the prevalence of gdm and its correlation with maternal age and gestational blood pressure (bp) in a study among women with singleton pregnancy in manipuri population . This was a cross - sectional study carried out in the department of medicine, regional institute of medical sciences (rims), in collaboration with the department of obstetrics and gynaecology, rims . The duration of the study is two calendar years from september 2010 to august 2012 . Three hundred cases of randomly selected pregnant women from the outpatient unit of the department of obstetrics and gynaecology, rims, with spontaneous singleton pregnancy in the 24 to 28 weeks of gestation having no history of gdm before the index pregnancy were made to undergo a detailed clinical examination . Women with a history of gdm in the previous pregnancy, known case of diabetes mellitus and systemic hypertension, and unwilling subjects were excluded from the study . All the selected participants underwent a glucose challenge test (gct) where 50 g of glucose was given to them, without considering the timing of the last meal . Cases with blood sugar level 140 mg / dl after 1 h proceeded to 100 g 3-h oral glucose tolerance test (ogtt). A patient is considered to have gdm if two or more of the values obtained during the 100 g glucose load 3-h ogtt are abnormal according to either or both the national diabetes data group (nddg) criteria and american diabetes association (ada) (2004) criteria . Women were categorized according to bp level recommended by american heart association, outside of pregnancy, that is, <120/80 mmhg as normal, 120 - 139/80 - 89 mmhg as prehypertension,> 140/90 mmhg as hypertension . Pregnancy bp was the bp measured at 24 - 28 weeks of gestation and pregravid bp is the bp measured closest to but before last menstrual period and no more than 5 years before pregnancy; not included if it was from an emergency visit . The data were analyzed using spss v. 16 and correlation was done using fisher's exact test with a confidence interval of 95% and the significance level at p <0.05 . This was a cross - sectional study carried out in the department of medicine, regional institute of medical sciences (rims), in collaboration with the department of obstetrics and gynaecology, rims . The duration of the study is two calendar years from september 2010 to august 2012 . Three hundred cases of randomly selected pregnant women from the outpatient unit of the department of obstetrics and gynaecology, rims, with spontaneous singleton pregnancy in the 24 to 28 weeks of gestation having no history of gdm before the index pregnancy were made to undergo a detailed clinical examination . Women with a history of gdm in the previous pregnancy, known case of diabetes mellitus and systemic hypertension, and unwilling subjects were excluded from the study . All the selected participants underwent a glucose challenge test (gct) where 50 g of glucose was given to them, without considering the timing of the last meal . Cases with blood sugar level 140 mg / dl after 1 h proceeded to 100 g 3-h oral glucose tolerance test (ogtt). A patient is considered to have gdm if two or more of the values obtained during the 100 g glucose load 3-h ogtt are abnormal according to either or both the national diabetes data group (nddg) criteria and american diabetes association (ada) (2004) criteria . Women were categorized according to bp level recommended by american heart association, outside of pregnancy, that is, <120/80 mmhg as normal, 120 - 139/80 - 89 mmhg as prehypertension,> 140/90 mmhg as hypertension . Pregnancy bp was the bp measured at 24 - 28 weeks of gestation and pregravid bp is the bp measured closest to but before last menstrual period and no more than 5 years before pregnancy; not included if it was from an emergency visit . The data were analyzed using spss v. 16 and correlation was done using fisher's exact test with a confidence interval of 95% and the significance level at p <0.05 . One hundred and thirty - three women out of the 300 (43.3%) have both systolic and diastolic bp in the prehypertensive range of above 120/80 mmhg . One hundred and seventy - nine (59.7%) women have systolic bp in the prehypertensive range of 120 - 139 mmhg and 191 (63.7%) women have diastolic bp in the prehypertensive range of 80 - 89 mmhg . Distribution of age and blood pressure during pregnancy out of 300 pregnant women screened, 263 (87.67%) had gct <140 mg% and 37 (12.33%) subjects had gct> 140 mg% . The majority of the women with positive screening on 50 g gct were in the age range 26 - 30 years [table 2]. A 50 g glucose challenge test in different age groups among these 37 participants screened positive for gct, there was a statistically significant association with raised systolic bp but similar association was not seen with diastolic bp . The distribution of bp among women with normal or impaired 50 g gct is shown in table 3 . Blood pressure and 50 g glucose challenge test with 100 g 3-h ogtt among the 37 subjects with positive 50 g gct, the median blood glucose fasting level was 82 12.14 mg%, for 1-h ogtt 170 24.3 mg%, for 2-h ogtt 132 17.7 mg%, and 104 15.8 mg% for 3-h ogtt . None of them fulfilled the nddg diagnostic criteria of gdm in the 100 g 3-h ogtt, although two patients had fasting blood glucose level above the cutoff level of> 105 mg% [table 4a]. A 100 g 3-h oral glucose tolerance test and blood pressure . National diabetes data group criteria however, on using the ada (2004) criteria, among the 37 participants with 50 g gct level> 140 mg%, three (8.1%) were diagnosed to have gdm with 3-h 100 g ogtt . This represents 8.1% increase in gdm diagnoses, from 0% (0 of 37) to 8.1% (3 of 37), using the more inclusive criteria . The bp of the three gdm patients was 120/88, 128/80, and 130/88 mmhg, which fell within the prehypertensive range [table 4b]. A 100 g 3-h oral glucose tolerance test and blood pressure . Ada (2004) criteria according to the nddg criteria with the cutoff point for a positive fasting blood sugar level> 105 mg%, two (5.4%) participants showed a positive result and both of them had systolic bp within the range 120 - 139 mmhg and diastolic bp within the range 80 - 89 mmhg (prehypertensive range). On using the ada (2004) criteria, five (13.51%) participants showed a positive result at> 95 mg% and they all fell in the prehypertensive range of bp, that is, 120 - 139 mmhg for systolic bp and 80 - 89 mmhg for diastolic bp . In the 1 h ogtt, none of the participants reached the cutoff level for positive test, that is,> 190 mg% on using the nddg criteria . But on using the ada (2004) criteria, four (10.81%) participants have blood sugar level above the cutoff level of> 180 mg% . Among these four women, three had a systolic and diastolic bp within the prehypertensive range (120 - 139 and 80 - 89 mmhg). Again in the 2-h ogtt result, none of the participants reached the nddg cutoff level of blood sugar> 165 mg%, whereas on using the ada criteria, three (8.1%) women had blood sugar level> 155 mg% . All three of them had systolic bp between 120 and 139 mmhg; two of them had diastolic bp between 80 and 89 mmhg . One (2.7%) participant showed a positive 3-h ogtt result of> 140 mg% as per the ada (2004) criteria and her bp fell within the prehypertensive range for both systolic and diastolic pressures . In manipuri population, the prevalence of diabetes mellitus has been reported to be 4.03% in 1997 and the prevalence of gestational diabetes was reported to be 1.6% in 1999 . A recent study in a tertiary care institution from north india several studies from various parts of india have reported the prevalence of gdm ranging from 3.8% to 17.8% using different criteria . In the present study, out of the 300 participants, 37 women had blood glucose level more than 140 mg% in the 50 g gct with the majority older than 26 years . The same finding was observed by seshiah et al . Who mentioned in their study that age> 25 years is one of the independent risk factor for gdm . Similarly, hoseini et al . Have also included maternal age> 26 years as one of the significant risk factors for gdm . Ada (2010) guideline also mentioned age <25 years as a low - risk group who do not require screening for gdm unless they have other risk factors for gdm . The distribution of the bp among the participants in relation to positive screening of gct showed a significant relationship . Although the participants do not have hypertension, those with positive gct still have a higher bp range compared with those with negative gct . Solomon et al . Observed abnormal glucose loading test as a significant predictor of development of hypertension and nonproteinuric hypertension in pregnancy . Vembergre et al . Also noted that pregnancy - induced hypertension appears to be linked to the level of glucose intolerance during pregnancy, independently of the other known risk factors of hypertension . During early pregnancy, women with prehypertension had a small increased risk of gdm, and women with hypertension had a twofold increased risk of gdm compared with women with normal bp after adjusting for age, race / ethnicity, gestational week of bp, body mass index, and parity . Similar results were seen among the subset of women with bp levels measured before pregnancy . Several randomized clinical trials had compared various standard criteria for screening and diagnosis of gdm, but the opinion about the policy of detection and diagnosis has not been settled . The fourth international workshop conference on gdm indicated that the infants of women who meet the lower carpenter coustan criteria are at similar risk for perinatal morbidity as those patients identified using the nddg criteria . Thus, using the more inclusive ada (2004) criteria may help us to identify an additional number of women with gdm who otherwise may have gone undiagnosed and untreated although they have similar risk as that of the treated women . Mild gestational hyperglycemia (mgh), which is defined as a positive gct followed by a negative ogtt, is characterized both by insulin resistance and a near - normal pattern of glucose - triggered insulin release . Vemberge et al . Defined gestational mild hyperglycemia as one abnormal value of ogtt in their study and showed the rate of pregnancy - induced hypertension in gdm, gestational mild hyperglycemia, and control were, respectively, 17.0%, 10.8%, and 4.6% . Pregnancy - induced hypertension was linked to the level of glucose intolerance during pregnancy, independently of the other known risk factors of hypertension . Had also mentioned that women with gestational hyperglycemia and gdm have higher risk of hypertension in addition to that of type 2 diabetes . Similarly, retnakaran et al . Have concluded in their study that both gdm and mild glucose intolerance predict an increased likelihood of metabolic syndrome at three months postpartum, supporting the concept that women with gestational dysglycemia may have an underlying latent metabolic syndrome . On that note, our study has observed 37 participants with abnormal gct but having normal 3-h ogtt according to the nddg criteria . Two participants showed an abnormal fasting blood sugar level but their 3-h ogtt remained within the normal limit . Apart from those who met two of the cutoff values in the 3-h ogtt to be diagnosed as gdm as per the ada (2004) guideline, an additional five women met one of the cutoff values among the three ogtt cutoffs . Three of them had a fasting blood glucose level> 95mg% and another two had a 1-h ogtt level> 180 mg% . So, in the present study, out of the 300 women, three were diagnosed with gdm, five women have gestational mild hyperglycemia and 37 women have mgh . In the present study, three (1%) women were diagnosed with gestational diabetes on using the ada (2010) criteria . Although none of the women in the study have hypertension, 83.8% of those who screened positive for gestational diabetes have systolic bp in the prehypertensive range; 16.2% of them have diastolic bp in the prehypertensive range showing an association of bp with gestational dysglycemia in manipuri women . Because of the small sample size and cross - sectional nature of the study, there is limitation in the interpretation of this study . Also, with 11.33% women detected to have gestational dysglycemia, a larger study with adequate sample size, follow - up, and duration is needed to give us more insight about the prevalence of gestational diabetes and its correlation with bp, its effect on the maternal and fetal outcome, and also the impact of diagnosing and treating the women who were diagnosed only on using the more inclusive criteria.
Sparganosis is a rare parasitic infection caused by a migrating plerocercoid tapeworm larva (= sparganum) of the genus spirometra . The sparganum is a wrinkled, whitish, ribbon - shaped organism that is a few millimeters wide and up to several centimeters long . Sparganosis is transmitted to humans by drinking water contaminated with copepods containing spargana, consuming the raw flesh of a second intermediate host such as frogs or snakes, or placing a poultice infected with spargana on an open wound . Although sparganosis may involve internal organs, such as the eyes, brain, and spinal cord, the clinical presentation of sparganosis usually occurs after the larvae have migrated to a subcutaneous location . Furthermore, because the mass is usually non - tender in early migratory stages and sometimes triggers a painful inflammatory reaction in the surrounding tissues, it is quite difficult to differentiate sparganosis from a soft tissue tumor based on preoperative radiographic and laboratory data . We report here a rare case of intramuscular sparganosis involving the gastrocnemius muscle in an elderly patient who was diagnosed by ultrasonography and mri and treated by performing surgical excision . A 77-year - old man was referred to our clinic from a private clinic because of a suspected soft tissue tumor . The patient had a 6-month history of a mass over the right posteromedial aspect of the proximal tibia . Physical examination revealed a 3 cm1 cm, soft, non - tender mass at the posteromedial aspect of the right proximal tibia, and the mass was freely mobile and easily compressed . He had pulmonary tuberculosis when he was 30 years old, for which he was treated and believed to have been cured . He also had the history of lobectomy because of a lung cancer in the left lower lobe at the age of 47 years and received a total gastrectomy due to an advanced gastric cancer at the age of 71 years . Plain radiographs of the right leg revealed multiple calcifications extending from the medial part of the knee to the calf . Ultrasonography revealed a heterogeneous, hyperechoic mass with a hypoechoic tubular lesion in the gastrocnemius muscle (fig . 1), and mri revealed multiple tubular and cystic lesions in the gastrocnemius muscle . The lesions showed high signal intensity on t2-weighted images and multilobulated, peripheral enhancing low signal intensity on t1-weighted images (fig . The patient was a farmer, and he had occasionally ingested raw snake meat when he was young . Preoperative serodiagnosis of human sparganosis by using a monoclonal antibody - based competitive elisa was positive (3.79 in od value) (negative <1.00). After preoperative ultrasound - guided marking for suspected cystic lesions, a longitudinal incision was made on the posterior aspect of the proximal tibia . A white, shiny, synovium - like piece of tissue emerged from the incision, and this was identified as a sparganum that measured 30 cm in length and 0.5 cm in width and was wriggling after removal from the patient (fig . The calcified foci near the calf were also removed, and these tracts possibly represented the pathway along which the larvae had passed . Sixteen months after the operation, the patient revisited our hospital because of pain and paresthesia of the right thigh and a mass at the posterior aspect of the right distal thigh . Physical examination revealed generalized pain and a tingling sensation on the right thigh and multiple soft, mobile, non - tender masses at the posterior aspect of the right proximal tibia . Mri revealed a 1.8 cm - sized, well - circumscribed round mass arising at the right distal sciatic nerve . The lesion showed heterogeneous high signal intensity on t2-weighted images and intermediate signal intensity on t1-weighted images with a target sign or a fascicular sign, and a benign neurogenic tumor, such as a schwannoma or neurofibroma, was suspected (fig . 4). Multifocal tubular and cystic lesions were also detected around the mass, showing high signal intensity on t2-weighted images and low signal intensity on t1-weighted images (fig . An excisional biopsy was performed, and a histologic analysis revealed a schwannoma showing spindle tumor cells in a palisading pattern (fig . Approximately 1 cm near the mass, several spargana worms were detected and removed at the same time . Cho et al . Reported that 54 of 70 cases of sparganosis in korea were found in the subcutaneous tissue or intermuscular fascia, 7 cases in the body cavity, 6 cases in the orbit, 2 cases in the urinary tract, and 1 case in the submucosal tissue, but no cases occurred in muscles . However, kim et al . Reported a case of intramuscular sparganosis in the sartorius muscle in 2001, and kim et al . Recently, lee et al . Reported a case of living sparganum for 20 years migrating from the knee joint to the ankle joint along the calcified tract in 2010 . Ha et al . Also reported a case of sparganosis combined with bursitis involving the medial gastrocnemius muscle of the proximal lower extremity in 2011 . Here we report a case of intramuscular sparganosis involving the gastrocnemius muscle in a 77-year - old man . The clinical manifestations of sparganosis are diverse, such as palpable masses, non - specific discomfort, vague pain, headache, or even no symptoms, depending on the organs involved . Thus, it is quite difficult to differentiate sparganosis from a soft tissue tumor based on the clinical manifestation and preoperative radiographic findings . Although the precise function of eosinophils is unclear, they are anyway involved in destroying the parasitic organisms . A competitive elisa that uses sparganum - specific monoclonal antibodies has been developed to improve the diagnostic specificity of sparganosis . Even though our patient did not show definite eosinophilia, a monoclonal antibody - based competitive elisa was positive . Radiologic evaluation, such as plain radiography, ultrasonography, or mri, is also useful for the diagnosis of sparganosis . In our patient, preoperative ultrasonographic findings showed a heterogeneous, hyperechoic mass with a hypoechoic tubular lesion in the gastrocnemius muscle . This heterogeneous, hyperechoic mass is considered to be a granuloma that contains the living sparganum, and the internal hypoechoic tubular lesion is considered to be the empty migrating tract of the larva . Multiple calcifications seen on plain radiographs can also be considered the pathway along which the sparganum passed . In this case, we performed preoperative ultrasound - guided marking for suspected lesions . Therefore, we could easily find intramuscular sparganum during the surgery, although it is difficult to determine the location of the spargana with naked eyes in the operative field . Human sparganosis is a surgical disease because the diagnosis depends almost entirely on detecting larvae from the lesions . Thus, in areas where sparganosis is endemic, the presumptive diagnosis can be made preoperatively by an experienced surgeon if the patient has a history of eating raw meat, together with the size, migration, and crepitation of the mass . However, it is quite difficult to differentiate sparganosis from a soft tissue tumor based solely on the clinical manifestations and preoperative radiographic findings . Furthermore, because the incidence of this disease is extremely low, even in endemic areas, it is difficult for surgeons to suspect sparganosis based only on the preoperative data . Thus, an immunodiagnosis for sparganosis is recommended when soft tissue tumors are detected in patients living in endemic areas . In addition, sparganosis should be included in the differential diagnosis of soft tissue tumors, especially among patients who frequently consume mountain water or raw snake or frog meat . In our case, although definite eosinophilia was not detected, we could suspect intramuscular sparganosis involving the gastrocnemius muscle based on the patient's habit of ingesting raw snake meat during his adolescence, multiple calcifications on plain radiography, a heterogeneous, hyperechoic mass with a hypoechoic tubular lesion on ultrasonography, multiple tubular and cystic lesions in the gastrocnemius muscle on mri, and the positive elisa using sparganum - specific monoclonal antibodies . In addition, we could easily remove the intramuscular sparganum with the aid of preoperative ultrasound - guided marking for suspected lesions . In our case, the patient revisited our hospital because of pain and paresthesia of the right thigh, and in the operative field, approximately 1 cm near the schwannoma at the right distal sciatic nerve, several sparganum were detected and removed . Schwannomas are tumors arising from the schwann cells of the neural sheaths of the motor and sensory nerves, and if schwannomas occur in the lower limb, they are found in association with either the sciatic nerve (1%) or the posterior tibial nerve . All ages and both sexes may be affected, but schwannomas are most frequently seen in patients between 40 and 50 years of age . Benign schwannomas are generally slow - growing tumors arising from the schwann cells of the peripheral nerve sheath, so patients with a sciatic schwannoma are usually asymptomatic until a local mass compresses the sciatic nerve . These masses are more often associated with the sensory aspect of the nerve; thus, surgical excision is the treatment of choice in symptomatic sciatic schwannomas . Although the etiology of these benign neurogenic tumors is unknown, in our patient, we considered that intramuscular sparganum might irritate the neighboring sciatic nerve, and we removed all the adjacent spargana worms.
Reversible phosphorylation of serine, threonine, and tyrosine residues is one of the most common and important regulatory posttranslational modifications of proteins and plays a crucial role in many biological processes, including cellular signal transduction, regulation of enzyme activities, metabolic regulation, molecular recognition and biomolecular interaction, protein localization, cell differentiation and proliferation [15]. Therefore, identifying phosphorylated proteins is an important step to elucidate the structural and functional role of the protein's phosphorylation regulation . Up to now, mass spectrometry, including matrix - assisted laser desorption / ionization time - of - flight mass spectrometry (maldi - tof - ms) and electrospray ionization mass spectrometry (esi - ms) have been key technologies in characterizing protein phosphorylation and phosphoproteome, and each approach presented strengths and limitations [615]. However, because of the low and dynamic stoichiometry of phosphorylation on proteins [16, 17] and the low ionization efficiency of phosphopeptides, it remains challenging to thoroughly characterize phosphorylation sites using mass spectrometry . Considerable effort has been made recently to improve the ionization efficiency of phosphopeptides by selecting and optimizing appropriate matrices with maldi - tof - ms . Several materials have been employed in phosphopeptide analysis, including 2, 5-dihydroxybenzoic acid (dhb) [1921], 2, 4, 6-trihydroxyacetophenone (thap), and ionic liquid matrices . In addition, ammonium salts, including diammonium hydrogen citrate (dahc) [2224], ammonium acetate and monoammonium phosphate, and phosphoric acid [1921] have been used as matrix additives and have shown some ability to enhance phosphopeptide ionization during maldi - tof - ms . 2, 6-dihydroxyacetophenone (dhap) was first used by gorman et al . As a matrix for maldi analysis of fragile peptides, disulfide bonding, and small proteins, including phosphopeptides . In recent years, xu et al . Reported that the dhap / dahc matrix combination was used as an effective maldi matrix in detecting methyl esterified phosphopeptides by maldi - tof - ms [26, 27]. After systematic optimization in this study, we found that contrary to previous reports, the matrix forms at a lower ratio of dhap / dahc and that this ratio shows greater efficiency in improving ionization ability of phosphopeptides . We investigated the characteristics of an optimal dhap / dahc matrix, and analyzed morphology and detection sensitivity of phosphopeptides . Using tryptic digests of - and -casein, we demonstrated a cid ms / ms analysis of phosphopeptides by maldi - tof / tof - ms with an optimized dhap / dahc matrix . Finally, we used this approach to analyze phosphorylation sites on human histone h1 treated with cyclin - dependent kinase-1 (cdk1). The chemicals 2, 6-dihydroxyacetophenone (dhap), 2, 5-dihydroxy - benzoic acid (dhb), diammonium hydrogen citrate (dahc), ammonium bicarbonate, trifluoroacetic acid (tfa), alkaline phosphatases, -casein and -casein (both from bovine milk, purity by electrophoresis) were all purchased from sigma aldrich (st . Louis, mo, usa). Cyclin - dependent kinase-1(cdk1) and human histone h1 were purchased from new england biolabs, inc . The standard peptides vnqigptlsesik (pt, purity> 95%) and sgslhripythqs (py, purity> 85%) were synthesized in beijing scilight biotechnology ltd . Sequencing grade modified trypsin (porcine) was purchased from promega co. (madison, wi, usa). Hplc - grade acetonitrile and acetic acid were purchased from mallinckrodt baker, inc, (phillipsburg, nj, usa). Ethanol and ammonium hydroxide solutions (nh3 h2o) were purchased from beijing chemical company (beijing china); ultrapure water was obtained from a milli - q system (millipore, bedford, ma, usa). The dhap matrix stock solution was prepared by dissolving 10 mg of dhap in 1 ml of anhydrous ethanol . A series of modified dhap / dahc matrix solutions were prepared by mixing dhap stock solution with dahc stock solution at different ratios from 10: 1 to 1: 80 (v / v). The dhb / phosphoric acid (pa) matrix was prepared as previously reported; we modified this previous protocol by dissolving 20 mg of dhb in 1 ml of 50% aqueous acetonitrile containing 1% phosphoric acid . -casein and -casein (100 nm in 50 mm ammonium bicarbonate buffer at ph 8.3) were digested overnight at 37c with trypsin at an enzyme / substrate ratio of 1: 25 (w / w). After digestion, tfa was added into the sample solution at 1.0% to quench the reaction . The digest was diluted with 60% acetonitrile containing 0.1% tfa to produce a series of sample solutions at concentrations of 1 pmol/l, 200 fmol/l, 50 fmol/l, 20 fmol/l, and 10 fmol/l . A stock solution containing standard peptides pt (1 mm) and py(1 mm) was prepared using ultra pure water . The solution was diluted with 60% acetonitrile containing 0.1% tfa to 1 pmol/l, 200 fmol/l, 50 fmol/l, 20 fmol/l, and 10 fmol/l . All of the peptide solutions prepared for dhb / pa measurements were diluted with 1% phosphoric acid . Briefly, the reaction solution was prepared with 3 l 10 cdk1 reaction buffer, 0.15 l cdk1 (150 u), 4 l histone h1 (1 g/l), 100 m atp, and 13 l water, then incubated at 30c for 45 minutes . The phosphorylated histone h1 was then separated using sds - page (12%) and stained with coomassie brilliant blue using microwave - assisted methods . The in - gel digestion of phosphorylated histone h1 was performed as follows: the band of histone h1 was excised from the polyacrylamide gel, washed twice with water, and destained with 40% acetonitrile/50 mm nh4hco3 . The gel pieces were dehydrated with 100% acetonitrile and dried for 5 min using a speedvac . Disulfide bonds were reduced with dtt (10 mm, 56c, 45 minutes), and the free sulfhydryl groups were alkylated with iodoacetamide (55 mm, 25c, 60 minutes in the dark). Gel pieces were washed with 50 mm nh4hco3, 50% acetonitrile/50 mm nh4hco3, and dehydrated with 100% acetonitrile . After drying with a speedvac, the gel was rehydrated using 100 ng/l trypsin (50 mm nh4hco3, ph 8.3) on ice for 30 minutes, and the digestion was carried out at 37c for 60 minutes and then quenched with 1.0% tfa . The tryptic peptides were extracted twice with 60% acetonitrile containing 0.1% tfa, and then the combined digest solution was concentrated to 10 l under vacuum . Maldi - tof - ms analysis of peptides was performed using an axima - cfr plus maldi - tof mass spectrometer (shimadzu / kratos, manchester, uk) equipped with a pulsed nitrogen laser operated at 337 nm . Positive / negative ion maldi mass spectra were acquired in the reflectron mode under the following parameters: ion source, 20/20 kv, lens, 6.3/6.3 kv, pulsed extraction, 2.5/2.5 kv, reflection, 25/25 kv . High - energy cid ms / ms was performed with the axima - tof maldi mass spectrometer (shimadzu / kratos, manchester, uk), equipped with the same pulsed 337-nm nitrogen laser . Operation parameters were: ion source, 20.0 kv; lens, 6.5 kv; pulsed extraction, 2.5 kv; and reflectron, 24.4 kv . To prepare for maldi - tof - ms analysis, 1 l of peptide solution was mixed with 3 l of matrix solution in an eppendorf tube, and 1 l of the peptide / matrix solution was spotted onto the maldi sample plate and then crystallized either under vacuum (when dhap / dahc was used as matrix) or in the air (when dhb / pa was used as matrix). Measurements using dhap / dahc were performed by detecting 1520 different positions on the broad outer zone (see discussion below) for each sample to accumulate a total of 200 profiles (5 shots per profile). For measurements with dhb / pa, a total of 200 profiles (5 shots per profile) were accumulated by searching for 1015 hot spots for each sample . The standard peptide pt (250 fmol on target) was used to determine sample homogeneity of the matrix . On each sample spot, 196 (14 14) positions were selected manually to ensure a full overview over the sample preparation . For each position, 50 profiles (5 shots per profile) were accumulated in positive ion mode . Mass spectrometry data were processed with launchpad 2.7.1 software (shimadzu / kratos, manchester, uk). The data of the cid ms / ms spectra of phosphopeptides from histone h1 were compared to the swissprot 56.6 protein sequence database (homo sapiens) using the on - line mascot database search engine (matrix science, london, uk) with the following parameters: trypsin with three missed cleavages; average mass values; peptide mass tolerance: 0.8 da; fragment mass tolerance: 1.0 da; fixed modifications: carbamidomethyl (c), variable modifications: oxidation (m), phosphor (st). Only the peptides with ions score above 38 the solution composition of the matrix is a factor that significantly influences the ionization efficiency and the quality of maldi mass spectra . Therefore, we prepared a series of different ratios of dhap (10 mg / ml) and dahc (20 mg / ml) matrix solutions (10: 1, 5: 1, 1: 1, 1: 4, 1: 8, 1: 16, 1: 20, 1: 30, 1: 40, 1: 50, 1: 60, and 1: 80 v / v) and investigated these matrix combinations using the tryptic digest of -casein, which contains nine phosphopeptides (t1-t9) and some nonphosphopeptides (such as t4, t10, t11, t18) (see table 1). Figure 1 shows a comparison of the maldi - tof - ms analysis of the tryptic digest of -casein (250 fmol on target) using matrix dhap / dahc at different ratios in positive ion mode . The signal intensity of phosphopeptides (especially for multiple phosphorylated peptides) changed dramatically with the ratio of dhap / dahc . It was clear that all phosphopeptides could be easily detected with a low dhap / dahc matrix ratio in the range of 1: 30 to 1: 60 . In an optimal matrix solution, the concentration of dhap was 1.63.3 mg / ml, which is lower than the concentration of 1015 mg / ml used by other groups [24, 26, 27]. Once the dhap / dahc ratio was down to 1: 80, most multiple phosphorylated peptides could no longer be detected, likely because there were not sufficient numbers of dhap molecules to transfer the laser energy to the peptides to be protonated . Another interesting phenomenon apparent in figure 1: the signal intensity of nonphosphopeptides was stable (t3), enhanced (t10) or weakened (t11, t4, t18) with the change of the dhap / dahc ratio . We calculated the bull and breese index (bb index) of these peptides, an index generally used to reflect the hydrophobic / hydrophilic nature of peptides . Higher bb index values are associated with more hydrophilic peptides . For the phosphopeptide, we calculated the bb index by replacing the phosphoserine or phosphothreonine residues with glutamic acid . By comparing the bb index of peptides listed in table 1, we found that the phosphopeptides were nearly hydrophilic peptides with high bb indices and that the nonphosphopeptides for which signal intensity was weakened at low dhap / dahc ratio were highly hydrophobic with lower bb index (t4: 5530, t11: 6480, t18: 6900). The nonphosphopeptide t10 for which the ion signal was enhanced at lower ratios of dhap / dahc was found to be slightly hydrophobic with a bb index of 750 . These findings indicated that the optimal dahc / dhap matrix tended to detect more hydrophilic peptides . Most phosphopeptides are hydrophilic, so we propose that the low ratio of dhap / dahc matrix could increase cocrystallization between matrix and phosphopeptides and further improve ionization efficiency of phosphopeptide in maldi ms . In this study, the phosphopeptide pt (250 fmol on target) was used to investigate the sample homogeneity of optimized dhap / dahc matrix, and for a comparison, we chose matrix combination of 2,5-dhb with 1% phosphoric acid, a currently popular matrix used for detection of phosphopeptide [3032]. It was apparent from the photographs of the maldi sample spots that there was a dramatic difference between the optimized dhap / dahc (figure 2(a)) and dhb / pa (figure 2(b)). Small crystals formed in a dhap / dahc sample spot while larger, needle - like crystals formed in a dhb / pa sample spot . Furthermore, 196 (14 14) positions on the target were measured covering the crystalline layer for a full overview (figure 2(e)), and the ms signal intensities of pt were monitored . The majority of the outer zones of the crystalline samples exhibited high signal intensities, except for a small center zone when dhap / dahc used as matrix (figure 2(c)). In comparison, the crystalline dhb / pa matrix exhibited the hot - spot phenomenon, signals were primarily observed in the narrow, inhomogeneous ring at the edge of the sample layer, and the inner position almost did not exhibit any signal (figure 2(d)). Therefore, the samples prepared with dhap / dahc were more homogeneous than those prepared with a dhb / pa matrix, reducing the need for time - consuming hot - spot searches . Based on the sample homogeneity of dhap / dahc and dhb / pa, in the next experiments, the data acquisition with dhap / dahc was carried out by randomly measuring the outer zone of the sample layer, while data were acquired from the dhb / pa matrix by searching for hot spots of sample . In order to further evaluate the measurement sensitivity of phosphopeptides with the optimal dhap / dahc matrix, the tryptic digest of -casein and -casein with 2.5250 fmol on the target were detected by maldi - tof ms in both positive and negative ion mode . Figure 3 shows that all eleven phosphopeptides from -casein and -casein could be detected with signal - to - noise (s / n) ratios greater than 3 at the low amount of 12.5 fmol in the positive mode (a) and (b) and detected at a lower amount of 5 fmol in the negative mode (c) and (d). The monophosphopeptides (t1 and t1) could still be observed at 2.5 fmol on target in negative ion mode (figure 3(c)). Phosphopeptides generally exhibited better s / n ratios in the ms spectra in negative ion mode than that in positive ion mode, although, consistent with previous reports, the absolute signal intensity of phosphopeptide was usually lower in negative ion mode . Because all tryptic phosphopeptides from -casein and -casein are serine - phosphorylated peptides, a threonine - phosphorylated peptide pt and a tyrosine - phosphorylated peptide py (2.5250 fmol on target) were used to investigate whether the dhap / dahc matrix had a generic nature in detection of phosphopeptides . As expected, pt and py could be readily detected down to 5 fmol on the target in both positive and negative modes (figure 4). We further directly compared optimized dhap / dahc to dhb / pa . Tryptic digests of -casein and -casein (12.5250 fmol on target) were detected using these two matrices (figure 5). When using the dhap / dahc matrix, all the phosphopeptides could readily be detected at three different sample amounts . By comparison, when dhb / pa was used as the matrix, although all the phosphopeptides in 250 fmol digest (on target) could be detected with dhb / pa, the amount of digest went down to 50 fmol (on the target), and only t1, t2, t3, t6, t1, and t2 could be observed . Furthermore, when the amount of digest went down to 12.5 fmol, only t1 and t1 were detected, with weak signals . Comparing these results, it was clear that compared with dhb / pa, the optimized dhap / dahc had comparative sensitivity in the detection of singly phosphorylated peptides (t1 and t1), but greater sensitivity in the detection of multiply phosphorylated peptides, such as t2 (4p) and t4 (5p). Herein, we suggest that sample should be analyzed immediately after sample preparation when optimized dhap / dahc matrix used, as the crystals might not last too long under atmospheric conditions and in vacuo . We employed the axima - tof maldi mass spectrometer (shimadzu / kratos, manchester, uk) to perform high energy cid ms / ms of phosphopeptides to characterize phosphorylation sites . Figure 6 shows the ms / ms spectra of three phosphopeptides t1, t3, and t2 from -casein and -casein (250 fmol on target) with dhap / dahc as matrix . The number of [mh-98] or [mh-80] peaks in the ms / ms spectra indicated the number of phosphate groups in each phosphorylated peptide, and the fragment ions including a, b, and y ions clearly show the phosphorylation sites of the phosphopeptides t1, t3, and t2 . These results demonstrated the feasibility of maldi - tof / tof ms with the dhap / dahc matrix for the analysis of the phosphorylation sites of phosphopeptides, including singly and multiply phosphorylated peptides . Further investigating the practicality of the dhap / dahc matrix experimentally, phosphorylated histone h1 was separated by sds - page and in - gel digested by trypsin . In order to generate appropriate peptide sizes for maldi - ms analysis, since the sequence of histone proteins is known to contain many lysine and arginine residues, the trypsin digestion time was optimized and reduced to 1 hour . Figure 7(a) shows the results of the maldi - tof - ms analysis of the tryptic digest of the phosphorylated histone h1 without the desalting step, directly detected with the optimized dhap / dahc matrix in positive ion mode . After performing data processing using the on - line mascot search engine, 31 peptides, including 2 possible phosphopeptides (in figure 7(a) labeled with *), were assigned to human histone h1, and 81% of the sequence was covered . These two peptides corresponded to the two monophosphopeptides of histone h1, comprising amino acids 117127 (vatpkkaskpk, 1234.7 m / z; possible phosphorylation sites indicated with underlined letters) and 133145 (aptkkpkatpvkk, 1473.9 m / z). The tryptic sample of h1 was also analyzed with dhb / pa, but the two peaks of the phosphopeptide were lower than those in figure 7(a) (see figure s1). To validate the mascot searching result for the two phosphorylated peptides, we compared the mass spectra of tryptic peptides of h1 before and after treatment with alkaline phosphatase, which can cleave the phosphate group from phosphopeptides . It was clear that the two peptide peaks (marked with asterisks in figure 7(a)) had disappeared and two new peaks with a mass shift of 80 da (hpo3 = 80 da) were visible (figure 7(b)). This demonstrated that the two peptides (marked with asterisks in figure 7(a) were singly phosphorylated peptides . To identify the phosphorylation sites, we used maldi - tof / tof ms to perform ms / ms analysis of the two peptides (figure 7(c) and 7(d)). Only one neutral - loss peak corresponding to [mh - h3po4] indicated that both phosphopeptides were monophosphopeptides . After a mascot search, the phosphorylation sites of the two phosphopeptides were determined to be aptkkpkaptpvkk (pt indicating the phosphothreonine) and vaptpkkaskpk with ion scores of 53 and 50, respectively (scores exceeding 38 were accepted as significant matches). In general, the protein phosphorylation sites by a particular protein kinase shared a set of consensus sequence, which is necessary and sufficient for recognition by the kinase . To further validate the phosphorylation sites of histone h1 identified with maldi - tof / tof ms, we compared the two phosphorylated peptide sequences with the consensus sequence ps / pt - p - x - r / k, most frequently recognized by cdk1 [34, 35], and found that both phosphopeptide sequences were consistent with the consensus sequence . These results thus demonstrate that the dhap / dahc matrix was robust and effective in analyzing the protein phosphorylation of the biological sample by maldi - ms . By optimizing the matrix solution composition, we found that a low ratio of dhap / dahc formulation was more efficient in detecting phosphopeptides than earlier protocols . Compared with dhb / pa, the optimized dhap / dahc exhibited higher sample homogeneity, and the phosphopeptides in tryptic digests of - and -casein without desalting and phosphopeptide enrichment could be measured with a higher sensitivity . Further, the optimized dhap / dahc showed high energy cid ms / ms analysis of the phosphorylation sites of phosphopeptides, including multiple phosphorylated peptides, and we successfully applied this method to the characterization of the phosphorylation sites of cdk1-treated human histone h1.
Methyleugenol (chart 1a, cas #93 - 15 - 2) is the methyl ether of eugenol (chart1b, cas #97 - 53 - 0), which is itself a major constituent of spices such as clove, allspice, and bay leaves and is known to induce contact allergies . Methyleugenol (meu) and eugenol (eug) are allylalkoxybenzene derivatives that are present as flavoring constituents in foodstuffs or are added as flavor extenders . Their chemical structures resemble those of safrole, a known carcinogen, and estragole . A safety assessment of allylalkoxybenzene derivatives was prepared by the expert panel of the flavor and extract manufacturers association, which summarized previous findings about the safety of meu . They conclude that the present human exposure to meu does not pose a significant cancer risk . By contrast, the twelfth report on carcinogens of the national toxicology program concludes that meu is reasonably anticipated to be a human carcinogen . This finding is based on the observation of increased incidence of malignant tumors in the liver, bile duct, glandular stomach, kidney, mesothelium, mammary gland, and skin in experimental animals . The mechanism of hepatic carcinogenesis appears to result from the bioactivation of meu to dna - reactive intermediates via hydroxylation of the 1 position of the allylic side chain and subsequent sulfation to generate 1-sulfoxymethyleugenol, conversions that can occur in the liver and that have also been demonstrated to occur in the human liver.in vitro studies have identified the human liver cytochrome p450 1a2 as the enzyme that is the likely bioactivator of meu . A 5-fold difference in activities occurred among 15 human microsome samples in a correlation study, which suggested that individual differences, arising from lifestyle factors such as smoking or drug use, can influence the likelihood that meu exposure will lead to harmful effects . Additionally, the european commission s scientific committee on food formulated an opinion on meu, based largely on the rodent studies, concluding it to be a multisite, multispecies genotoxin and carcinogen for which no threshold could be assumed and for which no safe exposure limit could be established . The use of pure meu has been banned in the european union but not in the u.s.a . The biological and biochemical mechanisms by which alkenylbenzenes cause mutagenesis and carcinogenesis are areas of active research . Bioactivation of these molecules to their ultimate carcinogens takes place through cytochromes p450 and sulfotransferases . Kinetic studies in vitro of [c]meu and [c]eug in human, mouse, and rat liver and lung tissue fractions showed that the compounds are metabolized differently: meu is converted to significant amounts of the 1-hydroxy proximate carcinogen, but eug is glucuronidated, thus avoiding the formation of 1-hydroxyeugenol . In a study of meu and its oxidative metabolites in chinese hamster lung fibroblasts, marginal cytotoxic effects were not observed, but meu and 1-hydroxymethyleugenol at concentrations of 10 m caused dna strand breaks . Meu bioactivation and detoxification were studied by a physiologically based kinetic approach comparing human and rat models, and the results obtained revealed that there were no substantial species differences in the formation of the reactive ultimate carcinogenic l-sulfoxymethyleugenol metabolite . Meu is a ubiquitous environmental component with annual use in excess of 30,000 kg in the united states food, perfume, and pesticide industries . It is present in soaps and perfumes as a fragrance, in commercial food products as a flavorant, and in pesticides as an insect attractant . High resolution mass spectrometric analyses of serum samples from 213 nonfasting adult participants in the third national health and nutrition examination survey detected methyleugenol in 98% of samples at a mean concentration of 24 pg / g (range <3.1390 pg / g). Fasted human volunteers who consumed controlled amounts of meu had prefeeding serum concentrations averaging 16.2 pg / g meu . Mean serum levels increased following meu consumption and then decreased over time with metabolism: 53.9 pg / g (15 min); 42.9 (30 min); 37.0 (60 min); and 25.2 (120 min) with an elimination half - life of ca . The total human oral intake of meu has been estimated as 0.77 g / kg - bw / d, of which 14% is added meu . Since meu is carcinogenic in extrahepatic tissues which have lower levels of cytochrome p450s and sulfotransferases, we decided to investigate the oxidation of meu and the related eug by peroxidases, which are widely distributed in extrahepatic tissues . Although phenoxyl radicals of eug have been investigated by electron paramagnetic resonance spectroscopy (epr), no reports of meu radicals have appeared . Okada, et al ., satoh et al ., and fujisawa et al . Observed phenoxyl radicals of eug in alkaline solution but do not report any epr hyperfine analysis . Nakagawa et al . Reported resolved and assigned epr spectra for sesmolyl and related phenoxyl radicals that were generated by continuous uv - irradiation of benzene solutions of the corresponding phenols . Oxidized eug to its phenoxyl radical using hrp in a fast - flow system and report epr parameters similar to those for related methoxy - substituted phenoxyl radicals . Although there have been no previous reports of epr observations of meu radical cations, there have been literature reports of chemical or electrochemical production of methoxybenzene radical cations . Zweig and co - workers used controlled potential electrolysis to generate methoxy - substituted benzene cation radicals in situ for epr studies . Siero and co - workers observed highly resolved epr spectra of radical cations generated by phenyl iodine(iii)bis(trifluoroacetate) [pifa] as the electron acceptor in a charge transfer complex formed by methoxybenzenes and pifa in hexafluoropropanol solution . Also kersten and co - workers reported the biochemical generation of methoxybenzene radical cations of 10 of the 12 possible methoxybenzene congeners, including cis- and trans-1,4-dimethoxybenzene, 1,2,3,4-tetramethoxybenzene, and 1,2,4,5-tetramethoxybenzene by ligninase peroxidase and hydrogen peroxide . By contrast, hrp was only able to oxidize the four congeners with the lowest half - wave oxidation potentials . Biochemical oxidation of these molecules produced methoxy - substituted benzoquinones and methanol and demonstrated that the quinone oxygen atoms were derived from the solvent water . In this study, we report the first observation of radical cations of meu produced by hrp metabolism . Observation of radical cations of several related compounds is also reported, and the epr spectral assignments for this series of radicals form a consistent pattern . Free radical metabolites have been widely implicated as contributing to disease states and may be involved in meu carcinogenicity in extrahepatic tissues . Methyleugenol (4-allyl-1,2-dimethoxybenzene); eugenol (4-allyl-2-methoxyphenol); 3,4-dimethoxytoluene; 2-methoxy-4-methylphenol; 3,4-dimethoxyphenol; cerium(iv) sulfate; sulfuric acid; dtpa; hemoglobin; and hrp (type vi) were purchased from sigma - aldrich (st . Louis, mo). The buffer for those reactions using it was 50 mm, ph 7.4, phosphate buffer prepared by diluting a 500 mm solution treated with chelex 100 resin (bio - rad, hercules, ca) to remove adventitious transition metal ions . These solutions also contained either 1.0 mm or 0.1 mm dtpa to further hinder trace metal catalysis . The h2o2 concentration was confirmed by absorbance measurements at 240 nm (240 = 39.4 m cm). In some experiments, the commercial preparation of hrp was loaded onto a pd-10 gel filtration column and eluted with 50 mm phosphate buffer, ph 7.4, treated with chelex before use . The concentration of hrp was determined from the soret maximum at 412 nm (native hrp = 102 mm cm). Unstable intermediates were detected by means of fast - flow epr experiments performed in a manner similar to that in our previous reports . Reagents placed in solution reservoirs were pumped though plastic hoses by means of a masterflex l / s microprocessor - controlled peristaltic pump (cole - parmer, vernon hills, il) to a quartz fast - flow mixing chamber flat cell (type wg-804, 10 mm width, wilmad glass co., buena, nj). Flow rates for each solution could be selected from 10 ml / min to greater than 100 ml / min . Epr measurements were made using an elexsys epr spectrometer operated at a frequency near 9.7 ghz and a magnetic field near 348 mt (bruker biospin, billerica, ma). The magnetic field at the er4122 super hi - q microwave cavity was modulated at 100 khz, affording the first - derivative epr spectra that were recorded as computer files using bruker s software . Subsequent analysis, interpretation, and simulation of the spectra used locally produced software . Absorption spectra were recorded on a cary 100 uv visible spectrometer (varian, victoria, australia) using 1.0 cm - path length quartz cuvettes . Hydroperoxide tests used quantofix peroxide test strips (sigma - aldrich, inc ., milwaukee, wi) that have a sensitivity reported to detect a lower limit of 1 mg / l peroxide . Northampton, ma) or coreldraw (corel corporation, ottawa, canada) software packages . Meu oxidized by hrp activated by hydrogen peroxide in a fast - flow system produced a well - resolved epr spectrum of the corresponding radical cation (figure 1a). Essentially the same spectrum resulted when meu was oxidized chemically by ce(iv) in sulfuric acid solution (data not shown). The same spectrum was also produced when meu was oxidized by hemoglobin and hydrogen peroxide in a fast - flow system (data not shown). When either meu (figure 2b) or hrp (figure 2c) was omitted, no epr spectrum was observed, as expected . However, when hydrogen peroxide was omitted, a strong epr signal was still observed (figure 2d), and the same spectrum was observed in the presence of catalase at a concentration 70 times that of the peroxidase (figure 2e). Epr fast - flow spectra of the meu radical produced in a system of meu, h2o2, and hrp . The concentrations of meu, h2o2, and hrp in the flat cell were 1.75 mm, 13 mm, and 0.46 m, respectively . Oxygen was removed from the reagent solutions by bubbling 99.9999% pure nitrogen gas through them . Equal volumes of a solution of meu / h2o2 and a solution of hrp in 100 mm phosphate buffer, ph 7.4, were mixed milliseconds before they entered the flat cell at a total flow rate of 40 ml / min . The final solution also contained 10% v / v ethanol and 0.10 mm dtpa . (b) simulated epr spectrum with the coupling constants given in table 1 . (c) residual plot produced when simulated spectrum b is subtracted from experimental spectrum a. epr spectra were recorded at 20 mw microwave power, 0.10 mt field modulation, 5.0 mt field sweep width, 164 ms time constant, 82 ms conversion time, and 33 scans of 1024 data points . Epr fast - flow spectra of the meu radical produced in a system of meu, h2o2, and hrp . The concentrations of meu, h2o2, and hrp in the flat cell were 1.75 mm, 13 mm, and 0.46 m, respectively . Oxygen was removed from the reagent solutions by bubbling 99.9999% pure nitrogen gas through them . Equal volumes of a solution of meu / h2o2 and a solution of hrp in 100 mm phosphate buffer, ph 7.4, were mixed milliseconds before they entered the flat cell at a total flow rate of 40 ml / min . The final solution also contained 10% v / v ethanol and 0.10 mm dtpa . (b) as in panel a but with no meu . (c) as in panel a but with no hrp . (e) as in panel a but with no h2o2 and 32 m catalase added . Numbers in parentheses are numbers of equivalent hydrogen atoms contributing to the stated hyperfine coupling . Coupling constants were not resolved and are less than the linewidths of epr lines, ca . Coupling constants in square brackets are literature values reported by ref (26). Alkenylbenzenes, such as meu, with allylic hydrogen atoms are known to be susceptible to autoxidation reactions that produce organic hydroperoxides . The quantofix test strip assay of stock meu indicated the presence of hydroperoxides in the range of 10 to 30 mg / l . The presence of endogenous hydroperoxides in stock meu and the ability of hydroperoxide to activate hrp and sustain the oxidation of meu to its radical were established by uv the spectrum obtained for native hrp before the addition of h2o2 and/or meu exhibits two characteristic bands at 502 and 642 nm and the soret band at 402 nm (figure 3a). The addition of h2o2 resulted in the immediate decrease in absorbance of the soret band and the appearance of peaks with maxima at approximately 565 and 650 nm, which are associated with the formation of compound i. the addition of 1.75 mm meu 2 min after the addition of h2o2 caused a shift of the soret peak from 402 to 419 nm and the appearance of two peaks at 528 and 557 nm that are characteristic of compound ii (figure 3b). During the first 4 min following the addition of meu, the soret intensity increased quickly, after which it remained at a constant level . Visible detection of enzymatic intermediates produced in a system of meu, h2o2, and hrp . The reaction mixtures consisted of hrp (5.3 m) in 50 mm phosphate buffer, ph 7.4, at 25 c . The dotted lines are for native hrp before the addition of h2o2 and/or meu . Absorption spectra were recorded every 2 min after the initiation of the reaction . (a) spectral changes from native hrp to compound i. the reaction was initiated by the addition of 10 m h2o2 . Compound i was prepared as described in a, and 1.75 mm meu was added 2 min after the addition of peroxide . (c) formation and spontaneous decomposition of compound ii in the absence of exogenously added h2o2 . In this case, the reaction was initiated by the addition of 2.5 mm meu, and scans were recorded every 2 min . The presence of endogenous hydroperoxide in the stock of meu was investigated by incubating hrp with meu in the absence of exogenously added h2o2 . The spectral variation observed between 2 and 10 min after the addition of meu is presented in figure 3c . The first scan following the addition of meu to the native enzyme showed the formation of species similar to compound ii, with absorption maxima at 419, 528, and 557 nm . The subsequent spectra showed that compound ii slowly reverted to the native form . None of the spectra observed showed the dramatic decrease in absorbance of the soret band and the absorption peaks at 565 and 650 nm that are characteristic of compound i, in accordance with the instability of this intermediate in the presence of one - electron reductants . That an endogenous hydroperoxide was effectively responsible for the accumulation of compound ii in the reaction system has been tested by the addition of catalase . Similar changes in spectra were obtained when catalase was added immediately before the injection of meu in the above reaction mixture (not shown). However, in the presence of the h2o2 scavenger, slightly less compound ii was generated, but the effect was small, indicating that the endogenous hydroperoxide did not react well with catalase (data not shown). The epr spectrum of the 3,4-dimethoxytoluene radical cation (figure 4) was recorded and interpreted to assist the assignment of epr hyperfine coupling constants in the meu radical cation with ce(iv) in 0.23 m sulfuric acid solution serving as the oxidizing agent . Oxidation of 3,4-dimethoxytoluene by hrp and hydrogen peroxide produced only a very weak epr signal under our fast flow conditions (data not shown). Epr fast - flow spectra of the 3,4-dimethoxytoluene radical cation produced in a system of 3,4-dimethoxytoluene, cerium(iv) sulfate, and h2so4 at concentrations in the flat cell of 1.0 mm, 0.9 mm, and 0.225 m, respectively . Oxygen was removed from the reagent solutions by bubbling 99.9999% pure nitrogen gas through them . Equal volumes of 3,4-dimethoxytoluene and ce(iv) solutions were mixed milliseconds before entering the flat cell at a total flow rate of 20 ml / min . (b) simulated epr spectrum with the coupling constants given in table 1 . (c) residual plot produced when simulated spectrum b is subtracted from experimental spectrum a. epr spectra were recorded at 20 mw microwave power, 0.025 mt field modulation, 6.0 mt field sweep width, 1.3 s time constant, 655 ms conversion time, and 36 scans of 1024 data points . A well - resolved epr spectrum of eugenol phenoxyl radical, reported in figure 5, was observed when eugenol was oxidized by reaction with hrp and hydrogen peroxide in a fast flow system . This spectrum was recorded with somewhat better signal - to - noise and resolution than that reported previously . Essentially the same spectrum was observed when eugenol was oxidized chemically by ce(iv) in 0.23 m sulfuric acid solution (data not shown). Epr fast - flow spectra of the eugenol phenoxyl radical produced in a system of eugenol, h2o2, and hrp at concentrations in the flat cell of 1.0 mm, 13 mm, and 0.23 m, respectively . Oxygen was removed from the reagent solutions by bubbling 99.9999% pure nitrogen gas through them . Equal volumes of a solution of eugenol / h2o2 and a solution of hrp in 100 mm, ph 7.4, phosphate buffer were mixed milliseconds before entering the flat cell at a total flow rate of 60 ml / min . The final solution also contained 10% v / v ethanol and 0.10 mm dtpa . (a) complete system with eugenol, h2o2, and hrp . (b) simulated epr spectrum with the coupling constants given in table 1 . (c) residual plot produced when simulated spectrum b is subtracted from experimental spectrum a. epr spectra were recorded at 20 mw of microwave power, 0.025 mt of field modulation, 5.0 mt of field sweep width, 164 ms time constant, 82 ms conversion time, and 12 scans of 1024 data points . The epr spectrum of 2-methoxy-4-methylphenol phenoxyl radical (figure 6) was recorded and interpreted to assist the assignment of epr hyperfine coupling constants in the eugenol phenoxyl radical . Epr fast - flow spectra of the 2-methoxy-4-methylphenol radical cation produced in a system of 2-methoxy-4-methylphenol, cerium(iv) sulfate, and h2so4 at concentrations in the flat cell of 1.0 mm, 0.9 mm, and 0.225 m, respectively . Oxygen was removed from the reagent solutions by bubbling 99.9999% pure nitrogen gas through them . Equal volumes of 2-methoxy-4-methylphenol and ce(iv) solutions were mixed milliseconds before they entered the flat cell at a total flow rate of 40 ml / min . (b) simulated epr spectrum with the coupling constants given in table 1 . (c) residual plot produced when simulated spectrum b is subtracted from experimental spectrum a. epr spectra were recorded at 20 mw of microwave power, 0.050 mt of field modulation, 6.0 mt of field sweep width, 164 ms time constant, 82 ms conversion time, and 33 scans of 1024 points . Kersten et al . Were unable to oxidize any of the dimethoxybenzene compounds (or any related compounds with oxidation potentials greater than about 1.36 v) with the hrp - hydrogen peroxide system . (all oxidation potentials have been corrected to volts vs the standard hydrogen electrode .) The oxidation potential of 1,2-dimethoxybenzene is reported as 1.69 v, while that for allylbenzene is about 2.6 v. the structurally similar compound (3,4-dimethoxy)methylcinnamate has an oxidation potential of 1.7 v. the oxidation potential of hrp compounds i and ii is about 0.9 v near ph 7.4 depending on the isozyme, which suggests that meu might be oxidized successfully by hrp . The meu radical cation has a well resolved epr spectrum (figures 1a and 2a) and is accurately simulated (figure 1b) by the hyperfine coupling constants reported in table 1 . Subtraction of the simulation from the experimental spectrum results in a residual pattern that is essentially noise (figure 1c). When either meu (figure 2b) or hrp (figure 2c) is omitted from the incubation, no epr spectrum is recorded . However, even when h2o2 is omitted from the incubation, a strong meu epr signal is seen (figure 2d), and the same spectrum is observed when the incubation lacks h2o2 but contains 32 m catalase (figure 2e). These observations are consistent with the presence of an organic hydroperoxide in meu; this was confirmed by peroxide test strips that provided a semiquantitative estimate of 1050 mg / l peroxide concentration . Treating meu with alumina to remove data presented in figure 3a and b are the first clear spectral evidence that the oxidation of meu proceeds by a cycle typical of peroxidase involving compounds i and ii . Hrp compound ii was the predominant form of peroxidase observed in the presence of a high concentration of meu, and this intermediate was detected even in the absence of exogenously added h2o2 . The intensity of the characteristic absorption peaks of hrp compound ii decreases over time as the reaction proceeds, and the resting state of hrp is regenerated (figure 3c). A similar experiment with catalase added to the incubation demonstrated that the hydroperoxide is not affected by the presence of catalase, a result that is consistent with the epr observation (figure 2e). This indicates that the hydroperoxide formed by autoxidation of meu is not a good substrate for catalase and explains why inclusion of catalase in the epr incubation of meu had no effect (figure 2e). Presumably glutathione peroxidase, which is nonspecific, will reduce this unknown hydroperoxide and thereby inhibit oxidation of meu . The meu radical epr spectrum hyperfine pattern arises from large hyperfine couplings of the two hydrogens of the methylene group attached at the 4 position and one hydrogen atom attached at the 5 position, as would be predicted from the simple model of benzene molecular orbital ordering that results from two ortho - methoxy electron - releasing substituents . That the methylene hyperfine couplings are large is also evident from the epr spectrum of the 3,4-dimethoxytoluene radical cation (see figure 4a). This epr spectrum is simulated successfully (figure 4b) with hyperfine parameters quite close to those for meu (see table 1), and the residual pattern that results from subtracting the simulated from experimental spectrum is essentially noise (figure 4c). Successful simulation of the spectra of both radicals also requires the inclusion of hyperfine coupling from two sets of methoxy hydrogen atoms, which indicates that we are seeing the initial free radical species before any demethoxylation reaction can occur . The epr spectrum of the eug phenoxyl radical (figure 5a) has been reported previously but recorded at somewhat lower resolution and poorer s / n . Hyperfine coupling constants for this radical and for the 2-methoxy-4-methylphenol phenoxyl radical (figure 6a) are reported in table 1 and are consistent with the related radicals . They are characterized by large hyperfine couplings to two and three hydrogen atoms, respectively, substituted at the number 4 carbon of the aromatic ring . Both spectra are accurately simulated by the assigned hyperfine parameters (figures 5b and 6b) and have residuals that are essentially noise (figures 5c and 6c). Methyleugenol undergoes autoxidation such that the commercial product contains 1030 mg / l hydroperoxide and is capable of activating peroxidases without the presence of added hydrogen peroxide . Our spectroscopic studies show that the hydroperoxide is not a good substrate for catalase, which suggests that glutathione peroxidase may be important in the inhibition of this pathway in vivo . Thus, we suggest that peroxidase metabolism may contribute to the observed carcinogenicity of meu in extrahepatic tissues . Whether the peroxidase metabolism may contribute to the observed carcinogenicity of meu in extrahepatic tissues remains to be investigated.
A cross - sectional study of 17 men and 17 women recruited from an urban and a rural residential area of cameroon was conducted . Ethical approval for the study was obtained from the cameroon national ethics committee, and all participants provided signed informed consent . Height and waist circumference body weight was measured using an electronic scale (tanita tbf-531; tanita u.k ., middlesex, u.k . ). Fat - free mass was calculated from tbw, assuming a hydration factor of 73%, and fat mass derived as the difference between body weight and fat - free mass . Resting energy expenditure (ree) was measured using the medgem handheld indirect calorimeter (healthetech inc ., golden, co). Total energy expenditure (tee) two baseline urine samples were collected on separate days before the administration of a standard dose of dlw (174 mg / kg body wt of oxygen-18 and 70 mg / kg body wt of deuterium). Tee was calculated using schoeller's estimation of co2 production, assuming a respiratory quotient of 0.85 . Paee (kj day kg) was calculated as 0.9 tee ree, taking the thermogenic effect of food into account . Aerobic fitness was estimated by linear extrapolation of the individually observed heart - rate response to a standardized step test up to the age - predicted maximum heart rate . Capillary blood glucose was measured after an overnight fast (fasting blood glucose [fbg]) and then 2 h after ingestion of 75 g glucose dissolved in 250300 ml water (2-h postload blood glucose [2-h bg]). This was done using a hemocue b - glucose analyzer (hemocue, ngelholm, sweden). Statistical analyses were done using stata (version 10 special edition; statacorp, college station, tx). Independent associations of paee with fbg and 2-h bg were assessed by multiple linear regression analyses . Body fat was significantly lower in men than in women (18.5 9.3 vs. 34.6 7.2%; p <0.001); however, the difference in bmi was of borderline significance (25.4 3.6 vs. 28.4 5.1 kg / m; p = 0.05). Tee (191.4 55.9 vs. 143 22.6 kj kg day; p = 0.002) and paee (73.9 49.7 vs. 45.4 17.4 kj kg day; p = 0.03) were significantly higher in men than in women . There was no difference in mean fbg (4.3 0.7 vs. 4.6 0.6 mmol / l; p = 0.21) or 2-h bg (5.9 1.8 vs. 6.3 1.1 mmol / l; p = 0.43) between men and women . Bmi, waist circumference, and body fat were not significantly correlated with fbg or 2-h bg . Paee was not significantly correlated with fbg (r = 0.11; p = 0.53) but inversely correlated with 2-h bg (r = 0.43; p = 0.011). In unadjusted linear regression analyses (table 1), paee was significantly negatively associated with 2-h bg (= 0.016 [95% ci 0.028 to 0.004]). This association remained largely unchanged when adjusted for age, sex, smoking, and alcohol consumption (0.018 [0.032 to 0.004]). Further adjustments for bmi, waist circumference, body fat, or vo2max (table 1) did not change the results . In a sample of nondiabetic cameroonian adults with bmi comparable with that of national survey data (2), we observed a strong inverse association between paee and 2-h plasma glucose but not fasting plasma glucose independent of obesity / adiposity . This association was unchanged after adjustment for age, sex, smoking, and alcohol consumption as confounders and also was not affected by adjustment for cardiorespiratory fitness . There were borderline significant associations between paee and body fat with fbg, but no significant associations were observed among bmi, waist circumference, or cardiorespiratory fitness with fbg or 2-h bg in this study . The difference in pattern of association between paee and 2-h bg or fbg may relate to the fact that these biochemical parameters reflect different pathophysiological processes (3). Because the risk of future diabetes, as well as cvd outcomes, is more closely associated with impaired glucose tolerance than with impaired fasting glucose (4), our observations have public health importance for the prevention of these metabolic disorders . The cross - sectional nature of these analyses limits any inference about the direction of causality . However, the results from trials are unequivocal about the role of physical inactivity in the development of diabetes (5). The small sample size of this study could have limited the statistical power to detect an association between paee and 2-h bg . However, the precise measurement of the exposure, as used in the present study, acts to increase statistical power and may be one explanation for our ability to detect this association in a small study . Our results are in agreement with other studies in caucasians using objective measurement of physical activity with adjustment for measurement error (6). They are also in agreement with studies in other ethnic groups, which have used less precise measures of paee but larger samples (7). This is the first report of objectively measured free - living paee and its association with glucose tolerance in an african population . These results suggest that low levels of paee may be a factor underlying the rise of diabetes in this population (8). Public health efforts to increase overall paee could play an important role in diabetes prevention and cvd reduction in developing countries.
Intraoperative monitoring (iom) of the motor pathways is a routine procedure for ensuring integrity of corticospinal tracts during scoliosis surgery . In combination with somatosensory evoked potentials, motor evoked potential (mep) monitoring is widely utilized in operations with significant risks of spinal cord damage [2, 14]. However, anesthetic agents, which cause suppression of cortical and spinal motor neuron excitability, affect them [4, 5]. While most iom protocols involving meps utilize total intravenous anesthesia (tiva), we have previously reported success with desflurane as a halogenated inhalational anesthetic agent . In iom, meps are elicited mostly with contralateral cortical electrical stimulation . Our paper describes observations of ipsilateral and contralateral meps with bilateral recordings, in conjunction with tiva or desflurane during iom of scoliosis surgery . We studied nine patients (mean age 16.2 years; range 1417 years; 1 male) over a 6-month period in a prospective manner . Multi - pulse transcranial electrical stimulation was performed using two constant - current stimulators connected in parallel configuration from a dantec keypoint emg machine (dantec, skovlunde, denmark). A train of five square wave stimuli 0.5 ms in duration was delivered at 4 ms (250 hz) interstimulus intervals . Stimulating electrodes consisted of 9 mm gold - plated disc electrodes at c3c4 (international 1020 system) affixed with collodion . Stimulation output was increased from 50 ma in steps of 5 ma until a reproducible mep was elicited . The intensity was then increased and fixed at 10% above this threshold intensity to obtain a supramaximal mep response . Each stimulator was capable of delivering a maximum output of 100 ma (200 ma in total). Mep recordings were obtained with 13 mm disposable subdermal needles (technomed europe, beek, netherlands) in the tibialis anterior (ta) bilaterally . Input impedance of stimulating and recording electrodes were maintained below 5 k. for induction of anesthesia, sodium thiopentone at 4 mg / kg and fentanyl at 2 mcg / kg was administered; 0.8 mg / kg of intravenous atracurium was used to facilitate endotracheal intubation . No further doses of neuromuscular blocking agents were used subsequently . In the desflurane group, anesthesia desflurane was introduced through a calibrated vaporizer up to an end - tidal concentration of 2.14.3%, with a mean concentration of 3.4% (approximately 0.5 maximum alveolar concentration). This was measured using an ohmeda respiratory gas monitor 5250 (boc group, louisville, usa). Closed circuit mechanical ventilation was adjusted to maintain end - tidal carbon dioxide levels between 32 and 35 mmhg . In the tiva group, anesthesia was maintained using the regime of 10 mg / kg of propofol for the first 10 min, 8 mg / kg for the next 10 min and 5 mg / kg for the subsequent length of operation; 50% air in oxygen was administered . In both groups, morphine was titrated as required for pain relief . After approximately 45 min post - induction, a train of four - twitch assessment was performed using a nerve stimulator (fischer paykel ns242, uk). Cortical stimulation was commenced only when the amplitude of the fourth was visibly similar to the first . Peak to peak amplitudes (between two largest peaks opposite in polarity) and onset latency was utilized for all mep responses recorded bilaterally . Hence, ipsilateral meps refer to meps recorded from the ta on the same side as cortical stimulation . Within each patient, ten consecutive supramaximal meps obtained before insertion of pedicle screws used as a baseline were averaged to obtain the first two parameters . The initial stimulation intensity was defined as the minimal intensity required to obtain five consistent visible mep responses at a vertical gain of 20 v per division . During insertion of pedicle screws and instrumentation, a 50% reduction of mep amplitude or 10% prolongation of latency statistical analyses using student s t - tests were obtained with microsoft spss for windows version 10.1 . There were no complaints of headache, seizures or skin burns postoperatively; all patients had normal neurological examination . Meps were successfully obtained from all patients with ta recordings bilaterally . Mean ages for desflurane (16.2) and tiva (15.7) groups were not significantly different (p = 0.6). So as to require immediate surgical attention during and after pedicel screw insertion and spinal instrumentation . The ipsilateral mep amplitudes (standard deviation) were significantly larger than contralateral mep amplitudes [68.9 (27.1) vs. 52.5 (15.7) v, p <0.01, paired t - test]. The initial stimulation intensity to obtain ipsilateral meps was significantly lower than for contralateral meps [66.9 (12.3) vs. 74.4 (10.1) ma, p <0.05, paired t - test]. However, there were no significant latency differences for ipsilateral and contralateral meps [32.0 (2.1) vs. 31.5 (2.0) ms, p = 0.3, paired t - test). There was no significant difference between use of tiva or desflurane anesthesia for mep amplitudes obtained, with ipsilateral (p = 0.06, unpaired t - test) and contralateral (p = 0.09, unpaired t - test) stimulation . Additionally, there was also no significant difference between right and left sided mep amplitudes, either with ipsilateral (p = 0.9, paired t - test) or contralateral (p = 0.7, paired t - test) stimulation . We consecutively studied an additional 17 subjects monitored for scoliosis surgery using an identical protocol (1 men, mean age 16.1 years, range 1422). All with right cortex stimulation, mean initial stimulation intensity to obtain ipsilateral meps [39.7 (9.9) ma] was significantly lower than to obtain contralateral meps [47.1 (11.3) ma, paired t - test, p <0.0005]. With left cortex stimulation, similar findings were obtained [40.6 (10.7) vs. 50.3 (11.8) ma, paired t - test, p <0.0005]. With right cortex stimulation, mean ipsilateral mep amplitudes [107.1 (35.7) v] were significantly larger than mean contralateral mep amplitudes [90 (37.1) v, paired t - test, p = 0.01]. With left cortex stimulation, similar findings were again observed [112.1 (37) vs. 82.3 (30.9) v, paired t - test, p = 0.0004]. With right cortex stimulation, mean ipsilateral mep latencies [30.2 (2.4) ms] were not significantly different from mean contralateral mep latencies [30 (2.3) ms, paired t - test, p = 0.3). With left cortex stimulation, similar findings were again observed [30 (2.1) vs. 30.4 (2.3) ms, paired t - test, p = 0.2]. Examples of meps obtained with both ipsilateral and contralateral stimulation are shown in schematically in fig . 1schematic diagram showing right cortical stimulation, resulting in ipsilaterally and transcallosally conducted corticospinal impulses activating the spinal cord anterior horn cell . Summation of ipsilaterally conducted and transcallosally generated descending impulses may thus result in larger ipsilateral meps from right cortical stimulationfig . 2actual consecutive meps obtained from a patient, showing larger amplitude responses with ipsilateral stimulation . Both recordings were made from the ta at 70% stimulation intensity schematic diagram showing right cortical stimulation, resulting in ipsilaterally and transcallosally conducted corticospinal impulses activating the spinal cord anterior horn cell . Summation of ipsilaterally conducted and transcallosally generated descending impulses may thus result in larger ipsilateral meps from right cortical stimulation actual consecutive meps obtained from a patient, showing larger amplitude responses with ipsilateral stimulation . The present study showed that ipsilateral meps have significantly larger amplitudes and were elicited with significantly lower stimulation intensities than contralateral meps . The origin of ipsilateral meps in humans is not well understood . In animal studies, cat corticospinal neurons have been shown to evoke ipsilateral actions via ipsilaterally descending reticulospinal tracts, as well as via contralaterally descending reticulospinal neurons, both by synapsing spinal interneurons . Tracer studies in rhesus monkeys have quantified ipsilateral corticospinal fibers as approximately 912% of the total descending corticospinal projections . Thus, current evidence points to contralateral corticospinal fibers as the predominant pathway for spinal motorneuron activation . Patients with congenital mirror movements, schizencephaly, and kallmann s syndrome show ipsilateral meps, which likely result from abnormal structure and function of ipsilateral corticospinal fibers . However, functional reorganization and unmasking of ipsilateral corticospinal pathways may contribute to the generation of ipsilateral meps after adult stroke and congenital hemiparesis . One study involving 50 normal children suggested that presence of ipsilateral meps might be a normal state of ontogeny . Their disappearance after 10 years old is likely due to increasing transcallosal inhibitory influences . In our study, all patients were above 10 years of age, and did not have clinical features to suggest presence of underlying conditions mentioned above . Another study comparing healthy adults with stroke patients has suggested ipsilateral meps may be conducted via corticoreticulospinal or corticopropriospinal pathways in normal subjects . Firstly, it is possible that ipsilateral meps may be solely due to transcallosal stimulation of the contralateral motor cortex . Additionally, the effects of anesthesia on corticospinal excitability may facilitate this, hence resulting in significantly lower initial stimulation intensity to obtain ipsilateral meps . While evidence to suggest this is scarce, rat brain studies have demonstrated widespread action of anesthesia at multiple binding sites . Magnetic resonance brain imaging has also demonstrated increased callosal t2 changes with anesthesia, suggesting structural alterations at a molecular level . Motor pathway reorganization and spinal cord plasticity have been well documented in response to cord injury in an activity - dependent manner . Thus, structural and postural changes of longstanding scoliosis may have resulted in reorganization of cortical or subcortical motor pathways, including ipsilateral corticoreticular fibres leading to our observations . However, lack of lateralization of mep amplitudes with ipsilateral or contralateral stimulation was not supportive of this hypothesis . Additionally, lack of significant ipsilateral and contralateral latency differences suggest bilateral motor cortex stimulation has resulted in ipsilateral meps, which may have comprised early ipsilaterally conducted components and late transcallosally stimulated components (fig this might also explain the larger amplitudes of ipsilateral meps obtained than meps derived from contralateral motor cortex stimulation . While the relative contributions of ipsilaterally and transcallosal conducted meps remain uncertain, bilateral mep recordings during spinal surgery iom may provide additional information regarding the integrity of descending motor tracts . Together with the electrophysiological findings presented here, future studies clarifying these aspects would be justified . It thus may be feasible to routinely monitor meps bilaterally in future iom protocols for spinal surgery.
In terms of stability and retention, attachment system provides superior effectiveness for complete dentures . Biological and technical (mechanical) are the two types of complications encountered in implant therapy . Prosthetic maintenance (the capability to adapt or repair) is of daily clinical interest . Technical aspects are the parameters to be considered now . Durable retention remains the subject to debate . Technical complications include mechanical damage to the implant and prosthetic components . Retention loss or adjustments are the most encountered problems (30% of the reported maintenance). The following keywords directed the search: complications, retention, wear, attachment biomaterial, overdenture attachments, attachment systems, implant - retained overdentures, implant - supported overdentures, and locator . Peer - reviewed articles reporting on investigations of retention, wear, or complications of overdenture attachment systems used specifically for mandibular two - implant overdentures were identified . The search included articles published in english up to july 2014 which contained all or part of the key words in their headings . Divers attachment systems are released often without evidence - based support for long - term maintenance or repair . Industry proposes to replace the well - documented ball and bars attachments by new connector type introduced in 2001, the locator attachment (zest anchors, escondido, ca, usa) combined the best features of a ball attachment, an era attachment (sterngold), and a cap attachment, with its appreciated dual retention and different retention values . It is classified as a resilient universal hinge, and is indicated for limited inter - arch distance . Limited in vitro reports on the locator retentive force are published . The dual retention (inner and outer) enhances its cross - section strength by mechanical and also frictional mode . The slightly oversized nylon male insert and the smaller diameter inner ring of the female abutment while the central stud of the nylon male insert of the locator attachment press fits within the inner metal ring of the female abutment, the outer margin engages the shallow undercut area present at the outer margin of the abutment simultaneously and completely . The color of the patrix (replaceable nylon insert) indicates the retention value . To correct implant angulation, implant manufacturers provide only little data about the retentive strength and wear of attachments . For a single individual unsplinted attachment, 4 n is mentioned as the minimum retentive force, whereas this retention may vary from as low as 1 n to as high as 85 n when mandibular overdentures are retained by two or more implants . When a rough estimate of 20 n of adequate retentive force has been proposed for a two - implant mandibular overdenture, pigozzo et al . Mechanical and frictional contacts, as well as magnetic forces of attraction between the patrix and matrix can be the basis of retentive force . Studies have classified the attachment systems into high (era gray), medium (locator lr white, spheroflex ball, hader bar and metal clip, era white), low (locator lr pink), and very low (shiner magnet, maxi magnet, magnedisc magnet) retention groups . Significantly higher retention and stability are provided by locator connectors and sterngold era, compared to nobel biocare ball connectors . Pointed that the locator white, pink, and blue connectors demonstrated higher retentive forces than either a 7.9-mm prototype ball attachment design or the standard 2.25-mm ball attachment . The greatest reported value for the peak load was for the zest anchor advanced generation (zaag) attachment, compared to the nobel biocare ball, the zest anchor, and the sterngold era . The peak load is defined as the maximum forces developed before complete separation of the attachment components from the implant abutments . The zaag attachment exhibited significantly the highest retentive vertical and oblique forces under dislodging tensile forces applied to the housings in two directions simulating function: vertical and oblique . The lowest vertical force corresponds to the zest anchor and the lowest oblique retentive force to the nobel biocare standard . Furthermore, according to lehmann, an attachment system must be able to maintain its retentive force during a 10-year shelf life . However, evidence from past and current studies demonstrates that the wear - induced structural changes undergone by an attachment system inevitably lead to a reduction or total loss of retention . A mechanical action alone or a combination of chemical and mechanical actions will induce loss of material from the surface . Components wear decrease ball attachment's retention . Deterioration and deformation, along with work hardening, can also lead to attachment fracture . The variations in the wear patterns seen with different attachment systems still need better understanding . Study designs that attempted to emulate the actual oral environment tried to investigate the effect of short- and long - term simulated function on the attachment systems retentive force . Attachment systems were then submitted to cyclic loading under either axial or paraxial forces in the range of 54010,000 cycles of repeated insertion and removal . This would represent 6 months to 9 years of clinical function, on the basis of three daily overdenture removals and insertions for hygienic purposes . A common trend toward reduction or total loss in retentive force this loss can be abrupt after approximately 500 cycles and may reach 80% of the initial value after 2000 cycles . To evaluate the effects of wear on overdenture resilient attachments, rutkunas et al . Determined the dimensional changes and surface characteristics using light microscopy and scanning electron microscopy (sem) of era orange and white (eo and ew), locator pink, white, and blue (lrp, lrw and lrb), and op anchor (op), respectively, after simulated 15,000 insertion removal cycles . The sudden decrease of retentive force of era attachments was opposed to the retentive force fluctuation of locator attachments throughout the wear simulation period . The dimensional changes and surface wear on the plastic rings of attachment males were less expressed than on plastic cores . A literature review confirmed the reduction of the retentive force of the majority of attachment systems under in vitro conditions . A dramatic loss of retention for era attachments was observed at the conclusion of wear simulation test . Upon microscopic measurement, gamborena noticed distinct wear patterns that arise due to distortion of the plastic patrices, with the metallic matrices remaining unchanged . Studies reported similar observations with four ball attachment systems, whereas some studies reported that significant and maximal amount of wear of the diameter of ball abutment was reached after 3 years of clinical use . This may indicate that severe mechanical wear on both surfaces may occur after long periods of use . While some studies sustained that attachment systems which possess a male and female component of different material composition exhibit smaller changes in the retention force, others reported the highest wear on the ball attachments for implant - retained overdentures . Moreover, even among samples of the same attachment systems, differences in the retentive forces were evident . To increase the wear compensation and retention force it is noteworthy that a minimal reduction in retentive force was achieved by the magnetic attachments compared to the gradual decrease of the stud attachments retention . When tested under identical conditions, less physical deterioration was found, despite microscopic corrosion signs observed within the stainless steel magnet case . In contrast, under long - term simulated function, in the case of telescopic attachments made of different alloys (titanium, gold, and cobalt - chromium), a steady increase in the retentive force has been observed . The authors explained their results by the increased mechanical adaptation of the attachment components under cyclic loading . Mechanical fatigue also induced some retention loss of the attachment systems during the experiment after 15,000 fatigue cycles, even though it was of little valuewhen compared to the initial retentive forces . Eight hundred cycles are needed to attain relatively stable retention of overdenture attachments, especially for the most retentive systems . The clinician has to place and remove the overdenture multiple times before delivery, although this reduction might not be noticeable to the patient but only to the examiner . Nevertheless, cakarer did not find any advantage of ball and bar designs over the locator in terms of retention . Also, the locator root pink remained the most retentive after fatigue, compared to the era orange and white . The wear patterns and their related attachment deformation generated by the mastication are different from those induced by insertion removal cycles . When occlusal loads are applied, the mucosa is displaced under the denture base resulting in denture rotation around the attachments . The amount of occlusal load transmitted to the attachments is a factor of their resiliency . An optimal stress distribution reduces the denture movement and, thereby, the forces on the implants . Simulated mastication reduced the locator retention to 40% of the baseline values with a non - linear descending curve . Kleis declared that after 12 months of overdentures delivery, the male parts of the locator have to be changed, as 75.5% loss of retention has been noticed . However, the reduction in peak load - to - dislodgement for these attachments is more apparent in case of non - parallel implants . Length, number, implant's angulation, opposing dentition, and parafunctional habits are among the factors that may increase susceptibility to mechanical complications . Excessive masticatory forces, as off - axis centric contacts, excursive contacts, and cantilevered loading, may be generated in severely resorbed mandible . Correct implant placement diminishes attachment systems maintenance . To overcome the inevitable continuous resorption of the underlying residual ridge, frequent rebasing of implant - retained overdentures (ovds) may restore the proper occlusion and reduce possible rotation of the denture around the retentive components . No major differences in prosthetic complications have been observed for bar or ball attachments, thus both are considered as reliable connectors . Most of them reported more prosthetic maintenance for separated attachments, whereas others found found more technical complications / repairs per patient around bar than ball attachments . Most of the separated attachments need more prosthetic maintenance; but for others, the frequency of technical complications / repairs per patient was higher around bar than ball attachments, with an increased failure rate for the cantilevered extended bars . Meanwhile, no difference was seen in the implant survival rate among splinted and unsplinted schemes . Recent studies sought to compare the incidence of mechanical complications of the locator attachments with that of the commonly used overdenture attachment systems . A recent survey showed that british general dental practitioners are not familiar with the locator attachment system and are reluctant to do implant - retained ovds maintenance . General dentist practitioner general practice dentists (gdps) would like further training in this area . Observed less prosthodontic complications and maintenance of the oral function for the locator system than for ball and bar attachments . 's findings were in agreement with this when they compared locator to southern plastic and straumann gold over a 3-year period . In contrast, kleis et al . Argued that the creep response of the matrices and the hardness of the patrices provoked extensive deformation and deterioration of the locator nylon parts, with a higher substantial need for maintenance, compared to ball attachments . Differences in the dimensions or material composition, and a large variation in retentive forces have been found between different batches of the same attachment system due to the poor manufacturing quality control . Researchers concluded that implant parallelism has more impact on the complications that occur than the choice of type of attachments . Any installation load greater than 0 n is recommended for the connection of ball, locator, or magnetic attachments to a denture base . By increasing this installation load, the resultant force acting on the implant may be decreased . However, when this load surpasses 100 n, the harmful denture movement may be increased . Implant manufacturers provide only little data about the retentive strength and wear of attachments . For a single individual unsplinted attachment, 4 n is mentioned as the minimum retentive force, whereas this retention may vary from as low as 1 n to as high as 85 n when mandibular overdentures are retained by two or more implants . When a rough estimate of 20 n of adequate retentive force has been proposed for a two - implant mandibular overdenture, pigozzo et al . Mechanical and frictional contacts, as well as magnetic forces of attraction between the patrix and matrix can be the basis of retentive force . Studies have classified the attachment systems into high (era gray), medium (locator lr white, spheroflex ball, hader bar and metal clip, era white), low (locator lr pink), and very low (shiner magnet, maxi magnet, magnedisc magnet) retention groups . Significantly higher retention and stability are provided by locator connectors and sterngold era, compared to nobel biocare ball connectors . Pointed that the locator white, pink, and blue connectors demonstrated higher retentive forces than either a 7.9-mm prototype ball attachment design or the standard 2.25-mm ball attachment . The greatest reported value for the peak load was for the zest anchor advanced generation (zaag) attachment, compared to the nobel biocare ball, the zest anchor, and the sterngold era . The peak load is defined as the maximum forces developed before complete separation of the attachment components from the implant abutments . The zaag attachment exhibited significantly the highest retentive vertical and oblique forces under dislodging tensile forces applied to the housings in two directions simulating function: vertical and oblique . The lowest vertical force corresponds to the zest anchor and the lowest oblique retentive force to the nobel biocare standard . Furthermore, according to lehmann, an attachment system must be able to maintain its retentive force during a 10-year shelf life . However, evidence from past and current studies demonstrates that the wear - induced structural changes undergone by an attachment system inevitably lead to a reduction or total loss of retention . A mechanical action alone or a combination of chemical and mechanical actions will induce loss of material from the surface . Components wear decrease ball attachment's retention . Deterioration and deformation, along with work hardening, can also lead to attachment fracture . The variations in the wear patterns seen with different attachment systems still need better understanding . Study designs that attempted to emulate the actual oral environment tried to investigate the effect of short- and long - term simulated function on the attachment systems retentive force . Attachment systems were then submitted to cyclic loading under either axial or paraxial forces in the range of 54010,000 cycles of repeated insertion and removal . This would represent 6 months to 9 years of clinical function, on the basis of three daily overdenture removals and insertions for hygienic purposes . A common trend toward reduction or total loss in retentive force . This loss can be abrupt after approximately 500 cycles and may reach 80% of the initial value after 2000 cycles . To evaluate the effects of wear on overdenture resilient attachments, rutkunas et al . Determined the dimensional changes and surface characteristics using light microscopy and scanning electron microscopy (sem) of era orange and white (eo and ew), locator pink, white, and blue (lrp, lrw and lrb), and op anchor (op), respectively, after simulated 15,000 insertion removal cycles . The sudden decrease of retentive force of era attachments was opposed to the retentive force fluctuation of locator attachments throughout the wear simulation period . The dimensional changes and surface wear on the plastic rings of attachment males were less expressed than on plastic cores . A literature review confirmed the reduction of the retentive force of the majority of attachment systems under in vitro conditions . A dramatic loss of retention for era attachments was observed at the conclusion of wear simulation test . Upon microscopic measurement, gamborena noticed distinct wear patterns that arise due to distortion of the plastic patrices, with the metallic matrices remaining unchanged . Studies reported similar observations with four ball attachment systems, whereas some studies reported that significant and maximal amount of wear of the diameter of ball abutment was reached after 3 years of clinical use . This may indicate that severe mechanical wear on both surfaces may occur after long periods of use . While some studies sustained that attachment systems which possess a male and female component of different material composition exhibit smaller changes in the retention force, others reported the highest wear on the ball attachments for implant - retained overdentures . When antagonist to titanium matrix . Moreover, even among samples of the same attachment systems, differences in the retentive forces were evident . To increase the wear compensation and retention force it is noteworthy that a minimal reduction in retentive force was achieved by the magnetic attachments compared to the gradual decrease of the stud attachments retention . When tested under identical conditions, less physical deterioration was found, despite microscopic corrosion signs observed within the stainless steel magnet case . In contrast, under long - term simulated function, in the case of telescopic attachments made of different alloys (titanium, gold, and cobalt - chromium), a steady increase in the retentive force has been observed . The authors explained their results by the increased mechanical adaptation of the attachment components under cyclic loading . Mechanical fatigue also induced some retention loss of the attachment systems during the experiment after 15,000 fatigue cycles, even though it was of little valuewhen compared to the initial retentive forces . Eight hundred cycles are needed to attain relatively stable retention of overdenture attachments, especially for the most retentive systems . The clinician has to place and remove the overdenture multiple times before delivery, although this reduction might not be noticeable to the patient but only to the examiner . Nevertheless, cakarer did not find any advantage of ball and bar designs over the locator in terms of retention . Also, the locator root pink remained the most retentive after fatigue, compared to the era orange and white . The wear patterns and their related attachment deformation generated by the mastication are different from those induced by insertion when occlusal loads are applied, the mucosa is displaced under the denture base resulting in denture rotation around the attachments . The amount of occlusal load transmitted to the attachments is a factor of their resiliency . An optimal stress distribution reduces the denture movement and, thereby, the forces on the implants . Simulated mastication reduced the locator retention to 40% of the baseline values with a non - linear descending curve . Kleis declared that after 12 months of overdentures delivery, the male parts of the locator have to be changed, as 75.5% loss of retention has been noticed . However, the reduction in peak load - to - dislodgement for these attachments is more apparent in case of non - parallel implants . Length, number, implant's angulation, opposing dentition, and parafunctional habits are among the factors that may increase susceptibility to mechanical complications . Excessive masticatory forces, as off - axis centric contacts, excursive contacts, and cantilevered loading, may be generated in severely resorbed mandible . Correct implant placement diminishes attachment systems maintenance . To overcome the inevitable continuous resorption of the underlying residual ridge, frequent rebasing of implant - retained overdentures (ovds) may restore the proper occlusion and reduce possible rotation of the denture around the retentive components . Study designs that attempted to emulate the actual oral environment tried to investigate the effect of short- and long - term simulated function on the attachment systems retentive force . Attachment systems were then submitted to cyclic loading under either axial or paraxial forces in the range of 54010,000 cycles of repeated insertion and removal . This would represent 6 months to 9 years of clinical function, on the basis of three daily overdenture removals and insertions for hygienic purposes . A common trend toward reduction or total loss in retentive force . This loss can be abrupt after approximately 500 cycles and may reach 80% of the initial value after 2000 cycles . To evaluate the effects of wear on overdenture resilient attachments, rutkunas et al . Determined the dimensional changes and surface characteristics using light microscopy and scanning electron microscopy (sem) of era orange and white (eo and ew), locator pink, white, and blue (lrp, lrw and lrb), and op anchor (op), respectively, after simulated 15,000 insertion the sudden decrease of retentive force of era attachments was opposed to the retentive force fluctuation of locator attachments throughout the wear simulation period . The dimensional changes and surface wear on the plastic rings of attachment males were less expressed than on plastic cores . A literature review confirmed the reduction of the retentive force of the majority of attachment systems under in vitro conditions . A dramatic loss of retention for era attachments was observed at the conclusion of wear simulation test . Upon microscopic measurement, gamborena noticed distinct wear patterns that arise due to distortion of the plastic patrices, with the metallic matrices remaining unchanged . Studies reported similar observations with four ball attachment systems, whereas some studies reported that significant and maximal amount of wear of the diameter of ball abutment was reached after 3 years of clinical use . This may indicate that severe mechanical wear on both surfaces may occur after long periods of use . While some studies sustained that attachment systems which possess a male and female component of different material composition exhibit smaller changes in the retention force, others reported the highest wear on the ball attachments for implant - retained overdentures . When antagonist to titanium matrix . Moreover, even among samples of the same attachment systems, differences in the retentive forces were evident . To increase the wear compensation and retention force it is noteworthy that a minimal reduction in retentive force was achieved by the magnetic attachments compared to the gradual decrease of the stud attachments retention . When tested under identical conditions, less physical deterioration was found, despite microscopic corrosion signs observed within the stainless steel magnet case . In contrast, under long - term simulated function, in the case of telescopic attachments made of different alloys (titanium, gold, and cobalt - chromium), a steady increase in the retentive force has been observed . The authors explained their results by the increased mechanical adaptation of the attachment components under cyclic loading . Mechanical fatigue also induced some retention loss of the attachment systems during the experiment after 15,000 fatigue cycles, even though it was of little valuewhen compared to the initial retentive forces . Eight hundred cycles are needed to attain relatively stable retention of overdenture attachments, especially for the most retentive systems . The clinician has to place and remove the overdenture multiple times before delivery, although this reduction might not be noticeable to the patient but only to the examiner . Nevertheless, cakarer did not find any advantage of ball and bar designs over the locator in terms of retention . Also, the locator root pink remained the most retentive after fatigue, compared to the era orange and white . The wear patterns and their related attachment deformation generated by the mastication are different from those induced by insertion when occlusal loads are applied, the mucosa is displaced under the denture base resulting in denture rotation around the attachments . The amount of occlusal load transmitted to the attachments is a factor of their resiliency . An optimal stress distribution reduces the denture movement and, thereby, the forces on the implants . Simulated mastication reduced the locator retention to 40% of the baseline values with a non - linear descending curve . Kleis declared that after 12 months of overdentures delivery, the male parts of the locator have to be changed, as 75.5% loss of retention has been noticed . However, the reduction in peak load - to - dislodgement for these attachments is more apparent in case of non - parallel implants . Length, number, implant's angulation, opposing dentition, and parafunctional habits are among the factors that may increase susceptibility to mechanical complications . Excessive masticatory forces, as off - axis centric contacts, excursive contacts, and cantilevered loading, may be generated in severely resorbed mandible . Correct implant placement diminishes attachment systems maintenance . To overcome the inevitable continuous resorption of the underlying residual ridge, frequent rebasing of implant - retained overdentures (ovds) may restore the proper occlusion and reduce possible rotation of the denture around the retentive components . No major differences in prosthetic complications have been observed for bar or ball attachments, thus both are considered as reliable connectors . Most of them reported more prosthetic maintenance for separated attachments, whereas others found found more technical complications / repairs per patient around bar than ball attachments . Most of the separated attachments need more prosthetic maintenance; but for others, the frequency of technical complications / repairs per patient was higher around bar than ball attachments, with an increased failure rate for the cantilevered extended bars . Meanwhile, no difference was seen in the implant survival rate among splinted and unsplinted schemes . Recent studies sought to compare the incidence of mechanical complications of the locator attachments with that of the commonly used overdenture attachment systems . A recent survey showed that british general dental practitioners are not familiar with the locator attachment system and are reluctant to do implant - retained ovds maintenance . General dentist practitioner general practice dentists (gdps) would like further training in this area . Observed less prosthodontic complications and maintenance of the oral function for the locator system than for ball and bar attachments . 's findings were in agreement with this when they compared locator to southern plastic and straumann gold over a 3-year period . In contrast, kleis et al . Argued that the creep response of the matrices and the hardness of the patrices provoked extensive deformation and deterioration of the locator nylon parts, with a higher substantial need for maintenance, compared to ball attachments . Differences in the dimensions or material composition, and a large variation in retentive forces have been found between different batches of the same attachment system due to the poor manufacturing quality control . Researchers concluded that implant parallelism has more impact on the complications that occur than the choice of type of attachments . Any installation load greater than 0 n is recommended for the connection of ball, locator, or magnetic attachments to a denture base . By increasing this installation load, the resultant force acting on the implant may be decreased . However, when this load surpasses 100 n, the harmful denture movement may be increased . However patient satisfaction is somehow, independent of the attachment system (3). In - depth studies following standardized criteria to compare different options for mandibular implant overdentures (iovd) treatment remain scarce . In particular, similar clinical protocols are still insufficient to allow the calculation of an overall complication incidence for implant overdentures iovds . A limitation of this literature review is that it includes studies with non - declared sample size, or measurement methods . Other studies are in vitro experiments, whereas non - randomized controlled studies are lacking . Well - structured clinical prospective studies remain essential, in addition to well - designed in vitro studies . The accuracy of parameters applied to the model, including geometry, constraints, and mechanical properties, determines the value of the finite element analysis experiment . Masticatory loading submits the prosthesis to a hardly reproducible scheme of three - dimensional movements . The clinical wear can be influenced by saliva, denture cleansers, or food particles . As a result factors should be investigated separately under well - controlled conditions, in order to limit the influence of confounding variables . Evidence - based studies do not allow us to select the most effective implant ovd connection . Equal atraumatic distribution of forces between mechanical and biological supporting structures, and minimal complications are the ultimate goals when placing any connector . The amount of retention desired and the specific clinical situation guide the clinician in selecting an appropriate attachment . The retention measurement values provided by the manufacturer at treatment beginning and after function would help to respond to individual needs of patients . Scientific evidence related to the material's clinical performance, objective oral function, and patients appreciation of the treatment should guide the clinician in making the ultimate choice of a specified attachment . Adequate aftercare may be difficult or impossible when treating aging patients . In the author's opinion and based on the presented data, the locator attachment system is an easy - to - use connector, with less post - insertion complications . The choice of implant location and retentive attachments in implant - retained overdentures is based on clinician's preference, expert opinion, and empirical information . Careful post - insertion maintenance of the prosthesis, attachment system, and mucosa is mandatory . Out - of - pocket treatment and post - treatment expenses should be less and the treatment should not be time consuming.
She was scratched on the right arm by a pet rat while visiting a friend who had several domestic rats (rattus norvegicus). One rat had been purchased at the end of december 2008 from a pet store . The rat became sick with sneezing, conjunctival hemorrhages, and epistaxis; it died 4 days after purchase . On january 4, 2009, the patient sought treatment at the emergency department of compigne hospital . Eight days later, the patient was admitted to the hospital with a black necrotic scab on the internal surface of the right arm, regional lymphangitis, and axillar lymphadenopathies (figure 1, panel a). After 3 weeks of unsuccessful antimicrobial drug treatment, she underwent surgery to remove the affected area . Cowpox virus infection in 3 persons in northern france caused by transmission from infected pet rats . Cutaneous lesions caused by cowpox virus are shown in patient 1 (a), patient 3 (b) and patient 4 (c, d). Panel c was obtained on january 30, 2009, panel d on february 6, 2009 . Negative - staining electron microscopy showed mulberry forms with conspicuous but short, randomly arranged surface tubules (e) and capsule forms with deeper stain penetration (f), both highly suggestive of poxvirus . Case - patient 2 was a 17-year - old woman who had purchased a domestic rat at the end of december 2008 . The rat died within 3 days of purchase and had respiratory symptoms identical to those of the rat that scratched case - patient 1 . Six days after the rat died, an inflammatory cutaneous macular lesion appeared at the base of the patient s neck, causing local pain and intense inflammatory reaction . Amoxicillin - clavulate was prescribed, but the necrotic scab continued to grow, and local pain increased along with fever (39c) (figure 1, panel b). She was admitted to the emergency department of compigne hospital on january 14, 2009 . On january 3, she had purchased a rat from the same pet store as case - patient 1 . Soon afterward, the rat began to cough and show signs of hemorrhagic lachrymal oozing; the rodent died on january 6 . On january 13, the patient had maculopapular lesions on the upper right eyelid, on her left shoulder, and at the base of her neck . Due to her deteriorating condition, she was admitted to the emergency department on january 17 with rash characterized by erythema and edema, and painful regional lymphangitis and lymphadenopathy . Case - patient 4 was a 29-year - old woman who reported having been scratched by a rat on january 21, 2009 . An inflammatory macule on her clavicle had progressed through papular, vesicular, and pustular stages; she also had fever and malaise . On january 14, she had purchased a domestic rat in the same store as the 3 previous case - patients . The rat had respiratory symptoms similar to the previously infected rats and died on january 21 . The patient was admitted to compigne hospital on january 30; examination showed a 20-mm black eschar with a crust, regional lymphangitis, and painful lymphadenopathies (figure 1, panels c and d). Biopsy specimens of case - patients 13 were sent to the national reference center for rickettsial diseases (marseille) on january 21 because anthrax and/or rickettsial disease was suspected . Broad range pcrs were performed for bacteria (16s rrna) (7) and fungi (18s rrna) (8). In the absence of etiology, and based on information provided by the institut de veille sanitaire, negative - stain electron microscopy was performed on january 26 . The biopsy samples showed typical poxvirus - like particles (figure 1, panels e and f). Molecular diagnosis was performed by using pcr targeting a 260-bp fragment in the cowpox hemagglutinin gene (forward primer 5-tacttttgttactaatatcattag-3, reverse primer 5-agcagtcaatgatttaattgt-3). Direct sequencing of the pcr product identified cowpox virus by blast analysis (http://blast.ncbi.nlm.nih.gov/blast.cgi) against the genbank database . The virus was isolated by using monolayers of vero cells in 12.5-cm flasks (9). When cytopathic effect was obvious, dna was extracted from the supernatant, and the complete hemagglutinin gene was sequenced from a pcr product (forward primer 5-ccattggaaaaaacacagtac-3, reverse primer 5-ccaaatatattcccatagtc-3), amplifying a 1,183-bp region . Electron microscopy and pcr were performed on formalin - fixed scabs from the lesion of case - patient 4 . Both test results were positive morphologically for a poxvirus and by pcr for cowpox virus . The virus was isolated from serum samples of case - patients 13 and assessed by cytopathic effect on vero cells, electron microscopic morphologic identification, positive pcr amplification, and subsequent direct sequencing . A 1,047-bp sequence was definitive and used for fine comparative genetic analysis with the full - length hemagglutinin gene sequences available in genbank . The most closely related sequence corresponded to clone cow ha24 of the catpox 5 isolate of cowpox virus, isolated from a cheetah in 1982 in the united kingdom (ay902254). Genetic heterogeneity with y902254 comprised 15 mutations; 10 were nonsynonymous, and 3 were insertions . Phylogenetic analyses indicated that sequences corresponding to these cases grouped together and were clearly distinct from other cowpox virus strains previously reported (figure 2). Sequence analysis indicated that these 4 infections were caused by a virus strain distinct from other cowpox virus sequences retrieved from genbank . Cpxv, cowpox virus; ectv, ectromelia virus; varv, variola virus; cmlv, camelpox virus; vacv, vaccinia virus; hspv, horsepox virus; mpxv, monkeypox virus . Sporadic human cases of cowpox virus infection have occurred in several european countries over the past few years . For most cases the source was domestic cats (46,10,11). Rat - to - human transmission of cowpox virus was described in the netherlands, but the source was a wild rat, not a pet rat (12). We know of only 1 previous case of human cowpox virus infection that may be linked to a pet rat (13). In the 4 cases reported here, all 4 patients reported scratches caused by rat claws, not bites, while handling the rats as pets . In 3 of the 4 cases, fever (39c) was noticed at the pustular stage, associated with lymphangitis and regional adenopathies . Interviews with the 4 case - patients showed all had purchased or had been in contact with domestic rats originating from the same pet store . Further investigations traced the origin of the cowpox virus - infected rats to a rat breeder in the czech republic (14). Recently, similar human cases linked to contact with pet rats have been reported in france, suggesting that the outbreak may involve more cases than were initially realized (14). The situations in france and germany mimic the monkeypox outbreak in the united states, i.e., human transmission of the virus by pet prairie dogs contaminated by probable contact with gambian rats imported from africa and directly associated with a us pet retailer (15). Our study and the us outbreak emphasize the need for extreme caution when humans adopt animals of exotic origin as pets . Our study justifies the establishment of a national diagnostic capability and the corresponding human expertise to enable rapid diagnosis and identification of human pathogens that can cause unimaginable levels of disease in our communities.
On september 12, 2001, a 9-year - old boy from la gamba, ro claro, in the brunca region of costa rica, was admitted to a local hospital with fever, cough, and malaise of 4 days duration . On september 19, he returned to the hospital because of muscular weakness; he and was referred to the regional hospital on september 23 with acute flaccid paralysis that rapidly progressed to respiratory failure . The patient was intubated and transferred to the national childrens hospital with a diagnosis of viral encephalomyelitis . His severe central nervous system involvement progressed to coma in <24 h with muscle hypotonia . A computed tomographic scan showed generalized brain edema, and an electroencephalogram indicated diffuse and severe cerebral damage . A nuchal skin biopsy tested negative for rabies virus antigen by an immunofluorescence antibody assay, but postmortem brain tissue tested positive . The house where the child had lived during the previous 4 months was in a wooded zone (figure 1). In an interview, two members of the family noted that a 62-year - old woman who was in charge of the child died in a similar manner . On september 17, she exhibited malaise, muscular pain, headache, insomnia, and anxiety . On september 23, she was admitted to the local hospital, but because of the severity of her disease, she was transferred to the regional hospital on september 26 . On september 29, she had sialorrhea, hemiplegia, and loss of muscular strength in her right arm; she also showed bizarre behavior and respiratory failure that progressed to apnea . Characterization of the agents, conducted at the centers for disease control and prevention, demonstrated a rabies virus variant associated with vampire bats (desmodus rotundus) from both patients, as described elsewhere in latin america (11). Patient s home, located in an area where the risk of being bitten by a bat was high . After the diagnosis of rabies was made, additional history was obtained about potential animal exposures . The family reported that during the middle of july 2001, their cat became very aggressive and demonstrated strange behavior . Their dog attacked the cat, and while the woman and child tried to stop the fight, the cat bit them . The dog killed the cat, and the carcass was discarded into a creek next to the house . After the human cases were reported, the dog was confined with close observation, and in december 2001, it was euthanized . Postmortem brain tissue samples from the dog tested negative for rabies virus antigen . Because rabies occurs in cattle in costa rica and because persons living in areas where contact with vampire bats is likely are at risk for of viral transmission, the program of animal health of the ministerio de agricultura y ganadera had implemented a geographic information system (gis) during 1980 . This animal surveillance system divided the country into 2,234 small areas (5 x 5 km). Trained veterinary technicians used a national census of animals to register the number of weekly vampire bat bites from the farms included in each area . According to the frequency of vampire bat bites in cattle, the presence of rabies virus in populations of bats, and ecologic variables (e.g., temperature, precipitation, altitude, and land use), the gis program defines areas of low, medium, and high - risk of disease (figure 2). During august and september 2001, the gis of animal health surveillance reported 169 cattle bites by vampire bats in the brunca region (rate 3.5%). Where the two patients lived, the rate of such attacks was 10.4%, and the area was classified as a high - risk zone for bat bites to cattle (figure 3). Rabies postexposure prophylaxis with a vero cell rabies vaccine was administered to the relatives of the patients and to veterinary and healthcare personnel, who were in contact with the saliva of the patients . A retrospective search of other possible human cases was performed by analyzing the deaths with unknown origin in the discharge registry of the hospitals of the brunca region and the registry of the national morgue . The case definition used was the following: any acute encephalopathy of unknown origin, with atypical focal neurologic signs or encephalomyelitis in any person who died between january 1 and september 30, 2001, in the brunca region . The search did not find any additional suspicious deaths associated with rabies in the region . After 31 years without a case of human rabies in costa rica, the disease reemerged . Its reappearance reinforces the importance of an integrated network between veterinary and human health programs to identify risk zones of bat bites and to prevent human and animal cases . The active participation of combined health services in the early warning of the occurrence of suspected cases of emergent diseases is essential for effective, integrated surveillance . Human rabies must always be considered in the differential diagnosis of acute flaccid paralysis with consciousness impairment, even in patients without a known history of animal bite (giving priority to those in zones where cattle are at high risk for bat bites). Rabies in bats was first reported during the 1920s . Since then, rabies has been confirmed in several species of bats . The historical association between vampire bats and livestock may tend to subvert the epidemiologic surveillance in other species, such as domestic animals such as cats and dogs (12). This lack of association with other species could be due to a lack of intensified surveillance or to unknown factors related to the virus, host, or ecology . Several studies have documented the transmission of rabies virus in the absence of a history of documented animal bite (13,14). No typical bite marks were observed in either of the patients, but the history of cat bite raises the possibility of an indirect manner of transmission, through the bite of another vector, previously infected by a bat . At the community level, the immigration of human populations throughout rural areas increases the risk of contact with hematophagous bats and facilitates the transmission of the virus to humans . The presence of rabies in humans in costa rica associated with bat bites has demonstrated the importance of strengthening the integrated surveillance of animal and human health care . The gis operation permits the identification of risk zones, so that measures for optimal control and prevention can be established . Participation of communities located in high - risk areas is a key strategy for the prevention and early detection of possible cases of rabies . Emergence of human rabies from infected wildlife, especially in countries where the disease has been eliminated in dogs, reinforces the need for maintaining awareness of both physicians and public health workers alike, as well as for improving laboratory tools needed for rapid rabies diagnosis.
Gaucher's disease is a lysosomal storage disorder characterized by the accumulation of glycosyl ceramide in cells of the macrophage / monocyte system . Gaucher's disease is due to mutations involving the -glucosidase gene, localized in the large arm of chromosome 1 (1q21). The glucocerebrosides, found inside the red blood cell and white blood cells membranes, are accumulated in large quantities in fibril - like elements in the bone marrow, liver, and spleen (gaucher's cells). Gaucher's cells inside the bone marrow will cause bone impairment, osteoporosis or fractures . In the liver and the spleen they will cause organ enlargement; in the blood system anemia, leucopenia and low platelet count are the most common profiles[1, 3, 5, 79]. Type 1 or the non - neuronopathic form, is the most common form of the disease, comprising 80% of all three types . According to the symptomatology and the clinical burden of the disease, only 30% of individuals with this type of the disease will represent clinical symptoms[24, 6, 9, 10]. Type 2 or the acute neuronopathic disease, is the most severe form and the first symptoms are present in the first six months of life, resulting in death during the second year of life . Type 3 or the chronic neuronopathic form, represents the same clinical symptomatology as the type 2, but it has a much slower progression . It is met in 5% of all cases with gaucher's disease, and its prevalence rates 1:100.000 individuals . This type is seen mainly in a local area of sweden, the norrbotten and vsterbotten region, forming thereby a particular picture of the disease[11, 12]. The majority of our patients (9/11) were suffering from type 1 of the disease, and two patients from type 3 . The most common mutations in the overall population are n370s, l444p, 84gg and ivs2 + 1 . These mutations are seen in 50 - 60% of the overall casuistic, and 90% of the ashkenazi jewish patients . As there is no connection between the mutation type and the clinical severity although no straight relation has been found between mutation type and severity, several studies suggest that some genotypes might indicate a better outcome[14, 15]. Dna analysis is the safest diagnostic method, because the other methods cannot discriminate the heterozygotic form from normal individuals[4, 7, 10, 12, 1619]. Dna analysis will find out about 85% of carriers in the overall population and 90% of ashkenazi jewish originated carriers . It is also the safest one in the prenatal diagnosis, either by searching the mutation when there is a known risk; or by the genetic connections analysis, when the mutation is not known[4, 6, 12, 19, 20]. The enzymatic replacement with cerezyme or imiglucerase, (a recombinant glucocerebrosidase administered intravenously), seems efficacious . The use of cerezyme in type 3 has not given satisfying results, especially in terms of improving the neurological signs . Cerezyme is a very expensive medicine; its annual cost varies from 100.000 to 550.000 usd, depending on the disease progression . Other alternative therapies are fare from approaching the efficacy of cerezyme[8, 10, 16, 21, 22]. During the period 2004 - 2009 in our service of pediatrics, uhc mother theresa of tirana, eleven children were hospitalized and treated for gaucher's disease . 9 children were suffering from type 1, and 2 children from type 3 of the disease . Chitotriosidase values, other biochemical markers and the genetic molecular analysis were performed in our study group . The group was treated with cerezyme and a thorough follow - up covering a two - year period of treatment was performed (tables 13). An informed consent was received in all cases (from respective parents when the patient was minor); the service of pediatrics and the university hospital center approved the methodology and the study . Furthermore, the local drug control office licensed the drug usage for this specific indication . The casuistic of our clinic consists of 11 patients treated with cerezyme. The patients were diagnosed as gaucher's disease; the onset of the disease anticipated the beginning of the enzymatic replacement treatment with an age span from 5 to 12 years . The criteria of diagnostic suspicion were: anamnesis, clinical signs such as tiredness, paleness, abdominal pain, hemorrhagic syndrome (ecchymosis, epistaxis or focal hemorrhages), liver and spleen enlargement, physical and/or mental retardation, low red blood cells count and hemoglobin value, low platelet and leukocyte count, radiological signs showing involvement of long bones (femur, humerus, vertebral column), myelogram, the liver biopsy, the values of the enzyme glucocerebrosidase and of the biomarker chitotriosidase . The diagnostic confirmation and the gene mutation localization were performed through the dna molecular examination in 9 of our patients . The peripheral blood examinations, the myelogram and the skeletal radiology were done in the clinical biochemical and imaging laboratories of uhc mother theresa the enzymatic examinations of the biomarker chitotriosidase were performed in sahlgren's university hospital, mlndal sweden; and the dna analyses were performed in children's hospital & regional medical center, seattle, usa . The treatment consisting in the enzyme replacement (cerezyme) was supported by genzyme corporation, usa . The treatment with cerezyme was applied parenterally and the drug was administered with a three hour - infusion through a venous line . The patients suffering from gaucher's disease type 1 received cerezyme with a dosage of 60 ui / kg of body weight every other week; the patients suffering from gaucher's disease type 3 received cerezyme with a dosage of 120 ui / kg of body weight every other week . The patients are actually under continuous treatment, since the preparation has to be administered ad vitam, apart from very rare cases of idiosyncrasy, an occurrence that was never encountered within our group of patients . Due to the consistent cost of the treatment, the respective dosage of cerezyme may be decreased after the pediatric age; at any case the treatment cannot be stopped merely on the ground of slight and transitory side effects . We did not registered serious side effects during the treatment that could warrant the treatment withdrawal . The dosages we applied are as well suggested from other authors, although there is a wide margin, namely of 30 60 ui / kg of cerezyme monthly per type 1; double dosages are used for type 3 of gaucher's disease together with maintenance infusions on a biweekly or even weekly basis . Informed consent was received from parents when patients were minor, and from adult patients . Albanian ethical committee approved the study and the hospital treatment was entirely performed in respect of albanian ethical and deontological code . Direction of the university hospital center was notified about the treatment and the results produced . Among our patients, 9 were suffering from the type 1 of gaucher's disease, and 2 others were suffering from the type 3 . All patients were born from normal pregnancies and normal birth deliveries . Within the patients group no one had any kinship relation with the others . After the diagnosis criteria were met, we initiated the specific treatment within a time delay that varied from 4 to 6 months . The most important clinical signs seen in our patients are shown in table 1 . In table 2 we describe the hematological values, the data of the myelogram, the enzymatic values of glucocerebrosidase and chitotriosidase performed in all our patients before the treatment was started, as well as the genetic mutations detected in 9 of them . Signs and symptoms before starting the treatment with cerezyme note: () sign or symptom is lacking, (+) minor sign or symptom, (+ +) moderate sign or symptom, (+ + +) prominent evidence, (+ + + +) massive evidence and/or requiring urgent treatment genetic, molecular and other baseline data note: myelogram () diagnostic negativity; myelogram (+) diagnostic positivity; hgb: hemoglobin; plt: platelet the parameters of the clinical improvement (liver and spleen reduction in size, bone signs improvement, epistaxis disappearance) and the biological improvement (hemoglobin value, platelet count and the levels of the biomarker chitotriosidase) after the treatment with the specific enzyme (cerezyme) was started, are shown in table 3 . Overall hematological, visceral, skeletal and chitotriosidase parameters improvement; two years after the treatment with cerezyme was started . Note: () no improvement; (+) slight improvement; (+ +) notable improvement; hgb: hemoglobin; plt: platelet in our case series, two years after the beginning of the treatment, the hemoglobin level was normalized, after it started to increase within the 6 month of the therapy . The platelet count was normalized in seven patients after one year of treatment, and in nine patients after two years of treatment . Chitotriosidase values decreased 10 - 20 times the initial value after one year of treatment, and in one case such a value reached the normal range . The decrease of chitotriosidase levels correlated with the improvement of the clinical signs, which justifies analyzing of this biomarker for a better evaluation of the disease progression, as well as in orientating the necessity for a specific cerezyme dosage, an extremely expensive drug . Oculomotor anomalies were less sensitive to the treatment, probably because the cerezyme given intravenously does not pass through the hematoencephalic barrier[5, 8, 16]. Spleen enlargement was seen in all our patients, but in six of them it was massive and was accompanied with a sharp pain in the left flank seen also after a light physical exercise, as a result of the splenic capsule tenderness . In three cases, the echography showed spleen nodules and hyperechogenic zones inside the parenchyma . Liver enlargement was found in all our patients, but it was less significant from the spleen enlargement, and it was not accompanied with an increase in the bilirubin and/or liver enzyme values, or with a decrease in the protrombine level as well as with no changes in the protein electrophoresis profile . Liver involvement might progress toward the cirrhosis, accompanied with portal hypertension, ascites and esophageal varicosities, but these manifestations were otherwise mainly absent in our group[5, 7, 10, 20]. The radiological examinations showed osteopenia of the femoral bone in all our patients, of the humerus in seven of the patients and of at least one vertebral body in three patients . Osteosclerotic changes were seen in four from our eleven patients, and osteolysis only in one patient . Bone pain was present in six children, and in two of them the pain was paroxysmal[3, 5, 16, 21, 22]. The bone marrow examination for the detection of gaucher's cells is important, but it is not pathognomonic . Sometimes a single bone marrow examination may not be sufficient, as the disease has a focal nature . In two of our cases the hematological disorders are one of the more constant manifestations of this disease . In our group, the hemoglobin level was decreased in six cases, the platelet count was decreased in nine cases and the white blood cell count was decreased in two cases . Platelet count never reached levels under 5010/mm in any of our patients, and no secondary infection due to leucopenia was met[8, 10, 20, 21]. According to majority of the authors, it was below 20% of the normal value in six of our patients, which were two cases of type 3 and four other patients of type 1 of the disease . Other biomarkers, which are related to biochemical changes and multisystemic involvement of the disease, can be evaluated as well . These biomarkers are chitotriosidase, -hexosaminidase, angiotensine converting enzyme (ace) and acid phosphatase . Among these, the most important is chitotriosidase, levels of which were increased in all our patients, varying from 1152 to 31220 kat / l (normal range 40 however, there is no correlation between these values and the clinical significance of the disease[10, 18, 21, 25]. The genetic molecular examinations were performed in eight patients of type 1 and in one patient of type 3 (table 2). All patients were affected of a double heterozygotic form, which means they were carrying simultaneously two different mutations . The dominant mutation in the type 1 of gaucher's disease in our group was n370s (8/9), which fully corresponds to the literature data . The second most common mutation in this type of the disease was d409h, which was present in four cases, while l444p was present in two cases, and r47x, r463h, s107l (rare mutations) were seen each in one patient . The very serious mutation l444p, characteristic for the types 1 and 3 of the gaucher's disease, was not seen in any case in homozygotic form, which does not correlate with other literature data[2, 4, 7, 8, 19]. The treatment with cerezyme, in the form of a recombinant enzyme provided good results in both types 1 and 3 . Liver and spleen reduction in size was achieved in our patients, in 40 - 50% and 50 - 60% respectively, two years after the therapy was started, and this corresponds to other casuistics[5, 8, 10]. In two cases bone alterations, such as osteopenia, were persisting even after two years of treatment . Cerezyme side effects such as hypersensitivity reactions, anaphylactic shock, pruritus, reddening, urticaria, angioedema, chest pain, dyspnea, hypotension, coughing and cyanotic reactions are rarely described in the literature and were not met in any of our patients [10, 11, 16, 22]. The results of the actual study are in accordance with the overall opinion of efficacy for cerezyme, although our study was an open - label, non randomized and non - blinded one . Another limitation probably is related to the fact that no control group receiving alternative treatment (such as substrate reduction therapy [srt] or miglustat) was instituted; other authors have suggested and applied such treatment in combination . Anyway, alternative treatments and the option of no treatment, although still in use in isolated cases, widely lack the level of efficacy offered from cerezyme. Gaucher's disease may be under - diagnosed in albania, due to the paucity of the sources for performing the genetic evaluation and analysis . The most common mutation in our patients group, formed from eleven patients, was n370s . Signs and symptoms in the group were registered at the baseline, and every six months after treatment institution, for a period of two years . The replacement enzyme, cerezyme resulted effective and safe in treating the gaucher's disease . The patients suffering from gaucher's disease type 1 received cerezyme with a dosage of 60 ui / kg of body weight every other week; the patients suffering from gaucher's disease type 3 received cerezyme with a dosage of 120 ui / kg of body weight every other week . The genetic profile was not decisive in the efficacy prediction; therefore such treatment has to be applied to all patients suffering from gaucher's disease type 1 and 3, whichever mutations are detected . The national state health system must provide some models to reimburse cerezyme in albania, due to proven efficacy of the treatment, as well as due to the extreme costs of the drug, and the necessity for a lifelong uninterrupted treatment . No funding was received for the present work . The actual treatment with cerezyme (enzyme replacement)

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