Split (1)

train · 133k rows

text stringlengths 0 868k
His vision, leadership, and hard work established the procedure of using the liver as a site for successful islet transplantation . The transplants normalized glycemia in rats previously made diabetic by streptozotocin injection (2). As he and his colleagues began publishing manuscripts, it did not take long for the surgical community to apply this technique to humans (fig . This proved to be a much more ambitious task than first imagined . Transporting pancreata isolated from brain - dead donors on life support, the inherent delays in islet isolation, the presence of autoimmune disease in the recipients, and the need to use powerful immunosuppressive drugs with significant side effects all presented significant barriers . Islets are removed from a donated pancreas by collagenase digestion, followed by purification to separate the islets from exocrine tissue . Islets are then infused by gravity into a catheter lodged in the hepatic portal vein . Blood flow within the vein carries the islets into the liver tributaries where they lodge within the sinusoids and establish vascular connections . Najarian and sutherland at the university of minnesota transplanted islets in nondiabetic recipients who were their own donors and reported reproducible successes beginning in 1980 (4). The patients had various forms of chronic, unrelentingly painful pancreatitis and each underwent a total pancreatectomy for pain relief and nutritional rehabilitation . Rather than dispose of the patient's resected pancreas, the clinical researchers used it to make a crude extract of islets that was returned to the operating room within 2 h. the islets were infused over 30 min into the patient's liver while hepatic portal venous pressure was monitored . A summary of the results reported that if over 300,000 autoislets could be transplanted, the success rate of preventing diabetes for more than 2 years was 74% (5). The autoislet experience was very encouraging because it proved that islet transplantation in nondiabetic humans was feasible . The stage for future successes in alloislet transplantation in patients with type 1 diabetes was set . Case reports and small patient series' revealed evidence for function of islets after transplantation and brief or partial improvement in glycemic control (611). One vexing variable was the irony that one of the drugs important generally for transplantation success, cyclosporine, had inhibitory effects on -cell function (1219). Oddly, this became the pattern for other important immunosuppressive drugs (3), especially glucocorticoids (14,18,2022). This led to the use of a glucocorticoid - free immunosuppressive regimen developed by shapiro et al . (1), which in turn led to their now historic series of successful islet transplants in type 1 diabetic patients . Another key aspect of the edmonton approach was to use an average of two sequential islet transplants to establish normoglycemia . Over time, however, the successes enjoyed by the edmonton group began to slowly diminish . In the year 2000, the first seven patients who were insulin free had normal or nearly normal levels of glucose and a1c . Over 2 years the series had grown to 17 patients, 14 (86%) of whom were c - peptide positive and 11 of whom remained insulin free, although two were using oral hypoglycemic agents (23). By 2005, the edmonton series had grown to 66 patients, 85% of whom were c - peptide positive but only 15% of whom were insulin - free (24). The other 85%, who were again using insulin, were reported to be using less than they had been pretransplantation . Using these data, the median time to a return to insulin therapy the immune tolerance network (itn) had undertaken a multicenter trial of alloislet intrahepatic transplantation using the edmonton protocol . The goal was to establish the fidelity with which nine centers in canada, the u.s ., and this consortium reported in 2006 (25) that, of 36 type 1 diabetic subjects studied, 44% achieved the primary end point of insulin independence with adequate glycemic control 1 year after islet transplantation . It is noteworthy that previous experience with islet transplantation at the various research sites was strongly associated with success in achieving the primary end points . Of the 18 subjects, 12 (67%) were successful at 1 year post - transplant at sites where 4 or more patients had been transplanted in the preceding 2 years . Only 4 of 18 subjects (22%) were successful at sites where there was a history of less than four transplantations previously . This outcome clearly signaled that the procedure itself has a steep learning curve . At this time, it remains more of a research procedure than one that is generally applicable to treating patients with type 1 diabetes . During the past decade other groups reported results that were generally in agreement with the edmonton and itn outcomes . As early as 2000, oberholzer et al . (11) reported that of 13 of 13 alloislet recipients had measureable blood c - peptide levels for at least 3 months, as did 7 of 11 recipients at 6 months and 5 of 8 recipients at 1 year post - transplant . (26) used the edmonton protocol and reported success rates of 79% for insulin independence at 1 year and 43% at 18 months for 16 recipients . Toso et al . (27) used sequential kidney followed by islet transplantation and the edmonton protocol . Of 8 patients transplanted, insulin - free status was achieved in all for at least 3 months, and 5 of 11 were insulin - free after an average of 24 months (1134). (28) reported that of six recipients, two were insulin - free at 24 months . (29) reported seven patients who underwent the islet after kidney transplantation approach; two achieved insulin - independence and six had persistent graft function at 1 year . (30) compared islet transplantation alone (ita) with islet after kidney (iak) transplantation . They observed partial function (c - peptide positivity) at 24 months in four of eight and six of six recipients in the ita and iak groups, respectively, as well as insulin - independence at 24 months in four of eight and five of six, respectively . These reports from groups in various parts of the world using more or less the same edmonton protocol, some with the added feature of studying islet after kidney transplantation, generally achieved similar results reported by the edmonton and itn groups . The overall conclusion of these studies is that islet transplantation can be considered largely successful at 1 year with a decline thereafter to an average success rate (insulin - independence and at least nearly normal glycemia) of 50% at various times during the 2nd year, and appreciably less at 5 years post - transplant . Do persistently measureable c - peptide levels combined with either a return to less intense insulin therapy and less hypoglycemia or, alternately, a state of insulin - independence but not completely normal a1c levels both represent successes, partial successes, partial failures, or failures? The analogy of whether a glass is half full or half empty seems unavoidable in an analysis of success versus failure in alloislet transplantation . The answer to this question is in the eye of the beholder . To the skeptic's eye, the procedure looks interesting, but is replete with problems . Given the current nearly normal life expectancy of people with type 1 diabetes who are well managed with insulin - based treatment, an invasive procedure with a 50% chance of success for only 12 years does not seem very useful . To the optimist's eye, a different conclusion might be reached . Certain patients with type 1 diabetes are difficult to manage medically and have a very poor quality of life because of recurrent hypoglycemia and rapid development of chronic complications secondary to chronic hyperglycemia . The diabetes care and complications trial (31) established that maintenance of a1c levels less than or equal to 7% is mandatory for minimizing complications . A procedure, such as alloislet transplantation, which shows promise of providing c - peptide positivity, nearly normal a1c levels, and strikingly fewer episodes of hypoglycemia for as long as 5 years, cannot be easily dismissed (24,3233). Furthermore, initial reports have appeared claiming that islet recipients have macro- and microvascular as well as quality of life benefits from restoration of c - peptide secretion even if a1c levels are not normalized (3436). An objective approach to evaluating the progress of islet transplantation is to compare its history to that of pancreas transplantation . The procedure of pancreas transplantation, originated at the university of minnesota, struggled greatly in its early days with high patient mortality and failure of grafts by 6 months (37). However, a return to the canine lab to reshape protocols and the advent of cyclosporine changed things dramatically . In the ensuing decades, a gratifying increase in pancreas survival and decrease in patient mortality took place . At its current zenith of success, pancreas transplantation enjoys virtually no patient mortality attributable to the surgery and an average of 80% success 3 years post - transplantation (38). 2), one sees that islet transplantation might not being doing quite as poorly as one might at first think . Pancreas allograft survival at 15 months post - transplant in the third epoch of this procedure (19841987) was precisely that of islet transplantation in its first successful years of 20002005, namely, 50% . Comparison of rates of progress of alloislet transplantation versus pancreas transplantation as therapy for type 1 diabetes . The first alloislet transplant to be reported as successful appeared in 1980, 20 years earlier than the edmonton series was reported . The first pancreas transplant to be reported as successful appeared in 1966, 18 years earlier than the 19841987 series was reported . At 15 months post - transplant, the edmonton series of alloislet transplants for the years 20002005 compared favorably with the success rate of pancreas transplants for the years 19841987 . A major deficiency of virtually all published studies of islet transplantation is the lack of a suitable control group of medically managed patients . While one might argue that islet transplantation improves the health and lives of islet recipients, this is not the central issue . Such an argument begs the question of whether the complicated and expensive procedure of islet transplantation performs better than standard insulin - based therapy . Without a randomized, nontransplanted control group patients who choose islet transplantation do so because they view their quality of life as poor, most often because of recurrent hypoglycemia . It is not always clear from published reports how objectively the diagnosis of recurrent hypoglycemia was made or how vigorously such patients underwent a period of insulin - based management by skilled diabetologists . Consequently, one cannot be certain of the claim that partially failed islet transplantation leads to the use of less insulin and less hypoglycemia on a cause - effect basis . It could just as easily be that patients who enter transplant programs come under close clinical scrutiny by interested diabetologists who begin managing them more skillfully . The sole study that did include a medically managed control group was reported by thompson et al . (36). In this study, 44 patients were candidates for islet transplantation; 27 became islet recipients whereas 17 continued on medical management . The group receiving transplants were reported to have less likelihood of progression of retinopathy than the medically managed patients . First, although the two groups had comparable durations of diabetes, no randomization procedure was used to determine which patients would be transplanted, and, second, a1c levels at entry into the study were significantly lower in the transplant group . Nonetheless, this is a valuable study that illustrates the possibility for randomized studies to ascertain whether islet transplantation accrues additional benefits compared with intensive medical management . With few exceptions, published reports of islet transplantation in type 1 diabetes have not adequately assessed the level to which endogenous insulin secretion is restored by the procedure . In most instances, basal levels and sometimes glucose - stimulated responses of c - peptide from recipients post - transplant are provided . Prior to transplant, only meager laboratory data about functionality of the islets to be transplanted are generally obtained . Typically, islets are stimulated with low and high concentrations of glucose in static incubations, and data are reported as a secretory index (insulin or c - peptide responses to the higher glucose concentration divided by the response to the lower glucose concentration). This practice at best relates only to whether the islets are alive or dead and imparts little about their degree of functionality . Consider, for example, that a fold response (usually 1.0 or a doubling is considered acceptable) to the higher glucose challenge will be dramatically affected by the baseline value . The lower the denominator used to divide the numerator, the more exaggeratedly high the quotient will look . What is needed is assessment of glucose - induced insulin secretion by either static or perifusion protocols . The former uses several glucose concentrations so that a half - maximal effective concentration can be calculated; the latter uses multiple samples during perifusion so that assessment of first- and second - phase responses to glucose stimulation can be obtained . These approaches would provide much more sophisticated information about the donor islets used for human transplantation and would go a long way in interpreting the clinical outcomes obtained in recipients . The usefulness of more intensive functional studies can be easily appreciated from the autoislet experience in humans . In this scenario, the number of islets transplanted correlates very well with the magnitude of insulin responses to several stimulii (39). Reported outcomes in the current literature have come from programs that made heroic efforts to procure pancreata as quickly as possible and then produce islet preparations that were as pure as possible . This involved exposure of islets to collagenase for variable periods of time followed by extensive centrifugation in cold temperatures . The 50% that are recovered after purification by cold centrifugation are, not surprisingly, often damaged . The method of counting viable islets afterward may or may not include damaged islets that are not totally dead . It seems very likely that the number of healthy, transplanted islets is significantly overestimated, meaning the recipients may not have received the stated goal of 6,000 healthy islets / kg body mass . Transplanting 6,000 islets / kg body mass delivers roughly 420,000 islets per person, 4050% the number of islets in a pancreas of a nondiabetic human . This number is similar to that contained in the remaining pancreatic segment in hemipancreatectomized donors and in the recipient of a hemipancreas, and is adequate to maintain normoglycemia for many years (40,41). An important difference is that islets used for autoislet transplantation are not purified and undergo much less stress pretransplantation . One answer to the alloislet problem may be to eliminate the cold centrifugation step (42). Cold centrifugation minimizes the total tissue mass used to infuse islets into the liver, which is thought to lessen the likelihood of complications such as hepatic portal hypertension or lobar infarction . However, this may be a case of too much caution because autoislet transplantation, in which there is no purification, has no significant history of either complication . Lacy's group originally recommended the liver, based on their rodent studies, and thereafter it was the site traditionally used for human auto- and alloislet transplantation . However, not only is the liver the site where ingested environmental toxins accumulate, but it is also where orally administered immunosuppressive drugs that are toxic to -cells are concentrated . Drug concentrations in hepatic portal venous blood are two- to threefold greater than in systemic circulation (43,44) and reach concentrations that inhibit -cell function in vitro (3,1219). This and other complications of currently available immunosuppressive drugs (infection, potential cancer) demand that the search for less toxic drugs continue unabated so that the islet, as well as pancreas, transplantation approach can become more clinically acceptable . 3), although they do respond normally to other stimuli, such as arginine . This has been demonstrated in human autoislet recipients (45,46), human alloiset recipients (47,48), and animals (49,50). A likely explanation is that glucose flux within the liver comes into contact with -cells on the periphery of transplanted islets . This is especially relevant during hypoglycemia when glycogenolysis is stimulated by catecholamines and central nervous system inputs . The intimate contact between intrahepatic glucose flux and -cells in the islet allograft abrogates the hypoglycemic signal delivered to the transplanted -cells from systemic blood coursing through the hepatic artery . This hypothesis was tested in animals that received alloislet transplantation in hepatic and nonhepatic sites and thereafter underwent insulin - induced hypoglycemic challenges before and after prolonged fasting (50). In all sites except the liver, when the intrahepatically transplanted animals underwent prolonged fasts and hepatic glycogen depletion, the initially absent glucagon responses to hypoglycemia were recovered (fig . 4). Refeeding the animals to replete liver glycogen caused the glucagon response to hypoglycemia to disappear once again . Nondiabetic control subjects have plasma glucagon responses to insulin - induced hypoglycemia during a stepped hypoglycemic clamp . This response is absent in patients with type 1 diabetes as well as patients who have received successful intrahepatic alloislet or autoislet transplantation . Restoration of absent glucagon responses to hypoglycemia from intraheptic islets in rats after prolonged fasting . Liver glycogen depletion caused by prolonged fasting results in restoration of the glucagon response to hypoglycemia . This abnormality in -cell function does not occur in fed animals if islets are placed into nonhepatic sites . It is very important to retain -cell responses to hypoglycemia in humans who undergo islet transplantation, return to insulin therapy, and become once again at risk for hypoglycemia . It is established that glucagon responses to hypoglycemia from the native pancreas of type 1 diabetic patients is defective because of absent signaling from neighboring -cells . Alloisets transplanted in nonhepatic sites will respond to hypoglycemia and will protect the patient with a partially successful transplant who uses insulin to manage hyperglycemia . What alternate sites might be considered? Preclinical literature supports consideration of celiac artery, intravenous access to lung, intrapancreas, intramuscular, subcutaneous, thymus, testis, intracisterna magna, omental pouches, bowel surfaces, peritoneal cavity, spleen, bone marrow, and kidney capsule (50,5254). I believe the use of liver should be reconsidered and a new look should be given to other sites first examined in 1972 but passed over for human use . With few exceptions, published reports of islet transplantation in type 1 diabetes have not adequately assessed the level to which endogenous insulin secretion is restored by the procedure . In most instances, basal levels and sometimes glucose - stimulated responses of c - peptide from recipients post - transplant are provided . Prior to transplant, only meager laboratory data about functionality of the islets to be transplanted are generally obtained . Typically, islets are stimulated with low and high concentrations of glucose in static incubations, and data are reported as a secretory index (insulin or c - peptide responses to the higher glucose concentration divided by the response to the lower glucose concentration). This practice at best relates only to whether the islets are alive or dead and imparts little about their degree of functionality . Consider, for example, that a fold response (usually 1.0 or a doubling is considered acceptable) to the higher glucose challenge will be dramatically affected by the baseline value . The lower the denominator used to divide the numerator, the more exaggeratedly high the quotient will look . What is needed is assessment of glucose - induced insulin secretion by either static or perifusion protocols . The former uses several glucose concentrations so that a half - maximal effective concentration can be calculated; the latter uses multiple samples during perifusion so that assessment of first- and second - phase responses to glucose stimulation can be obtained . These approaches would provide much more sophisticated information about the donor islets used for human transplantation and would go a long way in interpreting the clinical outcomes obtained in recipients . The usefulness of more intensive functional studies can be easily appreciated from the autoislet experience in humans . In this scenario, the number of islets transplanted correlates very well with the magnitude of insulin responses to several stimulii (39). Reported outcomes in the current literature have come from programs that made heroic efforts to procure pancreata as quickly as possible and then produce islet preparations that were as pure as possible . This involved exposure of islets to collagenase for variable periods of time followed by extensive centrifugation in cold temperatures . The 50% that are recovered after purification by cold centrifugation are, not surprisingly, often damaged . The method of counting viable islets afterward may or may not include damaged islets that are not totally dead . It seems very likely that the number of healthy, transplanted islets is significantly overestimated, meaning the recipients may not have received the stated goal of 6,000 healthy islets / kg body mass . Transplanting 6,000 islets / kg body mass delivers roughly 420,000 islets per person, 4050% the number of islets in a pancreas of a nondiabetic human . This number is similar to that contained in the remaining pancreatic segment in hemipancreatectomized donors and in the recipient of a hemipancreas, and is adequate to maintain normoglycemia for many years (40,41). An important difference is that islets used for autoislet transplantation are not purified and undergo much less stress pretransplantation . One answer to the alloislet problem may be to eliminate the cold centrifugation step (42). Cold centrifugation minimizes the total tissue mass used to infuse islets into the liver, which is thought to lessen the likelihood of complications such as hepatic portal hypertension or lobar infarction . However, this may be a case of too much caution because autoislet transplantation, in which there is no purification, has no significant history of either complication . Lacy's group originally recommended the liver, based on their rodent studies, and thereafter it was the site traditionally used for human auto- and alloislet transplantation . However, not only is the liver the site where ingested environmental toxins accumulate, but it is also where orally administered immunosuppressive drugs that are toxic to -cells are concentrated . Drug concentrations in hepatic portal venous blood are two- to threefold greater than in systemic circulation (43,44) and reach concentrations that inhibit -cell function in vitro (3,1219). This and other complications of currently available immunosuppressive drugs (infection, potential cancer) demand that the search for less toxic drugs continue unabated so that the islet, as well as pancreas, transplantation approach can become more clinically acceptable . 3), although they do respond normally to other stimuli, such as arginine . This has been demonstrated in human autoislet recipients (45,46), human alloiset recipients (47,48), and animals (49,50). A likely explanation is that glucose flux within the liver comes into contact with -cells on the periphery of transplanted islets . This is especially relevant during hypoglycemia when glycogenolysis is stimulated by catecholamines and central nervous system inputs . The intimate contact between intrahepatic glucose flux and -cells in the islet allograft abrogates the hypoglycemic signal delivered to the transplanted -cells from systemic blood coursing through the hepatic artery . This hypothesis was tested in animals that received alloislet transplantation in hepatic and nonhepatic sites and thereafter underwent insulin - induced hypoglycemic challenges before and after prolonged fasting (50). In all sites except the liver, when the intrahepatically transplanted animals underwent prolonged fasts and hepatic glycogen depletion, the initially absent glucagon responses to hypoglycemia were recovered (fig . 4). Refeeding the animals to replete liver glycogen caused the glucagon response to hypoglycemia to disappear once again . Nondiabetic control subjects have plasma glucagon responses to insulin - induced hypoglycemia during a stepped hypoglycemic clamp . This response is absent in patients with type 1 diabetes as well as patients who have received successful intrahepatic alloislet or autoislet transplantation . Reproduced from ref . Restoration of absent glucagon responses to hypoglycemia from intraheptic islets in rats after prolonged fasting . Liver glycogen depletion caused by prolonged fasting results in restoration of the glucagon response to hypoglycemia . This abnormality in -cell function does not occur in fed animals if islets are placed into nonhepatic sites . It is very important to retain -cell responses to hypoglycemia in humans who undergo islet transplantation, return to insulin therapy, and become once again at risk for hypoglycemia . It is established that glucagon responses to hypoglycemia from the native pancreas of type 1 diabetic patients is defective because of absent signaling from neighboring -cells . Alloisets transplanted in nonhepatic sites will respond to hypoglycemia and will protect the patient with a partially successful transplant who uses insulin to manage hyperglycemia . What alternate sites might be considered? Preclinical literature supports consideration of celiac artery, intravenous access to lung, intrapancreas, intramuscular, subcutaneous, thymus, testis, intracisterna magna, omental pouches, bowel surfaces, peritoneal cavity, spleen, bone marrow, and kidney capsule (50,5254). I believe the use of liver should be reconsidered and a new look should be given to other sites first examined in 1972 but passed over for human use . A valid argument against islet transplantation as a treatment for type 1 diabetes is the undeniable arithmetic that not nearly enough pancreas donors exist to treat patients with type 1 diabetes, let alone all people with type 1 and type 2 diabetes, especially in the face of the current diabetes epidemic . Does this mean we have been wasting our time and resources by studying islet transplantation? There will always be diabetic patients who need -cell replacement by transplantation of islets or the pancreas . One group of patients comprises those with rapid development of secondary complications despite optimal medical care . Another group is made up of patients with the neurological disorder of autonomic insufficiency, which is accompanied by a 50% death rate within 5 years of diagnosis . They are clearly candidates because successful pancreas transplantation converts this death rate from 50 to 10% . Our experiences with islet transplantation have taught us lessons that will be important for the use of -cell surrogates, be they stem cell derivatives or modified cell lines . We have learned about culturing cells, isolating islets and -cells, identifying safe and physiological sites for transplantation, avoiding immunosuppressive drugs that are toxic to -cells, meeting environmental needs for physiological -cell function, and selecting appropriate patients for -cell replacement . This is important information to use as we continue to meet the challenge of creating better means of controlling hyperglycemia and avoiding its complications . We just need to continue on with new scientific work until the transplantation glass is successfully filled.
Mumps, also known as epidemic parotitis, is a viral disease caused by the mumps virus . The virus usually causes parotitis but sometimes results in central nervous system (cns) complications such as acute aseptic meningitis or acute or chronic encephalitis . Cns involvement has been reported to occur in up to 65% of cases of mumps (12), and meningitis accounts for a major proportion of cns complications (1). The development of encephalitis is rare (3), with a reported incidence of 1 per 6000 mumps cases . However, encephalitis has a much graver prognosis 382than meningitis (12), with mortality rates ranging from 2.3% to 22% and long - term morbidity ranges of from 24% to 33% (1). Here, we describe an adult case of acute mumps meningoencephalitis that manifested as seizure and impaired consciousness with neuroimaging findings of bilateral hippocampal encephalitis and meningitis . The patient had no preceding clinical symptoms of acute parotitis, which made an early diagnosis of mumps difficult . Institutional review board approval was obtained, and the requirement for informed consent was waived due to the retrospective nature of this case report . A 32-year - old man was brought to the emergency room of our hospital with sudden - onset generalized seizures, which developed suddenly and stopped spontaneously after about three minutes without the administration of anticonvulsants; post - ictal stuporous mentality followed . The patient had suffered from headaches and fever and a sensation of chills for six days prior to his hospital visit . Clinical signs of meningitis, including nuchal rigidity and kernig and brudzinski signs, were observed . At this time, his temperature and blood pressure were 38.5 and 136/56 mm hg, respectively . His laboratory peripheral blood results revealed lymphocyte - predominant leukocytosis (white blood cell count, 16760/l; lymphocytes, 67.2%), and a cerebrospinal fluid (csf) examination revealed no remarkable findings . Antibodies for cytomegalovirus and orgherpes simplex virus were negative, and microorganisms including group b streptococcus, enterovirus, and mycobacterium tuberculosis were not found on csf examination; a serologic test for japanese encephalitis antibody was also negative . Initial brain ct and brain magnetic resonance imaging (mri) findings were normal . However, initial electroencephalography revealed continuous, irregular, slowing background activity and many spikes, waves, and bursts of semi - rhythmic delta activity in the bilateral fronto - temporal areas that suggested diffuse cerebral dysfunction . Day 3, a laboratory test was performed for mumps infection; the patient's serum mumps immunoglobulin m (igm) titer was 2.6 (normal range, <0.8), but his immunoglobulin g (igg) was equivocal (titer, 0.86; equivocal for 0.81.2). Although we could not establish our patient's vaccination history, these findings suggested active mumps infection, and we diagnosed mumps meningoencephalitis based on the patient's serological and brain imaging findings; notably, no symptoms of mumps infection (such as parotitis or orchitis) other than symptoms of meningoencephalitis were found . However, on admission day 7, his consciousness worsened to a deep stupor, and respiration became sufficiently unstable that the patient required endotracheal intubation and artificial ventilation . Follow - up csf examination revealed lymphocyte - predominant pleocytosis (18 nucleated cells / mm [74% lymphocytes]). On the same day, t2-weighted and diffusion - weighted brain mri images revealed increased signal intensity areas in the bilateral hippocampi, and postcontrast t1-weighted images revealed diffuse leptomeningeal enhancement but no contrast enhancement of the bilateral hippocampal lesions (fig . 1). On admission day 10, the patient became afebrile and alert, although intermittent seizure attacks persisted . Follow - up serology testing showed that his mumps igm was negative (titer, 0.49), which suggested treatment response, but that igg remained equivocal (titer, 0.83). The patient's brain mri on admission day 20 showed improvement of the bilateral hippocampal lesions and the disappearance of meningeal enhancement (fig . Complete recovery of neurologic symptoms was noted on admission day 36, and the patient was discharged . Herein, we report a unique case of mumps meningoencephalitis in an adult that involved the bilateral hippocampi without preceding parotitis . Bilateral hippocampal encephalitis and meningitis were observed by brain mri, and mumps was diagnosed based on elevated mumps - specific igm serum titers . We consider that this case was a pure form of mumps meningoencephalitis, which is an extremely uncommon form of mumps . Fortunately, the patient's neurologic symptoms fully abated, and follow - up serologic testing revealed negative conversion of mumps - specific serum igm, which suggested a treatment response . Symptoms of mumps are generally mild moderately elevated body temperature and parotid swelling (1). However, serious complications such as orchitis, pancreatitis, meningitis, and encephalitis occasionally occur (1). At most 10% of all patients with mumps parotitis develop clinical meningitis (45), which may develop without parotid gland swelling, as occurred in the present case . Mumps encephalitis is uncommon in adults (3), but when it is encountered, impaired consciousness, seizures, and psychological symptoms are often observed; in such cases, serological diagnosis or virus isolation is required for differential diagnosis . Furthermore, mumps may be infrequently complicated by acute encephalitis, which is generally mild and shows no focal signs of meningeal irritation (3). The first, primary mumps meningoencephalitis, involves direct invasion of the cns by the virus and is associated with the appearance of encephalitis during the early disease stage . Furthermore, autopsy findings of diffuse edema, perivascular cuffing, cytolysis, and satellitosis without evidence of demyelination have been ascribed to primary mumps meningoencephalitis (6). The second type results from immune - mediated demyelination, which occurs on average 10 days after disease onset . Acute disseminated encephalomyelitis (adem) and clinically mild encephalitis with reversible splenial lesions are included in this type (6). We consider that the meningoencephalitis in our case was caused by direct viral invasion rather than an autoimmune process because neurological abnormalities occurred on day six after disease onset and no white matter abnormalities were visualized on mri . However, direct viral infiltration of the cns was not confirmed by mumps virus mrna reverse transcription polymerase chain reaction (rt - pcr). Focal cns involvement is less common in mumps than is meningitis . In the english literature, mumps encephalitis affects the brainstem, basal ganglia, splenium of the corpus callosum, and white matter, and it is considered a form of adem (12345678910); however, bilateral hippocampal lesions resulting from mumps viral infection have not been previously reported . Other pathogens that should be included in the differential diagnosis of bilateral hippocampal lesions include herpes simplex virus, ebstein - barr virus, and cytomegalovirus . In the case focal cerebral edema secondary to persistent temporal lobe seizures might be considered imaging findings in our case, but clinical course and serological evidence of apparent mumps infection strongly suggested that the patient's condition resulted from the mumps . Notably, no established effective therapy is available for mumps encephalitis (2), although vaccination provides useful prophylaxis (2). Early accurate diagnosis is important, but in the present case, mumps infection was not easily detected . In its early stage, mumps meningoencephalitis may clinically mimic other acute brain disorders such as pyogenic brain abscess, acute multiple sclerosis, tuberculous meningitis, or drug intoxication (1), and radiologic findings are either normal or nonspecific . In our patient, clinical symptoms or signs (such as parotid swelling) that readily suggest mumps infection were absent . Mumps meningitis occurs in the absence of salivary gland involvement in up to 50% of cases, and it can precede salivary symptoms or occur after the appearance of parotitis (5). According to studies published in the 1950s and 1960s, the incidence of meningoencephalitis without salivary gland involvement was 4349% in epidemic mumps regions (10). One case report does describe brain stem involvement on a brain mri in an adult case of mumps encephalitis without salivary gland involvement (2). The rate of mumps viral infection has been greatly decreased by vaccination, and as a result, clinical suspicion of mumps meningoencephalitis is difficult, especially when there is no salivary gland involvement . Our patient only had symptoms of cns infection and abnormal neuroimaging findings, and it was immunoassays of serum that led to a diagnosis of acute mumps infection . Although brain parenchymal involvement in the mumps is extremely rare, symmetric hippocampal lesions could be considered when other pathogens of cns infection are not evident on laboratory examination . Although it is rare, mumps meningoencephalitis should be considered in the differential diagnosis of bilateral hippocampal lesions and in the presence of evidence of an infectious cns disorder.
Hypertension is a major risk factor for cardiovascular disease (cvd). In the majority of western countries, the cost of hypertension and its complications absorbs a large and growing share of health care resources.1 in italy, the expenditure for antihypertensives reached 991.35 million in 2003 . Subtracting expenditure due to use of the same drugs for diseases other than hypertension (around 15%), total expenditure for antihypertensive therapy (aht) may be considered to be approximately 840 million.2 moreover, at the national level, drugs used for cvd continued to show both the highest national health system (nhs) expenditure (37.5%) and the highest consumption (49.0%) in 2007.3 aht has been clearly shown to reduce both cardiovascular and cerebrovascular events47 and, consequently, to reduce overall health care costs because of better disease control and lower rates of adverse outcomes.813 the efficacy of pharmacologic treatment in reducing blood pressure has also been well demonstrated in regimem of chronic and adequately dosed drug use . However, evidence - based guidelines have often failed to impact clinical practice . In current practice, observational studies have shown that 30%70% of patients with treated hypertension does not persist or adhere to treatment.1419 these results and the considerable pressure on health care systems to provide high - quality care have led several public and private organizations to promote initiatives to monitor and improve hypertension control . Meanwhile, progressive reduction of available financial resources has ushered in a series of measures for controlling costs, such as reductions in drug prices, prescriptions charges for private citizens, and direct distribution of drugs . The aim of this work is to emphasize the role of adherence to aht as a key performance indicator in hypertension management . In addition to the existing literature, the present study adds a time - trend analysis of adherence during last years and a pharmacological treatment cost analysis . In italy the provider of health care for all citizens is the nhs, which is organized into local health units (lhu) throughout the country . Each lhu, as an autonomous body of the nhs, organizes and plans the health care system for a specific area so as to provide services in the community close to where people live . The lhus have an information network that routinely measures the volumes of expenditure generated by the use of health care services and collects them into computerized reimbursement databases . The data we used were retrieved from three different databases of the lhu of florence, an area located in the center of italy, which includes approximately 800,000 beneficiaries: a beneficiaries database, containing patient demographic data, a medications prescription database, providing information for each prescription (such as the prescribing physician s number, anatomical - therapeutic - chemical code of the drug purchased, number of packs, number of units per pack, dosages, unit cost per pack and prescription date), and a hospital discharge database, that includes all hospitalization data, with discharge diagnosis codes classified according to the international classification of diseases, ninth revision (icd-9). The lhu database affords a valuable opportunity to assess the use of aht in a real practice setting . To guarantee patient privacy this was a retrospective cohort study, which included only new aht users, 18 years of age or over, for whom all prescription and clinical outcome data over the study period were compiled . Patients were included if they had received at least one dispensed prescription of aht (diuretics [atc code c03, excluding loop diuretics, mainly used for heart failure], beta - blockers [c07], calcium channel blockers [c08], angiotensin - converting enzyme inhibitors [c09a / b], angiotensin receptor blockers [c09c / d]) between 01 january 2004 and 31 december 2007 . The date of the first purchased aht was defined as the enrolment date . Patients were defined as new users if they had not been prescribed any aht in the 12 months preceding the enrolment date . In line with previously published studies,20 and in order to include only patients treated for hypertension, we excluded any patient who had been diagnosed with heart failure (code 428.x), ischemic heart disease (code 410414.x), cerebrovascular disorders (code 430438.x) or other cardiovascular diseases (code 390400.x, 406459.x, excluding the aforementioned diagnosis codes) in the 12 months before enrolment . In addition, we excluded patients using nitrates in the 12 months preceding treatment initiation because these agents are also indicated in conditions other than hypertension . Only patients with continuous eligibility for at least 24 months (12 months before and 12 months after the enrolment date) were included . Adherence to aht was estimated by calculating the proportion of days (proportion of days covered [pdc]) on which a patient had pills available in the time interval of 12 months after the enrolment date (follow - up). The interval was separated into treatment episodes of continuous aht use based on the method of catalan.21 a treatment episode was measured as the time period between the starting date of the first aht prescription dispensed until the last day supplied on the final dispensed aht prescription . Prescriptions containing more than one drug contributed to both the sum of the days supply of all drugs from the same aht class (to accommodate any stockpiling) and the lower days supply drug value of drugs from different aht classes (identifying them as a combined therapy). The pdc corresponded to the total of number of days supply of medication dispensed within each episode divided by the total length of the interval and multiplied by 100 . Consistent with data in the literature,22,23 patients were divided into five different categories according to their pdc level, ie, low (pdc 20%), low - intermediate (21%40%), intermediate (41%60%), high - intermediate (61%80%), or high (> 80%). Cases of multiple class medications, such as calcium channel blockers and angiotensin receptor blockers, were identified as combined therapy . A four - year descriptive analysis was conducted to evaluate differences in baseline characteristics, use of aht, and adherence levels across the cohorts of patients identified by enrolment year from 2004 to 2007 . We summarized data as mean values with standard deviations for continuous variables and as numbers (percentages) of subjects for categoric variables . We used pearson s chi - square test and one - way analysis of variance (anova) to determine significant differences in baseline characteristics across adherence levels . A multiple logistic regression model was used to estimate odds ratios (or) and 95% confidence intervals (ci) and to identify possible factors significantly associated with the risk of nonadherence during 20042007 . Patients who met the inclusion criteria in more than one of the four previous cohorts were only included once . The pdc was dichotomized, setting a threshold of pdc 80% to identify nonadherent patients . The other covariates in the model were age, gender, presence of some medications (at least two prescriptions) evaluated in the 12 months before the enrolment date, antidiabetic agents (atc code a10), lipid - lowering drugs (c10), cardiac therapy (c01, nitrates excluded), drugs for obstructive airways disease (r03), platelet inhibitors (b01ac), and initial therapy drug class, with angiotensin receptor blockers as the reference . The year of treatment initiation was also controlled for to account for possible time trends in medication use . In fact, although it would be important for the decision - maker to consider other direct costs, eg, hospitalizations, control of pharmaceutical expenditure is often considered as the relevant maneuver from the public health care provider point of view, and hence the authors focused on that aspect.24 no information on indirect costs was available . Each prescription cost was calculated by multiplying the cost per pack by the total number of packs . Since each prescription is unequivocally linked to the patient through the personal health code, the exact direct cost per patient was also known . In italy the provider of health care for all citizens is the nhs, which is organized into local health units (lhu) throughout the country . Each lhu, as an autonomous body of the nhs, organizes and plans the health care system for a specific area so as to provide services in the community close to where people live . The lhus have an information network that routinely measures the volumes of expenditure generated by the use of health care services and collects them into computerized reimbursement databases . The data we used were retrieved from three different databases of the lhu of florence, an area located in the center of italy, which includes approximately 800,000 beneficiaries: a beneficiaries database, containing patient demographic data, a medications prescription database, providing information for each prescription (such as the prescribing physician s number, anatomical - therapeutic - chemical code of the drug purchased, number of packs, number of units per pack, dosages, unit cost per pack and prescription date), and a hospital discharge database, that includes all hospitalization data, with discharge diagnosis codes classified according to the international classification of diseases, ninth revision (icd-9). The lhu database affords a valuable opportunity to assess the use of aht in a real practice setting . To guarantee patient privacy this was a retrospective cohort study, which included only new aht users, 18 years of age or over, for whom all prescription and clinical outcome data over the study period were compiled . Patients were included if they had received at least one dispensed prescription of aht (diuretics [atc code c03, excluding loop diuretics, mainly used for heart failure], beta - blockers [c07], calcium channel blockers [c08], angiotensin - converting enzyme inhibitors [c09a / b], angiotensin receptor blockers [c09c / d]) between 01 january 2004 and 31 december 2007 . The date of the first purchased aht was defined as the enrolment date . Patients were defined as new users if they had not been prescribed any aht in the 12 months preceding the enrolment date . In line with previously published studies,20 and in order to include only patients treated for hypertension, we excluded any patient who had been diagnosed with heart failure (code 428.x), ischemic heart disease (code 410414.x), cerebrovascular disorders (code 430438.x) or other cardiovascular diseases (code 390400.x, 406459.x, excluding the aforementioned diagnosis codes) in the 12 months before enrolment . In addition, we excluded patients using nitrates in the 12 months preceding treatment initiation because these agents are also indicated in conditions other than hypertension . Only patients with continuous eligibility for at least 24 months (12 months before and 12 months after the enrolment date) were included . Adherence to aht was estimated by calculating the proportion of days (proportion of days covered [pdc]) on which a patient had pills available in the time interval of 12 months after the enrolment date (follow - up). The interval was separated into treatment episodes of continuous aht use based on the method of catalan.21 a treatment episode was measured as the time period between the starting date of the first aht prescription dispensed until the last day supplied on the final dispensed aht prescription . Prescriptions containing more than one drug contributed to both the sum of the days supply of all drugs from the same aht class (to accommodate any stockpiling) and the lower days supply drug value of drugs from different aht classes (identifying them as a combined therapy). The pdc corresponded to the total of number of days supply of medication dispensed within each episode divided by the total length of the interval and multiplied by 100 . Consistent with data in the literature,22,23 patients were divided into five different categories according to their pdc level, ie, low (pdc 20%), low - intermediate (21%40%), intermediate (41%60%), high - intermediate (61%80%), or high (> 80%). Cases of multiple class medications, such as calcium channel blockers and angiotensin receptor blockers, were identified as combined therapy . A four - year descriptive analysis was conducted to evaluate differences in baseline characteristics, use of aht, and adherence levels across the cohorts of patients identified by enrolment year from 2004 to 2007 . We summarized data as mean values with standard deviations for continuous variables and as numbers (percentages) of subjects for categoric variables . We used pearson s chi - square test and one - way analysis of variance (anova) to determine significant differences in baseline characteristics across adherence levels . A multiple logistic regression model was used to estimate odds ratios (or) and 95% confidence intervals (ci) and to identify possible factors significantly associated with the risk of nonadherence during 20042007 . Patients who met the inclusion criteria in more than one of the four previous cohorts were only included once . The pdc was dichotomized, setting a threshold of pdc 80% to identify nonadherent patients . The other covariates in the model were age, gender, presence of some medications (at least two prescriptions) evaluated in the 12 months before the enrolment date, antidiabetic agents (atc code a10), lipid - lowering drugs (c10), cardiac therapy (c01, nitrates excluded), drugs for obstructive airways disease (r03), platelet inhibitors (b01ac), and initial therapy drug class, with angiotensin receptor blockers as the reference . The year of treatment initiation was also controlled for to account for possible time trends in medication use . In fact, although it would be important for the decision - maker to consider other direct costs, eg, hospitalizations, control of pharmaceutical expenditure is often considered as the relevant maneuver from the public health care provider point of view, and hence the authors focused on that aspect.24 no information on indirect costs was available . Each prescription cost was calculated by multiplying the cost per pack by the total number of packs . Since each prescription is unequivocally linked to the patient through the personal health code, the exact direct cost per patient was also known . A total of 31,483 new aht patients were enrolled in 2004, 32,888 in 2005, 29,875 in 2006, and 27,456 in 2007, of whom 26.2%, 26.8%, 25.4%, and 25.7%, respectively, were excluded because of failure to meet our inclusion criteria . In 2004, 1786 patients were excluded as a result of having been hospitalized for a cardiovascular cause before the enrolment date (5.7% of enrolled subjects) and a further 2363 patients for having used nitrates or loop diuretics in the year prior to enrolment (7.5% of enrolled subjects). Corresponding respective figures for 2005 were 1848 (5.6% of enrolled subjects) and 2659 (8.1%); for 2006, 1568 (5.2%) and 2322 (7.8%); and for 2007, 1410 (5.1%) and 2234 (8.1%). Therefore, 27,334 (21.4%), 28,381 (20.9%), 25,985 (19.5%), and 23,812 (17.8%) subjects were included in the study in 2004, 2005, 2006, and 2007, respectively . Mean age, gender distribution, and use of hypoglycemic drugs, lipid - lowering drugs, cardiac agents, drugs for obstructive airways disease, and platelet inhibitors are shown in table 1 . From 2004 to 2007, low adherence decreased from 33.0% to 28.5%, while high adherence increased from 22.9% to 28.0% (table 2). Intermediate adherence remained stable across the study period (from 10.5% in 2004 to 10.7% in 2007). Low and intermediate - low adherence patients were younger (56.9 17.1 and 58.4 16.8 years, respectively) compared with other levels of adherence (table 3). Prevalence of use of hypoglycemic drugs, lipid - lowering drugs, and platelet inhibitors increased from low- to high - adherence patients . In contrast, combination therapy was rarely used as first - line therapy . From 2004 to 2007, the proportion of prescriptions for diuretics decreased (from 24.0% to 20.8%), increased slightly for beta - blockers (from 14.8% to 16.1%), decreased slightly for calcium channel blockers (from 9.5% to 7.7%), whilst use of angiotensin - converting enzyme inhibitors and combinations as starting therapy remained stable (35.5% to 35.3% and 4.9% to 5.5%, respectively), and use of angiotensin receptor blockers significantly increased (from 11.4% to 14.7%). In particular, low adherence was highest among subjects started on diuretics (54.7% of included subjects) and lowest among those started on angiotensin receptor blockers (13.0%). High adherence was highest among subjects initiated on angiotensin receptor blockers (33.4%) and lowest among subjects started on diuretics (10.1%). Subjects starting with angiotensin - converting enzyme inhibitors displayed high adherence in 29.9% of cases . Compared with subjects initiated on angiotensin receptor blockers, the risk of non - adherence was 19% higher in those initiated on angiotensin - converting enzyme inhibitors, 44% higher in those initiated on combination therapy, 56% higher in those initiated on beta - blockers, 67% higher in those initiated on calcium channel blockers, and more than four - fold (4.3 times) higher in those initiated on diuretics (table 6). The overall cost of aht for the one - year follow - up was 2,654,166 in 2004, 2,664,815 in 2005 (+ 0.4% compared with 2004), 2,339,704 in 2006 (12.2% compared with 2005), and 2,343,221 in 2007 (+ 0.2% compared with 2006 and 11.7% compared with 2004, table 7). The percentage of overall cost of aht allocated to high adherence patients ranged from 60.7% in 2004, to 59.5% in 2005, 60.6% in 2006, and 64.9% in 2007 . Overall costs allocated to low and intermediate - low adherence patients decreased during the study period . The mean cost of aht for the one - year follow - up decreased in each level of adherence . In high adherence, the mean cost decreased from 256.97 in 2004 to 228.21 in 2007 (table 8). From 2004 to 2007, low adherence decreased from 33.0% to 28.5%, while high adherence increased from 22.9% to 28.0% (table 2). Intermediate adherence remained stable across the study period (from 10.5% in 2004 to 10.7% in 2007). Low and intermediate - low adherence patients were younger (56.9 17.1 and 58.4 16.8 years, respectively) compared with other levels of adherence (table 3). Prevalence of use of hypoglycemic drugs, lipid - lowering drugs, and platelet inhibitors increased from low- to high - adherence patients . In contrast, combination therapy was rarely used as first - line therapy . From 2004 to 2007, the proportion of prescriptions for diuretics decreased (from 24.0% to 20.8%), increased slightly for beta - blockers (from 14.8% to 16.1%), decreased slightly for calcium channel blockers (from 9.5% to 7.7%), whilst use of angiotensin - converting enzyme inhibitors and combinations as starting therapy remained stable (35.5% to 35.3% and 4.9% to 5.5%, respectively), and use of angiotensin receptor blockers significantly increased (from 11.4% to 14.7%). Adherence levels varied significantly among drugs used for treatment initiation (table 5). In particular, low adherence was highest among subjects started on diuretics (54.7% of included subjects) and lowest among those started on angiotensin receptor blockers (13.0%). High adherence was highest among subjects initiated on angiotensin receptor blockers (33.4%) and lowest among subjects started on diuretics (10.1%). Subjects starting with angiotensin - converting enzyme inhibitors displayed high adherence in 29.9% of cases . Compared with subjects initiated on angiotensin receptor blockers, the risk of non - adherence was 19% higher in those initiated on angiotensin - converting enzyme inhibitors, 44% higher in those initiated on combination therapy, 56% higher in those initiated on beta - blockers, 67% higher in those initiated on calcium channel blockers, and more than four - fold (4.3 times) higher in those initiated on diuretics (table 6). The overall cost of aht for the one - year follow - up was 2,654,166 in 2004, 2,664,815 in 2005 (+ 0.4% compared with 2004), 2,339,704 in 2006 (12.2% compared with 2005), and 2,343,221 in 2007 (+ 0.2% compared with 2006 and 11.7% compared with 2004, table 7). The percentage of overall cost of aht allocated to high adherence patients ranged from 60.7% in 2004, to 59.5% in 2005, 60.6% in 2006, and 64.9% in 2007 . Overall costs allocated to low and intermediate - low adherence patients decreased during the study period . The mean cost of aht for the one - year follow - up decreased in each level of adherence . In high adherence, the mean cost decreased from 256.97 in 2004 to 228.21 in 2007 (table 8). Many studies have highlighted poor adherence in subjects newly treated with aht,1419 but few of them have evaluated how adherence levels vary over time.20 the present study provides some additional information about aht among newly treated patients . First, it measures the number of subjects initiating pharmacologic treatment and gives information on patient characteristics and cost of aht over time . From 2004 to 2007, there was a 12.9% decrease in the number of patients initiated on aht (from 21.4% to 17.8%) as well as an 11.7% decrease in the overall cost of aht for the one - year follow - up (from 2,654,166 in 2004 to 2,343,221 in 2007). This findings may be the result of reductions both in the number of patients started on aht and in drug prices, rather than attributable to a shift in prescribing preferences for cheaper therapeutic options (see table 4). These preliminary findings seem to show a trend towards economic savings in pharmaceutical expenditure by reducing the number of patients starting aht . In particular, the reduction of initiated patients refers to low adherence (from 9009 to 6790 patients) and to low - intermediate adherence (from 6068 to 4851 patients) but does not refer to higher levels of adherence (table 2). Because low and low - intermediate adherence patients indicate a lower risk compared with those with higher adherence (table 3), these findings seem to underline a therapeutic approach strategy which pays more attention to patient characteristics before initiating pharmacologic treatment . Understanding how patient adherence to pharmacologic treatment varies on the basis of demographic and clinical characteristics, decision - makers could better focus on those categories of patients who are more likely not to adhere to treatment, ie, younger men and those taking no concomitant cardiovascular medications (tables 3 and 4). This strategy could be particularly effective, especially if the resources saved could be reallocated to priority patients, ie, those at higher risk of cvd and those who need improvement in their adherence . Second, a high amount of aht expenditure was accounted for by nonadherent patients, although this decreased over time (from 39.3% of the overall cost in 2004 to 35.1% in 2007). Resources allocated to regimes not adhered to can be considered inappropriate both in the short term (because blood pressure is not effectively controlled) and in the long term (because the opportunity to decrease the costs of cardiovascular complications is missed). Third, the present four - year analysis of adherence shows a low rate of increase in adherence (from 22.9% in 2004 to 28.0% of newly treated subjects in 2007) suggesting the need for more effective interventions . Recently, the euroaspire iii (european action on secondary and primary prevention by intervention to reduce events iii) survey showed that blood pressure control did not improve with time, and indeed showed a 10% worsening over the three survey periods.25 the low increase in rates of adherence would indicate no increase in the proportion of patients achieving blood pressure control in clinical practice . As shown in recent studies, failures in reaching high adherence with aht implies avoidable cvd hospitalizations and deaths813 and increasing costs for the management of hypertension and related diseases.1,11,26 in 2003, the world health organization alerted health authorities and clinicians about poor adherence to chronic pharmacologic treatments in clinical practice and suggested the need for action.27 according to existing evidence, more effective interventions should be planned and developed . Discordance also between the ideal scenario and the results obtained from our study should increase awareness among health care providers of the need for better performance . From the perspective of enhancing treatment adherence, use of angiotensin receptor blockers as first - line therapy should be considered because these agents have shown the highest rates of high adherence and, above all, the lowest rate of early discontinuation (table 5). Our findings with regard to poor adherence should be considered both by clinicians when selecting first - line therapy and by health care providers when defining their pharmaceutical formularies . The principal limitation of this study is its lack of inclusion of patient clinical data . These ad hoc databases are comparable with the health care claims databases which have been utilized for outcomes research for years in the us and canada . 28,29 because these databases are normally used for administrative or accounting purposes, they omit information that would make it possible to determine the clinical status of patients . The absence of clinical outcomes data, in particular blood pressure control, could have generated bias, leading to comparisons of patients with different levels of disease severity . In addition, the data source does not provide any information about the diagnosis of hypertension, thus the possibility of disease misclassification cannot be excluded . Moreover, information about patient lifestyle habits and health status information, ie, physical activity levels, smoking status, and other determinants of cvd morbidity and mortality, were not available . The potential confounding effect of this lack of information on the association between adherence to antihypertensives and cvd events should be investigated . Another important limitation related to the source of the data is the lack of information on the indication for aht . To increase the likelihood that aht was used for hypertension, however, the data suggest a good correlation between pharmacy dispensing records and cumulative drug exposure.22 finally, only direct costs were included, but indirect and intangible costs have no actual financial implications for health care structure and should be considered only for a societal perspective.1 the findings of the present study show that adherence to aht improved slowly from 22.9% to 28.0% in newly treated subjects over four years . Local services should therefore be alerted to the need for routine monitoring of adherence to aht, because this is a key performance indicator for both management of hypertensive patients and overall cost minimization in cvd . Administrative databases, even if lacking patient lifestyle and health status information, offer a low - cost monitoring option, and include sufficiently accurate data for representative populations.
Stroke, in all of its forms, is one of the most frequent neurological causes of oropharyngeal dysphagia1 . More than 70% of patients exhibit swallowing alterations in the first days after a stroke2, and the manifestation of dysphagia is associated with the severity of the patient's neurological condition3 . Speech therapy in the hospital prevents nutritional deficits and dehydration and also helps to avoid aspiration pneumonia . Furthermore, such therapy can also reduce the time spent in hospital and thus speed the patient's return to independence1 . Video fluoroscopy, a radiological contrast examination, is used for more precise diagnosis of swallowing disorders and is considered to be the gold standard for assessment of the swallowing process . This examination allows visualization of the swallowing dynamics and identification of sub - clinical changes, which provides more comprehensive and reliable monitoring of the swallowing mechanism and helps to guide the treatment of any deficits4 . However, the combination of anamnestic information and clinical speech evaluation of swallowing continues to be the most widely used method for assessing swallowing function . Bedside clinical evaluation allows quick identification of alterations in oral functions, e.g., an increase in oral transit or incomplete swallowing of the food bolus, alterations in vocal quality, alterations to oral reflexes, reduction of laryngeal elevation during saliva swallowing, alterations in the sensitivity of the larynx, and signs suggestive of tracheal penetration / aspiration5 . Reduced oropharyngeal sensitivity is a common clinical finding upon evaluation of post - stroke neurogenic dysphagia . This reduction leads to delayed swallow triggering as well as motor and sensory alterations of the larynx6 . A lack of sensitivity in this region can result in delayed oral transit, delayed triggering of the swallowing reflex, and a decreased cough reflex . It can also increase the incidence of premature escape of food into the pharynx, which risks laryngeal penetration and/or tracheal aspiration7 . The plasticity of the central nervous system enables the adult neurological patient to recover at least partially . Repair and reorganization of the central nervous system begin spontaneously soon after the injury, and rehabilitation should therefore be started early in order to avoid further deterioration and rescue more - normal behavior patterns8 . Therapeutic techniques for rehabilitation of dysphagia can play an important role in neuroplasticity in stroke patients . However, at present, many such techniques are used concomitantly without adequate measurement of their efficiency . Direct tactile stimulation of the pharyngeal wall is considered to be an important technique for improving the sensitivity and even the mobility of the region9, which results in improvement of the swallowing reaction . Such tactile stimulation of the pharyngeal wall is commonly used in combination with cold stimulus during rehabilitation from dysphagia . Short touches of a cold laryngeal mirror on the lower third of the palatoglossal arch are used to increase the intra - oral input10 11 . Cryostimulation, i.e., cooling or decreasing the temperature of the tissue for therapeutic purposes, has proven to be an efficient way of regulating the sensitivity of damaged areas, including those affected by neurological injuries12 13 . There is a lack of published studies of the efficiency of the various techniques used in speech therapy or how to use them for better results . Therefore, the objective of this study was to determine the effects of cryostimulation on the sensitivity of the oropharyngeal region, swallowing reaction, and premature escape of food in subjects with post - stroke neurogenic dysphagia . This study was approved by the research ethics committee of santa maria federal university (ufsm) under the number 0055.0.243.000 - 07 and received institutional authorization from the hospital of passo fundo (hcpf), where the data were collected . The participants in the study were adults hospitalized at hcpf and diagnosed with stroke who met the criteria of the study, and either the patients themselves or their responsible family members consented to participate and signed the statement of consent . The inclusion criteria were: age up to 65 years (in order to avoid additional alterations to swallowing function due to aging), absence of oropharyngeal sensitivity to touch, presence of mild - to - moderate oropharyngeal dysphagia, sufficient alertness and understanding to participate in the therapy, premature escape of food during video fluoroscopic examination, and satisfactory tongue mobility (ability to lateralize, raise, and lower) without significant compromise of the oral route . The exclusion criteria were: tracheostomy, previous history of swallowing difficulties, and presence of another neurological disorder that could contribute to dysphagia . Sample selection was based on analysis of medical records, anamnesis14 of a family member or, if possible, the subject him- or herself, clinical evaluation of dysphagia15, and video fluoroscopic examination16 . These procedures were performed sequentially, and any potential subject excluded by one of them did not proceed to the next . Initially, 29 subjects with computed tomography (ct)-based diagnoses of ischemic or hemorrhagic stroke were selected . Of these, 15 met the established inclusion and exclusion criteria, and only 7 agreed to participate in the study; these were therefore designated as the sample for convenience . The refusal of the other 8 subjects to participate was due to the increased hospitalization time that would have been required to complete the course of therapy . The study sample was composed of 7 adults, 6 men and 1 woman aged 28 to 64 years, with a ct - based diagnosis of stroke and a diagnosis of oropharyngeal dysphagia without any other underlying disease . The data were collected from the evaluations used for the sample selection, which are described in detail below . Therapy was performed over 4 days, beginning on the day of the initial video fluoroscopic examination, and all evaluations were repeated on the fifth day using the same parameters considered in the initial evaluation . Clinical speech evaluation was performed by a single trained evaluator in order to establish the presence of dysphagia and verify which orofacial structures were involved in the swallowing difficulties, with particular emphasis on the sensitivity and mobility of these structures . Swallowing was evaluated both indirectly (without food) and directly with 5 ml each of liquid and paste . The indirect evaluation emphasized the evaluation of oropharyngeal sensitivity, which was assessed by touching a wooden spatula to the pillars of the fauces, back of the tongue, soft palate, and posterior oropharynx . The absence of a reaction (such as elevation of the soft palate, a swallowing reaction or, especially, a gag reflex) to the touch was considered suggestive of hyposensitivity or complete non - sensitivity in the region17 18 . During the direct evaluation parameters related to the state of alertness and understanding, oral language, speech articulation, and vocal quality were also considered . Video fluoroscopic examination was performed at hcpf by a radiology technician and a speech therapist using a shimadzu image intensifier (pleno model) and following a published proposed evaluation protocol16 . The images displayed by this equipment are slower than real time . During the examination, the liquid and paste swallowing tests were performed with the subject seated at a 90o angle, and the images were taken in the vertical plane and captured the oral cavity, lips, posterior pharyngeal wall, nasopharynx, and top opening of the upper esophageal sphincter12 19 . The examination procedure was explained to the subject, and the first image was taken with the patient at rest for calibration purposes . During the first attempt at swallowing each consistency, the patient was instructed to keep the contrast in the oral cavity until swallowing was requested in order to observe the patient's capacity to retain the contrast as well as any premature escape of food into the pharynx . In the rest of the swallows, each subject performed a total of 10 swallows, 5 of liquid (barium water) and 5 of paste (vanilla cream with barium), for a total of 50 ml . The images were subsequently analyzed by 3 speech therapists experienced in video fluoroscopy using the viewerlite 2 philips inturis suite lite, v.2.1.1 (2001) program . The oral phase comprised the retention capacity, movement of the tongue, premature loss of food into the pharynx, residue in the furrows, and the dorsum of the tongue, floor of the mouth, velopharyngeal closure, and oral transit time . The pharyngeal phase comprised the swallowing reaction, elevation of the larynx, movement of the hyoid, posterior pharyngeal wave, residue in the vallecula and posterior pharyngeal wall, time of pharyngeal transit, multiple swallows, laryngeal penetration, and tracheal aspiration . The moment at which the swallowing reaction was triggered was defined based on the literature, which indicates that the end of the oral phase is defined radiologically as the moment at which the food passes the posterior mandible, at which point the pharyngeal phase begins with anterior hyoid excursion12 19 . Premature escape of food into the pharynx was defined as food passing the posterior mandible into the pharynx without triggering a swallowing reaction . Immediately after the end of the videofluoroscopic examination, cryostimulation was performed with the objective of stimulating the oropharyngeal region and improving its sensitivity . The procedure was performed 3 times a day17, at approximate minimum intervals of 4 hours, for 4 consecutive days . The 3 daily sessions included the faucial pillars, posterior oropharyngeal wall, soft palate, and back tongue . The stimulus was applied 10 times to each structure in each session, for a subtotal of 30 applications to each structure each day and a total of 480 applications over the entire course of therapy . Cryostimulation was performed using a cold laryngeal mirror that was immersed in crushed ice for 10 minutes before the start of each stimulation session in order to attain a near - optimal temperature, which was approximately 11oc for this purpose . After 3 seconds, the mirror was re - immersed in the ice for a maximum of 3 seconds and then re - applied to the structure . After each set of 10 applications to each structure, the subject was stimulated to induce saliva swallowing17 . The handle of the mirror was insulated with polystyrene foam material to prevent conduction of the speech therapist's body heat and thus premature warming of the mirror during the manipulation . Such a lining is necessary to maintain a neutral temperature for up to 5 seconds after removal of the mirror from the ice20 . The 3-second application time was chosen to minimize the risk that the mirror would warm beyond the desired temperature12 21 . Cryostimulation depends on physical factors related to the conduction of heat from one body to another . The time for which the object must be immersed in the ice to achieve the desired temperature for the particular set of working conditions and object material used in this study was determined to be 10 minutes according to thermodynamic analysis by a mechanical engineer in a specific laboratory . The following parameters were considered in the analysis: the approximate room temperature, human body temperature, size of the mirror handle area, size of the mirror area in contact with the oral cavity, quantity of ice for cooling the object, quantity needed to reduce the heat of the instrument, flow of heat from the ice to the tool, and flow of heat from the tool to the body . The data were analyzed by comparing the findings of the clinical and videofluoroscopic evaluations before and after cryostimulation . The statistical significance of these results was determined using fisher's exact test for small samples with a p value <0.05 considered to indicate significance . For the analysis of the swallow time from the video fluoroscopic examination, 3 of the 5 swallows the following aspects of each swallow were analyzed: the total time of swallowing (ttd), time for oral transit until the swallowing reaction (tto - rd), and time for pharyngeal transit from the swallowing reaction until pharyngeal clearance (ttf - lf). In order to identify the anatomic site at which the swallowing reaction was initiated during the visualization of the images by the analysis program on the laptop computer, the site of swallow initiation was marked on the screen using a green pen compactor ohp permanente m. the ttd, tto - rd, and ttf - lf were determined as follows . The times were measured using a casio chronometer that allowed measurement of centiseconds (1/100 s). To obtain the most accurate results, then, there were three timed swallowing tests and their values were used to average the times obtained . It is important to highlight that the values obtained in seconds and centiseconds are longer than the actual swallowing times, as the images were not obtained in real time, and are therefore only used for comparing the times before and after cryostimulation therapy . We determined the ttd, tto - rd, and ttf - lf for each swallow consistency for each subject and compared the values obtained before and after cryostimulation . The values were compared and their statistical significance determined using student's t - test with a significance level of 5% (p <0.05). A second trained evaluator repeated the entire analysis of a randomly selected subject (subject 2), and these values were used to calculate the level of interobserver concordance . A point - to - point correlation of the 2 sets of measurements was performed, and the values obtained indicated acceptable inter - trial reliability . The inter - evaluator reliability was measured 20 days later using a random sample of 10% of the total number of swallows . Analysis of this set of swallows by the same criteria used for the original analysis revealed that the measurements were comparable . The data were collected from the evaluations used for the sample selection, which are described in detail below . Therapy was performed over 4 days, beginning on the day of the initial video fluoroscopic examination, and all evaluations were repeated on the fifth day using the same parameters considered in the initial evaluation . Clinical speech evaluation was performed by a single trained evaluator in order to establish the presence of dysphagia and verify which orofacial structures were involved in the swallowing difficulties, with particular emphasis on the sensitivity and mobility of these structures . Swallowing was evaluated both indirectly (without food) and directly with 5 ml each of liquid and paste . The indirect evaluation emphasized the evaluation of oropharyngeal sensitivity, which was assessed by touching a wooden spatula to the pillars of the fauces, back of the tongue, soft palate, and posterior oropharynx . The absence of a reaction (such as elevation of the soft palate, a swallowing reaction or, especially, a gag reflex) to the touch was considered suggestive of hyposensitivity or complete non - sensitivity in the region17 18 . During the direct evaluation parameters related to the state of alertness and understanding, oral language, speech articulation, and vocal quality were also considered . Video fluoroscopic examination was performed at hcpf by a radiology technician and a speech therapist using a shimadzu image intensifier (pleno model) and following a published proposed evaluation protocol16 . The images displayed by this equipment are slower than real time . During the examination, the liquid and paste swallowing tests were performed with the subject seated at a 90o angle, and the images were taken in the vertical plane and captured the oral cavity, lips, posterior pharyngeal wall, nasopharynx, and top opening of the upper esophageal sphincter12 19 . The examination procedure was explained to the subject, and the first image was taken with the patient at rest for calibration purposes . During the first attempt at swallowing each consistency, the patient was instructed to keep the contrast in the oral cavity until swallowing was requested in order to observe the patient's capacity to retain the contrast as well as any premature escape of food into the pharynx . In the rest of the swallows, each subject performed a total of 10 swallows, 5 of liquid (barium water) and 5 of paste (vanilla cream with barium), for a total of 50 ml . The images were subsequently analyzed by 3 speech therapists experienced in video fluoroscopy using the viewerlite 2 philips inturis suite lite, v.2.1.1 (2001) program . The oral phase comprised the retention capacity, movement of the tongue, premature loss of food into the pharynx, residue in the furrows, and the dorsum of the tongue, floor of the mouth, velopharyngeal closure, and oral transit time . The pharyngeal phase comprised the swallowing reaction, elevation of the larynx, movement of the hyoid, posterior pharyngeal wave, residue in the vallecula and posterior pharyngeal wall, time of pharyngeal transit, multiple swallows, laryngeal penetration, and tracheal aspiration . The moment at which the swallowing reaction was triggered was defined based on the literature, which indicates that the end of the oral phase is defined radiologically as the moment at which the food passes the posterior mandible, at which point the pharyngeal phase begins with anterior hyoid excursion12 19 . Premature escape of food into the pharynx was defined as food passing the posterior mandible into the pharynx without triggering a swallowing reaction . Immediately after the end of the videofluoroscopic examination, cryostimulation was performed with the objective of stimulating the oropharyngeal region and improving its sensitivity . The procedure was performed 3 times a day17, at approximate minimum intervals of 4 hours, for 4 consecutive days . The 3 daily sessions included the faucial pillars, posterior oropharyngeal wall, soft palate, and back tongue . The stimulus was applied 10 times to each structure in each session, for a subtotal of 30 applications to each structure each day and a total of 480 applications over the entire course of therapy . Cryostimulation was performed using a cold laryngeal mirror that was immersed in crushed ice for 10 minutes before the start of each stimulation session in order to attain a near - optimal temperature, which was approximately 11oc for this purpose . After 3 seconds, the mirror was re - immersed in the ice for a maximum of 3 seconds and then re - applied to the structure . After each set of 10 applications to each structure, the subject was stimulated to induce saliva swallowing17 . The handle of the mirror was insulated with polystyrene foam material to prevent conduction of the speech therapist's body heat and thus premature warming of the mirror during the manipulation . Such a lining is necessary to maintain a neutral temperature for up to 5 seconds after removal of the mirror from the ice20 . The 3-second application time was chosen to minimize the risk that the mirror would warm beyond the desired temperature12 21 . Cryostimulation depends on physical factors related to the conduction of heat from one body to another . The time for which the object must be immersed in the ice to achieve the desired temperature for the particular set of working conditions and object material used in this study was determined to be 10 minutes according to thermodynamic analysis by a mechanical engineer in a specific laboratory . The following parameters were considered in the analysis: the approximate room temperature, human body temperature, size of the mirror handle area, size of the mirror area in contact with the oral cavity, quantity of ice for cooling the object, quantity needed to reduce the heat of the instrument, flow of heat from the ice to the tool, and flow of heat from the tool to the body . The data were analyzed by comparing the findings of the clinical and videofluoroscopic evaluations before and after cryostimulation . The statistical significance of these results was determined using fisher's exact test for small samples with a p value <0.05 considered to indicate significance . For the analysis of the swallow time from the video fluoroscopic examination, 3 of the 5 swallows the following aspects of each swallow were analyzed: the total time of swallowing (ttd), time for oral transit until the swallowing reaction (tto - rd), and time for pharyngeal transit from the swallowing reaction until pharyngeal clearance (ttf - lf). In order to identify the anatomic site at which the swallowing reaction was initiated during the visualization of the images by the analysis program on the laptop computer, the site of swallow initiation was marked on the screen using a green pen compactor ohp permanente m. the ttd, tto - rd, and ttf - lf were determined as follows . The times were measured using a casio chronometer that allowed measurement of centiseconds (1/100 s). To obtain the most accurate results, each swallow was analyzed and each part of the swallow timed 20 times . Then, there were three timed swallowing tests and their values were used to average the times obtained . It is important to highlight that the values obtained in seconds and centiseconds are longer than the actual swallowing times, as the images were not obtained in real time, and are therefore only used for comparing the times before and after cryostimulation therapy . We determined the ttd, tto - rd, and ttf - lf for each swallow consistency for each subject and compared the values obtained before and after cryostimulation . The values were compared and their statistical significance determined using student's t - test with a significance level of 5% (p <0.05). A second trained evaluator repeated the entire analysis of a randomly selected subject (subject 2), and these values were used to calculate the level of interobserver concordance . A point - to - point correlation of the 2 sets of measurements was performed, and the values obtained indicated acceptable inter - trial reliability . The inter - evaluator reliability was measured 20 days later using a random sample of 10% of the total number of swallows . Analysis of this set of swallows by the same criteria used for the original analysis revealed that the measurements were comparable . Table 1 shows the clinical characteristics of the study sample obtained from the anamnesis and records, including each subject's sex, age, underlying disease, affected hemisphere, expressive and comprehensive language ability, presence of pneumonia, mode of alimentation, and food consistency . Legend: stroke (i): ischemic stroke; stroke (h): hemorrhagic stroke . The results obtained before and after the performance of cryostimulation therapy for all of the subjects are shown according to the evaluation . Tables 3 and 4 show the results obtained by video fluoroscopic evaluation of the oral and pharyngeal phases of swallowing, respectively, and table 5 shows the swallowing time data . Level of significance, 5% (p <0.05); legend: avl = labial alteration, eoa = oral anterior escape, ttoa = increased time for oral transit, rda = delayed swallowing reaction, rel = reduction in laryngeal elevation, md = multiple swallows, rn = nasal reflux, aac = alteration in cervical auscultation, t / e = coughing / choking, vm = wet voice, sspl = signs suggestive of laryngeal penetration, and ssat = signs suggestive of tracheal aspiration . Level of significance, 5% (p <0.05); legend: to = oral transit, rco = residue in oral cavity . Level of significance, 5% (p <0.05); legend: tf = pharyngeal transit, ep = premature escape, rd = swallowing reaction, el = laryngeal elevation, e = stasis, pl = laryngeal penetration, at = tracheal aspiration level of significance, 5% (p <0.05); legend: ttd = total swallowing time, tto - rd = oral transit time until the swallowing reaction, and ttf - lf = pharyngeal transit time of the swallowing reaction until pharyngeal clearance . Evaluation of oropharyngeal sensitivity before and after cryostimulation . Oropharyngeal sensitivity was absent in all of the subjects at the time of the initial evaluation, as this was one of the inclusion criteria for the study . This parameter improved significantly after cryostimulation (p = 0.002), as shown in figure 1 . Clinical evaluation (table 2) showed that the swallowing reaction and cervical auscultation results for both of the tested consistencies improved significantly after cryostimulation . The frequencies of wet voice, coughing / choking, and signs suggestive of tracheal aspiration associated with swallowing the liquid also decreased . Video fluoroscopic examination showed no significant differences in the oral phase of swallowing after cryostimulation (table 3). In contrast, improvements in several aspects of the pharyngeal phase during swallowing of both liquid and paste (table 4), including pharyngeal transit, the swallowing reaction, and the occurrence of premature escape of food, as well as elimination of laryngeal penetration of liquids in all of the patients in whom it had previously occurred, were observed after cryostimulation therapy . Table 5 shows the distribution of the means and standard deviations of the swallowing times for both consistencies, divided into the total swallowing time, oral transit time until the swallowing reaction, and pharyngeal transit time from the swallowing reaction until pharyngeal clearance . The mean total swallowing time (ttd) and the oral transit time until the swallowing reaction (tto - rd) for the mean time for pharyngeal transit from the swallowing reaction until pharyngeal clearance (ttf - lf) for the liquid swallows increased after cryostimulation . The clinical manifestations of dysphagia after stroke vary according to the size and location of the lesion, but some of the more frequently encountered signs are reduced sensitivity in the oropharyngeal region, delayed triggering of the swallowing reflex, and reduced pharyngeal contraction . Another important manifestation is a decreased coughing reflex, which can also occur in response to a loss of laryngeal sensitivity22 . If not recognized and treated in a timely fashion, all of these manifestations can cause have serious consequences, such as aspiration pneumonia, malnutrition, and even death, in patients with dysphagia after stroke . Therefore, a deep knowledge of the effects of various therapeutic techniques on each stage of swallowing is essential to shorten the rehabilitation process and reduce the risks of dysphagia . The results of this study showed that cryostimulation, a thermal - tactile technique, improved the oropharyngeal sensitivity, swallowing reaction, and premature escape of food in patients with dysphagia after stroke . These findings agree with published results that cryostimulation can increase local sensitivity and probably reduce the premature escape of food by decreasing the swallowing reaction time10 11 . The plasticity of the cells of the central nervous system during both development and pathological processes is known to be affected by the peripheral stimulation captured and conducted by sensory systems . The sensory area receives, decodes, and analyzes the stimuli, the motor area controls the conscious and voluntary movements, and the associative area integrates the information from the sensory and motor areas to plan the types of movements and behaviors most appropriate for the situation23 . Therefore, the objective of the therapy is to provide the needed stimuli so that the central nervous system can interpret the information coming from the periphery and process and integrate it with the other areas in a way that enables reorganization of afferents and lowers the thresholds of response8 23 24 . Therefore, oropharyngeal sensitivity, as represented by the gag reflex elicited during the indirect clinical evaluation, was considered the most relevant variable of the study . After therapy, the gag reflex in response to touch reappeared in 6 subjects and remained absent in only 1 . These results confirmed the hypothesis that cold thermal stimulation as well as sour taste influence the dynamic modulation of swallowing and may positively impact the rehabilitation of individuals with oropharyngeal dysphagia25 . An abnormal or absent gag reflex is considered a sign of dysphagia26 27, and such signs occur frequently in patients with stroke sequelae28 . However, the literature shows no direct relationship between oropharyngeal sensitivity and decreased gag reflex as factors that can contribute to delaying the swallowing reaction . The results of the functional clinical evaluation of swallowing of liquid and paste generally showed that the therapy produced important changes in the variables studied . Before cryostimulation, the swallowing reaction during alimentation with liquid and paste was delayed in all 7 subjects . This parameter improved after therapy, indicating that cryostimulation increased the swallowing response and corroborating the findings of other studies12 17 29 30 . However, some authors report that cold used in isolation does not affect swallowing and show that combined techniques, such as cold and citric acid31 or cold, mechanical, and gustatory21 stimuli applied together would be more efficient . Today, we know that the beginning of the pharyngeal phase of swallowing may vary even among healthy adult individuals and may be located in the oral cavity, oropharynx, or hypopharynx32 33 . Therefore, we question the conventional wisdom that the palatoglossal pillars are the main location of the receptors responsible for initiating the pharyngeal phase of swallowing, which is the basis for the use of the mechanical - thermal maneuver to stimulate the receptors in these pillars34 . Pharyngeal responses to the beginning of swallowing can be induced by stimulating different sensory sites32 . However, the results of the present study show that although the palatoglossal pillars are not the only or even the predominant sensory site, stimulation of these structures improves pharyngeal sensitivity in patients with neurological deficits after stroke . Cervical auscultation findings also improved for swallowing of both liquid and paste, which is attributable to the improvement in the swallowing reaction enabling the food to be swallowed without the laryngeal penetration observed during the initial evaluations . This would also explain the reduction in the occurrence of wet voice, a related parameter that also significantly improved after cryostimulation . We believed that these latter aspects have not had significant improvement in intake of pasty consistency, because the paste consistency had less change than the other consistencies, already in the initial evaluation . The video fluoroscopic examination showed no difference in the oral phase of swallowing (as represented by the oral transit time and residue in the oral cavity) after cryostimulation therapy . Although the literature reports that cold stimulation reduces the oral transit time12, we did not observe this effect . These findings can probably be explained by the fact that in the oral phase, reduced tongue mobility can limit the manipulation of food and even complicate the propulsion of the food bolus, actions that are not targeted by cryostimulation as performed in this study . In the pharyngeal phase, the video fluoroscopic examination after cryostimulation showed improvements in the pharyngeal transit, swallowing reaction, and occurrence of premature escape of food for both the liquid and the paste as well as the elimination of laryngeal penetration of the liquid . The improvement in the pharyngeal transit time in response to cryostimulation shown in this study was also observed in other studies29 35 . This finding can be explained by the improvement in the swallowing reaction, as these parameters are related19 . The premature escape of food and the delayed swallowing reaction were present for both food consistencies in all of the study subjects in the initial evaluation and improved significantly after cryostimulation, probably due to the increased oropharyngeal sensitivity17 . These results agree with published data showing that cold therapy is an efficient technique for improving the swallowing reaction12 17 29 30 . The results of videofluoroscopy concurred with those of the clinical evaluation, showing agreement between the evaluation tools used in this study . Cryostimulation significantly decreased the laryngeal penetration of liquid, as the final evaluation did not show laryngeal penetration in any of the study subjects . This can be explained as a consequence of the improvement in the swallowing reaction and reduction of the premature escape of food, because a delay in the triggering of swallowing and the premature escape of food can allow laryngeal penetration and/or tracheal aspiration . We analyzed the total swallowing time, oral transit time until the swallowing reaction, and time for pharyngeal transit from the swallowing reaction until pharyngeal clearance . These measures are important because adult patients with dysphagia after stroke usually exhibit a prolonged transition between the end of the oral phase and the beginning of the pharyngeal phase with premature escape and delayed swallowing reactions, which facilitate aspiration19 . We observed that the average total swallowing time and the oral transit time until the swallowing reaction decreased after cryostimulation only for the swallowing of paste . These results show that cryostimulation was able to improve the swallowing reaction and agree with the findings of previous studies that used cryostimulation in subjects with oropharyngeal dysphagia caused by stroke12 17 . The average pharyngeal transit time from the swallowing reaction until pharyngeal clearance for the liquid swallows increased after cryostimulation therapy . This apparently contradictory delay may be related to the persistence of liquid stasis even after therapy . This is attributable to the fact that even after improvement in the swallowing reaction, the subjects probably required a greater number of swallows to completely clear food residue from the pharynx, thus increasing the pharyngeal transit time from the swallowing reaction until complete pharyngeal clearance . It should be noted that we evaluated neither the number of swallows nor the occurrence of multiple swallows, which is a limitation of this study . The small size of the sample can also be considered a limitation of this study; however, the results showed statistically significant improvements in the parameters that were the main focus of this research . The study shows that this technique, the efficacy of which has been questioned, can be an important resource for speech therapy of post - stroke patients . It also shows the importance of the criteria used in the methodology, the tools selected, and, more specifically in this case, the achievement of the appropriate temperature based on the physical science of heat conduction . Spontaneous recovery of the central nervous system during the therapy period may have contributed to the study subjects' improvements in swallowing8 23, but we believe that early intervention aided and enhanced the improvement in this period by providing peripheral stimulation to assist synaptic recovery . In this study, cryostimulation therapy produced recovery of oropharyngeal sensitivity as well as improvements in the swallowing reaction and the premature escape of food during swallowing of both liquid and paste in patients with neurological dysphagia after stroke.
To investigate the relation between diet and chronic disease, several dietary assessment methods have been developed and evaluated . The food frequency questionnaire (ffq) has been used most frequently in large - scale epidemiologic studies, because ffq is less expensive to administer than are other dietary assessment methods, and the ffq evaluates long - term diet rather than 24-h dietary recall or food records . In addition, dietary intakes estimated by ffqs have shown clear associations with coronary heart disease, type 2 diabetes, and blood lipids [1 - 3]. However, several studies [4 - 7] suggested that other types of dietary assessment need to be used in epidemiologic studies of diet and diseases to overcome the limitations of ffqs . These limitations include weak associations with dietary biomarkers and a lack of consistency across studies examining diet and cancer risk . Food records ask participants to record all foods and beverages consumed over a specific period of time, usually 3 to 7 days or during multiple periods within a year . Because food records do not rely on memory, food records have been used as a reference method to validate other dietary assessment methods . However, due to day - to - day variations and seasonal variations in food records, multiple - day food records over four seasons have been used as a reference standard to evaluate other dietary assessment methods . Food records have revealed relationships not observed in the ffq [8 - 9]. A significant relationship between dietary fat and the risk of breast cancer was found based on multiple - day food records, but was not seen in the ffq [8 - 9]. However, multiple - day food records require highly motivated participants, and they are expensive to administer in large samples, thus 3-day food records have been commonly used in practical settings . Given these aspects, it has been debated which dietary assessment method would be appropriate in large - scale epidemiologic studies . In the present study, we investigated the relative validities of 3-day food records and the ffq, respectively, by comparing them with 9-day food records . Subjects were recruited from december 2002 to may 2004 in the health examination center at hallym university sacred heart hospital located in anyang, korea . Full details of the methods used in the study are given elsewhere and are summarized below . A total of 199 subjects between the ages of 40 to 70 years consented to join the study and completed the first food record (fr). Among them, 130 subjects completed the 3-day frs over four seasons, as well as the ffq . Six people who changed their diet for weight - loss purposes during the study period were excluded . This food - based ffq was developed with 24-h data from the korea national health and nutrition examination survey in 1998 . The procedures of development and evaluation of the ffq are described in detail elsewhere [10 - 11]. Briefly, food items were selected based on the cumulative percent contribution of each food and the cumulative r of multiple regression of each nutrient . The consumption frequency was classified into nine categories: never or seldom, one a month, two to three times a month, one to two times a week, three to four times a week, five to six times a week, once a day, twice a day, or three or more times a day . For the food items with different seasonal availability, like fruits, participants were additionally asked to mark one of four categories with respect to how many months they ate each particular item: three, six, nine and 12 months . Several photos of serving sizes of certain foods were presented to help in the understanding of portion sizes . Each participant was asked to keep 12-day frs for one year . To capture seasonal variations and weekly variations, participants were asked to keep non - consecutive 3-day frs including one weekend day or holiday during each of the four seasons . The participants were asked to record the amounts of foods consumed with multiples of household tableware in order to increase the accuracy of portion size . A standardized protocol was developed by a research dietitian supervisor, which included the manual providing information for fr procedure in detail . Dietitians trained participants with the manual and reviewed unclear descriptions, errors, omissions, or doubtful entries in frs and asked the participants to clarify them . Twelve - day frs were collected to validate the ffq . In the previous validation study between 12-day frs and the ffq, the pearson's correlation coefficients between 12-day frs and the second ffq were between 0.10 and 0.46 (median for all nutrients 0.33). Pearson's correlation coefficients between the first ffq and the second ffq ranged between 0.24 (carbohydrate) and 0.58 (cholesterol). In the current study, 3-day frs and the remaining 9-day frs nutrient intake in the ffq was calculated using a weighted frequency per day and a portion size per unit of each food item . The daily nutrient intakes of each participant were the sum of the nutrient intakes of each food item . The seventh edition of the food composition table of korea was used as the nutrient database . Three - day frs from each season and a randomly selected season were compared with the remaining 9-day frs, respectively . The mean difference of each nutrient between the 12-day frs and the ffq was tested by paired t - test . Correlations of nutrient intake were assessed by pearson's correlation coefficient after adjustment for sex . Most nutrient distributions were skewed, thus, all nutrients were natural log transformed prior to analysis . De - attenuated correlation coefficients were applied to correct the within - person error in the measurements of the frs . The observed correlations were multiplied by the de - attenuation factor (1+/n), where is the ratio of the withinand between - person variances and n is the number of repeats . Using the sas varcomp procedure, within- and between - person variances the agreement of 3-day frs with 9-day frs and the agreement of 9-day frs with the ffq were compared by cross - classification analysis . Subjects were classified into quartiles based on nutrient intake, and the percentages of agreement and disagreement were calculated . All statistical analyses were performed using the sas software (version 9.1 sas institute inc ., cary, nc), and p values <0.05 were considered to be significant . Subjects were recruited from december 2002 to may 2004 in the health examination center at hallym university sacred heart hospital located in anyang, korea . Full details of the methods used in the study are given elsewhere and are summarized below . A total of 199 subjects between the ages of 40 to 70 years consented to join the study and completed the first food record (fr). Among them, 130 subjects completed the 3-day frs over four seasons, as well as the ffq . Six people who changed their diet for weight - loss purposes during the study period were excluded . This food - based ffq was developed with 24-h data from the korea national health and nutrition examination survey in 1998 . The procedures of development and evaluation of the ffq are described in detail elsewhere [10 - 11]. Briefly, food items were selected based on the cumulative percent contribution of each food and the cumulative r of multiple regression of each nutrient . The consumption frequency was classified into nine categories: never or seldom, one a month, two to three times a month, one to two times a week, three to four times a week, five to six times a week, once a day, twice a day, or three or more times a day . For the food items with different seasonal availability, like fruits, participants were additionally asked to mark one of four categories with respect to how many months they ate each particular item: three, six, nine and 12 months . Several photos of serving sizes of certain foods were presented to help in the understanding of portion sizes . Each participant was asked to keep 12-day frs for one year . To capture seasonal variations and weekly variations, participants were asked to keep non - consecutive 3-day frs including one weekend day or holiday during each of the four seasons . The participants were asked to record the amounts of foods consumed with multiples of household tableware in order to increase the accuracy of portion size . A standardized protocol was developed by a research dietitian supervisor, which included the manual providing information for fr procedure in detail . Dietitians trained participants with the manual and reviewed unclear descriptions, errors, omissions, or doubtful entries in frs and asked the participants to clarify them . The research dietitian supervisor checked all completed records for accuracy . Twelve - day frs were collected to validate the ffq . In the previous validation study between 12-day frs and the ffq, the pearson's correlation coefficients between 12-day frs and the second ffq were between 0.10 and 0.46 (median for all nutrients 0.33). Pearson's correlation coefficients between the first ffq and the second ffq ranged between 0.24 (carbohydrate) and 0.58 (cholesterol). In the current study, 3-day frs and the remaining 9-day frs nutrient intake in the ffq was calculated using a weighted frequency per day and a portion size per unit of each food item . The daily nutrient intakes of each participant were the sum of the nutrient intakes of each food item . The seventh edition of the food composition table of korea was used as the nutrient database . Three - day frs from each season and a randomly selected season were compared with the remaining 9-day frs, respectively . The mean difference of each nutrient between the 12-day frs and the ffq was tested by paired t - test . Correlations of nutrient intake were assessed by pearson's correlation coefficient after adjustment for sex . Most nutrient distributions were skewed, thus, all nutrients were natural log transformed prior to analysis . De - attenuated correlation coefficients were applied to correct the within - person error in the measurements of the frs . The observed correlations were multiplied by the de - attenuation factor (1+/n), where is the ratio of the withinand between - person variances and n is the number of repeats . Using the sas varcomp procedure, within- and between - person variances were calculated . The agreement of 3-day frs with 9-day frs and the agreement of 9-day frs with the ffq were compared by cross - classification analysis . Subjects were classified into quartiles based on nutrient intake, and the percentages of agreement and disagreement were calculated . All statistical analyses were performed using the sas software (version 9.1 sas institute inc ., cary, nc), and p values <0.05 were considered to be significant . Men made up 26.6% of the total number of subjects, and the mean age of the subjects was 47.4 years . The mean body mass index (bmi) of the subjects was 23.4 kg / m . The proportions of current alcohol drinkers and current smokers were 41.1% and 12.9%, respectively . Mean daily intakes of nutrients estimated by four 3-day frs and the ffq are given in table 2 . When four 3-day frs were used to estimate energy and nutrient intakes, mean intakes of energy, protein, fat, carbohydrate, vitamin b1 and b2, niacin, phosphorus, na, fe, zinc, and cholesterol in men were higher than those of women . Whereas, mean intakes of vitamin c, folate, and calcium in women were higher than those of men . When the ffq was applied to estimate energy and nutrient intakes, there were no differences in nutrient intakes between men and women . The intakes of fat, fiber, vitamins a, e, and b6, -carotene, and na in four 3-day frs were higher than those of the ffq . The intake of carbohydrate in the ffq was higher than that of the four 3-day frs . The crude pearson's correlation coefficients after adjustment for sex between 3-day frs and 9-day frs for spring, summer, fall, and winter were 0.18 - 0.54, 0.20 - 0.50, 0.16 - 0.56, and 0.14 - 0.49, respectively . The pearson's correlation coefficients after adjustment for sex between the ffq and the 9-day frs with exclusions of spring, summer, fall, and winter were 0.07 - 0.39, 0.12 - 0.41, 0.11 - 0.41, and 0.09 - 0.38, respectively . The crude pearson's correlation coefficients between the 3-day frs and the 9-day frs were higher than the crude pearson's correlation coefficients between the ffq and the 9-day frs in most nutrients (table 3). Among the crude correlation coefficients between the 3-day frs and the 9-day frs, energy, carbohydrate, fiber, folate, phosphorus, sodium, and iron showed higher correlations (0.4) than did the other nutrients . Vitamin a, retinol, and -carotene showed seasonal differences in the correlations . Among the crude correlation coefficients between the ffq and the 9-day frs, vitamin a, vitamin e, vitamin b2, and sodium showed lower correlations (0.2) than did the other nutrients . De - attenuated correlation coefficients in most nutrients were improved in the comparisons (3-day frs vs. 9-day frs and ffq vs. 9-day frs). De - attenuated correlation coefficients of vitamin a, retinol, and -carotene between 3-day frs and 9-day frs constantly showed seasonal differences . After adjusting for sex and energy intake, the correlation coefficients of most nutrients in the comparison were decreased . The crude, de - attenuated, and energy - adjusted correlation coefficients between the 3-day frs and the 9-day frs were constantly higher than those between the ffq and the 9-day frs . The average proportions of classification into the same quartiles, adjacent quartiles, and distant quartiles between the 3-day frs and the 9-day frs were 35.8%, 40.5%, and 5.2%, respectively . The nutrients which showed 10% seasonal differences in the classification into the same quartiles or adjacent quartiles between the 3-day frs and 9-day frs were energy, fiber, vitamin a, retinol, vitamin b2, phosphorus, iron, and zinc . On average, the proportions of classification into the same quartiles, adjacent quartiles, and distant quartiles between the ffq and 9-day frs were 31.1%, 39.4%, and 6.9%, respectively . In this validation study, we evaluated the relative validities of 3-day food records (frs) and the food frequency questionnaire (ffq) by comparing them with that of the 9-day frs . Correlation coefficients between the 3-day frs and the 9-day frs were higher than those between the ffq and the 9-day frs . Average proportions of classification into the same quartiles and adjacent quartiles between the 3-day frs and the 9-day frs were higher than those between the ffq and the 9-day frs, but cross - classifications of two methods (3-day frs and ffq) with 9-day frs showed that both the 3-day frs and the ffq were able to reasonably categorize individuals by nutrient intake . Three - day frs showed higher correlations and higher agreement proportions of quartile classification with the 9-day frs than did the ffq, but both of the relative validities of 3-day frs and ffq were acceptable for use as dietary assessment tools . In the present study, correlations between 3-day frs and 9-day frs were higher than correlations between 9-day frs and the ffq . The ffq used in our study was a food - based ffq and, thus, seasonings and cooking oils were omitted, which might affect the estimations of some nutrients and correlations with frs . In the study by yun et al ., excluding oils and seasonings from a ffq underestimated vegetable fat, vitamin e, and sodium intake, and shim et al . Reported that seasonings contributed 8.4% of energy intake, 34.4% of fat intake, 20.5% of iron intake, and 17.9% of -carotene intake . Thus, intakes of seasonings and oils should be considered in the selection of the items of the ffq . A dish - based ffq can be a solution for including seasonings and oils in the estimation of nutrients, but due to a lack of standard korean recipes, the development of standard recipes for korean dishes is required first . The correlations of the ffq with the 9-day frs in this study appear to be lower than western countries [16 - 17], but comparable to other korean studies [18 - 20]. A typical korean meal consists of cooked rice, soup, and multiple side dishes and a meal is often served family style and side dishes are shared with multiple persons . Thus, koreans may have difficulties in answering the consumption frequency and portion size for a specific food item . Since the consumption of carbohydrate in koreans is higher than that of western countries, more food items containing high carbohydrate were listed in the ffq, which may result in higher mean of carbohydrate intake in the ffq than frs . Lower correlations of the ffq with the 9-day frs than those of the 3-day frs with the 9-day frs in the present study may be partially attributed to a concept of ffqs . The basic assumption of ffqs is that the average long - term diet is the conceptually important factor rather than dietary intake over a few specific days . In ffqs participants might have trouble remembering a consumption frequency or the amount of food consumed in a year . In a qualitative study using cognitive interviews for the ffq used in the present study, participants had difficulties in remembering events over the course of a full year . Elements contributing to those difficulties included the consumption of diverse foods, calculating the averages for seasonal foods, estimating consumption amounts from photos, adjusting frequency when the amount consumed is higher or lower than the quantities presented, and combining the frequency and quantity of each food item when several food items are clustered into one category, etc . Lee et al . To overcome the limitations of ffq, several approaches including detailed questions regarding preparation, questions on seasonal intakes for several foods, inclusion of portion size ranges, additional response categories for frequency of intake, and addition of the food glossary and written cues were suggested [23 - 24]. However, despite these limitations of the ffq, cross - classification showed that the ffq classified approximately 71% of the subjects within the same or adjacent quartiles and only 7% of the subjects were classified into distant quartiles . With respect to expediency of administration and day - to - day variation, the ffq can be a useful tool for estimating usual intake in a large epidemiologic study . Three - day frs showed a relatively higher validity than did the ffq, but 3-day frs have a weakness when it comes to characterizing an individual's usual diet due to within - person variations of day - to - day dietary intake . Reported components of variance in nutrient intake with the same data used in the current study . The major components of variance were within - individual variations (57.2 - 87.1%) and between - individual variations (12.2 - 37.4%) for all nutrients . Weekly and seasonal variations contributed small components for most nutrients, but vitamin a, retinol, and -carotene showed seasonal differences in the correlations between the 3-day frs and 9-day frs in the current study . Suggested that to estimate usual individual intakes within 20% of the true mean with 90% confidence level, energy, protein, carbohydrate, phosphorus, and iron required 3 - 9 days of dietary survey, fat and calcium required 13 - 19 days, and vitamins a and c required 25 - 29 days . Depending on the nutrients of interest and the purpose of a study, the number of days for frs should be taken into account . Despite the relatively higher validity of 3-day frs, to overcome the limitations of 3-day frs including day - to - day variation of intake and cost of administration, the ffq needs to be combined in a large epidemiologic study . Subjects were recruited in the health examination center, thus, there is a restriction in generalizing the results of the study . The reference measurement should be independent from the evaluated method, but since 9-day frs share common limitations with 3-day frs, the correlation between 9-day frs and 3-day frs may be affected . Reliable biomarkers should be introduced to validate food intake measurements for further studies . In conclusion, 3-day frs showed a relatively higher validity and agreement than ffq, but the ffq still has strength in large epidemiologic studies in terms of convenience of administration and estimation of usual intake . Therefore, many factors such as research objective and study design should be considered when selecting a dietary assessment method.
Mammary paget's disease (pd) of the nipple was first described by sir james paget in 1874 . Extramammary pd (empd) was first recognised and reported as a distinct clinical entity by radcliffe crocker in 1889 . Empd is morphologically and histologically identical to mammary pd of the nipple, the primary difference being the anatomical location . Unlike the mammary paget's disease, the extramammary type is less well known and much rarer, representing just 6.5% of all paget's disease and is more common among women over 50-years of age . Approximately 25% of empd (range 9 - 32%) are associated with an underlying in situ or invasive neoplasm . In all patients, this report describes a very rare case of primary bilateral axillary empd treated by wide local excision . The reconstruction of the resulting soft tissue defect was successfully achieved by using a thoraco - dorsal artery perforator - based limberg's flap . A 65-year - old woman was referred to the plastic and reconstructive department of the university of perugia in may 2012 for the management of an inflammatory skin disease of both axillae clinically unresponsive to long - term conventional topical therapy . The patient was initially treated with a combination of topical corticosteroids and antibiotics for two weeks without improvement . This was followed by topical imiquimod 5% cream for about 4 weeks with out benefit . On physical examination, the lesion in the left axilla measured 4 4 cm and the other side one was much bigger, measuring 12 10 cm . Both of them appeared as pink erythematous patches with irregular borders and with localised desquamated erosions together with leukokeratotic foci [figure 1]. Clinical presentation of the well - demarked pink erythematous patch in the left axilla bilateral breast examination revealed normal breast consistency without any nodules, nipple modification, excoriation or discharge; mammograms showed a normal breast parenchyma confirming the nodules absence . The patient underwent operation under general anaesthesia: in the right side we were able to repair the secondary skin defect by an intradermal suture, due to its moderate dimension . For the left lesion the patient was positioned on the contralateral side; left arm was abducted in 90. after excising the skin lesion with an apparent tumour - free margin of 2 cm, we provided the skin defect reconstruction using a thoraco - dorsal perforator flap, according to limberg's design [figures 2 and 3]. Pre - operative planning of the limber's flap intra - operatory view showing the resulting wide skin defect and the arch of rotation of the limberg's flap previously designed during the operation we removed two lymph nodes appearing lightly enlarged and of increased consistency: histopathological examination revealed no tumour invasion but just signs of reactive histiocytosis secondary to sovra - infections of the lesion . The excision specimen of the cutaneous lesion was sent for histopathological examination: there were nests of atypical cells with melanin pigmentation within their cytoplasm showing pagetoid involvement of the epidermis together with cells with abundant basophilic cytoplasm, with signet ring forms . These cells stained positively with alcian blue and pas - diastase stains, confirming the presence of mucin . On immunohistochemistry, the pagetoid cells stained strongly positively for cytokeratin 7 (ck7), cytokeratin 20 (ck20), carcinoembryonic antigen (cea), ema and cgdfp-15 . These cells also stained positively for melanocytic markers s-100, melan - a, condition quite rare with very few cases described in the literature . The presence of positivity for ck 7 and negativity for ck 5/6 (an excellent marker of squamous differentiation, characteristically expressed strongly and diffusely by either squamous carcinomas or benign squamous epithelium) allowed us to rule out, together with the specimen histopathological features (intracellular mucin, signet cells and glandular structures), the hypothesis of bowen's disease . The acinar formation, the intracellular mucin and the positivity of the anti - ema and anti - cea antibodies (usually not labelled by melanocytes) were strongly indicative for paget's disease rather than melanoma . The flap survived completely preserving a complete range of motion of the upper limb and the aesthetic result after 24 months was good . Both functional and aesthetic results are satisfactory the perineal area was also evaluated but was free from any pathological signs such as redness, swelling, dryness, and the patient denied any discomfort or itching in that area . The patient is currently alive without any sign / symptoms of recurrence after 15 months: she continues to be under follow - up . Axillary location of empd is very rare . A review of the literature revealed only 23 cases reported so far, the majority in japanese population . Out of these, 10 cases had isolated axillary involvement while 13 demonstrated simultaneous axillary and genital disease (so called triple empd). Presence of an underlying carcinoma in axillary empd has been reported in eight of the 23 cases (35%). One theory proposes that stem cells present in the basal cell layer of the epidermis change into paget cells as a result of faulty development of these cells in their attempt to mature into an apocrine structure . The second theory hypothesises that epidermal paget cells originate from cells that have migrated from an adjacent malignant region either an adnexal sweat gland or anorectal mucosa or from another contiguous structure . A third theory suggests that paget's cells may represent a form of metastasis, but not in extramammary paget's disease . Some authors suggest that p53 may play a role in the progression of vulvar paget's disease and may be a terminal event in some cases, especially those associated with invasive disease . A novel tumour marker rcas1 has been identified in cases of extramammary paget's disease and has the potential of becoming a biomarker for monitoring therapeutic efficacy . The contemporary expression of ck20, s100 and melan - a in the tumour cells makes this case interesting . These atypical findings may be compatible with the presence of a pigmented area and may be referred as a " locally " pigmented extramammary paget's disease . Patients commonly present with hyper- or hypopigmented pruritic patches or plaques with a non - specific clinical appearance, thus leading to a significant delay between initial presentation and diagnosis . They may be associated with a palpable mass in the subcutaneous tissue but are often limited to the skin . Diagnosis includes malignant melanoma, bowen's disease, mycosis fungoides and langherans cells hystiocytosis . Prognosis of the axillary paget's disease is better than other localisations because of the clear anatomy of the axillary region and the possibility to perform a wide excision thanks to the great numbers of reconstructive approaches using either local fasciocutaneous or muscolocutaneous flaps . One study showed a mortality of 18% for patients without associated carcinoma and 46% for those with underlying carcinoma . The perineal examination was done in order to evaluate the, although very rare, co - existence of a perineal empd in what is so called triple or quadruple empd . We decided not to perform any perianal skin biopsy motivated by the high rarity of this co - existence, counting less than 30 cases described in the literature and the majority among asian men, the absolute absence of any signs and symptoms . Although the gold standard of treatment of empd is mohs micrographic surgery (mms) providing margin control in tumour known to have diffuse spread beyond the clinically apparent margins, in cases in which mms is not available, a wide local excision with margins of 1 - 5 cm have been quoted to have excellent results . The management of primary extramammary paget's disease of the axilla was based on a locally wide excision but now the treatment ofchoice for empd is mohs micrographic surgery (mms) providing margin control . The role of sentinel lymph node evaluation is still unclear and the role of adjuvant therapy is controversial . However, because of the multifocal nature of this disease, recurrence rates are quite high.
Diabetes mellitus is the most common metabolic disease and becomes a heavy burden of public health systems . In china, deterioration of beta cell function and insulin resistance are two fundamental pathophysiologic defects of type 2 diabetes mellitus (t2 dm). It has been proven that at the time when t2 dm was established, the loss of beta cell function was shown to reduce by 50% and this decline of beta cell function progressed over time although traditional antihyperglycemic therapy had been applied . In order to postpone the progress of disease, new therapies are required to persistently act on beta cell failure and insulin resistance . In our previous studies, intensive insulin interventions, especially continuous subcutaneous insulin infusion (csii), induced near - normoglycemia over 1 year without antihyperglycemic agents in nearly half of the patients with newly diagnosed t2 dm with favorable recovery of beta cell function [3, 4]. The reason for glycemic remission in these patients was considered to be alleviation of glucotoxicity, lipotoxicity, and insulin resistance [5, 6]. However, the therapy, which lasted for only 2 - 3 weeks, had its limitations in covering the multiple pathophysiological defects in the long term . In another trial investigating the effect of combination of metformin or rosiglitazone with csii, the combination of metformin for 3 months had better effects on insulin secretion function measured by acute insulin response (air) and homa - b while the combination with rosiglitazone better improved muscle insulin resistance . Since the two medicines used in that study mainly were targeted at insulin resistance, it would be of great interest whether combining csii with medicine intervening beta cell failure, the critical pathophysiology mechanism of t2 dm, might provide better clinical outcomes compared with short - term csii alone . Liraglutide, a glucagon - like peptide-1 (glp-1) analog with a 97% homology with endogenous glp-1, lowers blood glucose by enhancing glucose - dependent insulin secretion of beta cells and suppressing glucagon secretion of alpha cells . In some rodent studies, liraglutide reduced beta cell apoptosis and promoted its proliferation, which might potentially modify the progression of t2 dm [9, 10]. Moreover, liraglutide also reduced body weight in a dose dependent manner, ameliorated lipid profiles, lowered blood pressure, and reduced cardiovascular risk markers such as adipokines and proinflammatory factors, all of which are favorable in management of t2 dm . We hypothesized that combining csii with liraglutide might have better effects over csii alone . Therefore, we conducted this randomized controlled trial investigating whether liraglutide in combination with short - term csii therapy has better effect over csii alone on beta cell function and sustained glycemic control . Thirty - nine newly diagnosed t2 dm patients diagnosed according to the 1999 world health organization diagnostic criteria, without previous usage of antihyperglycemic and antihyperlipidemic medication, were enrolled . The included patients were between 20 and 65 years of age and had a body mass index of 2035 kg / m, with fasting plasma glucose (fpg) between 7.0 and 16.7 mmol / l . Patients were excluded if they had severe acute or severe chronic diabetic complications and severe intercurrent illness and were positive for autoimmune antibodies against islets or with a recent history of being treated with corticosteroid, immunosuppressing drugs, or cytotoxic drugs . All patients were admitted to the hospitals after a 35-day run - in period and assigned to one of the following two groups by sequentially opening sealed, opaque envelopes arranged in a computer - generated random order . During hospitalization, patients in csii alone group received insulin aspart (novorapid, novo nordisk, bagsvrd, denmark) or insulin lispro (humalog, eli lilly, usa) with an insulin pump (minimed 712, medtronic, northridge, ca) as csii therapy, while the csii + lira group received liraglutide (victoza, novo nordisk, bagsvaerd, denmark) 0.6 mg per day in addition to aforementioned csii regimen . The initial insulin dosage was 0.50.7 iu / kg / d, with the total daily dosage divided into 50/50 as basal and bolus infusion . In order to achieve euglycemia, basal rates and premeal boluses of insulin were adjusted every day according to capillary blood glucose values which were monitored at least 7 times per day . The glycemic goal was defined as fasting blood glucose less than 6.0 mmol / l and postprandial blood glucose less than 8.0 mmol / l . After the glycemic targets were achieved, csii treatments were maintained for additional 14 days . After being discharged from the hospital, all patients were guided with diet and physical exercise . Patients in csii + lira group continued to use liraglutide 1.2 mg per day until the 12-week treatment period was finished . All recruited patients provided written informed consent for participation, and the study protocol was approved by the medical research and ethics committee of the first affiliated hospital of sun yat - sen university (guangzhou, china). Baseline anthropometric data such as blood pressure, height, weight, and waist and hip circumferences were measured, while fasting blood samples were collected for measurements of fpg and hba1c . An intravenous glucose tolerance test (ivgtt) using 25 g of glucose (50 ml of 50% glucose) was conducted to assess air which was used to estimate the first - phase beta cell insulin secretion . Serum insulin levels before and 1, 2, 4, 6, and 10 min after glucose injection were measured, and air was calculated as the incremental trapezoidal area during the first 10 min of the ivgtt . Homeostasis model assessment was used to estimate insulin resistance (homa - ir) and beta cell function (homa - b). After csii suspension, all baseline measurements were repeated at least 15 hours after cessation of insulin infusion and before liraglutide injection for csii + lira group . At the 12-week visit, the assessments were performed after 12 weeks of csii suspension for csii alone group or 7 days after liraglutide suspension . Normally distributed data were presented as mean sd, and nonnormally distributed variables (triglyceride, air, homa - b, and homa - ir) were expressed as median (interquartile range). The differences of normally distributed data between two groups were compared by independent - sample t - tests, while the comparisons of nonnormally distributed variables were using mann - whitney u tests . Paired - sample t - tests or wilcoxon signed ranks tests were performed to estimate the changes before and after intervention . The tests were applied to analyze the differences of proportions . A 2-sided value of p <0.05 was defined statistically significant . The enrolled patients were 45.91 8.7 years in age, with a bmi of 25.7 2.8 kg / m, fpg of 11.4 3.2 mmol / l, ppg of 17.4 5.9 mmol / l, and hba1c of 10.7 2.2% . They were assigned to csii alone group (n = 19) and csii + lira group (n = 20) and finished csii therapy . At the subsequent 12-week visit 8 patients (20.5%, 4 in csii alone group, 4 in csii + lira group) dropped out due to withdrawal of consent . At baseline there were no significant differences in clinical characteristics, fpg, and hba1c between two groups except for ppg, which was slightly higher in csii + lira group (15.2 6.1 mmol / l versus 14.4 4.1 mmol / l, p = 0.025). Markers of beta cell function (air and homa - b) and insulin sensitivity (homa - ir) were also comparable (table 1). Patients in csii + lira group reached target glycemic control in less time than those in csii alone group (2 (3) days versus 1 (0) days, for csii alone group and csii + lira group, resp . The 14 days of csii for maintaining euglycemia was divided into three stages: early stage (days 15), medium stage (days 610), and late stage (days 1114). Average daily insulin dosage was similar in both groups, while the proportions of daily bolus dosage in total daily insulin dosage were lower in csii + lira group throughout the csii therapy (figure 1). Air was restored in all patients after short - term csii therapy compared with baseline . At csii suspension, air improved from 6.60 (26.2) umin / ml to 52.05 (100.55) umin / ml in csii alone group and from 6.98 (21.71) umin / ml to 168.62 (350.95) umin / ml in csii + lira group . The increment of air was significantly higher in csii + lira group than that in csii alone group (177.58 (351.57) umin / ml versus 58.15 (51.30) umin / ml, p <0.001). However, after withdrawal of liraglutide after the 12-week treatment, the improvement in air rapidly disappeared in csii + lira group (168.62 (350.95) umin / ml versus 50.43 (70.40) umin / ml, for csii suspension and 12-week visit, resp ., p <0.001). Therefore, air between two groups at the end of follow - up was similar (p = 0.921) (figure 2(a)). In both groups, homa - b was ameliorated significantly after csii treatment compared with baseline . Similar to air, homa - b in csii + lira group was higher than that in csii alone group at the end of csii (67.64 (46.31) versus 40.00 (35.53), p = 0.007), but the improvement was not sustained after stop of liraglutide at 12-week visit (41.28 (21.62), p = 0.003, compared with that after csii suspension) and became similar to csii alone group (55.65 (56.27), p = 0.110) (figure 2(b)). But at 12-week visit, homa - ir was significantly elevated from csii suspension in both groups (figure 2(c)). Hba1c level was slightly lower in csii + lira group at the end of the 12-week follow - up compared with csii alone group but did not reach statistical significance (6.0 0.5% versus 6.3 0.7%, p = 0.325), with similar proportions of patients who achieved hba1c 6.5% (73% (11/15) versus 94% (15/16), for csii alone group and csii + lira group, resp . Considerable reduction in fpg and ppg from baseline was observed at csii suspension . However, at 12-week visit there was a slight but statistically significant elevation of fpg in csii + lira group from csii suspension (from 6.1 0.9 mmol / l to 6.9 1.1 mmol / l, p = 0.01), which was not seen in csii alone group (figure 3(a)). There was a tendency of higher hyperglycemia relapse rate (> 7.0 mmol / l) in csii + lira group at 12-week visit (20% (3/15) versus 43.75% (7/16), for csii alone group and csii + lira group, resp ., suspension, certain body weight loss was recorded in both groups (1.6 2.0 kg versus 1.2 2.3 kg, for csii alone group and csii + lira group, resp . Continuous decline of body weight during the 12-week visit was recorded in csii + lira group (69.8 7.5 kg versus 66.2 9.3 kg, for csii suspension and 12-week visit, resp ., p = 0.005) but not in csii alone group; however, the reduction of body weight during the 12-week visit in the two groups did not reach statistical significance (1.6 3.5 kg versus 3.3 4.1 kg, for csii alone group and csii + lira group, resp . During short - term csii therapy phase, the incidence of hypoglycemia which was defined as capillary blood glucose level <3.9 mmol / l was similar in csii alone group and csii + lira group (4 (5) versus 2 (3) times per patient, p = 0.120). Most of the hypoglycemic episodes were mild and could be corrected after ingestion of carbohydrate . Gastrointestinal symptoms happened in 35% of patients in csii + lira group in the first few days of liraglutide injections, and most of these symptoms were well tolerated . Intensive insulin treatment was introduced in the management of newly diagnosed t2 dm since 1997 . By fast correction of glucotoxicity and lipotoxicity, intensive insulin treatment is able to induce long - term glycemic remission and thereby be suggested by the latest chinese guideline for t2 dm [3, 4, 15]. In this study, a glp-1 analog, liraglutide, was used as an add - on therapy of csii and lasted for additional 12 weeks . As expected, liraglutide facilitated the achievement of euglycemia by shortening the time required for insulin dose titration before reaching glycemic targets . Liraglutide was also reported to reduce the daily insulin requirement in patients with more advanced t2 dm treated with insulin . Although the total daily insulin dosage throughout csii treatment did not significantly differ between the two treatment groups, liraglutide significantly decreased the proportion of daily premeal bolus . Furthermore, there was also a tendency of better average glycemic control in csii + lira group during the 12-week extended therapy phase, as indicated by a lower hba1c level than that in csii alone group at the end of the follow - up . These findings were probably attributed to a better amelioration of beta cell function in csii + lira group compared with that in csii alone group . These data were in accordance with previous reports on liraglutide, which showed that it reduced hyperglycemia, especially postprandial glycemic fluctuation, by glucose - dependent insulinotropic effect . However, to our surprise, shortly after the suspension of liraglutide, its effect on beta cell function rapidly faded with 1 week, leading to an elevation of fasting blood glucose . Recently retnakaran et al . Reported that 48 weeks of liraglutide administration in patients with mean diabetes duration of 2 - 3 years after 4 weeks of insulin therapy also robustly increased beta cell function measured by issi-2 . Similar to this study, they also found a rapid deterioration of beta cell function shortly after cessation of liraglutide . However, an earlier observation showed that, in patients whose blood glucose was insufficiently controlled by metformin, a prolonged treatment with exenatide for 3 years had a slight but statistical significant benefit in beta cell function 4 weeks after stopping the medicine, which was not seen in the 1-year follow - up . There are several possible explanations for the discrepancy between short - term and long - term glp-1 analogs therapies . Firstly, because of the beneficial effects of glp-1 analogs on beta cell proliferation and apoptosis from rodent models, liraglutide was expected to further improve functional beta cell mass [9, 10]. However, the renewal rate of beta cells in human islets was so slow that a prolonged therapy targeted at pancreatic beta cells might be necessary for an overt change in islet architecture . Less than 1 year, according to the results from both retnakaran et al . And secondly, part of the effects of glp-1 analogs is attributed to their effect on body weight which could help to relieve insulin resistance and restore beta cell function . In lead-3 monostudy, the maximum weight loss in 1.8 mg liraglutide treatment group existed in 20 weeks . Although ongoing weight loss was observed in csii + lira group rather than csii alone group in this study, the difference of weight loss between the two groups was not statistically significant in a relatively short treatment period (12 weeks) in a lower dose (1.2 mg / d). Thirdly, it has been well documented that glp-1 analogs could suppress inappropriate secretion of glucagon from alpha cells . However, despite certain controversy, there are some reports showing that incretin therapy may induce hyperplasia of pancreatic alpha cells in human and rodent models [1921]. The importance and clinical consequence of alpha cell hyperplasia are largely unknown due to lack of data, but it is not impossible that, after stopping liraglutide, the previous suppressed glucagon secretion could rebound, resulting in relapse of hyperglycemia . Previous observations suggested that persistent improvement in insulin sensitivity was critical for long - term maintenance of near - normoglycemia [5, 6]. Our previous studies also showed that combining csii with insulin sensitizers, that is, metformin or rosiglitazone, increases short - term remission rate by improving both insulin sensitivity and beta cell function patients with newly diagnosed t2 dm . As shown in this study, 12-week treatment with liraglutide was not sufficient to cause prominent effect on insulin resistance . Enhancement of insulin action may decrease insulin demand and subsequent beta cell overload, endoplasmic reticulum stress, or oxidative stress, leading to a longer duration of glycemic remission [22, 23]. In this point of view, insulin sensitizer, other than insulin secretagogues, should be tested as combination therapy to csii in future studies . First of all, as a pilot study, the relatively small sample size may reduce the statistical power when analyzing some clinical parameters . Second, ivgtt and homeostasis model were used to evaluate beta cell function and insulin resistance . Using clamp technique as well as physiologic challenge tests such as ogtt or mix - meal test could facilitate the achievement of glycemic targets and further improve beta cell function in patients with newly diagnosed t2 dm . Rapid waning of beneficial effects of liraglutide implied that a prolonged treatment period might be required to obtain a sustained favorable outcome.
With improved treatments for brain tumours leading to longer survival, late complications of therapy are more frequently observed . The development of new neurological symptoms following brain tumour treatment is primarily suggestive for tumour recurrence, but when this has been ruled out, adverse treatment effects as a cause of the symptoms should be considered . Amongst late complications of radiation therapy, progressive leukoencephalopathy with cognitive decline and focal radiation necrosis mimicking recurrent tumour growth we report on four patients with reversible clinical and radiological features occurring years after radiation for brain tumours, suggestive for the so - called smart syndrome: stroke - like migraine attacks after radiation therapy [24]. In 1999 at age 32 years, this man had progressive headache and diplopia due to an obstructive hydrocephalus caused by a pineal tumour . Following resection a pineoloma with malignant histological features he was subsequently treated with craniospinal irradiation (36 gy in 20 fractions with a boost of 18 gy to the original tumour site). Following a minor head trauma 4 years after rt he developed increasing headache over the following days and a left hemianopia . No tumour recurrence was observed on mri, nor in the cerebrospinal fluid (csf) (table 1). However, on mri, gyral thickening of the right parieto - occipital cortex with contrast enhancement was seen (fig . 1a, b). During hospitalisation he developed several generalized seizures with persistent post - ictal weakness of the left arm and confusion, lasting for several weeks . The observed mri abnormalities recovered over the weeks thereafter, with concomitant clinical improvement (fig . 1c, d).table 1clinical details of reported cases with the smart syndromeage (years)tumourcsf studiesmri radiation toxicityinterval (years)symptomsoriginal tumour siteradiation type and dosepatient 1 male, 36pinealomaprotein 0.33 leukocytes 1none4headache neurologic deficit seizurespineal regioncraniospinal axis 36 gy (20 1.8) tumour booster 18 gy (10 1.8)patient 2 male, 60brain metastases from small - cell lung cancerprotein 0.81 leukocytes 1diffuse white matter changes10headache neurologic deficit seizuresboth hemisphereswbrt 30 gy (10 3)patient 3 male, 42 oligodendrogliomaprotein 0.43 leukocytes 1deep white matter disease 2 and 5headache neurologic deficit seizures left parietal lobefocal 60 gy (30 2)patient 4 male, 39astrocytomaprotein 0.71 leukocytes 2none5 and 10neurologic deficit seizuresright temporal lobefocal 50 gy (20 2.5)protein level in g / linterval, years between irradiation and the first symptoms; gy, radiation dose in grays; x, number of radiation fractions; wbrt, whole - brain radiation therapy; focal, focal radiation on the original tumour sitefig . 136-year - old man who was treated 4 years previously for pineal tumor with radiation therapy, now presenting with headache and left hemianopia . A t2 weighted, fluid attenuated inversion recovery (flair) mr image showing diffuse gyral swelling and signal increase in the right occipital lobe . Mr images obtained 9 month later showing non residual signal abnormalities on flair (c) and no abnormal enhancement (d) clinical details of reported cases with the smart syndrome interval, years between irradiation and the first symptoms; gy, radiation dose in grays; x, number of radiation fractions; wbrt, whole - brain radiation therapy; focal, focal radiation on the original tumour site 36-year - old man who was treated 4 years previously for pineal tumor with radiation therapy, now presenting with headache and left hemianopia . A t2 weighted, fluid attenuated inversion recovery (flair) mr image showing diffuse gyral swelling and signal increase in the right occipital lobe . B t1 weighted mr image after gadolinium administration showing diffuse gyral and leptomeningeal enhancement . Mr images obtained 9 month later showing non residual signal abnormalities on flair (c) and no abnormal enhancement (d) two years later he developed a similar clinical episode for which he was admitted once more, this time without seizures . The same imaging abnormalities were observed which resolved once again over the following months without any treatment . However, in 2008, 8 years after the initial presentation he did develop spinal leptomeningeal metastases, for which he was treated with focal rt and systemic chemotherapy . In 1997 at age 50 years, this man was treated for small cell lung cancer and two brain metastases with systemic chemotherapy and whole - brain radiation (30 gy in 10 fractions). Ten years later he developed slowly progressive headache, and a few weeks later he woke up with an expressive dysphasia . During hospitalization he developed a right sided hemiparesis with right hemianopia and he suffered from frequent focal epileptic seizures of the right arm and leg, for which he received valproate and levetiracetam . Mri showed focal gyral thickening and enhancement of the left parieto - occipital lobe where metastases had not been observed previously (fig . Apart from diffuse leukoencephalopathy, presumably due to the earlier brain radiotherapy, there were no signs of recurrent brain metastases . Repeated csf analysis showed a slightly elevated protein level, but no leptomeningeal metastases (table 1). A follow - up mri 3 months after discharge also showed complete resolution of the abnormalities in the left parieto - occipital cortex (fig . 2e, f).fig . 260-year - old man who was treated 10 years previously for brain metastases of bronchial carcinoma with chemo- and radiation therapy, now presenting with gradually progressive headache and acute dysphasis . A t2 weighted mr image showing diffuse gyral swelling and signal increase in the left parietal lobe . B t1 weighted mr image after gadolinium administration showing abnormal regional leptomeningeal enhancement . C t2 weighted flair mr image showing diffuse gyral swelling and signal increase in the left occipital lobe . D t1 weighted mr image after gadolinium administration showing abnormal leptomeningeal enhancement . Mr images after 4 months . F t1 weighted mr image after gadolinium administration showing no abnormal leptomeningeal enhancement 60-year - old man who was treated 10 years previously for brain metastases of bronchial carcinoma with chemo- and radiation therapy, now presenting with gradually progressive headache and acute dysphasis . A t2 weighted mr image showing diffuse gyral swelling and signal increase in the left parietal lobe . B t1 weighted mr image after gadolinium administration showing abnormal regional leptomeningeal enhancement . C t2 weighted flair mr image showing diffuse gyral swelling and signal increase in the left occipital lobe . D t1 weighted mr image after gadolinium administration showing abnormal leptomeningeal enhancement . Mr images after 4 months . F t1 weighted mr image after gadolinium administration showing no abnormal leptomeningeal enhancement in 1997 at the age of 42 years, this man suffered from an epileptic seizure caused by an anaplastic oligodendroglioma in the left hemisphere . The tumour was resected and he subsequently received focal brain radiation (60 gy in 30 fractions). Two years later he was admitted to the hospital twice within a short time period with reversible neurological deficits: dysphasia, right hemianopia, headache and neglect of his right arm . Imaging showed no signs of recurrent tumour, but possibly a small infarction in the left occipital region and some diffuse white matter disease, possibly due to radiation . Three years thereafter he was again taken into hospital with focal epileptic seizures of the right side of his face . He suffered from headache, progressive aphasia, right hemianopia and mild paresis of his right arm and leg . An mri scan now showed diffuse cortical enhancement of the left occipital and temporal cortex (fig . This enhancement disappeared on a subsequent mri performed 8 days later (fig . 347-year - old man who was treated 5 years previously for a left parietal oligodendrolioma with chemo- and radiation therapy, now presenting with right hemianopia and mild paresis of his right arm and leg . T2 weighted flair mr images a showing gyral swelling and signal increase in the temporal lobe . B after gadolinium administration, diffuse gyral enhancement is seen in the occipital, temporal and insular lobe . Proton density (c) and t1 post gadolinium (d) mr imaging 8 days later showing normalization of signal intensity in the temporal lobe and marked reduction in abnormal gyral enhancement . Mr imaging again shows signal increase in the temporal lobe on pd weighted images (e) while on t1 post gadolinium imaging (f) abnormal gyral enhancement is again visible . Like after the previous episode, the mr abnormalities disappeared (g, h) 47-year - old man who was treated 5 years previously for a left parietal oligodendrolioma with chemo- and radiation therapy, now presenting with right hemianopia and mild paresis of his right arm and leg . T2 weighted flair mr images a showing gyral swelling and signal increase in the temporal lobe . B after gadolinium administration, diffuse gyral enhancement is seen in the occipital, temporal and insular lobe . Proton density (c) and t1 post gadolinium (d) mr imaging 8 days later showing normalization of signal intensity in the temporal lobe and marked reduction in abnormal gyral enhancement . Mr imaging again shows signal increase in the temporal lobe on pd weighted images (e) while on t1 post gadolinium imaging (f) abnormal gyral enhancement is again visible . Like after the previous episode, the mr abnormalities disappeared (g, h) three years later he was again admitted to hospital because of a new episode of worsening aphasia and paresis of his right arm and leg . Mr imaging, similar to 3 years earlier, demonstrated diffuse cortical enhancement of the left occipital and temporal cortex (fig . Although csf examination showed some atypical cells, he again partially recovered spontaneously and almost completely, making leptomeningeal seeding unlikely (table 1). A follow - up mri scan performed 1 month later again showed complete disappearance of the contrast enhancement (fig . 3 g, h), and he recovered afterwards in a rehabilitation centre . On longer follow - up, he was treated successfully with temozolomide chemotherapy for local tumour recurrence . In 1999 at the age of 34 years, this man suffered from an epileptic seizure caused by a right temporal anaplastic astrocytoma . The tumour was resected and he subsequently had focal brain radiation (50 gy in 20 fractions). In the following 5 years he had infrequent focal seizures of the left arm but no tumour recurrence was observed on repeated mri . Five years after initial treatment, however, he developed a progressive clumsy left hand, left - sided facial paresis and dysarthria in combination with an increase of focal seizures . The mri demonstrated no tumour recurrence but a gyriform enhancement and swelling of the right parieto - temporal cortex (fig . 438-year - old man who was treated 5 years previously for a right temporal anaplastic astrocytoma with chemo- and radiation therapy, now presenting with increasing epileptic activity and gradually progressive left sided hemiparesis and dysarthria . T2 weighted flair mr images (a, b) showing gyral swelling and signal increase in the temporal lobe . T2 weighted mr image (c, d) showing gyral swelling and signal increase in the temporal lobe . A post - operative parenchymal defect is seen in the area of the previously treated astrocytoma . T1 weighted mr images after gadolinium administration (e, f) abnormal gyral and leptomeningeal enhancement in the temporal lobe and in the basal frontal areas . No focal enhancement in post - operative parenchymal defect g, h t1 weighted mr image after gadolinium administration showing persistent gyral abnormalities but disappearance of abnormal enhancement . I t2 weighted flair mr image and j t1 weighted mr image after gadolinium administration showing persistent gyral abnormalities but disappearance of abnormal enhancement . K t2 weighted mr image and l t1 weighted mr image after gadolinium administration resolution of t2 abnormalities and disappearance of abnormal enhancement in the deep temporal area 38-year - old man who was treated 5 years previously for a right temporal anaplastic astrocytoma with chemo- and radiation therapy, now presenting with increasing epileptic activity and gradually progressive left sided hemiparesis and dysarthria . T2 weighted flair mr images (a, b) showing gyral swelling and signal increase in the temporal lobe . T2 weighted mr image (c, d) showing gyral swelling and signal increase in the temporal lobe . A post - operative parenchymal defect is seen in the area of the previously treated astrocytoma . T1 weighted mr images after gadolinium administration (e, f) abnormal gyral and leptomeningeal enhancement in the temporal lobe and in the basal frontal areas . No focal enhancement in post - operative parenchymal defect g, h t1 weighted mr image after gadolinium administration showing persistent gyral abnormalities but disappearance of abnormal enhancement . I t2 weighted flair mr image and j t1 weighted mr image after gadolinium administration showing persistent gyral abnormalities but disappearance of abnormal enhancement . Mr images 6 months after treatment for recurrent anaplastic astrocytoma using chemotherapy . K t2 weighted mr image and l t1 weighted mr image after gadolinium administration resolution of t2 abnormalities and disappearance of abnormal enhancement in the deep temporal area csf examination was, besides a slightly elevated protein level, completely normal (table 1). Over the following weeks the focal seizures disappeared and the left sided weakness improved . The patient was discharged from hospital on valproate and nearly complete resolution of the clinical picture . The abnormalities on mri three months later, a new mri was suggestive for recurrent tumour in the right temporal lobe, showing several nodular enhancements . A biopsy confirmed recurrent high - grade glioma, for which he was treated with temozolomide . Following chemotherapy treatment, the abnormal gyral and leptomeningeal enhancement temporarily disappeared, but recurred for which pcv chemotherapy was started . Ten years after the initial diagnosis of anaplastic astrocytoma he developed left hemianopia and a clumsy left hand, but had no seizures . On mri a right occipital contrast enhancing tumour was found, suggestive for recurrent tumour . Resection was planned, but pre - operative imaging showed the lesion to be smaller and an open biopsy did not confirm active tumour growth . Postoperatively he suffered from very frequent focal seizures of the left side of his face and a clumsy left hand, which did not subside until he was put into a propofol induced coma . Follow - up mri demonstrated the occipital contrast - enhancing to have disappeared completely (fig . The patient died a year later from tumour recurrence, 11 years after the initial diagnosis . In 1999 at age 32 years, this man had progressive headache and diplopia due to an obstructive hydrocephalus caused by a pineal tumour . Following resection a pineoloma with malignant histological features he was subsequently treated with craniospinal irradiation (36 gy in 20 fractions with a boost of 18 gy to the original tumour site). Following a minor head trauma 4 years after rt he developed increasing headache over the following days and a left hemianopia . No tumour recurrence was observed on mri, nor in the cerebrospinal fluid (csf) (table 1). However, on mri, gyral thickening of the right parieto - occipital cortex with contrast enhancement was seen (fig . 1a, b). During hospitalisation he developed several generalized seizures with persistent post - ictal weakness of the left arm and confusion, lasting for several weeks . The observed mri abnormalities recovered over the weeks thereafter, with concomitant clinical improvement (fig . 1c, d).table 1clinical details of reported cases with the smart syndromeage (years)tumourcsf studiesmri radiation toxicityinterval (years)symptomsoriginal tumour siteradiation type and dosepatient 1 male, 36pinealomaprotein 0.33 leukocytes 1none4headache neurologic deficit seizurespineal regioncraniospinal axis 36 gy (20 1.8) tumour booster 18 gy (10 1.8)patient 2 male, 60brain metastases from small - cell lung cancerprotein 0.81 leukocytes 1diffuse white matter changes10headache neurologic deficit seizuresboth hemisphereswbrt 30 gy (10 3)patient 3 male, 42 oligodendrogliomaprotein 0.43 leukocytes 1deep white matter disease 2 and 5headache neurologic deficit seizures left parietal lobefocal 60 gy (30 2)patient 4 male, 39astrocytomaprotein 0.71 leukocytes 2none5 and 10neurologic deficit seizuresright temporal lobefocal 50 gy (20 2.5)protein level in g / linterval, years between irradiation and the first symptoms; gy, radiation dose in grays; x, number of radiation fractions; wbrt, whole - brain radiation therapy; focal, focal radiation on the original tumour sitefig . 136-year - old man who was treated 4 years previously for pineal tumor with radiation therapy, now presenting with headache and left hemianopia . A t2 weighted, fluid attenuated inversion recovery (flair) mr image showing diffuse gyral swelling and signal increase in the right occipital lobe . Mr images obtained 9 month later showing non residual signal abnormalities on flair (c) and no abnormal enhancement (d) clinical details of reported cases with the smart syndrome interval, years between irradiation and the first symptoms; gy, radiation dose in grays; x, number of radiation fractions; wbrt, whole - brain radiation therapy; focal, focal radiation on the original tumour site 36-year - old man who was treated 4 years previously for pineal tumor with radiation therapy, now presenting with headache and left hemianopia . A t2 weighted, fluid attenuated inversion recovery (flair) mr image showing diffuse gyral swelling and signal increase in the right occipital lobe . B t1 weighted mr image after gadolinium administration showing diffuse gyral and leptomeningeal enhancement . Mr images obtained 9 month later showing non residual signal abnormalities on flair (c) and no abnormal enhancement (d) two years later he developed a similar clinical episode for which he was admitted once more, this time without seizures . The same imaging abnormalities were observed which resolved once again over the following months without any treatment . However, in 2008, 8 years after the initial presentation he did develop spinal leptomeningeal metastases, for which he was treated with focal rt and systemic chemotherapy . In 1997 at age 50 years, this man was treated for small cell lung cancer and two brain metastases with systemic chemotherapy and whole - brain radiation (30 gy in 10 fractions). Ten years later he developed slowly progressive headache, and a few weeks later he woke up with an expressive dysphasia . During hospitalization he developed a right sided hemiparesis with right hemianopia and he suffered from frequent focal epileptic seizures of the right arm and leg, for which he received valproate and levetiracetam . Mri showed focal gyral thickening and enhancement of the left parieto - occipital lobe where metastases had not been observed previously (fig . 2a d). Apart from diffuse leukoencephalopathy, presumably due to the earlier brain radiotherapy repeated csf analysis showed a slightly elevated protein level, but no leptomeningeal metastases (table 1). A follow - up mri 3 months after discharge also showed complete resolution of the abnormalities in the left parieto - occipital cortex (fig . 2e, f).fig . 260-year - old man who was treated 10 years previously for brain metastases of bronchial carcinoma with chemo- and radiation therapy, now presenting with gradually progressive headache and acute dysphasis . A t2 weighted mr image showing diffuse gyral swelling and signal increase in the left parietal lobe . C t2 weighted flair mr image showing diffuse gyral swelling and signal increase in the left occipital lobe . D t1 weighted mr image after gadolinium administration showing abnormal leptomeningeal enhancement . F t1 weighted mr image after gadolinium administration showing no abnormal leptomeningeal enhancement 60-year - old man who was treated 10 years previously for brain metastases of bronchial carcinoma with chemo- and radiation therapy, now presenting with gradually progressive headache and acute dysphasis . A t2 weighted mr image showing diffuse gyral swelling and signal increase in the left parietal lobe . C t2 weighted flair mr image showing diffuse gyral swelling and signal increase in the left occipital lobe . D t1 weighted mr image after gadolinium administration showing abnormal leptomeningeal enhancement . Mr images after 4 months . In 1997 at the age of 42 years, this man suffered from an epileptic seizure caused by an anaplastic oligodendroglioma in the left hemisphere . The tumour was resected and he subsequently received focal brain radiation (60 gy in 30 fractions). Two years later he was admitted to the hospital twice within a short time period with reversible neurological deficits: dysphasia, right hemianopia, headache and neglect of his right arm . Imaging showed no signs of recurrent tumour, but possibly a small infarction in the left occipital region and some diffuse white matter disease, possibly due to radiation . Three years thereafter he was again taken into hospital with focal epileptic seizures of the right side of his face . He suffered from headache, progressive aphasia, right hemianopia and mild paresis of his right arm and leg . An mri scan now showed diffuse cortical enhancement of the left occipital and temporal cortex (fig . This enhancement disappeared on a subsequent mri performed 8 days later (fig . 347-year - old man who was treated 5 years previously for a left parietal oligodendrolioma with chemo- and radiation therapy, now presenting with right hemianopia and mild paresis of his right arm and leg . T2 weighted flair mr images a showing gyral swelling and signal increase in the temporal lobe . B after gadolinium administration, diffuse gyral enhancement is seen in the occipital, temporal and insular lobe . Proton density (c) and t1 post gadolinium (d) mr imaging 8 days later showing normalization of signal intensity in the temporal lobe and marked reduction in abnormal gyral enhancement . Mr imaging again shows signal increase in the temporal lobe on pd weighted images (e) while on t1 post gadolinium imaging (f) abnormal gyral enhancement is again visible . Like after the previous episode, the mr abnormalities disappeared (g, h) 47-year - old man who was treated 5 years previously for a left parietal oligodendrolioma with chemo- and radiation therapy, now presenting with right hemianopia and mild paresis of his right arm and leg . T2 weighted flair mr images a showing gyral swelling and signal increase in the temporal lobe . B after gadolinium administration, diffuse gyral enhancement is seen in the occipital, temporal and insular lobe . Proton density (c) and t1 post gadolinium (d) mr imaging 8 days later showing normalization of signal intensity in the temporal lobe and marked reduction in abnormal gyral enhancement . Mr imaging again shows signal increase in the temporal lobe on pd weighted images (e) while on t1 post gadolinium imaging (f) abnormal gyral enhancement is again visible . Like after the previous episode, the mr abnormalities disappeared (g, h) three years later he was again admitted to hospital because of a new episode of worsening aphasia and paresis of his right arm and leg . Mr imaging, similar to 3 years earlier, demonstrated diffuse cortical enhancement of the left occipital and temporal cortex (fig . Although csf examination showed some atypical cells, he again partially recovered spontaneously and almost completely, making leptomeningeal seeding unlikely (table 1). A follow - up mri scan performed 1 month later again showed complete disappearance of the contrast enhancement (fig . 3 g, h), and he recovered afterwards in a rehabilitation centre . On longer follow - up, he was treated successfully with temozolomide chemotherapy for local tumour recurrence . In 1999 at the age of 34 years, this man suffered from an epileptic seizure caused by a right temporal anaplastic astrocytoma . The tumour was resected and he subsequently had focal brain radiation (50 gy in 20 fractions). In the following 5 years he had infrequent focal seizures of the left arm but no tumour recurrence was observed on repeated mri . Five years after initial treatment, however, he developed a progressive clumsy left hand, left - sided facial paresis and dysarthria in combination with an increase of focal seizures . The mri demonstrated no tumour recurrence but a gyriform enhancement and swelling of the right parieto - temporal cortex (fig . 438-year - old man who was treated 5 years previously for a right temporal anaplastic astrocytoma with chemo- and radiation therapy, now presenting with increasing epileptic activity and gradually progressive left sided hemiparesis and dysarthria . T2 weighted flair mr images (a, b) showing gyral swelling and signal increase in the temporal lobe . T2 weighted mr image (c, d) showing gyral swelling and signal increase in the temporal lobe . A post - operative parenchymal defect is seen in the area of the previously treated astrocytoma . T1 weighted mr images after gadolinium administration (e, f) abnormal gyral and leptomeningeal enhancement in the temporal lobe and in the basal frontal areas . No focal enhancement in post - operative parenchymal defect . G, h t1 weighted mr image after gadolinium administration showing persistent gyral abnormalities but disappearance of abnormal enhancement . I t2 weighted flair mr image and j t1 weighted mr image after gadolinium administration showing persistent gyral abnormalities but disappearance of abnormal enhancement . K t2 weighted mr image and l t1 weighted mr image after gadolinium administration resolution of t2 abnormalities and disappearance of abnormal enhancement in the deep temporal area 38-year - old man who was treated 5 years previously for a right temporal anaplastic astrocytoma with chemo- and radiation therapy, now presenting with increasing epileptic activity and gradually progressive left sided hemiparesis and dysarthria . T2 weighted flair mr images (a, b) showing gyral swelling and signal increase in the temporal lobe . T2 weighted mr image (c, d) showing gyral swelling and signal increase in the temporal lobe . A post - operative parenchymal defect is seen in the area of the previously treated astrocytoma . T1 weighted mr images after gadolinium administration (e, f) abnormal gyral and leptomeningeal enhancement in the temporal lobe and in the basal frontal areas . No focal enhancement in post - operative parenchymal defect g, h t1 weighted mr image after gadolinium administration showing persistent gyral abnormalities but disappearance of abnormal enhancement . Mr images 6 months after treatment for recurrent anaplastic astrocytoma using chemotherapy . I t2 weighted flair mr image and j t1 weighted mr image after gadolinium administration showing persistent gyral abnormalities but disappearance of abnormal enhancement . Mr images 6 months after treatment for recurrent anaplastic astrocytoma using chemotherapy . K t2 weighted mr image and l t1 weighted mr image after gadolinium administration resolution of t2 abnormalities and disappearance of abnormal enhancement in the deep temporal area csf examination was, besides a slightly elevated protein level, completely normal (table 1). Over the following weeks the focal seizures disappeared and the left sided weakness improved . The patient was discharged from hospital on valproate and nearly complete resolution of the clinical picture . Three months later, a new mri was suggestive for recurrent tumour in the right temporal lobe, showing several nodular enhancements . A biopsy confirmed recurrent high - grade glioma, for which he was treated with temozolomide . Following chemotherapy treatment, the abnormal gyral and leptomeningeal enhancement temporarily disappeared, but recurred for which pcv chemotherapy was started . Again, a complete response ensued . Ten years after the initial diagnosis of anaplastic astrocytoma he developed left hemianopia and a clumsy left hand, but had no seizures . On mri a right occipital contrast enhancing tumour was found, suggestive for recurrent tumour . Resection was planned, but pre - operative imaging showed the lesion to be smaller and an open biopsy did not confirm active tumour growth . Postoperatively he suffered from very frequent focal seizures of the left side of his face and a clumsy left hand, which did not subside until he was put into a propofol induced coma . Follow - up mri demonstrated the occipital contrast - enhancing to have disappeared completely (fig . The patient died a year later from tumour recurrence, 11 years after the initial diagnosis . All four patients had (partly) reversible neurological symptoms as well as reversible imaging findings several years after radiation therapy for their brain tumours . The combination of headaches in three of the four patients, as well as development of new neurological deficit and seizures in all four patients was clinically suggestive for tumour recurrence . However, neither local tumour recurrence, nor leptomeningeal disease were found to be the cause of the clinical deterioration . The radiological differential diagnosis of diffuse gyral signal increase and leptomeningeal enhancement as observed in these cases also includes vascular disorders such as ischemia, venous sinus thrombosis and dural arteriovenous fistula, and infection [6, 7]. Using csf examination and additional radiological techniques such as diffusion and perfusion weighted mri and digital subtraction angiography, also post - ictal imaging changes (swelling and enhancement) and posterior reversible encephalopathy syndrome (pres) as observed in patients with hypertensive encephalopathy or following the use of immunosuppressive agents should be considered [8, 9]. The episodic nature of the deficits with spontaneous resolution over weeks in combination with unilateral enhancement and thickening of the posterior cortex on mri might suggest focal epileptic activity with long - lasting post - ictal deficit as the cause of the clinical symptoms . Yaffe and ferreiro described eight patients who had all been treated with systemic chemotherapy with comparable reversible mri changes following seizures . The majority of the lesions involved grey and white matter, predominantly in the posterior vascular boundary zones . Brain barrier by systemic chemotherapy in combination with seizures . In our patients, however, damage to the parieto - occipital blood brain barrier was presumably due to radiation therapy . Irradiation affects primarily the vasculature or the trigeminovascular system and engender a reversible disturbance of homeostasis . Most previously presented cases [24] had been treated with posterior fossa irradiation, and it is presumed that the posterior lobes are specific vulnerable for radiation damage . Regarding epileptic seizures as the underlying mechanism, in two of our patients the seizures occurred either before the development of mri abnormalities or after the mri abnormalities had subsided . Also, repeated eeg in three of four patients did not show epileptic activity during the symptoms . Additionally, post - ictal mri abnormalities in patients without previous radiation therapy or chemotherapy are not confined to the parieto - occipital region . We therefore argue that the mri findings as observed in our patients cannot be explained by post - ictal enhancement alone . Reversible white matter enhancement, but not cortical enhancement has been described in patients with migraine [11, 12]. The underlying mechanisms of these transient imaging abnormalities in migraine include meningeal and parenchymal hyperperfusion, edema or inflammatory plasma protein extravasation after disruption of the blood brain barrier . Besides, the prolonged duration of the symptoms in our patients, who had no history of migraine headaches, are unlikely due to migraine . Otherwise, the clinical and radiological picture of these patients is compatible with the smart syndrome . Smart syndrome involves transient, reversible neurological dysfunction which may include migrainous headache, at times preceded by aura, prolonged hemispheric neurological impairment and sometimes seizure activity [2, 3]. Neuroimaging studies of patients with smart syndrome typically show focal gyral thickening of the affected cortex and gyriform contrast enhancement . Before smart was defined as a distinct syndrome, similar patients with sort - like reversible mri changes and clinical symptoms after radiation were reported [5, 1315]. . A specific vulnerability of the parieto - occipital cortex for radiation or chemotherapy, similar to that observed in pres, may explain why the imaging findings are preferentially observed in this region . Patient 2 had been treated with whole - brain radiation and developed parieto - occipital abnormalities in the left hemisphere, not related to the original site of the brain metastases, which supports this concept . The clinical presentation of pres has similarities with smart, including headaches, neurological deficits and, more frequently than in smart, seizures . The pathophysiology of pres is combined vasoconstriction and vasodilation and has the same patterns as seen in vasculopathy . This results in blood brain barrier disruption leading to symmetric hemispheric edema and contrast enhancement on mri . Since radiation may preferentially damage endothelial cells, the smart syndrome might be a reversible radiation vasculopathy comparable with pres . An alternative hypothesis is that post - radiation neuronal dysfunction is the underlying mechanism, such as in migraine or epilepsy, with impairment of the trigeminovascular system or a lowered threshold for cortical spreading depression .the parieto - occipital cortical damage might even be the cause of epileptic seizures rather than being the result of seizures . Development of the smart syndrome has been related to a radiation dose of at least 50 gy, as described in most of the previous cases [35]. In our cases, a dose exceeding 50 gy had only be applied to patients 1 and 3 . In the other two patients, however, the daily fraction doses were relatively high (3 gy in patient 2 and 2.5 gy in patient 4, respectively), which may have attributed to the radiation toxicity . Late effects of radiotherapy have mainly been blamed on vascular endothelial injury and demyelination, and include diffuse white matter changes or focal necrosis with mass effect [1618]. In contrast with the smart syndrome these late effects of radiation therapy are gradual in onset and may have a progressive nature [19, 20]. If these reversible, stroke - like migraine attacks are related to remote radiation therapy, this relationship may be under recognized so far because many patients did not live long enough to experience these late treatment effects.
Gallstone disease remains a common and significant cause of suffering in most parts of the world . It has been reported that there are 5.5 million gallstone sufferers in the uk and that over 50 000 cholecystectomies are performed each year with 15% of patients being symptomatic over a period of 15 years . Treatment of gallstone disease therefore continues to remain an important area of service in the peripheral district general hospital setting . Paradigm shifts in the concepts governing the management of gallstone disease are necessary to provide prompt and definitive care in most cases to enable patients to return to the community and to their productive lives . We discuss our philosophy and experience of having treated 1332 patients with gallstone disease in a national health service (nhs) district general hospital over the last 8 years . An evidence - based multidisciplinary approach together with incorporation of imaging and endoscopic and minimal access surgical techniques is the key to providing expected standards of care to patients . Gallstone disease can present in a variety of clinical scenarios, and the mode of management needs to be tailored to the manifestation of the condition . Our basis of management of gallstone disease has been the evidence - based consensus that definitive treatment should be provided as early as is feasible if not in the very same admission to prevent symptom recurrence or complications . We have endeavored to build a team of professionals with interest and experience in this field . An option to refer cases to this team was also provided to other surgeons in the hospital who formed part of the accident and emergency (a&e) cover but had other areas of special interest . Patients were treated based on a predetermined protocol depending on their mode of presentation (table 1). Protocol followed in management of patients with gallstone disease patients referred as outpatients with a diagnosis of gallstones or with symptoms suggestive of gallstone disease were investigated for the presence of abnormalities in liver function, common bile duct stones, and biliary tract obstruction before being booked for surgery . Those presenting through our a&e services with symptoms or signs suggestive of gallstone disease were investigated with blood investigations including liver function tests (lft), resuscitated, and treated supportively . They had ultrasound scans (us) and/or magnetic resonance imaging (mri) if required . Patients with dilated extrahepatic biliary tract or common bile duct stones had endoscopic retrograde cholangiopancreatography (ercp) for clearance of obstruction before laparoscopic cholecystectomy at the earliest opportunity . One 10-mm epigastric port and three 5-mm ports were situated in the umbilicus, right subcostal, and right flank regions, respectively . Pneumoperitoneum was achieved by veress needle through the umbilicus with a standard insufflation pressure of 14 mm hg . Lower pressures (10 mm to 12 mm) were used for patients with cardiovascular insufficiency . Medium large clips (horizon ligating clips, pilling weck closure systems, canada) were used to clip the cystic duct and cystic artery preferring to stay closer to the gallbladder . Gallbladder was hook dissected off the liver bed and retrieved through the epigastric port incision using a bert bag (vernon - carus ltd, lancashire, uk). In patients with a frozen calot's triangle secondary to adhesions, risk of iatrogenic injury to the adjoining biliary ducts and blood vessels in the hilar region during dissection to identify the cystic duct and artery is high in either laparoscopic or open surgery . These patients had laparoscopic subtotal cholecystectomy (lsc) through the most proximal accessible part of hartman's pouch or gallbladder neck by suturing closed or ligation of the stump using vicryl endoloop (ethicon, johnson & johnson, cincinnati, oh) after visual inspection of the stump lumen for any residual calculi and free flow of bile on milking with an atraumatic instrument . Anterior and posterior branches of the cystic artery were identified separately if possible and clipped . Failing this, they were ligated en mass with the cystic stump . Patients were allowed to eat and drink immediately postoperatively and discharged as soon as comfortable on regular oral analgesics as needed . They were followed up as outpatients for 3 weeks before being finally discharged unless any outstanding findings were identified . Data were prospectively collected by our team and regularly audited and discussed in relevant forums . Our team had the opportunity to provide treatment to 1332 patients with various presentations of gallstone disease over the 7-year period between september 1999 and december 2007 . From the community, 1083 patients were referred to us with symptoms of gallstones or with asymptomatic gallstones having being diagnosed on imaging . Acute symptoms of gallstone disease were present in 249 patients who were admitted for treatment . Eight patients had empyema of the gallbladder, and 124 patients in the series had evidence to suggest biliary obstruction necessitating further imaging with mri (30 patients). Endoscopic intervention with ercp was needed in 92 patients before they could be schueduled for lc . The surgery could be completed laparoscopically in all except 8 patients who needed conversion to open surgery; 3 of these patients had intraabdominal adhesions and 5 had difficult anatomy . The overall morbidity was 2.33% . Among patients who underwent lc, 5 had residual stones in the common bile duct (cbd) necessitating further imaging and ercp . One patient with a wide cystic duct had a bile leak from the sutured stump 48 hours after surgery . This was corrected by ercp and stent insertion as well as laparoscopic lavage and drainage of the peritoneal contamination . Another patient had a bile leak from a loose endoloop around the cystic stump following lsc . This was managed by laparoscopy and endoloop reapplication with additional suturing of the stump to ensure a repair . One patient developed peritonitis from a bile leak from an accessory bile duct that necessitated a laparotomy for adequate drainage and peritoneal lavage . One patient was re - referred with an incidentally noted derangement of lft 13 months after emergency lc . Investigations did not reveal any damage to the biliary tract, but an mri revealed features of right segmental liver atrophy suspected to be due to vascular injury . The patient was therefore referred to a specialist liver unit where a hepaticojejunostomy was advised and performed . The patient is well and asymptomatic but continues to have deranged lfts for reasons that are still not fully understood . Sixty - seven of 186 patients with acute cholecystitis underwent surgery within 96 hours of acute presentation . The rest were delayed due to the need for recovery from pancreatitis and the need for further imaging or interventions . Of 1332 patients who had lc, 684 were discharged within 23 hours of surgery; 253 were discharged home on the same day (figure 2); 379 patients required inpatient lc either because they were treated as emergencies during an acute attack or because they did not fulfill criteria for a short stay or outpatient treatment . One patient had to be admitted from the outpatient surgery unit due to unexplained persistent postoperative hypotension that later resolved spontaneously . Twelve patients from the 23-hour cases stayed longer due to social and care - related issues including pain management . Mode of delivery of care and admission and discharge pattern for laparoscopic cholecystectomy . A: 684 cases; 12 discharge failures . The cost of providing the service was kept low by reducing the use of disposables to a minimum and a low complication rate . The only disposable instruments used were the scissor tips (endocut scissor tips, microline pentax inc, beverly, ma, usa) and endoscopic retrieval (bert) bags . We consider this a good policy given the present need for economic and ecological prudence . The prevalence of gallstone disease makes it imperative for responsibility of care to remain the domain of the generalist in the district general hospital, and referral to a specialist center is practicable only for difficult clinical cases . In spite of the uniform availability and growing skill and safety of lc, the provision of an acceptable standard of care in the nhs district general hospital setting based on current evidence requires multidisciplinary team - based management strategies to treat gallstone disease in all its forms using minimal access techniques . It would also benefit the patient if a surgeon agreeing to care for a patient with gallstone disease as part of his on - call commitment had an in - hospital special - interest team to refer the patient onward for definitive care . The specialist team would be able to give greater priority to the case than the receiving surgeon who is quite likely to be committed to targets specific to his area of special interest . This team will also be keen to keep abreast of new skills and methods evolving in the treatment of gallstones and therefore continue to provide optimal care to every patient seeking treatment from his local hospital . Cholecystectomy has evolved over a span of a century from the initial open cholecystectomy described by langenbuck in 1882 to lc described by french surgeon phillipe mouret of lyon in march 1987 . The introduction of modern imaging techniques and the widespread availability of lc has brought about a paradigm shift with regards to management of gallstone disease . Surgical options can now be offered even to elderly individuals who were previously denied open cholecystectomy due to comorbid features . Best left alone if not troublesome to one that says, leave alone only if you have to! The recognition that a significant proportion of patients suffer repeated symptoms and complications has prompted this change . Lc allows inspection of the gallbladder without the need for commitment to completion or a large abdominal incision . Patients referred through the community have different treatment needs compared with those presenting as acute emergencies . Patients presenting through a&e had a varied spectrum of gallstone symptoms like acute cholecystitis, cbd stones, jaundice, pancreatitis, and other complications of gallstone disease and needed control of these before they could undergo lc . The challenge is to achieve these ideally within 4 days of onset of acute symptoms, which is by and large accepted as the best time for cholecystectomy in the acute setting though this can vary based on the surgeon's level of experience and the individual patient . The common problems that prevent immediate lc are obstruction of the cbd by stones, empyema of the gallbladder with sepsis, and pancreatitis . We were also bound by certain inherent methods in the health system with being able to coordinate investigations, surgical theater booking, and the availability of specialists to perform procedures due to tight advance scheduling of the above resource allocations . Patients with cbd stones confirmed by imaging underwent ercp at the earliest and then lc at the next available opportunity during regular hours . Those with pancreatitis without cbd stones were observed for clinical and biochemical recovery before having lc . Empyema of the gallbladder was treated by us - guided percutaneous drainage and interval cholecystectomy . This allows for resolution of systemic sepsis that could add to the morbidity if any surgery is undertaken under such circumstances . We feel that emergency lc is only useful in suspected perforation or gangrene of the gallbladder where drainage of the gallbladder alone would not be sufficient . Most outpatient referrals for gallstone disease can be safely treated either as outpatient cases or as short overnight (23 hour) in - hospital cases . Inpatient care would only be needed for those with medical comorbidities and social care needs for a smooth pre- and postoperative journey . Whilst we have endeavored to provide surgery at the earliest in this subgroup of patients, we were bound by list availability and fair prioritization inherent to the best institutions in the public health system . One of the commonest reasons for conversion from lc to open cholecystectomy used to be the discovery of a frozen, it would not be untrue to say that the risk of iatrogenic collateral biliary or vascular injury involved with dissection of a difficult frozen calot's triangle is no higher with laparoscopic surgery than with conventional open surgery . We join many other peers in continuing with this concept of lsc, because we do not see any benefits in conversion to open surgery towards achieving a better result in such cases . Being able to choose and deliver effective treatment thus needs training, experience, and a team approach to the problem involving experts from the field of imaging, upper gi endoscopy, and minimal access surgery . We believe that these principles can be easily adopted in most peripheral institutions with little additional cost and good results . Outpatient or short - stay treatment for most patients (71.5% in our cohort) reduced bed occupancy in the main hospital . This has a positive impact on the quality of life, the cost of health care provision, and the economy of the society at large . The promptness of therapy reduces the need for repeated outpatient visits and admissions due to recurrent attacks of symptoms from gallstone disease whilst returning patients quickly to their life and careers . The treatment of gallstone disease has evolved from the singular option of having a major abdominal operation to present practice based on the latest interventional and minimal access surgery techniques . The treatment of gallstone disease continues to remain the bane of the generalist in district general hospitals . Though treatments by individuals with general interests may appear to give satisfactory results, optimum results and minimal complications are best achieved when treatment is delivered by a specialized multidisciplinary team of experts in the district hospital, who have good experience, commitment, and continued training in the management of gallstone disease . Our results over the 8-year period of our study show that this approach can deliver good results, minimum morbidity, and maximum resource efficiency in an nhs district general hospital setting . Prompt investigations with the appropriate modern imaging and endoscopic techniques to elucidate the problem with lc performed at the earliest safe opportunity is a safe and definitive method of treating gallstone disease . Our experience with over 1000 patients has offered us the courage of conviction that long - awaited justice is finally here for gallstone sufferers.
Combined hepatocellular - cholangiocarcinoma (chcc - cc) is a rare form of primary liver cancer composed of cells with histopathologic features of both hepatocellular carcinoma (hcc) and cholangiocarcinoma (cc). It accounts for up to 14.3% of primary liver cancers, with incidence varying between multiple studies (1 - 4). Chcc - cc was first described in 1949 by allen and lisa (2), but the demographics and clinical features of these tumors remain ill - defined . In addition, the diagnostic features are not well established, which may have contributed to the variability in clinical outcomes (1,3 - 5). To our knowledge, surgery is the only treatment modality offering possibility for a cure (6), and there are at present no published reports describing non - surgical treatment options for chcc - cc . For patients with disseminated disease, systemic chemotherapy may be an option, but no known treatment to date has demonstrated any chance of significantly improving survival . We report a case of chcc - cc exhibiting a favorable response to systemic chemotherapy with doxorubicin and cisplatin . A 62-year old man was admitted to our hospital for recurrence of chcc - cc . He was diagnosed as stage ii hcc (t2n0m0) by tumor marker and imaging study in february 2004 . The patient did not have any known risk factors for hcc and thus underwent a right lobectomy at that time . Four years after the operation, a computed tomography (ct) scan of the chest showed multiple lung metastasis, along with a whole body bone scan (wbbs) revealing multiple bone metastasis (t - spine, rib, scapula). At the time of recurrence, laboratory tests including a liver function test were normal and his overall performance status was categorized as good . He received 1st line palliative therapy with sorafenib from july 2008 to september 2008, but a follow up imaging study showed disease progression with further lung metastasis (fig . 2) and a pivka - ii elevated to more than 2,000 mau / ml . He then received 2 cycles of palliative therapy with doxorubicin and cisplatin (ac regimen, doxorubicin 60 mg / m day 1, cisplatin 70 mg / m day 1 of a 3-week cycle) as 2nd line treatment . Surprisingly, a ct scan of the chest after the 2nd cycle of therapy showed markedly decreased size and number of his multiple hematogenous lung metastases (fig . After 6 more cycles of chemotherapy, the maintenance of response was confirmed by follow up imaging studies . Toxicities associated with the ac regimen (grade 2 neutropenia and grade 2 neuropathy) were manageable with supportive care . After a total of 8 cycles of therapy with the ac regimen, he presented with a grade 3 neuropathy, but no symptomatic cardiac function abnormalities . However, considering the accumulation dose of doxorubicin and grade 3 neuropathy associated with cisplatin, we changed his regimen to intravenous fluorouracil (5-fu) mono therapy (1,000 mg / m day 1 to 4 continuous infusion for a 3-week cycle). To date, he has completed his 15th cycle of 5-fu mono therapy with the disease status remaining stable during 18 months of follow - up . Almost all primary liver carcinomas are broadly classified as either hcc, derived from hepatocytes, or cc, arising from intrahepatic bile duct epithelium . Cases involving both hepatocellular and cholangiocellular components in the same tumor have been designated as chcc - cc . According to the world health organization tumor classification system, chcc - cc is defined as a tumor in which both hcc and cc components coexist in either the same tumor or the same liver (1,7). As hcc and cc differ in their clinical characteristics including etiology and epidemiology, chcc - cc is thought to be a result of dual differentiation of hepatic precursor cells toward hepatocytes and biliary epithelia based on clinicopathologic studies (8). Allen and lisa classified chcc - cc into the three categories: 1) separate tumors, each composed of only one type of cell; 2) contiguous tumors, each of a different cell type that may mingle as they grow; and 3) individual lesions that have both types of cells and are interpreted to have arisen from the same site (2). (1) also classified three types: 1) type i or " collision tumors "; 2) type ii or " transitional tumors "; 3) type iii or " fibrolamellar tumors . " In our case, there is no transition from the hepatocellular components to the cholangiocellular components and thus we may define this case as type i or collision tumors (fig . 1). The incidence of chcc - cc varies between studies, as 1.0 to 6.3% in asia (3,8 - 10) and 2.4 to 14.3% in western countries (1 - 4). Because of its low incidence, information on the prognosis of patients with chcc - cc is very limited and the reported clinical outcomes vary and may not accurately represent the actual prognosis of patients with chcc - cc (3,5,6). Surgery is the only treatment offering the possibility of a cure at the present time (6). However, commonly combined liver cirrhosis has the potential for serious complications, so strict selection of patients is required, taking into consideration any pre - existing cirrhosis, general physical condition, tumor extent as examples (6,11,12). Currently, aggressive treatments including liver transplantation have been attempted on chcc - cc patient (13). However, further data regarding long term outcomes with liver transplantation in the treatment of chcc - cc is required . Transarterial chemoembolization (tace) and percutaneous treatments such as percutaneous ethanol injection (pei) and radiofrequency ablation (rfa) are the most widely used management treatments for unresectable hcc and post resection recurrence . However, to our knowledge, chcc - ccs are more fibrotic and less vascular than hcc (14). As a result, they are less responsive to local treatments like tace or pei (15). For disseminated disease, systemic chemotherapy may be an option, but there is little data supporting its role in the treatment against chcc - cc . In our case, the patient was diagnosed with chcc - cc after a right lobectomy was performed for a liver mass . After a 4-year postoperative period, chcc - cc recurred presenting as multiple lung metastasis with high levels of pivka - ii . The patient first received sorafenib as a 1st line palliative therapy, but the disease was refractory to this chemotherapy . We then chose a combination of doxorubicin and cisplatin as a 2nd line chemotherapeutic regimen with favorable results as almost all multiple lung nodules disappeared . After completing 8 cycles of therapy with the ac regimen, he was then amended to receive 15 cycles of 5-fu mono therapy due to the toxicities associated with ac . This long period of disease control is a very encouraging result and requires further investigation in clinical trials.
The lien foundation's commissioned studies in 2010 and 2015 rate india poorly in the quality of dying . One of the reasons for this is the slow progress in palliative and end - of - life care . A notable exception is the state of kerala in southern india characterized by high development indicators . Unfortunately, the state is witnessing high morbidity levels, a graying population, and high prevalence of chronic diseases such as hypertension, diabetes, stroke, and cancer . It is in this context that the palliative care (pc) policy introduced to serve the needs of the community, especially those residing in rural resource - strapped areas, needs re - examination . Kerala announced the pain and pc policy in 2008 with an emphasis on community - based care and became the first state in india in this respect . The cornerstone of the policy is the home - care projects by local self - government institutions (lsgis - the primary level administrative system in india) in association with primary health - care system . The policy envisaged a public health approach and to provide holistic care to those with life - limiting illnesses through home - based care with their family as the core unit of care . It proposed extensive training of existing doctors, nurses, multipurpose health workers such as junior health inspectors (jhis) and junior public health nurses (jphns) and their supervisors, and elected members to lsgis to provide an integrated care with active community engagement . Pc was to be integrated into field level activities of existing multipurpose health workers and accredited social health activists (ashas). Essential medicines including oral morphine were to be available through pc units / primary health centers (phcs) and other government hospitals . There was an expressed need for monitoring for quality assurance (kerala pain and pc policy draft-2.2.b . 6). The policy speaks of involvement of all administrative health tiers in pc . Medical officers of phcs and community health centers (chcs) were to coordinate with community - based organizations (cbos) and lsgis in developing a common platform for palliative service delivery at the primary level (3.2.a and 3.2.b). An important feature of the program was developing a rational drug policy (5: 5.1, 5.2, 5.3) and providing greater treatment choices through integrative models using plural medical systems such as ayurveda and homeopathy (6.1). The policy was implemented as part of the national rural health mission (nrhm). The state health society of nrhm that has been named arogyakeralam, in collaboration with institute of palliative medicine, kozhikode, did pilot district level projects . Encouraged by the results of the project, nrhm (kerala) initiated state level pc project in 2008 . Subsequently, to the policy document, the government of kerala issued orders encouraging the involvement of lsgis in pc . It also gave orders for facilitating the collaboration of government hospitals including phcs in the process . Kerala institute of local administration, thrissur, an autonomous institution with the objective of training, research, and consultancy in decentralized governance and administration, published the guidelines for lsgis after a review of the policy implementation in 2011 . The guidelines were revised in 2013 and according to which pc became a mandatory project for all lsgis in kerala . Thus, the lsgis are primarily responsible for the overall implementation of the program, and not merely a funding agency or facilitator . These guidelines primarily focused on the following: preparation of annual plan including the budget allocation for each financial year, forming home care team and training, coordination between the services provided by nongovernmental organizations (ngos) and other cbos, and monitoring and evaluation of the pc program . The present study has two - fold objectives: first, to understand the structure of pc currently offered to rural population, and second, to assess how far the program organization and service structures conform to the policy and suggested guidelines for lsgis formulated by the state after the first review of the policy implementation in 2011 . Kerala announced the pain and pc policy in 2008 with an emphasis on community - based care and became the first state in india in this respect . The cornerstone of the policy is the home - care projects by local self - government institutions (lsgis - the primary level administrative system in india) in association with primary health - care system . The policy envisaged a public health approach and to provide holistic care to those with life - limiting illnesses through home - based care with their family as the core unit of care . It proposed extensive training of existing doctors, nurses, multipurpose health workers such as junior health inspectors (jhis) and junior public health nurses (jphns) and their supervisors, and elected members to lsgis to provide an integrated care with active community engagement . Pc was to be integrated into field level activities of existing multipurpose health workers and accredited social health activists (ashas). Essential medicines including oral morphine were to be available through pc units / primary health centers (phcs) and other government hospitals . There was an expressed need for monitoring for quality assurance (kerala pain and pc policy draft-2.2.b . 6). The policy speaks of involvement of all administrative health tiers in pc . Medical officers of phcs and community health centers (chcs) were to coordinate with community - based organizations (cbos) and lsgis in developing a common platform for palliative service delivery at the primary level (3.2.a and 3.2.b). An important feature of the program was developing a rational drug policy (5: 5.1, 5.2, 5.3) and providing greater treatment choices through integrative models using plural medical systems such as ayurveda and homeopathy (6.1). The policy was implemented as part of the national rural health mission (nrhm). The state health society of nrhm that has been named arogyakeralam, in collaboration with institute of palliative medicine, kozhikode, did pilot district level projects . Encouraged by the results of the project, nrhm (kerala) initiated state level pc project in 2008 . Subsequently, to the policy document, the government of kerala issued orders encouraging the involvement of lsgis in pc . It also gave orders for facilitating the collaboration of government hospitals including phcs in the process . Kerala institute of local administration, thrissur, an autonomous institution with the objective of training, research, and consultancy in decentralized governance and administration, published the guidelines for lsgis after a review of the policy implementation in 2011 . The guidelines were revised in 2013 and according to which pc became a mandatory project for all lsgis in kerala . Thus, the lsgis are primarily responsible for the overall implementation of the program, and not merely a funding agency or facilitator . These guidelines primarily focused on the following: preparation of annual plan including the budget allocation for each financial year, forming home care team and training, coordination between the services provided by nongovernmental organizations (ngos) and other cbos, and monitoring and evaluation of the pc program . The present study has two - fold objectives: first, to understand the structure of pc currently offered to rural population, and second, to assess how far the program organization and service structures conform to the policy and suggested guidelines for lsgis formulated by the state after the first review of the policy implementation in 2011 . Poovar and azhur are two rural areas provided home - based pc by the lsgis (called gram panchayats) and an ngo . The government pc service started in azhur and poovar in 2013 and 201011, respectively . At present, the ngo, as well as poovar chc, in collaboration with a medical officer at government ayurvedic health centre provide pc services in poovar . At azhur, the regional cancer center, thiruvananthapuram, also conducts home visits and provides follow - up palliative services to cancer patients undergoing treatment . The jurisdictional areas served by the lsgis have a population of 20,056 (poovar) and 28,331 (azhur) with a high concentration of scheduled castes 2586 in poovar and 5535 in azhur . The residents are mainly occupied in fishing and manual labor in poovar, and in coir industry and other manual labor in azhur . Around 47% of the residents in azhur although precise estimates for poovar are unavailable, in azhur, there are 2008 males and 2045 females above the age of sixty . The present study used a descriptive research design, focusing on primary level pc services offered in the above - mentioned two lsgis . The structure, organization, and pattern of home care delivery by both the government and ngo were assessed, and data were collected from health workers (doctors, nurses, and pc nurses) from both the ngo and public sector . Two officials from the national health mission in charge of pc program were also interviewed . Next, the compliance of the lsgis to the policy was understood by comparing the existing health delivery with the stated ideals as suggested in the policy draft and guidelines to lsgis . The information on the home - based pc provided by ngo was collected through direct interviews with the chief executive officer and director of the organization and field visits with their team as well . There were about 139 patients in poovar and 239 patients in azhur taking pc services provided by government health centers . Among them, 28 patients with catheter or bedsores received palliation in poovar and 71 in azhur . Most of them were suffering from serious multiple morbidities including noncommunicable diseases, mobility issues, and sensory and motor impairments . Table 1 provides a comparative description of the nature and organization of pc services provided by two lsgis and the ngo operating in poovar and azhur . Palliative care program run by three service providers: a comparative picture it is clear from a comparison of the three service providers that the palliative program in the region does not have a uniform structure in terms of workforce deployment, training of health workers, infrastructure availability, and composition of the palliative team . It is evident from the study that government palliative teams were merely nurse - led, while the ngo operating in that region had a more professional approach in terms of the composition of the team . Perhaps the reason for the dominance of nurse - led teams in government programs is the sheer workload of the doctors and poor motivation due to the lack of extra honorarium for palliative services . This is a major drawback of the policy itself since it relied on medical officers who are already in service rather than appointing new staffs . The nodal officer from nhm also emphasized the need of a dedicated palliative physician to ensure continuity of care thereby improving program efficiency each lsgi . The study found that a rational structure of work allocation has not been made for allied health workers, particularly for pc nurses who seemed to be overworked . It may be mentioned that initially, the policy had proposed augmenting field level and subcenter level services . Male and female multipurpose health workers were expected to provide comprehensive primary health - care services at the household level through the subcenters and at the phcs; they were to be given necessary orientation cum skill development training to play a major role along with the cbo volunteers and family members . However, in reality, the entire burden seems to have fallen on the palliative nurses who were also found to be overloaded with responsibilities for which they did not have the requisite skills, for instance, formulating budgets . The study showed poor frequency of home care visits in both places: 46 home visits in poovar and 89 home visits in azhur in a month . It is impossible to reach all patients at this rate in both lsgis . For those patients who needed urgent attention, the family members called the pc nurse and had to bear the cost of vehicle charges (back and forth). These informal visits were more frequent in poovar since the schedule of home visit was arranged according to the convenience of jhi or jphn accompanying the team rather than the needs of the families . Patients with urinary catheters, bedsores, or those needing special nursing care could be visited only once in a month indicating a serious care deficit . Others were visited once in 2 or 3 months according to the severity of their illnesses . One important reason could be a lack of workforce and dedicated transport for such a purpose . The revised guidelines for lsgis mention that transport facilities for home care need to be arranged from the budget earmarked for pc . The vehicle owned by the health center could be used if available, otherwise hired for the same purpose . In poovar, home care team was found to use the hospital vehicle during scheduled home visits; however, for unscheduled visits, the patient's family had to pay for the vehicle charge back and forth . Thus, except for the ngo, which had its own vehicle, transportation was a big problem in maintaining the quality of services . However, providing better services resulted in huge escalation of cost per home visit, as in the case of the ngo . This indicates the need for more serious cost analysis to ascertain whether home palliation in its present form, may be considered as the best model when resources are scarce . A great strength of the program medicines dispersed at free of cost, whether run by the ngo or the government . Making essential medicines for pc available to patients covered by pc services through pc units / phcs / other government hospitals are an important component of the policy . However, field observations show that not all patients in need get the medicines from their service providers; the logistics for disbursement also remains rather cumbersome except for the ngo . Families had to collect medicines from the facilities rather than having them delivered at home by the home care team, thus entailing extra time and effort . The palliative home care as provided to the two rural areas lacks an adequate social welfare provision for the poor . It also lacks alternative institutional structures for those who are living and dying alone and under desperate circumstances . Table 2 compares the two lsgis in terms of their adherence to the guidelines formulated in 2013 after a review of implementation of the pc policy . Adherence to selected guidelines for the local self - government institutions: a comparison of two local self - government institutions table 2 shows glaring gaps in adherence to the guidelines formulated for the lsgis . The policy had envisaged an integrated service provision with family as the core unit of care . Although the mandatory home care project is run by both the lsgis, the home care team does not follow the suggested composition, and it is rarely planned in the review meetings . Pc management committee meetings, though regular, do not enlist wide participation involving members enlisted in the guidelines . The revised guidelines for lsgis clearly mention involvement of lsgi members, medical officers, nurses, field workers, and ashas from the health centers under the jurisdiction of lsgi and representatives from ngos, volunteers from the community, representatives of kudumbasree community development societies (community - based organization), and teachers in charge of students police cadet, national service scheme, and national cadet corps (schools- and college - based youth development schemes). In contravention of the suggested guidelines, the meetings did not involve wide participation . The role of lsgis appears mainly restricted to funding its allocation and spending, rather than actively devising an organizational and care logistics geared toward improved service quality . The amount, meant primarily for dispensing medicines, meeting home care costs, and for providing remuneration to nurses, was reported as insufficient by pc nurses of both the lsgis . Another important finding from the field was that health inspectors, who had little training in higher - level management functions, often devised the annual outlays based on the report by pc nurses who were also poorly qualified . Moreover, nrhm instructions on annual planning and budgeting were overlooked resulting in a weak health delivery system and poor outcomes . While both lsgis provide some social support services but administrative delays prevent good outcomes with the result that a mere clinical approach to palliation has resulted without any rehabilitation activities . There is a little initiative to garner more funds from external sources and engage in continuing education activities and rehabilitation . Most of the educational activities are conducted by the national health mission rather than by the lsgis ., there were open conflicts between lsgi members and health workers regarding fund utilization, membership, and representation in meetings . Conflict between ngos and government providers was very overt to a level causing open arguments . In this sense, the initial aim of the government to utilize the experience of cbos / ngos and actively work with them seems to have failed . Clear instructions were provided to lsgis on how to monitoring the program and give suggestions for improvement . However, in practice, the meetings usually remain focused on fund expenditure rather than on quality of services . Moreover, a guideline for quality control was proposed to be set up at the state level with a monitoring / evaluating mechanism at the district level . Further, it envisaged developing a system to document and compile data on the pc - related activities and patient population at district and state level . However, field research shows serious flaws with irregular meetings and poor record of proceedings to guide action . Central to assessment of quality of services is the question of training of health workers . The nodal officer admitted that the basic qualification of a pc nurse was insufficient to equip her with good caring skills . Health coordinators such as the jhi / jphn were also overloaded with multiple duties in ongoing health programs . Continuing education was occasionally provided, but no volunteers were selected or trained in both lsgis . Contrary to the policy guidelines, family empowerment as an outcome measure for the success of palliation is also rather weak . Although the need for care became more pronounced as patients became completely bedridden and dependent, family involvement in caring was restricted, superficial, and confined to days when home visits were expected . Supportive care to families through pension / welfare inputs is also weak due to slow administrative system . Demand assessment of medicines and equipment within a proper logistics cycle to ensure just distribution . In addition, the pc structure, despite its clinical focus, lacked many specialist doctors such as psychiatrists, geriatricians, and physiotherapists . Only one lsgi could accommodate indigenous medicine; the other two providers failed to integrate this with a dominant biomedical approach despite ayurveda's confirmed therapeutic potential in treating bedsores, joint pains, etc . Despite having made much progress, the program in two lsgis is still short of a public health approach, and major guidelines of the palliative policy seem to have been given a miss . It also lacks the flavor of a community - owned program with a committed organizational structure, dedicated staff and delivery mechanism, seamless care through continuous monitoring, high frequency of visits, and adequate referrals . Finally, it appears too fragmented and restricted in its scope to meet the needs of the poor, the homeless, and those without caregivers unless it is located within an inclusive long - term care strategy involving a mix of health and social security measures . Evidently, this would require a huge structural reconfiguration of the delivery system a task whose magnitude and profundity necessitate greater state responsibility and political will in including palliation within a broader social organization of care . This study gives a clear lens toward the limitations of the pc program to provide holistic care in resource - poor settings . The present study is based on the data collected in march and april 2015 and findings are based on revised guidelines of 2013 . Since the study covers only two villages of kerala, we cannot generalize the findings to the whole state . This study gives a clear lens toward the limitations of the pc program to provide holistic care in resource - poor settings . The present study is based on the data collected in march and april 2015 and findings are based on revised guidelines of 2013 . Since the study covers only two villages of kerala, we cannot generalize the findings to the whole state.
The rare sugar 2-acetamido-2-deoxyfucose (fucnac) is a constituent monosaccharide of several bacterial capsular polysaccharides (cps). Both d- and l - enantiomers are found in nature, and they can be linked through either - or -glycosidic linkages (see chart 1). For example, the repeating trisaccharide of the type 5 cps of staphylococcus aureus features both a -d - fucnac residue and an -l - fucnac moiety, while the type 8 cps of s. aureus has d- and l - fucnac constituents that are both 1,2-cis - linked . The s. aureus strain m is built up from trisaccharide repeats, which are composed of two galactosaminuronic acid (galnaca) residues and an -d - fucnac monosaccharide . Various o - antigens of escherichia coli contain fucnac residues as exemplified by the structures in chart 1 . Well - defined fragments of bacterial polysaccharides have been used extensively in the development of (semi)-synthetic vaccines, as part of diagnostic tools, to unravel binding and interactions with carbohydrate binding receptors and as probes for bacterial cps - biomachinery enzymes . Organic synthesis can deliver these fragments as well - defined single molecules, devoid of any bacterial impurity and functionalized at predetermined sites with, for example, a conjugation handle for further manipulation . The synthesis of complex oligosaccharides, such as those depicted in chart 1, however, can be an arduous task, requiring a significant time and labor investment . This is largely due to the complexity associated with the stereoselective construction of glycosidic linkages . Few studies have been directed at the incorporation of fucosamine residues in oligosaccharides, and there is no general method to install the challenging -fucosamine linkage . There have been reports on the assembly of the trisaccharide repeating units of s. aureus type 5 and 8, but the syntheses reported were developed to target a single trisaccharide providing little insight into the reactivity and selectivity of fucosamine building blocks in a broader context, thus making it difficult to transpose the outcome of these studies to other relevant oligosaccharide targets or synthetic approaches . To facilitate the effective assembly of fucosamine - containing bacterial oligosaccharides, we here report an in - depth study of the reactivity and selectivity of a variety of fucosazide building blocks with the goal to understand and control the stereoselectivity of these donors . We investigated reactive intermediates formed upon activation of fucosazide donor synthons and we have formulated a mechanistic rationale to account for the stereoselectivity observed in fucosaminylation reactions . We applied the generated insight in the construction of several relevant 1,2-cis - fucosamine linkages as well as a modular synthesis of the s. aureus type 5 trisaccharide . To achieve the stereoselective introduction of 1,2-cis glycosamine linkages, the c2 amino functionality of a donor glycoside is generally masked as the nonparticipating azide . To generate a series of fucosazide (fucn3) donors, we decided to target phenylseleno fucosazides because selenoglycosides are generally very potent glycosyl donors and phenylseleno fucosazides can be effectively generated from readily available fucal precursors . To map the reactivity and selectivity of fucosazide donors, we investigated a set of donors having different protecting groups . Whereas the glycosylating properties of fucosazide donors have received relatively little attention, there is a large body of data available on the stereoselective introduction of fucosyl linkages . It appears that the -fucosyl linkage can be installed with relative ease . For the stereoselective construction of this linkage, fucosyl building blocks, bearing acyl protecting groups at c3 and/or c4, are commonly used, and it is often assumed that these groups are capable of remote participation . Of note, tri - o - benzyl - protected fucosyl donors have also been employed, and these have also been reported to provide the desired 1,2-cis fucosyl linkages with good selectivity . No mechanistic rationale has been forwarded to account for this striking selectivity . For our study, we generated six l - fucn3 donors (16, chart 2) from l - fucal, featuring benzyl, benzoyl, or tert - butyldimethylsilyl groups . We probed these donor fucosides in a series of glycosylation reactions using a preactivation protocol in which the donor glycosides were activated with the diphenyl sulfoxide (ph2so)triflic anhydride (tf2o) reagent couple . This reagent combination provides a very powerful electrophile for activation of thio- and selenoglycosides, and it allows for the detection of reactive intermediates by low - temperature nmr spectroscopy to provide insight into the glycosylation mechanism of the preactivated donor glycosides . Homogeneous azidoselenylation of easily accessible l - fucal installed the azide and the anomeric phenylseleno moiety in one step in the desired -fucosyl configuration, accompanied by minor amounts of inseparable isomers . Deacetylation of the crude product mixture allowed separation, yielding diol 9 in 58% yield over two steps . Donors 1, 2, and 5 could be accessed in one step each from diol 9 by benzylation (bnbr, nah in dmf, 85% yield), benzoylation (bzcl and a catalytic amount of dmap in a mixture of ch2cl2 and pyridine, 90% yield), and silylation (tbsotf and a catalytic amount of dmap in pyridine at elevated temperature, 85% yield), respectively . In the last case, standard silylation conditions employing tbscl as the silylating agent and either imidazole in dmf, or dmap and pyridine, failed to give the disilylated product . Donor 3, bearing c3-o - benzyl and c4-o - benzoyl protection, was procured by bu2sno - mediated, regioselective benzylation on the c3-o position followed by benzoylation of the remaining c4-o position using similar conditions as described for 2 to give 3 in 47% yield over two steps . A more elaborate protection sequence was required to access c4-o - benzyl donors 4 and 6, owing to the less reactive nature of the c4 position . Thus, regioselective, bu2sno - mediated p - methoxybenzylation of the c3-o position, benzylation of the remaining free c4 alcohol, followed by acid - mediated cleavage of the c3-o - pmb ether, using hcl in a mixture of ch2cl2 and hexafluoroisopropanol (hfip), gave key intermediate 10 in 47% yield over three steps . The use of oxidative conditions to remove the pmb group was avoided, owing to the potentially oxidation - sensitive phenylseleno moiety . With 10 in hand, donors 4 and 6 were obtained after benzoylation and silylation using conditions described above, in 96% and 92% yield, respectively . We started our investigation with the detection of the reactive intermediates, generated upon activation of two different donor synthons: di - o - benzyl- and di - o - benzoyl fucosazides 1 and 2, respectively . Thus, a mixture of 1 and ph2so (1.3 equiv) in cd2cl2 was treated with tf2o (1.3 equiv) at 80 c (figure 1a). After a h nmr spectrum (figure 1b) was recorded, two new anomeric signals appeared (6.06 and 6.10 ppm), which were assigned as -triflate 11 (j = 3.2 hz) and -oxosulfonium triflate 13 (j = 3.2 hz), respectively, based on their chemical shift . While the formation of the anomeric triflate was anticipated, oxosulfonium triflate formation under these conditions is quite surprising . The oxosulfonium species likely arises from reaction of the anomeric triflate with ph2so present in the reaction mixture . Because the amount of oxosulfonium fucosazide 13 is higher than what could be expected based on the excess of ph2so (0.3 equiv), it appears that the selenodonor 1 does not require a full equivalent of ph2so for complete activation . To account for complete activation of donor 1, we assume that the electrophile, generated upon reaction of the anomeric phenylselenol group with the diphenylsulfonium bis - triflate activator (phse sph2otf), is reactive enough to activate the nucleophilic phenylselenium moiety . Addition of more ph2so to the reaction mixture resulted in an increase of the signal at 6.10 ppm (figure 1c), reinforcing the presence of oxosulfonium triflate 11 . In order to assess the stability of the two reactive intermediates, the activation of dibenzoyl donor 2 proceeded in a similar manner to provide -triflate 12 and oxosulfonium triflate 14 (figure 1d). These reactive intermediates proved to be more stable than their dibenzyl counterparts, with decomposition setting in around 0 c . Partial h nmr spectra (400 mhz, 193 k) of reactive species from 1 using 1.3 and 2.0 equiv of ph2so (b and c, respectively) and 2 (1.3 equiv of ph2so, d). Next, we investigated the behavior of donor fucosazides 16 in a series of glycosylation reactions . To this end, we applied a unified glycosylation protocol to all condensation reactions, involving preactivation of the donor glycoside at low temperature (in the presence of the non - nucleophilic base 2,4,6-tri - tert - butylpyrimidine (ttbp)), then acceptor addition, subsequently warming the reaction mixture slowly to 40 c, and finally quenching the reaction at this temperature . We used the set of model acceptors depicted in chart 2 to map the selectivity of the fucosazide donors 16 . To study the dependency of acceptor nucleophilicity on the outcome of the glycosylation reactions a set of partially fluorinated ethanols in addition, three secondary alcohol acceptors were used: cyclohexanol, mannoside 7, having an axial c2-oh, and mannoside 8, with an equatorial c3-oh . Glycosylation of donors 16 with the series of ethanols (table 1, rows a d) revealed a clear dependency of the stereochemical outcome of the glycosylations on the nucleophilicity of the acceptor alcohols . All donors showed the same trend: with decreasing nucleophilicity (increasing amount of fluorine atoms in the acceptors) -selectivity increased . While the more reactive donors (1, 5, and 6) reacted in a nonselective manner with the most nucleophilic acceptor, ethanol (row a), the less reactive, benzoyl - bearing fucosazide donors reacted with moderate -selectivity . With the reactive secondary alcohol, cyclohexanol (row e), a similar picture emerged: less reactive donors provided more -product than the reactive fucosaminylating agents . The condensations of the secondary carbohydrate acceptors 7 and 8 (rows f and g) all proceeded with good to excellent -selectivity, again with the more reactive donors providing better -selectivity than their less reactive counterparts . Across the board, donors 1, 5, and 6 outperformed the benzoylated donors 24 in terms of yield of the glycosylation reactions . The observed -selectivity in the condensation reactions of the benzoylated fucosazide donors with ethanol and cyclohexanol strongly argue against a remote participation scenario for these donors . The selectivity in these reactions is better explained with the -anomeric triflates or oxosulfonium triflates 16 as glycosylating species (scheme 2). The presence of benzoyl groups on the fucosazide donors stabilizes these intermediates, as judged from the higher decomposition temperature found in the variable temperature nmr measurements . Strong nucleophiles can substitute the covalent -triflates / oxosulfonium triflates with inversion of configuration to provide the -linked products (18). Weaker nucleophiles, such as di- and trifluoroethanol and the carbohydrate alcohols (also featuring two or three electron withdrawing atoms at a -position with respect to the alcohol function), are unable to directly displace a covalently bound leaving group and require a more electrophilic glycosylating agent to react . The covalent triflate / oxosufonium species can serve as a reservoir for a more reactive oxocarbenium ion 17 with a loosely associated triflate counterion . It is now well established that the geometry of an oxocarbenium ion can be decisive for the stereochemical course of a glycosylation reaction . The fucosazide oxocarbenium ions that can form from the covalent triflates / oxosulfonium triflates can adopt a h4-like conformation (as in 17) in which the substituents at c2 and c4 are positioned properly to allow for stabilization of the electron depleted anomeric center, while the groups at c3 and c5 are positioned in sterically favorable pseudo - equatorial positions . This oxocarbenium ion is preferentially attacked on the diastereotopic face that leads to the product via an energetically favorable chairlike transition state, leading to the 1,2-cis product 18. this reaction trajectory is sterically relatively unhindered, and it can account for the selective formation of the 1,2-cis - products as observed here . The fact that more electron - rich donors provide higher -selectivity strongly supports this rationale . It also provides an adequate explanation for glycosylations of highly reactive per - benzylated fucosyl donors previously reported in literature . The reactivity study described above has revealed a clear dependence of the stereochemical course of the glycosylations on both the reactivity of the donor glycoside and the reactivity of the acceptor alcohol . The best 1,2-cis selectivity is obtained with reactive fucosazide donors, bearing benzyl or silyl ether protecting groups and relatively weak nucleophiles, such as secondary carbohydrate alcohols . Building on this knowledge, we set out to investigate the construction of a series of relevant glycosidic linkages, present in capsular polysaccharides of s. aureus . The repeating unit of the s. aureus strain m cps (figure 1) contains an -d - fucnac unit linked to two -linked n - acetylgalactosaminuronic acid (galnaca) residues . We anticipated that the glycosylation between a galacturonic acid c4-oh acceptor, generally considered to be a weak nucleophile, and a reactive fucosamine donor, would likely lead to a highly -selective glycosylation . Indeed, when d - fucosazide donor d-6 was coupled to acceptor 19, the -linked disaccharide 20 was obtained as the sole product in 65% yield (scheme 3). The repeating unit of s. aureus type 8 cps contains two -linked n - acetyl fucosamine units (figure 1). To investigate the construction of the -linkage between the two fucosamine residues, we first generated an -d - fucosazide acceptor bearing a spacer at its reducing end . Because the reactivity study described above indicated that nucleophilic primary alcohols react in a non- or -selective manner with fucosazide donors we turned our attention to the use of tetrabutylammonium iodide (bu4ni) as a stereochemistry - directing additive in the condensation of aminopentanol 21(57) and fucosazide donor d-6 . Bennett and co - workers have previously reported a ph2so / tf2o - based activation protocol (in the presence of the electrophilic scavenger n - methylmaleimide (nmm)), utilizing an excess of bu4ni to generate an intermediate anomeric iodide as a reactive species . As first conceived by lemieux and co - workers, an equilibrium is established between the - and -iodides, with the latter species being less stable but much more reactive . Nucleophiles can displace the -iodide in an sn2-like fashion, leading to the selective formation of the -product . When d-6 was glycosylated with 21 using a slight modification of bennett s protocol, product 22 was obtained in 85% yield with good -selectivity . Removal of the tbs group facilitated separation of the anomers, giving pure 23 in 65% yield . In the next glycosylation event, the reactive l - fucosazide donor 6 was paired with d - fucosazide acceptor 23 in a ph2so / tf2o - mediated preactivation glycosylation event to provide disaccharide 24 in 73% yield and high stereoselectivity . Removal of the tbs group under the agency of bu4nf allowed chromatographic separation of the two anomers, yielding the -linked disaccharide 25 in 71% yield . As a final endeavor, we set out to synthesize the repeating unit of the s. aureus type 5 cps repeating unit (scheme 4). Target trisaccharide 26 consists of a rare n - acetylmannosaminuronic acid (mannaca), a central -linked l - fucnac residue, and a terminal -d - fucnac connected to an aminopentanol spacer for future conjugation purposes . The synthesis of this repeating unit has previously been reported by the groups of adamo, boons, and very recently, demchenko . Adamo and co - workers relied on a strategy starting from the nonreducing end and using glucosyl and rhamnosyl synthons to form the mannaca and fucnac units, respectively . The final glycosylation between the l - fucn3-containing disaccharide and a d - fucnac unit proceeded with modest stereoselectivity . Demchenko and co - workers used a similar approach with glucosyl and fucosyl synthons . Boons and co - workers built the trisaccharide repeating unit, starting from the reducing end, using fucn3 building blocks . The installation of the glycosidic linkage between the two fucn3 units proved problematic, proceeding in low yield or with relatively poor stereoselectivity . Our strategy is presented in scheme 4 . In order to differentiate the c3-o position from the other alcohols in the trisaccharide, this position was protected as an ester in fully protected intermediate 27, while the others were masked as benzyl ethers . Based on the reactivity / selectivity study described above, we reasoned that the -fucosamine linkage could be constructed using a disarmed fucosazide donor, such as d-4 . For the pivotal -glycosidic linkage between the l - fucn3 and d - fucn3 moieties, the use of reactive 3,4-di - o - tbs donor 5 was anticipated because of the highly -selective glycosylations of this donor (table 1). We thus aimed to use a fucn3 donor for the installation of both the 1,2-cis and 1,2-trans fucosamine linkages . This will shorten the sequence of protecting group manipulations at the trisaccharide stage . For the introduction of the mannosaminuronic unit, we selected 2-azidomannuronate donor 28 because of the excellent -selectivity observed with this class of donors as we have disclosed previously . The use of a pre - oxidized mannosaminuronic acid synthon circumvents the necessity of a late - stage oxidation step in the assembly sequence . To effect the -selective glycosylation between donor d-4 and spacer 21 in the absence of a participating group on the c2-position of the donor, several modifications of our standard glycosylation conditions were tested (data not shown). It was found that the use of ether as a cosolvent effectively increased the -selectivity of the glycosylation . This is somewhat surprising given the fact that ether is commonly used to promote the formation of -glycosidic linkages . It can, however, be rationalized with the mechanistic scheme depicted in scheme 1 . The low polarity of ether (in comparison to dichloromethane) stabilizes the covalent anomeric triflate / oxosulfonium triflate because it disfavors charge separation as in oxocarbenium ion pairs . If the incoming alcohol acceptor is nucleophilic enough, it can displace the covalent reactive species in an sn2-manner leading to the stereoselective formation of the -fucn3 bond . The use of an 1:1 mixture of ch2cl2 and et2o in the glycosylation between aminopentanol 19 and disarmed fucn3 donor d-4 led to a spacer containing d - fucosamine building block 29 in 80% yield and 1:7 /-selectivity (scheme 5). Removal of the benzoyl group using zempln conditions afforded the d - fucn3 acceptor 30 in 95% yield . Next, the pivotal glycosylation between l - fucn3 donor 5 and d - fucn3 acceptor 30 was performed . Using the standard preactivation glycosylation protocol removal of both tbs ethers was followed by regioselective benzoylation of the c3-o position, using taylor s diphenylborinate catalyst 32, to give disaccharide acceptor 33 in 67% yield over two steps . The final glycosylation between mannosaminuronic acid donor 28 and dimer 33 proved challenging . Mannuronic acids are relatively reactive, and it was difficult to pair the reactive mann3a donor with the weakly nucleophilic fucn3 alcohol . It was found that the use of an excess donor and almost an equimolar amount of lewis acid promotor was most effective, allowing for the generation of trisaccharide 27 in 75% yield with complete stereoselectivity . With trisaccharide 27 in hand, first, the methyl mannuronate and the benzoyl ester on the central l - fucn3 unit were removed to protect the mannuronic acid moiety for potential lactamization upon exposure of the c2-amino group . It was found, however, that the subsequent o- and n - acetylation reaction resulted in a complex mixture of products, with lactam 36 as the major product . We therefore moved to an alternative reaction sequence, in which we first acetylated the free c3oh . Next both azides were transformed into the corresponding acetamido functionalities using thioacetic acid (acsh). This step likely proceeds via a one - step process and circumvents formation of the free amine . The synthesis of the s. aureus type 5 trisaccharide 26 was finalized by hydrogenation of 35 using pearlman s catalyst (pd(oh)2 on carbon) to remove all benzyl groups and the benzyloxycarbonyl carbamate . In conclusion, we have mapped the reactivity and selectivity of a panel of phenylseleno fucosazide donors . Low - temperature nmr studies on activated donors revealed the formation of the covalent -glycosyl triflates and oxosulfonium triflates, the stability of which depended on the protecting group pattern of the donor glycosides . Using a series of glycosylations involving a set of partially fluorinated ethanols, we were able to pinpoint how the stereoselectivity of the glycosylations of the different donors depends on the nucleophilicity of the acceptor alcohols . A mechanistic rationale was established that accounts for the stereoselectivity in glycosylations featuring fucosazide donors . Disarmed donors bearing acyl - protecting groups can selectively provide -linked products when paired with reactive nucleophiles in an sn2-like glycosylation reaction . Armed donors, having benzyl or silyl ether groups, on the other hand, are well suited for the installation of the challenging 1,2-cis fucosamine linkages, and this is rationalized with a h4-oxocarbenium ion like reactive intermediate that is selectively attacked on its -face . It is likely that reactions using reactive fucosyl donors proceed via similar pathways, providing a rationale for the high stereoselectivity obtained with these donors . It is anticipated that the use of the family of partially fluorinated ethanols to map reactivity selectivity relationships for other donor types will provide valuable insight into glycosylation mechanism of these donors and significantly increase our insight how effective stereoselective glycosylation reactions can be achieved . The insight into the reactivity selectivity of fucosazide donors generated here has paved the way for the construction of a variety of relevant glycosidic linkages and the modular assembly of the s. aureus type 5 repeating unit . All reactions were carried out in oven - dried glassware (85 c). Prior to reactions, traces of water and solvent reactions sensitive to air or moisture were carried out under an atmosphere of argon (balloon). Solvents for reactions were of reagent grade and stored over 4 molecular sieves (3 for ch2cl2, meoh, and mecn), except pyridine and dmf . Tf2o used in glycosylations was dried over p2o5 (3 h), followed by distillation, and stored in a schlenk flask at 20 c . Reaction progress was monitored using aluminum - supported silica gel tlc plates (with fluorescent indicator); visualization was carried out by irradiation with uv light (: 254 nm), followed by spraying with 20% h2so4 in etoh (w / v) or hanessian s stain ((nh4)6mo7o244h2o, 25 g / l; (nh4)4ce(so4)42h2o, 10 g / l; in 10% aq h2so4). Nmr spectra were recorded on 400/100 mhz (for h and c, respectively) or 500/125 mhz spectrometers . Chemical shifts () are reported in ppm relative to me4si (: 0.00 ppm) or residual solvent signals . Nmr spectra were recorded at ambient temperature, and samples were prepared in cdcl3 unless noted otherwise . Coupling constants of anomeric carbon atoms (jh1,c1) were determined using hmbc - gated experiments . Infrared spectra were recorded with an ftir instrument with wavenumbers () reported in cm . Lc ms analyses were performed on an hplc system equipped with a c-18 column (50 4.6 mm) connected to an ion - trap mass spectrometer with esi . Hrms spectra were recorded on a ltq - orbitrap instrument equipped with esi (source voltage 3.5 kv, sheath gas flow 10, capillary temperature 275 c) with resolution r 60.000 at m / z 400 (mass range: 1504000) and dioctyl phthalate (m / z 391.28428) as a lock mass . A solution of 3,4-di - o - acetyl - l - fucal (12.5 g, 58.4 mmol, 1.0 equiv) and (phse)2 (18.2 g, 58.4 mmol, 1.0 equiv) in ch2cl2 (300 ml, 0.2 m) was degassed by sonication (30 min) before being cooled to 30 c . Phi(oac)2 (18.8 g, 58.4 mmol, 1.0 equiv) and tmsn3 (15 ml, 116.8 mmol, 2.0 equiv) were added . The mixture was stirred for 1 h at 30 c and subsequently at 20 c overnight . To the mixture was added cyclohexene (15 ml), and the mixture was allowed to warm to room temperature . The bright orange solution was concentrated in vacuo, and the brown residual oil was subjected to column chromatography (pe / etoac, 1:0 9:1 the latter was concentrated and suspended in meoh (190 ml, 0.3 m), after which naome (0.31 g, 5.8 mmol, 0.1 equiv) was added . The mixture was stirred overnight, after which tlc analysis (pe / etoac, 1:1 v / v) showed complete conversion of the starting material . The reaction mixture was neutralized by addition of ion - exchange resin (amberlite ir-120, h form). The solid thus obtained was crystallized from toluene to obtain the title compound as an amorphous solid (11.1 g, 33.8 mmol, 58%). H nmr (400 mhz, acetone - d6): 7.627.57 (m, 2h, charom); 7.327.28 (m, 3h, charom); 5.96 (d, 1h, j = 5.2 hz, h-1); 4.29 (q, 1h, j = 6.4 hz, h-5); 4.40 (dd, 1h, j = 5.2 hz, 10.4 hz, h-2); 3.823.79 (m, 2h, h-3, h-4); 1.17 (d, 3h, j = 6.4 hz, h-6). C - apt nmr (100 mhz, acetone - d6); 135.4 (charom); 130.1 (cq, arom); 129.8, 128.3 (charom); 86.7 (c-1); 72.4, 72.2 (c-3, c-4); 70.2 (c-5); 62.6 (c-2); 16.5 (c-6). Ir (neat): 3279, 2100, 1578, 1252, 1094, 1059 . To a stirred solution of 9 (0.66 g, 2.0 mmol, 1.0 equiv) in dmf (8 ml, 0.25 m) were added bnbr (0.71 ml, 6.0 mmol, 3.0 equiv) and bu4ni (0.15 g, 0.4 mmol, 0.2 equiv). The mixture was cooled in an ice bath, and nah (60% w / w in oil, 0.32 g, 8.0 mmol, 4.0 equiv) was added . The mixture was stirred until tlc analysis (pe / etoac, 9:1 v / v) indicated complete consumption of the starting material (3 h). Excess nah was quenched by slow addition of cold water until gas evolution ceased . The mixture was diluted with water and et2o, and the aqueous phase was washed twice with et2o . The combined ethereal phases were washed with brine (1), dried over mgso4, filtered, and concentrated in vacuo . The residue was purified by column chromatography (pe / et2o 1:0 9:1) to furnish the title compound as an oil which solidified on standing, in 85% yield (0.87 g, 1.7 mmol). H nmr (400 mhz): 7.577.47 (m, 2h, charom); 7.457.22 (m, 13h, charom); 5.93 (d, 1h, j = 5.2 hz, h-1); 4.92 (d, 1h, j = 11.2 hz, phchh); 4.804.73 (m, 2h, phch2); 4.61 (d, 1h, j = 11.6 hz, phchh); 4.35 (dd, 1h, j = 5.2 hz, 9.8 hz, h-2); 4.22 (q, 1h, j = 6.4 hz, h-5); 3.753.72 (m, 2h, h-3, h-4); 1.13 (q, 3h, j c - apt nmr (100 mhz): 138.1, 137.4 (cq, arom); 134.3, 129.0, 128.6, 128.3, 128.1, 128.0, 127.8, 127.7, 127.6 (charom); 85.5 (c-1); 80.6, 75.7 (c-3, c-4); 75.0, 72.5 (phch2); 69.4 (c-5); 60.9 (c-2); 16.5 (c-6). Ir (neat): 2882, 2112, 1474, 1298, 1101, 1063, 1047 . N2 + h] calcd for c26h28no3se 482.1229, found 482.1229 . To a stirred solution of 9 (0.66 g, 2.0 mmol, 1.0 equiv) in ch2cl2/pyridine (3:1 v / v, 8 ml, 0.2 m) was slowly added bzcl (0.7 ml, 6.0 mmol, 3.0 equiv), followed by dmap (0.05 g, 0.4 mmol, 0.2 equiv). The mixture was stirred until tlc analysis (pe / etoac, 4:1 v / v) indicated complete conversion of the starting material (3 h). The reaction was quenched with meoh, and the mixture was diluted with ch2cl2, washed (1 m aq hcl, 2; satd aq nahco3, 1; brine, 1), dried over mgso4, filtered, and concentrated in vacuo . The residue was subjected to column chromatography (pe / etoac, 1:0 4:1) to furnish the title compound in 90% yield (0.96 g, 1.79 mmol). H nmr (400 mhz): 7.258.15 (m, 15h, charom), 6.12 (d, 1h, j = 5.2 hz, h-1), 5.76 (d, 1h, j = 2.8 hz, h-4), 5.51 (dd, 1h, j = 3.2 hz, 10.8 hz, h-3), 4.53 (dd, 1h, j = 5.6, 10.8 hz, h-2), 4.73 (q, 1h, j = 6.4 hz, h-5), 1.19 (d, 3h, j = 6.4 hz, h-6); c - apt nmr (100 mhz): 165.7, 165.4 (cobz), 134.9127.2 (charom), 84.6 (c-1), 72.4 (c-3), 70.8 (c-4), 68.0 (c-5), 59.6 (c-2), 16.0 (c-6). Ir (thin film): 3061, 2984, 2108, 1724, 1450, 1273, 1257, 1109, 1080, 1067, 1024 . Compound 9 (0.66 g, 2.0 mmol, 1.0 equiv) was suspended in toluene (7 ml, 0.3 m). Bu2sno (0.50 g, 2.0 mmol, 1.0 equiv) was added, and the mixture was heated to 140 c for 3 h, during which time a clear reaction mixture was obtained . The mixture was concentrated in vacuo and coevaporated once with dry toluene . The mixture was dissolved in dmf (9 ml, 0.2 m), bnbr (0.26 ml, 2.2 mmol, 1.1 equiv), and csf (0.33 g, 2.2 mmol, 1.1 equiv), and the mixture was stirred overnight, after which tlc analysis indicated conversion of the starting material (pe / etoac, 7:3 v / v). The reaction was diluted with h2o and extracted (et2o, 3), and the combined ethereal phases were washed (brine, 1), dried over mgso4, filtered, and concentrated in vacuo . The residue was passed over a small column (pe / etoac, 1:0 4:1 v / v) to obtain the 3-o - benzylated intermediate (0.42 mmol, 1 mmol, 50%). H nmr (400 mhz): 7.597.56 (m, 2h, charom); 7.427.24 (m, 8h, charom); 5.89 (d, 1h, j = 5.2 hz, h-1); 4.76 (d, 1h, j = 11.2 hz, phchh); 4.69 (d, 1h, j = 11.2 hz, phchh); 4.30 (q, 1h, j = 6.8 hz, h-5); 4.17 (dd, 1h, j = 5.2 hz, 10.4 hz, h-2); 3.88 (s, 1h, h-4); 3.70 (dd, 1h, j = 3.2 hz, 10.4 hz, h-3); 2.36 (s, 1h, 3-oh); 1.26 (d, 3h, j = 6.8 hz, h-6). C - apt nmr (100 mhz, cdcl3): 137.1 (cq, arom), 134.5, 129.2, 128.9, 128.5 (charom), 128.2 (cq, arom), 127.9 (charom), 85.3 (c-1), 79.3 (c-3), 72.3 (ch2 bn), 68.7, 68.6 (c-4, c-5); 60.3 (c-2); 16.2 (c-6). The intermediate was dissolved in ch2cl2/pyridine (4:1 v / v, 5 ml, 0.2 m), and bzcl (0.14 ml, 1.2 mmol, 1.2 equiv) and dmap (12 mg, 0.1 mmol, 0.1 equiv) were added at 0 c . After tlc analysis (pe / etoac, 9:1 v / v) indicated complete conversion of the starting material (1 h), the mixture was quenched by the addition of water . The mixture was diluted with ch2cl2 washed (1 m aq hcl, 2; satd aq nahco3 1; h2o 1; brine 1), dried over mgso4, filtrated, and concentrated under reduced pressure . Purification by column chromatography (pe / etoac, 17:3 v / v) afforded the title compound (0.49 g; 0.93 mmol; 47% over two steps). H nmr (400 mhz): 8.098.04 (m, 4h, charom), 7.647.20 (m, 11h, charom), 6.00 (d, 1h, j = 5.2 hz, h-1), 5.71 (d, 1h, j = 2.8 hz, h-4), 4.85 (d, 1h, j = 10.8 hz, phchh), 4.57 (d, 1h, j = 10.8 hz, phchh), 4.52 (q, 1h, j = 6.4 hz, h-5), 4.26 (dd, 1h, j = 5.2 hz, j = 10.4 hz, h-2), 3.90 (dd, 1h, j = 3.2 hz, j = 10.0 hz, h-3), 1.16 (d, 3h, j = 6.4 hz, h-6). C - apt nmr (100 mhz): 166.0 (cobz), 136.9 (cq, arom), 134.7127.7 (charom), 85.1 (c-1), 77.5 (c-3), 71.6 (phch2), 69.4 (c-4), 68.1 (c-5), 60.5 (c-2), 16.2 (c-6). Ir (thin film): 3061, 2984, 2897, 2108, 1719, 1452, 1263, 1109, 1078, 1062, 1024 . N2 + h] calcd for c26h26no4se 496.1022, found 496.1023 . In a three - necked flask equipped with a dean stark trap, a suspension of phenyl 2-azido-2-deoxy-1-seleno--l - fucopyranoside (4.27 g, 13 mmol, 1.0 equiv) and bu2sno (3.40 g, 13.7 mmol, 1.05 equiv) in toluene (65 ml, 0.2 m) was heated to 140 c for 1 h. the resultant clear, brown solution was cooled to 60 c, and bu4nbr (4.42 g, 13.7 mmol, 1.05 equiv), csf (2.08 g, 13.7 mmol, 1.05 equiv), and pmbcl (1.9 ml, 13.7 mmol, 1.05 equiv) were added . The mixture was heated to 120 c for 2 h, after which tlc analysis (pe / etoac, 3:2 v / v) indicated complete conversion of the starting diol . The mixture was cooled to room temperature, kf (10% in h2o, w / v) was added, and the resulting misture was stirred vigorously for 15 min . The aqueous phase was extracted (etoac, 2), and the combined organic fractions were washed (brine 1), dried over mgso4, filtered, and concentrated in vacuo . Purification by column chromatography (pe / etoac, 1:0 4:1) furnished the 3-o - pmb - protected intermediate as a yellow oil in 81% yield (4.71 g, 10.5 mmol). H nmr (400 mhz): 7.587.56 (m, 2h, charom); 7.347.24 (m, 5h, charom); 6.936.87 (m, 2h, charom); 5.87 (d, 1h, j = 5.2 hz, h-1); 4.68 (d, 1h, j = 10.8 hz, phchh); 4.62 (d, 1h, j = 10.8 hz, phchh); 4.28 (q, 1h, j = 6.4 hz, h-5); 4.14 (dd, 1h, j = 5.2 hz, 10.2 hz, h-2); 3.83 (d, 1h, j = 2.4 hz, h-4); 3.81 (s, 3h, och3); 3.68 (dd, 1h, j = 3.2 hz, 10.4 hz, h-3); 1.25 (d, 3h, j = 6.4 hz, h-6). C - apt nmr (100 mhz): 159.6 (cq, arom); 134.4, 134.3,129.7, 129.0 (charom); 129.0, 128.6 (cq, arom); 127.7, 114.0 (charom); 85.2 (c-1); 78.8 (c-3); 71.7 (phch2); 68.5, 68.4 (c-4, c-5); 60.0 (c-2); 55.2 (och3); 16.0 (c-6). Ir (thin film): 3441, 2897, 2106, 1612, 1512, 1246, 1088, 1063, 1031 . Hrms: [m + h] calcd for c20h24n3o4se 450.0927, found 450.0923 . A solution of the intermediate building block (1.56 g, 3.48 mmol, 1.0 equiv) and bnbr (0.83 ml, 6.96 mmol, 2.0 equiv) in dmf (12 ml, 0.3 m) was cooled to 0 c . Nah (60% dispersion in oil, 0.21 g, 5.22 mmol, 1.5 equiv) was added, and the mixture was allowed to reach room temperature . After 3 h, tlc analysis (pe / etoac, 9:1 v / v) indicated complete conversion of the starting material, and the reaction was quenched by slow addition of water . After gas evolution had ceased, the mixture was partitioned between water and et2o . The aqueous phase was extracted (et2o, 2), and the combined ethereal phases were washed (brine, 1), dried over mgso4, filtered, and concentrated in vacuo . Purification by column chromatography (pe / et2o, 1:0 9:1) delivered the fully protected intermediate as a colorless oil (1.68 g, 3.12 mmol, 90%). H nmr (400 mhz): 7.577.54 (m, 2h, charom); 7.367.23 (m, 10h, charom); 6.92 (d, 2h, j = 8.8 hz, charom); 5.91 (d, 1h, j = 5.2 hz, h-1); 4.94 (d, 1h, j = 11.6 hz, phchh); 4.724.66 (m, 2h, phch2); 4.60 (d, 1h, j = 11.6 hz, phchh); 4.32 (dd, 1h, j = 5.2 hz, 10.2 hz, h-2); 4.21 (q, 1h, j = 6.4 hz, h-5); 3.82 (s, 3h, och3); 3.733.68 (m, 2h, h-3, h-4). C - apt nmr (100 mhz): 159.5, 138.2 (cq, arom); 134.3, 129.5, 129.0 (charom); 128.7 (cq, arom); 128.3, 128.1, 127.7, 127.6, 114.0 (charom); 85.6 (c-1); 80.3, 75.8 (c-3, c-4); 74.9, 72.2 (phch2); 69.4 (c-5); 60.8 (c-2), 55.3 (och3); 16.5 (c-6). Ir (thin film): 2868, 2104, 1612, 1512, 1246, 1099, 1063, 1034 . Hrms: [m + h] calcd for c27h30n3o4se 540.1396, found 540.1394 . To a stirred solution of the fully protected 2-azidofucoside (1.56 g, 2.9 mmol, 1.0 equiv) and et3sih (0.73 ml, 8.7 mmol, 3.0 equiv) in ch2cl2 (15 ml, 0.2 m) was added a solution of hcl (0.25 ml of an 37% solution, w / v in water) in hfip (15 ml). After 1 min, the mixture was poured into a solution of nahco3 (satd aq). After separation of the layers, the aqueous phase was extracted (ch2cl2, 1), and the combined organic phases were washed (brine, 1), dried over mgso4, filtered, and concentrated in vacuo . After column chromatography (toluene / etoac, 1:0 9:1), the c3-oh intermediate was obtained as an oil in 64% yield (0.78 g, 1.9 mmol). H nmr (400 mhz): 7.587.56 (m, 2h, charom); 7.387.25 (m, 8h, charom); 5.91 (d, 1h, j = 5.2 hz, h-1); 4.81 (d, 1h, j = 11.6 hz, phchh); 4.72 (d, 1h, j = 11.6 hz, phchh); 4.33 (q, 1h, j = 6.4 hz, h-5); 4.02 (dd, 1h, j = 5.2 hz, 10.2 hz, h-2); 3.853.79 (m, 1h, h-3); 3.69 (d, 1h, j = 2.8 hz, h-4); 2.26 (d, 1h, j = 8.8 hz, 3-oh); 1.25 (d, 3h, j = 6.8 hz, h-6). C - apt nmr (100 mhz): 137.7 (cq, arom); 134.3, 129.1, 128.7, 128.2, 128.1, 127.7 (charom); 85.2 (c-1); 79.3 (c-4); 71.9 (c-3); 69.3 (c-5); 62.5 (c-2); 16.6 (c-6). Ir (thin film): 3468, 2882, 2106, 1263, 1094, 1057, 1022 . N2 + h] calcd for c19h22no3se 392.0759, found 392.0759 . To a stirred solution of 10 (0.21 g, 0.5 mmol, 1.0 equiv) in ch2cl2/pyridine (1.6 ml, 0.3 m, 1:1 v / v) were added bzcl (0.12 ml, 1.0 mmol, 2 equiv) and dmap (6 mg, 0.05 mmol, 0.1 equiv) at 0 c . After tlc analysis indicated complete conversion of the starting material (typically, the reaction mixture was left overnight), the reaction was quenched by addition of meoh . The mixture was diluted with ch2cl2, washed with cuso45h2o (in h2o, 10% w / v, 2), water (1), and brine (1), dried over mgso4, filtered, and concentrated in vacuo . Purification by column chromatography (pe / et2o, 1:0 9:1) furnished the title compound in 96% yield (0.25 g, 0.48 mmol). H nmr (400 mhz): 8.09 (d, 2h, j = 7.6 hz, charom); 7.637.58 (m, 3h, charom); 7.48 (t, 2h, j = 7.6 hz, charom); 7.417.23 (m, 8h, charom); 6.01 (d, 1h, j = 5.2 hz, h-1); 5.29 (dd, 1h, j = 2.8 hz, 11.0 hz, h-3); 4.67 (d, 1h, j = 11.2 hz, phchh); 4.58 (dd, 1h, j = 5.2 hz, 11.2 hz, h-2); 4.53 (d, 1h, j = 11.6 hz, phchh); 4.43 (q, 1h, j = 6.4 hz, h-5); 4.01 (d, 1h, j = 2.0 hz, h-4); 1.17 (d, 3h, j = 6.4 hz, h-6). C - apt nmr (100 mhz): 165.7 (cobz); 137.4 (cq, arom); 134.5, 133.7, 129.9, 129.1 (charom); 129.0 (cq, arom); 128.6 (charom); 128.4 (cq, arom); 128.3, 128.1, 127.9, 127.8 (charom); 84.9 (c-1); 76.6 (c-4); 75.6 (phch2); 75.1 (c-3); 69.1 (c-5); 59.6 (c-2); 16.3 (c-6). Ir (thin film): 2936, 2108, 1722, 1267, 1107, 1086, 1070 . Hrms: [m + na] calcd for c26h25n3o4sena 546.0903, found 546.0902 . A 100 ml, three - necked flask was equipped with a septum, a gas inlet, and a liebig condenser fitted with a drying tube . Under a flow of n2 gas, the flask was charged with a solution of phenyl 2-azido-2-deoxy-1-seleno--l - fucopyranoside (1.31 g, 4.0 mmol, 1.0 equiv) in pyridine (20 ml, 0.2 m). At 0 c, dmap (98 mg, 0.8 mmol, 0.2 equiv) was added followed by tbsotf (3.7 ml, 16.0 mmol, 4.0 equiv, in a dropwise fashion). The mixture was heated to 70 c and stirred for 16 h, after which tlc analysis (pe / et2o, 19:1 v / v) showed complete conversion of the starting material . The reaction mixture was cooled to rt, quenched with meoh, and diluted with etoac . The mixture was washed with 10% aq cuso4 solution (2), h2o, and brine, dried over mgso4, filtered, and concentrated in vacuo . Purification by column chromatography (pe / et2o, 1:0 49:1 v / v) furnished the title compound as a light - yellow oil in 85% yield (3.4 mmol, 1.90 g). H nmr (cd2cl2, 193 k): 7.53 (d, 2h, j = 7.6 hz, charom); 7.277.25 (m, 3h, charom); 5.89 (d, 1h, j = 5.2 hz, h-1); 4.21 (q, 1h, j = 6.4 hz, h-5); 4.06 (dd, 1h, j = 4.8 hz, 10.0 hz, h-2); 3.703.67 (m, 2h, h-3, h-4); 1.06 (d, 3h, j = 6.0 hz, h-6); 0.90, 0.82 (s, 9h, ch3,tbu); 0.14, 0.11, 0.09, 0.03 (s, 3h, ch3,me). C - apt nmr (cd2cl2, 193 k): 134.3, 128.7 (charom); 128.0 (cq, arom); 127.4 (charom); 85.3 (c-1); 73.6, 72.9 (c-3, c-4); 69.5 (c-2); 61.6 (c-5); 25.5, 25.3 (ch3,tbu); 18.1, 17.8 (cq, tbu); 16.6 (c-6); 4.3, 4.7, 5.3, 5.3 (ch3,me). Ir (thin film): 2953, 2930, 2856, 2106, 1472, 1252, 1115, 1067, 1022 . A 50 ml, three - necked flask was equipped with a septum, a gas inlet, and a liebig condenser fitted with a drying tube . Under a flow of n2 gas, the flask was charged with a solution of 10 (0.63 g, 1.5 mmol, 1.0 equiv) in pyridine (7.5 ml, 0.2 m). At 0 c, dmap (4 mg, 0.3 mmol, 0.2 equiv) was added followed by tbsotf (0.69 ml, 3.0 mmol, 2.0 equiv, in a dropwise fashion). The mixture was heated to 70 c and stirred for 16 h, after which tlc analysis (pe / et2o, 19:1 v / v) showed complete conversion of the starting material . The reaction mixture was cooled to rt, quenched with meoh, and diluted with etoac . The mixture was washed with 10% aq cuso4 solution (2), h2o and brine, dried over mgso4, filtered and concentrated in vacuo . Purification by column chromatography (pe / et2o, 1:0 19:1 v / v) furnished the title compound as a light yellow oil in 92% yield (0.73 g, 1.38 mmol). H nmr (400 mhz): 7.577.55 (m, 2h, charom); 7.397.26 (m, 8h, charom); 5.96 (d, 1h, j = 4.8 hz, h-1); 5.06 (d, 1h, j = 11.2 hz, phchh); 4.59 (d, 1h, j = 11.2 hz, phchh); 4.27 (q, 1h, j = 6.4 hz, h-5); 4.22 (dd, 1h, j = 5.2 hz, 10.0 hz, h-2); 3.88 (dd, 1h, j = 2.4 hz, 10.0 hz, h-3); 3.53 (bs, 1h, h-4); 1.15 (d, 3h, j = 6.4 hz, h-6); 0.99 (s, 9h, (ch3)3csi); 0.25, 0.22 (s, 3h, ch3si). C - apt nmr (100 mhz): 138.5 (cq, arom); 134.3, 129.0, 128.3, 127.8, 127.7, 127.6 (charom); 85.6 (c-1); 80.1 (c-4); 75.6 (phch2); 74.2 (c-3); 69.4 (c-5); 62.9 (c-2); 26.0 ((ch3)3csi); 16.5 (c-6). Ir (thin film): 2953, 2930, 2886, 2857, 2106, 1472, 1260, 1111, 1080, 1062, 1042, 1022 . Hrms: [m + h] calcd for c25h36n3o3sesi 534.1686, found 534.1688 . A mixture of glycosyl donor (0.038 mmol, 1.0 equiv) and ph2so (10 mg, 0.049 mmol, 1.3 equiv; 15 mg, 0.076 mmol, 2.0 equiv; or 31 mg, 0.152 mmol, 4.0 equiv) was dried by coevaporation with toluene (3) followed by three vacuum / argon purges . The mixture was dissolved in cd2cl2 (0.75 ml, 0.05 m) and transferred to a dry nmr tube, which was subsequently capped with a septum . The tube was placed in the probe of a nmr magnet and cooled to 80 c, after which a h nmr spectrum was recorded . The tube was removed from the magnet and placed in a acetone / n2 (l) bath (temperature 80 c). Tf2o (8 l, 0.049 mmol, 1.3 equiv) was added with a microliter syringe, and after rapid mixing and recooling, the tube was placed back in the nmr instrument . A h nmr spectrum was recorded, which revealed the formation of reactive intermediate(s). Cosy, and hsqc) the temperature of the sample was increased by increments of 10 c until decomposition of the intermediate(s) was observed . To a mixture of donor (0.1 mmol, 1.0 equiv), ph2so (26 mg, 0.13 mmol, 1.3 equiv), and ttbp (62 mg, 0.25 mmol, 2.5 equiv) in dry ch2cl2 (2 ml, 0.05 m) were added flame - dried 3 molecular sieves . The mixture was subsequently stirred for 30 min before being cooled to 80 c . At this temperature, tf2o (22 l, 0.13 mmol, 1.3 equiv) was added via syringe, and the temperature was raised to 60 c over the course of 30 min . After the temperature was recooled to 80 c, the acceptor (0.2 mmol, 2.0 equiv, 0.4 ml of a 0.5 m stock solution in ch2cl2) was added at 80 c, and the reaction mixture was allowed to warm to 40 c, after which the reaction was quenched by addition of net3 (0.1 ml) and subsequently diluted with ch2cl2 . The mixture was filtered through a small bed of celite, the residue was washed with ch2cl2, and the filtrate was washed once with brine, dried over mgso4, filtered, and concentrated in vacuo . Purification by ordinary column chromatography and/or size - exclusion chromatography afforded the corresponding o - glycoside(s). The title compounds (/ 1:1) were obtained after column chromatography (hexane / et2o, 1:0 9:1) in 88% yield (35 mg, 0.088 mmol). H nmr (400 mhz): 7.437.25 (m, 18h, charom); 4.954.92 (m, 1.8h, phchh, phchh); 4.90 (d, 0.8h, j = 4.0 hz, h-1); 4.744.60 (m, 5.4h, phch2, phch2); 4.18 (d, 1h, j = 8.0 hz, h-1); 3.993.78 (m, 4.2h, h-2, h-2, h-3, h-5, chhch3); 3.753.67 (m, 1.8h, h-4, chhch3); 3.603.50 (m, 2.8h, h-4, chhch3, chhch3); 3.41 (q, 1h, j = 6.4 hz, h-5); 3.30 (dd, 1h, j = 2.8 hz, 10.4 hz, h-3); 1.281.16 (m, 10.8h, h-6, h-6, ch2ch3, ch2ch3). C - apt nmr (100 mhz): 138.3, 137.7 (cq, arom); 128.5, 128.5, 128.4, 128.3, 128.2, 127.9, 127.8, 127.7, 127.6 (charom); 102.0 (c-1); 97.9 (c-1); 81.1 (c-3); 78.0 (c-3); 76.3 (c-4); 74.9 (c-4); 74.9, 74.6, 72.6, 72.4 (phch2); 70.5 (c-5); 66.5 (c-5); 65.3 (ch2ch3); 63.7 (ch2ch3); 63.0 (c-2); 59.6 (c-2); 16.9, 16.7 (c-6); 15.0, 15.0 (ch2ch3). Ir (thin film): 2893, 2106, 1454, 1356, 1099, 1063 . The title compounds (/ 1:4) were obtained after column chromatography (hexane / etoac, 1:0 4:1 v / v) in 39% yield (25 mg, 0.059 mmol). H nmr (400 mhz): 8.098.03 (m, 10h, charom); 7.897.86 (m, 9h, charom); 7.647.59 (m, 6h, charom); 7.537.46 (m, 17h, charom); 7.357.31 (m, 10h, charom); 5.78 (dd, 1h, j = 3.2 hz, 10.8 hz, h-3); 5.71 (dd, 1h, j = 1.2 hz, 3.2 hz, h-4); 5.59 (dd, 4h, j = 0.8 hz, 3.2 hz, h-4); 5.17 (dd, 4h, j = 3.6 hz, 10.8 hz, h-3); 5.13 (d, 1h, j = 3.6 hz, h-1); 4.51 (d, 4h, j = 8.0 hz, h-5); 4.10 (dq, 4h, j = 7.2 hz, 9.6 hz, chhch3); 3.983.90 (m, 8h, h-2, h-5); 3.883.84 (m, 2h, h-2, chhch3); 3.763.63 (m, 6h, chhch3, chhch3); 1.371.21 (m, 30h, h-6, h-6, ch2ch3, ch2ch3). C - apt nmr (100 mhz): 165.8, 165.8, 165.4 (cobz); 133.4, 133.4, 133.3, 133.2, 129.9, 129.7 (charom); 129.2, 129.1 (cq, arom); 128.5, 128.5, 128.3, 128.3 (charom); 102.2 (c-1); 98.1 (c-1); 72.0 (c-3); 71.4 (c-4); 70.3 (c-4); 69.5 (c-5); 69.3 (c-3); 66.2 (ch2ch3); 65.1 (c-5); 64.3 (ch2ch3); 61.2 (c-2); 57.9 (c-2); 16.3 (c-6); 16.1 (c-6); 15.1 (ch2ch3); 15.0 (ch2ch3). Ir (thin film): 1980, 2927, 2110, 1724, 1450, 1261, 1175, 1109, 1094, 1067, 1026 . The title compounds (/ 1:3) were obtained after column chromatography (hexane / et2o, 1:0 4:1 v / v) in 61% yield (25 mg, 0.061 mmol). H nmr (400 mhz): 8.158.07 (m, 8h, charom); 7.607.56 (m, 4h, charom); 7.487.44 (m, 8h, charom); 7.367.24 (m, 20h, charom); 5.68 (d, 1h, j = 2.4 hz, h-4); 5.54 (dd, 3h, j = 0.8 hz, 3.2 hz, h-4); 4.98 (d, 1h, j = 3.6 hz, h-1); 4.83 (d, 1h, j = 10.8 hz, phchh); 4.79 (d, 3h, j = 11.6 hz, phchh); 4.564.53 (m, 4h, phchh, phchh); 4.28 (d, 4h, j = 8.0 hz, h-5); 4.11 (dd, 1h, j = 3.2 hz, 10.4 hz, h-3); 4.063.99 (m, 3h, chhch3); 3.783.60 (m, 11h, h-2, h-2, h-5, chhch3, chhch3); 3.45 (dd, 3h, j = 3.2 hz, 10.4 hz, h-3); 1.591.26 (m, 21h, h-6, ch2ch3, ch2ch3); 1.22 (d, 3h, j = 6.8 hz, h-6). C - apt nmr (100 mhz): 166.2 (cobz); 137.2, 137.1 (cq, arom); 133.3, 133.2, 130.0, 129.8 (charom); 129.4 (cq, arom); 128.5, 128.4, 128.4, 128.4, 128.2, 128.4, 1f27.9, 127.8 (charom); 102.0 (c-1); 97.9 (c-1); 77.6 (c-3); 74.4 (c-3); 71.5 (phch2); 71.5 (phch2); 70.0 (c-4); 69.5 (c-5); 68.9 (c-4); 65.9 (ch2ch3); 65.1 (c-5); 64.0 (ch2ch3); 62.6 (c-2); 59.3 (c-2); 16.5 (c-6); 16.3 (c-6); 15.1 (ch2ch3); 15.0 (ch2ch3). Ir (thin film): 2980, 2870, 2108, 1721, 1452, 1265, 1175, 1111, 1065, 1026 . The title compounds (/ 2:5) were obtained after column chromatography (hexane / et2o 1:0 9:1) in 58% yield (24 mg, 0.058 mmol). H nmr (400 mhz): 8.108.06 (m, 14h, charom); 7.627.58 (m, 7h, charom); 7.497.45 (m, 14h, charom); 7.267.19 (m, 35h, charom); 5.57 (dd, 2h, j = 3.0 hz, 11.0 hz, h-3); 5.00 (d, 2h, j = 3.6 hz, h-1); 4.95 (dd, 5h, j = 3.0 hz, 11.0 hz, h-3); 4.724.68 (m, 7h, phchh, phchh); 4.574.52 (m, 7h, phchh, phchh); 4.37 (d, 5h, j = 8.0 hz, h-5); 4.053.94 (m, 14h, h-2, h-2, h-4, chhch3); 3.823.74 (m, 7h, h-4, chhch3); 3.683.56 (m, 12h, h-5, chhch3, chhch3); 1.311.20 (m, 42h, h-6, h-6, ch2ch3, ch2ch3). C - apt nmr (100 mhz): 165.9 (co bz); 137.6, 137.5 (cq, arom); 133.5, 133.5, 129.9 (charom); 129.2 (cq, arom); 128.5, 128.3, 128.2, 128.1, 127.8, 127.8, 127.6 (charom); 101.9 (c-1); 98.0 (c-1); 77.4 (c-4); 76.0 (c-4); 75.5 (phch2); 75.4 (phch2); 75.0 (c-3); 72.3 (c-3); 70.5 (c-5); 66.2 (c-5); 65.6 (ch2ch3); 63.9 (ch2ch3); 61.2 (c-2); 58.0 (c-2); 16.6, 16.4, 15.0, 15.0 (ch2ch3, c-6, c-6). Ir (thin film): 2978, 2932, 2108, 1721, 1452, 1265, 1175, 1094, 1069, 1026 . The title compounds (/ 2:5) were obtained after column chromatography (hexane / et2o, 1:0 19:1) along with a minor amount of inseparable, hydrolyzed donor in 63% yield (28 mg, 0.063 mmol). H nmr (400 mhz): 4.91 (d, 2h, j = 3.6 hz, h-1); 4.013.95 (m, 7h, h-3, ochhch3); 3.88 (q, 2h, j = 6.4 hz, h-5); 3.733.70 (m, 6h, h-2, h-4, ochhch3); 3.623.52 (m, 17h, h-2, h-4, ochhch3; ochhch3); 3.45 (q, 5h, j = 6.4 hz, h-5); 3.35 (dd, 5h, j = 2.4 hz, 10.4 hz, h-3); 1.291.18 (m, 42h, h-6, h-6, ch2ch3, ch2ch3); 0.960.93 (m, 126h, (ch3)3csi, (ch3)3csi); 0.190.09 (m, 84h, ch3si, ch3si). C - apt nmr (100 mhz): 102.5 (c-1); 97.7 (c-1); 75.2 (c-4); 74.5 (c-3); 74.0 (c-4); 71.3 (c-3); 71.2 (c-5); 67.7 (c-5); 65.5 (och2ch3); 63.8 (c-2); 63.4 (och2ch3); 61.1 (c-2); 26.3, 26.2, 26.1 ((ch3)3csi); 18.6, 18.5 (cqsi); 17.6, 17.3 (och2ch3); 15.1, 15.0 (c-6, c-6); 3.5, 3.6, 4.2, 4.4 (ch3si). Ir (thin film): 2928, 2857, 2112, 1252, 1117, 1069 . Hrms: [m + nh4] calcd for c20h47n4o4si2 463.3130, found 463.3129 . The title products (/ 1:1) were obtained after column chromatography (hexane / et2o, 1:0 19:1 v / v) in 81% yield (34 mg, 0.081 mmol). H nmr (400 mhz): 7.397.26 (m, 10h, charom); 5.055.02 (m, 2h, phchh, phchh); 4.91 (d, 1h, j = 3.6 hz, h-1); 4.614.56 (m, 2h, phchh, phchh); 4.19 (d, 1h, j = 8.0 hz, h-1); 4.12 (dd, 1h, j = 2.8 hz, 10.0 hz, h-3); 3.983.93 (m, 2h, h-5, ochhch3); 3.743.50 (m, 8h, h-2, h-2, h-3, h-4, h-5, ochhch3, 2 ochhch3); 3.37 (d, 1h, j = 2.4 hz, h-4); 1.271.19 (m, 12h, h-6, h-6, och2ch3, och2ch3); 0.98, 0.96 (s, 9h, (ch3)3csi); 0.24 (s, 3h, ch3si); 0.18 (m, 6h, ch3si); 0.15 (s, 3h, ch3si). C - apt nmr (100 mhz): 138.6, 138.6 (cq, arom); 128.3, 128.1, 128.1, 127.9, 127.6, 127.5 (charom); 102.1 (c-1); 97.8 (c-1); 80.9 (c-4), 79.2 (c-4); 75.6, 75.3 (phch2); 74.9 (c-3 or c-5); 71.5 (c-3); 70.3 (c-3 or c-5); 66.5 (c-5); 65.4 (och2ch3); 64.6 (c-2); 63.6 (och2ch3); 61.5 (c-2); 25.9, 25.9 ((ch3)3csi); 18.2, 18.1 (cqsi); 16.8, 16.7 (c-6, c-6); 15.1, 15.0 (och2ch3, och2ch3); 4.0, 4.3, 4.7, 5.0 (ch3si). Ir (thin film): 2930, 2891, 2857, 2108, 1254, 1171, 1117, 1067, 1047 . The title products (/ 1:1) were obtained after column chromatography (hexane / etoac, 1:0 4:1) in 72% yield (30 mg, 0.072 mmol). H nmr (400 mhz): 7.667.63 (m, 2h, charom); 7.467.25 (m, 18h, charom); 4.954.91 (m, 3h, h-1, 2 phchh); 4.744.50 (m, ch2f, ch2f, 2 phchh; 4x phchh); 4.26 (d, 1h, j = 8.0 hz, h-1); 4.023.73 (m, 9h, h-2, h-2, h-3; h-4, h-5, ch2ch2f, ch2ch2f); 3.54 (d, 1h, j = 2.4 hz, h-5); 3.31 (dd, 1h, j = 2.8 hz, 10.4 hz, h-3); 1.201.17 (m, 6h, h-6, h-6). C - apt nmr (100 mhz): 138.2, 137.6 (cq, arom); 131.0, 129.9, 128.5, 128.5, 128.4, 128.3, 128.2, 127.9, 127.8, 127.7, 127.7, 124.7 (charom); 102.3 (c-1); 98.4 (c-1); 82.7 (d, j = 168 hz, ch2f); 82.5 (d, j = 168 hz, ch2f); 80.9 (c-3); 77.8 (c-3); 76.1 (c-4); 74.9 (phch2); 74.8 (c-4); 72.7, 72.4 (phch2); 70.6 (c-5); 68.3 (d, 20 hz, ch2ch2f) 67.1 (d, 20 hz, ch2ch2f); 66.7 (c-5); 62.9 (c-2); 59.5 (c-2); 16.8, 16.7 (c-6, c-6). Ir (thin film): 2876, 2108, 1726, 1358, 1109, 1062, 1045 . The title compounds (/ 1:2) were obtained after column chromatography (hexane / etoac, 1:0 4:1) in 34% yield (15 mg, 0.034 mmol). H nmr (400 mhz): 8.088.03 (m, 6h, charom); 7.897.86 (m, 6h, charom); 7.637.60 (m, 3h, charom); 7.537.46 (m, 9h, charom); 7.357.31 (m, 6h, charom); 5.78 (dd, 1h, j = 3.2 hz, 11.2 hz, h-3); 5.72 (d, 1h, j = 3.2 hz, h-4); 5.195.16 (m, 3h, h-1, h-3); 4.774.60 (m, 6h, ch2f, ch2f, h-1); 4.58 (d, 2h, j = 8.4 hz, h-5); 4.253.89 (m, 11h, h-2, h-2, h-5, ch2ch2f, ch2ch2f); 1.31 (d, 6h, j = 6.4 hz, h-6); 1.25 (d, 3h, j = 6.4 hz, h-6). C - apt nmr (100 mhz): 165.8, 165.4 (cobz); 133.5, 133.4, 133.3, 133.3, 129.9, 129.8 (charom); 129.3, 129.2, 129.0 (cq, arom); 128.6, 128.3 (charom); 102.5 (c-1); 98.6 (c-1); 82.6 (d, j = 169 hz, ch2ch2f); 82.4 (d, j = 170 hz, ch2ch2f); 72.0 (c-3); 71.3 (c-4); 70.2 (c-4); 69.7 (c-5); 69.2 (c-3); 69.1 (d, j = 21 hz, ch2ch2f); 67.5 (d, j = 20 hz, ch2ch2f); 65.4 (c-5); 61.3 (c-2); 58.0 (c-2); 16.3 (c-6); 16.1 (c-6). Ir (thin film): 2984, 2924, 2110, 1721, 1450, 1260, 1169, 1107, 1094, 1067, 1026 . Hrms: [m + na] calcd for c22h22fn3o6na 466.1385, found 466.1384 . The products (/ 1:1) were obtained after column chromatography (hexane / etoac, 1:0 9:1) and size - exclusion chromatography (ch2cl2/meoh, 1:1 v / v) in 56% yield (24 mg, 0.056 mmol), accompanied by a small amount of inseparable, hydrolyzed donor . H nmr (400 mhz): 8.148.07 (m, 4h, charom); 7.607.56 (m, 2h, charom); 7.494.44 (m, 4h, charom); 7.357.25 (m, 10h, charom); 5.70 (d, 1h, j = 2.8 hz, h-1); 4.854.53 (m, 8h, ch2f, ch2f, phch2, phch2); 4.36 (d, 1h, j = 8.0 hz, h-5); 4.153.71 (m, 8h, h-2, h-2, h-3, h-5, ch2ch2f, ch2ch2f); 3.47 (dd, 1h, j = 3.2 hz, 10.2 hz, h-3); 1.281.21 (m, 6h, h-6, h-6). C - apt nmr (100 mhz): 166.2, 166.1 (cobz); 137.1, 137.0 (cq, arom); 133.4, 133.3, 130.2, 130.0, 129.8, 129.6 (charom); 129.4 (cq, arom); 128.5, 128.4, 128.2, 128.0, 127.9, 127.8 (charom); 102.3 (c-1); 98.4 (c-1); 82.7 (d, j = 169 hz, ch2f); 82.5 (d, j = 170 hz, ch2f); 77.5 (c-3); 74.3 (c-3); 71.6 (phch2); 71.5 (phch2); 69.8 (c-4); 69.6 (c-5); 68.8 (d, j = 20 hz, ch2ch2f); 68.8 (c-4); 67.4 (d, j = 20 hz, ch2ch2f); 65.4 (c-5); 62.6 (c-2); 59.2 (c-2); 16.5, 16.3 (c-6, c-6). Ir (thin film): 2926, 2110, 1721, 1452, 1267, 1169, 1111, 1067, 1026 . The products (/ 2:3) were obtained after column chromatography (hexane / etoac, 1:0 9:1) and size - exclusion chromatography (ch2cl2/meoh, 1:1 v / v) in 60% yield (26 mg, 0.060 mmol). H nmr (400 mhz): 8.108.06 (m, 10h, charom); 7.627.58 (m, 5h, charom); 7.497.45 (m, 10h, charom); 7.267.20 (m, 25h, charom); 5.58 (dd, 2h, j = 2.8 hz, 11.2 hz, h-3); 5.05 (d, 2h, j = 3.6 hz, h-1); 4.95 (dd, 3h, j = 3.2 hz, 10.8 hz, h-3); 4.734.52 (m, 20h, chch2f, phch2); 4.43 (d, 3h, j = 8.0 hz, h-1); 4.173.78 (m, 22h, h-2, h-2, h-4, h-4, h-5, ch2ch2f); 3.67 (q, 3h, j = 6.4 hz, h-5); 1.261.21 (m, 15h, h-6, h-6). C - apt nmr (100 mhz): 165.8 (cobz); 137.5, 137.4 (cq, arom); 133.6, 133.5, 129.9 (charom); 129.3, 129.1 (cq, arom); 128.6, 128.3, 128.3, 128.1, 127.9, 127.9 (charom); 102.3 (c-1); 98.5 (c-1); 82.6 (d, j = 168 hz, ch2ch2f); 82.4 (d, j = 169 hz, ch2ch2f); 77.2 (c-4); 75.9 (c-4); 75.6 (phch2); 75.4 (phch2); 74.9 (c-3); 72.2 (c-3); 70.6 (c-5); 68.6 (d, j = 21 hz, ch2ch2f); 67.3 (d, j = 20 hz, ch2ch2f); 66.5 (c-5); 61.2 (c-2); 58.0 (c-2); 16.6 (c-6); 16.4 (c-6). Ir (thin film): 2934, 2110, 1721, 1452, 1267, 1171, 1096, 1069, 1026 . Hrms: [m + nh4] calcd for c22h28fn4o5 447.2038, found 447.2038 . The products (/ 2:3) were obtained after column chromatography (hexane / et2o, 1:0 9:1) in 82% yield (38 mg, 0.082 mmol). H nmr (400 mhz): 4.94 (d, 2h, j = 3.6 hz, h-1); 4.664.51 (m, 10h, ch2f, ch2f); 4.26 (d, 3h, j = 8.0 hz, h-1); 4.154.01 (m, 5h, h-3, chhch2f); 3.943.68 (m, 13h, h-2, h-4, h-5, ch2ch2f, chhch2f); 3.593.55 (m, 6h, h-2, h-4); 3.47 (q, 3h, j = 6.4 hz, h-5); 3.36 (dd, 3h, j = 2.4 hz, 10.2 hz, h-3); 1.23 (d, 9h, j = 6.4 hz, h-6); 1.19 (d, 6h, j = 6.4 hz, h-6), 0.960.93 (m, 90h, (ch3)3csi); 0.190.08 (m, 60h, ch3si). C - apt nmr (100 mhz): 102.8 (c-1); 98.3 (c-1); 82.8 (d, j = 168 hz, ch2ch2f); 82.6 (d, j = 168 hz, ch2ch2f); 75.1 (c-4); 74.4 (c-3); 73.9 (c-4); 71.3 (c-5); 71.2 (c-3); 68.3 (d, j = 20 hz, ch2ch2f); 66.9 (d, j = 20 hz, ch2ch2f); 63.8 (c-2); 61.1 (c-2); 26.3, 26.1, 26.1 ((ch3)3csi); 18.6, 18.6, 18.5 (cqsi); 17.5 (c-6), 17.3 (c-6); 3.5, 3.5, 3.6, 3.7, 4.3, 4.5, 4.5, 4.7 (ch3si). Ir (thin film): 2930, 2857, 2108, 1252, 1177, 1119, 1069, 1045, 1028 . The title products (/ 1:1) were isolated after column chromatography (hexane / et2o, 1:0 9:1) in 80% yield (35 mg, 0.080 mmol). H nmr (400 mhz): 7.397.26 (m, 10h, charom); 5.055.02 (m, 2h, 2 phchh); 4.94 (d, 1h, j = 3.6 hz, h-1); 4.13 (dd, 1h, j = 2.8 hz, 10.4 hz, h-3); 4.003.76 (m, 7h, h-2, h-2; h-5; ch2ch2f, ch2ch2f); 3.523.48 (m, 3h, h-3, h-4, h-5); 3.38 (d, 1h, j = 2.8 hz, h-4); 1.211.18 (m, 6h, h-6, h-6); 0.98, 0.97 (s, 9h, (ch3)3csi); 0.24 (s, 3h, ch3si); 0.20 (s, 6h, 2 ch3si); 0.16 (s, 3h, ch3si). C - apt nmr (100 mhz): 138.5, 138.5 (cq, arom); 128.3, 128.2, 128.1, 127.9, 127.6, 127.6 (charom); 102.5 (c-1); 98.4 (c-1); 82.7 (d, j = 168 hz, ch2f); 82.6 (d, j = 168 hz, ch2f); 80.7 (c-4); 79.0 (c-4); 75.6, 75.4 (phch2); 74.8 (c-5); 71.4 (c-3); 70.5 (c-3); 68.4 (d, j = 20 hz, ch2ch2f); 67.1 (d, j = 20 hz, ch2ch2f); 66.8 (c-5); 64.6 (c-2); 61.4 (c-2); 25.9, 25.9 ((ch3)3csi); 18.1, 18.0 (cqsi); 16.7, 16.6 (c-6, c-6); 4.0, 4.3, 4.8, 5.1 (ch3si). Ir (thin film): 2930, 2886, 2857, 2108, 1254, 1169, 1119, 1065, 1045 . The products (/ 3:2) were obtained after column chromatography (toluene / etoac, 1:0 9:1 v / v) in 81% yield (35 mg, 0.081 mmol). H nmr (400 mhz): 7.437.25 (m, 50h, charom); 6.085.78 (m, 5h, chf2, chf2); 4.954.91 (m, 7h, h-1, phchh, phchh); 4.744.59 (m, 14h, phch2); 4.24 (d, 2h, j = 8.0 hz, h-1); 4.013.73 (m, 24h, h-2, h-2, h-3, h-4, h-5, ch2chf2, ch2chf2); 3.54 (d, 2h, j = 2.4 hz, h-5); 3.31 (dd, 2h, j = 2.8 hz, 10.0 hz, h-3); 1.201.17 (m, 15h, h-6, h-6). C - apt nmr (100 mhz): 138.1, 138.0, 137.5, 137.5 (cq, arom); 128.6, 128.5, 128.3, 128.3, 128.2, 128.0, 127.8, 127.8, 127.8, 127.6 (charom); 114.3 (chf2); 113.9 (chf2); 102.4 (c-1); 99.0 (c-1); 80.8 (c-3); 77.6 (c-3 or c-5); 75.9 (c-4); 75.0 (phch2); 74.7 (phch2); 74.6 (c-4); 72.8 (phch2); 72.4 (phch2); 70.9 (c-5); 68.3 (t, j = 29 hz, ch2chf2); 67.2 (c-3 or c-5); 67.2 (t, j = 29 hz, ch2chf2); 62.8 (c-2); 59.4 (c-2); 16.7, 16.6 (c-6, c-6). Ir (thin film): 2926, 2110, 1738, 1454, 1360, 1109, 1069 . The title compounds (/ 3:2) were obtained after column chromatography (toluene / etoac, 1:0 9:1 v / v) in 74% yield (34 mg, 0.074 mmol). 8.078.02 (m, 5.4h, charom); 7.897.86 (m, 5.4h, charom); 7.647.60 (m, 2.7h, charom); 7.537.46 (m, 8.1h, charom); 7.357.31 (m, 5.4h, charom); 6.175.87 (m, 2.7h, chf2, chf2); 5.765.73 (m, 3.4h, h-3, h-4); 5.60 (d, 1h, j = 3.2 hz, h-4); 5.195.16 (m, 2.7h, h-1, h-3); 4.56 (d, 1h, j = 8.0 hz, h-5); 4.173.86 (m, 9.1h, h-2, h-2, h-5, ch2ch2f, ch2ch2f); 1.321.23 (m, 8.1h, h-6, h-6). C - apt nmr (100 mhz): 165.7, 165.7, 165.4 (cobz); 133.5, 133.5, 133.4, 133.3, 129.9, 129.8 (charom); 129.2, 129.1, 129.0 (cq, arom); 129.0, 128.6, 128.4, 128.3 (charom); 114.0 (t, j = 240 hz, chf2); 113.7 (t, j = 240 hz, chf2); 102.6 (c-1); 99.1 (c-1); 71.9 (c-3); 71.1 (c-3 or c-4); 70.0, 69.9 (c-4, c-5); 69.0 (c-3 or c-4); 68.8 (t, j = 30 hz, ch2chf2); 67.4 (t, j = 30 hz, ch2ch2f); 65.8 (c-5); 61.2 (c-2); 57.9 (c-2); 16.2 (c-6), 16.0 (c-6). Ir (thin film): 2926, 2110, 1726, 1450, 1261, 1163, 1107, 1094, 1067 . Hrms: [m + na] calcd for c22h21f2n3o6na 484.1291, found 484.1289 . The products (/ 3:1) were obtained after column chromatography (toluene / etoac, 1:0 19:1) in 76% yield (34 mg, 0.076 mmol), accompanied by a small amount of inseparable, hydrolyzed donor . H nmr (400 mhz): 8.128.07 (m, 8h, charom); 7.607.58 (m, 4h, charom); 7.497.44 (m, 10h, charom); 7.357.25 (m, 18h, charom); 6.125.82 (m, 4h, chf2, chf2); 5.70 (d, 3h, j = 2.8 hz, h-1); 4.83 (d, 3h, j = 10.8 hz, phchh); 4.79 (d, 1h, j = 11.6 hz, phchh); 4.564.53 (d, 4h, phchh, phchh); 4.33 (d, 1h, j = 8.0 hz, h-5); 4.08 (dd, 3h, j = 3.2 hz, 10.8 hz, h-3); 4.033.69 (m, 13h, h-2, h-2, h-5, ch2chf2, ch2chf2); 3.47 (dd, 1h, j = 3.6 hz, 10.8 hz, h-3); 1.311.22 (m, 12h, h-6, h-6). C - apt nmr (100 mhz): 166.1, 166.0 (cobz); 137.0, 136.9 (cq, arom); 133.4, 133.3, 130.0, 129.9, 129.5, 129.4, 129.2 (charom); 128.6, 128.5 (cq, arom); 128.4, 128.4, 128.3, 128.2, 128.0, 127.9, 127.6 (charom); 114.2 (t, j = 240 hz, chf2); 113.8 (t, j = 240 hz, chf2); 102.4 (c-1); 99.0 (c-1); 77.5 (c-3); 74.1 (c-3); 71.6 (phch2); 71.5 (phch2); 69.8 (c-5); 69.6 (c-4); 68.6 (c-4); 64.6 (t, j = 30 hz, ch2chf2); 67.4 (t, j = 28 hz, ch2chf2); 65.8 (c-5); 62.5 (c-2); 59.1 (c-2); 16.4, 16.3 (c-6, c-6). Ir (thin film): 2924, 2110, 1721, 1452, 1265, 1167, 1109, 1067, 1053, 1026 . The title compounds (/ 1:1) were obtained after column chromatography (toluene / etoac, 1:0 9:1) in 80% yield (36 mg, 0.080 mmol). H nmr (400 mhz): 8.108.06 (m, 4h, charom); 7.637.59 (m, 2h, charom); 7.497.46 (m, 4h, charom); 7.267.20 (m, 10h, charom); 6.115.82 (m, 2h, chf2, chf2); 5.54 (dd, 1h, j = 3.2 hz, 11.2 hz, h-3); 5.04 (d, 1h, j = 3.6 hz, h-1); 4.95 (dd, 1h, j = 2.8 hz, 10.8 hz, h-3); 4.724.69 (d, 2h, j = 11.6 hz, 2 phchh); 4.574.52 (m, 2h, 2 phchh); 4.42 (d, 1h, j = 8.8 hz, h-1); 4.144.3.78 (m, 9h, h-2, h-2, h-4, h-4, h-5, ch2chf2, ch2chf2); 3.68 (q, 1h, j = 6.4 hz, c - apt nmr (100 mhz): 165.8 (cobz); 137.4, 137.3 (cq, arom); 133.6, 133.6, 130.2, 129.9 (charom); 129.3 (cq, arom); 129.0, 128.6, 128.5, 128.4, 128.3, 128.2, 128.0, 127.9 (charom); 114.1 (t, j = 240 hz, chf2); 113.8 (t, j = 240 hz, chf2); 102.4 (c-1); 99.0 (c-1); 77.0, 75.7 (c-4, c-4); 75.6, 75.5 (phch2); 74.7 (c-3); 71.9 (c-3); 70.9 (c-5); 68.767.3 (m, 2c, ch2chf2, ch2chf2); 66.9 (c-5); 61.1 (c-2); 57.9 (c-2); 16.5, 16.3 (c-6, c-6). Ir (thin film): 2924, 2110, 1721, 1452, 1265, 1169, 1096, 1069, 1026 . The title products (/ 5:2) were obtained after column chromatography (hexane / et2o, 1:0 19:1 v / v) in 75% yield (36 mg, 0.075 mmol). H nmr (400 mhz): 6.095.79 (m, 7h, chf2, chf2) 4.93 (d, 5h, j = 3.2 hz, h-1); 4.013.73 (m, 27h, h-2, h-3, h-5, och2chf2, och2chf2); 3.71 (d, 5h, j = 2.0 hz, h-4); 3.583.53 (m, 4h, h-2, h-4); 3.47 (q, 2h, j = 6.4 hz, h-5); 3.36 (dd, 2h, j = 2.4 hz, 10.4 hz, h-3); 1.23 (d, 6h, j = 6.4 hz, h-6); 0.960.89 (m, 126h, ((ch3)3csi); 0.180.09 (m, 84h, ch3si). C - apt nmr (100 mhz): 114.4 (t, j = 240 hz, chf2); 114.1 (t, j = 240 hz, chf2); 102.8 (c-1); 98.9 (c-1); 75.0 (c-4); 74.3 (c-3); 73.8 (c-4); 71.5 (c-5); 71.1 (c-3); 68.5 (c-5); 68.2 (t, j = 29 hz, ch2chf2); 67.1 (t, j = 29 hz, ch2chf2); 63.8 (c-2); 61.0 (c-2); 26.3, 26.1 ((ch3)3csi); 18.6, 18.5 (cqsi); 17.4, (c-6); 17.3 (c-6); 3.5, 3.5, 3.8, 4.4, 4.5, 4.7 (ch3si). Ir (thin film): 2930, 2859, 2108, 1252, 1177, 1113, 1069, 1043, 1028 . The title products (/ 2:1) were obtained after chromatography (hexane / et2o, 1:0 9:1 v / v) in 87% yield (40 mg, 0.087 mmol). H nmr (400 mhz): 7.397.25 (m, 7.5h, charom); 6.085.81 (m, 1.5h, chf2, chf2); 5.055.02 (d, 1.5h, j = 11.2 hz, phchh, phchh); 4.93 (d, 1h, j = 3.6 hz, h-1); 4.624.56 (m, 1.5h, phchh, phchh); 4.25 (d, 0.5h, j = 8.0 hz, h-1); 4.08 (dd, 1h, j = 2.8 hz, 10.4 hz, h-3); 3.94 (q, 1h, j = 6.4 hz, h-5); 3.823.73 (m, 4h, h-2, ch2chf2, ch2chf2); 3.66 (dd, 0.5h, j = 8.0 hz, 10.4 hz, h-2); 3.523.50 (m, 2h, h-3, h-4, h-5); 3.39 (d, 0.5h, j = 2.4 hz, h-4), 1.261.18 (m, 4.5h, h-6, h-6); 0.98 (s, 9h, (ch3)3csi); 0.97 (s, 4.5h, (ch3)3csi); 0.24 (s, 3h, ch3si); 0.20 (s, 4.5h, ch3si, ch3si); 0.16 (s, 1.5h, ch3si). C - apt nmr (100 mhz): 138.4, 138.4 (cq, arom); 128.3, 128.2, 128.1, 127.9, 127.7, 127.7 (charom); 114.3 (t, j = 240 hz, chf2); 114.0 (t, j = 240 hz, chf2); 102.6 (c-1); 99.0 (c-1); 80.5 (c-4); 78.9 (c-4); 75.7, 75.5 (phch2); 74.7 (c-3 or c-5); 71.3 (c-3); 70.7 (c-3 or c-5); 68.4 (t, j = 27 hz, ch2chf2); 67.3 (t, j = 29 hz, ch2chf2); 67.3 (c-5); 64.5 (c-2); 61.3 (c-2); 25.9, 25.8 ((ch3)3csi); 18.1, 18.0 (cqsi); 16.7, 16.6 (c-6, c-6); 4.1, 4.3, 4.8, 5.1 (ch3si). Ir (thin film): 2930, 2110, 1260, 1169, 1115, 1070, 1047 . Hrms: [m + nh4] calcd for c21h37f2n4o4si 475.2546, found 475.2547 . The title compound was obtained after column chromatography (hexane / et2o 1:0 9:1 v / v) in 80% yield (36 mg, 0.080 mmol, 80%). H nmr (400 mhz): 7.447.25 (m, 10h, charom); 4.964.92 (m, 2h, phchh, h-1); 4.75 (s, 2h, phch2); 4.60 (d, 1h, j = 11.6 hz, phchh); 3.993.88 (m, 5h, h-2, h-3, h-5, ch2cf3); 3.54 (d, 1h, j = 2.4 hz, h-4); 1.18 (d, 3h, j = 6.4 hz, h-6). C - apt nmr (100 mhz): 138.0, 137.5 (cq, arom); 128.6, 128.3, 128.3, 128.0, 127.8, 127.8 (charom); 123.6 (q, j = 277 hz, cf3); 99.0 (c-1); 77.4 (c-3 or c-5); 75.9 (c-4); 75.0, 72.5 (phch2); 67.5 (c-3 or c-5); 64.9 (q, j = 35 hz, ch2cf3); 59.2 (c-2); 16.7 (c-6). C - gated nmr (100 mhz): 99.0 (d, j = 170 hz, c-1). Ir (thin film): 2927, 2108, 1454, 1356, 1279, 1163, 1082, 1051 . Hrms: [m + na] calcd for c22h24f3n3o4na 474.1611, found 474.1609 . The title compounds (/ 10:1) were isolated after column chromatography (hexane / etoac . H nmr (400 mhz): 8.048.02 (m, 2h, charom); 7.897.86 (m, 2h, charom); 7.667.61 (m, 1h, charom); 7.547.45 (m, 3h, charom); 7.367.32 (m, 2h, charom); 5.777.73 (m, 2h, h-3, h-4); 5.20 (d, 1h, j = 3.6 hz, h-1); 4.37 (q, 1h, j = 6.4 hz, ch2cf3); 3.95 (dd, 1h, j = 3.2 hz, 11.4 hz, h-2); 1.26 (d, 3h, j = 6.8 hz, h-6). C - apt nmr (100 mhz): 165.6, 165.3 (cobz); 133.5, 133.4, 129.9, 129.8 (charom); 129.2, 129.0 (cq, arom); 128.6, 128.3 (charom); 123.4 (q, j = 276 hz, cf3); 99.2 (c-1); 71.0 (c-3); 68.8 (c-4); 65.8 (c-5); 65.3 (q, j = 35 hz, ch2cf3); 57.7 (c-2); 16.0 (c-6). Ir (thin film): 2928, 2110, 1724, 1452, 1273, 1261, 1157, 1109, 1094, 1069, 1026 . Hrms: [m + na] calcd for c22h20f3n3o6na 502.1196, found 502.1195 . The title compound was obtained after column chromatography (hexane / et2o 1:0 9:1 v / v) in 45% yield (21 mg, 0.045 mmol, / 19:1). H nmr (400 mhz): 8.08 (d, 2h, j = 7.2 hz, charom); 7.59 (t, 1h, j = 7.6 hz, charom); 7.46 (t, 2h, j = 8.0 hz, charom); 7.347.24 (m, 5h, charom); 5.72 (d, 1h, j = 2.8 hz, h-4); 5.05 (d, 1h, j = 3.6 hz, h-1); 4.85 (d, 1h, j = 10.4 hz, phchh); 4.55 (d, 1h, j = 10.8 hz, phchh); 4.19 (q, 1h, j = 6.4 hz, h-5); 4.11 (dd, 1h, j = 2.8 hz, 10.4 hz, h-3); 4.01 (q, 2h, j = 8.4 hz, ch2cf3); 3.80 (dd, 1h, j = 3.6 hz, 10.4 hz, h-2); 1.24 (d, 3h, j = 6.8 hz, h-6). C - apt nmr (100 mhz): 166.0 (cobz); 137.0 (cq, arom); 133.4, 129.8 (charom); 129.5 (cq, arom); 128.5, 128.4, 128.3, 127.9 (charom); 123.5 (q, j = 277 hz, cf3); 99.1 (c-1); 74.1 (c-3); 71.6 (phch2); 69.6 (c-4); 66.2 (c-5); 65.3 (q, j = 35 hz, ch2cf3); 58.9 (c-2); 16.3 (c-6). Ir (thin film): 2924, 2110, 1721, 1452, 1267, 1157, 1111, 1084, 1055, 1026 . Hrms: [m + h] calcd for c22h23f3n3o5 466.1584, found 466.1581 . The title compounds (/ 7:1) were obtained after column chromatography (hexane / et2o 1:0 4:1 v / v) in 77% yield (36 mg, 0.077 mmol). H nmr (400 mhz): 8.108.06 (m, 2.3h, charom); 7.637.59 (m, 1.2h, charom); 7.497.46 (m, 2.5h, charom); 7.297.21 (m, 6h, charom); 5.54 (dd, 1h, j = 2.8 hz, 11.2 hz, h-3); 5.07 (d, 1h, j = 3.6 hz, h-1); 4.95 (dd, 0.15h, j = 2.8 hz, 11.2 hz, h-3); 4.724.69 (m, 1.15h, phchh, phchh); 4.574.52 (m, 1.15h, phchh, phchh); 4.48 (d, 0.15h, j = 8.0 hz, h-1); 4.11 (q, 1h, j = 6.8 hz, h-5); 4.073.95 (m, 4.45h, h-2, h-4, ch2cf3, h-2, ch2cf3); 3.82 (d, 0.15h, j = 2.8 hz, h-4); 3.68 (q, 0.15h, j = 6.8 hz, c - apt nmr (100 mhz): 165.8 (co bz); 137.3 (cq, arom); 133.6, 129.9 (charom); 129.1 (cq, arom); 128.6, 128.4, 128.3, 128.2, 128.0 (charom); 123.5 (q, j = 277 hz, cf3); 102.0 (c-1); 99.1 (c-1); 76.9 (c-4); 75.6 (phch2); 75.6 (c-4); 75.5 (phch2); 74.6 (c-3); 71.8 (c-3); 71.0 (c-5); 67.2 (c-5); 65.0 (q, j = 35 hz, ch2cf3); 61.1 (c-2); 57.6 (c-2); 16.4 (c-6); 16.3 (c-6). Ir (thin film): 2924, 2110, 1721, 1452, 1267, 1155, 1105, 1096, 1070, 1045, 1026 . The title compounds (/ 19:1) were isolated after column chromatography (hexane / et2o, 1:0 49:1) in 84% yield (42 mg, 0.084 mmol). H nmr (400 mhz): 4.97 (d, 1h, j = 3.2 hz, h-1); 4.01 (dd, 1h, j = 2.0 hz, 10.4 hz, h-3); 3.983.88 (m, 3h, h-5, ch2cf3); 3.79 (dd, 1h, j = 3.6 hz, 10.4 hz, h-2); 3.72 (d, 1h, j = 1.2 hz, h-4); 1.20 (d, 3h, j = 6.4 hz, h-6); 0.96, 0.94 (s, 9h, (ch3)3csi); 0.19 (s, 3h, ch3si); 0.160.15 (m, 6h, ch3si); 0.07 (s, 3h, ch3si). C - apt nmr (100 mhz): 123.7 (q, j = 276 hz, cf3); 98.8 (c-1); 74.9 (c-4); 70.9 (c-3); 68.8 (c-5); 64.7 (q, j = 35 hz, ch2cf3); 60.8 (c-2); 26.2, 26.1 ((ch3)3csi); 18.6, 18.5 (cqsi); 17.2 (c-6); 3.5, 3.8, 4.5, 4.8 (ch3si). Ir: 2932, 2859, 2108, 1279, 1256, 1177, 1045 . The title product was obtained after column chromatography (hexane / et2o, 1:0 9:1 v / v) in 90% yield (43 mg, 0.090 mmol). H nmr (400 mhz): 7.397.25 (m, 5h, charom); 5.04 (d, 1h, j = 11.2 hz, phchh); 4.96 (d, 1h, j = 3.6 hz, h-1); 4.57 (d, 1h, j = 11.2 hz, phchh); 4.10 (dd, 1h, j = 2.4 hz, 10.2 hz, h-3); 3.973.91 (m, 3h, h-5, ch2cf3); 3.81 (dd, 1h, j = 3.6 hz, 10.0 hz, h-2); 3.53 (d, 1h, j = 2.0 hz, h-4); 1.20 (d, 3h, j = 6.4 hz, h-6); 0.98 (d, 9h, (ch3)3csi); 0.24, 0.20 (s, 3h, ch3si). C - apt nmr (100 mhz): 138.4 (cq, arom); 128.3, 128.1, 127.9 (charom); 123.6 (q, j = 277 hz, cf3); 99.0 (c-1); 8.4 (c-4); 75.7 (phch2); 71.1 (c-3); 67.7 (c-5); 64.9 (q, j = 35 hz, ch2cf3); 61.1 (c-2), 25.9 ((ch3)3csi); 18.1 (cqsi); 16.5 (c-6); 4.1, 5.1 (ch3si). Ir (thin film): 2930, 2859, 2108, 1279, 1261, 1163, 1121, 1084, 1045 . The title compounds (/ 1:2) were obtained after column chromatography (hexane / et2o 1:0 9:1 v / v) in 75% yield (34 mg, 0.075 mmol). H nmr (400 mhz): 7.447.25 (m, 30h, charom); 5.02 (d, 1h, j = 3.6 hz, h-1); 4.944.91 (m, 3h, phchh, phchh); 4.774.60 (m, 12h, phch2, phch2); 4.28 (d, 2h, j = 8.0 hz, h-1); 4.023.96 (m, 2h, h-3, h-5); 3.81374 (m, 4h, h-2, h-2, h-4); 3.65 (tt, 2h, j = 7.6 hz, 9.6 hz, chcy); 3.58 (tt, 1h, j = 7.6 hz, 9.6 hz, chcy); 3.51 (d, 2h, j = 2.4 hz, h-5); 3.26 (dd, 2h, j = 2.8 hz, 10.4 hz, h-3), 1.901.62 (m, 12h, ch2,cy); 1.501.34 (m, 10h, ch2,cy); 1.251.15 (m, 15h, h-6, h-6, ch2,cy). C - apt nmr (100 mhz): 138.2, 137.8 (cq, arom); 128.5, 128.5, 128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 127.4, 127.6 (charom); 100.3 (c-1); 96.6 (c-1); 80.9 (c-3); 77.6 (c-3); 77.2 (chcy); 76.2 (c-4); 76.1 (chcy); 74.8 (c-4); 74.5, 72.6, 72.2 (phch2); 70.4 (c-5); 66.5 (c-5); 63.2 (c-2); 59.4 (c-2); 33.3, 31.5, 31.4, 25.6, 25.5, 24.1, 23.9, 23.8 (ch2,cy); 17.0 (c-6); 16.7 (c-6). Ir (thin film): 2932, 2855, 2106, 1454, 1359, 1107, 1067, 1038 . Hrms: [m + nh4] calcd for c26h37n4o4 469.2809, found 469.2810 . The title compounds (/ 1:9) were obtained after column chromatography (hexane / etoac 1:0 9:1 v / v) in 38% yield (18 mg, 0.038 mmol). H nmr (400 mhz): 8.098.07 (m, 2h, charom); 7.897.85 (m, 2h, charom); 7.647.60 (m, 1h, charom); 7.527.45 (m, 3h, charom); 7.32 (t, 2h, j = 7.6 hz, charom); 5.56 (d, 1h, j = 3.6 hz, h-4); 5.15 (dd, 1h, j = 3.6 hz, 10.8 hz, h-3); 4.61 (d, 1h, j = 8.0 hz, h-1); 3.953.88 (m, 2h, h-2, h-5); 3.79 (tt, 1h, j = 7.6 hz, 9.6 hz, chcy); 2.032.01 (m, 2h, ch2,cy); 1.811.79 (m, 2h, ch2,cy); 1.571.43 (m, 3h, ch2,cy); 1.371.22 (m, 8h, h-6, ch2,cy). C - apt nmr (100 mhz): 165.9, 165.4 (cobz); 133.4, 133.3, 129.9, 129.7 (charom); 129.3, 129.1 (cq, arom); 128.5, 128.3 (charom); 100.5 (c-1); 78.3 (chcy); 71.9 (c-3); 70.3 (c-4); 69.4 (c-5); 61.5 (c-2); 33.5, 31.6, 25.5, 24.1, 23.9 (ch2,cy); 16.4 (c-6). Ir (thin film): 2934, 2857, 2110, 1724, 1450, 1281, 1263, 1173, 1107, 1096, 1069, 1026 . The title compounds (/ 1:4) were obtained after column chromatography (hexane / et2o 1:0 9:1 v / v) in 71% yield (33 mg, 0.071 mmol). H nmr (400 mhz): 8.148.07 (m, 2.5h, charom); 7.607.56 (m, 1.25h, charom); 7.487.44 (m, 2.5h, charom); 7.357.24 (m, 6.25h, charom); 5.70 (d, 0.25h, j = 2.4 hz, h-4); 5.52 (dd, 1h, j = 0.8 hz, 3.2 hz, h-4); 5.11 (d, 0.25h, j = 3.6 hz, h-1); 4.84 (d, 0.25h, j = 10.4 hz, phchh); 4.78 (d, 1h, j = 11.6 hz, phchh); 4.564.52 (m, 1.25h, phchh); 4.39 (d, 1h, j = 8.4 hz, h-1); 4.26 (q, 0.25h, j = 7.2 hz, h-5); 4.13 (dd, 0.25h, j = 3.6 hz, 10.6 hz, h-3); 3.743.60 (m, 3.5h, h-2, h-2, h-5, chcy); 3.41 (dd, 1h, j = 3.2 hz, 10.2 hz, h-3); 1.981.77 (m, 5h, ch2,cy); 1.551.43 (m, 4h, ch2,cy); 1.311.20 (m, 7.25h, h-6, h-6, ch2,cy). C - apt (100 mhz): 166.30 (cobz); 137.2 (cq, arom); 133.3, 133.2, 130.1, 129.8 (charom); 129.5 (cq, arom); 128.4, 128.4, 128.2, 128.1, 127.8, 127.8 (charom); 100.3 (c-1); 96.7 (c-1); 78.0 (chcy); 77.5 (c-3); 74.1 (c-3); 71.5 (phch2); 71.5 (phch2); 70.1 (c-4); 69.4 (c-5); 69.0 (c-4); 65.2 (c-5); 62.9 (c-2); 59.2 (c-2); 33.5, 33.3, 31.6, 31.5, 25.5, 24.1, 23.9, 23.8 (ch2,cy); 16.6 (c-6); 16.3 (c-6). Ir (thin film): 2922, 2110, 1720, 1446, 1265, 1107, 1068 . Hrms: the title compounds (/ 1:4) were obtained after column chromatography (hexane / et2o 1:0 9:1 v / v) in 71% yield (35 mg, 0.075 mmol). H nmr (400 mhz): 8.108.06 (m, 10h, charom); 7.627.58 (m, 5h, charom); 7.487.45 (m, 11h, charom); 7.257.18 (m, 28h, charom); 5.59 (dd, 1h, j = 2.4 hz, 11.2 hz, h-3); 5.13 (d, 1h, j = 3.2 hz, h-1); 4.93 (dd, 4h, j = 2.8 hz, 10.8 hz, h-3); 4.714.69 (m, 5h, phchh); 4.574.52 (m, 5h, phchh); 4.47 (d, 4h, j = 8.0 hz, h-5); 3.983.93 (m, 5h, h-2, h-4); 3.88 (dd, 1h, j = 3.4 hz, 11.2 hz, h-2); 3.763.70 (m, 8h, h-4, chcy); 3.663.61 (m, 5h, h-5, chcy); 1.931.75 (m, 22h, ch2,cy); 1.521.43 (m, 18h, ch2,cy); 1.321.18 (m, 38h, h-6, h-6, ch2,cy). C - apt nmr (100 mhz): 165.8 (co bz); 137.6, 137.5 (cq, arom); 133.5, 129.9 (charom); 129.2 (cq, arom); 128.5, 128.3, 128.2, 128.2, 127.8, 127.8 (charom); 100.3 (h-1); 96.7 (h-1); 77.6 (chcy); 77.4 (h-4); 76.5 (chcy); 75.9 (c-4); 75.5 (phch2); 75.3 (phch2); 74.8 (c-3); 72.0 (c-3); 70.4 (c-5); 66.2 (c-5); 61.4 (c-2); 57.8 (c-2); 33.4, 33.3, 31.4, 29.7, 25.5, 23.9, 23.8 (ch2,cy); 16.7 (c-6); 16.4 (c-6). Ir (thin film): 2932, 2857, 2108, 1452, 1265, 1173, 1096, 1069, 1038, 1026 . Hrms: [m + nh4] calcd for c26h35n4o5 483.2602, found 483.2602 . The products (/ 1:3) were obtained after column chromatography (hexane / et2o, 1:0 9:1) in 80% yield (40 mg, 0.080 mmol). H nmr (400 mhz): 5.04 (d, 1h, j = 3.6 hz, h-1); 4.05 (dd, 1h, j = 2.4 hz, 10.4 hz, h-3); 3.94 (q, 1h, j = 6.8 hz, h-5); 3.693.56 (m, 6h, h-2, h-4, ochcy, ochcy); 3.55 (d, 3h, j = 2.4 hz, h-4); 3.51 (dd, 3h, j = 8.0 hz, 10.2 hz, h-2); 3.42 (q, 3h, j = 6.4 hz, h-5); 3.33 (dd, 3h, j = 2.4 hz, 10.4 hz, h-3); 1.971.25 (m, 40h, ch2,cy); 1.22 (d, 9h, j = 6.4 hz, h-6); 0.960.90 (m, 72h, (ch3)3csi); 0.180.08 (m, 48h, ch3si). C - apt nmr (100 mhz): 100.6 (c-1); 96.3 (c-1); 77.4 (chcy); 75.6 (chcy); 74.5 (c-3); 74.0 (c-4); 71.2 (c-3); 71.0 (c-5); 67.8 (c-5); 64.2 (c-2); 60.9 (c-2); 33.5, 33.3, 31.8, 31.4 (ch2,cy); 26.2, 26.1 ((ch3)csi); 25.7, 25.6, 24.1, 24.0, 23.9, 23.7 (ch2,cy); 18.6, 18.5 (cqsi); 17.7 (c-6); 17.3 (c-6); 3.4, 3.6, 4.3, 4.4, 4.5, 4.7 (ch3si). Ir (thin film): 2930, 2857, 2110, 1252, 1115, 1069, 1026 . Hrms: [m + na] calcd for c24h49n3o4si2na 522.3154, found 522.3151 . The title compounds (/ 1:2) were obtained after column chromatography (hexane / et2o, 1:0 19:1) in 80% yield (38 mg, 0.080 mmol). H nmr (400 mhz): 7.397.26 (m, 15h, charom); 5.035.01 (m, 4h, h-1, phchh, phchh); 4.614.55 (m, 3h, phchh, phchh); 4.28 (d, 2h, j = 8.0 hz, h-1); 4.14 (broad doublet, j = 8.4 hz, h-3); 4.00 (q, 1h, j = 6.4 hz, h-5); 3.673.43 (m, 11h, h-2, h-2, h-3, h-4, h-5, ochcy, ochcy); 3.36 (bs, 2h, h-4); 1.891.11 (m, 39h, ch2,cy/, h-6, h-6); 0.98 (s, 9h, (ch3)3csi, 0.96 (s, 18h, (ch3)3csi); 0.230.15 (m, 18h, ch3si/).c - apt nmr (100 mhz): 138.7 (cq, arom); 128.2, 128.2, 128.1, 127.9,127.5, 127.5 (charom); 100.4 (c-1); 96.6 (c-1); 81.0 (c-4); 79.2 (c-4); 77.3 (ochcy); 76.0 (ochcy); 75.6 (phch2); 75.3 (phch2); 74.9 (c-3 or c-5); 71.3 (c-3); 70.2 (c-3 or c-5); 66.6 (c-5); 64.9 (c-2); 61.2 (c-2); 33.4, 33.3, 31.5 (ch2,cy); 25.9, 25.6 (ch3)3csi/); 24.1, 23.9, 23.8 (ch2,cy); 16.9 (c-6); 16.7 (c-6); 3.9, 4.3, 4.7, 5.0 (ch3si). Ir (thin film): 2930, 2857, 2108, 1254, 1115, 1067, 1040 . The product was obtained after size - exclusion chromatography (ch2cl2/meoh, 1:1 v / v) in 68% yield (49 mg, 0.068 mmol). H nmr (400 mhz): 7.537.50 (m, 2h, charom); 7.447.23 (m, 18h, charom); 5.64 (s, 1h, phch); 4.95 (d, 1h, j = 3.6 hz, h-1); 4.90 (d, 1h, j = 11.2 hz, phchh); 4.794.69 (m, 5h, h-1, phch2); 4.59 (d, 1h, j = 11.6 hz, phchh); 4.37 (q, 1h, j = 6.4 hz, h-5); 4.25 (dd, 1h, j = 4.8 hz, 10.2 hz, h-6); 4.194.13 (m, 3h, h-2, h-4, h-3); 3.98 (dd, 1h, j = 3.6 hz, 10.0 hz, h-3); 3.89 (t, 1h, j = 10.4 hz, h-6); 3.78 (dt, 1h, j = 4.8 hz, 9.2 hz, h-5); 3.74 (d, 1h, j = 1.2 hz, h-4); 3.70 (dd, 1h, j = 3.2 hz, 10.8 hz, h-2); 3.36 (s, 3h, och3); 1.70 (d, 3h, j = 6.8 hz, h-6). C - apt nmr (100 mhz): 138.2, 137.6, 137.5 (cq, arom); 128.8, 128.5, 128.3, 128.2, 128.2, 128.1, 127.9, 127.9, 127.7, 127.6, 127.5, 126.1 (charom); 101.5 (phch); 98.8 (c-1); 97.3 (c-1); 78.5 (c-4); 76.5 (c-2 or c-3); 76.0 (c-4); 75.0 (phch2); 74.8 (c-3); 73.5 (c-2 or c-3); 72.7, 71.9 (phch2); 68.8 (c-6); 67.1 (c-5); 64.1 (c-5); 58.9 (c-2); 55.0 (och3); 16.7 (c-6). C - gated (100 mhz): 98.8 (j = 168 hz, c-1); 97.3 (j = 170 hz, c-1). Ir (thin film): 2909, 2108, 1454, 1371, 1101, 1059, 1040, 1003 . The title compounds (/ 4:1) were obtained after size - exclusion chromatography (ch2cl2/meoh, 1:1 v / v), followed by column chromatography (toluene / acetone, 1:0 49:1 v / v) in 38% yield (29 mg, 0.038 mmol). H nmr (400 mhz, for the -isomer): 8.128.09 (m, 2h, charom); 7.607.22 (m, 18h, charom); 5.725.69 (m, 2h, h-3, phch); 5.03 (d, 1h, j = 3.6 hz, h-1); 4.85 (d, 1h, j = 12.0 hz, phchh); 4.77 (d, 1h, j = 1.2 hz, h-1); 4.714.65 (m, 2h, phch2); 4.56 (q, 1h, j = 6.4 hz, h-5); 4.51 (d, 1h, j = 11.2 hz, phchh); 4.304.20 (m, h-4, h-6); 4.17 (dd, 1h, j = 1.6 hz, 3.2 hz, h-2); 3.98 (dd, 1h, j = 3.6 hz, 10.2 hz, h-3); 3.923.87 (m, 2h, h-4, h-6); 3.853.75 (m, 2h, h-2, h-5); 3.76 (s, 3h, och3); 0.98 (d, 3h, j = 6.4 hz, h-6). Diagnostic peaks for the -anomer: 5.62 (s, 0.25h, phch); 3.54 (q, 0.25h, j = 6.4 hz, h-5), 1.22 (d, 0.75h, j = 6.4 hz, h-6). C - apt nmr 100 mhz, for the -isomer): 165.8 (cobz); 137.7, 137.4 (cq, arom); 135.5, 129.9 (charom); 129.2 (cq, arom); 128.7, 128.5, 128.2, 128.1, 128.1, 127.8, 127.6, 127.5, 127.3, 127.1, 126.0 (charom); 101.4 (phch); 98.9 (c-1); 97.4 (c-1); 78.8 (c-4); 77.5 (c-4); 75.6 (phch2); 74.6 (c-3); 74.3 (c-2); 73.0 (phch2); 71.2 (c-3); 68.7 (c-6); 66.7 (c-5); 64.1 (c-5); 57.6 (c-2); 54.9 (och3); 16.1 (c-6). Ir (thin film): 2936, 2110, 1726, 1452, 1273, 1261, 1101, 1070, 1045, 1026, 1006 . Hrms: [m + na] calcd for c41h41n3o11na 774.2631, found 774.2633 . The title compound (/ 10:1) was obtained after size - exclusion chromatography (ch2cl2/meoh, 1:1 v / v) and column chromatography (toluene / acetone, 1:0 49:1 v / v) in 58% yield (43 mg, 0.058 mmol). H nmr (400 mhz): 8.078.05 (m, 2h, charom); 7.587.25 (m, 18h, charom); 5.66 (s, 1h, phch); 5.65 (d, 1h, j = 2.4 hz, h-1); 4.884.80 (m, 2h, phchh 2, h-1); 4.72 (d, 1h, j = 12.0 hz, phchh); 4.65 (q, 1h, j = 6.4 hz, h-5); 4.54 (d, 1h, j = 10.8 hz, phchh); 4.294.23 (m, 2h, h-6, h-3); 4.204.15 (m, 2h, h-2, h-4); 4.02 (dd, 1h, j = 3.2 hz, 10.0 hz, h-3); 3.90 (t, 1h, j = 10.4 hz, h-6); 3.80 (dt, 1h, j = 4.8 hz, 9.6 hz, h-5); 3.59 (dd, 1h, j = 3.6 hz, 10.8 hz, h-2); 3.39 (s, 3h, och3); 1.04 (d, 3h, j = 6.8 hz, h-6). C - apt nmr (100 mhz): 166.1 (cobz); 138.1, 137.6, 137.1 (cq, arom); 133.2, 130.0, 129.8 (charom); 129.6 (cq, arom); 128.9, 128.4, 128.4, 128.3, 128.2, 128.2, 127.8, 127.8, 127.7, 127.1, 126.1 (charom); 101.5 (phch); 98.8 (c-1); 97.3 (c-1); 78.7 (c-2 or c-4); 74.5 (c-3); 74.2 (c-2 or c-4); 73.2 (c-3); 73.1 (phch2); 71.3 (phch2); 69.8 (c-4); 68.8 (c-6); 65.7 (c-5); 64.1 (c-5); 58.7 (c-2); 55.0 (och3); 16.2 (c-6). Ir (thin film): 2932, 2108, 1721, 1452, 1373, 1267, 1175, 1101, 1074, 1061, 1045, 1026, 1003 . The disaccharides (/ 4:1) were isolated after size - exclusion chromatography (ch2cl2/meoh, 1:1 v / v) and column chromatography (toluene / acetone, 1:0 49:1 v / v) in 68% yield (50 mg, 0.068 mmol). H nmr (400 mhz, for the -anomer): 8.11 (d, 2h, j = 7.2 hz, charom); 7.607.22 (m, 18h, charom); 5.725.69 (m, 2h, h-3, phch); 5.03 (d, 1h, j = 3.6 hz, h-1); 4.85 (d, 1h, j = 4.85 hz, phchh); 4.70 (d, 1h, j = 1.2 hz, h-1); 4.704.65 (m, 2h, phch2); 4.56 (q, 1h, j = 6.4 hz, h-5); 4.51 (d, 1h, j = 11.2 hz, phchh); 4.304.20 (m, 2h, h-4, h-6); 4.17 (dd, 1h, j = 1.6 hz, 3.2 hz, h-2); 3.98 (dd, 1h, j = 3.2 hz, 10.2 hz, h-3); 3.923.87 (m, 2h, h-4, h-6); 3.923.75 (m, 2h, h-2, h-5); 3.38 (s, 3h, och3); 0.98 (d, 3h, j = 6.4 hz, h-6). Diagnostic peaks for the -anomer: 5.62 (s, 0.25h, phch); 3.54 (q, 0.25h, j = 6.4 hz, h-5); 3.35 (s, 0.75h, och3); 1.22 (d, 0.75h, j = 6.4 hz, h-6). C - apt nmr (100 mhz, for the -anomer): 165.8 (cobz); 138.4, 137.7, 137.4 (cq, arom); 133.5, 129.9 (charom); 129.2 (cq, arom); 128.8, 128.5, 128.2, 128.1, 127.8, 127.6, 127.5, 127.3, 127.1, 126.0 (charom); 101.4 (phch); 98.9 (c-1); 97.4 (c-1); 78.8 (c-4); 77.5 (c-4); 75.6 (phch2); 74.6 (c-3); 74.3 (c-2); 73.0 (phch2); 71.2 (c-3); 68.7 (c-6); 66.7 (c-5); 64.1 (c-5); 57.6 (c-2); 54.9 (och3); 16.1 (c-6). Ir (thin film): 2934, 2909, 2110, 1722, 1452, 1373, 1269, 1103, 1074, 1043, 1028 . The title compound was obtained after column chromatography (hexane / et2o, 1:0 9:1) as the sole product in 67% yield (52 mg, 0.067 mmol). H nmr (400 mhz): 7.507.49 (m, 2h, charom); 7.397.25 (m, 13h, charom); 5.59 (s, 1h, phch); 4.95 (d, 1h, j = 3.6 hz, h-1); 4.80 (d, 1h, j = 12.4 hz, phchh); 4.744.70 (m, 2h, h-1, phchh); 4.294.25 (m, h-5, h-6); 4.17 (dd, 1h, j = 2.4 hz, 10.4 hz, h-3); 4.124.05 (m, h-2, h-4); 3.96 (dd, 1h, j = 3.2 hz, 9.8 hz, h-3); 3.863.76 (m, 2h, h-5, h-6); 3.71 (dd, 1h, j = 3.2 hz, 10.4 hz, h-2); 3.61 (d, 1h, j = 1.6 hz, h-4); 3.36 (s, 3h, och3); 1.010.99 (m, 12h, h-6, (ch3)3csi); 0.91 (s, 9h, (ch3)3csi); 0.22, 0.16, 0.15, 0.14 (4x s, 3h, ch3si). C - apt nmr (100 mhz): 138.4, 137.6 (cq, arom); 128.8, 128.3, 128.1, 127.5, 127.4, 126.1 (charom); 101.5 (phch); 99.1 (c-1); 97.3 (c-1); 79.0 (c-4); 75.3 (c-4); 74.5 (c-3); 73.6 (c-2); 72.3 (phch2); 70.9 (c-3); 69.0 (c-6); 68.4 (c-5); 64.0 (c-5); 60.9 (c-2); 55.0 (och3); 26.2, 26.1 ((ch3)3csi); 18.6, 18.6 (cqsi); 17.2 (c-6); 3.4, 3.4, 4.6, 4.7 (ch3si). Ir (thin film): 2930, 2857, 2108, 1254, 1177, 1103, 1061, 1042, 1028, 1004 . The title disaccharide was isolated after column chromatography (hexane / et2o, 1:0 4:1) as the sole product in 74% yield (55 mg, 0.074 mmol). H nmr (400 mhz): 7.517.50 (m, 2h, charom); 7.407.24 (m, 13h, charom); 5.62 (s, 1h, phch); 5.00 (d, 1h, j = 11.2 hz, phchh); 4.95 (d, 1h, j = 3.2 hz, h-1); 4.814.70 (m, 3h, h-1, phch2); 4.53 (d, 1h, j = 11.2 hz, phchh); 4.37 (q, 1h, j = 6.4 hz, h-5); 4.294.24 (m, 2h, h-3, h-6); 4.144.09 (m, 2h, h-2, h-4); 3.97 (dd, 1h, j = 3.2 hz, 10.0 hz, h-3); 3.86 (t, 1h, j = 10.4 hz, h-6); 3.78 (dt, 1h, j = 4.4 hz, 9.6 hz, h-5); 3.64 (dd, 1h, j = 3.2 hz, 10.4 hz, h-2); 3.44 (d, 1h, j = 2.4 hz, h-4); 3.36 (s, 3h, och3); 1.04 (d, 3h, j = 6.4 hz, h-6); 0.99 (s, 9h, (ch3)3csi); 0.26 (s, 3h, ch3si); 0.21 (s, 3h, ch3si). C - apt nmr (100 mhz): 138.6, 138.3, 137.6 (cq, arom); 128.8, 128.3, 128.2, 128.1, 127.8, 127.6, 127.5, 127.0, 126.1 (charom); 101.5 (phch); 98.9 (c-1); 97.4 (c-1); 81.0 (c-4); 78.8 (c-4); 75.6 (phch2); 74.6 (c-3); 73.5 (c-2); 72.5 (phch2); 70.7 (c-3); 68.9 (c-6); 67.1 (c-5); 64.1 (c-5); 61.0 (c-2); 55.0 (och3); 25.8 ((ch3)3csi); 18.1 (cqsi); 16.6 (c-6); 3.6, 5.0 (ch3si). Ir (thin film): 2928, 2857, 2106, 1454, 1371, 1258, 1171, 1101, 1040, 1004 . Hrms: [m + na] calcd for c40h53n3o9sina 770.3443, found 770.3441 . The title compound was obtained after size - exclusion chromatography (ch2cl2/meoh, 1:1 v / v) and column chromatography (toluene / acetone, 1:0 9:1 v / v) in 72% yield (52 mg, 0.072 mmol). H nmr (400 mhz): 7.397.22 (m, 20h, charom); 4.954.91 (m, 2h, h-1, phchh); 4.86 (d, 1h, j = 11.6 hz, phchh); 4.72 (d, 1h, j = 12.0 hz, phchh); 4.694.63 (m, 3h, h-1, phch2); 4.53 (d, 1h, j = 11.6 hz, phchh); 4.23 (dd, 1h, j = 4.0 hz, 9.6 hz, h-6); 4.164.09 (m, 3h, h-3, h-4, h-5); 4.00 (m, 2h, h-2, h-3); 3.883.77 (m, 3h, h-2, h-5, h-6); 3.56 (s, 1h, h-4); 3.31 (s, 3h, och3); 0.88 (d, 3h, j = 6.4 hz, h-6). C - apt nmr (100 mhz): 138.3, 138.1, 137.7, 137.6 (cq, arom); 128.9 . 128.6 . 128.4, 128.4, 128.2, 128.1, 127.8, 127.8, 127.6, 127.4, 126.2, 125.9 (charom); 101.8 (phch); 100.3 (c-1); 95.7 (c-1); 78.3 (c-3); 77.1 (c-3 or c-4); 76.3 (c-4); 75.4 (c-2); 74.8, 73.8 (phch2); 73.4 (c-2); 72.1 (phch2); 68.9 (c-6); 66.6 (c-5); 64.2 (c-5); 59.9 (c-2); 54.8 (och3); 16.3 (c-6). Ir (thin film): 2930, 2108, 1454, 1098, 1057, 1026 . The title compound was obtained after size - exclusion chromatography (ch2cl2/meoh, 1:1 v / v) and column chromatography (toluene / acetone, 1:0 49:1 v / v) in 64% yield (48 mg, 0.064 mmol). H nmr (400 mhz): 8.007.98 (m, 2h, charom); 7.907.87 (m, 2h, charom); 7.607.24 (m, 16h, charom); 5.79 (dd, 1h, j = 3.2 hz, 10.0 hz, h-3); 5.64 (s, 1h, phch); 5.55 (d, 1h, j = 2.0 hz, h-4); 5.14 (d, 1h, j = 3.6 hz, h-1); 4.96 (d, 1h, j = 12.0 hz, phchh); 4.78 (d, 1h, j = 12.0 hz, phchh); 4.70 (d, 1h, j = 1.6 hz, h-1); 4.57 (q, 1h, j = 6.4 hz, h-5); 4.27 (dd, 1h, j = 4.4 hz, 9.6 hz, h-6); 4.224.18 (m, 2h, h-3, h-4); 4.10 (dd, 1h, j = 3.6 hz, 11.0 hz, h-2); 3.933.88 (m, 3h, h-2, h-5, h-6); 3.35 (s, 3h, och3); 0.76 (d, 3h, j = 6.4 hz, h-6). C - apt nmr (100 mhz): 165.7, 165.3 (cobz); 138.1, 137.4 (cq, arom); 133.3, 133.2, 129.7 (charom); 129.5, 129.2 (cq, arom); 129.1, 128.5, 128.4, 128.3, 128.2, 128.0, 127.7, 126.3 (charom); 102.1 (phch); 100.4 (c-1); 96.3 (c-1) 77.1 (c-3 or c-4); 75.8 (c-2); 74.8 (c-3 or c-4); 73.9 (phch2); 71.4 (c-4); 70.1 (c-3); 68.8 (c-6); 65.2 (c-5); 64.4 (c-5); 58.8 (c-2); 54.9 (och3); 15.4 (c-6). Ir (thin film): 2932, 2108, 1724, 1452, 1275, 1261, 1098, 1065, 1026, 1005 . The title compound was obtained after size - exclusion chromatography (ch2cl2/meoh, 1:1 v / v) and column chromatography (toluene / acetone, 1:0 49:1 v / v) in 54% yield (40 mg, 0.054 mmol). H nmr (400 mhz): 8.088.06 (m, 2h, charom); 7.617.59 (m, 1h, charom); 7.477.19 (m, 17h, charom); 5.59 (s, 1h, phch); 5.52 (d, 1h, j = 2.8 hz, h-1); 4.94 (d, 1h, j = 11.6 hz, phchh); 4.814.75 (m, 2h, phch2); 4.68 (d, 1h, j = 1.2 hz, h-1); 4.50 (d, 1h, j = 10.8 hz, phchh); 4.41 (q, 1h, j = 6.4 hz, h-5); 4.25 (dd, 1h, j = 3.6 hz, 9.4 hz, h-6); 4.174.12 (m, h-3, h-4, h-3); 3.913.80 (m, 4h, h-2, h-5, h-6, h-2); 3.33 (s, 3h, och3); 0.83 (d, 3h, j = 6.4 hz, h-6). C - apt nmr (100 mhz): 166.1 (cobz); 138.1, 137.6, 137.1 (cq, arom); 133.2, 129.8 (charom); 129.7 (cq, arom); 129.2, 128.4, 128.3, 128.3, 128.1, 127.8, 127.8, 26.2 (charom); 102.1 (phch); 100.4 (c-1); 96.0 (c-1); 77.1, 75.6, 75.0, 74.2 (c-2, c-3, c-4, c-3); 73.8, 71.4 (phch2); 70.0 (c-4); 68.9 (c-6); 65.2 (c-5); 64.3 (c-5); 59.6 (c-2); 54.9 (och3); 15.8 (c-6). Ir (thin film): 2930, 2110, 1721, 1454, 1269, 1110, 1099, 1059, 1026, 1003 . Hrms: [m + na] calcd for c41h43n3o10na 760.2841, found 760.2839 . The products (/ 10:1) were obtained after size - exclusion chromatography (ch2cl2/meoh, 1:1 v / v) and column chromatography (toluene / acetone, 1:0 49:1 v / v) in 64% yield (47 mg, 0.064 mmol). H nmr (400 mhz): 8.088.06 (m, 2h, charom); 7.617.17 (m, 18h, charom); 5.615.57 (m, 2h, h-3, phch); 5.03 (d, 1h, j = 3.6 hz, h-1); 4.93 (d, 1h, j = 12.0 hz, phchh); 4.73 (d, 1h, j = 12.0 hz, phchh); 4.66 (d, 1h, j = 1.6 hz, h-1); 4.58 (d, 1h, j = 11.2 hz, phchh); 4.46 (d, 1h, j = 11.2 hz, phchh); 4.32 (q, 1h, j = 6.4 hz, h-5); 4.25 (dd, 1h, j = 4.0 hz, 9.8 hz, h-6); 4.214.13 (m, 3h, h-3, h-4, h-2); 3.88 (t, 1h, j = 10.0 hz, h-6); 3.833.78 (m, 3h, h-2, h-5, h-4); 3.32 (s, 3h, och3); 0.82 (d, 3h, j = 6.4 hz, h-6). C - apt nmr (100 mhz): 165.8 (cobz); 138.1, 137.6 (cq, arom); 133.5, 129.8 (charom); 129.3 (cq, arom); 128.9, 128.7, 128.5, 128.3, 128.2, 128.1, 128.0, 127.8, 127.7 (charom); 102.0 (phch); 100.4 (c-1); 96.2 (c-1); 77.5 77.1, 75.7, 74.2 (c-2, c-3, c-4, c-4); 75.6, 73.8 (phch2); 73.0 (c-3); 68.8 (c-6); 66.4 (c-5); 64.3 (c-5); 58.8 (c-2); 54.8 (och3; 15.8 (c-6). 2932, 2108, 1722, 1452, 129, 1098, 1067, 1026 . [m + na] calcd for c41h43n3o10 760.2841, found 760.2836 . The title product was obtained after column chromatography (hexane / et2o, 1:0 4:1) in 73% yield (56 mg, 0.073 mmol). H nmr (400 mhz): 7.457.25 (m, 10h, charom); 5.58 (s, 1h, phch); 4.984.95 (m, 2h, h-1, phchh), 4.78 (d, 1h, j = 11.6 hz, phchh); 4.69 (d, 1h, j = 1.6 hz, h-1); 4.24 (dd, 1h, j = 4.0 hz, 9.6 hz, h-6); 4.144.12 (m, 2h, h-3, h-4); 4.074.02 (m, 2h, h-3, h-5); 3.883.82 (m, h-2, h-2, h-5, h-6); 3.55 (d, 1h, j = 1.6 hz, h-4); 3.32 (s, 3h, och3); 0.94 (s, 9h, (ch3)3csi); 0.90 (s, 9h, (ch3)3csi); 0.79 (d, 3h, j = 6.4 hz, h-6); 0.16, 0.13, 0.13, 0.03 (4x s, 3h, ch3si). C - apt nmr (100 mhz): 138.4, 137.8 (cq, arom); 128.9, 128.3, 128.1, 127.9, 127.7, 126.2, 125.8 (charom); 101.9 (phch); 100.7 (c-1); 96.1 (c-1); 77.3 (c-3, c-4); 75.7 (c-2, c-5); 75.3 (c-4); 73.9 (c-3, c-4); 73.9 (phch2); 71.4 (c-3, c-5); 68.9 (c-6); 68.0 (c-3, c-5); 64.4 (c-2, c-5); 61.8 (c-2); 54.8 (och3); 26.3, 26.1 ((ch3)3csi); 18.6, 18.5 (cqsi); 16.8 (c-6); 3.4, 4.0, 4.5, 4.6 (ch3si). Ir (thin film): 2953, 2857, 2106, 1254, 1179, 1121, 1099, 1049, 1028 . The title compounds (/ 10:1) were obtained after column chromatography (hexane / et2o, 1:0 4:1) in 64% yield (48 mg, 0.064 mmol). H nmr (400 mhz): 7.467.24 (m, 15h, charom); 5.58 (s, 1h, phch); 5.004.92 (m, 3h, h-1, 2 phchh); 4.78 (d, 1h, j = 11.6 hz, phchh); 4.70 (d, 1h, j = 1.6 hz, h-1); 4.50 (d, 1h, j = 10.8 hz, phchh); 4.42 (dd, 1h, j = 4.0 hz, 9.6 hz, h-6); 4.164.06 (m, 4h, h-3, h-3, h-4, h-5); 3.90 (dd, 1h, j = 3.2 hz, 10.2 hz, h-2); 3.883.79 (m, 3h, h-2, h-5, h-6); 3.363.32 (m, 4h, h-4, och3); 0.96 (s, 9h, (ch3)3si); 0.80 (d, 3h, j = 6.4 hz, h-6); 0.21 (s, 3h, ch3si); 0.15 (s, 3h, ch3si). C - apt nmr (100 mhz): 138.7, 138.3, 137.7 (cq, arom); 128.9, 128.6, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.5, 127.4, 126.2, 125.8 (charom); 101.9 (phch); 100.5 (c-1); 96.5 (c-1); 81.0 (c-4); 77.2 (c-4); 75.8 (c-2); 75.6 (phch2); 74.4 (c-3 or c-3); 73.8 (phch2); 71.8 (c-3 or c-3); 68.9 (c-6); 66.9 (c-5); 63.3 (c-5); 62.3 (c-2); 54.8 (och3); 25.9 ((ch3)3csi); 18.0 (cqsi); 16.1 (c-6); 4.2, 5.0 (ch3si). Ir (thin film): 2930, 2857, 2106, 1454, 1364, 1260, 1117, 1098, 1047, 1028 . Hrms: [m + na] calcd for c40h53n3nao9si 770.3443, found 770.3442 . To a solution of d-6 (0.29 g, 0.54 mmol, 2.0 equiv), ph2so (0.11 g, 0.54 mmol, 2.0 equiv), and ttbp (0.27 g, 1.08 mmol, 4.0 equiv) in ch2cl2 (5.4 ml, 0.1 m relative to donor) were added flame - dried, rod - shaped, 3 ms . After 30 min, the mixture was cooled to 80 c, tf2o (91 l, 0.54 mmol, 2.0 equiv) was added, and the mixture was warmed to 70 c . The mixture was recooled to 80 c, and a solution of acceptor 19 (0.17 g, 0.27 mmol, 1.0 equiv) in ch2cl2 (0.54 ml, 0.5 m) was added via the wall of the flask . The mixture was warmed to 40 c and kept at this temperature for 2 h, after which the reaction was quenched by addition of pyridine (0.4 ml), filtered over a pad of celite, washed with brine, dried over mgso4, filtered, and concentrated in vacuo . Purification by column chromatography (hexane / etoac, 19:1 7:3 v / v) yielded the title disaccharide in 68% yield (0.17 g, 0.167 mmol). H nmr (500 mhz, 323 k): 7.437.23 (m, 20h, charom); 5.16 (s, 2h, phch2); 5.05 (d, 1h, j = 3.0 hz, h-1); 4.994.97 (m, 2h, h-1, phchh); 4.82 (d, 1h, j = 11.5 hz, phchh); 4.66 (d, 1h, j = 12.0 hz, phchh); 4.60 (s, 1h, h-4); 4.514.48 (m, 3h, phch2); 4.31 (s, 1h, h-5); 4.17 (q, 1h, j = 6.5 hz, h-5); 4.07 (dd, 1h, j = 2.5 hz, 10.5 hz, h-3); 3.90 (bd, 1h, j = 10.5 hz, h-3); 3.81 (s, 3h, co2ch3); 3.74 (dd, 1h, j = 3.5 hz, 10.5 hz, h-2); 3.63 (bs, 1h, ochhpentyl); 3.61 (dd, 1h, j = 3.5 hz, 10.5 hz, h-2); 3.47 (bs, 1h, ochhpentyl); 3.40 (s, 1h, h-4); 3.22 (bs, 2h, nch2,pentyl); 1.52 (bs, 4h, ch2,pentyl); 1.341.27 (m, 2h, ch2,pentyl); 0.990.93 (m, 12h, h-6, c(ch3)3si); 0.21, 0.18 (s, 3h, ch3si). C - apt nmr (125 mhz, 323 k): 168.6 (c-6); 138.7, 138.0, 137.3 (cq, arom); 128.8, 128.5, 128.4, 128.4, 128.2, 127.9, 127.8, 127.8, 127.7, 127.5, 127.2 (charom); 99.2 (c-1); 98.2 (c-1); 81.0 (c-4); 75.6 (phch2); 75.0 (c-3); 73.8 (c-4); 72.0 (phch2); 71.1 (c-3); 70.1 (c-5); 68.8 (och2,pentyl); 67.6 (c-5); 67.1 (phch2); 62.0 (c-2); 59.4 (c-2); 52.4 (co2ch3); 50.5 (phch2); 29.0 (ch2,pentyl); 25.9 (c(ch3)3si); 23.2 (ch2,pentyl); 18.1 (cqsi); 16.5 (c-6); 3.9, 5.0 (ch3si). C - gated nmr (125 mhz, 323 k): 99.2 (d, j = 170 hz, c-1); 98.2 (d, j = 172 hz, c-1). Ir (thin film): 2930, 2106, 1730, 1697, 1454, 1254, 1125, 1067, 1042 . Hrms: [m + na] calcd for c53h69n7nao11si 1030.4717, found 1030.4721 . To a stirred solution of donor d-6 (0.80 g, 1.5 mmol, 1.0 equiv), ph2so (0.39 g, 1.95 mmol, 1.3 equiv), n - methylmaleimide (0.25 g, 2.25 mmol, 1.5 equiv), and ttbp (0.93 g, 3.75 mmol, 2.5 equiv) in ch2cl2 (30 ml, 0.05 m) were added, flame - dried, rod - shaped, 3 ms . After being stirred for 30 min, the mixture was cooled to 80 c, tf2o (0.33 ml, 1.95 mmol, 1.3 equiv) was added, and the mixture was warmed to 70 c, after which tlc analysis (toluene / etoac, 9:1 v / v) indicated complete activation of the donor . The mixture was recooled to 80 c, after which bu4ni (as a 1 m solution in ch2cl2, 7.5 ml, 7.5 mmol, 5.0 equiv) was added, upon which the reaction mixture assumed a maroon color . After 5 min at 80 c, a solution of acceptor 21 (as a 0.5 m solution in ch2cl2/1,4-dioxane, 1:1 v / v, 3.0 mmol, 2.0 equiv) was slowly added via the wall of the flask, and the mixture was allowed to warm to room temperature . After the mixture was stirred for 18 h, tlc analysis (toluene / etoac, 9:1 v / v) indicated complete conversion of the starting material . The reaction was quenched by addition of net3, diluted with ch2cl2, filtered over a pad of celite, washed (brine, 1), dried over mgso4, filtered, and concentrated in vacuo . Purification by column chromatography (toluene / et2o, 1:0 9:1) delivered 22 (/ 7:1) as an inseparable mixture in 85% yield (0.90 g, 1.27 mmol). To a stirred solution of 22 (0.90 g, 1.27 mmol, 1.0 equiv) in thf (4 ml, 0.3 m) was added bu4nf (as a 1 m solution in thf, 2.5 ml, 2.5 mmol, 2.0 equiv). After tlc analysis (pe / etoac, 7:3 v / v) indicated complete consumption of the starting material (2 h), the reaction was quenched by addition of nahco3 (satd aq) and extracted with etoac (3). The combined organic phases were washed (h2o 1, brine 1), dried over mgso4, filtered, and concentrated in vacuo . Purification by column chromatography (pe / etoac, 19:1 4:1 v / v) delivered the title compound as an oil in 63% yield (0.47 g, 0.80 mmol). H nmr (500 mhz, 323 k): 7.367.20 (m, 15h, charom); 5.17 (s, 2h, phch2); 4.82 (d, 1h, j = 3.0 hz, h-1); 4.77 (d, 1h, j = 11.5 hz, phchh); 4.70 (d, 1h, j = 11.5 hz, phchh); 4.48 (s, 2h, phch2); 4.03 (bd, 1h, j = 8.0 hz, h-3); 3.92 (q, 1h, j = 6.0 hz, h-5); 3.623.56 (m, 2h, h-4, ochhpentyl); 3.153.39 (m, h-2, ochhpentyl); 3.24 (bs, nch2,pentyl); 1.54 (bs, 4h, ch2,pentyl); 1.31 (bs, 2h, ch2,pentyl); 1.24 (d, 3h, j = 6.5 hz, h-6). C - apt nmr (125 mhz, 323 k): 138.0, 136.9 (cq, arom); 128.6, 128.5, 128.4, 128.0, 127.9, 127.8, 127.5, 127.2, 126.9 (charom); 98.2 (c-1); 80.3 (c-4); 76.1 (phch2); 68.5 (c-3); 68.1 (och2,pentyl); 67.2 (phch2); 66.5 (c-5); 61.0 (c-2); 50.5 (nch2,pentyl); 29.1, 23.4 (ch2,pentyl); 16.7 (c-6). C - gated nmr (125 mhz, 323 k): 98.2 (d, j = 170 hz, c-1). Ir (thin film): 2934, 2108, 1690, 1454, 1421, 1229, 1171, 1067 . Hrms: [m + h] calcd for c33h41n4o6 589.3021, found 589.3022 . To a stirred solution of donor 6 (0.43 g, 0.80 mmol, 2.0 equiv), ph2so (0.16 g, 0.80 mmol, 2.0 equiv), and ttbp (0.40 g, 1.60 mmol, 4.0 equiv) in ch2cl2 (8 ml, 0.1 m relative to donor) were added flame - dried, rod - shaped, 3 ms . After being stirred for 30 min, the mixture was cooled to 80 c, and tf2o (0.13 ml, 0.80 mmol, 2.0 equiv) was added . After the mixture was allowed to warm to 70 c, it was recooled to 80 c, and a solution of acceptor 23 (0.40 mmol, 1.0 equiv, in 0.8 ml ch2cl2, dried by triple coevaporation with toluene) was added via the wall of the flask . The mixture was warmed to 60 c, left at this temperature for 15 min, and the reaction was quenched by addition of net3 . The bright yellow solution was filtered over celite, washed (brine, 1), dried over mgso4, filtered, and concentrated in vacuo . Purification by size - exclusion chromatography (ch2cl2/meoh, 1:1 v / v) yielded 24 as a mixture of disaccharides (/ 7:1) in 73% yield (0.27 g, 0.28 mmol). H nmr for the -anomer (500 mhz, 323 k): 7.417.21 (m, 20h, charom); 5.24 (d, 1h, j = 3.5 hz, h-1); 5.16 (bs, 2h, phch2); 5.03 (d, 1h, j = 11.5 hz, phchh); 4.91 (d, 1h, j = 11.5 hz, phchh); 4.88 (d, 1h, j = 3.5 hz, h-1); 4.62 (d, 1h, j = 11.0 hz, phchh); 4.55 (d, 1h, j = 11.0 hz, phchh); 4.48 (bs, 2h, phch2); 4.10 (dd, 1h, j = 3.0 hz, 10.5 hz, h-3); 4.06 (dd, 1h, j = 3.0 hz, 11.0 hz, h-3); 3.99, 3.92 (q, 1h, j = 6.5 hz, h-5, h-5); 3.843.81 (m, 2h, h-2, h-2); 3.603.56 (m, 2h, h-4, ochhpentyl); 3.47 (d, 1h, j = 1.5 hz, h-4); 3.40 (bs, 1h, ochhpentyl); 3.23 (bs, 2h, nch2,pentyl); 1.53 (m, 4h, ch2,pentyl); 1.30 (m, 2h, ch2,pentyl); 1.20, 1.16 (d, 3h, j = 6.5 hz, h-6, h-6); 0.96 (s, 9h, (ch3)3csi); 0.20, 0.14 (s, 3h, ch3si). C - apt nmr (125 mhz, 323 k): 138.7, 138.5, 138.1 (cq, arom); 128.5, 128.4, 128.4, 128.3, 127.9, 127.9, 127.8, 127.7, 127.6, 127.6, 127.3 (charom); 99.5 (c-1); 98.3 (c-1); 80.8 (c-4); 79.9 (c-4); 76.2 (c-3); 75.6, 75.2 (phch2); 71.5 (c-3); 68.3 (och2,pentyl); 67.8 (c-5 or c-5); 67.2 (phch2); 66.9 (c-5 or c-5); 61.8, 60.4 (c-2, c-2); 29.2 (ch2,pentyl); 25.9 ((ch3)3csi); 23.4 (ch2,pentyl); 18.1 (cqsi); 16.8, 16.7 (c-6, c-6); 4.1, 5.0 (ch3si). Diagnostic h nmr signal for the -anomer (500 mhz, 323 k): 4.32 (d, 1h, j = 8.0 hz, h-1). Disaccharide 24 was dissolved in dry thf (1.4 ml, 0.2 m), and bu4nf (as 1 m solution in thf, 0.34 ml, 0.34 mmol, 1.2 equiv) was added . After 4 h, tlc analysis (pe / etoac, 7:3 v / v) indicated complete consumption of the starting material and the appearance of two more polar products . The reaction was quenched by addition of satd aq nahco3, the mixture was extracted (ch2cl2, 3), and the combined organics were washed (h2o, 1; brine, 1), dried over mgso4, and concentrated in vacuo . Purification by column chromatography (hexane / etoac, 19:1 4:1 v / v) furnished the title disaccharide in 71% yield (0.17 g, 0.20 mmol). H nmr (500 mhz, 323 k): 7.367.19 (m, 20h, charom); 5.20 (d, 1h, j = 4.0 hz, h-1); 5.17 (bs, 2h, phch2); 4.89 (d, 1h, j = 3.0 hz, h-1); 4.79 (d, 1h, j = 11.5 hz, phchh); 4.75 (d, 1h, j = 11.5 hz, phchh); 4.724.67 (m, 2h, 2 phchh); 4.49 (bs, 2h, phch2); 4.04 (dd, 1h, j = 3.0 hz, 10.8 hz, h-3); 3.933.90 (m, 3h, h-3, h-5, h-5); 3.84 (dd, 1h, j = 3.5 hz, 10.5 hz, h-2); 3.59 (bs, 1h, ochhpentyl); 3.55 (d, 1h, j = 2.5 hz, h-4); 3.52 (d, 1h, j = 2.5 hz, h-4); 3.49 5(dd, 1h, j = 3.5 hz, 10.8 hz, h-2); 3.41 (bs, 1h, ochhpentyl); 3.24 (bs, 2h, nch2pentyl); 1.55 (bs, 4h, 2 ch2,pentyl); 1.31 (bs, 2h, ch2,pentyl); 1.231.20 (m, 6h, h-6, h-6). C - apt nmr (125 mhz, 323 k): 138.4, 138.0, 137.9, 136.9 (cq, arom); 128.6, 128.5, 128.4, 128.4, 128.1, 128.1, 127.9, 127.8, 127.7, 127.6, 127.3 (charom); 99.8 (c-1); 98.2 (c-1); 80.4 (c-4); 79.8 (c-4); 76.2 (c-3); 76.0, 75.5 (phch2); 68.7 (c-3, c-5 or c-5); 68.2 (och2,pentyl); 67.4 (c-3, c-5 or c-5); 67.2 (phch2); 67.0 (c-3, c-5 or c-5); 61.0 (c-2); 60.4 (c-2); 29.1 (2 ch2,pentyl); 23.4 (ch2,pentyl); 16.8, 16.8 (c-6, c-6). C - gated nmr (125 hz): 99.8 (d, j = 170 hz, c-1); 98.2 (d, j = 168 hz, c-1). Ir (thin film): 2936, 2106, 1694, 1454, 1422, 1092, 1036, 1028 . Hrms: [m + nh4] calcd for c46h59n8o9 867.4400, found 867.4403 . To a stirred, ice - cooled solution of phenyl 2-azido-3-o - benzyl-4,6-o - benzylidene-2-deoxy-1-thio--d - mannopyranoside (4.9 g, 10.3 mmol, 1.0 equiv) in ch2cl2 (30 ml, 0.3 m) was added, under argon, bh3.thf (as 1 m solution in thf, 30 ml, 3.0 equiv), followed by bu2botf (as 1 m solution in ch2cl2, 10 ml, 1.0 equiv) and the reaction mixture was kept at 0 c . After 1 h, tlc analysis (pe / etoac, 4:1 v / v) indicated complete conversion of the starting material, and the reaction was quenched by sequential addition of net3 (4 ml) and meoh (added dropwise until gas evolution ceased). The mixture was concentrated in vacuo and the residue was coevaporated with meoh (3). Purification by column chromatography (pe / etoac, 19:1 9:1 v / v) delivered phenyl 2-azido-3,4-di - o - benzyl-2-deoxy-1-thio--d - mannopyranoside as a colorless oil (4.6 g, 9.7 mmol, 94% yield). H nmr (400 mhz): 7.427.22 (m, 15h, charom); 5.40 (s, 1h, h-1); 4.90 (d, 1h, j = 10.8 hz, phchh); 4.75 (s, 2h, phch2); 4.66 (d, 1h, j = 11.2 hz, phchh); 4.154.04 (m, 3h, h-2, h-3, h-5); 3.92 (t, 1h, j = 9.6 hz, h-4); 3.77 (bs, 2h, h-6); 1.88 (bs, 1h, 6-oh). C - apt nmr (100 mhz): 137.9, 137.3, 132.9 (cq, arom); 132.1, 129.2, 128.6, 128.4, 128.1, 128.0, 127.9 (charom); 86.2 (c-1); 79.8 (c-2, c-3 or c-5); 75.4 (phch2); 74.2 (c-4); 73.2 (c-2, c-3 or c-5); 72.7 (phch2); 62.7 (c-2, c-3 or c-5); 61.7 (c-6). Ir (thin film): 2872, 2102, 1454, 1265, 1096, 1084, 1026 . Hrms: [m + na] calcd for c26h27n3nao4s 500.1615, found 500.1611 . The primary alcohol (1.17 g, 2.6 mmol, 1.0 equiv) was dissolved in ch2cl2 (8 ml), and h2o (4 ml) and tert - butyl alcohol (1 ml, final concentration 0.2 m) were added . Under vigorous stirring were added acoh (15 l, 0.26 mmol, 0.1 equiv), tempo (81 mg, 0.52 mmol, 0.2 equiv), and phi(oac)2 (2.09 g, 6.5 mmol, 2.5 equiv), and the resulting red mixture was stirred until tlc analysis (pe / etoac / acoh, 75:20:5 v / v / v) indicated complete conversion of the starting material into a lower running spot (90 min). The reaction was quenched by addition of satd aq na2s2o3, and the resulting light yellow mixture was extracted (ch2cl2, 2). The combined organic fractions were washed (h2o 1, brine 1), dried over mgso4, filtered, and concentrated in vacuo . After coevaporation with toluene (1), the residue was dissolved in dmf (9 ml, 0.3 m), and mei (0.32 ml, 5.2 ml, 2.0 equiv) and k2co3 (0.72 g, 5.2 mmol, 2.0 equiv) were added . The reaction was stirred overnight, after which tlc analysis (pe / etoac / acoh, 70:30:5 v / v / v) indicated complete conversion of the starting material . The reaction mixture was partitioned between et2o and h2o, and after separation, the aqueous phase was extracted with et2o (2). The combined ethereal phases were washed (brine 1), dried over mgso4, filtered, and concentrated in vacuo . The residue was purified by column chromatography (pe / etoac, 1:0 9:1 v / v) to deliver methyl (phenyl 2-azido-3,4-di - o - benzyl-2-deoxy-1-thio--d - mannopyranosiduronate) as an oil in 79% yield (1.04 g, 2.06 mmol). H nmr (400 mhz): 7.627.60 (m, 2h, charom); 7.387.25 (m, 13h, charom); 5.61 (d, 1h, j = 7.6 hz, h-1); 4.694.59 (m, 5h, h-5, phch2); 4.21 (dd, 1h, j = 4.4 hz, 5.6 hz, h-4); 3.93 (dd, 1h, j = 3.2 hz, 5.6 hz, h-3); 3.72 (dd, j = 2.4 hz, 7.6 hz, h-2); 3.54 (s, 3h, och3). C - apt nmr (100 mhz): 169.3 (c-6); 137.3, 136.8 (cq, arom); 132.4 (charom); 132.0 (cq, arom); 128.9, 128.5, 128.4, 128.1, 128.0, 127.8, 127.7); 74.6 (c-4); 73.0 (phch2); 72.9 (c-5); 58.7 (c-2); 52.2 (och3). The c-1 and c-3 were not observable at room temperature due to signal broadening . Ir (thin film): 2870, 2102, 1749, 1454, 1439, 1265, 1119, 1094, 1078, 1024 . Hrms: [m + na] calcd for c27h27n3nao5s 528.1564, found 528.1559 . To a solution of the methyl uronate (0.56 g, 1.12 mmol, 1.0 equiv) in acetone (8.4 ml) was added h2o (2.8 ml, final concentration 0.1 m). At 0 c, nbs (0.60 g, 3.35 mmol, 3.0 equiv) was added, and the reaction mixture assumed an orange - brown color . After 90 min, a second portion of nbs (0.60 g, 3.35 mmol, 3.0 equiv) was added, and the mixture was stirred for an additional 15 min, after which tlc (pe / etoac, 7:3 v / v) indicated complete conversion of the starting material . The reaction was quenched by addition of satd aq na2so3, and the mixture was extracted (etoac, 3). The combined organic phases were washed (h2o 1, brine 1), dried over mgso4, filtered, and concentrated in vacuo . Methyl 2-azido-3,4-di - o - benzyl-2-deoxy-/-d - mannopyranosiduronate was obtained after column chromatography (pe / etoac, 9:1 7:3 v / v), with the -product predominating (/ 1:9) in 82% yield (0.38 g, 0.92 mmol). H nmr (400 mhz, 323 k): 7.297.22 (m, 10h, charom); 5.42 (d, 1h, j = 3.6 hz, h-1); 4.65 (s, 2h, phch2); 4.624.54 (m, 2h, phch2); 4.49 (d, 1h, j = 5.6 hz, h-5); 4.33 (bs, 1h, 1-oh); 4.12 (t, 1h, j = 5.6 hz, h-4); 3.96 (bd, 1h, j = 3.2 hz, h-3); 3.76 (bs, 1h, h-2); 3.55 (s, 3h, och3).c - apt nmr (100 mhz, 323 k): 169.7 (c-6); 137.5, 137.3 (cq, arom); 128.5, 128.4, 128.3, 128.1, 127.9, 127.8, 127.7 (charom); 92.1 (c-1); 77.4 (c-3); 75.2 (c-4); 73.5, 72.8 (phch2); 72.3 (c-5); 61.0 (c-2); 52.1 (och3). C - gated nmr (100 mhz, 323 k): 92.1 (d, j = 170 hz, h-1). Ir (thin film) 3439, 2104, 1747, 1454, 1437, 1281, 1240, 1123, 1093, 1072, 1024 . Hrms: [m + na] calcd for c21h23n3nao6 436.1479, found 436.1476 . To a solution of the reducing sugar (0.30 g, 0.73 mmol, 1.0 equiv) in acetone (2.5 ml, 0.3 m) were added n - phenyl 2,2,2-trifluoroimidoyl chloride (0.17 ml, 1.1 mmol, 1.5 equiv) and cs2co3 (0.29 g, 0.88 mmol, 1.2 equiv), and the mixture was stirred until tlc analysis (pe / etoac, 4:1 v / v) indicated complete conversion of the starting material (2 h). The reaction mixture was diluted with acetone, filtered, and concentrated in vacuo . Purification of the residue by column chromatography (pe / etoac / net3, 100:0:1 90:10:1) delivered the title compound as a mixture of anomers and/or conformers, in 88% yield (0.38 g, 0.64 mmol). H nmr (major isomer, 400 mhz, 323 k): 7.347.25 (m, 13h, charom); 7.11 (t, 1h, j = 7.6 hz, charom); 6.80 (d, 2h, j = 8.0 hz, charom); 6.30 (bs, 1h, h-1); 4.774.64 (m, 4h, phch2); 4.38 (d, 1h, j = 7.2 hz, h-4); 4.02 (dd, 1h, j = 2.8 hz, 7.6 hz, h-3); 3.87 (bs, 1h, h-2); 3.66 (s, 3h, och3). C - apt nmr (100 mhz, 323 k): 168.5 (c-6); 143.1, 137.5, 137.1 (cq, arom); 128.8, 128.6, 128.5, 128.3, 128.2, 128.1, 128.0, 127.8, 124.6, 124.4, 119.4 (charom); 94.4 (c-1); 77.8 (c-3); 74.9 (c-4); 74.6 (phch2); 73.8 (c-5); 73.5 (phch2); 59.8 (c-2); 52.4 (och3). Diagnostic h nmr signals for the minor isomer (400 mhz, 323 k): 6.00 (bs, 0.2h, h-1); 4.30 (t, 0.2h, j = 6.8 hz, h-4); 3.91 (bs, 0.2h, h-2); 3.62 (s, 0.6h, och3). Ir (thin film): 2110, 1751, 1717, 1317, 1207, 1161, 1115, 1026 . Hrms: [m + na] calcd for a three - necked, 250 ml flask, equipped with a thermometer, rubber septa, an argon balloon, and a gas outlet, was charged with a solution of donor d-4 (2.86 g, 5.5 mmol, 1.0 equiv), ph2so (1.44 g, 7.1 mmol, 1.3 equiv), and ttbp (3.39 g, 13.4 mmol, 2.5 equiv) in ch2cl2/et2o (110 ml, 1:1 v / v, 0.05 m). Flame - dried 3 ms were added, and the mixture was stirred at room temperature for 30 min . After the mixture was cooled to 80 c, tf2o (1.2 ml, 7.1 mmol, 1.3 equiv) was added and the mixture was allowed to warm to 60 c . After the mixture was recooled to 80 c, a solution of 21 (3.58 g, 10.9 mmol, 2.0 equiv) in ch2cl2/et2o (20 ml, 1:1 v / v) was added via cannula transfer (reaction mixture temperature did not exceed 70 c during addition). The mixture was allowed to warm to 40 c, after which the reaction was quenched by addition of net3 (4 ml), filtered over a pad of celite, diluted with ch2cl2, washed with brine, dried over mgso4, filtered, and concentrated in vacuo . Column chromatography (pe / etoac, 49:1 9:1 v / v) first furnished a mixture of / products (0.77 g, 1.1 mmol, 20% yield, / 1:3); when all higher - running -product had eluted off, further elution (pe / etoac, 4:1 v / v) delivered pure -product as a colorless oil (2.43 g, 3.5 mmol, 60%). H nmr (400 mhz, 323 k): 8.06 (d, 2h, j = 7.6 hz, charom); 7.59 (t, 1h, j = 7.6 hz, charom); 7.46 (t, 2h, j = 7.6 hz, charom); 7.307.17 (m, 14h, charom); 5.17 (bs, 2h, phch2); 4.93 (dd, 1h, j = 2.8 hz, 10.8 hz, h-3); 4.68 (d, 1h, j = 11.6 hz, phchh); 4.55 (d, 1h, j = 11.6 hz, phchh); 4.49 (s, 2h, phch2); 4.29 (d, 1h, j = 8.0 hz, h-1); 3.963.91 (m, 2h, h-2, ochhpentyl); 3.78 (d, 1h, j = 2.0 hz, h-4); 3.63 (q, 1h, j = 6.4 hz, h-5); 3.48 (bs, 1h, ochhpentyl); 3.23 (ch2npentyl); 1.61 (bs, 4h, 2 ch2,pentyl); 1.35 (bs, 2h, 2h, ch2,pentyl); 1.25 (d, 3h, j = 6.4 hz, h-6). C - apt nmr (100 mhz, 323 k): 165.9 (cobz); 138.1, 137.7 (cq, arom); 133.5, 129.9 (charom); 129.4 (cq, arom); 128.6, 128.5, 128.4, 128.3, 128.2, 127.9, 127.8, 127.3 (charom); 102.3 (c-1); 76.3 (c-4); 75.5 (phch2); 75.0 (c-3); 70.6 (c-5); 69.8 (och2,pentyl); 67.2 (phch2); 61.6 (c-2); 50.7 (phch2); 29.2, 23.3 (ch2,pentyl); 16.7 (c-6). Ir (thin film): 2937, 2110, 1719, 1695, 1452, 1421, 1265, 1096, 1069, 1026 . Hrms: [m + na] calcd for c40h44n4nao7 715.3102, found 715.3097 . To a stirred solution of 29 (2.43 g, 3.5 mmol, 1.0 equiv) in dry meoh (18 ml, 0.2 m) was added a chip of na metal . The reaction mixture was stirred at room temperature until tlc analysis (pe / etoac, 7:3 v / v) indicated complete conversion of the starting material . The reaction mixture was neutralized by addition of amberlite ir-120 (h form), filtered, and concentrated in vacuo . The title compound was obtained after column chromatography (pe / etoac) as a colorless oil in 95% yield (1.94 g, 3.3 mmol). H nmr (400 mhz, 323 k): 7.367.22 (m, 15h, charom); 5.17 (s, 2h, phch2); 4.80 (d, 1h, j = 11.6 hz, phchh); 4.72 (d, 1h, j = 11.6 hz, phchh); 4.48 (s, 2h, phch2); 4.16 (d, 1h, j = 7.2 hz, h-1); 3.86 (bs, 1h, ochhpentyl); 3.533.43 (m, 5h, ochhpentyl, h-2, h-3, h-4, h-5); 3.23 (bs, 2h, nch2,pentyl); 2.17 (d, 1h, j = 9.2 hz, 3-oh); 1.571.53 (m, 4h, ch2,pentyl); 1.441.43 (m, 2h, ch2,pentyl); 1.26 (d, 3h, j = 7.2 hz, h-6). C - apt nmr (100 mhz, 323 k): 138.1 (cq, arom); 128.5, 128.5, 128.4, 128.2, 128.0, 127.9, 127.3 (charom); 102.3 (c-1); 78.6 (c-3, c-4, or c-5); 76.0 (phch2); 73.2, 70.9 (c-3, c-4 or c-5); 69.7 (och2,pentyl); 67.2 (phch2); 64.8 (c-2); 29.3, 23.3 (ch2,pentyl); 16.9 (c-6). Ir (thin film): 2934, 2108, 1690, 1454, 1421, 1229, 1171, 1067 . Hrms: [m + na] calcd for c33h40n4nao6 611.2840, found 611.2833 . To a stirred solution of 5 (1.22 g, 2.2 mmol, 2.0 equiv), ph2so (0.44 g, 2.2 mmol, 2.0 equiv), and ttbp (1.09 g, 4.4 mmol, 4.0 equiv) in ch2cl2 (22 ml, 0.1 m relative to donor) were added flame - dried, rod - shaped, 3 molecular sieves . After 30 min at room temperature, the reaction mixture was cooled to 80 c, after which tf2o (0.37 ml, 2.2 mmol, 2.0 equiv) was added . The reaction mixture was allowed to warm to 70 c, after which it was recooled to 80 c . A solution of acceptor 30 (dried by triple coevaporation with toluene, 0.65 g, 1.1 mmol, 1.0 equiv) in ch2cl2 (2.2 ml) was added via the wall of the flask, and the mixture was allowed to warm to 60 c, after which it was stirred at this temperature for 15 min . The reaction was stopped by addition of net3 (1 ml), and the mixture filtered over a pad of celite, washed with brine (1), dried over mgso4, filtered, and concentrated in vacuo . Purification of the residue by column chromatography (toluene / etoac, 1:0 9:1 v / v) and size - exclusion chromatography (ch2cl2/meoh, 1:1 v / v) furnished the title compound as an oil (0.83 g, 0.84 mmol, 76%). H nmr (400 mhz, 323 k): 7.347.21 (m, 15h, charom); 5.36 (d, 1h, j = 3.6 hz, h-1); 5.16 (s, 2h, phch2); 4.97 (d, 1h, j = 11.6 hz, phchh); 4.58 (d, 1h, j = 11.2 hz, phchh); 4.48 (s, 2h, phch2); 4.17 (d, 1h, j = 8.0 hz, h-1); 4.99 (bd, 1h, j = 10.0 hz, h-5); 3.853.80 (m, 2h, ochhpentyl, h-2); 3.773.71 (m, 2h, h-2, h-4); 3.523.42 (m, 4h, ochhpentyl, h-3, h-4, h-5), 3.23 (bs, 2h, nch2,pentyl); 1.53 (bs, 4h, 2 ch2,pentyl); 1.34 (bs, 2h, ch2,pentyl); 1.23 (d, 3h, j = 6.4 hz, h-6 or h-6); 1.19 (d, 3h, j = 6.4 hz, h-6 or h-6); 0.93, 0.93 (s, 9h, ch3,tbusi); 0.16, 0.14, 0.11, 0.09 (s, 3h, ch3,mesi). C - apt nmr (100 mhz, 323 k): 151.5 (cocbz); 138.3, 138.1 (cq, arom); 128.5, 128.4, 128.3, 127.9, 127.9, 127.8, 127.7, 127.2 (charom); 102.8 (c-1); 99.3 (c-1); 78.8, 78.7 (c-4, c-5); 75.0 (phch2); 74.9 (c-4); 71.4 (c-3); 70.8 (c-3); 69.7 (och2,pentyl); 69.0 (c-5); 67.2 (phch2); 63.8 (c-2); 61.2 (c-2); 50.5 (phch2); 29.2 (ch2,pentyl); 26.2, 26.1 (ch3,tbusi); 23.3 (phch2); 18.6, 18.5 (cq, tbusi); 17.3, 16.9 (c-6, c-6); 3.4, 3.6, 4.5, 4.6 (ch3,mesi). Ir (thin film): 2930, 2857, 2114, 1697, 1252, 1177, 1105, 1067, 1042, 1026 . Hrms: [m + na] calcd for c51h77n7nao9si2 1010.5214, found 1010.5216 . To a stirred solution of disaccharide 31 (0.83 g, 0.84 mmol, 1.0 equiv) in thf (4 ml, 0.2 m) was added bu4nf (as 1 m solution in thf, 2.0 ml, 2.4 equiv), and the resulting yellow reaction mixture was stirred until tlc analysis (toluene / etoac, 4:1 v / v) indicated complete conversion of the starting material (2 h). The mixture was diluted with etoac, and the reaction quenched by addition of satd aq nahco3 . After separation of the layers, the aqueous layer was extracted with etoac (1), and the combined organics were washed (h2o, 1; brine, 1), dried over mgso4, filtered, and concentrated in vacuo . The residue was coevaporated once with dry mecn before dissolution in mecn (4 ml, 0.2 m). Added were, in succession, 2-aminoethyl diphenylborinate 30 (18 mg, 0.08 mmol, 0.1 equiv), dipea (0.3 ml, 1.68 mmol, 2.0 equiv), and bzcl (0.2 ml, 1.68 mmol, 2.0 equiv). The reaction vessel was stirred, under exclusion of light, until tlc analysis (toluene / etoac, 4:1 v / v) indicated complete conversion of the starting material (16 h). The reaction mixture was diluted with etoac, washed (h2o, 2; brine, 1), dried over mgso4, filtered, and concentrated in vacuo . Purification by column chromatography (toluene / etoac, 1:0 17:3 v / v) furnished the title compound as a colorless oil in 67% yield over two steps (0.48 g, 0.56 mmol). H nmr (500 mhz, 323 k): 8.108.09 (m, 2h, charom); 7.59 (t, 1h, j = 7.5 hz, charom); 7.487.21 (m, 17h, charom); 5.46 (dd, 1h, j = 3.0 hz, 11.5 hz, h-3); 5.42 (d, 1h, j = 3.5 hz, h-1); 5.16 (bs, 2h, phch2); 4.93 (d, 1h, j = 12.0 hz, phchh); 4.77 (d, 1h, j = 11.5 hz, phchh); 4.48 (bs, 2h, phch2); 4.20 (d, 1h, j = 7.5 hz, h-1); 3.99 (bs, 1h, h-4); 3.95 (q, 1h, j = 6.5 hz, h-5); 3.903.86 (m, 2h, h-2, ochhpentyl); 3.82 (dd, 1h, j = 3.5 hz, 11.3 hz, h-2); 3.553.45 (m, 4h, h-3, h-4, h-5, ochhpentyl); 3.22 (bs, 2h, nch2,pentyl); 1.591.21 (m, 6h, 3 ch2,pentyl); 1.22 (d, 3h, j = 6.5 hz, h-6); 1.14 (d, 3h, j = 6.5 hz, h-6). C - apt nmr (125 mhz, 323 k): 165.5 (cobz); 138.2, 138.1 (cq, arom); 133.5, 129.9 (charom); 129.5 (cq, arom); 128.6, 128.5, 128.4, 128.1, 127.9, 127.8, 127.3 (charom); 102.8 (c-1); 99.8 (c-1); 78.9, 78.3 (c-3, c-4 or c-5); 75.2 (phch2); 71.4 (c-3); 71.0 (c-3, c-4 or c-5); 70.2 (c-4); 69.8 (och2,pentyl); 67.2 (phch2); 66.5 (c-5); 63.8 (c-2); 57.4 (c-2); 29.3, 23.3 (ch2,pentyl); 17.1 . 16.1 (c-6, c-6). Ir (thin film): 2117, 1717, 1273, 1072 . Hrms: [m + na] calcd for c46h53n7nao10 886.3746, found 886.3743 . To a stirred solution of donor 28 (0.75 g, 1.28 mmol, 4.0 equiv) and acceptor 33 (0.28 g, 0.32 mmol, 1.0 equiv) in ch2cl2 (3.2 ml, 0.1 m) were added flame - dried, rod - shaped, 3 ms . After 30 min, the mixture was cooled to 80 c, and tbsotf (60 l, 0.26 mmol, 0.8 equiv) was added . The reaction mixture was allowed to warm to 55 c and was stirred at this temperature using an immersion cooler . After the mxiture was stirred for 5.5 h, tlc analysis (toluene / acetone, 4:1 v / v) indicated complete disappearance of the acceptor, and the reaction was quenched by addition of net3 (0.2 ml). The mixture was diluted with ch2cl2, filtered over a small bed of celite, washed (brine, 1), dried over mgso4, filtered, and concentrated in vacuo . Purification by size - exclusion chromatography (ch2cl2/meoh, 1:1v / v) followed by column chromatography (toluene / etoac, 1:0 9:1) furnished the title trisaccharide as a colorless oil in 65% yield (260 mg, 0.21 mmol). H nmr (500 mhz, 323 k): 8.118.09 (m, 2h, charom); 7.57 (t, 1h, j = 7.5 hz, charom); 7.477.14 (m, 27h, charom); 5.415.39 (m, 2h, h-1, h-3); 5.16 (bs, 2h, phch2); 4.95 (d, 1h, j = 11.5 hz, phchh); 4.76 (d, 1h, j = 11.0 hz, phchh); 4.724.68 (m, 3h, phch2); 4.55 (d, 1h, j = 11.0 hz, phchh); 4.494.48 (m, 3h, h-1, phch2); 4.21 (d, 1h, j = 3.0 hz, h-4); 4.18 (d, 1h, j = 8.0 hz, h-1); 4.023.96 (m, 3h, h-2, h-2, h-5); 3.94 (t, 1h, j = 9.0 hz, h-4); 3.86 (bs, 1h, ochhpentyl); 3.83 (dd, 1h, j = 8.0 hz, 10.5 hz, h-2); 3.55 (d, 1h, j = 10.0 hz, h-5); 3.523.46 (m, 5h, h-3, h-3, h-4, h-5, ochhpentyl); 3.22 (bs, 2h, nch2,pentyl); 3.13 (s, 3h, och3); 1.581.38 (m, 6h, ch2,pentyl); 1.30 (d, 3h, j = 6.0 hz, h-6); 1.16 (d, 3h, j = 7.0 hz, h-6). C - apt nmr (125 mhz, 323 k): 166.9, 166.1 (c-6, cobz); 138.2, 138.1, 137.4 (cq, arom); 133.0, 130.1 (charom); 129.9 (cq, arom); 128.6, 128.5, 128.5, 128.4, 1283, 128.2, 128.1, 127.9, 127.9, 127.7, 127.7, 127.5, 127.3 (charom); 102.8 (c-1); 101.2 (c-1); 99.5 (c-1); 79.9 (c-3); 79.0 (c-3); 78.3 (c-4); 76.5 (c-4); 75.3, 75.2 (phch2); 75.2, 75.2 (c-4; c-5); 72.3 (phch2); 70.9 (c-5); 69.8 (och2,pentyl); 69.5 (c-3); 67.2 (phch2); 66.4 (c-5); 63.8 (c-2); 61.3 (c-2); 57.4 (c-2); 51.9 (och3,methyl); 50.9 (nch2,pentyl); 29.3, 23.3 (ch2,pentyl); 17.1 (c-6); 16.6 (c-6). C - gated nmr (125 mhz, 323 k): 102.8 (d, j = 159 hz, c-1); 101.2 (d, j = 160 hz, c-1); 99.5 (d, j = 173 hz, c-1). Ir (thin film): 2934, 2110, 1749, 1717, 1699, 1273, 1103, 1069, 1038 . Hrms: [m + na] calcd for c67h74n10nao15 1281.5227, found 1281.5228 . A solution of 27 (50 mg, 0.040 mmol, 1.0 equiv) in thf was treated with a freshly prepared solution of kooh (h2o2, 30% w / w in h2o, was added to 0.5 m aq koh solution), and the slightly turbid mixture was allowed to stir for 2 days until tlc analysis (toluene / etoac / acoh, 60:40:5 v / v / v) indicated complete conversion of the starting material . The mixture was acidified to ph 3 with 1 m aq hcl and extracted (ch2cl2, 5), and the combined organic fractions were washed (brine, 1), dried over mgso4, filtered, and concentrated in vacuo . 70:30:5 v / v / v) gave free uronic acid 34 as a milky solid (30 mg, 0.026 mmol, 66%). H nmr (500 mhz, 323 k, cd3cn + acetic acid - d4): 7.407.18 (m, 25h, charom); 5.22 (d, 1h, j = 3.5 hz, h-1); 5.12 (s, 2h, phch2); 4.85 (d, 1h, j = 11.0 hz, phchh); 4.774.75 (m, 3h, h-1, phch2); 4.664.61 (m, 3h, phch2); 4.46 (s, 2h, phch2); 4.34 (d, 1h, j = 2.5 hz, h-2); 4.24 (d, 1h, j = 7.0 hz, h-1); 4.03 (q, 1h, j = 6.5 hz, h-5); 3.96 (d, 1h, j = 9.0 hz, h-5); 3.943.89 (m, 2h, h-3, h-4); 3.87 (t, 1h, j = 9.5 hz, h-4); 3.78 (dd, 1h, j = 3.5 hz, 9.0 hz, h-3); 3.753.74 (m, 1h, ochhpentyl); 3.633.54 (m, 4h, h-2, h-3, h-4, h-5); 3.483.36 (m, 1h, ochhpentyl); 3.39 (dd, 1h, j = 3.5 hz, 10.5 hz, h-2); 3.27 (t, 2h, j = 7.5 hz, nch2,pentyl); 1.531.50 (m, 4h, ch2,pentyl); 1.341.20 (m, 8h, h-6, h-6, ch2,pentyl). Hrms: [m + h] calcd for c59h69n10o14 1141.4989, found 1141.4994 . The uronic acid 32 (17 mg, 0.015 mmol, 1.0 equiv) was dissolved in pyridine (0.6 ml), and at 0 c, ac2o (0.15 ml) was slowly added . The mixture was stirred until tlc analysis (toluene / etoac / acoh, 60:40:5 v / v / v) indicated complete conversion of the starting material . The reaction was quenched by slow addition of water, and after 15 min, the mixture was extracted (ch2cl2, 5). The organic phases were washed (brine, 1), dried over mgso4, filtered, and concentrated in vacuo . The residue was coevaporated with toluene (2) to remove excess acetic acid and pyridine . H nmr (500 mhz, cd3cn + acetic acid - d4): 7.397.22 (m, 25h, charom); 5.32 (d, 1h, j = 3.5 hz, h-1); 5.14 (dd, 1h, j = 3.0 hz, 11.5 hz, h-3); 5.10 (bs, 2h, phch2); 4.87 (d, 1h, j = 10.0 hz, phchh); 4.794.74 (m, 2h, phch2); 4.67 (d, 1h, j = 1.0 hz, h-1); 4.654.60 (m, 2h, phch2); 4.54 (d, 1h, j = 11.0 hz, phchh); 4.44 (s, 2h, phch2); 4.31 (d, 1h, j = 2.5 hz, h-2); 4.24 (bs, 1h, h-1); 4.11 (d, 1h, j = 3.0 hz, h-4); 4.08 (q, 1h, j = 6.5 hz, h-5); 3.833.70 (m, 5h, h-2, h-3, h-4, h-5, ochhpentyl); 3.583.53 (m, 4h, h-2, h-3, h-4, h-5); 3.44 (bs, 2h, ochhpentyl); 3.20 (bs, 2h, nch2,pentyl); 2.01 (s, 3h, ch3,ac); 1.511.49 (m, 4h, ch2,pentyl); 1.321.19 (m, 8h, h-6, h-6, ch2,pentyl). The crude o - acetate (12 mg, 0.01 mmol, 1.0 equiv) was dissolved in pyridine (0.5 ml, degassed by sonication before use), and added was freshly distilled acsh (0.5 ml). The reaction was stirred for 3 days after which lc ms analysis (mecn / h2o / tfa, 50:50:0.1 90:10:0.1 v / v / v, tr: 6.80 min) indicated complete conversion of the starting material . The mixture was diluted with pyridine (1 ml) and concentrated in vacuo . The crude mixture was subjected to size - exclusion chromatography (ch2cl2/meoh, 1:1 v / v) to isolate the intermediate 35 in 57% yield (7 mg, 0.0057 mmol). H nmr (500 mhz, 323 k, cd3cn + acetic acid - d4): 7.427.21 (m, 25h, charom); 5.11 (s, 2h, phch2); 4.974.90 (m, 4h, h-1, h-2, h-3, phchh); 4.794.77 (m, 2h, phch2); 4.694.67 (m, 2h, h-1, phchh); 4.58 (d, 1h, j = 10.5 hz, phchh); 4.484.46 (m, 3h, phch2); 4.41 (dd, 1h, j = 3.5 hz, 11.5 hz, h-2); 4.25 (d, 1h, j = 9.0 hz, h-1); 4.104.05 (m, 3h, h-2, h-4, h-5); 3.813.60 (m, 6h, h-3, h-3, h-4, h-5, h-5, ochhpentyl); 3.56 (d, 1h, j = 2.5 hz, h-4); 3.373.35 (m, 1h, ochhpentyl); 3.21 (t, 1h, j = 7.0 hz, nch2,pentyl); 2.03, 1.95, 1.91, 1.81 (ch3,ac); 1.501.45 (m, 4h, ch2,pentyl); 1.271.18 (m, 8h, h-6, h-6, ch2,pentyl). C - apt nmr (125 mhz, 323 k, cd3cn + acetic acid - d4): 171.9 (c-6); 129.6, 129.5, 129.4, 129.4, 129.3, 129.0, 129.0, 128.8, 128.8, 128.6, 128.3 (charom); 102.3 (c-1); 101.5 (c-1); 100.4 (c-1); 80.9 (c-3, c-4 or c-5); 80.4 (c-4); 78.8 (c-3); 77.3, 77.0 (c-4 and c-3, c-4 or c-5); 76.2, 75.8 (phch2); 75.6 (c-3, c-4 or c-5); 72.0 (phch2); 71.5 (c-5); 71.0 (c-3); 69.9 (och2,pentyl); 68.1 (c-5); 67.9 (phch2); 53.0 (c-2); 50.3 (c-2); 47.8 (c-2); 30.1, 24.1 (ch2,pentyl); 23.5, 23.3, 23.3, 21.3 (ch3,ac); 17.5, 16.8 (c-6, c-6). The trisaccharide 33 (4 mg, 0.003 mmol) was dissolved in thf, tert - butyl alcohol, and h2o (1:1:3 v / v / v, 0.002 m). Added was a drop of acoh and the mixture was degassed (freeze thaw, 3 cycles) and backfilled with argon . Pd(oh)2 (20 weight% on carbon, 50% h2o) was added and the mixture was purged with argon (balloon), followed by h2 (balloon). After purging, the mixture was filtered over a fritted syringe filled with celite, the residue was washed with thf / h2o (1:1 v / v) and concentrated in vacuo . After h nmr analysis revealed no aromatic signals, the crude trisaccharide was purified by passing over a c18 solid - phase extraction column (mecn / h2o, 0:1 1:9) and subsequently lyophilized to obtain the product 26 (2.5 mg, 0.003 mmol) as a white solid . H nmr (500 mhz, d2o): 5.03 (dd, 1h, j = 3.0 hz, 12.0 hz, h-3); 5.01 (d, 1h, j = 4.0 hz, h-1); 4.74 (bs, 1h, h-1); 4.59 (d, 1h, j = 4.5 hz, h-2); 4.41 (d, 1h, j = 8.5 hz, h-1); 4.38 (dd, 1h, j = 4.0 hz, 11.5 hz, h-2); 4.22 (d, 1h, j = 2.5 hz, h-4); 4.19 (q, 1h, j = 6.5 hz, h-5); 3.99 (t, 1h, j = 8.5 hz, h-2); 3.913.86 (m, 1h, ochhpentyl); 3.823.77 (m, 4h, h-3, h-3, h-4, h-5); 3.65 (t, 1h, j = 9.5 hz, h-4); 3.603.56 (m, 2h, h-5, ochhpentyl); 2.14, 2.09, 2.01, 1.99 (s, 3h, ch3,ac); 1.66 (t, 2h, j = 7.5 hz, ch2,pentyl); 1.621.56 (m, 2h, ch2,pentyl); 1.411.38 (m, 2h, ch2,pentyl); 1.28 (d, 3h, j = 6.5 hz, h-6); 1.25 (d, 3h, j = 6.5 hz, h-6). C - apt nmr (125 mhz, d2o): 175.7, 175.6, 174.3, 174.1, 173.9 (c-6, coac); 101.6 (c-1); 99.9 (c-1); 99.1 (c-1); 78.7 (c-5); 71.1 (c-3, c-3, c-4 or c-5); 71.7, 70.8, 70.5 (c-3, c-3, c-4, c-5); 70.1 (och2,pentyl); 69.9 (c-3); 69.6 (c-4); 66.9 (c-5); 53.1 (c-2); 51.4 (c-2); 47.2 (c-2); 39.4 (nch2,pentyl); 28.2, 26.5 (ch2,pentyl); 22.2, 22.2, 22.1 (ch3,ac); 22.0 (ch2,pentyl); 20.4 (ch3,ac); 15.4, 15.3 (c-6, c-6).
Acute hepatitis a virus (hav) infection is common in the developing countries among children, but hydrops of gallbladder due to hepatitis a infection is an uncommon presentation . A five - year - old boy was admitted in namazi hospital, shiraz, iran due to jaundice and severe abdominal pain for 10 days . Physical examination revealed a mass in the right upper quadrant with severe tenderness . Liver function tests were abnormal while other laboratory data such as blood urea nitrogen, serum creatinine, sodium, and potassium were within the normal range . Abdominal ultrasonography showed that the gallbladder was very much distended and its fundus was near the iliac crest . After five days, without any specific treatment, his symptoms improved and he was discharged with good condition . Acute acalculous gallbladder disease is a rare complication of hav infection which should be suspected in any child with right upper quadrant abdominal pain, tenderness, and mass which can lead to surgical emergency in rare conditions . The distinction between these two conditions may be difficult . These diseases may present with a spectrum of symptoms ranging from transient gallbladder distention with spontaneous resolution to acute acalculous cholecystitis (aac) with necrosis of the gallbladder wall (1). Acute hydrops is defined by marked gallbladder distention in the absence of calculi, bacterial infection, or congenital gallbladder anomaly . The absence of a significant inflammatory component and its typically benign prognosis are the features that distinguish hydrops from acalculous cholecystitis (1). One of the conditions related to gallbladder hydrops is viral hepatitis and gallbladder hydrops due to viral hepatitis a is rarely reported in children (2). Here is the presentation of infection in a five - year - old boy with hydrops of gallbladder due to hepatitis a virus (hav), which is rarely reported in the pediatric age group . A five - year - old boy was admitted in namazi hospital, shiraz, iran with severe abdominal pain, jaundice and low grade fever . He had abdominal pain and vomiting from 20 days before admission; his jaundice worsened and developed from 10 days ago . At the time of admission he was very sick with fever and yellowish discoloration of skin and sclera . He was very irritable due to abdominal pain . In the physical examination he had normal growth parameters and his vital signs were as follows: temperature: 38c, pulse rate: 100/min, respiratory rate: 30/min and blood pressure: 100/60 mmhg . In the abdominal examination, a large mass was palpated in the right upper quadrant (ruq) with severe tenderness . Initial laboratory investigations showed elevated liver enzymes: ast: 516 u / l, alt: 722 u / l, alkaline phosphatase: 1930 u / l, total bilirubin: 5.3 mg / dl, direct bilirubin: 3.9 mg / dl, total protein: 7.7 g / dl, albumin: 3.8 g / dl, wbc: 8000/mm, hb: 11.9 g / dl, platelet: 426000/mm, cpr: 6 mg / dl, esr: 40 mm / h . Coagulation studies revealed pt: 13 sec, ptt: 34 sec and inr: 1 . Serum amylase, blood sugar and serum sodium, potassium, bun and creatinine were within normal range . Venous blood gas analysis revealed: ph: 7.5, hco3: 30, and pco2: 37.9 . Serology of viral hepatitis was positive for hav igm, and other viral markers were negative . Abdominal ultrasonography showed normal size and echogenicity of the liver, with significantly distended gallbladder (span 130 50 mm) with the fundus of gallbladder near iliac crest . The hydrops of gallbladder was confirmed by ultrasonography (figure 1). Due to fever and toxic condition after the third day of admission, his fever and blood culture became negative and antibiotics were stopped . After five days, general condition and abdominal pain of and jaundice improved and he was discharged with close follow up . Abdominal ultrasonography showed normal size and echogenicity of the liver, with significantly distended gallbladder (span 130 50 mm); fundus of gallbladder was near iliac crest . Hepatitis a viral infection is a wide spread infection throughout the world . In the developing countries, hepatitis 92% to 100% of the 18-year - old people have serologic evidence of past infection (1). Infants and toddlers are more likely to be asymptomatic (anicteric hepatitis), whereas the majority of adults will develop clinically evident hepatitis (1). One of every 12 young children develops jaundice, and children are more likely than adults (60% vs. 20%) to have diarrhea, often leading to the mistaken diagnosis of infectious gastroenteritis (1). Gallbladder hydrops due to hepatitis a viral infection is uncommon and is exceptionally described in the literature (2 - 4). Abdominal pain, vomiting, and ruq mass in abdominal examination are the typical presentations of gallbladder hydrops . Kawasaki syndrome (5), mesenteric adenitis with pressure over cystic duct (5), streptococcal and staphylococcal infections with the associated toxin production (6), viral hepatitis, henoch - schoenlein purpura (7) and hypokalemia (8) had reported association with hydrops of gallbladder . Other etiologies include tumors, polyps or malignancy of the gallbladder, spontaneously resolved acute cholecystitis, congenital narrowing of the cystic duct, parasites such as ascaris (occasionally), prolonged parenteral nutrition, typhoid, leptospirosis, and appropriate response to ceftriaxone therapy (1). Although the etiology is unclear, invasion of the gallbladder and bile duct epithelial by hepatitis a viral infection and cell - mediated immunologic response are proposed in the pathogenesis of cholecystitis resulted from hav infection (9). Low grade fever, colicky abdominal pain, vomiting, right sub costal tenderness, and a palpable tender mass in ruq are common (1). A mild, direct hyperbilirubinemia may also be present . If the infection left untreated, it can rapidly progress to gallbladder gangrene and perforation leading to peritonitis, sepsis, and shock . In uncomplicated acute cholecystitis, liver function tests are normal or there is only slightly liver enzyme elevation . Mild cholestatic abnormalities (increased bilirubin up to 4 mg / dl and elevated alkaline phosphatase) are common, probably indicating the mechanical obstruction of the cystic duct resulting from intrinsic inflammation of the biliary tract (10). The diagnosis of hydrops is generally made by abdominal ultrasonography demonstrating a markedly distended, echo - free gallbladder with normal wall thickness and a normal - caliber biliary tree . Serial ultrasonographic examination is useful to monitor the patient and detect the resolution (10). Most cases are self - limited and size of the gallbladder may spontaneously become normal with treatment of the underlying disease within approximately two weeks (1). In conclusion, in the case of hepatitis a viral infection with severe abdominal pain and specially ruq abdominal mass, hydrops of gallbladder should be kept in mind as a possible complication and abdominal ultrasonography should be done accordingly.
This unusual dental anomaly showing an accessory cusp like structure projecting from the cingulum to the cutting edge was first described by mitchell in 1892 . It was thereafter named a talon cusp by mellor and ripa due to its resemblance to an eagle's talon . The exact etiology is not known, but it is suggested to be a combination of genetic and environmental factors . It is thought to arise during the morpho - differentiation stage of tooth development, as a result of outfolding of the enamel organ or hyper - productivity of the dental lamina . It is composed of enamel, dentine, and a varying amount of pulp tissue . Reports of a mandibular talon cusp are rare in the literature . To the best of our knowledge, only 14 cases have been reported [table 1], of which only 2 cases are in mandibular left central incisors . We report the second instance of a talon cusp in the lingual aspect of the mandibular left central incisor and the first such report in a patient of libyan origin . Reported cases of mandibular talon's cusp an apparently healthy 11-year - old boy reported to the outpatient department in the faculty of dentistry, al jabal - algharbi - zawia university, zawia city, libya, for a routine dental checkup . Clinical examination revealed an accessory cusp on the lingual aspect of the mandibular left central incisor [figure 1]. It was projecting from the cemento - enamel junction and extended towards the incisal edge . There was evidence of attrition on the lingual aspect of the maxillary left central incisor . An intra - oral periapical radiograph revealed an inverted v shaped radiopaque structure on the mandibular left central incisor [figure 2]. The extent of pulp tissue into the cusp could not be determined from the radiograph . Treatment planning included periodic and gradual reduction of the accessory cusp to relieve the occlusal interference followed by application of topical fluoride . Clinical picture showing the type 1 talon cusp on lingual aspect of mandibular left permanent central incisor intra oral periapical radiograph showing the inverted v - shaped radiopaque accessory cusp on mandibular permanent central incisor it has been defined as a supernumerary accessory talon - shaped cusp projecting from the lingual or facial surface of the crown of a tooth and extending for at least half the distance from the cemento - enamel junction to the incisal edge . There is a wide variation in the size and shape of this anomaly . Due to the variation, and in order to have diagnostic criteria, it has been classified into three types by hattab et al . Radiographically, it may appear typically as a v - shaped radiopaque structure, as in true talon or semi- talon, or be tubercle - like, as in trace talon, originating from the cervical third of the root . The radiopaque v - shaped structure is superimposed over the normal image of the crown of the tooth . This appearance varies with the shape and size of the cusp, and the angle at which the radiograph is taken . It is composed of enamel, dentine, and a varying amount of pulp tissue . The extent of pulp extension into the cusp is however difficult to determine because of its superimposition over the main pulp chamber . The presence of pulp tissue within the accessory cusp has been controversial with contrasting reports of the presence and no evidence of pulp extension into the cusp . Cases with large cusps presenting away from the tooth had been shown to contain an extension of the pulp, superimposition of the image of the cusp over the main tooth made it difficult to determine the extent of pulp tissue in the anomalous cusp . The exact etiology is not known, but it is suggested to be a combination of genetic and environmental factors . There was no associated systemic or local condition in this patient as is the case in most previous reports . Mays reported a statistically significant bias in favor of males and our patient is an 11-year - old boy which is concurrent with the literature . It is more common in the permanent dentition (75%) than in the primary dentition, while 92% affect the maxillary teeth . The maxillary lateral incisor is the most frequently affected in the permanent dentition, while the maxillary central incisor is the most affected in the primary dentition . Reports of the mandibular talon cusps are rare in the literature . To the best of our knowledge, only 14 cases have been reported of which only 2 cases are of mandibular left central incisors [table 1]. We report the second instance of the talon cusp in the lingual aspect of the mandibular left central incisor and the first such report in a patient of libyan origin . There are several reports in the literature that suggest the hereditary character of a talon cusp . The presence of a talon cusp is not always an indication for dental treatment unless it is associated with clinical problems . The complications of the talon cusp are diagnostic, functional, aesthetic, and pathological . It includes compromised aesthetics, periodontal problems, or irritation of the soft tissues during speech or mastication . Functional complications include occlusal interference, trauma to the lip and tongue, speech problems, and displacement of teeth . The deep grooves which join the cusp to the tooth may also act as stagnation areas for plaque and debris, become carious, and cause subsequent periapical pathology . Occlusal interference can damage the periodontium, cause infra - occlusion of the opposing tooth and also temporo - mandibular joint pain . Severe attrition or fracture of the enamel surface can cause exposure of the dentine - pulp complex and consequently, pulp necrosis . In our patient, the prominent lingual accessory cusp had occlusal interference with attrition facet on the opposing maxillary central incisor . Where there are deep developmental grooves, simple prophylactic measures such as fissure sealing and composite resin restoration can be carried out . An essential step, especially in the case of occlusal interference, is to reduce the bulk of the cusp gradually and periodically and application of topical fluoride such as duraphat or acidulated phosphate fluoride (apf) gel to reduce sensitivity and stimulate reparative dentine formation for pulp protection, or outright total reduction of the cusp and calcium hydroxide pulpotomy . It may also become necessary sometimes, to fully reduce the cusp, extirpate the pulp, and carry out root canal therapy . Orthodontic correction may become necessary when there is tooth displacement or malalignment of affected or opposing teeth . Our patient was treated with periodic and gradual reduction of the accessory cusp to relieve the occlusal interference followed by application of topical fluoride . We report the second instance of the talon cusp in the lingual aspect of the mandibular left central incisor and the first such report in a patient of libyan origin . Although such instances are rare, early identification and prompt treatment of accessory cusps can prevent the complications of occlusal interference and malocclusion.
A 68-year - old woman presented with decreased visual acuity (va), and 20 hours earlier had noticed a visual field defect in the superior half of her right eye . Slit lamp biomicroscopy and fundus examination revealed intraretinal edema with inferior retinal whitening in addition to a visible hollerenhorst plaque . Fluorescein angiography (fa) revealed an arterial filling defect along the inferotemporal arcade, distal to the corresponding embolus with foveal ischemia (fig . The initial treatment involved anterior chamber paracentesis, use of an iop - lowering agent, and ocular massage . A goldmann three mirror lens was used to focus an nd: yag laser onto the arterial embolus . The laser energy level commenced at 0.5 mj and was increased to 1 mj (about 12 pulses) until the embolus was partially or completely shattered . After shattering the embolus, a small amount of vitreous hemorrhage was noted inferior to the optic disc . Five days after treatment, fa showed no evidence of the emboli, and retinal arteriolar blood flow had been restored . Bcva improved to 20/80 . At two months after nye, bcva was 20/25 with no remaining vitreous hemorrhage . At three months after nye, bcva was 20/20 and fa showed that the branch retinal artery and all arterioles were patent with good flow . At ten months after nye, an 82-year - old woman presented with a four - day history of progressive decrease in va in her left eye . Her medical history was unremarkable . At presentation, bcva was 20/200, which was lower than the reading of 20/32 taken six months earlier . Slit - lamp biomicroscopy revealed a large plaque in the associated branch retinal artery on the disc of the left eye . Fa showed an arterial filling defect along the superotemporal arcade distal to the corresponding embolus (fig . 2). A macular contact lens was used to deliver the nd: yag laser to the plaque . Pulsing commenced with one pulse at 0.5 mj, and the energy was increased to 1 mj, at which a total of eight pulses were applied to the plaque . A small amount of vitreous hemorrhage over the arteriole was noted which resulted in cessation of the laser treatment despite the remaining embolus . The following day, vitreous hemorrhage decreased slightly and laser treatment was recommenced (1 mjten pulses). However, a partial embolus fragment remained in the associated arteriole . On the second day after nye, bcva had improved to 20/100, and the vitreous hemorrhage remained present . On the third day after nye, fa showed restoration of the retinal arteriolar blood flow . Forty days after nye, bcva had improved to 20/32 . At four months after nye, fa showed no retinal neovascularizations, and bcva remained at 20/32 . A 68-year - old woman presented with decreased visual acuity (va), and 20 hours earlier had noticed a visual field defect in the superior half of her right eye . Slit lamp biomicroscopy and fundus examination revealed intraretinal edema with inferior retinal whitening in addition to a visible hollerenhorst plaque . Fluorescein angiography (fa) revealed an arterial filling defect along the inferotemporal arcade, distal to the corresponding embolus with foveal ischemia (fig . The initial treatment involved anterior chamber paracentesis, use of an iop - lowering agent, and ocular massage . A goldmann three mirror lens was used to focus an nd: yag laser onto the arterial embolus . The laser energy level commenced at 0.5 mj and was increased to 1 mj (about 12 pulses) until the embolus was partially or completely shattered . After shattering the embolus, a small amount of vitreous hemorrhage was noted inferior to the optic disc . Five days after treatment, fa showed no evidence of the emboli, and retinal arteriolar blood flow had been restored . Bcva improved to 20/80 . At two months after nye, bcva was 20/25 with no remaining vitreous hemorrhage . At three months after nye, bcva was 20/20 and fa showed that the branch retinal artery and all arterioles were patent with good flow . At ten months after nye, an 82-year - old woman presented with a four - day history of progressive decrease in va in her left eye . Her medical history was unremarkable . At presentation, bcva was 20/200, which was lower than the reading of 20/32 taken six months earlier . Slit - lamp biomicroscopy revealed a large plaque in the associated branch retinal artery on the disc of the left eye . Fa showed an arterial filling defect along the superotemporal arcade distal to the corresponding embolus (fig . 2). A macular contact lens was used to deliver the nd: yag laser to the plaque . Pulsing commenced with one pulse at 0.5 mj, and the energy was increased to 1 mj, at which a total of eight pulses were applied to the plaque . A small amount of vitreous hemorrhage over the arteriole was noted which resulted in cessation of the laser treatment despite the remaining embolus . The following day, vitreous hemorrhage decreased slightly and laser treatment was recommenced (1 mjten pulses). The large embolus was ruptured, and recirculation around the macula was noted . However, a partial embolus fragment remained in the associated arteriole . On the second day after nye, bcva had improved to 20/100, and the vitreous hemorrhage remained present . On the third day after nye, fa showed restoration of the retinal arteriolar blood flow . Forty days after nye, bcva had improved to 20/32 . At four months after nye, fa showed no retinal neovascularizations, and bcva remained at 20/32 . Recent reports showed immediate restoration of retinal blood flow after nye, in addition to rapid restoration of bcva . However, complications such as vitreous hemorrhage and subretinal hemorrhage were observed.3,4 in the present cases, laser treatment resulted in dramatic improvement in bcva and restoration of retinal blood flow despite a small amount of vitreous hemorrhage . Mason et al.5 reported that visual prognosis after brao appeared to correlate with presenting va, and that eyes with an initial va of 20/40 or better usually remained at 20/40 or better . Individuals with poor va (e.g., 20/100 or worse) generally did not show significant improvement . They concluded that it may be misleading to indicate that 80% of eyes with brao improve to 20/40 or better (as described in previous reports) when final bcva is so closely related to presenting bcva . In conclusion therefore, nye should be considered an effective treatment in patients with brao with visible emboli.
The iridoids are a distinct class of approximately 600 monoterpenes that display a broad range of pharmacological and agrochemical activities (tundis et al ., 2008; dewhirst et al ., 2010; dinda et al ., we recently reported the discovery of iridoid synthase, the enzyme that produces nepetalactol (1a), the common biosynthetic precursor for all iridoids (geu - flores et al ., 2012). Notably, this enzyme is mechanistically distinct from canonical terpene synthases (uesato et al ., 1983; uesato et al ., 1984; 1987). Instead of forming a reactive cationic species from geranyl pyrophosphate (figure 1a) (degenhardt et al ., 2009; chen et al ., 2011; kim et al ., 2012), iridoid synthase catalyzes cyclization that is triggered by reduction of 8-oxogeranial (2) to form enol or enolate intermediate 3 . Intermediate 3 is poised to cyclize to form nepetalactol (1a) by either an inverse electron demand hetero diels - alder (for examples of enzymatic diels - alder reactions, see kim et al ., 2012) or a michael reaction (for examples of enzymatic michael reactions, see kusebauch et al ., 2009; we describe the synthesis and enzymatic assay of two substrate analogs designed to probe which reaction pathway iridoid synthase favors for cyclization . On the basis of these studies, along with clues from the product profile of the native substrate, it appears that iridoid synthase utilizes a michael addition reaction mechanism for cyclization of the iridoid class of natural products . This provides an essential piece of the mechanistic puzzle of how the iridoid scaffold is constructed . After iridoid synthase reduces 8-oxogeranial (2) using nicotinamide adenine dinucleotide phosphate (nadph) as hydride (h) donor, enol or enolate intermediate 3 is formed (figure 1b). The existence of reaction intermediate 3 is supported by the identification of reduced aldehyde 4, the more stable tautomer of 3, as a minor product in the iridoid synthase catalyzed reaction (geu - flores et al ., 2012). Moreover, the formation of an enol intermediate is entirely consistent with the proposed mechanism of progesterone--reductase (thorn et al . 2010), which displays high sequence similarity to iridoid synthase (67% amino acid identity compared with digitalis purpurea p5br2). Once formed, 3 can cyclize to form the characteristic bicyclic 5 - 6 ring iridoid framework of nepetalactol (1a). Cyclization could occur by a stepwise michael reaction, forming the 5-membered ring first, with subsequent cyclization to the lactol (figure 1b, blue arrows). Alternatively, the reaction could proceed via an inverse electron demand hetero diels - alder reaction (figure 1b, red arrows). To distinguish between these two mechanistic possibilities, one substrate, compound 5, was designed to disfavor the michael mechanism while favoring a diels - alder reaction; the other, compound 6, strongly disfavored the diels - alder reaction while favoring the michael reaction (figure 2). Provided that both can be accommodated within the enzyme active site, cyclization of only one of these substrates by the enzyme would suggest the more likely reaction mechanism for the native substrate . Intermediate 7 harbors a diene with electron withdrawing groups (fluorine) and a dienophile with an electron donating group (oh or o) and could therefore undergo an inverse electron demand diels - alder to form product 8 (figure 2a); precedent for fluorinated dienes in enhancing diels - alder reactions exists (kazmina et al ., 1984; roversi et al ., 2002; vogel et al ., 2007). In contrast, the michael addition with substrate 5 entails formation of a carbanion species (figure 2a), which is far less stable than the enol or enolate species that would occur with the native substrate (figure 1b). Although the carbanion could be somewhat stabilized because of the inductive electron - withdrawing effects of the fluorine atoms, this effect is greatly mitigated because of electron - pair repulsions between the carbanion and the fluorine lone pairs (zhang et al ., 2012). Altogether, compared with the native substrate 2, 5 is a much weaker candidate for a michael addition . Thus the formation of cyclization product 8 upon incubation of iridoid synthase with substrate 5 would suggest that the enzyme utilizes a pericyclic reaction mechanism . Candidate 6 is intended to undergo conjugate reduction in the enzyme to form intermediate 10, which is primed to perform an sn2 conjugate addition to give cyclization product 11 (figure 2b). It is highly unlikely for intermediate 10 to undergo a diels - alder equivalent reaction (a halo - alder - ene); we have found no precedent for such a reaction . Therefore, formation of cyclized product 11 would suggest that the enzyme utilizes a michael reaction mechanism . The synthesis of both 5 and 6 started with the acetalization of citral (14), followed by the allylic oxidation of one methyl group using stoichiometric amounts of seo2 to yield 16 (figure 3; supplemental information available online). Ensuing difluoromethylenation with sodium chlorodifluoroacetate followed by hydrolysis of the acetal led to the diels - alder test substrate, 8-(difluoromethylene)geranial (5). The michael test substrate, 8-chlorogeranial (6), was obtained from 16 by reduction of the aldehyde using sodium borohydride, chlorination with tosyl chloride followed by deacetalization (figure 3; supplemental information). Compounds 5 and 6 were incubated with iridoid synthase and product formation was assessed by gas chromatography - mass spectrometry (gc - ms). The major products for both enzymatic reactions were also isolated, purified, and then characterized by nuclear magnetic resonance (nmr), further validating the structures of the enzymatic products (si). Upon incubation of iridoid synthase with substrate 5, the linear reduction product 12 was observed (figure 2a, red compound). This indicates that iridoid synthase is catalytically competent with 5, despite the perturbations to the native substrate structure . Additionally, the lack of observable cyclized product 8 shows that the enzyme does not favor the diels - alder cyclization mechanism for which this substrate was designed . In contrast, when iridoid synthase was incubated with compound 6, cyclized product 11 could be cleanly isolated (figure 2b, blue compound). Given that a pericyclic reaction for compound 6 is highly disfavored, it seems most likely that the observed cyclization occurs via the michael reaction . Nuclear overhauser effect spectroscopy nmr spectra suggest that the product has the relative stereochemistry shown (supplemental information), which matches that of the native enzyme product 1b . Compounds 5 and 6 were subjected to steady - state kinetic analysis (supplemental information). Compound 5 (km = 485 160 m, kcat = 6.4 0.8 s, kcat / km = 0.013 s m; supplemental information) had a catalytic efficiency 8-fold less than that observed for compound 6 (km = 81.9 5.6 m, kcat = 8.1 0.5 s, kcat / km = 0.099 s m; supplemental information). Although both 5 and 6 had lower catalytic efficiencies than that observed for natural substrate 2 (km = 9.9 2.1 m, kcat = 1.4 0.1 s, kcat / km = 0.14 s m), the steady - state kinetic measurements confirm that both 5 and 6 are competent substrates, though only substrate 6 was cyclized . For these studies, an enzyme with a truncation at the n terminus was used, which increases the structural stability of the nearest iridoid synthase homolog, progesterone beta - reductase . This truncation has recently been shown to affect kinetic parameters for progesterone beta - reductase (rudolph et al ., 2014). Therefore, kinetic parameters for 2 were remeasured using this truncated enzyme . For comparison, kinetic parameters for the full - length enzyme with 2 are km = 4.5 0.2 m, kcat = 1.6 0.1 s, kcat / km = 0.36 s m . For example, both analogs 5 and 6 had a higher km than native substrate 2, but the difference was more marked for 5, which might be indicative of an impaired binding to the active site . Additionally, the electronic properties of both analogs could be modulated by hydrogen bonding interactions with the enzyme, thereby altering the propensity of the compounds to cyclize via a diels - alder or michael reaction . Ideally, comparison of nonenzymatic cyclization reactions with enzyme - catalyzed reactions would provide more insight into the baseline reactivity of these compounds . After chemical reduction of a more stable and synthetically accessible analog of 5 (9,9-difluoro-2,6-dimethylnona-2,6,8-trienal) using l - selectride to generate the reactive enol / enolate, we only obtained the alcohol (9,9-difluoro-2,6-dimethylnona-2,6,8-trien-1-ol). Reduction using stryker s reagent in combination with licl or tmscl led to an unidentifiable product mixture . Efforts to generate a protected enol species that could be subjected to chemical conditions favorable for a diels - alder reaction were unsuccessful . Efforts to assess whether 6 could cyclize nonenzymatically were complicated by the propensity of 6 to rearrange in solution . Despite these caveats, the results from enzymatic assay with the two substrate analogs are consistent: substrate 5, which is primed for a diels - alder reaction, failed to cyclize, whereas substrate 6, primed for a michael reaction, did cyclize . Therefore, it seems reasonable to conclude that the cyclization step of iridoid synthase likely operates via a michael addition reaction mechanism . Finally, it is prudent to consider whether the product distribution that results from native substrate 2 also supports this mechanism . The michael reaction proceeds via the open form of nepetalactol (1a), iridodial (1b), while the diels - alder proceeds directly to the closed form 1a (figure 1b). The native iridoid synthase cyclization product appears as a mixture of the closed and open forms, 1a and 1b, as evidenced by gc - ms and tlc (geu - flores et al ., 2012). We reported in our initial experiments that 1a and 1b are in equilibrium (geu - flores et al ., 2012), which would mean that the presence of both the open and closed forms provides no insight into a mechanistic hypothesis . We have now performed a more detailed analysis of the product distribution of 1a and 1b, which demonstrates that the amount of open form observed is in fact greatly dependent upon the temperature of the gc inlet (low gc inlet temperatures have shown the stability of the open form), as well as on its usage history (dawson et al ., 1989). With this knowledge at hand, it is clear that the open and closed forms of 1 equilibrate on a much slower timescale than previously assumed (see the supplemental information for detailed information and data). Therefore, we can now conclude that both the open and closed forms are produced in the enzymatic reaction . Because the diels - alder mechanism does not involve the open form 1b, we would be less likely to observe the open form if the enzyme used a pericyclic cyclization . The presence of both open and closed forms of 1 is also more consistent with a cyclization mechanism utilizing the michael reaction . Although the slow equilibrium would suggest that the stereochemistry at the hemi - acetal carbon of the closed form 1a could provide mechanistic insight into the nature of the cyclization reaction, epimerization can also occur via acid - catalyzed loss of lactolic oh to give an oxocarbenium intermediate . Therefore, we have not considered the stereochemistry of 1a at this carbon as supportive of one mechanism over the other . In synthetic systems, intramolecular cyclization of dicarbonyl substrates to form the iridoid scaffold has utilized both diels - alder and michael addition mechanisms . For example, a domino knoevenagel - hetero - diels - alder reaction has been employed to form the iridoid scaffold (tietze and bartels, 1991), and an enol ether derivative of a trialdehyde substrate also cyclized immediately via an intramolecular inverse electron demand diels - alder to yield an iridoid derivative (tietze et al ., 1980 however, intramolecular cyclization of dicarbonyl substrates to yield iridoids has also been achieved via michael reaction using a jrgensen - hayashi catalyst (marqus - lpez et al ., 2009), and a reductive michael cyclization of a keto aldehyde has been reported (yang et al ., 2005). Although the inherent chemical reactivity of the linear iridoid precursor is compatible with both reactions, our studies suggest that nature utilizes the michael reaction . Iridoid synthase joins a growing list of diverse enzymes that catalyze unusual terpene cyclization reactions (itoh et al ., 2010; although substrate probes and product identities cannot be used to definitively prove the course of an enzymatic mechanism, the collective results described here provide consistent evidence that iridoid synthase catalyzes cyclization of the iridoids via a michael addition rather than a diels - alder reaction . Additionally, this work demonstrates that iridoid synthase can cyclize substrates other than 8-oxogeranial (2), suggesting the potential utility of this enzyme for enzymatic synthesis of new compounds . Understanding the mechanism of iridoid synthase cyclization now enables us to better predict which substrates this enzyme can cyclize . Further studies exploring the potential of this enzyme to synthesize new cyclic compounds from nonnatural substrates are currently under way . The design and synthesis of two substrate analogs are used to probe the mechanism of iridoid synthase . Enzymatic assay of these substrate analogs, along with clues from the product profile of the native substrate, strongly suggest that iridoid synthase utilizes a michael reaction to achieve cyclization, rather than a diels - alder reaction . Additionally, this work demonstrates that iridoid synthase can cyclize nonnative substrates, suggesting the potential utility of this enzyme for enzymatic synthesis of new compounds . This improved mechanistic understanding will facilitate the exploitation of the potential of iridoid synthase to synthesize new cyclic compounds from nonnatural substrates . All enzyme assays were carried out using 20 mm mops (ph 7.0) as buffer . The substrates were kept as 50 mm stocks in tetrahydrofuran (thf) at 20c . Care was taken not to exceed thf concentrations higher than 0.5% in the presence of enzyme, as concentrations above 1% thf were found to affect activity adversely . The milligram - scale enzyme assays were carried out using an nadph generation / regeneration system consisting of glucose-6-phosphate (g6p), glucose-6-phosphate dehydrogenase (g6pdh), and nadp . Enzymatic rates for steady - state kinetic analysis of the iridoid synthase reactions were measured spectrophotometrically, monitoring nadph consumption at 340 nm . For gc - ms analysis, reactions (200 l) were set up in glass vials using 200 m substrate, 600 m nadph, and 0.5 g of purified protein and were terminated after 1 hr by adding 250 l ch2cl2 . Standard gc - ms spectra were recorded on an agilent 6890n gc system equipped with a split / splitless injector and coupled to an agilent 5973 ms detector . Gc - ms - based accurate mass determination was performed on a waters gct system consisting of an agilent 6890 series gc system fitted with a split / splitless injector and coupled to a waters gct classic mass spectrometer . For analysis by tlc, 150 l of the organic phase was vacuum - concentrated to approximately 10 l, spotted onto normal - phase tlc plates, run using 10:1 hexanes / ethyl acetate, and visualized with anisaldehyde stain . For kinetic studies, the absorbance at 340 nm of 200 l assays was measured using a 96-well plate reader . Procedures for the synthesis of substrates 5 and 6, along with all spectral characterization for synthetically and enzymatically generated products, are reported in the supplemental information . S.l . Carried out all syntheses, enzyme assays of 5 and 6, and characterization of the enzymatic products . F . Cloned and expressed the enzyme version used in the assays, assayed substrate 2, and performed the equilibrium experiments with open / closed forms of product 1 . N.h.s . Conceived the design of substrates 5 and 6 as well as the initial synthetic strategy.
In the previous issue of critical care an interesting observational study suggests a promising avenue of research that has the potential to improve clinical outcomes . The early identification and rapid treatment of haemodynamic shock is widely acknowledged as a vital step towards improving survival . In prehospital care, this process is particularly challenging . Limitations of time, equipment, available skill set and environment render the objective diagnosis of haemodynamic shock difficult . The utility of serum lactate as a tool to identify the most seriously ill patients and to monitor their response to treatment has long been recognised [3 - 5]. This latest investigation describes the prognostic value of peripheral venous or capillary blood lactate concentration, measured in 124 patients before hospital arrival by paramedic ambulance staff using hand - held battery - powered technology . The findings confirm the expected relationship between the prehospital serum lactate concentration and subsequent hospital mortality . These data should encourage further research into the prehospital use of serum lactate to facilitate prompt identification and treatment of haemodynamic shock and/or to indicate those patients who might benefit from advanced activation of medical staff in the destination hospital . The authors suggest that a single value of serum lactate measured in the prehospital environment predicts hospital mortality in this population . Whilst lactate levels are clearly much greater in those patients who die, this variable does not appear to have been included in the multivariate analysis . It is the change in serum lactate, between the first measurement in the community and the second on hospital arrival, that is independently associated with death . The importance of this distinction would depend upon how these findings are applied in clinical practice . If lactate measurement is incorporated into routine prehospital care, it would probably be as part of a specific treatment algorithm . Indeed, biomarkers can only be used to improve clinical outcome when used as a trigger for a specific intervention, or less commonly when used as a therapeutic target . Accurate data on threshold values are essential if lactate measurement is to be used in this way . In this study, receiver operator characteristic curve analysis suggests a lactate concentration of 3.5 mmol / l as the optimal cutoff value for mortality prediction . If lactate is not an independent predictor of outcome, however the sample population is also too small and too heterogeneous to support specific conclusions regarding threshold values for specific subgroups of patients (for example, septic shock patients). For similar reasons, the accuracy of lactate measurement in peripheral venous or capillary blood samples must be carefully considered . This is a simple and attractive approach that allows the measurement of serum lactate in the great majority of patients attended in the prehospital environment . The relationship between the lactate concentration in such samples and those drawn from an arterial or central venous catheter, however, has not been established . Anecdotal experience suggests that lactate concentrations are often greater in peripheral blood samples but not by a constant or predictable margin . The authors are to be congratulated for completing this first phase of a promising line of investigation . Future research should further clarify the clinical significance of lactate concentrations in patients with haemodynamic shock . Interventional trials may then confirm the efficacy of serum lactate measurements to aid the identification of these patients and to guide their subsequent treatment.
Ovarian cancer is called the silent killer because of its not - so - obvious symptoms such as fatigue, weight change, abdominal distention and pain . The lack of efficient and early detection is the reason for the high mortality rate . The gold standard of ovarian cancer treatment consists of surgery followed by carboplatin alone, or a combination of chemotherapy with carboplatin and paclitaxel (ptx). Currently, first line therapy in early disease (stages i iia) is only surgery . In 25% of patients, this protocol does not cure, and the disease will recur . In advanced stages (iia iv), the current standard of care is cytoreduction followed by platinum - based chemotherapy . The most common adverse effects include haematological disorders, nausea, vomiting, disruption of the bone cycle and pain . According to most patients, alopecia is one of the most distressing side effects . Drugs commonly used in ovarian cancer treatment (paclitaxel, docetaxel) cause severe alopecia which markedly lowers the quality of patients lives [5, 6]. There is a need to investigate and find new drugs which will selectively and effectively treat ovarian cancer, and also be able to overcome multidrug resistance (mdr) the factor responsible for many cases of recurrent epithelial ovarian cancer (eoc). Trabectedin, also known as yondelis or et-743, is an anti - tumour drug, originally derived from the marine tunicate ecteinascidia turbinata . 1). Two of the rings covalently bind the n2 amino group in the guanine residue in the dna minor groove, in contrast to traditional alkylating drugs that bind guanine at the n7 or o6 position in the dna major groove . The adducts are stabilized by van der waals interactions and hydrogen bonds between rings a and b and dna [7, 8]. Trabectedin induces dna alkylation and dna - protein crosslinks which cause formation of dna strand breaks [7, 9, 10]. Trabectedin causes disruption of transcription by inhibition of the transcription - dependent nucleotide excision repair system (ner), followed by g2/m arrest and activation of the extrinsic and intrinsic apoptotic pathways, occurring through p53-independent process [7, 11, 12]. Data on hela, chinese hamster and human leukaemia cells obtained by soares et al . Indicated that dna synthesis and cellular viability were reduced by 50% after 1 h exposure to, respectively, 30 nm and 20 nm trabectedin . It was associated with formation of difficult - to - repair drug - dna adducts, converted later into dna double - strand breaks (dsbs). Furthermore, trabectedin toxicity was 8-fold higher toward brca2 deficient cells compared with the parental cell lines . Unrepaired dsbs in brca2-deficient cells led to chromosomal aberrations . Due to the promising outcomes of trabectedin, trabectedin was approved in the european union in july 2007 for the treatment of soft - tissue sarcomas (sts) in adults after failure of conventional therapy including anthracyclines and ifosfamide [7, 9, 11]. In a preclinical study, it demonstrated antineoplastic activity in vitro and in vivo in ovarian, breast, prostate, renal, melanoma and non - small cell lung cancer . It is effective in ovarian carcinoma xenografts and ovarian cancer explants and can be combined with cisplatin . Unfortunately, in vitro studies with mammalian cells transfected with mdr1 genes and an in vivo study in mice with p - gp overexpression showed that trabectedin is a substrate of p - glycoprotein (p - gp), the molecule responsible for multidrug resistance in human cancer cells . Anti - tumour activity of trabectedin in ovarian cancer has been studied in phase i and phase ii clinical trials . The phase i trial study included women with platinum - sensitive and platinum - resistant ovarian cancer after therapy based on platinum and taxanes . Trabectedin caused 43% response rates in patients with platinum - sensitive ovarian cancer, with a time to progression of 7 to 9 months . There were also observed two partial responses among women with platinum - resistant ovarian cancer . The most common adverse effects were nausea and vomiting (78%), neutropenia (41%), asthenia (78%) and thrombocytopenia (7.5%). Phase ii clinical trials included women with recurrent ovarian cancer after one or two platinum - based chemotherapy regimens . Trabectedin showed 29% and 6.3% response rates in women with platinum - sensitive and platinum - resistant ovarian cancer respectively, with a median progression time of between 5.1 and 2 months . The side effects were neutropenia (8%), nausea, vomiting, and fatigue (5%). Anti - tumour effectiveness of trabectedin in monotherapy was evaluated by mabuchi et al . In vitro using 3 lines of human ovarian clear cell carcinoma (ccc) and human ovarian serous adenocarcinomas (sac) 3 lines . Then, the anti - tumour effect of trabectedin was evaluated in vivo using a xenograft model with mice inoculated with ccc cells . After 4 weeks of treatment the tumour mass was reduced by greater than 70%, compared with pbs - treated mice . It is metabolized in the liver with a half - life of approximately 90 hours . The major tissue toxicities are connected with the bile duct (elevated plasma bilirubin, bile acids and aspartate transaminase (ast), alanine transaminase (alt), -glutamyl transferase (ggt) and alkaline phosphatase activities). The most common side effects are fatigue, nausea, anorexia, vomiting, constipation, atelectasis, dyspnoea, neutropenia, haemorrhoids, and intestinal obstruction . There were no indications of renal toxicity in rats (or mice and dogs) [7, 14, 17, 18]. There is also no cardiac toxicity and no ecg changes (considered as prolongation of qt / qtc interval) after treatment with trabectedin . Very interesting data were obtained for the combination of trabectedin with pegylated liposomal doxorubicin (pld, doxil). This modification prevents plasma protein adsorption to the liposome surface and showed that in contrast to non - pegylated liposomes, plds are able to remain in the circulation much longer . Due to the enhanced permeability and retention (epr) effect, the liposomes delivered drugs more specifically to the cancer tissues and limited exposure of normal cells to the drugs . The use of pld for ovarian cancer treatment gave promising results [19, 20]. Based on the results of a randomized phase iii trial (et-743-ova-301) (comparing pld alone with a combination of pld and trabectedin), on 672 patients diagnosed with a recurrent ovarian cancer from 21 countries in 2009, the european commission approved the combination with pld: 30 mg / m (as a 3 h infusion), every 3 weeks, for the treatment of patients with a recurrent platinum - sensitive ovarian cancer, for whom a first line platinum - based chemotherapy had failed [14, 21, 22]. The study demonstrated that trabectedin in combination with pld improves progression - free survival (pfs) and overall response rate (orr) in comparison to doxil alone as a second - line treatment of recurrent ovarian cancer . Among the 672 patients, 522 (77.7%) deaths were observed (including 258 in the trabectedin and doxil arm and 264 in the doxil arm). The median overall survival (os) for trabectedin plus doxil and surprisingly, the progression - free interval (pfi) favouring the pld arm (pld pfi) was 13.3 months, whereas trabectedin + pld pfi was 10.6 months . Additionally, this combination was well tolerated, with manageable toxicity . Furthermore, a decrease of pld - associated toxicity was observed, which supports the thesis that trabectedin in combination with pld is a good solution for patients with recurrent ovarian cancers [2123]. Angiogenesis the creation of new blood and lymphatic vessels is a crucial process for tumour development . Most types of tumours respond to a hypoxic environment by secreting a key pro - angiogenic growth factor called vascular endothelial growth factor (vegf). These particles bind to its receptors (vegfr-1, vegfr-2, vegfr-3) on the surface of cancer cells, leading to metastasis . Showed that the overexpression of vegfr-2 occurred among patients with early ovarian cancer stages (figo i, iia), which may indicate the important role of apoptosis during this phase, whereas an increased level of vegfr-3 was detected in advanced stages of cancer, and correlated with a positive response to chemotherapy . A high level of vegfr-3 is also connected with aggressiveness of ovarian cancer and indicates poor prognosis . Vasculogenic mimicry is a phenomenon connected with the formation of fluid - conducting channels, not lined with endothelium . This process occurs during tumour development and concerns undifferentiated cancers especially in the advanced stage of the disease . Additionally, some researchers claim that vasculogenic mimicry may protect the neoplasm against anticancer agents (as tumours demonstrating this phenomenon are often drug - resistant). Currently the most studied agent among anti - angiogenic drugs is bevacizumab a humanized igg1 monoclonal antibody which selectively inhibits vegf activity . Two phase iii clinical trials, gog-218 and icon7, showed a significant benefit in pfs when the standard (carboplatin - paclitaxel) chemotherapy was combined with bevacizumab . Based on these results, the european medicines agency (ema) approved this combination of drugs for the front line treatment of advanced epithelial ovarian cancer . . Indicated that bevacizumab in combination with trabectedin and oxaliplatin causes complete remission of recurrent ovarian clear cell carcinoma, with manageable general toxicity . It is thought that bevacizumab blocks vascular repair and survival, which enhances the activity of trabectedin and oxaliplatin . The latest studies indicate that angiogenesis and vasculogenic mimicry are important in tumour development; therefore deeper understanding of the individual angiogenic patient's phenotype may be helpful in designing an appropriate and effective anticancer therapy.
Two proven vector control strategies are currently advocated to reduce transmission of malarial disease in africa, namely indoor residual spraying (irs) [1 - 5] and insecticide - treated bednets (itns) [6 - 9]. Both methods are based on the use of residual insecticides in the intra - domiciliary domain and target mosquito vectors either before (itns) or after host - feeding (irs). Impressive reductions in childhood morbidity and mortality have been demonstrated in a variety of epidemiological settings, and it can be expected that irs / itns will remain in the forefront of malaria vector control for at least the remainder of this decade . In spite of their proven effectiveness [5 - 9], both methods have some drawbacks and limitations, such as insecticide resistance [10 - 13], environmental or human health concerns and socio - economic or cultural acceptance by communities . It is also clear that these powerful tools are not sufficient on their own to eliminate or drastically reduce the malaria burden from the most intensely endemic regions of the tropics, notably sub - saharan africa . An expansion of this limited arsenal of vector - control tools, with new strategies to reduce human exposure, the size of mosquito populations, or transmissibility of disease, is therefore needed, and preferably appropriate for use in an integrated fashion with irs / itns [19 - 21]. New innovative strategies, involving the release of genetically - engineered mosquitoes, aimed at rendering vector populations less susceptible to infection by human pathogens have seen enormous developments over the past few years [22 - 27]. If transposable genetic elements can be used to drive genes coding for refractoriness into fixation in wild vector populations, a substantial reduction in disease transmission may result . However, advances to date have been confined to laboratory settings and many questions relating to the fitness, behaviour, ecology and phenotypic characteristics of transformed insects remain unanswered [27 - 29]. The spread of desired traits, such as refractoriness to plasmodium infection, will depend on the reproductive fitness, evolutionary cost of the introduced trait, and manifestation of life - history behaviours, such as dispersal and mating, by engineered specimens . For instance, given the likelihood of assortative mating, transgenic males and females may face strong competition upon release, which necessitates an increased understanding of the behavioural and ecological determinants of gene flow in mosquito populations . The characteristics of genetically - engineered mosquitoes should preferably be similar to those of their wild con - specifics but may be compromised by genetic modification, selection for specific traits or routine laboratory maintenance and difficult to assess realistically under standard laboratory conditions . Many of the ecological and population biology issues thus remain serious challenges to the application of genetically - engineered mosquitoes . Moreover, until such time that the probability of potential public health benefits can be maximised, it will be unlikely that approval for release can and will be granted . The use of large contained field - based research settings is now widely advocated to face the shortfalls in our understanding of the behaviour and ecology of genetically - engineered vectors, prior to their release in the wild . Contained semi - field systems have been used for a variety of studies on mosquitoes, albeit outside africa [37 - 40]. In kenya, we have recently rejuvenated this approach and developed semi - field systems to study the behavioural ecology of malaria vectors [32,41 - 44]. By doing so we present the first attempts to complete the life - cycle of this important vector in such systems, as a first step towards studies involving genetically - engineered specimens . We transformed an existing greenhouse (cambridge glass house co. ltd ., uk), measuring 11.4 7.1 m (fig . 1a,1b,1c,1d) into a' malariasphere' (an enclosed environment with all components of a natural anopheles ecosystem) by replacing all glass parts with dark - green shade netting (density 90%) permitting airflow (wind) and precipitation to enter the system . Consequently, a sliding door provides entrance to the sphere, after passing a double layer of similar shade netting to prevent escape of released mosquitoes and entry of wild ones . The sphere is located on the shores of lake victoria, west kenya at the mbita point research & training centre (0025's, 3413'e) of the international centre of insect physiology and ecology (icipe). The area experiences two rainy seasons; the long rains from mid - march through june and short rains from october through december . Relatively high temperatures prevail throughout the year, ranging from 16c to 34c . During the rainy season, there are ample breeding sites in the mbita area for an . Suba district is inhabited by some 156,000 people, mostly belonging to the luo ethnic group, who practice mainly fishing and subsistence farming . ; data - loggers are shown as grey cubes) and photographs of the hut (b, note the white arrow showing the breeding site in front of the hut (c, d). Further details see text . Inside the sphere, a traditional luo house (3.2 2.8 1.7 m, fig . A mixture of wood ash and clay was used for plastering and smoothing the wall surfaces . The roof (2.8 m at its apex) was made of grass thatch mounted on a wooden frame . The house has a single door and no windows, which is typical for a local' simba' house . Eaves (height 15 cm) all around provide ventilation and serve as the predominant entry point for mosquitoes . We maintained the depth of the site at 15 cm by replenishing it with water collected from lake victoria . In each site we suspended a hobooptic stowaway tidbit data logger just below the water surface, and recorded the temperature at 30 min intervals . In order to monitor climatic conditions inside the system, we fitted six more hoboh8 data loggers, three inside the hut (at 0.5, 1.5 and 2.5 m from ground level) and three outside the hut on a pole at similar heights (fig . Climatic data were collected in february (peak summer) and june (onset of the cold season). We allowed plants to emerge from seeds present in the soil brought into the sphere, and in addition to this we planted a variety of food crops normally found around local homesteads (table 1). Prior to the release of mosquitoes and during construction of the house and planting of crops, a wide variety of other organisms entered the sphere, including some known mosquito predators such as ants (formicidae), spiders (e.g. Salticidae) and geckos (geckonidae). Mosquitoes used in the experiments originates from njage village (70 km from ifakara), south - east tanzania, and has been maintained under laboratory conditions since april 1996 . Adult insects were kept in standard 30 30 30 cm netting cages and offered 6% glucose as a carbohydrate source . The cages were kept under ambient climatic conditions and females given the opportunity to feed on an arm of a volunteer for 10 min three times per week . Eggs were collected on wet filter paper disks (9 cm diameter) and transferred to plastic containers containing water from lake victoria . Larvae were fed daily on tetraminfish food . Upon pupation, insects were transferred to cages for emergence . In order to assess whether all major life - history behaviours (i.e. Mating, sugar feeding, host seeking and oviposition) occurred successfully in the sphere, we attempted to complete the life - cycle during three separate experiments by introducing i) a group of 100 blood - fed females, ii) groups of 500 virgin females and 1500 males or iii) batches of 500 eggs in both breeding sites: i) in the first experiment we introduced 100 three - day - old females (f0), which had been held in cages with males since the time of emergence . They were blood fed (for the first time) on the forearm of a volunteer for 15 min and subsequently released (at 21.30 hrs) from a paper cup placed on the bed inside the hut . We then monitored the presence and development of eggs, larvae and pupae by inspecting the breeding sites at daily intervals . Following emergence of the first adults, we deliberately waited for six days before entering the greenhouse at night, in order to assess whether mosquitoes would successfully mate and survive / feed on the plants in the system . On day 17, 19, 20 and 21 following the introduction of females, a volunteer slept inside the hut from 21.30 hrs until 07.00 the following day, which allowed the f1 population, and any of their parents that had survived, to feed on human blood . Ii) as some of the parental (f0) females could have survived until day 17, it needed to be ascertained that virgin (newly emerged) insects survived, mated and blood - fed successfully too . We therefore introduced 500, 3 to 5 day - old virgin females, which had emerged from the pupa individually in glass vials, together with 1500, 5 to 7 day - old males at 21.00 hrs . Starting three days afterwards, a volunteer slept in the hut for 5 consecutive nights . We observed daily whether eggs were laid in the breeding sites to ensure that insemination, blood feeding and oviposition had taken place for two weeks following the release . All pupae were collected from the breeding sites as they appeared so that assessments of survival by the f0 generation would not be confounded by the emergence of an overlapping f1 generation . Iii) a third experiment was started by introducing 500 eggs at night (22.30 hrs) in each of the two breeding sites . Concurrently we reared one thousand eggs from the same batch under standard laboratory conditions described above . This enabled us to determine the sex ratio and thus the number of males and females released . A volunteer occupied the hut for four consecutive nights, starting on day 22 after the start of the experiment . Thereafter, the breeding sites were monitored daily for the presence of eggs / larvae until day 32 . A research protocol for the above experiments was submitted to the kenya national ethical review committee, based at the kenya medical research institute (kemri), in which the discomfort and potential risks of (non - infectious) mosquito bites to volunteers was explained . Bnn, bgjk and gfk were involved in the experiments, and do not object to their names being revealed for publication . A parasite - free environment was ensured through a) regular screening of the volunteers' peripheral blood for plasmodium parasites and b) non - occupancy of the sphere beyond 5 days after the mosquitoes were given the first opportunity to obtain a blood meal . As malaria infections in mosquitoes take at least 10 days to reach the sporozoite - infective stage, this procedure minimised risks of volunteers being infected within the experimental set up . We transformed an existing greenhouse (cambridge glass house co. ltd ., uk), measuring 11.4 7.1 m (fig . 1a,1b,1c,1d) into a' malariasphere' (an enclosed environment with all components of a natural anopheles ecosystem) by replacing all glass parts with dark - green shade netting (density 90%) permitting airflow (wind) and precipitation to enter the system . Consequently, a sliding door provides entrance to the sphere, after passing a double layer of similar shade netting to prevent escape of released mosquitoes and entry of wild ones . The sphere is located on the shores of lake victoria, west kenya at the mbita point research & training centre (0025's, 3413'e) of the international centre of insect physiology and ecology (icipe). The area experiences two rainy seasons; the long rains from mid - march through june and short rains from october through december . Relatively high temperatures prevail throughout the year, ranging from 16c to 34c . During the rainy season, there are ample breeding sites in the mbita area for an . Suba district is inhabited by some 156,000 people, mostly belonging to the luo ethnic group, who practice mainly fishing and subsistence farming . ; data - loggers are shown as grey cubes) and photographs of the hut (b, note the white arrow showing the breeding site in front of the hut (c, d). Further details see text . Inside the sphere, a traditional luo house (3.2 2.8 1.7 m, fig . A mixture of wood ash and clay was used for plastering and smoothing the wall surfaces . The roof (2.8 m at its apex) was made of grass thatch mounted on a wooden frame . The house has a single door and no windows, which is typical for a local' simba' house . Eaves (height 15 cm) all around provide ventilation and serve as the predominant entry point for mosquitoes . We maintained the depth of the site at 15 cm by replenishing it with water collected from lake victoria . In each site we suspended a hobooptic stowaway tidbit data logger just below the water surface, and recorded the temperature at 30 min intervals . In order to monitor climatic conditions inside the system, we fitted six more hoboh8 data loggers, three inside the hut (at 0.5, 1.5 and 2.5 m from ground level) and three outside the hut on a pole at similar heights (fig . Climatic data were collected in february (peak summer) and june (onset of the cold season). We allowed plants to emerge from seeds present in the soil brought into the sphere, and in addition to this we planted a variety of food crops normally found around local homesteads (table 1). Prior to the release of mosquitoes and during construction of the house and planting of crops, a wide variety of other organisms entered the sphere, including some known mosquito predators such as ants (formicidae), spiders (e.g. Salticidae) and geckos (geckonidae). Mosquitoes used in the experiments originates from njage village (70 km from ifakara), south - east tanzania, and has been maintained under laboratory conditions since april 1996 . Adult insects were kept in standard 30 30 30 cm netting cages and offered 6% glucose as a carbohydrate source . The cages were kept under ambient climatic conditions and females given the opportunity to feed on an arm of a volunteer for 10 min three times per week . Eggs were collected on wet filter paper disks (9 cm diameter) and transferred to plastic containers containing water from lake victoria . In order to assess whether all major life - history behaviours (i.e. Mating, sugar feeding, host seeking and oviposition) occurred successfully in the sphere, we attempted to complete the life - cycle during three separate experiments by introducing i) a group of 100 blood - fed females, ii) groups of 500 virgin females and 1500 males or iii) batches of 500 eggs in both breeding sites: i) in the first experiment we introduced 100 three - day - old females (f0), which had been held in cages with males since the time of emergence . They were blood fed (for the first time) on the forearm of a volunteer for 15 min and subsequently released (at 21.30 hrs) from a paper cup placed on the bed inside the hut . We then monitored the presence and development of eggs, larvae and pupae by inspecting the breeding sites at daily intervals . Following emergence of the first adults, we deliberately waited for six days before entering the greenhouse at night, in order to assess whether mosquitoes would successfully mate and survive / feed on the plants in the system . On day 17, 19, 20 and 21 following the introduction of females, a volunteer slept inside the hut from 21.30 hrs until 07.00 the following day, which allowed the f1 population, and any of their parents that had survived, to feed on human blood . Ii) as some of the parental (f0) females could have survived until day 17, it needed to be ascertained that virgin (newly emerged) insects survived, mated and blood - fed successfully too . We therefore introduced 500, 3 to 5 day - old virgin females, which had emerged from the pupa individually in glass vials, together with 1500, 5 to 7 day - old males at 21.00 hrs . Starting three days afterwards, a volunteer slept in the hut for 5 consecutive nights . We observed daily whether eggs were laid in the breeding sites to ensure that insemination, blood feeding and oviposition had taken place for two weeks following the release . All pupae were collected from the breeding sites as they appeared so that assessments of survival by the f0 generation would not be confounded by the emergence of an overlapping f1 generation . Iii) a third experiment was started by introducing 500 eggs at night (22.30 hrs) in each of the two breeding sites . Concurrently we reared one thousand eggs from the same batch under standard laboratory conditions described above . This enabled us to determine the sex ratio and thus the number of males and females released . A volunteer occupied the hut for four consecutive nights, starting on day 22 after the start of the experiment . Thereafter, the breeding sites were monitored daily for the presence of eggs / larvae until day 32 . A research protocol for the above experiments was submitted to the kenya national ethical review committee, based at the kenya medical research institute (kemri), in which the discomfort and potential risks of (non - infectious) mosquito bites to volunteers was explained . Bnn, bgjk and gfk were involved in the experiments, and do not object to their names being revealed for publication . A parasite - free environment was ensured through a) regular screening of the volunteers' peripheral blood for plasmodium parasites and b) non - occupancy of the sphere beyond 5 days after the mosquitoes were given the first opportunity to obtain a blood meal . As malaria infections in mosquitoes take at least 10 days to reach the sporozoite - infective stage, this procedure minimised risks of volunteers being infected within the experimental set up . Figure 2 shows the climatic conditions recorded in the greenhouse over a 3-week period in june 2000, coinciding with the time of the third experiment (onset cold dry season). Similar data sets for february 2000 (peak of the main hot and dry season) showed an average temperature at the water surface of 24.0c (range 20.029.8c). Other studies have recorded slightly lower average temperatures and larger ranges over which these fluctuate, both in artificially constructed and natural sites . Haddow recorded a range of 19.034.5c in pans of similar size that were supplemented with soil and had water of similar depth near kisumu (80 km from mbita point). Gimnig et al . Recorded an average of 26.4 0.7c from natural sites between march and august 1998 in that same area, as did koenraadt et al . (pers . Comm . ), with 25.8 4.2c, in two subsequent years . The lower temperatures recorded from sites in the sphere were probably caused by reduced infiltration of sunlight through the roof's shade netting . Temperature (a) and relative humidity (b) data recorded in one of the two the breeding sites and different heights (0.5, 1.5 and 2.5 m) inside the hut over a 3-week period in june 2000 . Arrows on y - axis show maximum and minimum recorded and accompanying figures show the same data for data - loggers outside the hut at those same heights . 2a) inside the hut at various heights fluctuated much more considerably but nevertheless remained relatively consistent throughout the observational periods . Average temperatures increased both inside (23.1, 23.8 and 23.9c for 0.5, 1.5 and 2.5 m respectively) and outside the hut with height as did the range over which these fluctuated daily . Corresponding data for february inside the hut showed higher averages (24.8, 25.1 and 25.2c for 0.5, 1.5 and 2.5 m respectively). Between the seasons, maximum variation between temperatures was found outside the hut at 2.5 m, from 37.0c (february - maximum) to 16.4c (june - minimum). The smallest variation was observed inside the hut at 0.5 m, from 28.7c (february - maximum) to 19.7c (june - minimum). As such, the range over which temperatures fluctuated between seasons was 2.3 times larger outside (2.5 m) than inside (0.5 m) the hut . Haddow recorded mean temperatures between october and december in local huts (at 1 m height) in the kisumu area and found an average temperature of 24.2c (range 21.027.0c). Our own measurements inside the hut in the sphere between 18 october and 15 november (2000) at 1.5 m height gave values of 24.0 1.76c (range 19.829.1c) whereas measurements inside 4 local houses in mbita of similar design during that same period gave higher average values (0.50.8c) and absolute maxima (0.8c). Ambient conditions inside local houses are more constant than outdoor climatic conditions (this study and) and it would seem that the sphere itself exerts a similar, albeit small, insulating effect: slightly lower temperatures and smaller ranges over which these fluctuate, both inside and outside the hut . Relative humidity (rh) data (fig . Relative humidity is fairly constant and averages inside the hut during june ranged from 63.5% (1.0 m) to 69.3% (2.5 m). Minimum values were always higher inside the hut than outside, providing more suitable microclimatic conditions for resting mosquitoes . Measurements in october / november, both in the sphere and a local house of similar design in mbita point, showed slightly higher average rh values outdoors in both settings than indoors with minimal differences between the sphere and the village hut . Overall, as with temperatures, the range over which the rh fluctuated was smaller inside than outside the hut and minima inside the sphere were always 3 to 4% higher than those measured in village huts . Although small, these climatic differences may affect development of immature stages and survival of adults, and research findings from experiments inside the sphere should be compared with field conditions at slightly higher altitudes . The introduction of blood - fed females into the greenhouse resulted in the presence of eggs in the breeding sites on day 3 (2.5 days after release), and eggs continued to be observed in the sites until day 7 (fig . 3). Larvae (from l1 to l4 stage) were seen feeding at the water surface until day 23, when the last l4 larva pupated . The first five pupae were seen in the breeding sites in the evening of day 10, meaning that the variation in maturation time from egg to pupa was 720 days . In spite of having conditions with higher larval density and smaller water surface area, gimnig et al . Recorded much reduced periods to pupation, from 5 to 12 days . In total, 57 pupae were counted in the breeding site 3.8 m in front of the hut and 130 in the site 1.1 m behind it, and on average they harboured only 0.08 and 0.18 larva / cm, respectively . These densities are lower than those observed in natural habitats, and given the fact that we observed algal growth, considered important for larval growth, it seems that the prolonged time to pupation in this trial may be attributed to the relatively small range over which temperatures fluctuate . Alternatively, re - introducing mosquitoes that had been maintained under laboratory conditions for several years in a more natural setting may have caused these effects, and poor adaptation to these conditions may have stunted their development . Completion of the anopheles gambiae life - cycle in the greenhouse over a 27-day period . Blood - fed females were released on day 1, and arrows show time periods during which the various developmental stages were observed . The first adults were seen inside the hut on day 11, and continued to be present until the end of the experiment (day 27). Starting in the morning of day 22, we observed new eggs in the breeding sites and subsequent larval development . From the above it can be deduced that specific behaviours of the adult insects occurred during certain time periods (fig . Oviposition activity took place twice during this trial, meaning that females survived until eggs were mature, that they successfully located a breeding site, accepted it for oviposition, and laid eggs . Other potential breeding sites, like the leaf axils of the banana trees in the sphere, were examined but were not found to harbour eggs, larvae or pupae . The period for reaching sexual maturity for males may be at least one day and for females up to 60 hrs, so mating of the f1 generation may not have taken place until dusk on day 14 . In spite of regular observations during dusk, we did not see any swarming activity typically associated with mating in an . This is a particularly interesting point, because in contrast with other settings (e.g. ), where mating swarms were frequently observed we failed to do so over a 3-year period in the mbita area as did charlwood (pers.comm .) Who observed only one anopheline swarm during 4 years in the kilombero valley of tanzania, raising the question as to whether swarming is an obligate component of the an . Alternatively, maintenance of mosquitoes in laboratory cages for several years forced this strain to become stenogamous (i.e. The ability to mate in small cages), and this adaptation may have interfered with its ability to swarm when introduced into the sphere . As newly emerged adults rarely survive for more than 48 hrs without the availability of an energy source, mosquitoes must have supplemented their energy reserves with carbohydrates from the plants in the sphere (table 1) for up to 6 days before they were allowed access to a blood source, in the form of a human volunteer sleeping in the hut . Some plants like castorbean (ricinus communis l.) were flowering at the time of the experiment, and may have provided nectar sources . In cage experiments (impoinvil et al ., in prep .) We have observed a mean survival time of 7.0 0.2 days on this plant, which is comparable to mosquitoes given 6% glucose (8.7 0.2 days). Gambiae mosquitoes emerge from the pupal stage with a deficit not only in carbohydrates, but also lipid and protein, which usually is compensated for by consuming a (small) blood meal within the first few days of adult life, it is likely that mortality during the 6-day post - emergence period in the current trial was too high to have a good number of the 8090 females that emerged survive long enough to obtain their first blood meal . Within 15 min of entering the hut at night, the volunteer noticed the sound of mosquitoes and subsequently felt mosquito bites on his exposed lower limbs . This implies that females were receptive to host cues, entered the hut, probably through the eaves, and then successfully located and fed on the human host . At sunrise, several engorged females were seen resting on the walls, indicating successful blood feeding and endophily (indoor resting), which is typical for this species . Following maturation of eggs, the second oviposition began during the night of day 21, thus completing the life - cycle . We continued to observe both immature and adult insects (presumably mostly from the f1 generation) until day 27, when the experiment was terminated (by refraining from entering the sphere for about 1 month). The second experiment, in which we released 500 virgin females together with 1500 males demonstrated that mating does occur in a relatively small, semi - field system . After the third night that a volunteer had slept in the hut, we observed eggs in the breeding sites . The production of offspring, though, was low, and we only collected 40 pupae by the end of the trial period . This may have been caused by heavy rainfall during three consecutive nights (day 24), which may have affected the survival of the adults and/or larvae or washed away the larvae from the breeding sites due to overflow . Since we observed few mosquitoes, we decided to conduct a human landing catch during two nights inside the hut, starting two nights after sleeping in the hut had ended . Nevertheless, the life - cycle was completed, as manifested by the harvested pupae, which were removed from the breeding sites to prevent emergence of the f1 generation which would have compromised interpretation of survival of the f0 generation . The third experiment started by introducing 500 eggs into each of the breeding sites, whilst 1000 eggs (from the same original batch) were reared under laboratory conditions . In the laboratory, larvae developed at the same rate and most reached maturity by day 10, when the first pupae were observed (fig . This was similar for the breeding sites in the sphere, except that development was highly asynchronous, i.e. Some larvae pupated by day 10, whereas others took up to day 24 before pupation . These times to pupation are similar to those observed in the first trial, but are again in contrast with other studies . On day 7 we counted all larvae and observed 887 in the insectary, as opposed to 652 in the sphere . Overall, the laboratory batch yielded 804 pupae, versus 495 from the breeding sites . On the basis of these data, the average daily survival was 0.90 for the laboratory larvae, and 0.83 for the larvae in the sphere . With nearly half the larvae surviving to the adult stage, these results contrast sharply with much higher mortalities (up to 90%) observed in the kisumu area and so tome (charlwood, pers . The sex ratio of emerging adults in the laboratory was 2:3, which translated into a female population in the sphere of 297, on the assumption that no insects died during emergence . On day 28, after the release of eggs in the breeding sites, we observed that eggs had been laid by the f1 generation, but with only 6 and 3 eggs in the two breeding sites respectively . Cumulative percentage of pupation of eggs introduced in the two breeding sites inside the greenhouse () or under insectary conditions (). Our results have shown that by starting either at the post - blood feeding, pre - mating, or egg stage, a new generation of insects can be reared under these semi - field conditions, and that all life - history behaviours were successfully completed to a lesser or greater extent . This therefore represents the first and promising step towards continuous maintenance of parasite - free an . Gambiae populations under semi - natural conditions that can be experimentally manipulated in studies of malaria vector ecology and transmission control . Russell and rao used a large outdoor cage, based on a design by hackett and bates, to study swarming and oviposition behaviour of an . Culicifacies giles in india, and showed that such systems can be used to unravel aspects of the behavioural ecology of anophelines . Our study shows that such systems can now also be developed for studies on african malaria vectors in order to start filling the gaps in our understanding of the behavioural ecology of these insects . First and foremost, it provides a suitable intermediate between laboratory - based studies addressing mosquito behaviour and ecology, and the field situation . Too often, conclusions are drawn from results obtained under laboratory conditions that necessitate speculation as to what may or may not happen in the field . Fixed climatic conditions, cage - experiments, olfactometers and windtunnels, in which the mosquito strains used have been laboratory - reared for sometimes decades, may readily distort behavioural and ecological phenomena . Here we have shown that, beyond introducing f1 generation malaria - free mosquitoes from wild populations, it may be possible to rear vectors in situ within a semi - natural system that may minimize such artefacts . Conversely, the advantage of using insectary - reared mosquitoes is in the level of control that may be exerted that would not otherwise be possible: experiments can be conducted all - year - round, with fixed numbers of insects, of known age and physiological status, in a malaria parasite - free environment under ambient climatic conditions . This enables more direct inferences to be drawn from data analysis as compared to longitudinal field studies, because of constant conditions and simplified experimental design . We have recently evaluated the efficacy of several plants traditionally used by the luo community as repellents in a similar semi - field set - up, and simple logistic regression, on data collected during four nights per plant, yielded significant results . Within a year of nearly continuous experimentation, the repellency of 8 plant species and 3 combinations thereof was evaluated through thermal expulsion or direct burning, and 9 species and 2 combinations thereof were tested in potted form . Such studies would have taken several years under field conditions, and would be limited to times when mosquito densities would have been sufficiently high to permit meaningful experiments . Gambiae maintained on a variety of diets (blood or sugar alone, or a combination of both) and revealed similar results to those obtained under laboratory conditions . Within one year of starting studies on the behaviour of mosquitoes around bednets in a semi - field setup, we transformed a regular conical bednet into a trap that may catch up to 70% of the females released . Recent field evaluation of this trap shows it to be a promising replacement for the human landing catch (mathenge et al . With the trap now being considered for commercial manufacturing (it took a mere two years to reach this stage), this would have been impossible without the availability of a semi - field set - up in which continuous experimentation ensured rapid progress towards product development . Even though our system resembles more closely the field situation, it remains to be ascertained to what extent . Our current study was mainly qualitative in design and focused on life - cycle completion . For instance, observation of eggs in the breeding sites in the morning of day 22 during the first trial implies that these originated from females that fed once on day 19, as those that fed on day 17 should have laid before . However, it is likely that these females fed twice, on day 17 and day 19, and should be classified as pre - gravids, before fully maturing a batch of eggs . Furthermore, in the absence of a human host, mosquitoes survived up to six days after emergence, confirming field results that feeding on plant sugars does occur and may be an important feature of the life - cycle in this species (foster and knols, unpublished data). There may be other, yet unknown, factors that affect the behavioural ecology of the insects in such systems . Or, as bates wrote, following his outdoor cage studies in albania: " one still cannot be sure that the reactions of the mosquito are' natural' because there is always the barrier of wire liable to be encountered on extended flights; and when the flight of a mosquito has been interrupted by this wire barrier its further activity may be definitely unnatural " . Rightly so, and even though we did not observe any obvious distorted behaviours, it is imperative that findings from semi - field studies be verified under natural outdoor conditions . Additional studies in which the release and performance of field - collected, blood - fed mosquitoes in the sphere is compared with that of laboratory specimens in terms of egg - recovery, developmental periods and important behavioural characteristics (like swarming) will provide further insight to what extent such systems mimic the natural anopheles environment and colony adaptation impairs natural behaviours . Nevertheless, since bates' days, advances in science merit a renewed impetus towards semi - field studies in contained near - natural environments, particularly with respect to transgenic mosquitoes . Fitness evaluations of engineered strains of vectors are mandatory for transformation technology to become an established disease control tool in africa . Perhaps this alone, is ample justification for more intensive sphere studies, hopefully not only in kenya, but also in other african countries likely to be involved in this endeavour . Studies on gene flow, mating behaviour and reproductive fitness, combined with studies on the effects of laboratory maintenance on the genetic make - up of transformed strains to be released, can be conducted in semi - field systems . Such systems, particularly when used to study genetically - engineered mosquitoes will require more advanced containment levels than the system described here . Guidelines for facility location, physical and biological containment, safety practices and calamity control need to be developed and adapted from existing arthropod containment guidelines . There are several good reasons to further such studies in disease - endemic settings . Under such conditions it will be possible to transform offspring from wild mosquitoes, conduct experiments under local ambient climatic conditions and evaluate transgene spread and fixation in offspring from field - collected gravid females that emerge in a semi - field setup . Last, but not least, it will enable scientists from developing countries to become more directly involved in evaluating the potential use and application of transgenic mosquitoes for future malarial disease control . Figure 2 shows the climatic conditions recorded in the greenhouse over a 3-week period in june 2000, coinciding with the time of the third experiment (onset cold dry season). Similar data sets for february 2000 (peak of the main hot and dry season) showed an average temperature at the water surface of 24.0c (range 20.029.8c). Other studies have recorded slightly lower average temperatures and larger ranges over which these fluctuate, both in artificially constructed and natural sites . Haddow recorded a range of 19.034.5c in pans of similar size that were supplemented with soil and had water of similar depth near kisumu (80 km from mbita point). Gimnig et al . Recorded an average of 26.4 0.7c from natural sites between march and august 1998 in that same area, as did koenraadt et al . (pers . Comm . ), with 25.8 4.2c, in two subsequent years . The lower temperatures recorded from sites in the sphere were probably caused by reduced infiltration of sunlight through the roof's shade netting . Temperature (a) and relative humidity (b) data recorded in one of the two the breeding sites and different heights (0.5, 1.5 and 2.5 m) inside the hut over a 3-week period in june 2000 . Arrows on y - axis show maximum and minimum recorded and accompanying figures show the same data for data - loggers outside the hut at those same heights . 2a) inside the hut at various heights fluctuated much more considerably but nevertheless remained relatively consistent throughout the observational periods . Average temperatures increased both inside (23.1, 23.8 and 23.9c for 0.5, 1.5 and 2.5 m respectively) and outside the hut with height as did the range over which these fluctuated daily . Corresponding data for february inside the hut showed higher averages (24.8, 25.1 and 25.2c for 0.5, 1.5 and 2.5 m respectively). Between the seasons, maximum variation between temperatures was found outside the hut at 2.5 m, from 37.0c (february - maximum) to 16.4c (june - minimum). The smallest variation was observed inside the hut at 0.5 m, from 28.7c (february - maximum) to 19.7c (june - minimum). As such, the range over which temperatures fluctuated between seasons was 2.3 times larger outside (2.5 m) than inside (0.5 m) the hut . Haddow recorded mean temperatures between october and december in local huts (at 1 m height) in the kisumu area and found an average temperature of 24.2c (range 21.027.0c). Our own measurements inside the hut in the sphere between 18 october and 15 november (2000) at 1.5 m height gave values of 24.0 1.76c (range 19.829.1c) whereas measurements inside 4 local houses in mbita of similar design during that same period gave higher average values (0.50.8c) and absolute maxima (0.8c). Ambient conditions inside local houses are more constant than outdoor climatic conditions (this study and) and it would seem that the sphere itself exerts a similar, albeit small, insulating effect: slightly lower temperatures and smaller ranges over which these fluctuate, both inside and outside the hut . Relative humidity (rh) data (fig . Relative humidity is fairly constant and averages inside the hut during june ranged from 63.5% (1.0 m) to 69.3% (2.5 m). Minimum values were always higher inside the hut than outside, providing more suitable microclimatic conditions for resting mosquitoes . Measurements in october / november, both in the sphere and a local house of similar design in mbita point, showed slightly higher average rh values outdoors in both settings than indoors with minimal differences between the sphere and the village hut . Overall, as with temperatures, the range over which the rh fluctuated was smaller inside than outside the hut and minima inside the sphere were always 3 to 4% higher than those measured in village huts . Although small, these climatic differences may affect development of immature stages and survival of adults, and research findings from experiments inside the sphere should be compared with field conditions at slightly higher altitudes . The introduction of blood - fed females into the greenhouse resulted in the presence of eggs in the breeding sites on day 3 (2.5 days after release), and eggs continued to be observed in the sites until day 7 (fig . 3). Larvae (from l1 to l4 stage) were seen feeding at the water surface until day 23, when the last l4 larva pupated . The first five pupae were seen in the breeding sites in the evening of day 10, meaning that the variation in maturation time from egg to pupa was 720 days . In spite of having conditions with higher larval density and smaller water surface area, gimnig et al . Recorded much reduced periods to pupation, from 5 to 12 days . In total, 57 pupae were counted in the breeding site 3.8 m in front of the hut and 130 in the site 1.1 m behind it, and on average they harboured only 0.08 and 0.18 larva / cm, respectively . These densities are lower than those observed in natural habitats, and given the fact that we observed algal growth, considered important for larval growth, it seems that the prolonged time to pupation in this trial may be attributed to the relatively small range over which temperatures fluctuate . Alternatively, re - introducing mosquitoes that had been maintained under laboratory conditions for several years in a more natural setting may have caused these effects, and poor adaptation to these conditions may have stunted their development . Completion of the anopheles gambiae life - cycle in the greenhouse over a 27-day period . Blood - fed females were released on day 1, and arrows show time periods during which the various developmental stages were observed . The first adults were seen inside the hut on day 11, and continued to be present until the end of the experiment (day 27). Starting in the morning of day 22, we observed new eggs in the breeding sites and subsequent larval development . From the above it can be deduced that specific behaviours of the adult insects occurred during certain time periods (fig . Oviposition activity took place twice during this trial, meaning that females survived until eggs were mature, that they successfully located a breeding site, accepted it for oviposition, and laid eggs . Other potential breeding sites, like the leaf axils of the banana trees in the sphere, were examined but were not found to harbour eggs, larvae or pupae . The period for reaching sexual maturity for males may be at least one day and for females up to 60 hrs, so mating of the f1 generation may not have taken place until dusk on day 14 . In spite of regular observations during dusk, we did not see any swarming activity typically associated with mating in an . This is a particularly interesting point, because in contrast with other settings (e.g. ), where mating swarms were frequently observed we failed to do so over a 3-year period in the mbita area as did charlwood (pers.comm .) Who observed only one anopheline swarm during 4 years in the kilombero valley of tanzania, raising the question as to whether swarming is an obligate component of the an . Alternatively, maintenance of mosquitoes in laboratory cages for several years forced this strain to become stenogamous (i.e. The ability to mate in small cages), and this adaptation may have interfered with its ability to swarm when introduced into the sphere . As newly emerged adults rarely survive for more than 48 hrs without the availability of an energy source, mosquitoes must have supplemented their energy reserves with carbohydrates from the plants in the sphere (table 1) for up to 6 days before they were allowed access to a blood source, in the form of a human volunteer sleeping in the hut . Some plants like castorbean (ricinus communis l.) were flowering at the time of the experiment, and may have provided nectar sources . In cage experiments (impoinvil et al ., in prep .) We have observed a mean survival time of 7.0 0.2 days on this plant, which is comparable to mosquitoes given 6% glucose (8.7 0.2 days). Gambiae mosquitoes emerge from the pupal stage with a deficit not only in carbohydrates, but also lipid and protein, which usually is compensated for by consuming a (small) blood meal within the first few days of adult life, it is likely that mortality during the 6-day post - emergence period in the current trial was too high to have a good number of the 8090 females that emerged survive long enough to obtain their first blood meal . Within 15 min of entering the hut at night, the volunteer noticed the sound of mosquitoes and subsequently felt mosquito bites on his exposed lower limbs . This implies that females were receptive to host cues, entered the hut, probably through the eaves, and then successfully located and fed on the human host . At sunrise, several engorged females were seen resting on the walls, indicating successful blood feeding and endophily (indoor resting), which is typical for this species . Following maturation of eggs, the second oviposition began during the night of day 21, thus completing the life - cycle . We continued to observe both immature and adult insects (presumably mostly from the f1 generation) until day 27, when the experiment was terminated (by refraining from entering the sphere for about 1 month). The second experiment, in which we released 500 virgin females together with 1500 males demonstrated that mating does occur in a relatively small, semi - field system . After the third night that a volunteer had slept in the hut, we observed eggs in the breeding sites . The production of offspring, though, was low, and we only collected 40 pupae by the end of the trial period . This may have been caused by heavy rainfall during three consecutive nights (day 24), which may have affected the survival of the adults and/or larvae or washed away the larvae from the breeding sites due to overflow . Since we observed few mosquitoes, we decided to conduct a human landing catch during two nights inside the hut, starting two nights after sleeping in the hut had ended . Nevertheless, the life - cycle was completed, as manifested by the harvested pupae, which were removed from the breeding sites to prevent emergence of the f1 generation which would have compromised interpretation of survival of the f0 generation . The third experiment started by introducing 500 eggs into each of the breeding sites, whilst 1000 eggs (from the same original batch) were reared under laboratory conditions . In the laboratory, larvae developed at the same rate and most reached maturity by day 10, when the first pupae were observed (fig . This was similar for the breeding sites in the sphere, except that development was highly asynchronous, i.e. Some larvae pupated by day 10, whereas others took up to day 24 before pupation . These times to pupation are similar to those observed in the first trial, but are again in contrast with other studies . On day 7 we counted all larvae and observed 887 in the insectary, as opposed to 652 in the sphere . Overall, the laboratory batch yielded 804 pupae, versus 495 from the breeding sites . On the basis of these data, the average daily survival was 0.90 for the laboratory larvae, and 0.83 for the larvae in the sphere . With nearly half the larvae surviving to the adult stage, these results contrast sharply with much higher mortalities (up to 90%) observed in the kisumu area and so tome (charlwood, pers . . The sex ratio of emerging adults in the laboratory was 2:3, which translated into a female population in the sphere of 297, on the assumption that no insects died during emergence . On day 28, after the release of eggs in the breeding sites, we observed that eggs had been laid by the f1 generation, but with only 6 and 3 eggs in the two breeding sites respectively . Cumulative percentage of pupation of eggs introduced in the two breeding sites inside the greenhouse () or under insectary conditions (). Our results have shown that by starting either at the post - blood feeding, pre - mating, or egg stage, a new generation of insects can be reared under these semi - field conditions, and that all life - history behaviours were successfully completed to a lesser or greater extent . This therefore represents the first and promising step towards continuous maintenance of parasite - free an . Gambiae populations under semi - natural conditions that can be experimentally manipulated in studies of malaria vector ecology and transmission control . Russell and rao used a large outdoor cage, based on a design by hackett and bates, to study swarming and oviposition behaviour of an . Culicifacies giles in india, and showed that such systems can be used to unravel aspects of the behavioural ecology of anophelines . Our study shows that such systems can now also be developed for studies on african malaria vectors in order to start filling the gaps in our understanding of the behavioural ecology of these insects . First and foremost, it provides a suitable intermediate between laboratory - based studies addressing mosquito behaviour and ecology, and the field situation . Too often, conclusions are drawn from results obtained under laboratory conditions that necessitate speculation as to what may or may not happen in the field . Fixed climatic conditions, cage - experiments, olfactometers and windtunnels, in which the mosquito strains used have been laboratory - reared for sometimes decades, may readily distort behavioural and ecological phenomena . Here we have shown that, beyond introducing f1 generation malaria - free mosquitoes from wild populations, it may be possible to rear vectors in situ within a semi - natural system that may minimize such artefacts . Conversely, the advantage of using insectary - reared mosquitoes is in the level of control that may be exerted that would not otherwise be possible: experiments can be conducted all - year - round, with fixed numbers of insects, of known age and physiological status, in a malaria parasite - free environment under ambient climatic conditions . This enables more direct inferences to be drawn from data analysis as compared to longitudinal field studies, because of constant conditions and simplified experimental design . We have recently evaluated the efficacy of several plants traditionally used by the luo community as repellents in a similar semi - field set - up, and simple logistic regression, on data collected during four nights per plant, yielded significant results . Within a year of nearly continuous experimentation, the repellency of 8 plant species and 3 combinations thereof was evaluated through thermal expulsion or direct burning, and 9 species and 2 combinations thereof were tested in potted form . Such studies would have taken several years under field conditions, and would be limited to times when mosquito densities would have been sufficiently high to permit meaningful experiments . Gambiae maintained on a variety of diets (blood or sugar alone, or a combination of both) and revealed similar results to those obtained under laboratory conditions . Within one year of starting studies on the behaviour of mosquitoes around bednets in a semi - field setup, we transformed a regular conical bednet into a trap that may catch up to 70% of the females released . Recent field evaluation of this trap shows it to be a promising replacement for the human landing catch (mathenge et al ., in preparation . ). With the trap now being considered for commercial manufacturing (it took a mere two years to reach this stage), this would have been impossible without the availability of a semi - field set - up in which continuous experimentation ensured rapid progress towards product development . Even though our system resembles more closely the field situation, it remains to be ascertained to what extent . Our current study was mainly qualitative in design and focused on life - cycle completion . For instance, observation of eggs in the breeding sites in the morning of day 22 during the first trial implies that these originated from females that fed once on day 19, as those that fed on day 17 should have laid before . However, it is likely that these females fed twice, on day 17 and day 19, and should be classified as pre - gravids, before fully maturing a batch of eggs . Furthermore, in the absence of a human host, mosquitoes survived up to six days after emergence, confirming field results that feeding on plant sugars does occur and may be an important feature of the life - cycle in this species (foster and knols, unpublished data). Obviously, there are limitations associated with these studies . Some phenomena, like dispersal, there may be other, yet unknown, factors that affect the behavioural ecology of the insects in such systems . Or, as bates wrote, following his outdoor cage studies in albania: " one still cannot be sure that the reactions of the mosquito are' natural' because there is always the barrier of wire liable to be encountered on extended flights; and when the flight of a mosquito has been interrupted by this wire barrier its further activity may be definitely unnatural " . Rightly so, and even though we did not observe any obvious distorted behaviours, it is imperative that findings from semi - field studies be verified under natural outdoor conditions . Additional studies in which the release and performance of field - collected, blood - fed mosquitoes in the sphere is compared with that of laboratory specimens in terms of egg - recovery, developmental periods and important behavioural characteristics (like swarming) will provide further insight to what extent such systems mimic the natural anopheles environment and colony adaptation impairs natural behaviours . Nevertheless, since bates' days, advances in science merit a renewed impetus towards semi - field studies in contained near - natural environments, particularly with respect to transgenic mosquitoes . Fitness evaluations of engineered strains of vectors are mandatory for transformation technology to become an established disease control tool in africa . Perhaps this alone, is ample justification for more intensive sphere studies, hopefully not only in kenya, but also in other african countries likely to be involved in this endeavour . Studies on gene flow, mating behaviour and reproductive fitness, combined with studies on the effects of laboratory maintenance on the genetic make - up of transformed strains to be released, can be conducted in semi - field systems . Such systems, particularly when used to study genetically - engineered mosquitoes will require more advanced containment levels than the system described here . Guidelines for facility location, physical and biological containment, safety practices and calamity control need to be developed and adapted from existing arthropod containment guidelines . There are several good reasons to further such studies in disease - endemic settings . Under such conditions it will be possible to transform offspring from wild mosquitoes, conduct experiments under local ambient climatic conditions and evaluate transgene spread and fixation in offspring from field - collected gravid females that emerge in a semi - field setup . Last, but not least, it will enable scientists from developing countries to become more directly involved in evaluating the potential use and application of transgenic mosquitoes for future malarial disease control . The introduction of blood - fed females into the greenhouse resulted in the presence of eggs in the breeding sites on day 3 (2.5 days after release), and eggs continued to be observed in the sites until day 7 (fig . 3). Larvae (from l1 to l4 stage) were seen feeding at the water surface until day 23, when the last l4 larva pupated . The first five pupae were seen in the breeding sites in the evening of day 10, meaning that the variation in maturation time from egg to pupa was 720 days . In spite of having conditions with higher larval density and smaller water surface area, gimnig et al . Recorded much reduced periods to pupation, from 5 to 12 days . In total, 57 pupae were counted in the breeding site 3.8 m in front of the hut and 130 in the site 1.1 m behind it, and on average they harboured only 0.08 and 0.18 larva / cm, respectively . These densities are lower than those observed in natural habitats, and given the fact that we observed algal growth, considered important for larval growth, it seems that the prolonged time to pupation in this trial may be attributed to the relatively small range over which temperatures fluctuate . Alternatively, re - introducing mosquitoes that had been maintained under laboratory conditions for several years in a more natural setting may have caused these effects, and poor adaptation to these conditions may have stunted their development . Completion of the anopheles gambiae life - cycle in the greenhouse over a 27-day period . Blood - fed females were released on day 1, and arrows show time periods during which the various developmental stages were observed . The first adults were seen inside the hut on day 11, and continued to be present until the end of the experiment (day 27). Starting in the morning of day 22, we observed new eggs in the breeding sites and subsequent larval development . From the above it can be deduced that specific behaviours of the adult insects occurred during certain time periods (fig . Oviposition activity took place twice during this trial, meaning that females survived until eggs were mature, that they successfully located a breeding site, accepted it for oviposition, and laid eggs . Other potential breeding sites, like the leaf axils of the banana trees in the sphere, were examined but were not found to harbour eggs, larvae or pupae . The period for reaching sexual maturity for males may be at least one day and for females up to 60 hrs, so mating of the f1 generation may not have taken place until dusk on day 14 . In spite of regular observations during dusk, we did not see any swarming activity typically associated with mating in an . This is a particularly interesting point, because in contrast with other settings (e.g. ), where mating swarms were frequently observed we failed to do so over a 3-year period in the mbita area as did charlwood (pers.comm .) Who observed only one anopheline swarm during 4 years in the kilombero valley of tanzania, raising the question as to whether swarming is an obligate component of the an . Alternatively, maintenance of mosquitoes in laboratory cages for several years forced this strain to become stenogamous (i.e. The ability to mate in small cages), and this adaptation may have interfered with its ability to swarm when introduced into the sphere . As newly emerged adults rarely survive for more than 48 hrs without the availability of an energy source, mosquitoes must have supplemented their energy reserves with carbohydrates from the plants in the sphere (table 1) for up to 6 days before they were allowed access to a blood source, in the form of a human volunteer sleeping in the hut . Some plants like castorbean (ricinus communis l.) were flowering at the time of the experiment, and may have provided nectar sources . In cage experiments (impoinvil et al ., in prep .) We have observed a mean survival time of 7.0 0.2 days on this plant, which is comparable to mosquitoes given 6% glucose (8.7 0.2 days). Gambiae mosquitoes emerge from the pupal stage with a deficit not only in carbohydrates, but also lipid and protein, which usually is compensated for by consuming a (small) blood meal within the first few days of adult life, it is likely that mortality during the 6-day post - emergence period in the current trial was too high to have a good number of the 8090 females that emerged survive long enough to obtain their first blood meal . Within 15 min of entering the hut at night, the volunteer noticed the sound of mosquitoes and subsequently felt mosquito bites on his exposed lower limbs . This implies that females were receptive to host cues, entered the hut, probably through the eaves, and then successfully located and fed on the human host . At sunrise, several engorged females were seen resting on the walls, indicating successful blood feeding and endophily (indoor resting), which is typical for this species . Following maturation of eggs, the second oviposition began during the night of day 21, thus completing the life - cycle . We continued to observe both immature and adult insects (presumably mostly from the f1 generation) until day 27, when the experiment was terminated (by refraining from entering the sphere for about 1 month). The second experiment, in which we released 500 virgin females together with 1500 males demonstrated that mating does occur in a relatively small, semi - field system . After the third night that a volunteer had slept in the hut, we observed eggs in the breeding sites . The production of offspring, though, was low, and we only collected 40 pupae by the end of the trial period . This may have been caused by heavy rainfall during three consecutive nights (day 24), which may have affected the survival of the adults and/or larvae or washed away the larvae from the breeding sites due to overflow . Since we observed few mosquitoes, we decided to conduct a human landing catch during two nights inside the hut, starting two nights after sleeping in the hut had ended . Nevertheless, the life - cycle was completed, as manifested by the harvested pupae, which were removed from the breeding sites to prevent emergence of the f1 generation which would have compromised interpretation of survival of the f0 generation . The third experiment started by introducing 500 eggs into each of the breeding sites, whilst 1000 eggs (from the same original batch) were reared under laboratory conditions . In the laboratory, larvae developed at the same rate and most reached maturity by day 10, when the first pupae were observed (fig . This was similar for the breeding sites in the sphere, except that development was highly asynchronous, i.e. Some larvae pupated by day 10, whereas others took up to day 24 before pupation . These times to pupation are similar to those observed in the first trial, but are again in contrast with other studies . On day 7 we counted all larvae and observed 887 in the insectary, as opposed to 652 in the sphere . Overall, the laboratory batch yielded 804 pupae, versus 495 from the breeding sites . On the basis of these data, the average daily survival was 0.90 for the laboratory larvae, and 0.83 for the larvae in the sphere . With nearly half the larvae surviving to the adult stage, these results contrast sharply with much higher mortalities (up to 90%) observed in the kisumu area and so tome (charlwood, pers . . The sex ratio of emerging adults in the laboratory was 2:3, which translated into a female population in the sphere of 297, on the assumption that no insects died during emergence . On day 28, after the release of eggs in the breeding sites, we observed that eggs had been laid by the f1 generation, but with only 6 and 3 eggs in the two breeding sites respectively . Cumulative percentage of pupation of eggs introduced in the two breeding sites inside the greenhouse () or under insectary conditions (). Our results have shown that by starting either at the post - blood feeding, pre - mating, or egg stage, a new generation of insects can be reared under these semi - field conditions, and that all life - history behaviours were successfully completed to a lesser or greater extent . This therefore represents the first and promising step towards continuous maintenance of parasite - free an . Gambiae populations under semi - natural conditions that can be experimentally manipulated in studies of malaria vector ecology and transmission control . Russell and rao used a large outdoor cage, based on a design by hackett and bates, to study swarming and oviposition behaviour of an . Culicifacies giles in india, and showed that such systems can be used to unravel aspects of the behavioural ecology of anophelines . Our study shows that such systems can now also be developed for studies on african malaria vectors in order to start filling the gaps in our understanding of the behavioural ecology of these insects . First and foremost, it provides a suitable intermediate between laboratory - based studies addressing mosquito behaviour and ecology, and the field situation . Too often, conclusions are drawn from results obtained under laboratory conditions that necessitate speculation as to what may or may not happen in the field . Fixed climatic conditions, cage - experiments, olfactometers and windtunnels, in which the mosquito strains used have been laboratory - reared for sometimes decades, may readily distort behavioural and ecological phenomena . Here we have shown that, beyond introducing f1 generation malaria - free mosquitoes from wild populations, it may be possible to rear vectors in situ within a semi - natural system that may minimize such artefacts . Conversely, the advantage of using insectary - reared mosquitoes is in the level of control that may be exerted that would not otherwise be possible: experiments can be conducted all - year - round, with fixed numbers of insects, of known age and physiological status, in a malaria parasite - free environment under ambient climatic conditions . This enables more direct inferences to be drawn from data analysis as compared to longitudinal field studies, because of constant conditions and simplified experimental design . We have recently evaluated the efficacy of several plants traditionally used by the luo community as repellents in a similar semi - field set - up, and simple logistic regression, on data collected during four nights per plant, yielded significant results . Within a year of nearly continuous experimentation, the repellency of 8 plant species and 3 combinations thereof was evaluated through thermal expulsion or direct burning, and 9 species and 2 combinations thereof were tested in potted form . Such studies would have taken several years under field conditions, and would be limited to times when mosquito densities would have been sufficiently high to permit meaningful experiments . Gambiae maintained on a variety of diets (blood or sugar alone, or a combination of both) and revealed similar results to those obtained under laboratory conditions . Within one year of starting studies on the behaviour of mosquitoes around bednets in a semi - field setup, we transformed a regular conical bednet into a trap that may catch up to 70% of the females released . Recent field evaluation of this trap shows it to be a promising replacement for the human landing catch (mathenge et al . With the trap now being considered for commercial manufacturing (it took a mere two years to reach this stage), this would have been impossible without the availability of a semi - field set - up in which continuous experimentation ensured rapid progress towards product development . Even though our system resembles more closely the field situation, it remains to be ascertained to what extent . Our current study was mainly qualitative in design and focused on life - cycle completion . For instance, observation of eggs in the breeding sites in the morning of day 22 during the first trial implies that these originated from females that fed once on day 19, as those that fed on day 17 should have laid before . However, it is likely that these females fed twice, on day 17 and day 19, and should be classified as pre - gravids, before fully maturing a batch of eggs . Furthermore, in the absence of a human host, mosquitoes survived up to six days after emergence, confirming field results that feeding on plant sugars does occur and may be an important feature of the life - cycle in this species (foster and knols, unpublished data). Obviously, there are limitations associated with these studies there may be other, yet unknown, factors that affect the behavioural ecology of the insects in such systems . Or, as bates wrote, following his outdoor cage studies in albania: " one still cannot be sure that the reactions of the mosquito are' natural' because there is always the barrier of wire liable to be encountered on extended flights; and when the flight of a mosquito has been interrupted by this wire barrier its further activity may be definitely unnatural " . Rightly so, and even though we did not observe any obvious distorted behaviours, it is imperative that findings from semi - field studies be verified under natural outdoor conditions . Additional studies in which the release and performance of field - collected, blood - fed mosquitoes in the sphere is compared with that of laboratory specimens in terms of egg - recovery, developmental periods and important behavioural characteristics (like swarming) will provide further insight to what extent such systems mimic the natural anopheles environment and colony adaptation impairs natural behaviours . Nevertheless, since bates' days, advances in science merit a renewed impetus towards semi - field studies in contained near - natural environments, particularly with respect to transgenic mosquitoes . Fitness evaluations of engineered strains of vectors are mandatory for transformation technology to become an established disease control tool in africa . Perhaps this alone, is ample justification for more intensive sphere studies, hopefully not only in kenya, but also in other african countries likely to be involved in this endeavour . Studies on gene flow, mating behaviour and reproductive fitness, combined with studies on the effects of laboratory maintenance on the genetic make - up of transformed strains to be released, can be conducted in semi - field systems . Such systems, particularly when used to study genetically - engineered mosquitoes will require more advanced containment levels than the system described here . Guidelines for facility location, physical and biological containment, safety practices and calamity control need to be developed and adapted from existing arthropod containment guidelines . There are several good reasons to further such studies in disease - endemic settings . Under such conditions it will be possible to transform offspring from wild mosquitoes, conduct experiments under local ambient climatic conditions and evaluate transgene spread and fixation in offspring from field - collected gravid females that emerge in a semi - field setup . Last, but not least, it will enable scientists from developing countries to become more directly involved in evaluating the potential use and application of transgenic mosquitoes for future malarial disease control . Bgjk conceived of the study, and developed the system and experiments together with bnn, emm and wrm . Jcb and gfk actively contributed to the interpretation of the findings and drafting of the final manuscript . Bgjk and emm are engaged in commercialising the mbita bednet trap, developed in semi - field systems similar in nature to that described in this article, in collaboration with the vestergaard frandsen group (denmark). Bos of wageningen university and research centre (the netherlands) for assistance with plant nomenclature . This research was supported by the national institutes of health, usa (grant numbers u19 ai45511, d43 tw01142, d43 tw00920). Emm and wrm receive financial support from the undp / world bank / who special programme for research and training in tropical diseases (tdr) under grants i d 980794 and 980692 respectively.
Mycobacterium avium complex (mac) consists predominantly of two species: m. avium subspecies avium and m. avium subspecies intracellulare . With growing incidence of human immunodeficiency virus (hiv) infection, disease due to the mac is one of the most important opportunistic pulmonary infections most prevalent in immunocompromised patients . In more then 95% patients, aids related disseminated mac infection is caused by m. avium subspecies avium . However, recently, incidence of mac infection has been found to increase even in immunocompetent pulmonary disease patients . Studies have reported that pulmonary diseases caused by mac in non - hiv infected persons are as common as pulmonary tuberculosis (tb) in many areas . Early differential diagnosis of m. avium is becoming increasingly important because of growing frequency of mac infection in immunocompetent patients, impeding availability of new drugs, clinical features resembling tb and difficult chemotherapy as compared to mycobacterium tuberculosis and poor prognosis of disseminated mac disease . The diagnostic criteria currently followed by revised national tuberculosis control program of india, in accordance with american thoracic society guidelines for m. avium are based on the radiological findings, sputum smear examination along with clinical manifestations . All the features are variable, non - specific and can also be produced by co - existing lung diseases or by other opportunistic infections in hiv seropositive individuals or in early stages of tb by m. tuberculosis . Moreover, demonstration of acid - fast bacilli in sputum and bronchoalveolar lavage is same in both m. tuberculosis and m. avium infections, but due to different chemotherapy for the two diseases, prompt distinction is essential and warrants serious consideration for the development of rapid and specific methods for the differential diagnosis of m. avium disease from m. tuberculosis . Till date, several diagnostic methods and techniques for differential diagnosis of m. avium infection like biochemical tests based diagnostic procedures, i - labeled cdna probe assay, phenotypic identification of culture by hybridization protection assay, nucleic acid based approaches and esat- 6 polymerase chain reaction (pcr) primers have been reported. [1014] these methods have their own limitations; they are complex, not very specific, require the growth of mycobacterial cultures from patient's specimen which is time consuming or require radioisotopic facilities, restriction enzymes and extensive instrumentation . So, the development of simple and rapid diagnostic method for differential diagnosis of m. avium infection is the need of the hour . Serodiagnostic techniques, based upon the recognition of species - specific proteins secreted by actively growing myobacterial bacilli in the culture filtrate (cf), have been advocated to be simple, easy, cost - effective and rapid methods . In addition, this is a highly sensitive technique capable of detecting mycobacterial antigens at a concentration of 10/ml . Till date, 14 kda protein and 81.6 kda (katg) protein have been recognized to be helpful in the differential diagnosis of m. avium infection . Taking a clue from these findings, the present study was designed to identify, isolate and purify m. avium specific secretory proteins and to evaluate the role of these proteins in the diagnosis of m. avium infection, particularly in patients with mac infection . The present study was an ethically designed study, approved by institutional animal ethical committee and institutional human ethical committee . All animal experiments were conducted in accordance with the guidelines laid down by institutional animal ethical committee . Care of animals was according to the ethical committee guidelines and guidelines issued by cpcsea . Culture filtrate proteins (cfps) of m. avium subspecies avium (mtcc 1723 imtech, nctc 8551 london), m. tuberculosis h37rv (nctc london) and mycobacterium bovis bacille calmette guerin (bcg) were isolated by growing the bacilli in modified youman's liquid synthetic medium as a stationary pellicle culture . Bacilli were harvested after 45 weeks, supernatants were filter sterilized (0.22 m pore size membrane filter), desalted and concentrated 100 times by ultrafiltration on an amicon ym-3 membrane (millipore, bedford, ma, usa). These mycobacterial cfps were analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (sds - page) using 1012% resolving gel followed by silver staining. [2021] forty balb / c mice of either sex (2025 g body weight, 45 weeks) were housed in cages kept in negative pressure regulated animal isolators and were fed on standard pellet diet (hindustan lever ltd ., antisera against various mycobacterial species for comparative enzyme - linked immunosorbent assay (elisa) and western immunoblot studies were obtained by infection of mice with the above - mentioned bacilli . Briefly, mice (10 in each group) were infected with 110 bacilli through intravenous route and bled in the 2, 4, 6, 8 and 10 weeks post infection . M. avium cfps were separated on the basis of charge using anion exchange column chromatography technique using deae sepharose cl-6b matrix . After the washing of column with two - bed volume of equilibration buffer to remove the unbound proteins, the bound proteins were eluted using step gradients of nacl . Approximately 510 mg protein of the desired chromatography gradient containing specific protein of interest was resolved and purified by high - resolution preparative sds - page (hoefer se 600, amersham pharmacia, biotec inc ., san francisco, ca, usa) (16 cm4 cm1.5 mm) at 250 v using tris - glycine (25 mm, 192 mm) as electroelution buffer . Louis, mo, usa) method and purified protein was analyzed by sds - page followed by silver staining and stored at 20c till further use . The purified protein was subjected to n - terminal sequencing (ladder sequencing, concentration dependent) using liquid chromatography - mass spectrometry (lc - ms - ms; bruker daltonics inc ., the sequence so obtained was carried for blast search to detect the homology with other mycobacterial species . Hiv seronegative, pulmonary tb patients (n=100) with sputum / bronchoalveolar lavage sample smear positive for acid - fast bacilli (hiv tb patients) hiv seropositive patients (n=54) with disseminated or extra pulmonary tb having cd4 t cells <100 cells /l (hiv tb patients) hiv seropositive patients (n=20) without any radiological or bacteriological evidence of tuberculosis and with cd4 t cell count> 100 cells /l (hiv tb patients) bcg vaccinated, hiv seronegative healthy volunteers (n=20) for detection of antibodies present against m. avium specific proteins, 1 ml of blood was withdrawn from all participants, after taking informed written consent . To check the significance of m. avium specific proteins in the diagnosis of m. avium infection in the clinical setup, m. avium infected patients were selected from hiv positive population on the basis of culture positivity on the culture media and by a battery of biochemical tests . The m. avium specific secretory proteins were subjected to indirect elisa with 1:100 dilution of sera of hiv tb patients, hiv tb patients, hiv tb patients, m. avium antisera, m. tuberculosis antisera and healthy individual's sera to check their specificity for the diagnosis of m. avium infection . Culture filtrate proteins (cfps) of m. avium subspecies avium (mtcc 1723 imtech, nctc 8551 london), m. tuberculosis h37rv (nctc london) and mycobacterium bovis bacille calmette guerin (bcg) were isolated by growing the bacilli in modified youman's liquid synthetic medium as a stationary pellicle culture . Bacilli were harvested after 45 weeks, supernatants were filter sterilized (0.22 m pore size membrane filter), desalted and concentrated 100 times by ultrafiltration on an amicon ym-3 membrane (millipore, bedford, ma, usa). These mycobacterial cfps were analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (sds - page) using 1012% resolving gel followed by silver staining. [2021] forty balb / c mice of either sex (2025 g body weight, 45 weeks) were housed in cages kept in negative pressure regulated animal isolators and were fed on standard pellet diet (hindustan lever ltd ., mumbai, india) and water ad libitum . Antisera against various mycobacterial species for comparative enzyme - linked immunosorbent assay (elisa) and western immunoblot studies were obtained by infection of mice with the above - mentioned bacilli . Briefly, mice (10 in each group) were infected with 110 bacilli through intravenous route and bled in the 2, 4, 6, 8 and 10 weeks post infection . M. avium cfps were separated on the basis of charge using anion exchange column chromatography technique using deae sepharose cl-6b matrix . After the washing of column with two - bed volume of equilibration buffer to remove the unbound proteins, the bound proteins were eluted using step gradients of nacl . Approximately 510 mg protein of the desired chromatography gradient containing specific protein of interest was resolved and purified by high - resolution preparative sds - page (hoefer se 600, amersham pharmacia, biotec inc ., san francisco, ca, usa) (16 cm4 cm1.5 mm) at 250 v using tris - glycine (25 mm, 192 mm) as electroelution buffer . Louis, mo, usa) method and purified protein was analyzed by sds - page followed by silver staining and stored at 20c till further use . The purified protein was subjected to n - terminal sequencing (ladder sequencing, concentration dependent) using liquid chromatography - mass spectrometry (lc - ms - ms; bruker daltonics inc ., the sequence so obtained was carried for blast search to detect the homology with other mycobacterial species . Hiv seronegative, pulmonary tb patients (n=100) with sputum / bronchoalveolar lavage sample smear positive for acid - fast bacilli (hiv tb patients) hiv seropositive patients (n=54) with disseminated or extra pulmonary tb having cd4 t cells <100 cells /l (hiv tb patients) hiv seropositive patients (n=20) without any radiological or bacteriological evidence of tuberculosis and with cd4 t cell count> 100 cells /l (hiv tb patients) bcg vaccinated, hiv seronegative healthy volunteers (n=20) for detection of antibodies present against m. avium specific proteins, 1 ml of blood was withdrawn from all participants, after taking informed written consent . To check the significance of m. avium specific proteins in the diagnosis of m. avium infection in the clinical setup, m. avium infected patients were selected from hiv positive population on the basis of culture positivity on the culture media and by a battery of biochemical tests . The m. avium specific secretory proteins were subjected to indirect elisa with 1:100 dilution of sera of hiv tb patients, hiv tb patients, hiv tb patients, m. avium antisera, m. tuberculosis antisera and healthy individual's sera to check their specificity for the diagnosis of m. avium infection . The 4-week stationary culture of m. avium subspecies avium, m. tuberculosis h37rv and m. bovis bcg isolated the cfps with a mean yield of 152 mg / l of the secretory proteins . The cfps resolved on 12% denaturing gel showed the presence of protein bands ranging from a molecular weight of 6 to 97 kda on sds - page, but in m. avium cf, the protein band in the regions of 4550 kda seemed to be specifically present and absent from rest of mycobacterial species . Immunoblotting of m. avium antisera with m. bovis bcg, m. tuberculosis h37rv and m. avium cfps also indicated the presence of a number of common protein bands in all the mycobacterial species . However, m. avium antisera immunoblotted with m. avium cf showed a specific protein band of 4550 kda that was not evident in others [figure 1]. Lanes 1 and 2: with m. bovis bcg cf; lanes 3 and 4: with m. tuberculosis cf; lanes 5 and 6: with m. avium cf; lane 7: standard molecular weight markers . (the black arrow within immunoblot indicates the presence of specific protein band of 4550 kda) anion exchange column chromatography resulted in the elution of an immunodominant protein of ~48 kda in 150 mm elution gradient . Comparative immunoblotting of ~48 kda protein of 150 mm elution gradient with m. avium antisera (taken as positive control) and tb patient's sera resulted in the recognition of these proteins only with m. avium antisera and not with tb patient's sera or m. tuberculosis antisera, thus depicting the specificity of these proteins for m. avium infection [figure 2]. Comparative immunoblotting of 150 mm elution gradient of m. avium cf with m. avium antisera and tb patient's sera . Lane 1: standard pre - stained molecular weight markers; lane 2: immunoblotting with m. avium antisera; lanes 37: immunoblotting with tb patient's sera . (the black arrow within the immunoblot indicates the presence of specific protein band of ~48 kda) on lc - ms - ms, the ~48 kda protein was found to be a complex protein demonstrating three peaks in the mass spectra . Molecular mass of these three proteins was 47.3, 50.9 and 55.7 kda with the number of amino acids being 463, 469 and 515, respectively . The first protein designated as aro a homologue protein (aro) showed 92% homology with m. avium paratuberculosis and 75.59% homology with m. tuberculosis starting at 31 amino acid . The second protein designated as tal protein (putative transaldolase) showed 79.53% homology with m. avium paratuberculosis and 66.73% homology with m. tuberculosis starting at 97 amino acid, while with the third protein, i.e. Aspartate transaminase (ast), the homology was 82.91% and 68.93% with m. avium paratuberculosis and m. tuberculosis, respectively, starting at the 88 amino acid [table 1]. These data show that the n - terminal sequence of all the three proteins are different from other mycobacterial species . Characterization of purified m. avium specific aro - tal - ast secretory protein immunoreactivity studies of purified aro - tal - ast complex protein with m. avium and m. tuberculosis infected mice sera demonstrated the presence of aro - tal - ast antibodies only in m. avium antisera [figure 3] and confirmed the specificity of m. avium aro - tal - ast complex protein for the diagnosis of m. avium infection . Recognition of m. avium aro - tal - ast complex protein in m. avium infection in mouse model kinetic expression studies performed by immunoblotting m. avium cf with m. avium antisera collected in 2, 4, 6, 8, 10, 12 weeks after experimental m. avium infection in mouse model showed that aro - tal - ast complex protein was recognized as early as from 2 week post infection [figure 3]. Significance of purified m. avium aro - tal - ast complex protein in the diagnosis of mac in the clinical setup was confirmed by indirect elisa with the patients with mac bacteremia, selected from hiv population with disseminated mycobacterial disease on the basis of blood culture by lysis centrifugation method, followed by a battery of biochemical tests . Out of 54 blood samples of hiv patients with disseminated mycobacterial infection, 14 samples were mycobacterial culture positive, and of these 14 samples, 10 were mac and 4 were m. tuberculosis positive on the basis of biochemical tests . When purified m. avium aro - tal - ast complex protein was subjected to indirect elisa with the serum samples (1:100 dilutions), of these 14 patients, 9 out of 10 mac positive patients had antibodies to recognize the protein, giving a percent recognition of 90% . Four samples that were positive for m. tuberculosis did not recognize aro - tal - ast complex protein, confirming that this protein is specific to m. avium infection [figure 4]. Detection of antibodies in serum samples of hiv seropositive mycobacterial culture positive patients against aro - tal - ast complex proteins . The solid horizontal line shows the cut - off value, i.e. Mean absorbance of hiv positive tb negative, hiv negative tb positive, hiv negative tb negative patients + 2.5 sd further, m. avium aro - tal - ast complex protein based elisa in the serum samples from 100 pulmonary tb patients demonstrated that 99/100 patients showed no reactivity, thus confirming the specificity of m. avium aro - tal - ast complex based serodiagnostic assay [figure 5]. Also, the absence of anti - aro - tal - ast complex antibodies in healthy bcg vaccinated individuals further confirmed the recognition of this protein only during active disseminated mac disease and showed that it is not affected by prior bcg vaccination / exposure to environmental mycobacteria . Evaluation of seroreactivity of aro - tal - ast complex protein with the serum samples of smear positive tb patients . The solid horizontal line shows the cut - off value [mean od of healthy individuals + 2.5 sd, i.e. 0.0675 + 2.5 (0.068)] aro - tal - ast complex protein based elisa has an overall sensitivity of 90% and specificity of 99% in diagnosing m. avium infection . Moreover, this test can also be applied for hiv positive / aids patients, who do not respond to t - cell based assays due to very low cd4 t - cell counts . These data clearly demonstrate that the m. avium aro - tal - ast complex protein is able to differentially diagnose disseminated mac disease in hiv infected population at an early stage and the results obtained with antibody - based easy and rapid elisa test correlate with those of conventional time - consuming blood culture and biochemical tests . Of the 40 patients with disseminated mycobacterium infection but blood culture negative, 4 had anti - aro - tal - ast antibodies in their serum, but as they were not differentially diagnosed to be m. avium infected clinically, nothing can be acertained . As no other test can specifically differentiate m. avium from m. tuberculosis, it may be possible that these patients were m. avium infected, but neither were they diagnosed clinically nor they were blood culture positive . Therefore, presently, we cannot say surely from the available data if aro - tal - ast can be considered a marker for mac infection in patients with negative biochemical tests . This study clearly indicates that the development of m. avium aro - tal - ast complex protein based diagnostic elisa could be of great help to the clinicians in the timely diagnosis and treatment of disseminated mac disease in hiv patients.
The reported incidence ranges from 0 to 30%,2 3 4 5 depending on the study population and procedure type, with overall mean incidence 5% via meta - analysis.4 dcps most commonly present within the first 3 days following surgery but have been reported up to 2 months after surgery.2 6 fortunately, the majority resolve within 6 months, though residual deficits may persist in up to 20%.2 despite identification of the predisposing factors and plausible theories, the true etiology remains unknown . The majority of the proposed theories can be categorized as either mechanical or vascular and include nerve root traction due to postoperative cord shift or change in alignment, spinal cord ischemia, and reperfusion injury.4 5 7 8 9 10 excessive root traction is the most commonly cited mechanical etiology, supported by differences in preoperative foraminal width, laminectomy trough width, preexisting rotation of the spinal cord, and postlaminectomy cord drift posteriorly in patients who developed c5 palsies compared with controls . Root traction is also supported by a decreased incidence of dcps following prophylactic foraminotomies.11 12 13 autoimmune and inflammatory etiologies such as idiopathic brachial neuritis (parsonage - turner syndrome) and postsurgical inflammatory neuropathy (psin) have less frequently been cited but have certainly been described after cervical decompression and fusion.14 15 other variables that have been associated with an increased incidence of dcps include male gender and the underlying pathology (ossification of the posterior longitudinal ligament> cervical spondylomyelopathy).6 the heterogeneity of previously established risk factors and lack of an all - encompassing theory indicate that the etiology may be multifactorial . Additionally, most previous studies focused on asian cohorts developing dcps following laminoplasty, specifically confined to the c5 level . Thus, the incidence in patients undergoing either laminectomy alone or cervical decompression with instrumented fusion in north american populations is not well established, as is the frequency of involvement of other cervical levels . The present study evaluated the overall incidence of dcps in all cervical myotomes following cervical decompression with and without instrumented fusion for degenerative disease of the cervical spine . The presence of previously established risk factors for psin outlined by staff et al was also assessed relative to internal controls.15 all aspects of this study were approved by the institutional review board and in adherence with ethical standards . We retrospectively reviewed 1,768 consecutive, current procedural terminology (cpt)-coded cervical decompressions with and without instrumented fusion at mayo clinic rochester between january 2008 and december 2013 for postoperative weakness within 6 weeks of the operative date . Dcps were defined as either unilateral or bilateral, new onset or worsened motor function confined to one or more cervical myotomes . Both the operative note and the immediate postoperative exam record were carefully evaluated for weakness or pain consistent with iatrogenic injury . Patients who experienced weakness directly attributable to intraoperative injury or upper extremity pain or sensory symptoms without accompanying weakness were excluded . We also excluded all tumor and trauma cases following review of all the pertinent preoperative documentation to specifically evaluate the incidence of dcps in patients treated for degenerative disease . The electronic medical record of every patient was reviewed for baseline data including age, gender, and length of follow - up . Dcps were further characterized by myotomal involvement, either unilateral or bilateral, and worse / end motor grade . Finally, the presence of suggested risk factors for psin were recorded as outlined by staff et al (intraoperative transfusions, history of diabetes, comorbid cancer, concomitant history of infection, and history of smoking).15 several categorical variables were further analyzed in more specific subcategories including transfusion (intraoperative versus postoperative prior to discharge), comorbid cancer (active metastatic disease or active tumor burden; remote no evidence of disease), concomitant history of infection (active current use of antibiotics or chronic infection; remote acute infection within three months), and smoking (active smoking within 3 months of operative date; remote any other history). A history of autoimmune disease was also recorded as positive if the patient had a documented history of 1 or more of the 81 autoimmune diseases as reported in a recent comprehensive review by hayter and cook (appendix a).16 seronegative spondyloarthropathies (ankylosing spondylitis, reactive arthritis, and psoriatic arthritis) are notable diseases of the spine with a potential autoimmune origin excluded by hayter and cook and thus were evaluated as a separate variable . The history of autoimmune disease was further classified into one of four categories: diabetes mellitus type i, inflammatory bowel disease (crohn disease, ulcerative colitis), rheumatoid arthritis, and other . Underlying ossification of the posterior longitudinal ligament was not specifically evaluated given its low incidence among our study cohort, a finding consistent with epidemiologic studies completed in north america.17 additional analysis was performed on operative differences including approach (anterior, posterior, or both) and use of instrumentation . The intraoperative somatosensory and motor evoked potentials were reviewed when available only in patients who developed a dcp . When both cpt coding and reviewer classification were concordant, procedures were classified into one of several nonexclusive categories: anterior fusion, anterior diskectomy and fusion, arthroplasty, corpectomy, foraminotomy, laminectomy without fusion, laminoplasty, osteotomy, posterior fusion, and revision . Procedure categories including more than 100 cases were analyzed as a categorical variable for the entire cohort, as well as examined separately for procedure specific risk factors . Postoperative electromyograms (emgs) within 6 months of the operative date were also evaluated for patients with persistent weakness . These studies were evaluated for bilateral involvement, multiple myotomes, and patterns consistent with a brachial plexopathy . Reprinted from hayter sm, cook mc . Autoimmunity reviews 2012;11:754765, with permission from elsevier.16 incidence data (no prevalence studies identified). Anti - candidal enolase, anti - pituitary, anti - calcium sensing receptor protein, anti - aromatic l - amino acid decarboxylase, anti - tyrosine hydroxylase . Histidyl trna, aminoacyl trna synthetase, dna - dependent nucleosome - stimulated atpase, exosc10 protein, chromodomain - helicase - dna - binding protein 4 . Anti - scl70,anti - pm / scl, anti - rna polymerase iii, anti - centromere . Anti - dsdna, anti - u1a, anti - u2b, anti - pcna, anti - smith, anti - ssa, anti - ssb . U2 snrnp b, cardiolipin, fibronectin, ro, la, histone h2a h2b, vimentin . All statistical analysis was performed with jmp 10 (sas institute inc ., cary, north carolina, united states) in consultation with an institutional biostatistician . For patients undergoing multiple procedures during the period, only the first event was analyzed to exclude bias introduced by multiple representations of patient - specific factors on statistical analysis . The backward selection method was utilized for the multivariate analysis, retaining each variable significant on univariate analysis . All aspects of this study were approved by the institutional review board and in adherence with ethical standards . We retrospectively reviewed 1,768 consecutive, current procedural terminology (cpt)-coded cervical decompressions with and without instrumented fusion at mayo clinic rochester between january 2008 and december 2013 for postoperative weakness within 6 weeks of the operative date . Dcps were defined as either unilateral or bilateral, new onset or worsened motor function confined to one or more cervical myotomes . Both the operative note and the immediate postoperative exam record were carefully evaluated for weakness or pain consistent with iatrogenic injury . Patients who experienced weakness directly attributable to intraoperative injury or upper extremity pain or sensory symptoms without accompanying weakness were excluded . We also excluded all tumor and trauma cases following review of all the pertinent preoperative documentation to specifically evaluate the incidence of dcps in patients treated for degenerative disease . The electronic medical record of every patient was reviewed for baseline data including age, gender, and length of follow - up . Dcps were further characterized by myotomal involvement, either unilateral or bilateral, and worse / end motor grade . Finally, the presence of suggested risk factors for psin were recorded as outlined by staff et al (intraoperative transfusions, history of diabetes, comorbid cancer, concomitant history of infection, and history of smoking).15 several categorical variables were further analyzed in more specific subcategories including transfusion (intraoperative versus postoperative prior to discharge), comorbid cancer (active metastatic disease or active tumor burden; remote no evidence of disease), concomitant history of infection (active current use of antibiotics or chronic infection; remote acute infection within three months), and smoking (active smoking within 3 months of operative date; remote any other history). A history of autoimmune disease was also recorded as positive if the patient had a documented history of 1 or more of the 81 autoimmune diseases as reported in a recent comprehensive review by hayter and cook (appendix a).16 seronegative spondyloarthropathies (ankylosing spondylitis, reactive arthritis, and psoriatic arthritis) are notable diseases of the spine with a potential autoimmune origin excluded by hayter and cook and thus were evaluated as a separate variable . The history of autoimmune disease was further classified into one of four categories: diabetes mellitus type i, inflammatory bowel disease (crohn disease, ulcerative colitis), rheumatoid arthritis, and other . Underlying ossification of the posterior longitudinal ligament was not specifically evaluated given its low incidence among our study cohort, a finding consistent with epidemiologic studies completed in north america.17 additional analysis was performed on operative differences including approach (anterior, posterior, or both) and use of instrumentation . The intraoperative somatosensory and motor evoked potentials were reviewed when available only in patients who developed a dcp . When both cpt coding and reviewer classification were concordant, procedures were classified into one of several nonexclusive categories: anterior fusion, anterior diskectomy and fusion, arthroplasty, corpectomy, foraminotomy, laminectomy without fusion, laminoplasty, osteotomy, posterior fusion, and revision . Procedure categories including more than 100 cases were analyzed as a categorical variable for the entire cohort, as well as examined separately for procedure specific risk factors . Postoperative electromyograms (emgs) within 6 months of the operative date were also evaluated for patients with persistent weakness . These studies were evaluated for bilateral involvement, multiple myotomes, and patterns consistent with a brachial plexopathy . Autoimmunity reviews 2012;11:754765, with permission from elsevier.16 incidence data (no prevalence studies identified). Anti - candidal enolase, anti - pituitary, anti - calcium sensing receptor protein, anti - aromatic l - amino acid decarboxylase, anti - tyrosine hydroxylase . Histidyl trna, aminoacyl trna synthetase, dna - dependent nucleosome - stimulated atpase, exosc10 protein, chromodomain - helicase - dna - binding protein 4 . Anti - scl70,anti - pm / scl, anti - rna polymerase iii, anti - centromere . Anti - dsdna, anti - u1a, anti - u2b, anti - pcna, anti - smith, anti - ssa, anti - ssb . U2 snrnp b, cardiolipin, fibronectin, ro, la, histone h2a h2b, vimentin . All statistical analysis was performed with jmp 10 (sas institute inc ., cary, north carolina, united states) in consultation with an institutional biostatistician . For patients undergoing multiple procedures during the period, only the first event was analyzed to exclude bias introduced by multiple representations of patient - specific factors on statistical analysis . The backward selection method was utilized for the multivariate analysis, retaining each variable significant on univariate analysis . There were 1,669 patients who underwent 1,768 procedures during the study period, with dcps following the initial procedure in 56 individuals (3.4%). The majority of dcps presented in the first 72 hours following surgery (87.5%) with maximal deficit occurring within 72 hours of onset (85.7%). The c5 myotome was involved in 40 (71.4%) cases, and 31 (55.4%) cases had multiple myotomes involved . A complete analysis of temporal profile and myotomal involvement is available in table 1 . Of note, 5 (8.9%) cases of dcps occurred at levels not operated upon . Of these first operations, there were 842 (50.4%) cervical decompressions with fusion and 827 (49.6%) cervical decompressions only . In addition to the procedure categories analyzed in table 2, a minority of patients underwent arthroplasty (n = 6), laminoplasty (n = 6), osteotomy (n = 1), or revision (n = 21). The analysis of demographics and the risk factors for psin are also summarized in table 2 . We found significant associations between the incidence of dcp with age (positive dcp: 62.2 versus negative dcp: 57.1, p = 0.0061), intraoperative transfusion (positive dcp: 16.1% versus negative dcp: 6.9%, p = 0.0231), and history of other autoimmune disease (positive dcp: 10.7% versus negative dcp: 3.0%, p = 0.0107). Significant procedural factors included posterior fusion (positive dcp: 48.2% versus negative dcp: 22.0%, p <0.0001), sitting (positive dcp: 7.0% versus negative dcp: 21.2%, p = 0.0037), and number of levels (positive dcp mean: 3.52 versus negative dcp: 2.26, p <0.0001). Table 3 summarizes the significant variables identified on univariate and multivariate analysis as well as provides a calculated odds ratio for each associated risk factor . On multivariate analysis, the number of operative levels (p = 0.0053, odds ratio [or] = 1.27, 95% confidence interval [ci] 1.075 to 1.496) and nonspecific autoimmune disease (p = 0.0416, or 2.95, 95% ci 1.047 to 7.092) remained significant . Table 4 summarizes the risk factors for specific procedure categories with a sufficient number of cases for analysis . The risk factors identified for the entire cohort were not significant for anterior diskectomy and fusion or corpectomy . Anterior fusions (number of levels, intraoperative transfusion), posterior fusions (number of levels), foraminotomies (age, sitting, number of levels, other autoimmune disease), laminectomies without fusion (author autoimmune disease), and all nonfusion procedures (sitting, other autoimmune disease) had at least one significantly correlated risk factor . Foraminotomies were performed in 677 cases and a dcp occurred in 24 individuals (3.5%) and at one of the roots expressly decompressed in 18 cases (2.7%). Comparing those not undergoing a foraminotomy to those who did, there was no significant difference in the rate of a dcp . Chi - square analysis was done both including cases occurring at a root not decompressed with a foraminotomy (fisher exact test p = 0.78) and excluding cases where a dcp occurred at a root not decompressed with a foraminotomy (fisher exact test p = 0.56). Only one patient exhibited any abnormality during the procedure, but the motor evoked potential instability was not consistent with the myotome affected postoperatively . Emg results were available for 17 patients (30%) postoperatively (table 5). The incidence of bilateral (24%) and multilevel level (88%) involvement was increased when compared with clinician evaluation consistent with the dcp . Additionally, 5 (29%) were consistent with a brachial plexopathy and less indicative of a process occurring at the nerve root . The median neurologic follow - up for our cohort is 15.6 months (range 0.1 to 65.84 months). Unfortunately, there has been no recovery of motor function in 4 (7.1%) patients . However, for those with documented improvement, the first increases in motor grade were observed a median of 22.5 days postoperatively (range, 1 to 424). At last follow - up, the majority of individuals had regained normal (n = 27, 48.2%) or near normal (n = 12, 21.4%) strength . Postoperative dcp is a known complication of cervical spine surgery with a reported incidence around 5% based on a recent meta - analysis.4 in the current study, we demonstrated that dcp occurred in 3.4% of our cohort, and the majority of these cases involved the c5 myotome . Although abnormal transcranial electrical stimulation - induced evoked potentials are highly sensitive and specific for radiculopathy that manifests immediately upon waking from anesthesia, dcp injury does not exhibit any signs of a potentially injurious event.18 although nerve root irritation remains a possible consideration, our limited intraoperative monitoring results support the suggestion that these palsies were not likely the result of intraoperative trauma or positioning . At final follow - up, 7% had stable or worsening weakness . The rate of improvement reported in our cohort is slightly better than the 80% noted in the literature,4 though likely attributable to variability in follow - up and the definition of improvement . Some authors have suggested prophylactic foraminotomies may lead to decreased rates of dcp, particularly after laminoplasty.3 19 20 although we were unable to determine whether a foraminotomy was prophylactic from a retrospective review of operative reports, the rate of dcp at nerve roots decompressed with foraminotomies was not statistically different from those not operated upon . In fact, there was a slight trend toward an increased prevalence of dcp when a foraminotomy was performed . Although the protective effect of prophylactic foraminotomies would be better studied prospectively, this difference may suggest that the benefit of foraminotomy noted following laminoplasty in asian cohorts may not be applicable to north american populations undergoing laminectomy and fusion . Interestingly, none of the risk factors for psin as suggested by staff et al were correlated with the development of a dcp15; however, nonspecific autoimmune disease remained statistically significant on multivariate analysis (p = 0.0416). This finding, in addition to the clinical presence of bilateral (18%) and multilevel (55%) involvement and involvement of roots that were not at the operative levels (9%), suggests that autoimmune reactions or nonmechanical factors potentially play a role . Further consideration of the emg results suggest that the reported incidences of bilaterality and multilevel involvement may be underestimated . However, the higher rates of bilateral and multimyotomal palsies observed in this subpopulation may be the result of more significant or prolonged disease requiring nerve conduction studies for further evaluation . The present study also identified increased age to be significantly correlated with the development of a dcp, which is consistent with a large series by nassr et al, which reports an association with age and c5 palsy after corpectomy and laminoplasty.2 we also describe several procedural risk factors that are correlated with an increased rate of dcps on univariate analysis including posterior fusions (p <0.0001), prone versus sitting (0.0036), and number of operative levels (p <0.0001). Recently, the development of dcps was reported by yamanaka et al to be higher in patients undergoing instrumented fusion after cervical laminoplasty,21 a finding that was replicated in this study . Although the difference in cases selected for anterior and sitting approaches may explain this finding, another potential explanation is that the anterior approach and the sitting / semisitting position could offer some anatomical advantage or minimizes manipulation of the nerve roots . Other studies have suggested that the correction of cervical lordosis that is targeted following laminectomy and fusion can close the foramen and exacerbate the compression of the c5 root in cases of preexisting foraminal stenosis.6 it may be possible to evaluate potential differences in positioning with an anatomic study, but a complete understanding of this finding may prove elusive . The number of operative levels was extremely significant on univariate analysis (p <0.0001) and the only mechanical variable that remained significant on multivariate analysis (0.0053). This result suggests that the extent of the operation is the primary risk factor for this complication, and supports the theory that mechanical factors significantly contribute to the development of this complication . Specifying risk factors by procedure categories further elucidates evidence regarding the etiology of risk factors . The prevalence of dcp across procedure categories in the present study is similar to values reported previously and suggests that the highest prevalence of dcp is observed during posterior fusion.6 again, the number of operative levels appears as the dominant risk factor for both anterior and posterior fusions, which may be the result of increased traction on the nerve roots from changes in alignment / cord position and higher rates of transfusion . There is also evidence from odate et al suggesting that one can reduce the incidence of dcps in anterior cervical decompression and fusion cases by restricting the decompression to 15 mm and avoiding asymmetric decompressions.22 interestingly, sitting was correlated with a lower incidence for all nonfusion cases . Due to the limited applicability of the sitting position for anterior and instrumented cases, there was a valid concern that sitting may be a proxy variable for posterior noninstrumented cases; however, the result of this study validates that sitting may be protective for this complication . The most interesting outcome of this section is the strong correlations of nonspecific autoimmune disease and age with foraminotomies . The development of a dcp seems to correlate with those undergoing direct manipulation of the nerve root in those with preexisting autoimmune disease . Although it is purely conjecture, manipulation of the nerve roots may expose immune - privileged antigens, which could further propagate an autoimmune response in an individual with heightened immunity . The bilateral and multiple myotomal presentation as well as emg findings supporting plexopathy in some patients lend further support to this hypothesis . Further evaluation in larger cohorts may be appropriately powered to more specifically evaluate this general result . Although there is significant evidence for a multifactorial etiology, this study identifies the number of operative levels as the most specific etiologic factor . Age and history of nonspecific autoimmune disease are identified as risk factors for this complication, which could be useful when selecting patients for surgery and counseling them as to the risks of the procedure . From a surgical perspective, the association of extent of surgery with development of dcp may lead the surgeon to be more conservative with the decompression to decrease the risk of this complication; however, this risk must be weighed against the risk of inadequate decompression resulting in continued symptoms postoperatively . Additionally, the positioning concerns may lead to specific planning considerations to minimize risk . Finally, this study is important because it represents a western cohort of patients undergoing cervical decompression with or without fusion . Many previous studies have focused on asian cohorts undergoing laminoplasty operations, with only c5 myotomes . This study is limited by its retrospective design, study population, methodology, and inclusion criteria . Although verifying procedure specifics with both cpt codes and independent chart review increases our confidence in the data set, each method has its own inherent limitations that do not disappear when they are combined . The chosen study cohort also may prevent the broad applicability of this study's results, as nearly all of the cases were originally indicated for the treatment of degenerative spine disease . This cohort is only a segment of the larger cervical spine surgery population, and the current study does not sufficiently evaluate the potentially important variable of the original underlying pathology . Additionally, in an attempt to identify all atypical dcps, it was necessary to use rather loose exclusion criteria . Neurologic deterioration resulting from a myelopathy or exacerbation of the original pathology could present similarly to a multimyotomal dcp . Each misclassification of other similar pathologies weakens the results and conclusions of the present study . There has also been increasing evidence that radiographic measurements including anteroposterior diameter, foramina diameter, and/or cord lamina angle can be predictive of dcps . These and other radiographic measurements should continue to be evaluated in similar cohorts to identify predicative factors that could be easily applied to clinical practice.23 although a mechanical etiology is partially supported as a cause for dcp, notable correlations with autoimmune risk factors as well as bilateral and multimyotomal involvement does support the hypothesis that some dcps may result from an autoimmune response.
Fractures of the upper face and anterior skull base are a challenging neurosurgical, plastic, maxillofacial surgery problem . After clinical and radiographic evaluation of the fracture, prompt surgical intervention should be immediately instituted to excise any necrotic tissues inside or outside the cranial cavity, brain isolation by meticulous dural closure ablation of the frontal air sinuses and bony coverage of the region by either immediate or delayed frontal bone reconstruction . If frontal bone is comminuted, it is difficult to replace the small bony fragment by rigid bone plate fixation . In such cases, it is prudent to leave the bony fragments where they are and camouflage the defect . These procedures include use of hydroxyapatite cement, hydroxyapatite block, hydroxyapatite granules, carbonated calcium phosphate bone cement, norian craniofacial repair system (crs) or carbonated calcium phosphate plate (ccpp), high - density porous polythene implants or bioactive glass ceramic implant and acrylic (methylmethacrylate). We have used polymethylmethacrylate or pmma for correction of frontal bone defect in one patient and has found it to be a convenient, safe and simple method . A 28-year - old male patient reported to our department with h / o of rta . On clinical examination, there was depressed frontal bone and zygoma fracture [figure 1a and b]. Pre operative lateral view of depressed frontal bone fracture pre operative depressed frontal bone close up view axial and spiral computerized tomography (ct) scanning of head was done to rule out any head injury . Radiograph revealed depressed comminuted fracture of frontal bone on left side leading to obliteration of frontal sinus on that side . As the comminuted bony fragments of frontal bone could not be brought back into the normal position, it was decided to camouflage the defect with gentamicin impregnated pmma (poly methylmethacrylate) bone cement . After proper scrubbing of operating field and draping, 2% lignocaine with 1:200000 adrenaline was infiltrated in the area to achieve vasoconstriction and to get fluid dissection . The fracture site was exposed via extending the existing lacerated wound [figure 2]. Since it was not associated with any head injury, the defect was flushed and dried to make it ready to receive the bone cement . Exposure of the fracture site through the lacerated wound the bone cement was then mixed as per manufacturer's instructions [figure 3]. Once it reached dough stage, it was used to fill up the defect and manipulated to the desired shape [figure 4]. Bone cement being prepared as per manufacturer instructions bone cement placed and adopted over the defect immediate post operative picture as the comminuted bony fragments of frontal bone could not be brought back into the normal position, it was decided to camouflage the defect with gentamicin impregnated pmma (poly methylmethacrylate) bone cement . Patient was operated under general anesthesia . After proper scrubbing of operating field and draping, 2% lignocaine with 1:200000 adrenaline was infiltrated in the area to achieve vasoconstriction and to get fluid dissection . The fracture site was exposed via extending the existing lacerated wound [figure 2]. Since it was not associated with any head injury, the defect was flushed and dried to make it ready to receive the bone cement . Exposure of the fracture site through the lacerated wound the bone cement was then mixed as per manufacturer's instructions [figure 3]. Once it reached dough stage, it was used to fill up the defect and manipulated to the desired shape [figure 4]. Bone cement being prepared as per manufacturer instructions bone cement placed and adopted over the defect immediate post operative picture the wound healed very well and there was no sign of infection or any other complication at the time of discharge [figure 6]. Appropriate treatment depends on an accurate diagnosis, focusing on the physical examination and data from computed tomography scans . It is not uncommon for post craniofacial trauma patients to require augmentation of depressed craniofacial skeleton . Reconstruction and recontouring of these defects in natural esthetic fashion can pose challenge to clinician . Various autogenous bone graft and alloplastic materials have been in use for this purpose i.e. Titanium mesh, polymethyl methacrylate autogenous bone, hydroxyapatite, htr - pmi (hard tissue replacement patient matched implant) have been used with varying success . All materials and grafts have merits and demerits in their use . This may lead to donor site morbidity, inability to obtain adequate bone for large defect . Cranial defect caused by trauma can be satisfactorily treated using cranioplast implant made from htr - pmi process . Infection and foreign body reaction and availability are the main demerits of this alloplastic material . Other alloplastic materials that have been used for such defects are hydroxyapatite, silicon rubber, acrylic metal plates and proplast . Advantages of alloplast: availabilitynonresorbabilityease of surgical procedureexcellent post operative cosmetic result ease of surgical procedure excellent post operative cosmetic result disadvantages include: foreign body reactionpotential for infection which may produce fistula, slippage, extrusion, granuloma and erosion.polymethylmethacrylate is the most commonly used alloplastic material . Foreign body reaction potential for infection which may produce fistula, slippage, extrusion, granuloma and erosion . Tissue tolerancereliable recounstructive material reliable recounstructive material disadvantages include infection, limitation of growth and it may fracture and requires time for shaping and curing . The risk of infection may be reduced by adding antibiotic i.e. Gentamycin to the acrylic and using it under sterile conditions, beneath well - vascularized skin . Growth limitation may be obviated by not placing acrylic across sutures in children with enlarging skulls . This technique, apart from being affordable, also ensures shorter operative time and good esthetic result . So we have chosen this technique in our patient and did one patient using pmma and got initial promising results.
Infections by herpes viruses (herpes simplex type 1 or 2, varicella zoster virus [vzv]) are frequent in humans, and such viruses tend to persist within cranial nerves, dorsal roots, and autonomic ganglia causing latent infections by virtue of reactivation . Reactivation of vzv mainly presents with rash and pain affecting the entire dermatome and less frequently a zoster sine herpete . Vzv infection of the central nervous system (cns) such as encephalitis, meningitis, myelitis, or vasculitis occurs rarely but is feared because of the numerous unfavorable outcomes of such presentations . Cns infection with vzv in young immunocompetent adults is rare and unexpected, and only very few cases have been described so far in the world literature . We bring to limelight, the scenario of a young immunocompetent patient with vzv meningitis . A 33-year - old healthy indian male employed as a software engineer developed intermittent holocranial throbbing headache, which soon progressed to severe continuous headache, associated with fever, photophobia, and vomiting for a duration of 3 days . He also complained of rashes over the skin of his left shoulder blade and left side of his body which was of the same duration . There was no past history of similar illness, contact with people having similar complaints, or chicken pox . The patient was febrile, had neck stiffness and photophobia, and was found to have rashes along the cutaneous distribution of two lower intercostal nerves of the left side . His complete blood count was unremarkable except for raised erythrocyte sedimentation rate (30 mm/1 h). Renal function tests, liver function tests, and serum c - reactive protein were also reported normal . Serologies for dengue, typhoid, malaria, infectious mononucleosis, toxoplasma, rubella, cytomegalovirus, and herpes simplex antibodies were negative . Human immunodeficiency virus (hiv) serology was negative . The cerebrospinal fluid (csf) analysis showed cell count of 535 cells/l csf (differential count: 99% lymphocytes, 01% polymorphs), csf protein was 115 mg / dl, csf glucose was 34 mg / dl, and plasma glucose 76 mg / dl . Csf grams stain, ziehl nielsen's acid - fast bacilli stain, india ink stain, cryptococcal antigen, common bacterial antigen assay (haemophilus influenzae b, streptococcus group b, streptococcus pneumoniae, neisseria meningitidis a cyw 135, n. meningitidis b, and escherichia coli), venereal disease research laboratory, and polymerase chain reaction (pcr) for tuberculosis were negative . He was treated with a broad spectrum antibiotic (ceftriaxone) and intravenous acyclovir (dose of 10 mg / kg body weight every 8 h) for 6 days . Gradual improvement in the condition of the patient was observed from 3 day after initiation of parenteral acyclovir . Most neurological complications caused by vzv can occur in both primary and reactivated vzv although they seem to appear more frequently in herpes zoster than in vzv . Both immunocompetent and immunocompromised patients may suffer from these neurological complications, but they appear to be more frequent and more severe in the latter group . The common neurological complications associated with herpes zoster are postherpetic neuralgia, myelitis, encephalitis, ventriculitis, aseptic meningitis, and white - matter disease . Meningitis is a rare complication of vzv infection in a review of 859 patients with varicella - zoster infection; meningitis was reported in only 0.5% within 60 days of diagnosis incidence increases with age including 2.5 cases/1000 in ages 2150 versus 10.1 cases/1000 in those older than 80 years . The incidence is increased in hiv - seropositive patients including 29.4 cases/1000 person - years as compared to the hiv - seronegative patients with 2.0 cases/1000 person - years . Although the incidence of varicella and complications has decreased since the licensure of the vaccine, there may be breakthrough cases of varicella with aseptic meningitis and transient sensorineural hearing loss . Patients with vzv meningitis may suffer from high fever, severe headache, cervical rigidity, seizure, ataxia, hemiplegia, and even coma; these symptoms may appear within days after the appearance of the skin lesion and sometimes without skin lesions also . Csf shows increased cell counts and elevated protein levels in the csf using sensitive laboratory analyses (e.g., pcr and detection of intrathecal production of specific antibodies), recent epidemiological studies found a portion of 529% of vzv in aseptic meningitis and encephalitis and it has been suspected that vzv infections had been underestimated in earlier publications . Nevertheless, in immunocompetent patients without neurological deficits (as in our case), vzv meningitis is rare . We, therefore, highlight the importance of considering vzv as a possible cause for meningitis even in previously healthy young patients and the recommended diagnostic lumbar puncture . Detailed csf diagnostic procedures including pcr and detection of intrathecal synthesis of antiviral antibodies (especially for vzv and herpes simplex viruses) should be considered even though csf cell count and total protein seem to indicate a bacterial infection . Since herpes zoster is a viral disease, conservative care or acyclovir administration is the routine treatment, and steroid therapy is employed as an optional addition . The administration of acyclovir within 4872 h of the appearance of zoster effectively relieves acute pain and the vesicles, induces a higher rate of remission, and deters the virus from spreading throughout the patient's body, and they usually show a full recovery without developing other complications.
Hypertension is a complex multifactorial disorder with masses of genetic and environmental factors contributing to its occurrence . Although great effort has been devoted to uncover the genetic underpinnings of hypertension, there is no definite consensus on how many genes and which genetic determinants are actually involved in its development . Currently, evidence that links inflammation to the genesis of hypertension is proliferating [2, 3]. As an anti - inflammatory regulator, transforming growth factor beta-1 or tgf-1 (gene: tgfb1) plays a part in many different clinical processes, such as embryonal development, cellular proliferation and differentiation, wounding healing, and angiogenesis [4, 5]. In addition, augmented production of tgf-1, partly via the mediation of angiotensin ii, potentially contributes to target organ damage related to hypertension [69]. Since the genomic sequence of tgfb1 gene is highly polymorphic, it is of added interest to confirm which tgfb1 polymorphism(s) might have functional potentials to influence the final bioavailability of tgf-1, thus the development of hypertension . In particular, an exonic polymorphism, 869t / c (rs1982073) in tgfb1 gene, has been studied extensively; however, the results are not often reproducible with positive signals being reported in some, but not all [11, 12], studies . Generally, association studies with individually low statistical power might account for this lack of consistency . As meta - analysis is a reliable way to resolve discrepancies in association studies and in an effort to clarify earlier inconclusive results, i decided to evaluate the influence of tgfb1 gene 869t / c polymorphism on the occurrence of hypertension, while addressing between - study heterogeneity, as well as publication bias . Both english and chinese language publications were identified using pubmed and embase engines, as well as china biological medicine (http://sinomed.imicams.ac.cn/index.jsp) and wanfang (http://www.wanfangdata.com.cn) databases with the deadline at february 1, 2011 . Keywords used for search in the boolean expression were (transforming growth factor beta-1 or tgf-1 or tgfb1) and (hypertension or blood pressure) and (polymorphism or allele or genotype or variant or variation). Searching results were limited to human populations (rather than family - based populations). The full text of the retrieved articles was scrutinized to decide whether information on the topic of interest was included . In addition, reference lists of the retrieved articles and reviews were also checked for citations of publications that were not initially identified . If more than one geographic or ethnic groups were included in one publication, each group was treated separately . Qualified studies in this meta - analysis met the following criteria: (i) evaluation of the tgfb1 gene 869t / c polymorphism with hypertension; (ii) case - control or cross - sectional study using either a hospital - based or population - based design; (iii) sufficient information on 869t / c genotype counts between hypertensive patients and controls for estimating odds ratio (or) and its corresponding 95% confidence interval (95% ci). Hypertension was defined as systolic blood pressure equal to or above 140 mmhg or diastolic blood pressure equal to or above 90 mmhg or previous treatment with antihypertensive drugs . Studies evaluating secondary hypertension or other types of monogenic hypertension were excluded . Where there were multiple publications from the same study population, the following information was extracted from each qualified study: first author's name, publication date, population ethnicity, study design, diagnostic criteria, baseline characteristics of the study population (such as age, gender, and body mass index), and the 869t / c genotype counts in patients and controls . For consistency, continuous variables expressed as mean standard error (se) were converted to mean standard deviation (sd). Moreover, the units of measures used in this study are transformed into the standard measurement units . I assessed the association of tgfb1 gene 869c allele with hypertension relative to the 829 t allele (allelic model), as well as the homozygous contrast (869cc versus 869tt), the dominant model (869cc plus 869tc versus 869tt), and the recessive model (869cc versus 869tt plus 869tc). Unadjusted or and 95% ci were used to compare contrasts of alleles or genotypes between patients and controls . The random - effects model using the method of dersimonian & laird, instead of fixed - effects model, was implemented to bring the individual effect - size estimates together, and the estimate of heterogeneity was taken from the mantel - haenszel model . Satisfaction of 869t / c genotypes with hardy - weinberg proportions was calculated using the test or fisher's exact test in control groups . Between - study heterogeneity was assessed by the inconsistency index i statistic (ranging from 0 to 100%), which was documented for the percentage of the observed between - study variability due to heterogeneity rather than chance, with higher values of this index suggesting the existence of heterogeneity [15, 16]. In the case of between - study heterogeneity, i examined the study characteristics that can stratify the studies into subgroups with homogeneous effects . Cumulative meta - analysis was conducted to identify the influence of the first published study on the subsequent publications and the evolution of the combined estimates over time according to the ascending date of publication . Likewise to identify potentially influential studies, sensitivity analysis was undertaken by removing an individual study each time to check whether any of these estimates can bias the overall estimate . Additionally, to estimate the extent to which one or more covariates explain heterogeneity, metaregression, as an extension to random - effects meta - analysis, was employed . The metaregression model relates the treatment effect to the study - level covariates including averaged values of age, male percent, body mass index (bmi), glucose, triglyceride (tg), total cholesterol (tc), high - density lipoprotein cholesterol (hdlc), and low - density lipoprotein cholesterol (ldlc) between patients and controls, as well as the study design (population - based design versus hospital - based design) and ethnicity (asians versus whites). Egger's test can detect funnel plot asymmetry by determining whether the intercept deviates significantly from zero in a regression of the standardized effect estimates against their precision . Probability less than 0.05 was judged as significant with the exception of the i statistic and publication test, where a significance level of less than 0.1 was chosen . Further application of the identification criteria left 8 published papers [1012, 1923] involving 9 study populations (case - patients / controls: 2747/3404) in an attempt to evaluate the association of tgfb1 gene 869t / c polymorphism with hypertension . Thereof, three papers were written in english language [1012], and the remaining in chinese language . The study aims and main results of chinese language reports are presented in supplementary table 1 (see table 1 in supplementary material available online at doi:10.4061/2011/934265). Seven populations included chinese subjects [10, 1923], one included japanese subjects, and one included white population . Except one study involving patients with both hypertension and rheumatoid arthritis, others were focusing on essential hypertension patients . Genotyping for 869t / c polymorphism in all qualified studies, except one using roche lightcycler method, was conducted using polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp) followed by enzyme digestion . The frequencies of 869c allele in the case / control groups were 0.527/0.457 in all populations and were exceedingly low (0.347/0.367) in whites . Taking into account only the control groups, genotype distributions were in hardy - weinberg equilibrium across all studies . In allelic model, comparison of the mutant 869c allele with the wild 869 t allele generated a significant 30% increased risk for hypertension (95% ci: 1.111.51; p = 0.001), yet with strong evidence of between - study heterogeneity (i = 72.4%; p <0.0005) (figure 1). Besides the suggestive symmetry of funnel plot (figure 2), egger's test indicated no publication bias (p = 0.25). Further, this association was potentially strengthened in the homozygous comparison (869cc versus 869tt) with or nearly doubled to 1.62 (95% ci: 1.232.14; p = 0.001). Similarly, this association was still tingled by significant heterogeneity (i = 66.2%; p = 0.003), the risk estimates from individual studies were symmetric (figure 2), and the egger's test suggested a low probability of publication bias (p = 0.262). Additionally, in view of the heterozygous 869tc genotype, i considered two different models of inheritance . Overall, the ors from allelic model were almost similar in magnitude from both dominant (or = 1.35; 95% ci: 1.111.64; p = 0.003) and recessive (or = 1.41; 95% ci: 1.161.71; p <0.0005) models (figure 1). Although between - study heterogeneity was attenuated to a certain extent, statistical tests still reached significance (dominant: i = 57.4%; p = 0.016; recessive: i = 53.0%; p = 0.03). Moreover, there was evident publication bias only in dominant model as reflected by the funnel plot and statistical test (p = 0.06). In the cumulative meta - analysis, there was no evidence suggesting the first published study that reported a potentially significant result and then trigged the subsequent replication . Also generally the sensitivity analysis revealed that no single studies were observed to influence the pooled results significantly (data not shown). Considering the significant heterogeneity in the above comparisons, i considered it a better choice to try investigating its sources by first conducting subgroup analyses in homogeneous groups and then incorporating various study - level covariates in a metaregression model . To evaluate the possible effect of study design on the variability of overall estimates, studies were divided into population based and hospital based, and importantly the magnitude of association in population - based studies was gradiently potentiated in 869t / c allelic (or increased by 7.94%), homogeneous (10.13%), and recessive (18.61%) models compared with that in hospital - based studies (figure 3). Further ethnicity - stratified analysis indicated strikingly heterogeneous associations of 869t / c polymorphism with hypertension, by showing a contradictory association between asians and whites (figure 4). In asians, i consistently observed a risk - conferring effect of the 869c allele or 869cc genotype for hypertension, even upon stratification by countries, as well as chinese ethnic groups . Contrastingly, after restricting analysis to whites, although there was just one study, i hereto observed a protective effect (or = 0.840.92), and the corresponding wide confidence intervals in all genetic models gave an indication of insufficient study power in white populations . After metaregressing the explanatory variables of interest in this study (see section 2.4), i unfortunately failed to detect any statistical significance concerning 869t / c polymorphism across all genetic models (data not shown). Via a comprehensive evaluation of tgfb1 gene 869t / c polymorphism among 6151 subjects, i provided for the first time convincing evidence that individuals homozygous for the 869c allele were 62% more likely to develop hypertension with respect to homozygous for the 869tt subjects . Although between - study heterogeneity, albeit disturbing, could not be easily eliminated, this study indicated that tgfb1 gene could be a genetic marker for hypertension . In this meta - analysis, study design and ethnicity were regarded as potential sources of between - heterogeneity by subgroup but not metaregression analyses . Although i only focused on nine populations, which runs the risk of false - positive findings, my results can still drop several hints here . Firstly, subgroup analysis indicated that magnitude of association was potentially strengthened in population - based studies relative to in hospital - based studies . I agree that control for population stratification remains an important consideration in hospital - based studies, because in this meta - analysis, most studies have recruited subjects from only one hospital, and thus there might be a narrow socioeconomic profile for both patients and controls . Moreover, in hospital - based studies, poor comparability between cases and controls might exert a confounding effect on the true association in light of a regional specialty for the disease under study and the differential hospitalization rates between cases and controls . In contrast, subjects drawn from community or a fixed group might be representative of the true population, leading us to believe that results from population - based studies might hold the water . Considering the wider confidence intervals of estimates and small sample sizes in population - based studies, secondly, remarkable heterogeneous associations of 869t / c polymorphism with hypertension were identified across different ethnic populations . On one hand, hypertension is a complex disease, and different genetic profiles may cause this discrepancy, as indicated by the big difference of 869c allele frequencies across different populations . In this regard, it is important to construct a database of genetic variants related to hypertension in each ethnic group . On the other hand, this discrepancy is likely due to chance because there is only one study in whites, which might be statistically underpowered to detect a slight effect or may have generated a fluctuated risk estimate . It is thus obvious that more studies are required in subjects of caucasian descent in order to fully address this issue . Thirdly, as an alternative approach to subgroup analysis and a multivariate meta - analysis, the metaregression failed to provide any significant signals regarding the allelic / genotypic associations of 869t / c polymorphism with hypertension . However, it is important to bear in mind that metaregression analysis, although enabling covariates to be considered, does not have the methodological rigor of a properly designed study that is intended to test the effect of these covariates formally . Importantly, one limitation tarnishing this meta - analysis was the number of studies that are available for inclusion . In fact, some studies did not report the study - level covariates of interest, precluding a more robust assessment of sources of heterogeneity . Last but not least, despite the clear strength of this study including relatively large sample sizes, satisfaction of hardy - weinberg equilibrium, and lack of publication bias, interpretation of the current study, however, should be viewed in light of several technical limitations . Because only published studies were retrieved in this meta - analysis and the grey literature (papers in languages other than english and chinese) was not included, publication bias might be possible, even though the funnel plots and statistical tests did not show it . In addition, most studies in this meta - analysis have recruited subjects aged 50 years, for whom environmental factors are likely to contribute more prominently than the genetic component to the development of hypertension, suggesting that large association studies in a younger population of hypertensive subjects are of added interest . Moreover, the single - locus - based nature of meta - analysis precluded the possibility of gene - gene and gene - environment interactions, as well as haplotype - based effects, suggesting that additional studies assessing these aspects will be necessary . Furthermore, i only centered on tgfb1 gene 869t / c polymorphism and did not covered other genes or polymorphisms . It seems likely that the 869t / c polymorphism individually makes a moderate contribution to risk prediction in hypertensive subjects, but whether this variant integrated with other risk factors will enhance the prediction requires additional research . Thus, the jury must refrain from drawing a conclusion until large, well - performed studies confirm or refuse this result . Taken together, i expand previous individual findings on hypertension, indicating that the tgfb1 gene 869t / c polymorphism may influence the risk of hypertension, especially in asian populations . Also my observation leaves open the question of heterogeneous effect of 869c allele across different ethnic groups . I believed that this study provides an anchoring point for better understanding of the pathogenesis of hypertension . Nevertheless, for practical reasons, i hope that this study will not remain just another endpoint of research instead of a beginning to establish the background data for further investigation on mechanisms of the tgfb1 gene and hypertension.
Glaucoma, an optic neuropathy characterized by progressive visual field loss, is currently a leading cause of blindness in the world, regardless of the population studied.1,2 according to the world health organization, the disease accounts for 12.3% of blindness, cataracts being the leading cause.2 note that unlike cataracts, blindness from glaucoma is irreversible . Although a multifactorial disease, elevated intraocular pressure (iop) remains the most important known risk factor.1,35 the level of iop appears to play an important role in the development and progression of the disease even when in the statistically normal range.3,4 thus, it is evident that the main treatment for avoiding disease progression or development in patients with glaucoma or who are suspect is the reduction of iop.5 the three options for iop reduction are clinical treatment, laser, or incisional surgery . Although there are safe and effective drugs, most of them present side effects, which can be mild and local (eg, conjunctival hyperemia of prostaglandin analogs), or systemic and more serious (eg, cardiorespiratory effects of beta - blockers).6,7 in this context, although fixed combinations seem to be slightly less efficacious than their respective unfixed combinations, the former lead to a lower hyperemia risk.7 another important factor to be considered in relation to glaucoma treatment is patient adherence to proposed therapy . Unfortunately, studies of persistence and adherence in glaucoma treatment show that a significant proportion of patients discontinue their use of newly prescribed therapy within the first year of treatment.811 we know that the daily use of eye drops requires patient dedication, understanding of the disease severity, and ability to instill the medications . Even though there are educational materials for patients seeking to circumvent some of these problems, many report great difficulty during the instillation of the drops . The main reported hindrances include difficulty in keeping the eyes open, handling the bottle, avoiding the contact of the tip to the globe, and finally getting the drops in the right place . These difficulties often imply the need for additional help to instill the medication . In this context, a device that could simplify daily instillation of the eye drops would be useful to minimize some of these difficulties and increase adherence to topical therapy . The aim of this study was to evaluate the efficacy and safety of a new device (eyedrop; vanguard design, so paulo, brazil) for eye drop instillation in patients with and without glaucoma . This interventional protocol adhered to the tenets of the declaration of helsinki and was approved by the institutional review board of unifieo in november 2013 . In addition, written informed consent was obtained from all participants . In this prospective study, consecutive patients with glaucoma (presence of glaucomatous optic neuropathy associated with characteristic visual field defect) and healthy participants with and without prior experience in placing eye drops were included . Prior experience in eye drop instillation was defined as at least one year of continuous use of at least one topical medication . Exclusion criteria were history of prior ocular trauma or surgery, secondary glaucoma, uncontrolled iop, advanced disease (vertical cup - to - disk ratio 0.8 or fixation threat in visual field testing), presence of any other ocular disease besides glaucoma, and physical inability to handle the device (example: severe rheumatoid arthritis). Glaucomatous optic neuropathy was defined as cup- to - disk ratio> 0.6, asymmetry between eyes 0.2, presence of localized defects of the retinal nerve fiber layer, and/or neuroretinal rim in the absence of any other anomalies that could explain such findings . Characteristic glaucomatous visual field defect in standard automated perimetry (humphrey sita standard 24 - 2; carl zeiss meditec, dublin, ca, usa) was defined as three or more adjacent points with probability <5% (except those on the periphery of the field or directly above and below the blind spot) on the pattern deviation graphic, a pattern standard deviation index with a probability <5%, or glaucoma hemifield test with results outside normal limits . In the first stage, patients underwent a complete ophthalmic examination (visual acuity, goldmann applanation tonometry, and fundus examination). Possible signs of intolerance to medication use (conjunctival hyperemia, keratitis, and dry eye signs) were evaluated . The baseline iop for each patient was defined by the average of three measurements . At the end of the visit, bimatoprost 0.01% (lumigan rc; allergan, inc ., irvine, ca, usa), from a small round shaped bottle, was introduced in both eyes for all participants . For those already using hypotensive the eyedrop delivery device was made available to all participants for use in only one eye, in a randomized order . As on - site training prior to medication dispensing using the device, a 3-minute video clip demonstrating how to use the device properly was initially presented . Then, each patient used the device once at the office, supervised by a physician . Eyedrop is a plastic device with blunt edges, easy to handle, in which the bottle is inserted (figure 1). To instill drops, based on its design and working mechanism, the device could be useful to help patients to overcome some of the previously cited difficulties, such as keeping their eyes open, avoiding contact of the tip to the globe, and finally getting the drops in the globe, and not in the periocular region . In the second stage of study, held between 10 and 15 days after the first for glaucoma patients and on the same day (6 hours apart) for healthy volunteers, all patients were evaluated by an experienced examiner (blinded to which eye was chosen for applicator use) for iop determination and investigation of possible associated side effects . To evaluate the ease of instilling the eye drops, a visual analog scale (vas) (scores ranging from 010) was used . In order to investigate whether the drops were being properly instilled, on that same day, each patient was asked to instill fluorescein eye drops in both eyes, the applicator being used in only one eye (choice made at random). The examiner, again masked to which eye was chosen for applicator use, subjectively assessed the pattern of distribution of fluorescein (the presence or absence of the drops at the bottom of the lacrimal sac and possible inadequate instillation in the periocular region). The paired t - test was used to compare the iop values with and without the use of the applicator . Differences in vas scores between eyes were investigated using the wilcoxon (signed - rank) test . The frequency of side effects between eyes (with and without the applicator) was compared using the mcnemar test . For a sample power of 80% (value of 0.20) and value of 0.05, we would need 26 patients to detect an iop difference of 2 mmhg (assuming a standard deviation of 2.5 mmhg) between eyes with and without the applicator . In this prospective study, consecutive patients with glaucoma (presence of glaucomatous optic neuropathy associated with characteristic visual field defect) and healthy participants with and without prior experience in placing eye drops were included . Prior experience in eye drop instillation was defined as at least one year of continuous use of at least one topical medication . Exclusion criteria were history of prior ocular trauma or surgery, secondary glaucoma, uncontrolled iop, advanced disease (vertical cup - to - disk ratio 0.8 or fixation threat in visual field testing), presence of any other ocular disease besides glaucoma, and physical inability to handle the device (example: severe rheumatoid arthritis). Glaucomatous optic neuropathy was defined as cup- to - disk ratio> 0.6, asymmetry between eyes 0.2, presence of localized defects of the retinal nerve fiber layer, and/or neuroretinal rim in the absence of any other anomalies that could explain such findings . Characteristic glaucomatous visual field defect in standard automated perimetry (humphrey sita standard 24 - 2; carl zeiss meditec, dublin, ca, usa) was defined as three or more adjacent points with probability <5% (except those on the periphery of the field or directly above and below the blind spot) on the pattern deviation graphic, a pattern standard deviation index with a probability <5%, or glaucoma hemifield test with results outside normal limits . In the first stage, patients underwent a complete ophthalmic examination (visual acuity, goldmann applanation tonometry, and fundus examination). Possible signs of intolerance to medication use (conjunctival hyperemia, keratitis, and dry eye signs) were evaluated . The baseline iop for each patient was defined by the average of three measurements . At the end of the visit, bimatoprost 0.01% (lumigan rc; allergan, inc ., irvine, ca, usa), from a small round shaped bottle, was introduced in both eyes for all participants . For those already using hypotensive the eyedrop delivery device was made available to all participants for use in only one eye, in a randomized order . As on - site training prior to medication dispensing using the device, a 3-minute video clip demonstrating how to use the device properly was initially presented . Then, each patient used the device once at the office, supervised by a physician . Eyedrop is a plastic device with blunt edges, easy to handle, in which the bottle is inserted (figure 1). To instill drops, based on its design and working mechanism, the device could be useful to help patients to overcome some of the previously cited difficulties, such as keeping their eyes open, avoiding contact of the tip to the globe, and finally getting the drops in the globe, and not in the periocular region . In the second stage of study, held between 10 and 15 days after the first for glaucoma patients and on the same day (6 hours apart) for healthy volunteers, all patients were evaluated by an experienced examiner (blinded to which eye was chosen for applicator use) for iop determination and investigation of possible associated side effects . To evaluate the ease of instilling the eye drops, a visual analog scale (vas) (scores ranging from 010) was used . In order to investigate whether the drops were being properly instilled, on that same day, each patient was asked to instill fluorescein eye drops in both eyes, the applicator being used in only one eye (choice made at random). The examiner, again masked to which eye was chosen for applicator use, subjectively assessed the pattern of distribution of fluorescein (the presence or absence of the drops at the bottom of the lacrimal sac and possible inadequate instillation in the periocular region). The paired t - test was used to compare the iop values with and without the use of the applicator . Differences in vas scores between eyes were investigated using the wilcoxon (signed - rank) test . The frequency of side effects between eyes (with and without the applicator) was compared using the mcnemar test . For a sample power of 80% (value of 0.20) and value of 0.05, we would need 26 patients to detect an iop difference of 2 mmhg (assuming a standard deviation of 2.5 mmhg) between eyes with and without the applicator . A total of 32 participants (mean age 42.316.2 years) were evaluated . Of these the majority of participants were women (72%) and white (66%), and 56% had prior experience with eye drop instillation . Glaucomatous patients were significantly older (mean age, 62.212.1 years; range, 4282 years) than healthy individuals (mean age, 29.211.9 years; range, 2065 years; p<0.01). There was no significant difference in mean iop variation when comparing the eye on which the applicator was used (3.92.9 mmhg) and the eye on which traditional instillation was used (3.32.6 mmhg; p=0.36). The subjective rating of the facility of drops instillation was significantly higher with the use of applicator (vas = 7.61.6) than without it (vas = 6.21.8; p<0.01). There was a higher frequency of positive device - related evaluations (vas score> 5) among participants without prior experience with eye drop instillation (78.6% [11/14]) versus those already experienced (66.7% [12/18]). Conjunctival hyperemia was the most common side effect in both groups, being observed in 25% of the eyes for which the delivery device was used and in 21.9% of those for which the device was not used . Punctate keratitis was also frequent, being observed in 12.5% of the eyes for which the device was used and in 9.4% of those for which the device was not used . There were no side effects specifically related to the device (such as ocular or periocular trauma or corneal / conjunctival abrasion). No difference in the frequency of side effects or the distribution pattern of fluorescein between eyes was observed (p0.63). Finally, among participants with negative evaluations about the delivery device (vas score 5), the most common complaint was finding the right placement of the device on the orbit . When it comes to glaucoma management, patient adherence to topical therapy, which although essential to control the disease, is often insufficient . Evaluating the usefulness of a new device for placing drops in eyes of patients with and without glaucoma, we found a better response from the participants when asked to compare the use of the applicator to the conventional instillation of eye drops . No loss of the hypotensive effect of the medication or increased occurrence of side effects was observed . We believe that these results will add to the existing measures to increase patients adherence to glaucoma treatment, facilitating the correct use of medications, especially for those with greater difficulty and no previous experience, and hence aiding in disease control . This pilot study is the first clinical evaluation of this new device . In the literature, the main causes related to poor adherence include a lack of awareness about the disease and irreversible damage,12 number of drugs used, impact on family income,13,14 side effects, patients forgetfulness, older age, inability to instill eye drops, problems in handling the bottle, and not having someone to help.1416 at least some of these problems could be minimized with the use of a device for proper instillation of hypotensive eye drops . In the few studies carried out on the use of eye drops and improvement of patient compliance through a delivery device, the most studied device has been the xal - ease delivery system (pfizer ophthalmics, new york, usa). In comparison with the conventional use of xalatan and xalacom eye drops (both from pfizer ophthalmics), different studies showed significant benefits with the use of the device.17,18 semes and shaikh17 found better use of the drops with the device (greater number of drops per bottle) when compared to conventional use . The authors attributed their results to a more uniform individual volume of each drop obtained with the device.17 in another study, with a more robust design (multicenter, prospective, randomized, crossover), nordmann et al18 observed that the use of the device halved the need for additional help to instill eye drops as compared to conventional use . In addition, the risk of the tip of the eye drop bottle touching the globe was significantly reduced . After one month, more than 70% of the patients reported being satisfied with using xal - ease, without significant side effects.18 although our study has a different design and has focused on other aspects related to the use of a delivery device, we believe that our data corroborate those described above, as approximately 72% of our patients had a positive response to the use of the eyedrop device . It is noteworthy that unlike the xal - ease, which is designed only for the application of the two abovementioned medications (from pfizer ophthalmics), the eyedrop device can be used for instillation of different eye drops, with various bottle shapes . Although we consider this as a significant advantage, it should be emphasized that even though the device is suitable for most types of bottles in the market, it cannot be used either with multi - dose bottles with the abak filter system or with preservative - free single - dose units (small containers). Finally, although an internet search (google.com) revealed that there are other commercially available devices for instilling eye drops, we did not find any related scientific articles currently published . The study had a relatively small sample (although it was satisfactory from a statistical point of view through the sample calculation for a power of 80%), had a short follow - up period, and evaluated only one medication (monotherapy). Therefore, our results should not be extrapolated for patients under two or more topical medications . Moreover, there is the possibility of positive bias in favor of the device, as the individual stimulus generated by the introduction of new equipment could lead to a greater commitment to the use of the medication (regardless of the effectiveness of the applicator itself). Finally, we know that in studies of this type, the fact that the patients know that they are being monitored may lead to an improvement in adherence to treatment.19 although this is an important factor, we believe that the paired comparison nature of our study probably minimized this type of influence . In this pilot study, a better subjective response was reported regarding the ease of instillation of hypotensive eye drops using the eyedrop delivery device compared to traditional instillation, especially in patients with no previous experience with eye drops . The use of the device neither resulted in loss of the hypotensive effect of the medication, nor did it increase the frequency of side effects . Even though we have not directly assessed the long - term iop control and stability of the disease, we believe our findings can have a positive impact on these aspects, since the improvement in patient adherence to treatment could result in a better iop control and lower risk of progression over time . Further studies with larger sample sizes and longer follow - up are necessary in order to better investigate such aspects.
The index case was a 14-year - old boy transferred from a regional hospital to our unit 24 h after a laparoscopic appendicetomy for perforated appendicitis . Abdomen was distended and tender with purulent discharge coming from a peritoneal drain chest x ray demonstrated extensive right pleural effusion (fig . 1). Fluid resuscitation and aggressive broad - spectrum antibiotics were commenced and a pleural drain was placed with improvement in his respiratory status . The intraperitoneal drain was inadvertently removed and some days later he became febrile again . Abdominal computed tomography (ct) showed multiple locules in an extensive right - sided abdominal collection extending from the subhepatic space to the iliac fossa (fig . Non - operative management with intravenous antibiotics failed to resolve all of these and a laparotomy was performed with drainage of all except a small subhepatic collection . A large sump drain was left in - situ and, following cessation of drainage and a satisfactory clinical state, he was discharged . Five days later, he re - presented with severe left pleuritic chest pain and dyspnoea . Bilateral pleural effusions and a large high attenuation pericardial effusion were noted on imaging (helical ct and echocardiography) (figs . A previously demonstrated small right subphrenic collection had increased in size . Due to the size and possible infective nature of the pericardial effusion, the pericardial effusion was microscopically analysed revealing profuse polymorphs and profuse gram negative rods, gram positive cocci and gram positive rods . The culture of the effusion grew enteric gram negative rods and mixed anaerobes, of which streptococcus anginosus (s. milleri) was isolated . His clinical condition improved significantly and the drain was removed on the second post - operative day . Pericardial effusions in adults are most commonly idiopathic, followed by infection and malignancy,, . Rarer causes include radiation, uraemia and post - acute myocardial infarction . In paediatric cases, the leading causes are infective pericarditis, connective tissue disease, metabolic disorders and malignancies . We searched the ncbi pubmed and medline database for english literature from 1946 to present to determine the frequency and management of pericardial effusion as a complication arising from acute appendicitis . However, after reviewing the papers for their relevance to this case, only two case reports remained . Reported a 3-year - old girl with a perinephric abscess extending from a ruptured appendix and subsequent empyema and pericardial effusion both treated by percutaneous drains . The subsequent purulent pericardial effusion was drained by retrosternal pericardiotomy with subsequent pericardiectomy required . Up to 55 per cent of children with advanced appendicitis demonstrated the anatomical connections between the retroperitoneum and the mediastinum by depicting ectopic air from radiographs and suggested these connections as providing pathways for bidirectional spread of disease processes into the mediastinum and pleural cavities . The european society of cardiology guidelines recommend surgical drainage over percutaneous drainage in complicated purulent pericarditis . A case series from northern india reported 25 cases of pericardial effusions in children over three years . No cases of intraabdominal infections were found and nine cases were complicated by cardiac tamponade . Echo - guided percutaneous drainage and pigtail catheter insertion were found to be safe and effective treatments . Contrary to the guideline previously mentioned, this case series calls into question the need for surgical drainage in the treatment of complicated pericardial effusion . This case report and review has demonstrated the extreme rarity of clinically significant exudative pericardial effusion complicating ruptured appendicitis . Given the low incidence, it is unlikely that consensus as to the ideal management (percutaneous v pericardotomy) will be reached.
Cancer detection has always been a major issue for the pathologists and medical practitioners for diagnosis and treatment planning . The manual identification of cancer from microscopic biopsy images is subjective in nature and may vary from expert to expert depending on their expertise and other factors which include lack of specific and accurate quantitative measures to classify the biopsy images as normal or cancerous one . The automated identification of cancerous cells from microscopic biopsy images helps in alleviating the abovementioned issues and provides better results if the biologically interpretable and clinically significant feature based approaches are used for the identification of disease . Cancer is one of the common diseases in india which has responsibility to maximum mortality with about 0.3 million deaths per year . The chances of getting affected by this disease are accelerated due to change in habits in the people such as increase in use of tobacco, deterioration of dietary habits, lack of activities, and many more . The possibility of cure from cancer is increased due to recent combined advancement in medicine and engineering . Medical professionals use several techniques for detection of cancer . These techniques may include various imaging modalities such as x - ray, computer tomography (ct) scan, positron emission tomography (pet), ultrasound, and magnetic resonance imaging (mri) and pathological tests such as urine test and blood test . For accurate detection of cancer pathologists use histopathology biopsy images, that is, the examination of microscopic tissue structure of the patient . Thus biopsy image analysis is a vital technique for cancer detection [2, 3]. Histopathology is the study of symptoms and indications of the disease using the microscopic biopsy images . To visualize various parts of the tissue under a microscope, the main goal of staining is to reveal the components at cellular level and counterstains are used to provide color, visibility, and contrast . Hematoxylin - eosin (h&e) is staining component that has been used by pathologists for over few decades . Hematoxylin stains cell nuclei which are blue in color while eosin stains cytoplasm and connective tissues which are of pink color . The histology is related to the study of cells in terms of structure, function, and interpretations of the tissue and cells . Microscopic biopsies are most commonly used for both disease screenings because of the less invasive natures . The microscopic biopsy images are easier to analyze specimens compared to histopathology due to absence of noncomplicated structures . The accurate manual identification of cancer from microscopic biopsy images has always been a major issue by the pathologists and medical practitioners observing cell or tissue structure under the microscope . In histopathology, the cancer detection process normally consists of categorizing the image biopsy into cancerous one or noncancerous one . In microscopic biopsy image analysis doctors and pathologists observe many of the abnormalities and categorize the sample based on various characteristics of the cell nuclei such as color, shape, size, and proportion to cytoplasm . For the detection and diagnosis of cancer from microscopic biopsy images, the histopathologists normally look at the specific features in the cells and tissue structures . The various common features used for the detection and diagnosis of cancer from the microscopic biopsy images include shape and size of cells, shape and size of cell nuclei, and distribution of the cells . It has been observed that the overall shape and size of cells in the tissues are mostly normal . The cellular structures of the cancerous cells might be either larger or shorter than normal cells . Cancer cells usually do not function in a useful way and their shapes are often not even . (b) size and shape of the cell's nucleus . The shape and size of the nucleus of a cancer cell are often not normal . The image of the cell looks like an omelet, in which the central yolk is the nucleus and the surrounding white is the cytoplasm . The nuclei of cancer cells are larger than the normal cells and deviated from the centre of the mass . The segmentation step mainly focuses on separation of regions of interests (cells) from background tissues as well as separation of nuclei from cytoplasm . The function of each tissue depends on the distribution and arrangements of the normal cells . These adjectives of microscopic biopsy images have been included in shape and morphology based features, texture features, color based features, color gray level cooccurrence matrix (glcm), law's texture energy (lte), tamura's features, and wavelet features which are more biologically interpretable and clinically significant . The main aim of this paper is to design and develop a framework and a software tool for automated detection and classification of cancer from microscopic biopsy images using the abovementioned clinically significant and biologically interpretable features . This paper focuses on selecting an appropriate method for each design stage of the framework after making a comparative analysis of the various commonly used methods in each category . The various stages involved in the proposed methodology include enhancement of microscopic images, segmentation of background cells, features extraction, and finally the classification . Section 2 describes the related works, section 3 presents the methods and models, section 4 describes the results and discussions, and finally section 5 draws the conclusion of the work presented in this paper . In recent years, few works have been reported in the literature for the design and development of tools for automated cancer detection from microscopic biopsy images . Kumar and srivastava presented detailed reviews on the computer aided diagnosis (cad) for cancer detection from microscopic biopsy images . Presented a review on computer aided diagnosis system to detect cancer from microscopic biopsy images using image processing techniques . The quasisupervised learning algorithm operates on two datasets, the first one having normal tissues labeled only indirectly and the second one containing an unlabeled collection of mixed samples of both normal and cancer tissues . This method was applied on the dataset of 22,080 vectors with reduced dimensionality, 119 from 132 . The regions having the cancerous tissues were accurately identified having true positive rate 88% and false positive rate 19%, respectively, by using manual ground truth dataset . Used haralick's textures features, histogram of oriented gradients (hog), and color component based statistical moments (ccsm) features selection and extraction approaches to classify the cancerous cells from microscopic biopsy images . The various features used in this paper are contrast, correlation, energy, homogeneity, glcm texture features, rgb, gray level, and hsv . Huang and lai presented a methodology for segmentation and classification techniques for histology images based on texture features and by using svm the maximum classification accuracy obtained is 92.8% . Presented a method for morphologic characterization of cell neighborhoods in neoplastic and preneoplastic tissue of microscopic biopsy images . In this paper, authors presented watershed transforms to compute the cell and nuclei area and other parameters . The distance measure of the neighborhood value has been used for calculating the neighborhood complexity with reference to the v - cells . The best classification which has been obtained by knn classifier is 83% for dysplastic and neoplastic classes and sinha and ramkrishan extracted some features of microscopic biopsy images which include eccentricity, area ratio, compactness, average values of color components, energy entropy, correlation, and area of cells and nucleus . The classification accuracy obtained by bayesian, k - nearest neighbor, neural networks, and support vector machine was 82.3%, 70.60%, 94.1%, and 94.1%, respectively . Extracted the features of microscopic biopsy images including area, perimeter, convex area, solidity, major axis length, orientation filled area, eccentricity, ratio of cell and nucleus area, circularity, and mean intensity of cytoplasm . The knn and neural network classifier are used for classification accuracy 86% and 92%, respectively . In this paper, a framework for automated detection and classification of cancer from microscopic biopsy images using clinically significant and biologically interpretable features is proposed and examined . For segmentation of images the various hybrid features which are extracted from the segmented images include shape and morphological features, glcm texture features, tamura features, law's texture energy based features, histogram of oriented gradients, wavelet features, and color features . For classification purposes, k - nearest neighbor based method the efficacy of other classifiers such as svm, random forest, and fuzzy k - means is also examined . For testing purposes, 2828 microscopic biopsy images available from histology database it was observed that the proposed method is performing better in comparison to other methods discussed as above . The overall summary and comparison of the proposed method and other methods the detection and classification of cancer from microscopic biopsy images are a challenging task because an image usually contains many clusters and overlapping objects . The various stages involved in the proposed methodology include enhancement of microscopic images, segmentation of background cells, features extraction, and finally the classification . For the enhancement of the microscopic biopsy images, the contrast limited adaptive histogram equalization [19, 20] approach is used and for the segmentation of background cells k - means segmentation algorithm is used . In feature extraction phase, various biologically interpretable and clinically significant shape and morphology based features are extracted from the segmented images which include gray level texture features, color based features, color gray level texture features, law's texture energy (lte) based features, tamura's features, and wavelet features . Finally, the k - nearest neighborhood (knn), fuzzy knn, and support vector machine (svm) based classifiers are examined for classifying the normal and cancerous biopsy images . These approaches are tested on four fundamental tissues (connective, epithelial, muscular, and nervous) of randomly selected 1000 microscopic biopsy images . Finally, the performances of the classifiers are evaluated using well known parameters and from results and analysis, it is observed that the fuzzy knn based classifier is performing better for the selected features set . The main purpose of the preprocessing is to remove a specific degradation such as noise reduction and contrast enhancement of region of interests . The biopsy images acquired from microscope may be defective and deficient in some respect such as poor contrast and uneven staining, and they need to be improved through process of image enhancement which increases the contrast between the foreground (objects of interest) and background . The contrast limited adaptive histogram equalization (clahe) approach is used for enhancement of microscopic biopsy images . Several segmentation methods have been adapted for cytoplasm, cell, and nuclei segmentation from microscopic biopsy images like threshold based, region - based, and clustering based algorithms . However the selections of segmentation methods depend on the type of the features to be preserved and extracted . For the segmentation of roi (region of interest), the ground truth (gt) of the images is manually cropped and created from histology dataset . The k - means clustering based segmentation algorithms are used because of the preservation of the desired information . From the obtained results through experimentation it is observed that the clustering based algorithms specifically k - means based method are the best suited for microscopic biopsy images . Figure 3 shows the original and k - means segmented microscopic biopsy image . For testing and experimentation purpose, twenty (20) microscopic biopsy images available from histology the visual results of texture based segmentation, fcm segmentation, k - means segmentation, and color based segmentation [20, 2326] are presented in figures 3(a)to 3(d). Thus from the visual results obtained and reported in figures 3(a)to 3(d), it is observed that the k - means clustering based segmentation method performs better in most of the cases as compared to other segmentation approaches under consideration for microscopic biopsy image segmentation . Finally the roi segmented image of microscopic biopsy is compared to ground truth images for the quantitative evaluation of various segmentation approaches for all 20 sample images from histology dataset . The performance of the various segmentation approaches such as k - means, fuzzy c - means, texture based segmentation, and color based segmentation was evaluated in terms of various popular parameters of segmentation measures . These parameters include accuracy, sensitivity, specificity, false positive rate (fpr), probability random index (ri), global consistency error (gce), and variance of information (voi). The brief description of few of these performance measures used in this paper is as follows . Random index between test (s) and ground truth (g) is estimated by summing the number of pixel pairs with same label and number of pixel pairs having different labels in both s and g and then dividing it by total number of pixel pairs . Given a set of ground truth segmentations gk, the pri is estimated using (1) such that cij is an event that describes a pixel pair (i, j) having same or different label in the test image stest(1)pristest, gk=1n/2i, j&i <jcijpij+1cij1pij . The variation of information is a measure of the distance between two clusters (partitions of elements). Clustering with clusters is represented by a random variable x, x = {1,, k} such that pi = \|xi \| /n, i x, and n = ixi is the variation of information between two clusters x and y. thus voi(x, y) is represented using (2)voix, y = hx = hy2ix, y, where h(x) is entropy of x and i(x, y) is mutual information between x and y. voi(x, y) measures how much the cluster assignment for an item in clustering x reduces the uncertainty about the item's cluster in clustering y. (iii) global consistency error (gce). The gce is estimated as follows: suppose segments si and gj contain a pixel, say pk, such that s s, g g where s denotes the set of segments that are generated by the segmentation algorithm being evaluated and g denotes the set of reference segments . To begin with, a measure of local refinement error is estimated using (3) and then it is used to compute local and global consistency errors, where n denotes the set of difference operation and r(x, y) represents the set of pixels corresponding to region x that includes pixel y. using (3) the global consistency error (gce) is computed using (4) where n denotes the total number of pixels of the image . Gce quantify the amount of error in segmentation (0 signifies no error and 1 indicates no agreement):(3)esi, gj, pk = rsi, pkrgj, pkrsi, pk,(4)gces, g=1nminies, g, pi,ies, g, pi . Table 2 and figure 4 show the comparison of various segmentation algorithms on the basis of average accuracy, sensitivity, specificity, fpr, pri, gce, and voi for 20 sample images taken from histology dataset . From table 2 and figure 4, it is observed that k - means, color k - means, fuzzy c - means, and texture based methods are performing better at par in terms of accuracy, specificity, and pri segmentation measures but except for k - means based segmentation methods other methods are not performing better in terms of other important parameters . Only the k - means based segmentation algorithm is associated with larger value of accuracy, sensitivity, specificity, and random index (ri) and smaller value of fpr, gce, and voi in comparison to other methods and hence it is better in comparison to others . Hence, k - means based segmentation is the only method which performs better in terms of all parameters and that is why it is chosen as the segmentation method in the proposed framework for cancer detection from microscopic biopsy images . After segmentation of image features are extracted from the regions of interest to detect and grade potential cancers . The features are extracted at tissue level and cell level of microscopic biopsy images for better predictions . To better capture the shape information, we use both region - based and contour - based methods to extract anticircularity, area irregularity, and contour irregularity of nuclei as the three shape features to reflect the irregularity of nuclei in biopsy images . The cellular level feature focuses on quantifying the properties of individual cells without considering spatial dependency between them . In biopsy images for a single cell, the shape and morphological, textural, histogram of oriented gradients and wavelet features are extracted . The tissue level features quantify the distribution of the cells across the tissue; for that, it primarily makes use of either the spatial dependency of the cells or the gray level dependency of the pixels . Based on these characteristics, some important shape and morphological based features are explained as follows . The nucleus area can be represented by nucleus region containing total number of pixels; it is shown in (5)a=i=1n j=1mbi, j, where a is nucleus area and b is segmented image of i rows and j columns . The average value of intensity of the pixels belonging to the nucleus region is known as nucleus brightness . The largest circle's diameter circumscribing the nucleus region is known as nucleus longest diameter; it is shown in (6)nld = x1x22+y1y22,where x1, y1 and x2, y2 are end points on major axis . It is represented in (7)nsd = x2x12+y2y12,where x1, y1 and x2, y2 are end points on minor axis . This is represented by the ratio of the shortest diameter to the longest diameter of the nucleus region, shown in (8)nucleus elongation = nldperimeter . The length of the perimeter of the nucleus region is represented using (9)p = even count+2odd count unit . The ratio of the nucleus area to the area of the circle corresponding to the nucleus longest diameter is known as nucleus compactness, shown in (10)=ap=4areap2 . Solidity is ratio of actual cell / nucleus area to convex hull area shown in (11)solidity = areaconvex area . The ratio of major axis length and minor axis length is known as eccentricity and defined in (12)eccentricity = length of mejor axislength of minor axis . There are seven sets of features used for computing the feature vector of microscopic biopsy images explained as follows . [3234] autocorrelation, contrast, correlation, cluster prominence, cluster shade, difference variance, dissimilarity, energy, entropy, homogeneity, maximum probability, sum of squares, sum average, sum variance, sum entropy, difference entropy, information measure of correlation 1, information measure of correlation 2, inverse difference (inv), inverse difference normalized (inn), and inverse difference moment normalized are major texture features which can be calculated using equations of the texture features . (ii) morphology and shape feature (f23f32). In papers [35, the considered shape and morphological features in this paper are area, perimeter, major axis length, minor axis length, equivalent diameter, orientation, convex area, filled area, solidity, and eccentricity . Histogram of oriented gradient is one of the good features set to deify the objects . In our observation it will be included for better and accurate identification of objects present in microscopic biopsy images . The wavelets are useful in multiresolution analysis of microscopic biopsy images because they are fast and give better compression as compared to other transforms . The fourier transform converts a signal into a continuous series of sine waves, but the wavelet precedes it in both time and frequency . Daubechies wavelets have been used because they have fractal structures and they are useful in the case of microscopic biopsy images . In this paper mean, entropy, energy, contrast homogeneity, and sum of wavelet coefficients are taken into consideration . The components of these models are hue, saturation, and value (hsv). This is represented by the six sided pyramids, the vertical axis behaves as brightness, the horizontal distance from the axis represents the saturation, and the angle represents the hue . Here mean and standard deviation of hsv components are taken as features . Tamura's features are computed on the basis of three fundamental texture features: contrast, coarseness, and directionality . Coarseness is the measure of granularity of an image; thus coarseness can be represented using average size of regions that have the same intensity . These features are texture description features which mainly used average gray level, edges, spots, ripples, and wave to generate vectors of the masks . Law's mask is represented by the features of an image without using frequency domain . Laws significantly determined that several masks of appropriate sizes were very instructive for discriminating between different kinds of texture features present in the microscopic biopsy images . Thus its classified samples are based on expected values of variance - like square measures of these convolutions, called texture energy measures . The lte mask method is based on texture energy transforms applied to the image classification used to estimate the energy within the pass region of filters . Table 3 provides the distribution of name of the feature type and the number of features selected for the classification of microscopic biopsy images . The classification of microscopic biopsy images is the most challenging task for automatic detection of cancer from microscopic biopsy images . Classification might provide the answer whether microscopic biopsy is benign or malignant . For classification purposes, some commonly used classification methods are artificial neural networks (ann), bayesian classification, k - nearest neighbor classifiers, support vector machine (svm), and random forest (rf). Supervised machine learning approaches first step is to prepare the data (feature set), the second step is to choose an appropriate algorithm, the third step is to fit a model, the fourth step is to train the fitted model, and then the final step is to use fitted model for prediction . The k - nearest neighborhood (knn), fuzzy knn and support vector machine (svm), and random forest classifiers are used for classifying the normal and cancerous biopsy images . The proposed methodologies were implemented with matlab 2013b, on dataset of digitized at 5x magnification on pc with 3.4 ghz intel core i7 processor, 2 gb ram, and windows 7 platform . For the testing and experimentation purposes, a total of 2828 histology images from the histology image dataset (histologyds2828) and annotations the image distributions based on the fundamental tissue structures in the histology dataset include connective-484, epithelial-804, muscular-514, and nervous-1026 microscopic biopsy images with magnifications 2.5x, 5x, 10x, 20x, and 40x . Although the method is magnification independent, in this work the results are provided on samples digitized at 5x magnification . The features extracted from microscopic biopsy images must be biologically interpretable and clinically significant for better diagnosis of cancer . Table 4 provides the brief description of dataset used for identification of cancer from microscopic biopsy images . The proposed methodology for detection and diagnosis of cancer detection from microscopic biopsy images consists of the stages of images enhancement, segmentation, feature extraction, and classification . The contrast limited adaptive histogram equalization (clahe) is used for enhancement of microscopic biopsy images, because it has ability to better highlight the regions of interests in the images as tested through experimentation . To better preserve the desired information in microscopic biopsy images during segmentation process, the various clustering and texture based segmentation approaches were examined . For microscopic biopsy images it is required to discover as much as possible the nuclei information in order to make reliable and accurate detection and diagnosis based on cells and nuclei parameters . From results and analysis presented in section 4, k - means segmentation algorithm was used for segmenting the microscopic biopsy images as it performs better in comparison to other methods . During segmentation process of k - means clustering method, the number of clusters k was set to k = 3 . Further, to find the center of the clusters, squared euclidean distance measures are used as similarity measures . In feature extraction phase, various biologically interpretable and clinically significant shape and morphology based features were extracted from the segmented images which include gray level texture features (f1f22), shape and morphology based features (f23f32), histogram of oriented gradients (f33f68), wavelet features (f69f100), color based features (f101f106), tamura's features (f107f119), and law's texture energy (f110f115) based features . Finally a 2d matrix of 2828 115 feature matrix was formed using all the feature sets, where 2828 are the number of microscopic images in the dataset and 115 are the total number of features extracted . Randomly selected 1000 data / samples were used for testing various classification algorithms . The 10-fold cross validation approach was used to partition the data in training and testing sets . Thus 900 data / samples were used for training purposes and 100 data / samples were used for testing purposes . The k - nearest neighbor (knn) is a simple classifier in which a feature vector is assigned . For knn classification the numbers of nearest neighbor (k) were set to 5, and euclidean distance matrix and the nearest rule to decide how to classify the sample were used . The proposed method was also tested by using support vector machine (svm) based classifier for linear kernel function with 10-fold cross validation methods . In svm classification model, the kernel's parameters and soft margin parameter c play vital role in classification process; the best combination of c and was selected by a grid search with exponentially growing sequences of c and . Each combination of parameter choices was checked using cross validations (10-fold), and the parameters with best cross validation accuracy were selected . For svm's linear kernel function, quadratic programming (qp) optimization parameter was used to find separating hyperplane . In the case of random the performance of classifiers was calculated using confusion matrix of size 2 2 matrix and the value of tp, tn, fp, and fn was calculated . The performance parameters accuracy, sensitivity, and specificity were calculated using (14)(19). The classification accuracy of a technique depends upon the number of correctly classified samples (i.e., true negative and true positive) and is calculated as follows:(14)accuracy = tp+tnn100,where n is the total number of samples present in the microscopic biopsy images it can be calculated using (15)sensitivity = tptp+fn, where the value of sensitivity ranges between 0 and 1, where 0 and 1, respectively, mean worst and best classification . Specificity . The value of sensitivity is calculated using (16)specificity = tntn+fp.its value ranges between 0 and 1, where 0 and 1, respectively, mean worst and best classification the geometric mean of sensitivity and specificity is considered as balance classification rate [43, 44]. It is defined by using (18)precision = tptp+fp, recall = tptp+fn, f - measure=2precisionrecallprecision+recall.the value of f - measure ranges between 0 and 1, where 0 means the worst classification and 1 means the best classification . It can be calculated using the following formula: (19)mcc = tptnfpfntp+fntp+fptn+fntn+fp.its value ranges between 1 and + 1, where 1, + 1, and 0, respectively, correspond to worst, best, at random prediction . Table 5 shows classification results of the proposed framework for four different tissues of microscopic biopsy images containing cancer and noncancer cases tested using four popular classifiers like k - nearest neighbor, svm, fuzzy knn, and random forest . From table 5 and figure 5(a) the following observations are made for sample test cases containing connective tissues. (i)for the identification of cancer from biopsy images of connective tissues in the case of knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.921909, 0.940164, 0.819922, 0.880263, 0.759395, and 0.717455, respectively. (ii)for the identification of cancer from biopsy of connective tissues in the case of svm, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.89245, 0.888438, 0.948297, 0.918756, 0.538314, and 0.55879, respectively. (iii)for the identification of cancer from biopsy of connective tissues in the case of fuzzy knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.787879, 0.867476, 0.370074, 0.618789, 0.356613, and 0.231013, respectively. (iv)for the identification of cancer from biopsy of connective tissues, in the case of random forest classifier, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.907245, 0.993668, 0.493996, 0.743832, 0.647373, and 0.642137, respectively . For the identification of cancer from biopsy images of connective tissues in the case of knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.921909, 0.940164, 0.819922, 0.880263, 0.759395, and 0.717455, respectively . For the identification of cancer from biopsy of connective tissues in the case of svm, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.89245, 0.888438, 0.948297, 0.918756, 0.538314, and 0.55879, respectively . For the identification of cancer from biopsy of connective tissues in the case of fuzzy knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.787879, 0.867476, 0.370074, 0.618789, 0.356613, and 0.231013, respectively . For the identification of cancer from biopsy of connective tissues, in the case of random forest classifier, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.907245, 0.993668, 0.493996, 0.743832, 0.647373, and 0.642137, respectively . From table 5 and figure 5(b) the following observations are made for sample test cases containing epithelial tissues. (i)for the identification of cancer from biopsy images of epithelial tissues in the case of knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.884727, 0.916446, 0.801733, 0.859435, 0.795319, and 0.71626, respectively. (ii)for the identification of cancer from biopsy of epithelial tissues in the case of svm, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.796998, 0.7851, 0.898525, 0.842279, 0.472804, and 0.4587, respectively. (iii)for the identification of cancer from biopsy of epithelial tissues in the case of fuzzy knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.665834, 0.76465, 0.407057, 0.585984, 0.401181, and 0.17053, respectively. (iv)for the identification of cancer from biopsy of epithelial tissues, in the case of random forest classifier, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.849306, 0.966243, 0.555332, 0.760788, 0.675868, and 0.609494, respectively . For the identification of cancer from biopsy images of epithelial tissues in the case of knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.884727, 0.916446, 0.801733, 0.859435, 0.795319, and 0.71626, respectively . For the identification of cancer from biopsy of epithelial tissues in the case of svm, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.796998, 0.7851, 0.898525, 0.842279, 0.472804, and 0.4587, respectively . For the identification of cancer from biopsy of epithelial tissues in the case of fuzzy knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.665834, 0.76465, 0.407057, 0.585984, 0.401181, and 0.17053, respectively . For the identification of cancer from biopsy of epithelial tissues, in the case of random forest classifier, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.849306, 0.966243, 0.555332, 0.760788, 0.675868, and 0.609494, respectively . From table 5 and figure 5(c) the following observations are made for sample test cases containing muscular tissues. (i)for the identification of cancer from biopsy images of muscular tissues in the case of knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.897321, 0.923277, 0.650761, 0.787092, 0.543009, and 0.49783, respectively. (ii)for the identification of cancer from biopsy of muscular tissues in the case of svm, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.884379, 0.886718, 0.786303, 0.83681, 0.263764, and 0.320547, respectively. (iii)for the identification of cancer from biopsy of muscular tissues in the case of fuzzy knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.614958, 0.672503, 0.535894, 0.604364, 0.538571, and 0.208941, respectively. (iv)for the identification of cancer from biopsy of muscular tissues, in the case of random forest classifier, the accuracy, specificity, sensitivity, bcr, f - measure, and mcc are 0.889878, 0.995023, 0.193145, 0.594084, 0.313309, and 0.37318, respectively . For the identification of cancer from biopsy images of muscular tissues in the case of knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.897321, 0.923277, 0.650761, 0.787092, 0.543009, and 0.49783, respectively . For the identification of cancer from biopsy of muscular tissues in the case of svm, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.884379, 0.886718, 0.786303, 0.83681, 0.263764, and 0.320547, respectively . For the identification of cancer from biopsy of muscular tissues in the case of fuzzy knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.614958, 0.672503, 0.535894, 0.604364, 0.538571, and 0.208941, respectively . For the identification of cancer from biopsy of muscular tissues, in the case of random forest classifier, the accuracy, specificity, sensitivity, bcr, f - measure, and mcc are 0.889878, 0.995023, 0.193145, 0.594084, 0.313309, and 0.37318, respectively . From table 5 and figure 5(d) the following observations are made for sample test cases containing nervous tissues. (i)for the identification of cancer from biopsy images of nervous tissues in the case of knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.861763, 0.880866, 0.835733, 0.858482, 0.834116, and 0.716492, respectively. (ii)for the identification of cancer from biopsy of nervous tissues in the case of svm, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.769545, 0.723056, 0.946068, 0.834923, 0.630126, and 0.552038, respectively. (iii)for the identification of cancer from biopsy of nervous tissues in the case of fuzzy knn, the accuracy, specificity, sensitivity, bcr, f - measure, and mcc are 0.808453, 0.882722, 0.242776, 0.562835, 0.225886, and 0.11837, respectively. (iv)for the identification of cancer from biopsy of nervous tissues, in the case of random forest classifier, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.843102, 0.92827, 0.723262, 0.825766, 0.792403, and 0.676888, respectively . For the identification of cancer from biopsy images of nervous tissues in the case of knn, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.861763, 0.880866, 0.835733, 0.858482, 0.834116, and 0.716492, respectively . For the identification of cancer from biopsy of nervous tissues in the case of svm, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.769545, 0.723056, 0.946068, 0.834923, 0.630126, and 0.552038, respectively . For the identification of cancer from biopsy of nervous tissues in the case of fuzzy knn, the accuracy, specificity, sensitivity, bcr, f - measure, and mcc are 0.808453, 0.882722, 0.242776, 0.562835, 0.225886, and 0.11837, respectively . For the identification of cancer from biopsy of nervous tissues, in the case of random forest classifier, the average value of accuracy, specificity, sensitivity, bcr, f - measure, and mcc is 0.843102, 0.92827, 0.723262, 0.825766, 0.792403, and 0.676888, respectively . From the above discussions for all four categories of test cases, it is observed that the knn is performing better in comparison to other classifiers for the identification of cancer from biopsy images of nervous tissues . From all above observations, it is concluded that the knn classifier is producing better results in comparison to other methods for the case of biopsy images of connective tissues . The maximum values of the accuracy, sensitivity, and specificity are 0.9552, 0.9615, and 0.9543, respectively . The k - nearest neighbor classifier is also performing better for all cases as well as that was discussed above . Table 6 gives a comparative analysis of the proposed framework with other standard methods available in the literature . From table 6, it can be observed that the proposed method is performing better in comparison to all other methods . An automated detection and classification procedure was presented for detection of cancer from microscopic biopsy images using clinically significant and biologically interpretable set of features . The proposed analysis was based on tissues level microscopic observations of cell and nuclei for cancer detection and classification . For enhancement of microscopic biopsy images contrast after segmentation of images, a total of 115 hybrid sets of features were extracted for 2828 sample histology images taken from histology database . Out of 1000 samples of 115 features, 900 samples were selected for training purposes and 100 samples were selected for testing purposes . The various classification approaches tested were k - nearest neighborhood (knn), fuzzy knn, support vector machine (svm), and random forest based classifiers . From table 5 we are in position to conclude that knn is the best suited classification algorithm for detection of noncancerous and cancerous microscopic biopsy images containing all four fundamental tissues . Rf classifier provides very low sensitivity and down accuracy rate as compared to knn classifier for this dataset . Hence, from experimental results, it was observed that knn classifier is performing better for all categories of test cases present in the selected test data . These categories of test data are connective tissues, epithelial tissues, muscular tissues, and nervous tissues . Among all categories of test cases, further it was observed that the proposed method is performing better for connective tissues type sample test cases . The performance measures for connective tissues dataset in terms of the average accuracy, specificity, sensitivity, bcr, f - measure, and mcc are 0.921909, 0.940164, 0.819922, 0.880263, 0.759395, and 0.717455, respectively.
Fundus fluorescein angiography (ffa) is a procedure for the examination of chorioretinal circulation . It can be used to confirm the diagnosis, monitor the progress of posterior segment diseases, and assess the efficacy of certain treatments like laser photocoagulation and intravitreal injections . Although ffa is a safe procedure, some serious complications like allergic skin rash and anaphylaxis could be seen rarely . The effect of fluorescent molecule or ffa procedure on choroidal circulation is not known much . The dynamic contour tonometry (dct) is a noninvasive and direct intraocular pressure (iop) measuring device and is considered to accurately measure the iop independent of the corneal thickness or corneal elasticity . It also provides a continuous examination of the iop and measures ocular pulse amplitude (opa) which is accepted as an indirect measurement of the choroidal blood flow . The opa is generally considered as the difference between systolic and diastolic values of the pulsatile iop . It gives us an opinion about the choroidal blood flow corresponding with the heart pulse as a function of time . Choroidal circulation has a vital role in ocular physiology and may be affected in various ocular and systemic diseases [5, 6]. Ffa is an imaging procedure using fluorescence ability, and it has been thought to have little or no effect on ocular physiology . The rationale of this study was that fluorescein sodium passes freely through the choroidal vessels into the extravasal space and this would have an influence on choroidal blood flows, either by an osmotic effect or a hemorheological effect . Also, influence of fluorescein sodium on the erythrocyte aggregation was shown in the past literature, which might be related with alterations in retina - choroidal microcirculation . Although it was not possible to examine the tissue pathology in this study, we wanted to reveal the effects of ffa procedure on eye in terms of iop and opa . This study was a prospective cross - sectional case series and involved 60 eyes of 30 patients . The study was conducted in accordance with the ethical standards of declaration of helsinki and was approved by the institutional ethical committee . The diagnosis of npdrp was based on indirect retinoscopy and fundus fluorescein angiography (ffa). All of the subjects underwent an ophthalmic examination including visual acuity assessment, biomicroscopy, air - puff tonometer, indirect retinoscopy, pachymeter, and macular optical coherence tomography before their participation in the study . Exclusion criteria were any ocular surgery other than phacoemulsification, corneal irregularities, history of glaucoma, uveitis, and any other retinal disorders except npdrp . Also, the patients who had nausea and vomiting after ffa were excluded, since these situations could affect the opa and iop . Ffa measurements were done with topcon trc 501x angiography (topcon corporation, tokyo, japan). 5 ml 10% fluorescein sodium was injected rapidly from median antecubital vein or dorsal hand veins . Between postinjection 5 and 10 minutes, iop and opa were measured . In order to separate the effect of rapid photography from fluorescein sodium injection, we had taken immediate postcolored retinal photography opa and iop measurements of 10 eyes before the dye injection . Intraocular pressure (iop) and ocular pulse amplitude (opa) measurements were done with the pascal dynamic contour tonometer (pascal dct, swiss microtechnology ag, port, switzerland). This is a slit - lamp biomicroscopy mounted, self - calibrating, 7 mm tip diameter, and 1.2 mm pressure sensor diameter device . One drop of alcaine (proparacaine chloride 5 mg / ml; alcon, fort worth, tx) was instilled just prior to iop and opa measurements . Three dct measurements were taken in order to have one good - quality measurement . Only quality 1 and 2 measurements all opa and iop measurements were performed by the same investigator (gp) before and after 5 minutes from fluorescein dye injection . Optical coherence tomography (oct) measurements were done with spectral - domain oct (spectralis, heidelberg, germany). Corneal pachymetry measurements were done with nidek up-1000 ultrasonic pachymeter and central corneal thickness was recorded . The pachymeter probe was placed on the center of the cornea and the mean of five readings was calculated . Statistical analysis was performed with spss for windows statistical software version 17.0 (spss inc . Paired t - test between pre - ffa and post - ffa opas and iops were performed to evaluate the effect of ffa on opa and iop . The wilcoxon signed - rank test was used to analyze pre- and postretinal photography opa and iop values of 10 eyes . Age and macular thickness were assessed for a relationship with pre - ffa and post - ffa opa and iop differences using univariate linear regression analysis . The effects of lens status and gender on pre - ffa and post - ffa opa and iop differences were analyzed with independent samples t - test . The study evaluated 30 nonproliferative diabetic retinopathy patients (15 males, 15 females). The distribution of eyes according to npdrp severity was as follows: mild npdrp in 10 eyes, moderate npdrp in 38 eyes, and severe npdrp in 12 eyes . Pre - ffa mean opa value was 3.05 1.36 (range: 1.407.60, median: 2.60) mmhg and post - ffa mean opa value was 2.93 1.28 (range: 1.107.00, median: 2.60) mmhg (p = 0.071). Pre - ffa mean iop value was 17.97 1.99 (range: 12.8023.40, median: 18.10) mmhg and post - ffa mean iop value was 17.81 2.22 (range: 12.2024.10, median: 17.80) mmhg (p = 0.407). Best corrected visual acuity values evaluated with snellen chart ranged between 0.1 and 0.9 in decimals (mean 0.54 0.28). In order to evaluate the individual effect of successive retinal photography, we recorded post - photography opa and iop values of only ten eyes . Pre - retinal photography mean opa value was 2.70 0.66 mmhg and postretinal photography mean opa value was 2.63 0.61 mmhg (p = 0.45). Pre - retinal photography mean iop value was 18.36 1.49 mmhg and postretinal photography mean iop value was 18.28 1.99 mmhg (p = 0.78). No adverse effects like allergic skin rush or anaphylaxis occurred after intravenous fluorescein dye injection . Some demographic and clinical characteristics of the subjects studied, including mean age, gender, lens status, central corneal thickness, and central macular thickness, are shown in table 1 . When considered alone, age (p = 0.017; r = 0.094) showed statistically significant association with pre - ffa and post - ffa opa difference, while macular thickness (p = 0.119; r = 0.074) did not show significant association with opa change . Age (p = 0.001; r = 0.166) showed statistically significant association with pre - ffa and post - ffa iop difference, while macular thickness (p = 0.421; r = 0.020) did not show significant association with iop difference . In males, mean opa value increased 0.093 mmhg; whereas in females, mean opa value decreased 0.330 mmhg after ffa (p = 0.001). In males, mean iop value decreased by 0.027 mmhg and in females mean iop value decreased by 0.317 mmhg (p = 0.468). In phakic eyes, mean opa value decreased by 0.058 mmhg and in pseudophakic eyes, mean opa value decreased by 0.300 mmhg (p = 0.101). In phakic eyes, mean iop value decreased by 0.10 mmhg and in pseudophakic eyes, mean iop value decreased by 0.38 mmhg (p = 0.548). Since fluorescein molecule is relatively small, in a normal eye, it remains within retinal vessels but freely passes the walls of choroidal vessels . In a diseased retina like diabetic retinopathy there is lack of comprehensive data about the effects of fluorescein sodium and ffa procedure on ocular tissues . In this study, we examined iop and opa only in diabetic retinopathy, although there are many other ocular tissues, functions, and disorders that may be affected by ffa procedure . Possible factors related to ffa procedure that may affect ocular tissues and functions are not clear . But, the amount of injected fluid, the fluorescein leakage both in diseased retinal vessels and choroid, anxiety of patients related to the examination, the effect of sequential photograph taking, and/or the fluorescence ability of injected molecule might affect iop and opa . But more probable factors are the osmotic effects and hemorheological effects of fluorescein sodium . We did not have the opportunity to investigate individual contribution of these factors, but we take into consideration ffa procedure in general . Sargento et al . In their study about sodium fluorescein influence on the hematological properties of diabetic patients, revealed that sodium fluorescein injection during retinography increased whole blood viscosity, erythrocyte elongation index, blood ph, carboxyhemoglobin, and methemoglobin levels and caused a sudden structural or functional reduction of red cell acetylcholinesterase activity . All of these factors might have an effect on choroidal blood flow and hence ocular pulse amplitude . Another possible mechanism about sodium fluorescein effect on opa might be the osmotic effect, since fluorescite 10% has an osmolality of 572858 mosm / kg and plasma has an osmolality of 285295 mosm / kg . It is known that small changes in osmolality can have a marked effect on water transfer . However, it was reported that 30 minutes after sodium fluorescein injection, the increase in plasma osmolality was statistically insignificant . In this study, although both mean opa and iop values were decreased after ffa procedure, the difference was not statistically significant mean opa value of males increased, whereas mean opa value of females decreased after ffa . Possible explanation about this result might be the effects of gender on ffa procedure and opa, since gender differences in health issues could be seen . In the literature, some of the factors that decrease opa are carotid artery stenosis, accommodation, glaucoma, scleral buckling surgery, regional orbital anesthesia, graves' ophthalmopathy, panretinal photocoagulation, physical exercise, diurnal variation (lowest opa at 1:003:00 pm), advanced retinitis pigmentosa, and exudative age - related macular degeneration [1121]. Some factors that does not alter opa are smoking, intravitreal bevacizumab injection, trabeculectomy, valsalva maneuver, and systemic blood pressure [3, 2225]. Some factors that increase opa are water drinking and high iop [26, 27]. Since opa is accepted as an indirect measurement of choroidal blood flow and fluorescein dye freely passes from choroidal vessels into the extravasal space, we thought that ffa might have an influence on opa . They found that premenopausal females (age: 1742 years) had significantly higher rates of opa when compared with age - matched males, whereas postmenoposal females (age:> 55 years) had similar opa values with age - matched males ., the difference between males and females was not based on mean opa value; it was due to the different influence of ffa procedure on opa between genders . Although we could not explain the exact mechanism of this result, it is known that gender influences occurrence and outcome of some disorders . Also, we measured that pre - ffa and post - ffa mean iop difference was higher in females than in males . Since iop affects opa, this might also explain different opa measurements between males and females . Examined opa in diabetes mellitus and reported that choroidal circulation remained unaffected as diabetic retinopathy advanced . Their study group was composed of diabetic patients in three subgroups: no diabetic retinopathy, npdrp, and proliferative drp . In our study, only npdrp patients were included into the study in order to maintain better homogeneity . We also examined the effects of fluorescein leakage amount in terms of macular thickness and found that it had no significant effect on opa and iop . For example, ideal timing of opa and iop measurements after ffa is difficult to determine . Since taking photographs is very important in the first five minutes, it was not appropriate to disturb the examination . We did not perform ffa on normal subjects, but in order to maintain standardization of the patients, only npdrp patients were included in the study . One may say if there were an issue, 50 years and millions of fluorescein angiograms later, we would have learned of it . But, according to us, it is important to document these data about ffa for the first time in the literature . In conclusion but, age showed significant association with pre - ffa and post - ffa opa and iop differences . Interestingly, mean opa value of males increased, whereas mean opa value of females decreased after ffa . More comprehensive studies including various ocular and systemic diseases and measurements at different ffa stages are needed to reveal more effects of ffa on opa.
<45 years old, mechanical lbp is the most common cause of disability, and it is generally attributed to an acute traumatic event . However, cumulative trauma must also be considered in the etiology, such as occupational overuse injury.2,3 the best approach to treat lbp appears to be a combination of pharmacological and nonpharmacological strategies.4 there is a great variability, possibly genetically related, in the individual response to painkillers.5 nonsteroidal anti - inflammatory drugs (nsaids) are the most common type of medication used in the treatment of acute pain, exerting their effect by interfering in the inflammatory response.6 they inhibit the cyclooxygenase (cox) enzyme, reducing the synthesis of prostaglandins . The traditional nsaids inhibit both cox-1 and cox-2, and inhibiting cox-1 decreases platelet aggregation, irritates the gastric mucosa, and alters renal flow.7 ketorolac trometamol (kt; toragesic, ems sigma pharma ltd ., campinas, brazil) is an nsaid, which is a racemic mixture of the s- and r - enantiomeric forms . Kt inhibits cox that results in reduced synthesis of prostaglandins, thromboxanes, and prostacyclin as well as diminished platelet aggregation.8 compared with aspirin, which produces prolonged and irreversible antiplatelet effects that persist beyond drug administration, the anti - platelet activity of kt is not apparent after elimination from the plasma and is reversible . In addition, the platelet effects of kt are not related to dose.8 the s - enantiomer of kt is a rapid - acting and potent analgesic that possesses no anesthetic, sedative, or antianxiety effects and has no effect on gut motility.8 the effectiveness, safety, and analgesic efficacy and potency of kt are considered higher than those of ketoprofen in postoperative ear nose throat9 and dental surgeries.10 the aim of the current clinical trial (protocol number nct01471886) was to test the hypothesis that kt is not inferior to naproxen (na) in its analgesic efficacy and incidence of adverse effects for the treatment of moderate - to - severe acute lbp . This 10-day, double - dummy, randomized, prospective, noninferiority clinical trial was conducted at two research centers in so paulo, brazil, in accordance with the principles of the declaration of helsinki and under protocol number 0752/11, which was approved by the ethics committee of the university hospital at so paulo university . Because the package insert indicates that the upper limit of use for kt is 5 days, the treatment duration was not longer than 5 days, and the study finished with safety reassessment 10 days after treatment initiation . Outpatients diagnosed with moderate or severe acute lbp as determined by a visual analog scale (vas) score> 40 mm were screened . The exclusion criteria included the following: weight <50 kg; severe congestive heart failure; current alcoholism or illegal drug use; presence of fever or signs of infection; kidney disease; fracture; fibromyalgia; cancer; neuropsychiatric disease; rheumatologic disease; history of peptic ulcer disease, gastrointestinal bleeding, or hemorrhagic diathesis; cerebrovascular disease; hemostatic disorders or use of anticoagulants; pregnancy; lactation; postoperative patients at high risk of bleeding; history of hypersensitivity to any of the ingredients in the formula or other nsaids; nasal polyps; and asthma . Participants could not have participated in another experimental study in the 6 months prior to study entry . None of the 83 prospective participants were excluded; thus, these 83 volunteers were randomized . This noninferiority study compared the analgesic efficacy and incidence of adverse effects in the treatment of moderate - to - severe acute lbp for kt at a dosage of 10 mg given sublingually three times daily (tid) and na at a dosage of 250 mg administered orally tid . Eligible participants were randomly assigned to one of these two treatment groups by a computer - generated lottery . This scale was used to evaluate the investigators global assessment of efficacy on reducing the participant s pain through the following ratings: excellent, vas score reduction 50 mm or greater; very good, vas score reduction between 40 mm and 50 mm; good, vas reduction between 30 mm and 40 mm; regular, vas between 20 mm and 30 mm; bad, vas score 10 mm or less . During the initial visit (v0) on the first treatment day, the participant received two pills of either the tested or reference medication, followed by one tablet every 8 hours, for a total of 40 mg and 1,000 mg, respectively . Thereafter, a daily dosage of 10 mg tid was administered sublingually for the test medication and 250 mg tid for the reference medication . From the second to the fifth day of treatment, if the patient had vas> 40 mm, increased dosage to four times per day was allowed . Participants were reassessed after the initiation of treatment at 2 days (v1) and 4 days (v2), when the treatment was discontinued . Ten days (v3) after the initiation of the study, participants returned for a safety assessment and adverse events were recorded . The analgesic effect was evaluated on v0 before and 60 minutes after taking the drug (v060) as well as on v1 and v2 through a vas score categorized as follows: 0 mm, no pain; 0.140 mm, mild pain; 4170 mm, moderate pain; and 71100 mm, severe pain . The primary end point was the rate of pain relief (rpr) calculated by the following formula: rpr = vas(vi)vas(vf)vas(vf)(1)where vas(vi) is the vas on v0 (before taking the drug) and vas(vf) is the vas on v060 or v1 or v2 . The secondary end point was rpr1 and was calculated for each of the three drug administrations on each day as follows: rpr1=vas(vi)vas(vf)vas(vf)(2)where vas(vi) is the vas 1 hour before the administration of medication and vas(vf) is the vas 1 hour after the administration of medication . Adverse effects occurring up to 10 days after v0 (v3) were also included in the study . A sample size of at least 78 participants was deemed sufficient to detect differences in the rpr at a significance level of 5%, power of 80%, noninferiority margin of 10% (as per the recommendation of the us food and drug administration [fda]), and an average difference between 5% and 10%, considering a 20% dropout.11,12 a total of 63 (per protocol) of the 83 (intention - to - treat, itt) participants completed the study (there was no screening failure). The homogeneity of the demographic and clinical features between the groups was compared by tests, fisher s exact tests (for sex, and clinical changes), levene s test for variance equality, and t - test for two independent samples to compare means between two groups for the averages equality (age, weight, rolland - morris, and vas). The evaluation of the effectiveness (vas) was performed by levene s test for variance equality and t - tests for mean equality in the protocol population . The primary end point was the rpr between v060 and v0, between v1 and v0, and between v2 and v0 . The secondary end point was the pain relief following each of the three drug administrations on each day (rpr1). The primary end point for this analysis was the investigators global assessments of efficacy as measured using a likert scale . We used a logistic model with a stepwise backward approach to select the dependent variables among treatment, sex, age, current smoking status, alcohol use, and clinical changes (inclusion in the model p<0.05, and exclusion p>0.1). The secondary end point in this analysis was the percentage of participants with improvement in pain relief as assessed by the vas . For this analysis, we considered pain improvement as vas scores from 0 to 3 after the administration of medication . We also used a logistic model with a stepwise backward approach to select the dependent variables among treatment, sex, age, current smoking status, alcohol use, and clinical changes (inclusion in the model p<0.05, and exclusion p>0.1).13 eighty - three outpatients diagnosed with moderate or severe acute lbp as determined by a visual analog scale (vas) score> 40 mm were screened . The exclusion criteria included the following: weight <50 kg; severe congestive heart failure; current alcoholism or illegal drug use; presence of fever or signs of infection; kidney disease; fracture; fibromyalgia; cancer; neuropsychiatric disease; rheumatologic disease; history of peptic ulcer disease, gastrointestinal bleeding, or hemorrhagic diathesis; cerebrovascular disease; hemostatic disorders or use of anticoagulants; pregnancy; lactation; postoperative patients at high risk of bleeding; history of hypersensitivity to any of the ingredients in the formula or other nsaids; nasal polyps; and asthma . Participants could not have participated in another experimental study in the 6 months prior to study entry . None of the 83 prospective participants were excluded; thus, these 83 volunteers were randomized . This noninferiority study compared the analgesic efficacy and incidence of adverse effects in the treatment of moderate - to - severe acute lbp for kt at a dosage of 10 mg given sublingually three times daily (tid) and na at a dosage of 250 mg administered orally tid . Eligible participants were randomly assigned to one of these two treatment groups by a computer - generated lottery . This scale was used to evaluate the investigators global assessment of efficacy on reducing the participant s pain through the following ratings: excellent, vas score reduction 50 mm or greater; very good, vas score reduction between 40 mm and 50 mm; good, vas reduction between 30 mm and 40 mm; regular, vas between 20 mm and 30 mm; bad, vas score 10 mm or less . During the initial visit (v0) on the first treatment day, the participant received two pills of either the tested or reference medication, followed by one tablet every 8 hours, for a total of 40 mg and 1,000 mg, respectively . Thereafter, a daily dosage of 10 mg tid was administered sublingually for the test medication and 250 mg tid for the reference medication . From the second to the fifth day of treatment, if the patient had vas> 40 mm, increased dosage to four times per day was allowed . Participants were reassessed after the initiation of treatment at 2 days (v1) and 4 days (v2), when the treatment was discontinued . Ten days (v3) after the initiation of the study, participants returned for a safety assessment and adverse events were recorded . The analgesic effect was evaluated on v0 before and 60 minutes after taking the drug (v060) as well as on v1 and v2 through a vas score categorized as follows: 0 mm, no pain; 0.140 mm, mild pain; 4170 mm, moderate pain; and 71100 mm, severe pain . The primary end point was the rate of pain relief (rpr) calculated by the following formula: rpr = vas(vi)vas(vf)vas(vf)(1)where vas(vi) is the vas on v0 (before taking the drug) and vas(vf) is the vas on v060 or v1 or v2 . The secondary end point was rpr1 and was calculated for each of the three drug administrations on each day as follows: rpr1=vas(vi)vas(vf)vas(vf)(2)where vas(vi) is the vas 1 hour before the administration of medication and vas(vf) is the vas 1 hour after the administration of medication . Adverse effects occurring up to 10 days after v0 (v3) were also included in the study . A sample size of at least 78 participants was deemed sufficient to detect differences in the rpr at a significance level of 5%, power of 80%, noninferiority margin of 10% (as per the recommendation of the us food and drug administration [fda]), and an average difference between 5% and 10%, considering a 20% dropout.11,12 a total of 63 (per protocol) of the 83 (intention - to - treat, itt) participants completed the study (there was no screening failure). The homogeneity of the demographic and clinical features between the groups was compared by tests, fisher s exact tests (for sex, and clinical changes), levene s test for variance equality, and t - test for two independent samples to compare means between two groups for the averages equality (age, weight, rolland - morris, and vas). The evaluation of the effectiveness (vas) was performed by levene s test for variance equality and t - tests for mean equality in the protocol population . The primary end point was the rpr between v060 and v0, between v1 and v0, and between v2 and v0 . The secondary end point was the pain relief following each of the three drug administrations on each day (rpr1). The primary end point for this analysis was the investigators global assessments of efficacy as measured using a likert scale . We used a logistic model with a stepwise backward approach to select the dependent variables among treatment, sex, age, current smoking status, alcohol use, and clinical changes (inclusion in the model p<0.05, and exclusion p>0.1). The secondary end point in this analysis was the percentage of participants with improvement in pain relief as assessed by the vas . For this analysis, we considered pain improvement as vas scores from 0 to 3 after the administration of medication . We also used a logistic model with a stepwise backward approach to select the dependent variables among treatment, sex, age, current smoking status, alcohol use, and clinical changes (inclusion in the model p<0.05, and exclusion p>0.1).13 one participant was withdrawn due to adverse effects (acute cholecystitis, improbably associated with the test drug), four due to the use of banned medicine, and five due to a breach of protocol, while four chose to withdraw from the study, and six were withdrawn because of lack of follow - up (figure 1). Both sample groups were clinically homogeneous, and there was no significant difference between them (table 1). Comparison of the rpr between v0 and v060 showed a 5.6% gain in the rpr for kt compared with na, which was below the 10% limit (difference = 0.056; 95% confidence interval [ci], 0.166, 0.055) and within the noninferiority margin . Comparing the rpr for v1 and v0, and for v2 and v0, the secondary end point showed that kt was not inferior to na on day 1 for the first and third administrations of the drug, on day 2 for the second and third administrations, on days 3 and 4 for the first three administrations, and on day 5 for the first and third administrations (table 3). Because we observed a pattern of superiority in the rpr for kt compared with that for na (figure 2), we performed a post hoc analysis using likert scale scores of the investigators global assessment of efficacy on reducing the participants pain as the end point . The final logistic model included age as the only important independent variable after a stepwise approach . On v1, participants in the kt group had 193.1% higher odds of pain reduction compared with those in the na group, controlling for age (95% ci, 1.107.80). However, this was not observed on v2, at which no statistically significant difference in pain improvement was detected between the two treatment groups (table 4). However, the percentage of participants who reported an improvement in pain relief at v060 was higher in the kt group (24.2%) than in the na group (6.5%; p=0.049). The percentage of participants rated as excellent, very good, or good by the investigator for the kt treatment group (66.7%) was much higher than that for the na group (40.0%), with 95% cis for the differences of 0.493 and 0.041 . These results indicated that, compared with the na treatment, the kt treatment had a margin of superiority equal to 4.1% . There were 35 adverse events in the study population treated with na and 42 in the population treated with kt . The main adverse effects were diarrhea, stomach pain, drowsiness, nausea, and vomiting . The frequency of occurrence of each event between the two treatments was not statistically significant (table 5). The safety evaluation for changes in the laboratory results performed in the protocol population on v2 and v3 and compared with that on v0 did not show any difference between the two treatment groups (table 6). This study showed that the efficacy of kt was not inferior to that of na in the treatment of acute lbp of moderate - to - severe intensity, with no significant differences in the occurrence of adverse effects between the two treatment groups . However, participants who received kt exhibited a higher percentage of response after the initial administration and had higher odds of responding according to the investigator s assessment at the first visit, suggesting a faster pain relief in the kt group . The rate of pain relief should always be considered when choosing an analgesic in order to improve the quality of life for patients . There is evidence that kt is more effective than other nsaids in pain reduction from both inflammatory and non - inflammatory etiologies.9,10 its mechanism of action is to reduce prostaglandin production by blocking cox 1 and 2 . It has no sedative or anxiolytic properties.14 the fda approved ketorolac in november 1989 . In studies of postoperative pain, kt showed an opioid dose - sparing effect and consequently a decrease in the adverse events related to opioids.15,16 in addition, for intravenous administration, kt is more cost - effective than morphine in blunt limb injury.17 only two studies have evaluated kt for the treatment of acute lbp, and both studies used opioids as comparators.18,19 in both studies, kt had comparable efficacy and fewer side effects . The current study is the first to compare kt to another nsaid in the management of acute lbp . The treatment of acute lbp is an unmet need in which the variability of medical options, mostly uncontrolled, includes the use of nsaids, opioids, corticosteroids, and invasive procedures, such as epidural blockade . The results of our study comparing kt with the gold standard nsaid na indicated that kt is a valid option for the treatment of lbp . Kt is not inferior to na in efficacy, provides faster pain relief, and is a safe acute treatment option for acute pain relief.
Organophosphorus pesticides (ops) are a group of insecticides derived from the phosphoric or phosphorothioic acid; its use has increased in the recent years for the improvement of agriculture production, in the industry and prevention of human health through control and/or eradication of unwanted insects, plants, animals, and disease vectors . According to information published by the national pesticide information center (npic), the most used ops are chlorpyrifos, malathion, acephate, naled, dicrotophos, phosmet, phorate, diazinon, dimethoate, and azinphos - methyl . At a worldwide level, even though ops have limited persistence in the environment, they are highly toxic for humans and are responsible for most of accidental intoxications . The world health organization (who) estimated that, every year, almost 3 million people suffer acute intoxication due to ops; hence its use is considered a worldwide public health problem . The first one is acute toxicity, initiated by the inhibition of the acetylcholinesterase enzyme (ache) with the subsequent accumulation of acetylcholine (ach) in the nervous termination, provoking an overstimulation of muscarinic acetylcholine (machr) and nicotinic acetylcholine (nachr) receptors . The inhibition mechanism of ache is conducted through phosphorylation of the hydroxyl group in the serine of the active site of the enzyme; once phosphorylated it is extremely stable, which avoids its physiological action on the ach that consists in the degradation of this neurotransmitter to allow reuptake of acetate and choline in the nervous terminal [3, 6, 7]. The second effect is chronic and is denominated organophosphate - induced delayed polyneuropathy (opidp), which is characterized by ataxia and paralysis, signs that appear 2 - 3 weeks after exposure to ops [1, 8]. After its application on agricultural crops, residual ops enter water bodies as result of spray drift, soil leaching, and running off soils dedicated to agriculture, provoking adverse effects on the target species but also on a wide range of nontarget organisms, especially those that inhabit aquatic ecosystems such as invertebrates, birds, and fishes [9, 10]. Among the nontarget species exposed to ops, it is important to mention fishes, since these organisms are transcendental due to their status as top consumer species in the food chain, besides of playing an important role in the maintenance of the balance of aquatic ecosystems . From an evolutionary point of view, fishes are important organisms because they appeared over 560 million years ago; they are a group of vertebrates phylogenetically antique; there are over 25,000 species; therefore their great diversity stands out in comparison to other vertebrates [11, 12]. Among other species, some have stood out for their ecological or economic importance, while others have been used as study models in diverse areas of scientific research . Fishes are the first group of organisms that present an innate and adaptive immunity system; therefore the study of these organisms is of great relevance due to the information it gives about evolution of the immune system in vertebrates . The innate immune system is of paramount importance in fishes [1517]; among the components of humoral innate immunity that are mainly characterized in fishes are antibacterial peptides, lysozymes, lectins, acute - phase proteins, and molecules of the complement system, while innate immunity cells mostly characterized are macrophages, neutrophils, and eosinophils [1820]. On the other hand, adaptive immunity mechanisms in fishes play a vital role in the protection against recurrent infections, response that is mediated by t- and b - lymphocytes and antibodies . Fishes are the first vertebrates where clonal selection and genetic rearrangement in receptors of lymphocytes are present . Likewise, leucocytes with t cell activity have been reported, similar to the cooperative and cytotoxic t cells of mammals (cd4-like, cd8-like). Apart from that, based on the profile of cytokines, there have been reports of t cells subpopulations similar to the ones reported in mammals . In contrast, b cells in fishes have been characterized through the expression of antigen receptor (bcr). In fishes, igm is the main soluble antibody, which is tetrameric; on the other hand, igd, just like in mammals, is expressed in the surface of b cells . In addition, other isotypes have been identified, such as lgt and lgz, which are mainly found in mucosa, such as in intestine, in skin, and in gills [2124]. In recent years, an immunotoxic effect of ops has been reported in diverse organisms, including fishes . Immune system is the first defense line against pathogenic organisms; however, it is a very sensitive system to be altered by stressing factors present in the environment (biotic and abiotic) [1, 10]; thus it is vulnerable to any xenobiotic such as ops, which can cause structural or functional alterations in humoral or cell mechanisms (nonspecific or adaptative) of the immune response (table 1), which entails, among others, an increase in the susceptibility to infections . In fishes, molecules that are responsible of the innate and adaptative humoral response can be altered by ops, like chlorpyrifos, diazinon, and phosalone, among others [10, 2530]. Thus, lysozyme is an important molecule defense of the innate immune system of fishes that is frequently altered by ops . A study showed that the lysozyme activity increased significantly in liver and spleen of beluga (huso huso) exposed acutely to diazinon (1.5 mg / l). However, at subacute and subchronic exposure of this pesticide, lysozyme activity decreased in plasma, liver, kidney, and spleen . On the other hand, it has been reported that the acute exposure to diazinon (2.0 and 4.0 mg / l) in grass carp (ctenopharyngodon idella) induced a significant increase in the lysozyme activity present in kidney and spleen of this fish . Recent studies have reported a decrease in the lysozyme activity in plasma of rainbow trout (oncorhynchus mykiss) and common carp (cyprinus carpio l.) exposed to diazinon (0.1 and 0.2 mg / l) and phosalone (0.15, 0.30, and 0.60 mg / l), respectively [28, 30]. Also, it has been reported that chlorpyrifos provoke a diminishment in the enzyme activity present in plasma and spleen of common carp (c. carpio) exposed acutely to 75 g / l of pesticide . Recently, it was reported that exposure of nile tilapia (oreochromis niloticus) to chlorpyrifos (0.102 and 0.255 mg / l) provoked an increase in the activity of this enzyme in the plasma of these organisms; however, at a lower concentration (0.051 mg / ml) the pesticide did not cause any effect on the activity of this enzyme . Another important molecule of the innate immune system of fishes is the protein c3 of the complement, which is also altered by the exposition to ops . A deregulation at concentration and mrna expression of this molecule has been reported in anterior kidney, spleen, and plasma of common carp (c. carpio l.) exposed acutely to chlorpyrifos . Reactive c protein (rcp) is another molecule of the innate immune system of fishes affected by exposure to this type of pesticides . In this context, it has been reported that acute exposure to metrifonate (0.4 ppm) in rainbow trout (o. mykiss) provoked a significant increase of this protein in the plasma of organisms exposed during 3 days to the pesticide; however, at 10 and 18 days after exposure, protein activity diminished significantly . Some studies have reported that, in plasma of rainbow trout (o. mykiss), concentration of these proteins diminishes significantly when organisms are exposed acutely and subacutely to diazinon (0.1 and 0.2 likewise, a diminishment in the concentration of globulins in plasma of common carp (c. carpio l.) exposed acutely to phosalone has been shown . On the other hand, it has been reported that immunoglobulins are also affected by ops; in this sense, there are studies that show that these pesticides alter the concentration of igm, which is the most important gamma - globulin in fishes [33, 34]. In this context, it has been published that chlorpyrifos (0.051 mg / ml) diminish concentration of igm in plasma of nile tilapia (o. niloticus). Furthermore, it has also been reported that the exposure to chlorpyrifos (75 g / l) during 24 h provoked a significant decrease of igm in plasma of common carp, apart from a diminishment of igm present in spleen of fishes exposed acutely to 15 and 75 in addition, a significant increase has been reported in the concentration of igm in plasma of nile tilapia exposed acutely to diazinon (1.96 mg / l). Nevertheless, exposure to lower concentrations of this pesticide (0.78 and 0.39 mg / l) did not alter concentration of igm in plasma of these organisms [26, 35]. Regarding the effect of ops on the cytokines, it has been reported that the exposure to chlorpyrifos during 24 h (1.16, 11.6 and 116 g / l) induces an increase in the expression of mrna of il-1, il-1r, and ifn- in spleen of carp (c. carpio). The innate and adaptative cellular response of fishes can be deregulated by the exposure to diverse ops . Studies show that exposure of rainbow trout and common carp to diazinon provokes a diminishment in the white blood cell (wbc) in these species . The differential account of these cells showed a diminishment in the percentage of lymphocytes, monocytes, and basophils; however, the percentage of neutrophils and eosinophils increased significantly after exposure to the pesticide [28, 37]. A decrease in wbc in other species such as nile tilapia (o. niloticus) exposed to malathion (0.23 and 0.46 mg / l) and carp (c. carpio) exposed to phosalone (0.15, 0.30, and 0.60 mg / l) has also been reported . In common carp, lymphocytes diminished significantly at the three evaluated concentrations, even though the percentage of monocytes and neutrophils increased [1, 30]. In contrast to the results in the before mentioned studies, ural reported an increase in the wbc of common carp exposed to chlorpyrifos (0.04 and 0.08 mg hedayati and tarkhani reported that, in iridescent shark (pangasius hypophthalmus) exposed to diazinon (0.5 and 1 ppm), a significant increase in the total number of wbc, particularly in neutrophils, was shown, while the number of lymphocytes did not show any change due to the exposure to this pesticide . However, no eosinophils and monocytes were detected in the blood samples of the analyzed fishes [38, 39]. On the other hand, it was also reported that the ops not only induce alteration in the number of cells, but also in the morphology and functionality of them . Hence, it was reported that diazinon (15, 30, 45, 60, and 75 g / l) provoked changes in the size of macrophages of kidney and spleen of the fish lepomis macrochirus . In addition, it has been reported that the phagocytic activity of cells is also altered by the exposure to ops . Girn - prez et al . Showed that the phagocytic index of mononuclear cells of nile tilapia decreased by exposure in vivo to diazinon; however, an increase in the respiratory burst of these cells was observed [26, 35]. Regarding the effect of ops on the proliferative capacity of lymphocytes, it has been reported that chlorpyrifos at concentrations 0.051, 0.102, and 0.255 mg / l during 96 h did not affect the proliferative capacity of lymphocytes in nile tilapia . However, the lymphoproliferation of splenocytes of this fish diminishes significantly after exposure in vivo to diazinon (7.83, 3.91, and 1.95 mg / l) during 96 h . Nevertheless, the exposure in vivo of lymphocytes to diazinon and diazoxon (main metabolite of diazinon) did not affect the proliferative capacity of these cells . The effects mentioned above show that ops alter the function of certain elements of the immune system, even though the mechanisms of immunotoxicity of the ops are not clear . Such mechanism of ops is not direct but it works through indirect mechanisms, topics that will be discussed in this section, based on evidence shown in different animal models (figure 1). As previously mentioned, ops are substances that have as target molecule the enzyme ache, blocking its activity through the irreversible bound to the active site, which provokes an increase in the levels of the neurotransmitter ach in the nervous system . In this context, in mammals, the influence of the nervous system on the regulation of the immune system has been demonstrated years ago; thus the increase in the concentration of neurotransmitters, in this case neuronal ach, can deregulate the immune function . Apart from that, there is clear evidence that lymphocytes of mammals express machr and nachr in their membrane and possess all necessary enzymes to produce ach and autodegrade it through the ache enzyme; hence they possess a self - cholinergic system, denominated extraneuronal or nonneuronal cholinergic system [43, 44]. In this way, the existence of an extraneuronal cholinergic system in lymphocytes makes them susceptible to perturbation by ops . It has been suggested that ops can modulate lymphocytes through cholinergic receptors, evoking an immediate intracellular signalization of diverse molecules, among them c - fos, modulating therefore the levels of second messengers . Activation of cholinergic receptors can act upstream in the transduction of signals, causing the interruption of cellular homeostasis, decaying into apoptosis . Data obtained in our laboratory have proven that exposure in vitro to diazinon and diazoxon does not alter the lymphoproliferative capacity in fishes; nevertheless these substances induce an increase in the concentration of ach, which significantly diminishes lymphoproliferation . Besides inhibiting the enzyme ache, ops are capable of inhibiting serine hydrolases enzymes, such as molecules of the complement and thrombin system, which will influence directly the functionality of the immune system . In addition, the damage in the lymphoid tissue is the result of the phosphorylation, oxidative damage, and/or altered neuronal function, induced by ops . In this sense, in aquatic and human models, it has been reported that ops diminish nk cell, lak cell, and cytotoxic activities [4750]. Even though there are very few studies on the mechanisms of induced inhibition by ops in this type of cells, it has been proposed that this effect might be mediated by the inhibition of serine proteases (granzymes, perforins, and granulysins), molecules that are usually released by exocytosis [47, 5153]. In addition, ops have been reported not only to inhibit activity and release granules, but also to inhibit the expression of genes related with these molecules [49, 50]. On the other hand, the effect of ops in nk cells through fasl / fas pathway has been researched, and it has been reported that dichlorvos (ddvp) induces decrease on the expression of fas in cells yac-1 and the expression of fasl in lak cells . This suggests that ops reduce cytolytic capacity and proapoptotic signals through two mechanisms: (1) diminishment in exocytosis of granules and (2) fasl / fas pathway . Alterations of the components and immune functions have also been related to the sequence and intensity of phosphorylation and dephosphorylating of protein kinases, essential process to modulate the immune response . A key molecule in this process is the protein suppressor of cytokine signaling 3 (socs3), which regulates protein stat . Socs3 mediates inhibition of phosphorylation of stat5, which has been related with the diminishment of cellular proliferation . In this context, it has been reported that dialkylphosphates (daps), metabolites produced during biotransformation of ops, interact with leucocytes altering cellular signalization . There is evidence that diethyldithiophosphate (dedtp) and diethylthiophosphate (detp) interact and produce effects on the immune system, reducing the expression of cd25 and cd4 and secretion of il-2, altering signalization of il-2r, by modifying the phosphorylation status of stat5 proteins . Apart from that, it has been reported that dedtp increases phosphorylation of socs3 and dephosphorylation of stat5 and also induces phosphorylation of erk, jnk, and p38, depending events of pkc, plc-, and amp - responsive element - binding protein, which results in the nuclear translocation of nfat, ap1, and erk [46, 54, 55]. Lima and vega reported that dedtp induces the arrest of the cellular cycle, mediated by socs3, initiating a feedback mechanism associated with p21 and p53 [56, 57]. It has also been reported that ops (chlorpyrifos, sarin, and soman) can activate the plc- and after that the transduction via of signals mapk through pkc, as a consequence of accumulation of ip3 and dag . It is known that the initiation of apoptosis is regulated by external and internal signals, such as the activation of dead receptors, damage to dna, and perturbation of the mitochondrial membrane . These mechanisms carry the caspases activation and subsequently the destruction of the cell in a programmed way . Thus, it has been reported that some ops (monocrotophos, profenofos, chlorpyrifos, and acephate) induce apoptosis and necrosis in cultured human lymphocytes of peripheral blood . Reported that chlorpyrifos induce apoptosis in the cellular line u937 of human monocytes, besides inducing an increase of caspase 3 . On the other hand, it has been shown that parathion and paraoxon (parathion metabolite) induce apoptosis in the cellular line of lymphocytic leukemia t (el4) through activation of caspase-3 . Likewise, exposure in vitro and in vivo to paraoxon provoked cytochrome c translocation from the mitochondria to cytosol, activating proapoptotic molecules such as bax . It has been shown that exposure of cell line zc-7901 of grass carp fish (ctenopharyngodon idellus) to malathion (23.75 mg / l) during 2 h induces a decrease in the mitochondrial membrane potential (m), besides increasing the intracellular calcium flux . Fishes are the first vertebrates with innate and adaptative immune mechanisms, similar to mammals . Thus, fishes can be used as a model in biomedical research, allowing data in the immunotoxicology field in evolutionary terms . Besides, due to fishes being the most abundant vertebrate in the planet, a lot of them with commercial importance, data generated could have economic and ecological importance . Although, the immunotoxicity mechanisms are not completely clarified, evidence suggests that ops can target several molecules related to the immune system and execute the immunotoxic effect through the alteration of the neuroimmune communication, particularly the cholinergic neuronal and immune system . Nevertheless, further research is needed in order to understand the mechanisms of immunoregulation of this type of pesticides widely used in household and agricultural activities.
An estimated 66% of maternal deaths and 50% of deaths in children under the age of 5 occurred in sub - saharan africa in 2015 [1, 2]. Although ghana's health indicators largely surpass many african nations, the country has faced significant challenges in improving maternal and child survival . Despite nearly halving the number of maternal deaths since 1990, approximately 319 women die each year out of 100,000 live births from pregnancy- and birth - related complications a figure nearly twice that of the millennium development goal [1, 3]. Among children under five, while overall deaths have declined by 51% since 1990, neonatal deaths occurring within 28 days after birth have largely remained stagnant, changing only from 30 to 29 deaths per 1,000 live births [2, 4]. A vast majority of maternal, neonatal, and child deaths are preventable [1, 58]. However, limited access and utilization of skilled antenatal, delivery, and postpartum care services hinder the timely and safe delivery of life - saving interventions [69]. In addition, reliable and comprehensive data on the implementation of maternal, neonatal, and child health services are needed to monitor and ensure that life - saving services are provided across the care continuum [1012]. However, the inability of traditional health information systems to track new mothers and babies once they leave health facilities as well as inadequate communication between health workers at different levels of the health system has led to slower progress towards maternal, neonatal, and child health goals [1315]. Global penetration of mobile phones has the potential to optimize delivery of high quality maternal, neonatal, and child health information and access to services [1621]. In particular, mobile apps, or software applications developed specifically for use on small, wireless computing devices such as smartphones or tablets, have been shown to improve the quality of care provided to pregnant and recently delivered women through electronic decision support [17, 18, 22], health worker planning [23, 24], and data collection and reporting [19, 2527]. The reported ease of use and adaptability of mobile apps (or applications) for diverse populations also makes them an attractive, low - cost platform for developing health education and clinical care strategies [2830]. Nonetheless, to increase efficacy and sustainability of mobile health applications, previous studies have underscored the importance of ensuring high user acceptance and usability of the technological innovation [3133]. User - centered design and robust needs assessments are recognized as best practices for development technology . Equipping frontline health workers with mobile phone applications may enhance the delivery of maternal, neonatal, and child health services as well as the accuracy of data capture and recording [16, 26, 35, 36]. Yet, little research exists on health worker perceptions of mobile applications intended to improve maternal, neonatal, and child data management, despite user views being critical to product uptake and implementation [3739]. In addition, the opinions and experiences of frontline health workers in sub - saharan africa on using mobile applications to track pregnant women and mother - infant pairs are underreported . Such information is critical for improving the long - term impact and sustainability of mobile health (mhealth) strategies for clinical and community health settings . Evaluation objective . This evaluation aimed to qualitatively examine the feasibility, usability, and acceptability of the client data application (or client data app) that was part of the mobile technology for community health (motech) program to support frontline health workers in the delivery of maternal, neonatal, and child care in rural ghana . The motech program was initiated in 2009 as a partnership between the ghana health service, grameen foundation, and columbia university mailman school of public health to leverage mobile technology to increase quantity and quality of maternal and infant care in rural areas and ultimately improve health outcomes . The client data app enabled community health nurses (chns) to use mobile phones to digitize care provided to women and children and thereby track pregnant women and mother - infant pairs needing care (figure 1). Parallel care reminders and alerts, in addition to other actionable health information, were sent to pregnant women and mothers with infants less than 12 months of age as part of a mutually supportive mobile midwife program within motech . A separate evaluation, not discussed in this manuscript, is planned to examine health workers' views on motech's mobile midwife service . Motech was implemented in awutu senya in 2011 and then replicated in gomoa west and three other districts in 2013 . Both districts are located in ghana's central region and have among the highest rates of under - five and neonatal mortality in the country [4, 40]. This region additionally has significant human resource shortages . In the central region of ghana, there are over 25,000 individuals to one doctor compared to the nationwide ratio of nearly 12,000 individuals to one doctor [41, 42]. The region also suffers from high vacancy rates of nurse - midwives trained to manage basic and emergency obstetric care [4345]. Under these circumstances, mobile technologies that expand the reach of health workers although telecommunication connectivity in the central region can be unreliable and mobile phones are not ubiquitous, phone ownership and access are high among health workers . Maternal, neonatal, and child health services within ghana's health system are primarily delivered via public health centers and community health posts . The health centers are staffed primarily by skilled health professionals (such as nurses and midwives) who offer comprehensive preventive and basic curative services, including minor surgeries and uncomplicated deliveries . In contrast, as part of ghana's community - based health planning and services (chps) initiative, community health posts are staffed by lower - skilled chns who provide health education, outreach and counseling, and basic curative services to clients via home visits and facility - based care . Community health posts (referred to locally as chps facilities) are typically staffed by 2 to 3 chns who are required to have completed a two - year postsecondary certificate program in obstetrics as well as general and community health nursing . Motech ghana was originally designed for use in community health posts and later extended to the health centers, hospitals, and other private health facilities . Within the two evaluation districts, motech was implemented in a total of 46 facilities, including 35 community health posts and 11 health centers . The mobile client data app used in this evaluation was delivered by low - cost gsm mobile nokia 1680 and nokia asha 200 feature phones provided by ghana health service, which helped chns and other users to digitize and track care delivered to mother - infant pairs in their area . The system's architecture was based on field - tested open - source software, including openxdata for mobile data collection and openmrs for electronic medical records . The client data system used a java 2 platform micro edition (j2me) application to capture and store client data . All clients were assigned a unique motech identification (i d) number upon registration to protect confidentiality and enable tracking across multiple facilities . During client encounters, chns first recorded care provided using five simplified paper registers, which were developed by motech to condense more than a dozen registers and streamline data collection . Chns later entered data into digital forms on their mobile phones (figure 2(a)). General packet radio service (gprs) data channels were used to transfer these data from the phone to a central clinical data system that was stored on the motech server (figure 2(b)). The data app system then crosschecked uploaded clinical information on timing and type of care given with national guidelines to estimate specific due dates for routine care . As a result, health workers received a weekly list via short message service (sms) of pregnant clients and mother - infant pairs in their catchment area who were either due for or defaulted on care . Chns were also able to query client data, enabling them to retrieve lists of defaulters or women due to deliver in the upcoming week, and to search for details about individual clients (figure 2(c)). In addition, the client data app generated preselected monthly health reports that were required for national reporting, if client data were at least 85% complete and accurate three consecutive months . Previously, monthly health reports were numerous, redundant, and compiled by hand . The motech developers designed the client data app for low - skilled health providers in rural and resource - poor settings . To account for anticipated power and mobile network breaks, the java - enabled nokia handsets allowed for mobile forms to be completed and stored offline for uploading at a later time . Phones had dual subscriber identity module (sim) capacity and were equipped with sim cards from two different mobile operators in case of network or congestion problems . Field testing during the prototyping stage served to align the app's features with user needs, including the simplification of data entry using check boxes, radio buttons, lists, and number fields . Phones were password - protected, and user authentication schemes were built into java forms to maintain confidentiality of client data . Qualitative in - depth interviews and focus groups were used to examine health worker perceptions on the client data app's feasibility, usability, and acceptability . For purposes of this evaluation, we defined the assessment areas as follows: feasibility was defined as whether implementation of the client data app was easily and conveniently done, accounting for advantages and disadvantages to integrating the application into routine workflow . Usability was defined as whether the client data app could be used by chns to adequately record, track, and summarize data, including whether it functioned in a way that enhanced productivity or led to unproductive tasks due to errors . Acceptability was defined as whether chns and other stakeholders found the client data app likeable, including its interface and navigation features . These definitions were derived from similar prior research that qualitatively assessed user experiences for mhealth applications [28, 4955]. Data were collected at three levels of the health system: community health posts (referred to locally as chps facilities), health centers, and district health directorates . Semistructured interview guides were used at all levels . In - depth interviews with chns and midwives asked them to describe perceived benefits and drawbacks of the client data app, as well as their experience using it during clinic and community outreach activities . Chns and midwives were also asked to assess advantages and disadvantages of using the client data app for tasks such as recording care, tracking clients, and verifying data with automatic health reports . Skilled nurses working at the health centers did not use the client data app and therefore were not recruited for data collection . Interviews with district health directors and district health information officers explored how the client data app affected the quality of data provided by chns and their ability to use and supervise submission of monthly health reports . Other district health directorate staff, including disease control officers and public health officers, who did not engage with the client data app were not interviewed . Focus groups with chns were included to further investigate findings from the individual interviews and to obtain chns' recommendations for modifying the motech data management system . Focus groups with midwives are not conducted given the limited number of midwives available at participating sites . All interviews and focus groups were conducted in english by a local ghanaian and a us researcher, both with experience conducting qualitative research . The interviews were conducted at community health posts and health centers and ranged from 40 to 120 minutes . One health center and three community health posts were randomly selected from each of the two participating districts . Purposive sampling was then used to identify chns, midwives, district health information officers, and district health directors with a minimum of six months experience using motech's client data app . The target interview sample size was 14 individuals, representing one chn and one midwife per health center, one chn each from three community health posts in each district, and the district health director and district health information officer in each district . The target focus group sample size was two groups each with 7 to 8 chns . Given resources available, this sample size was expected to enable the evaluation to reach saturation in which no new findings emerged . Interview and focus group transcripts were manually coded using a priori topical codes according to the evaluation's three assessment areas: feasibility, usability, and acceptability . Emergent subcodes were then developed based on patterns within each concept and which were relevant to the literature . We then followed an iterative process of developing a codebook, identifying salient themes, and integrating core findings . When new themes were identified throughout this process, transcripts were reanalyzed to find evidence that verified or modified those themes . Later - stage interviews and we also confirmed findings based on feedback from motech implementation partners during various stages of data collection and analysis . The evaluation was approved by the institutional review board at the johns hopkins bloomberg school of public health in baltimore, maryland, usa . As part of the motech implementation plan, the ghana health service in accra, ghana, also approved the evaluation's activities . This manuscript complies with the mhealth evidence reporting and assessment (mera) checklist on reporting of health interventions using mobile technologies . Several findings emerged from our analyses, revealing both the achievements and challenges of motech's implementation of the client data app . The emergent themes within each of the three assessment areas (feasibility, usability, and acceptability) are illustrated below with exemplary quotations . A total of 14 individual interviews were conducted, representing eight chns and two midwives as first - line users of the client data app, as well as two district health information officers and two district health directors (table 1). Two focus groups were additionally carried out, consisting of a total of 15 chns . The majority of participants were female and within the ages of 26 to 30 years . We defined feasibility as whether implementation of the client data app was easily and conveniently achieved, accounting for advantages and disadvantages to integrating the application into routine care . Participants generally reported that the client data app was easily integrated into their workflow and data capture . Chns were able to incorporate registration of eligible individuals, including issuing their motech ids, into their client visits . Chns also noted being able to readily incorporate information provided by motech alerts into their weekly routines . Based on the ease of integration, users were able to envision the client data app becoming a permanent part of their work.when a pregnant woman comes here, you start reviewing her records . When there is a new client, you give her an anc booklet; you fill the form for her, taking the vitals of the client . (chn, health center) every monday morning, the motech system sends us alert messages to remind us on those who are due for care . And so, every monday morning i checked the phone and gets tracked on the data, and the details on those who are due for care and those who are defaulters and follow them and give the care to them . (chn, community health post)we just hope that the motech project comes to stay forever and incorporate it into ghana health service, forever . (district health information officer) when a pregnant woman comes here, you start reviewing her records . When there is a new client, you give her an anc booklet; you fill the form for her, taking the vitals of the client . (chn, health center) every monday morning, the motech system sends us alert messages to remind us on those who are due for care . And so, every monday morning i checked the phone and gets tracked on the data, and the details on those who are due for care and those who are defaulters and follow them and give the care to them . (chn, community health post) we just hope that the motech project comes to stay forever and incorporate it into ghana health service, forever . (district health information officer) however, limited mobile network connectivity in more rural areas of the district posed significant challenges to uploading client data . Consequently, chns had to preoccupy themselves with locating places with adequate connectivity in order to upload the mobile forms . For some chns, accommodating network connectivity challenges also required them to extend their workday.we also have problems with network connectivity . (district health information officer)we try to find a place where the network is good . Let's say, my house, the network is good, i have to take the phone and upload there . Sometimes they're typed, but it doesn't go through . Sometimes it doesn't go through at all . (district health information officer) we try to find a place where the network is good . For example, when the network is good, you can upload everything . Let's say, my house, the network is good, i have to take the phone and upload there . (chn, health center) chns and other users additionally expressed concerns about the hardware used, as the small size and tedious typing functionality of the nokia 1680 phones made data entry time - consuming . This rendered the client data app as a less feasible option in the long run, especially at high - volume health centers . Requests to be equipped with larger screen devices with keypads such as laptops or tablets were given by over half of interview participants, who stressed the challenges associated with their current handsets.the numbers are quite huge numbers . (district health information officer)they also should give us laptop, or tablet, as we are typing, we can't speed it . I would prefer a smart phone because in smart phone, when you type something you don't have type everything, it will just [automatically] fill . (district health information officer) they also should give us laptop, or tablet, as we are typing, we can't speed it . I would prefer a smart phone because in smart phone, when you type something you don't have type everything, it will just [automatically] fill . (chn, health center) another challenge involved the omitted generation of automated reports for some facilities because reporting completeness and accuracy had not reached 85% for the required three consecutive months . Limitations in meeting completion and accuracy thresholds and the corresponding failure to receive automated reports left many users frustrated.tomorrow i will be thinking i have to submit the report by monday . If we are supposed to submit on monday, there is a lot to do . Oh . We did three months and so automation will generate reports for you . (chn, community health post) tomorrow i will be thinking i have to submit the report by monday . If we are supposed to submit on monday, there is a lot to do . Oh . We did three months and so automation will generate reports for you . (chn, community health post) feasible implementation was also hindered by a shortage of chns or other data entry staff, especially in health centers where client volume outweighed staff availability . High client caseloads interfered with chns' capacity to submit clinical data and receive timely alerts on clients who were due for or missed care . The significant clinical responsibilities of trained midwives at health centers and the unpredictability of their workdays also posed challenges to the consistent use of the client data app . The imbalance between client loads and health worker availability was compounded by high chn attrition rates, which required retraining of staff on the client data app.because we are the health center, unlike the chps zone, concerning our volume, after the outreach, you have to do all the uploads maybe they can take out the health centers or have something done to help because our workload is intense . (chn, health center) by the end of the day, maybe you have a lady who haven't deliver[ed] yet, so after you close the antenatal phase, you have to go to the labor ward and be monitoring this labor case . Then by the end of the day, you are coming back to load the motech in fact it's a headache . (midwife, health center) in non - urban settings, like this place, staff attrition is very high . For the past about three years, we've been receiving an average of about 20 community health nurses every year . (district health director) because we are the health center, unlike the chps zone, concerning our volume, after the outreach, you have to do all the uploads . Maybe they can take out the health centers or have something done to help because our workload is intense . (chn, health center) by the end of the day, maybe you have a lady who haven't deliver[ed] yet, so after you close the antenatal phase, you have to go to the labor ward and be monitoring this labor case . Then by the end of the day, you are coming back to load the motech in fact it's a headache . (midwife, health center) in non - urban settings, like this place, staff attrition is very high . For the past about three years, we've been receiving an average of about 20 community health nurses every year . (district health director) many participants commented that the feasibility of the client data app would be improved if staff were designated to data management rather than responsible for multiple care tasks, especially during community outreach activities . Others suggested that health worker shortages could be overcome by training health extension workers (who had previously been discontinued) for routine data entry . In addition, some participants proposed recruiting other motech staff to assist with routine data entry during care provision.i do have some problems with recording the care given . Sometimes, at the child welfare clinic, i have to take the height and the muac [mid - upper arm circumference] of the babies . I will be doing the weighing, the immunizing and the recording, and a whole lot of it . Sometimes i even forgot to take the muac or the height there is no help . (chn, community health post)if we can get other cadre of staff, who are not necessarily well trained, so that they can do this .we did train some health extension workers, and then other staff [but]they are not professionals . We did train them, but unfortunately they've all been exited out of the system, so that has compounded the problem . (district health director)would that be possible? To train personnel from motech, and then they join us on the field? Or join us in our health facility? Because basically in ghana, our work is very broad (district health information officer) i do have some problems with recording the care given . Sometimes, at the child welfare clinic, i have to take the height and the muac [mid - upper arm circumference] of the babies . I will be doing the weighing, the immunizing and the recording, and a whole lot of it . Sometimes i even forgot to take the muac or the height there is no help . (chn, community health post) if we can get other cadre of staff, who are not necessarily well trained, so that they can do this .we did train some health extension workers, and then other staff [but]they are not professionals . We did train them, but unfortunately they've all been exited out of the system, so that has compounded the problem . (district health director) would that be possible? To train personnel from motech, and then they join us on the field? Or join us in our health facility? Because basically in ghana, our work is very broad . Very huge . (district health information officer) we defined usability as whether the client data app could be used by chns to adequately record, track, and sum data, including whether it functioned in a way that enhanced productivity or led to unproductive tasks due to errors . Chns reported that motech's simplified paper registers were a significant improvement over the previous data collection process . Chns reported that they were generally able to transfer clinical information from the simplified registers to the mobile forms in approximately two minutes . In addition, by enabling chns to avoid redundant paper registers, the client data app saved time and allowed for more case entries per day and week.i think motech has made our work much easier . Before there is a lot of writing, then after motech, maybe the important information will be portioned, so maybe when you write, it's easier . (chn, health center)we don't rule lines anymore and the immunizations, we can write the actual number, everything else is there . (chn, health center) i think motech has made our work much easier . Before there is a lot of writing, then after motech, maybe the important information will be portioned, so maybe when you write, it's easier . (chn, health center) we don't rule lines anymore and the immunizations, we can write the actual number, everything else is there . (chn, health center) nevertheless, the client data app's usability was hampered by technical errors which interfered with completing registration and data submission . The most commonly reported error involved identical motech i d numbers designated to different clients . User delays resulting from these errors were also exacerbated by difficulties reaching the technical support team . In addition, data entry was often interrupted when the software froze unexpectedly.they will say the motech i d is already in use . If i use those motech i d numbers, they will just send me errors . I uploaded, and i called the call center, but they don't respond . (chn, community health post)it's sometimes .an experience on the phone . I think sometimes it freeze, when you are working on some forms, you realize that it stops then you realize it's freeze . (chn, health center) they will say the motech i d is already in use . The motech i d says it's new . If i use those motech i d numbers, they will just send me errors . I uploaded, and i called the call center, but they don't respond . (chn, community health post) it's sometimes .an experience on the phone . I think sometimes it freeze, when you are working on some forms, you realize that it stops then you realize it's freeze . (chn, health center) usability challenges also arose because motech was not interoperable with the national health information system . While the integration of motech data with the district health information management system 2 (dhims-2) was beyond the program's resources and scope, participants expressed frustration with this disconnect . The process of recording and summarizing client data was also delayed because it was not possible for motech to automate all of the monthly reports for qualifying facilities.mind you, we have been advocating that if the motech system and the dhims-2 would be on the same platform - so that this one will fit into this . So you don't have to print out, before you re - enter . That will save a lot of time . (district health information officer)we do the entry at the sub - district level, all the facilities in motech . Change our phone and get us [a] tablet . Then we can do the dhims-2 ourselves at the sub - district level . (chn, community health post)there is nothing about antenatal care . Even though they take [mobile] data on antenatal care, which they upload . So, the core areas which we think should be there because of motech for the [mobile] reports are omitted . (district health information officer) mind you, we have been advocating that if the motech system and the dhims-2 would be on the same platform - so that this one will fit into this . (district health information officer) we do the entry at the sub - district level, all the facilities in motech . (chn, community health post) there is nothing about antenatal care . Even though they take [mobile] data on antenatal care, which they upload . So, the core areas which we think should be there because of motech for the [mobile] reports are omitted . (district health information officer) finally, some of the information needed to identify and track clients in their communities was not readily available through the client data app's alerts or queries . Chns suggested including client addresses and travel instructions to ease follow - up in remote areas and the overall usability of the application.the reminder is good, but the difficulty in tracing the defaulters is the address . To get the address, we still have to go back to the simplified registers to locate the defaulters . When the phone is in your pocket and you look at it, you see the person [but] it only comes with the name and the care . (chn, health center) the reminder is good, but the difficulty in tracing the defaulters is the address . To get the address, we still have to go back to the simplified registers to locate the defaulters . When the phone is in your pocket and you look at it, you see the person [but] it only comes with the name and the care . (chn, health center) the usefulness of the client data app was likewise affected by interruptions in network connectivity . The discrepancy between data collection and submission also hindered the client data app's tracking functionality and the accuracy of the aggregate numbers in the automated monthly reports.the internet connection is not that stable it is time - consuming when you are doing the uploads because you have to try it, try it, try it . When the network is good, we just do it once and it hits the system the network is not stable . So you have to wait, waiting, waiting, waiting (chn, community health post)because sometimes we upload the forms but it doesn't go through . It means it wouldn't be captured by the system . It is a challenge we have . Yesterday the phone i uploaded, it didn't go through . (chn, community health post) the internet connection is not that stable it is time - consuming when you are doing the uploads because you have to try it, try it, try it . When the network is good, we just do it once and it hits the system the network is not stable . So you have to wait, waiting, waiting, waiting (chn, community health post) because sometimes we upload the forms but it doesn't go through . It means it wouldn't be captured by the system . (chn, community health post) in order to minimize delays due to connectivity interruptions, one chn suggested that motech incorporate additional feedback loops between users and the central database . Maybe on mondays, if they can send us what we've uploaded for us to cross check . If it's not uploaded we will re - send it it will be easier and faster than just waiting for them . Otherwise we always complain about the automated generated report, because it's not tallying what's really happened . So given the reminders, they should also give us the reminders of the uploads . (chn, health center) maybe on mondays, if they can send us what we've uploaded for us to cross check . Otherwise we always complain about the automated generated report, because it's not tallying what's really happened . So given the reminders, they should also give us the reminders of the uploads . (chn, health center) acceptability was defined as whether chns and other stakeholders found the client data app likeable, including its interface and navigation features . The client data app was acceptable to users given that it introduced new tools to assist health workers in performing their assigned tasks . In particular, chns and those at community health posts found that the automated monthly reports eased their workload for data reporting and motivated them to capture and use data.sometimes, when you go to the outreach it's difficult for you to go to the register and find out which community you went to and which not . When you go to the reminders on motech, it will just tell you because you uploaded the information after the outreach . (chn, community health post)it is so rewarding when at the end of month, you receive your computer - generated reports . And then you don't have to go through the registers and do the targets . Even that, there's some incentive for those who receive the computer degrees and reports . It's difficult for you to go to the register and find out which community you went to and which not . When you go to the reminders on motech, it will just tell you because you uploaded the information after the outreach . (chn, community health post) it is so rewarding when at the end of month, you receive your computer - generated reports . And then you don't have to go through the registers and do the targets . Even that, there's some incentive for those who receive the computer degrees and reports . (district health information officer) conversely, chns who worked at facilities that were not receiving the automated reports due to incomplete submitted data were less enthusiastic about the client data app . They expressed a desire to see these new job aids materialize.i wish motech system could generate all the reports for me . (chn, community health post) i wish motech system could generate all the reports for me . (chn, community health post) the client data app was also valued for improving the efficiency of client follow - up through weekly reminders, which eliminated the need for chns to conduct time - consuming searches through paper registers . In addition, chns noted that the weekly defaulter lists helped structure their community outreach and home visit schedules, maximizing the follow - up care provided.before motech, sometimes we don't trace defaulters . It's very helpful . Because if you don't trace, you don't know what's happening to your clients . (chn, community health post)before the motech, we don't have the reminders, i write it somewhere else, sometimes i remember it . Sometimes you remember . Sometimes it makes it easier to have the reminders, because you can't remember everything . (chn, community health post)it helps trace our important clients and the message it gave, it tells them it saves mothers and pregnant women . (chn, health center) before motech, sometimes we don't trace defaulters . It's very helpful . Because if you don't trace, you don't know what's happening to your clients . (chn, community health post) before the motech, we don't have the reminders, i write it somewhere else, sometimes i remember it . Sometimes you remember . Sometimes it makes it easier to have the reminders, because you can't remember everything . (chn, community health post) it helps trace our important clients and the message it gave, it tells them it saves mothers and pregnant women . (chn, health center) administrators and data management staff working in the district health directorates were most satisfied with the client data app for its capacity to summarize data for timely decision - making . The automated monthly reports provided by motech enabled a new level of scrutiny for data collection and aggregation in health centers and community health posts.it's very good, for instance, the fact that the data, you can get everything at the facility level . But before that came into being, everything was allocated at the health center level or sub - district level . Now we can get more detailed data at the chps [health post] level than used to be . I think to me that is what i can put my finger on more details . (district health director) it's very good, for instance, the fact that the data, you can get everything at the facility level . But before that came into being, everything was allocated at the health center level or sub - district level . Now we can get more detailed data at the chps [health post] level than used to be . I think to me that is what i can put my finger on more details . (district health director) district health directors felt the client data app brought about a better awareness of how data were collected and used and, therefore, a better appreciation for accuracy and completeness . District health directors also noted that, for staff with limited data analytical skills, the client data app assisted in synthesizing data rapidly and in a user - friendly format . Having more valid, aggregated data additionally enabled administrators to monitor trends and determine what needed to be improved.to a large extent, because it is automated, i think i have to concede that the capacity of a good number of our staff at especially the lower levels are inadequate when you are talking about data analysis . So when it is ready - cooked like this, of course it's easy for them to if it's [a] graphical presentation you can visualize everything that you want . And that is very handy . (district health director)as you know, these things are needed to help with decision - making at that level . You can clearly see where you are doing well [and] where you are not doing well . (district health director) to a large extent, because it is automated, i think i have to concede that the capacity of a good number of our staff at especially the lower levels are inadequate when you are talking about data analysis . So when it is ready - cooked like this, of course it's easy for them to if it's [a] graphical presentation you can visualize everything that you want . And that is very handy . (district health director) as you know, these things are needed to help with decision - making at that level . You can clearly see where you are doing well [and] where you are not doing well . Our data indicate that motech's client data app was a feasible, usable, and acceptable tool to aid health workers in collecting and tracking data to improve maternal, neonatal, and child health services . Health workers agreed that the client data app simplified individual client data collection, was easily integrated into their workflow, and enhanced their capacity to deliver follow - up services across the care continuum . These findings align with other studies that have demonstrated the benefits of equipping health workers with data management technologies to improve the continuity of care in rural populations [27, 61]. At the same time, our results highlight several challenges that would need to be addressed to optimize the utility of a client data management system using mobile devices in resource - poor settings . First, our findings suggest that the successful integration of mobile applications into service delivery may necessitate targeted changes in human resources available at certain health facilities . The greatest obstacle to implementation of the client data app was the combined effect of high client volumes, staff shortages, and poor network connectivity . Chns tended to integrate mobile data entry into client care by waiting until the end of the day or week to upload data to the motech server . Yet, the number of clients seen by health workers at the health centers and the long delays in uploading data precluded real - time submission . Thus, while data uploads were manageable for chns working at the community health posts, they were generally not manageable at health centers . Similar issues emerged within the context of a telemedicine program in ghana's amansi - west district, in which providers assumed a greater workload without a reduction in other tasks or increased personnel . Our participants suggested training less skilled data staff or community health volunteers to assist with data entry, particularly for older providers who were sometimes less proficient with mobile apps . In the context of future mhealth deployments, it will be important for implementing partners to work closely with the national health system and other government agencies to assess health workers' capacity to absorb additional mobile - based responsibilities and to explore possibilities for hiring and maintaining data staff . Second, given the current cellular network in rural ghana, it may have been possible to minimize network challenges through a more advanced monitoring system . Our results revealed that the lack of network reliability compromised the efficiency and usability of the client data app, notwithstanding the technological systems put in place to address these complications . Other studies have shown that poor network coverage and signal strength are often a major limitation to implementation of mobile technologies [23, 27, 31, 65, 66]. While the motech team took careful measures to mitigate connectivity challenges, such as instituting a customer support call center and using high - storage devices with offline and multinetwork functionality, users still grew frustrated with the efforts required to upload data . The longs lags between data collection and submission, which chns reported as sometimes taking up to two weeks, limited the capacity of motech to reliably track all client needs . A mechanism that allowed health workers to directly access clinical data to fix errors from incomplete uploads, rather than relying on a customer support center, may have enabled more immediate troubleshooting . In addition, it is critical for implementers to carefully select what minimal clinical data are required for decision - making, rather than digitizing all paper registers or entry fields in order to maximize entry quality and speed . Third, our findings shed light on the interconnectedness of the evaluation's assessment areas . Acceptance of the client data app was closely linked to its perceived usefulness to generate automated reports . When no problems were encountered during uploading the motech team had anticipated this would be the primary incentive for chns to engage with the client data app . However, several health center - based chns were less motivated to enter data when the reports were not generated . These findings align with other research studies that have found technical issues such as screen freezes and delayed uploads impede efficient use and limit user uptake of mobile health applications [27, 55, 67]. A lower threshold of data completeness and accuracy for receiving automated reports may have improved usability . Similarly, engaging dedicated data staff to manage the client data app may have enhanced its usefulness and acceptance by increasing the number of facilities with minimum completeness standards and automated reports . Other strategies such as rewards and recognition may also have encouraged user uptake in the short term, yet their sustainability is questionable . Studies conducted with frontline health workers prior to the integration of new technologies into their workflow have revealed high levels of acceptance and willingness to learn, despite the lack of experience with such tools [64, 68, 69]. In many cases, such interest is related to the appeal of innovative technologies as representative of modern medicine [64, 67]. Thus, efforts should be taken to capitalize on and preserve existing positive attitudes during the implementation of mobile health applications by ensuring the usability of new devices . Specifically, incurring higher upfront costs for more advanced mobile handsets may improve data accuracy and user adoption as compared to inexpensive, less user - friendly devices . Motech selected the nokia 1680 model, in part, due to its lower cost (40 usd) for use in awutu senya . Yet, as our participants concluded, the small - sized keypad was more problematic than anticipated . In later deployments of motech in gomoa west as well as the other regions not explored in this evaluation, the phone was upgraded to a more expensive, user - friendly nokia asha 200 . The evolution of mobile phones used by motech highlights the value of continually assessing user experiences and reevaluating the trade - offs inherent in each decision . As the participants noted, using larger tablets or laptops in the future may further improve client data management at facility and community levels . Finally, greater global focus has been placed on ensuring the interoperability of mobile and digital health innovations . The proliferation of multiple disconnected mhealth systems in many countries has led to calls to examine how systems operating in the same country can exchange information across multiple platforms . Systems like openhie (https://ohie.org/) and similar shared health record alliances are being explored as a mechanism to allow smaller mhealth products to share connectivity to a central backbone of core information . In our evaluation, participants identified a lack of interoperability with the national dhims-2 as an important barrier to maximizing utility . Although often more expensive and time - consuming during development, the future benefits of building mobile applications to global health information standards quickly materialize with increased scale, through interoperability with other facility and national aggregate systems first, self - reported data is subject to social desirability bias, and thus participants may have exaggerated their positive reactions to the client data app . However, this bias may have been minimized by the fact that the interviewers were external consultants and not affiliated with motech . Second, the transferability of the findings to other motech districts may be limited by the unique situation of the awutu senya site . For example, the district health director from awutu senya was highly dedicated to and involved in project implementation, which may not be true in other settings . Nevertheless, by engaging with diverse user groups, this evaluation provided rich insights into the client data app's implementation challenges from multiple perspectives . The use of two types of qualitative methods further strengthened the credibility of the findings, as later - stage interviews and the focus groups were able to provide feedback on early analyses . Mobile phones hold great promise for overcoming health disparities among rural populations by bridging the gap between access to client health information and service provision . Motech's client data app is a promising tool to aid health workers in collecting and tracking data across the health care continuum . Results of this evaluation may be used to guide future research on mhealth innovations to address challenges related to infrastructure, human resources, and technology before and during program deployment . Qualitative assessments of user perceptions should remain a priority in efforts to optimize the use of mobile data applications to alleviate barriers to maternal, neonatal, and child care in ghana and beyond.
As developing countries begin to meet the first round of public health goals, they should put in place policies that anticipate the next . According to recent reports, progress on frontline issues has been good: both the maternal mortality rate and the mortality rate for children under five have nearly halved since 1990 . There is much progress yet to be made on these indicators, in developing countries, meagre resources, and weak healthcare systems create very different but equally challenging cost - benefit questions . Add to that the fact that some clearly cost - effective solutions do not work as anticipated when they come up against human behavior . Health seeking behavior is preceded by a decision - making process that is further governed by individuals and/or household behavior, community norms, and expectations as well as provider - related characteristics and behavior . Health or care seeking behavior has been defined as any action undertaken by individuals who perceive themselves to have a health problem or to be ill for the purpose of finding an appropriate remedy . For this reason, the nature of care seeking is not homogenous depending on cognitive and noncognitive factors that call for a contextual analysis of care seeking behavior . Context may be a factor of cognition or awareness, sociocultural as well as economic factors . The health belief model (hbm) proposes that whether a person performs a particular health behavior is influenced by two major factors: the degree to which the disease (negative outcome) is perceived by the person as threatening and the degree to which the health behavior is believed to be effective in reducing the risk of a negative health outcome . The first factor, i.e., perceived threat, is determined by whether someone believes he or she is susceptible to (that is, likely to get) the disease, and how severe that person believes it would be if it developed . The second factor, perceived effectiveness of the preventive behavior, takes into account not only whether the person thinks the behavior is useful, but how costly (in terms of money, time and effort) it is to carry out the preventive behavior . Hypothesis generated by the hbm have been generally supported by research . When health messages demonstrate to people that there is a real threat to their health and also convince them that a particular behavior can reduce their risk, the likelihood of behavior change is greatly increased . One family of seven members residing in tripuri - the field practice area of medical college, is considered as the present case study material . The family comprised of husband, wife, and five children (first two children are females, third male child, fourth female, and the last one is male). Husband though illiterate was vocationally trained painter and wife is matriculate but employed as house help . While the first four deliveries were conducted at home by traditional birth attendant (dai) only, for the fifth pregnancy she visited community health center and delivery was conducted there . A person having belief in traditional system for the first four pregnancies and resulting deliveries changed her behavior from a time - tested system to a newer approach and behavior . On asking for the reasons first, she had pain and so was scared of complications, the other reason being monetary . Many factors influence an individual's decision to engage in valuable health behaviors; not the least of these are beliefs, attitudes, and intentions . The female is matriculate and better placed than illiterate husband, especially in decision - making . However, she opted for traditional home delivery assisted by dai, may be for two reasons, i.e., rural habitat and her mother happened to be a dai . She lost her maternal support as her mother expired after the third child but the fourth child was also delivered by dai at home . She migrated from rural uttar pradesh to punjab urban slums only just before the delivery of the fourth child . However, the urban and media effect became pronounced by the time of the fifth child . Here comes the factors responsible for visible change in her health seeking behavior and reflects decade of experience in her family life, shift to urban slum from rural environs apart from the all invasive effect of media in publicizing the government's efforts in improving maternal health care by providing free and prompt ambulance service and monetary benefits in the form of janani suraksha yojana, etc ., for institutional deliveries . In this case, she was afraid of complications, knew about the availability of maternal and child health services at community health center coupled with economic reasons at that time led to change of her behavior . Thus, client based factors, provider - based factors caretaker perceptions; social and demographic factors, cost, social networks, and biological signs and symptoms work synergistically to produce a pattern of health seeking behavior . The case presently studied points to certain aspects such as these . Designing any health policy is essentially pivoted by these important feedbacks and the results rely upon timely and perfect implementation . It is not just making something new available but making it attractive enough for the stakeholders to seek . This is affected positively by wide publicity on media; especially digital one as print media attracts only the educated while digital media such as tv and radio penetrates into all sections of the population . Publicity of programs is not only need but a vital component in achieving the goal . This fact is evidenced in this case study; wherein the lady is impressed by the newer facilities provided by the government, but it took time . The offer of financial support is also taken when it is laced with needed services like free ambulance both ways, for example
Hematopoietic stem cell transplantation (hsct) has become a common procedure of therapy for patients with hematological malignancies and many other life threatening blood disorders . However, viral and fungal infections associated with the severe immunoablative conditioning used prior to hsct still represent a major challenge (martelli et al ., 2002; seggewiss and einsele, 2010). One approach to address this barrier is to use reduced intensity conditioning (ric). Hsct following ric relies on non - myeloablative preparatory regimen that spares a substantial level of the host immunity and thus reduce transplant related mortality (trm) by both improving post - transplant immune reconstitution and reducing the toxicity associated with the conditioning agents (ballen and spitzer, 2010; gyurkocza et al ., ric was first developed to enable allogeneic hsct in patients with advanced hematological malignancies who cannot withstand myeloablative conditioning because of age and/or performance status . Ric protocols also enable the use of hsct in patients with non - malignant disorders, associated with longer life expectancy . In these patients the aim of the conditioning prior to the allogeneic hsct is merely to support sustained donor cell s engraftment for correction of the disease (steward and jarisch, 2005; ringden et al ., 2006), or, as demonstrated in limited number of patients, for the induction of donor chimerism, as a platform for tolerance induction as prelude to organ transplantation (kawai et al ., nevertheless, currently used ric protocols are still relatively aggressive and therefore it is highly desirable to develop safer preparative regimen protocols that selectively achieve a state of donor - specific unresponsiveness without compromising the overall immune response . One way to achieve a state of donor - specific tolerance uses donor cells endowed with veto activity . The term veto, coined in 1980 by miller (miller, 1980), relates to the ability of cells to specifically delete t cells directed against antigens (ags) of the veto cells themselves, but not against third - party ag (muraoka and miller, 1983; claesson and miller, 1984). The suppression of effector t cells directed against the veto cells is both ag - specific and major histocompatibility complex (mhc) restricted, resulting from the unique manner by which the veto cell kills its target . Thus, veto activity results from unidirectional recognition of the veto cell by the responding t cell, but not vice versa . Therefore, the recognizing t cell, the t cell receptor (tcr) of which is directed against the mhc of the veto cell, is killed upon binding to its veto target, due to exchange of signals allowed following this interaction . Hence, the use of donor veto cells capable of specifically eliminating only the host anti - donor t cell clones that mediate the transplant rejection while sparing other t cells that can persist and fight infectious pathogens, could offer an effective modality for the induction of transplantation tolerance . Initially, veto activity was described for cells within the spleen of athymic nude mice (miller, 1980). Based on this initial observation, various cell types have been shown to mediate veto activity including t lymphocytes, natural killer cells, and dendritic cells . A very strong veto activity was documented for cd8 cytotoxic t cell (ctl) lines or clones (fink et al ., 1984; claesson and ropke, 1986; claesson and miller, 1989) and direct comparison of the veto reactivity of various cell types revealed that ctls have the strongest in vitro veto effect (reich - zeliger et al ., cells in murine bm and in t cell colonies grown from such bm were shown to mediate veto activity in vitro (muraoka and miller, 1980) and un - separated donor bm was shown to specifically reduce the frequency of anti - donor ctls in grafted mice (wood et al ., 1992). However, this in vivo tolerizing activity of the bm cells could be attributed to t cells which reside within the bm . These t cells, while potentially mediating beneficial veto activity, also cause a severe multi - system graft versus host disease (gvhd). Early attempts to avoid gvhd risk and to apply t cell - depleted bmt (tdbmt) in leukemia patients indeed revealed that this benefit of gvhd prevention is offset by increased risk for graft rejection, due to absence of donor t cells within the graft (gale and reisner, 1986; kernan et al ., 1987). However, veto activity could be also assigned to non - t cells within the bm . For example, a series of studies by the group of judy thomas described potent veto activity of cells within the cd8cd16dr subset in the bm of rhesus macaque primates (thomas et al ., 1991; cd8 surface expression was shown to play a pivotal role in the tolerogenic effect of these bm cells . Thus, these studies demonstrated that cd8 crosslinking following interaction with donor - reactive ctl precursors (ctlp), elicits upregulation of transforming growth factor-1 (tgf-1) and fas ligand (fasl) by these donor bm cells, leading to clonal deletion of the donor - reactive ctlp (asiedu et al . (1998) that human hematopoietic cd34 progenitors are endowed with marked veto activity (figure 1). Explaining in part how megadose of purified cd34 cells enables to overcome rejection in recipients of three hla - loci mismatched (haploidentical) hsct while avoiding the threat of gvhd (aversa et al . (2005) demonstrated that this veto activity is mediated through a tnf- based mechanism . (2002) demonstrated that veto activity is not only mediated directly by the infused cd34 cells but also by their cd33 progeny, which lose this tolerizing activity upon completion of maturation, at the level of cd14 monocytes or cd13 neutrophils . Furthermore, preliminary results suggest that bm - derived immature dendritic cells, previously shown to induce immune tolerance, exhibit marked veto activity on cd8 t cells, in addition to the non - specific suppression of cd4 t cells mediated by the no system (zangi et al ., 2009). Finally, nk cells which were shown to exhibit veto activity upon activation with il-2, develop and appear early during the post - transplant period (chrobak and gress, 2001; reich - zeliger et al ., 2004a). The regulatory activity of cd34 cells: evidence for target specificity . The average ctl response (sd) in the presence (black bars) or absence (white bars) of cd34 cells at a veto - to - responder cell ratio of 0.5 . The veto effect was tested by a limiting dilution assay as follows: equal numbers (1 10/ml) of responder cells and irradiated allogeneic stimulator cells from the donor of the cd34 cells and a third - party donor were co - cultured for 5 days . The responder cells were then cultured again for 7 days under limiting dilution, and the ctl activity was determined by cr - release assay . Data represent the average sd of 11 independent experiments using different donor and third - party pairs . A significant difference (p <0.001 on t - test compared with control cultures without cd34 cells) between control cultures and those including cd34 cells was found upon stimulation against donor cells (rachamim et al ., 1998). Thus, based on these observations, the following working hypothesis can be suggested to explain how megadose of cd34 cells can overcome rejection in human recipients of haploidentical hsct . Upon administration of purified cd34 cells, the graft supporting veto activity is initially mediated directly by the infused cd34 cells, and subsequently by the cd33 progeny of these cells which grow exponentially within the first few days post - transplant . This second phase of differentiating veto cells also includes cd11c+ immature dendritic cells and other graft facilitating cells . Clearly, the number of all these tolerizing cell types emerging after transplantation is proportional to the number of cd34cells infused . The increased engraftment of megadose of hsct is therefore greatly dependent not only on the ability of the initial inoculums of the cd34 cells to veto anti - donor t cells, but also on their ability to seed the bm and to generate as rapidly as possible the second or third derivatives which are required to complete the eradication of host anti - donor t cells . As described above, the use of purified megadose of cd34 hsct has enabled haploidentical transplantation in leukemia patients and was the first demonstration of the potent clinical potential of donor veto cells . However, this approach is currently limited to supra - lethal myeloablative and highly immunosuppressive conditioning protocols (martelli et al ., 2002). Indeed, studies in non - human primates revealed that any significant reduction of the conditioning, to levels acceptable for elderly patients or for patients with non - malignant disorders, would require veto inducing cd34 stem cell numbers which cannot be realistically collected from human donors (gan et al . Therefore, other populations of veto cells could have a crucial role in supporting and promoting successful engraftment of purified stem cell transplantation under relatively safer ric . As outlined above, cd8 ctls were shown in vitro to exhibit the strongest veto reactivity (reich - zeliger et al ., 2004a). Previous insights on the veto mechanism of cd8 veto ctls, combining anti - cd8 blockade and fasl - mutated veto cells, have suggested that co - expression of cd8 and fasl is required for the veto activity of these cells (reich - zeliger et al ., 2010,2004a). Such a mechanism involves initial recognition of the veto cell by the tcr of the effector t cells, leading to expression of fas by the effector t cell upon activation, and thereby enabling fas fasl mediated apoptosis to take place, once inhibitory molecules such as flice - inhibitory protein (flip) are down regulated in the effector cell (reisner et al ., 2006). In addition, the interaction between cd8 on veto ctl and the mhc class i alpha3 domain on the effector cell, is associated with phosphorylation of mek / erk in the latter cell, and with a significant reduction of x - linked inhibitor of apoptosis protein (xiap) levels which, in turn, enables even more potent triggering of fas fasl mediated apoptosis in the recognizing effector cell scheme 1 reich - zeliger et al ., 2010). More recent results have indicated that veto activity exhibited by ctls can also be mediated by an additional perforin based mechanism (milstein et al ., 2010). However, despite their potent veto activity, cd8 ctls cannot be used for safe tolerance induction in allogeneic hsct because of their marked gvh reactivity . Veto - based deletion of host anti - donor cd8 t cell clones by veto ctl . Cd8-mhc class i engagement induces perk, which is associated with reduction of xiap (apoptosis inhibitor) levels, thereby enabling fas one approach for generating veto ctls with reduced gvhd reactivity has been described by a series of studies by d. h. fowler (fowler and gress, 2000; erdmann et al ., 2004; mariotti et al ., 2008). In these studies, it was demonstrated that by using cd3/cd28 particles under il-2-, il-7-, and il-4-containing medium, veto ctls with tc2 phenotype can be generated . We have previously described an alternative approach to generate ctls with highly reduced gvh reactivity by means of stimulation against third - party stimulators in the absence of exogenous cytokines, followed by further ex vivo expansion using third - party stimulators and il-2 (bachar - lustig et al ., 2003). This approach was based on the observation that only activated ctlp are capable of surviving the cytokine deprivation in the primary culture, and that these anti - third - party clones further expand throughout the culture . These anti - third - party ctls indeed were shown to be depleted of gvhd while supporting bm engraftment in murine models . In accordance with the veto concept thus, a ctl line originating from a strain other than that of the bm donor failed to prevent graft rejection (bachar - lustig et al ., 2003). Importantly, anti - third - party veto ctls, upon adoptive transfer into tdbmt recipient mice, were shown to eliminate not only host anti - donor nave cells, but also host anti - donor memory cells (reich - zeliger et al ., 2007). Memory t cells, derived from prior exposure to alloantigen or generated by heterologous immunity or lymphopenia - induced proliferation, are believed to be an important part of the barrier preventing the translation of tolerance induction protocols from inbred rodent strains to the clinic (pantenburg et al . Therefore, the ability of veto ctls to overcome memory t cells - mediated rejection could be highly important for their action in clinical settings . Furthermore, a recent study of nguyen and geiger (2010) propose that veto ctl can also effectively promote b cell tolerance . Thus, in this study the authors demonstrated that murine cd8 ctls, in addition to their renowned veto activity upon recognizing t cells, can also induce ag - specific elimination of recognizing b cells, both in vitro and in vivo and thus veto ctls may selectively overcome both cellular and humoral graft rejection . Nevertheless, despite all these important attributes of the veto cd8 ctls, their in vivo activity was shown to be markedly inferior compared to that exhibited in vitro, requiring the administration of large number of ctls in conjunction with the immunosuppressive drug rapamycin in order to efficiently overcome tdbmt rejection in murine models (bachar - lustig et al ., 2003). The discrepancy between the ctls veto activity in vitro and in vivo could be explained when considering two limitations of the veto cells: first, the veto activity is mediated through cell to cell contact and, second, host t cells are prone to veto mainly in a window of opportunity of up to 48 h after their activation, thus, once these host t cells develop into mature effector t cells, veto cells can no longer exert their effect (anderson and zimring, 2006). Therefore, while in vitro the veto ctls are directly plated with their cognate targets, upon infusion in vivo the veto ctls need to co - localize with the host t cells within the first 48 h of the rejection process, or else their effect will be hampered . Indeed, we recently demonstrated that ctls attained upon long ex vivo culture in the presence of il-2 exhibit a migration pattern different from the one displayed by naive host t cells thereby precluding their co - localization . Thus, while nave host t cells home efficiently to the lymph nodes (lns) of bmt recipient mice, the veto ctls are excluded from the lns and tend to localize in peripheral sites (ophir et al ., in order to improve the lns homing potential of anti - third - party cd8 veto cells we developed a new protocol, using il-15, that favor the induction of central memory phenotype in anti - third - pary cd8 t cells (ophir et al ., 2010). Central memory t cells (tcm) express the ln homing receptors cd62l and ccr7 (sallusto et al ., 1999) and similarly to naive t cells, localize to the t cell area of all secondary lymphoid organs (weninger et al ., 2001). Indeed, we demonstrated that these ex vivo induced anti - third - party tcm, in contrast to anti - third - party ctls, home to lns of bmt recipients, where they co - localize with the recipient s endogenous host t cells . Moreover, the tcm displayed strong proliferation at the early post - transplant period and subsequently persisted in vivo for more than 1 year post - bmt, in line with their memory phenotype (ophir et al . Most importantly, we demonstrated that tcm derived from (host donor)f1 mice can specifically and efficiently delete in vivo host tcr - transgenic t cells carrying a tcr transgene with anti - donor specificity . In accordance with the veto concept, this efficient in vivo deletion of anti - donor host t cells, found to be mediated by apoptosis, did not occur when non - specific tcm, not expressing the donor h-2 haplotype, were used (figure 2). Thus, murine tcm perform in vivo veto activity, efficiently deleting host t cells only when the host t cells recognize antigens on the tcm, and not by a non - specific general immunosuppressive effect . Taken together, all these attributes of the tcm were shown to translate into improved efficacy in overcoming t cell mediated rejection of murine tdbmt, thereby enabling high survival rate and long - term donor chimerism, without causing gvhd . Thus, adoptive transfer of anti - third - party tcm, in the absence of rapamycin treatment significantly abolished host t cells - mediated rejection of fully mismatched nude - bm . This is in sharp contrast to anti - third - party ctls, which fail to enable engraftment unless administered in higher numbers and in conjunction with rapamycin treatment (bachar - lustig et al we concluded therefore that by generating anti - third - party cd8 cells with a central memory phenotype we were able to dramatically enhance their tolerizing veto activity in vivo . (a, b) lethally irradiated c57bl/6 (h-2) mice received 1 10 cd8 purified tcr - transgenic 2c cells (carrying tcr with anti h-2 specificity) and 5 10 irradiated balb / c (h-2) splenocytes . The following day, the mice were transplanted with 1 10 c57bl/6-nude bm cells or received, in addition, 5 10 specific, derived from cb6 (h-2, black bars), or non - specific, derived from c57bl/6 (h-2, gray bars) anti - third - party tcm . Recipients were sacrificed 8 days post - transplant, their spleens were harvested, and the deletion of anti - donor 2c t cells was monitored by facs . (a) representative result demonstrating the level of surviving (7aad) 2c cells in the absence (left panel, 2c alone) or presence of specific tcm (right panel 2c + specific tcm). (b) quantification of results demonstrating efficient inhibition of the 2c cells only by data represent average sd of percent inhibition from at least 10 animals in each group, pooled from two independent experiments . (c, d) in vivo model was established as in (a, b), but 5 10 purified cd8 2c cells and 2.5 10 irradiated balb / c splenocytes were administrated . Recipients were sacrificed 8 days post - transplant, their spleens were harvested and facs analysis of annexin v levels on living (7aad) cd81b2 + 2c cells was conducted . (c) representative result demonstrating apoptosis induction upon anti - donor 2c cells by the tcm as evident by annexin v levels on 2c cells in the absence (left panel, 2c alone) or presence of specific tcm (right panel, 2c + specific tcm). (d) quantification of results measuring annexin v levels on the 2c cells following interactions with specific and non - specific tcm . Data present average sd of percent annexin v levels in at least four animals from each group, in one representative experiment, out of three performed . * * p <0.01, * * * a second objective which could be attained by administration of donor - derived activated cd8 t cells in the context of hsct is related to the ability of these cells to control residual hematological malignancies . Considering that gvl is generally associated with gvhd it was surprising that autologous or allogeneic human and murine anti - third - party veto cd8 t cells, markedly depleted of gvh reactivity, exhibit vigorous responses against different b cell malignancies (arditti et al ., 2005; lask et al ., 2011). The killing of b cell tumors by anti - third - party ctls was shown to involve a unique tcr - independent two steps mechanism . First, long - lasting conjugates are formed between the ctl and the tumor cell . These conjugates are rapidly formed through binding of icam1 on tumor cells by lfa-1 expressed on the veto ctl . Second, a slower process of mhc class i - dependent apoptosis is mediated by binding of the mhc class i 2/3 constant region on the tumor cells to the cd8 molecule on the ctl membrane (arditti et al ., recent results from our laboratory indicate that murine anti - third - party veto tcm can also efficiently eliminate murine b cell lymphoma in vivo, through a similar tcr - independent mechanism (lask et al ., 2010). Hence, such single agent veto cell therapy has the potential to have a double benefit in the context of hsct, namely, promoting and supporting hsct engraftment through specific tolerizing veto activity while mediating effective anti - cancer response, in the absence of gvhd . Translation of this therapy, optimizing protocols for the generation of human anti - third - party tcm is now in progress and clinical evaluation will commence in the near future in patients with b cell malignancies . Donor derived veto cells represent an attractive and effective modality for the induction of specific tolerance toward donor ags . This approach has been already demonstrated in patients by using megadose of purified veto cd34 stem cells, that can overcome the host s residual immunity surviving the myeloablative conditioning and enable engraftment of hsct across major genetic barriers without the severe threat of gvhd . However, the number of veto cd34 cells that can be harvested is insufficient for overcoming the large numbers of host t cells remaining after ric . Therefore, combining megadose of cd34 hsct with other gvhd - depleted veto cells, potentially anti - third - party cd8 tcm, could enable facilitation of engraftment of hsct under ric without the adverse complication of gvhd - producing t cells and without the need for deleterious post - transplant gvhd prophylaxis . It is hoped that this approach could extend the use of hsct to elderly patients with b cell malignancies who cannot tolerate intensive protocols and to a variety of patients with non - malignant disorders, associated with longer life expectancy, in whom the current trm associated with hsct may not be ethically justified . Use of experimental animals statement: all experiments on live vertebrates were performed in accordance with the weizmann institutional animal care and use committee.
Studies have shown that laparoscopic cbd stone retrieval is as efficient and effective as endoscopic retrograde cholangiopancreatography (ercp). Usually, the transcystic approach is considered the first treatment of choice for laparoscopic cbd exploration because transcystic cbd exploration has more benefits than the choledochotomy approach . In the laparoscopic setting, it would be difficult for surgeons to manipulate the choledochoscope into the cbd from a small opening in the cystic duct . To overcome this shortcoming from april 2010 to june 2012, 9 consecutive patients diagnosed with cholelithiasis and cbd stones were enrolled in this study . There were 2 men and 7 women, with a median age of 57 years (range, 4371 years). The inclusion criteria were as follows: no upper abdominal surgical history, stones measuring <5 mm, no intrahepatic duct stones, number of stones <3, normal liver function test results, no jaundice, and normal leukocyte count . All the patients were fully informed about the characteristics of the procedure and its advantages over conventional choledochotomy exploration and 2-stage minimally invasive procedures . The patient was transferred to the operating room for laparoscopic management of gallbladder stones and cbd stones and placed in the supine position . At the time of general anesthesia, prophylactic antibiotic the first assistant, who was in charge of handing the laparoscope, stood at the surgeon's left . The procedure was carried out using a 4-trocar cholecystectomy technique . Instead of a 5-mm trocar, a 12-mm trocar was inserted about 3 cm from the right costal arch, at the midclavicular line (figure 1). After dissection of the calot triangle, the cystic artery and cystic duct came into view . Once the cystic artery was transected, dissection of the gallbladder from its bed was begun at the fundus and continued to the body and infundibulum . While the gallbladder was dissected free from the liver connections, the fundus of the gallbladder was extracted via the 12-mm port incision carefully under laparoscopic vision (figure 2). After a small incision was made in the fundus of the gallbladder extracorporeally, a suction device was inserted into the gallbladder cavity, aspirating the bile juice . A flexible choledochoscope was introduced into the gallbladder through the opening in the fundus of the gallbladder and gently advanced toward the cystic duct under the guidance of both laparoscopic imaging and endoscopic imaging (figure 3). Before introduction of the choledochoscope into the cbd, intraoperative cholangiography was performed to confirm the diagnosis of cbd stones and provide information about the number, size, and location of stones, as well as the anatomy of the cystic duct and cbd . In most situations, the cystic duct needs to be dilated by the choledochoscope itself or by a dilator for passage of the choledochoscope . By use of both endoscopic and laparoscopic imaging, the cbd was thoroughly examined, and the stones were retrieved under direct choledochoscopic vision (figure 4). Finally, the cystic duct was closed with clips or surgitie (covidien, mansfield, massachusetts), and the gallbladder, within a retrieval bag, was removed from the abdomen through the 12-mm port . Liver function tests and assessments of the amylase level and leukocyte count were performed in patients postoperatively . Successful transcystic cbd stone clearance was achieved in 7 of 9 patients, whereas treatment failure occurred in the other 2 patients . The reasons for failure were a narrow cystic duct in 1 patient and the cystic duct joining the cbd on the left side in the other patient . The duration of the operation in the 7 patients with successful transcystic cbd stone clearance was 126 minutes (range, 96141 minutes). Postoperative ercp was successfully performed in the 2 patients in whom failure occurred . No bile leakage, hemobilia, abdominal bleeding, or pancreatitis occurred in our series . Transient epigastric colic pain occurred in 2 patients and was relieved by use of anisodamine . A transient increase in the amylase level was observed in 3 patients, and the amylase level returned to normal on postoperative day 3 without any treatment . The external drainage tube was removed 48 hours postoperatively in the absence of surgical complications . Finally, the patients were discharged home on day 4 postoperatively once we were completely assured that the operation was successful and no complications had occurred . Short - term follow - up (median, 23 months; range, 1640 months) showed no recurrence of cbd stones by use of magnetic resonance cholangiography . In the era of minimally invasive surgery, various options for the treatment of cbd stones in patients with gallbladder stones are available, including ercp plus laparoscopic cholecystectomy, open surgery, and 1-stage laparoscopic cholecystectomy and cbd exploration . Recent reports have shown that laparoscopic clearance of cbd stones is as efficient and effective as that with ercp and can avoid the potential complications of ercp, such as cholangitis, pancreatitis, duodenal perforation, and bile duct injury . So, laparoscopic cholecystectomy with simultaneous transcystic cbd exploration has gained wide acceptance for 1-stage laparoscopic management of cholecystochodocholithiasis . There are 2 types of laparoscopic cbd exploration: the transcystic approach and the choledochotomy approach . More and more surgeons prefer the transcystic approach to choledochotomy for cbd stone clearance because the transcystic approach can avoid an incision in the cbd wall . To carry out laparoscopic transcystic cbd exploration, a small incision has to be made in the cystic duct . The key step for successful transcystic cbd exploration is introduction of the choledochoscope into the cystic duct . In the laparoscopic setting, surgeons often confront difficulties in manipulating the choledochoscope into the small cystic duct through its partial opening . To overcome these shortcomings, we developed a novel transcystic approach that would facilitate introduction of the choledochoscope into the cystic duct and then its advancement into the cbd . A narrow cystic duct and the unfavorable anatomy of the junction of the cystic duct and cbd resulted in losing access to the cbd and were responsible for the failure of the novel transcystic approach . No difficulties were met in the process of retracting stones in the selected patients . Except for 2 wound infections in the 12-mm port, no other complications were observed . We attributed the results to our inclusion criterion requiring stones to measure <5 mm . In the future, we would expand this technique to acute inflammatory settings, impacted stones, or stones measuring> 5 mm . In a recent study, chiarugi et al showed that laparoscopic transcystic exploration for single - stage management of common duct stones and acute cholecystitis was a simple technique with a high yield of cbd clearance in the acute setting because the dissection of the calot triangle is facilitated by the edema . Laparoscopic cbd exploration via a transcystic approach together with holmium laser lithotripsy can solve impacted or large solitary stones . Laser lithotripsy can serve as an additional tool for the laparoscopic surgeon when confronted with impacted or large stones . Laparoscopic cholecystectomy with simultaneous laparoscopic cbd exploration offers the advantage of avoiding an extra procedure and the potential complications of ercp . Our results, along with other reports, have shown that single - stage laparoscopic cbd stone retrieval was feasible and efficient . Our novel technique provides an alternative transcystic approach for single - stage laparoscopic cbd exploration . We believe that more and more cbd stones can be treated by the transcystic approach with advancements in equipment and surgical skill.
Abiotrophia defectiva is part of the normal human microbiota, colonizing the oral, genitourinary, and intestinal tracts . It is a rare, yet important, cause of infective endocarditis, and is estimated to cause approximately 5 - 6% of all cases of infective endocarditis, including being a major cause of blood culture - negative infective endocarditis . It affects diseased valves in 90% of cases and it is implicated in embolic complications and valvular destruction, despite being sensitive to antibiotics . Previous studies have shown mortality and relapse rates as high as 17% despite antibiotic treatment, and this makes accurate and quick identification important . Herein we report the first case of infective endocarditis caused by a. defectiva in korea . A 62-year - old female was admitted to the emergency department (ed) after two consecutive episodes of syncope . The patient was diagnosed with severe rheumatic mitral stenosis, and underwent a mitral valve replacement (mvr). Two months previously, the patient had undergone a simple extraction of her #16 tooth due to secondary dental caries and had taken prophylactic antibiotics (amoxicillin 2,000 mg). Upon admission to the ed the patient was alert and had a body temperature of 38.6c, a pulse rate of 66 beats / min, and blood pressure of 149/51 mmhg . Upon physical examination, no cardiac murmur was auscultated and no other evidence, such as clubbed fingers, laboratory studies showed a white blood cell count of 14,260/mm(neutrophil 79.6%), erythrocyte sediment rate of 92 mm / h and c - reactive protein of 111.1 mg / l . Chest x - ray revealed mild cardiomegaly and electrocardiography showed a newly developed complete atrioventricular block . Chest ct and abdominal - pelvic ct was performed to evaluate fever focus, which showed no significant finding . A transthoracic echocardiogram (tte) showed that the mechanical prosthetic mitral valve functioned well, and there was no visible vegetation, however, infective endocarditis could not be completely ruled out because of the patient s previous mvr . Thus, empirical antibiotics (vancomycin 1 g intravenous q12hr, gentamicin 60 mg intravenous q8hr and rifampin 600 mg per oral q24hr) were administered, and a transesophageal echocardiogram (tee) performed the day after admission showed mitral valve vegetation and mild transvalvular mitral regurgitation (fig . 1), leading to a conclusive diagnosis of infective endocarditis according to duke criteria (one major and three minor criteria). (a) long - axis view (135) of a transesophageal echocardiography showing two examples of vegetation attached to the prosthetic mitral valve . The larger one measures 0.7 0.5 cm (large arrow head) and the smaller one measures 0.5 0.3 cm (small arrow head). (b) two - chamber view (59) of a transesophageal echocardiography (zoomed view) showing vegetation attached to the prosthetic mitral valve (arrowhead). A color doppler shows the jet flow with mild transvalvular mitral regurgitation in systole (arrow). Three sets of blood cultures taken on admission showed tiny, non - hemolytic colonies on a blood agar plate (asan pharmaceutical, hwaseong, korea), and pleomorphic gram - positive cocci from smear preparation of the blood agar plate (fig . A. defectiva was identified by maldi - tof - ms (matrix - assisted laser desorption / ionization time - of - flight mass spectrometry, bruker daltonics inc ., ma, usa), not identified in vitek 2 (biomrieux, marcy l'etoile, france) systems . However, because the amount of bacteria was insufficient, cultures were sub - cultured on a medium containing vitamin b6 to assess antibiotic susceptibility . Tiny, non - hemolytic colonies on a blood agar plate (asan pharmaceutical, hwaseong, korea) after 48 hour of incubation at 35c with 5% co2 (left), and pleomorphic gram - positive cocci from smear preparation of the blood agar plate (gram stain, 1,000, right). Susceptibility to cefotaxime, penicillin g and vancomycin was tested by the e - test method; the isolate tested cefotaxime - sensitive (mic 0.75 g / ml), penicillin g - intermediate (mic 1.0 g / ml) and vancomycin - sensitive (mic 0.38 g / ml). An adjusted antibiotic regimen of vancomycin 1 g q12hr and gentamicin 60 mg q8hr was initiated, after which the patient s fever subsided . Vancomycin therapeutic drug monitoring was performed, and vancomycin dosage was adjusted to 400 mg q12hr . On day 12 of admission, the patient became feverish once more and in the subsequent tee the mitral valve vegetation on the medial side seemed to have resolved, but there was an increase in the size of the vegetation on the lateral annulus (0.5 0.3 cm 0.8 0.3 cm) which indicated a perivalvular infection (fig . Operation finding showed small vegetation in prosthetic mv and annulus, which was removed, and mvr was conducted using 23 mm ats medical open pivot heart valve (ats medical, inc ., pathology finding showed myxoid degeneration, acute and chronic inflammation, fibrosis and calcification, and tissue culture showed no growth of organism (it was on the 19 day on antibiotics, and blood culture was negatively converted at this time). On day 23 of admission, the patient was treated with antibiotics for 5 weeks and was discharged on day 36 . The patient was followed - up at an out - patient clinic for 3 months after discharge with no significant complications . Organisms of the genus abiotrophia were first classified as nutritionally variant streptococci (nvs) in 1961 . Bouvet et al . Identified two species of nvs; streptococcus defectivus and streptococcus adjacens . In 1995, kawamura et al . Nvs are part of the normal flora of the mouth and urogenital and intestinal tracts . Recently, an increasing number of cases have been described of a. defectiva isolates recovered from invasive and non - invasive infections following dental procedures . Has fastidious culturing and the unspecific colony morphology that they presents on primary detection, such strains have caused major diagnostic difficulties . Thus, it has been supposed that many culture - negative endocarditis could have been caused by these species, which could have lead to an underestimation as pathogens of infective endocarditis . In this case report maldi - tof - ms is a new technology for routine identification of bacteria in clinical microbiology laboratories . Much of the work using maldi - tof - ms for microbial identification has focused on demonstrating that reproducible mass spectra can be obtained using intact cells and developing algorithms for interpretation and comparison of these spectra . By testing colonies, it takes only a few minutes to have a correct identification which makes not only possible to identify the microorganisms at the species levels but sometimes at the sub - species and strains levels, allowing the detection of epidemic lineages . A. defectiva can cause serious infections such as bacteremia, osteomyelitis, brain abscess, pancreatic abscess, septic arthritis, crystalline keratopathy and in rare cases, infective endocarditis . Endocarditis caused by nvs is implicated in 56% of all streptococcal endocarditis cases, and <1% of all endocarditis cases are caused by a. defective . However, a. defectiva has a higher affinity for the endocardium because of its ability secrete exopolysaccharide, enabling it to adhere to fibronectin in the extracellular matrix . Of all the nvs - induced endocarditis patients, 90% suffered from heart disease, and in most cases, bacteremia associated with the pre - existing valvular heart disease led to the development of endocarditis with subacute prognoses . Infective endocarditis caused by a. defectiva and other nvs had higher mortality, morbidity and complication rates than those caused by other viridans streptococci . Most deaths were due to refractory congestive cardiac failure or major systemic emboli . A mortality rate of up to 17% has been reported, which is higher compared to endocarditis caused by viridians streptococci (012%). Previous studies have shown a treatment failure rate as high as 41%, despite the use of appropriate antibiotics . The antibiotic regimen to combat a. defectiva endocarditis includes penicillin or ampicillin, plus an aminoglycoside or vancomycin for cases of antimicrobial resistance, taken for 46 weeks . Infective endocarditis due to a. defectiva progresses slowly, but despite its sensitivity to antimicrobials ~50% of cases need surgical management . Surgical treatment combined with concurrent antimicrobial therapy result in better prognoses, and the main indications for surgery are persistent sepsis and vegetation, severe congestive heart failure and recurrent embolism . In korea, 2 cases of infective endocarditis caused by s. adjacens were first reported in 1996 . Since then, a case of infective endocarditis by grnulicatella adjacens was reported in 2010 . After classified as a. defectiva, this is the first report as the pathogen of infective endocarditis in korea . The case presented here is an example of the successful treatment of infective endocarditis caused by a. defectiva following a tooth extraction in a post - mvr patient, and represents the first reported case of infective endocarditis caused by a. defectiva in korea . This case shows that a. defectiva could be considered as a causative organism of infective endocarditis in korea.
The approach usually taken by the general population to phytomedicine is that such a therapy is natural and therefore safe, but several studies have demonstrated that this statement is not true . The relationship between the use of herbal compounds, alone or in combination with traditional drugs, and the occurrence of side effects have been described . Moreover, two cases of acute disseminated encephalomyelitis (adem) after exposure to intravenous herbal extracts have been reported . Herein we report the case of a patient who suffered an acute demyelinating disorder with severe and persistent neurologic impairment (finally leading to a fatal outcome) temporally related to the use of an oral therapy that contained herbal extracts . A 63-year - old woman was admitted to our hospital with a 7-day history of asthenia, postural instability and falls . Her past medical history included hypertension, hashimoto's thyroiditis (with detectable anti - thyroglobulin and anti - peroxidase autoantibodies), benign leukopenia and cervical arthrosis . Fifteen days before the onset of the symptoms she had started a homeopathic treatment for cervical pain . Immunostimulant) consisted of echinacea purpurea 45 mg, uncaria tomentosa 37.5 mg, and tabebuia avellanedae and plantago maritima 30 mg . On admission, neurological examination revealed the presence of left - eye horizontal nystagmus, mild dysarthria, and mild right arm weakness together with both axial and appendicular ataxia . Cranial t2-weighted and flair sequences on magnetic resonance image (mri) showed pseudonodular, subcortical and periventricular bilateral white matter lesions with heterogeneous gadolinium enhancement . High signal on an apparent diffusion coefficient map was compatible with vasogenic edema (fig . Cerebrospinal fluid (csf) analysis showed a mild increase in protein concentration (60 mg / dl, = 45 mg / dl) without pleocytosis . Csf and serum oligoclonal bands, csf vdrl and herpes simplex virus pcr were negative . Antinuclear antibodies were undetectable . On hospital day 6, central right facial nerve paralysis was observed . On the following day, the patient was admitted to the intensive care unit (icu) because of impaired consciousness, and intravenous methylprednisolone was started, due to the presumptive diagnosis of a central nervous system (cns) demyelinating process . The patient developed generalized tonic clonic seizures requiring anti - convulsant therapy, sedation and mechanical ventilation . Repeat mri showed progression of the previously described lesions and involvement of the cerebellum (fig . Computer tomography (ct)-guided stereotactic biopsy was performed, and pathology demonstrated features compatible with acute demyelination (fig . The biopsy specimen was reviewed for the presence of an immune cell infiltrate using immunohistochemical markers for t cells (cd3) and b cells (cd20). The tissue harbored a considerable immune cell infiltrate including significant numbers of t cells that were distributed throughout the tissue (fig . B cells were fewer in number than t cells and more sparsely distributed (fig ., oral steroids, five plasma exchange sessions and 120 g immunoglobulin i.v ., without major improvement of the symptoms that required treatment in our icu . The patient was discharged to a rehabilitation facility; she was quadriparetic, required help in daily activities, and was fed through a percutaneous endoscopic gastrostomy tube . Her level of consciousness was impaired and she was barely able to connect with her family members . A follow - up brain mri at 8 months showed atrophy and scarring; no new lesions had occurred (fig . To our knowledge, this is the second report that links the use of herbal extracts and demyelinating disorders of the cns, and the first with oral phytotherapy . Up to three quarters of the monophasic demyelinating disease of the cns may be regarded as post - infectious or post - immunization processes . Typically, the latency until the onset of neurological symptoms is 7 - 14 days after the causative insult . In our case, the patient had started the herbal extract (putative triggering event) 2 weeks earlier . Although our case showed many features compatible with adem (evidence of demyelination, monophasic nature, absence of oligoclonal bands in csf and grey matter involvement), the unremitting, progressive and persistent severe sequelae made us view it as an acute, otherwise unclassified immune - mediated demyelination event [5, 6]. The finding of foamy histiocytes, together with an intense t cell (and to a lesser extent b cell) infiltrate within the lesions, points to an autoreactive immune mechanism towards myelin as the main pathogenic mechanism . Many immunological players have been proven to take part in its pathophysiology, including th1, th17, regulatory t cells, antigen presenting cells and b cells, among many others . However, the exact factors that trigger autoimmunity have not yet been identified; ambient insults are thought to play a major role . Experimental data has shown that echinacea may have immunostimulant properties, such as an increase in cytokine production by human macrophages, but these results have not convincingly been proven in clinical studies . On the other hand, numerous adverse immune reactions, including anaphylactic responses, urticaria, lfgren's syndrome, vasculitis, and erythema multiforme, have been described . Immunostimulatory herbal supplements has also been reported . The long tradition and presumed natural origin of herbal medicines do not translate to a guarantee for treatment safety . Currently, more than 1,000 plants are sold worldwide, although clinical trials have been published only for 156 plants supporting specific pharmacological activities and therapeutic applications . Furthermore, the largest study on this topic shows that severe adverse reactions (hospitalization, life - threatening clinical events and death) account for over one third of the undesired effects . It is difficult to argue that the immune - mediated demyelinating disease detailed here was entirely due to the patient's use of herbal extracts . However, both previous reports and temporal concomitance led us to the conclusion that such an association may be in part true and should be taken into account in the evaluation of future patients . We, as well as others, believe in the urgent need of regulatory controls, scientific evaluation and active pharmacovigilance in phytomedicine in order to avoid undesired and often unpredictable side effects.
While breast cancer has long been recognized as a major public health burden in high - income countries, the majority of cases actually occur in low- and middle - income countries (lmcs), and it is expected that incidence rates will rise most rapidly in these locations . The relative burden of mortality is also higher in less developed countries than in more developed countries, as indicated by higher mortality: incidence ratios (0.44 versus 0.29, resp . ). Current global initiatives focus on developing and implementing resource - appropriate guidelines and strategies to improve breast health care and breast cancer outcomes in lmcs [35]. Common challenges cited for resource - poor countries include limited health care infrastructure, later stages at diagnosis, and competing health care priorities . The purpose of this paper is to examine the potential for community health worker (chw) programs to improve access to breast health resources in lmcs . To this end, we briefly review the effectiveness of chw programs in lmcs and identify key components of a chw - based breast health program . We focus on south africa as an example country to assess the feasibility of such a program . South africa represents a middle - income country, according to its world bank classification, has a growing cancer control infrastructure including many of the cancer centers in africa, with well - trained oncologists and radiologists [9, 10], has historical experience with community - based health worker programs [11, 12], and has a higher breast cancer mortality: incidence ratio compared to the world standard (table 1), indicating lower survival from breast cancer [2, 13]. Importantly, it also enjoys advocacy from its first lady, her excellency madam tobeka stacie madiba - zuma, who serves as vice chairperson of the newly - formed forum of african first ladies against breast and cervical cancer . Many women do not have access to the information and screening necessary to prolong survival, as evidenced by the high mortality: incidence ratio in south africa . These factors illustrate both the need and potential assets for a successful chw breast health program . Data from south africa's national cancer registry (ncr) show breast cancer as the leading cancer among women . South african women have a 1 in 29 lifetime risk of developing breast cancer, with an age - standardized incidence rate of 30.6 per 100,000 population . These rates vary by race group, with black women having the lowest (16.3) and white women the highest (69.4) rates of breast cancer diagnosis . The ncr is a pathology - based, rather than a population - based registry these statistics belie marked disparities in stages of cancers at diagnosis, survival rates and overall breast cancer in south africa . However, the indication of disparities along racial lines adds urgency to the call for expanded access to breast cancer screening, diagnostic services, and treatment, particularly through community - based approaches . The cancer control continuum is a commonly used public health framework that describes the various stages at which potential programs or interventions can be developed to improve cancer outcomes for population groups (figure 1). For chws to intercede with the objective of reducing stage of breast cancer at diagnosis and increasing survival, target areas along the cancer continuum should be early detection, diagnosis, and treatment . Below we present evidence to develop a program model for a successful breast health program focusing on these areas along the continuum . Community health workers are members of the communities where they work, should be selected by the communities, should be answerable to the communities for their activities, should be supported by the health system but not necessarily a part of it the most frequently identified roles of chws are health education, health services provision, and patient navigation and support . Health education is one of the most common roles of chws in all types of settings [11, 12, 1921]. Patient navigation helping patients find their ways through health systems to ensure timely screening, diagnosis, and treatment is also mentioned frequently as a role for chws (e.g.,) and has been successfully employed in the field of cancer . Because they work in their own communities, chws presumably have a shared life experience and understand the sociocultural context in which health services are received and health behaviors occur [11, 1921]. Many other terms are used to describe chws, including lay health workers, village health workers, or community care workers, specifically in south africa . Chws have an extensive history of action in communities throughout the world, but especially in lmcs . One important aspect of chw programs is the necessity of integration with the community [12, 21]. As highlighted in the who report, more important is an acknowledgement that the definition of chws must respond to local societal and cultural norms and customs to ensure community acceptance and ownership [21, page v]. In this same report, the authors note that the chw literature is unanimous in saying that the communities need to assume ownership for chw programs to work successfully and that such programs work best when the community has a strong investment in the program . In many areas, chws are the only source of health services . These services include, for example, malaria treatment, as indicated in studies about burkina faso, and antiretroviral medication administration . In haiti, researchers from partners in health have concluded that chws have strengthened the health system by providing services to rural communities that would otherwise not be reached . In a review of chws in africa, authors argue that in order to expand health services on the african continent, chws are a necessity . Lehmann and sanders note that the shortage of health workers is significant in places like sub - saharan africa (see) and that these gaps in services could be filled by chws . Several reviews have examined the impact of chw programs throughout the world across a range of health outcomes . In a recent review of 82 randomized controlled trials (rcts) of lay health workers (33% of the studies took place in lmcs), most demonstrated improved health behaviors or health outcomes as a result of chw interventions . In one rct in low socioeconomic communities in south africa, mothers who received this intervention were significantly more sensitive in their infant interactions at both 6 and 12 months (p <.05), and more infants had securely attached to mothers at 18 months (75% versus 63%, p <.05). A descriptive and historical review from the who included approximately 250 citations about chws . Authors concluded that robust evidence supports the positive impact chws can have on health outcomes [21, page 26]. In another review of chw interventions, a case management model for pneumonia led to a 24% reduction in overall mortality under age five across several countries . Apart from the rcts referenced above, a large body of evidence about chw effectiveness is available from observational and descriptive studies, including two studies in south africa highlighting the importance of chws . In a review of a developing national chw program, response by the south african government . In a longitudinal study of antiretroviral therapy in free state province, patients visited by chws at 6 months had significantly increased probability of having cd4 counts higher than 200 cells/l at 1 year (p <.05). Those visited at 12 months, compared to patients without chws, were significantly more likely to be considered treatment successes at 24 months . They argued that chws were part of the untapped community resource available to provide chronic disease care [31, page 1184]. A qualitative review across all provinces of south africa recognized the importance of chws' work . Authors found that chws increasingly provided health services, such as antiretroviral medication administration, in addition to health promoting activities . They helped to expand health services available, especially in impoverished areas of the country, and assisted patients in navigating the health system [12, page 3]. In another qualitative study, chws in kwazulu - natal excelled at identifying community problems because of their connection to community . However, they also quickly named the obstacles to providing services in these locations, raising the need for regular monitoring and support for chw programs . A cross - sectional study in cape town found that women contacted by a chw were more likely to return for a cervical cancer screening visit . Loss - to - follow - up was reduced from 21% to 6% for 6-month visits and reduced by half for 24-month visits . In summary, there is widespread evidence that chw programs can be an effective part of improving community health, particularly in limited - resource areas of the world . Community health worker approaches are proving beneficial in the areas of immunization uptake, breastfeeding, tuberculosis (tb) treatment, and child morbidity and mortality . Given the long history of the use of lay health workers in africa and lessons learned about the successes and challenges of creating effective chw programs in south africa and other lmcs [12, 23], the capacity to expand to breast cancer appears to be feasible . The use of chws should be considered as a key resource - appropriate strategy to bring culturally appropriate breast health services to women . Based on the literature, chws in south africa could assume three primary functions: health education, health service provision (i.e., breast exam), and patient navigation . Integrating breast cancer education into existing health education would be vitally important because knowledge is an essential starting place in establishing the need for breast cancer screening . Awareness varies widely among women [35, 36]; in some settings, breast cancer is stigmatized or considered contagious . Particularly in rural areas of south africa, breast cancer may be understood as a curse or poison sent by a sorcerer . Chws, because of their grounding in the community, are uniquely prepared to understand and acknowledge local beliefs or myths and provide information about causes of the disease, which could help to destigmatize breast cancer . Women are more likely to engage with chws from their own communities, who understand and respect their beliefs and concerns and have earned their trust . In addition to education, chws would also provide clinical breast exams (cbes) as a form of early detection . Although mammography is the standard screening for breast cancer in high - income countries, population - based screening is not feasible in many lmcs due to high costs of the required equipment and personnel . One study in india, for example, found that mammography was not as cost - effective as cbe . Cbe is a low - cost method of screening women for breast cancer in lower resource areas, such as parts of south africa . Cbe has been successfully taught to lay health workers in other settings and used on a large scale, similar to chw cervical cancer screening implemented in rural alaska, u.s.a . . Recent research supports the use of role play as a method of health skills training in limited - resource settings . Chws could be taught breast self - exam and then cbe by practicing on other chws in a the goal of chw - delivered cbe would be to downstage presentation of breast cancer in lmcs [1, 43]. Currently the majority of breast cancers found in africa are in stages iii and iv [44, 45]. Trials of cbe in cairo and mumbai, where cancer is also found at late stages, have shown that cbe as a primary screening tool can be provided by lay persons as a sustainable form of early detection . In malaysia, use of cbe was shown to increase downstaging . Chws can also serve as patient navigators through the continuum of breast cancer screening, diagnosis, and treatment . Bridges to local health systems, chws can be highly effective in assisting patients to maximize their access to existing systems, thereby reducing potential delays in care . Barriers to care in lmcs can include traveling to health centers, lack of affordable services, and cultural challenges with seeking care . For example, chws have successfully acted as treatment buddies in hiv treatment in south africa . As navigators, they provide emotional and logistic support, which can be the crucial element in enabling patients to access any form of care . In one study, rural african - american women, who had lay health advisors contacting them as an intervention, reported an 11-percentage - point increase in mammography compared to women who did not receive the intervention . A recent study from ethiopia illustrates the inefficient multistep health care journey of many breast cancer patients, supporting the need for streamlined patient navigation as a way to save resources and time in lmcs . Although many current chw programs are disease - specific, convincing arguments exist for integrating several health services within one chw program's domain . One economic exercise presented the potential benefits of bundling services for various health concerns together in lmcs . The authors found that packaging services together in this way would expand the possibilities for reaching populations in lmcs because of reduced cost . More recently, in treatment of hiv / aids and tb in africa, authors have argued that chws fit best into an entire community health team, operating most effectively in a generalized, instead of disease - specific, way . Screening for breast and cervical cancer could be fit into regular primary care visits conducted by health workers, such as chws . For example, knaul et al . Have suggested that breast cancer screening could be integrated into existing reproductive health programs . A cost - effectiveness simulation of screening in india, a lower middle - income country, indicated that cbe would have the greatest impact with women ages 4060 compared with ages 5070 . Furthermore, miller argues, based on early findings from the cairo breast screening trial, that all women ages 4069 should receive cbe . Although women with a family history of breast cancer are often targeted in other countries, this approach does not seem feasible in lmcs because of poor record keeping and accuracy of the reporting of breast cancer . Across all discussions of chws, . Such involvement can help to ensure that sociocultural norms and customs are recognized and respected . In the past, south african communities with a vested interest in chw programs benefited most from the programs . These communities participated in identifying their own needs and potential solutions, which increased the success of chw programs . The south african government notes the importance of encouraging community members to define their own needs . Once community members are invested in a chw program, they must be involved in nominating candidates to become chws . Doing so increases the likelihood that chw candidates are respected members of the community who will be effective communicators, educators, and service providers . Some communities in south africa have also incorporated traditional healers into chw programs, which can enhance programmatic success . The longer a chw program is established in a community, the more successful it can become . Maintaining chw programs requires the ongoing support of the community, in addition to ongoing resources and training . Trainings for chws have been especially effective in keeping programs productive and useful to their respective communities . Often in community nongovernment organization (ngo)government partnerships, confusion ensues over responsibilities for funding and program management . According to the south african department of health, governments ideally provide funding through ngos, which in turn employ chws . The most recent draft document from the departments of health and social development describes this approach using the general partnerships within south africa (e.g.,). However, changes in policy and leadership can lead to changes in funding, leaving ngos and chws without the essential resources and supplies to continue their work . Because of this challenge, chw programs that build capacity within communities are more sustainable and rely less on outside funding from ngos or parts of the government . The south african government notes the importance of increasing community capacity . For example, the concept of training up workers so that existing chws take on more skills, such as cbe, builds capacity within the community . Task shifting, in which each level of health worker takes on additional skills, such as a general nurse performing oncology duties . How would such a chw program benefit women and their families? By educating women about breast health, women in the community would be better poised to seek essential services . Because of misconceptions about breast cancer and fears about how it could affect their families, many women do not seek care until it is too late . Women who are diagnosed when their cancer is in earlier stages and connect with chws can successfully access and receive treatment in a timely manner and have better chances of survival . A chw program for breast cancer as described here must address problems specific to screening in lmcs . One of the predominant problems in screening for breast cancer is a shortage of trained personnel who can deliver breast health services, aggravated in africa by the brain drain, in which talented clinicians leave for better situations outside africa . The clinical breast exam has been used successfully for screening in other studies, and chws have been able to learn and successfully administer the exam . With chws performing routine screenings, medical staff can be freed for more skilled tasks . For patient navigation, chws should be trusted members of the women's communities and be able to link them to post - screening care after a positive finding . They should also offer guidance to help women understand their diagnoses and courses of treatment . Patients who work with chws are more likely to adhere to follow - up treatment because they have a better understanding of the health system and the course of their treatment . This enhanced understanding saves clinical time and resources and can lead to better outcomes for patients . The use of chws to perform cbe is fairly new in the field of breast cancer care, and more evidence - based studies or program evaluations are needed to detail methods that successfully incorporate this component . First, in other lmcs facing demanding health concerns such as access to clean water and infectious disease control, some may question the priority of the time and expense needed to initiate a successful program . Second, the lack of reliable population - based cancer data for most of africa is a barrier to understanding the full extent of the breast cancer burden . The limited data available suggest that breast cancer incidence rates may peak at younger ages . Third, sub - optimal resources can hamper the development of new programs in low- and middle - income countries . Lack of food, income, transportation, and other conditions of poverty limit the ability of people to access health programs and services . To successfully maintain a chw screening program we contend that breast cancer screening can be woven into existing infrastructure already prominent in many middle - income countries . South africa is presented here as an example because, as an upper middle - income country, mammography is available in some areas and the country has a history of chw programs . The present recommendations are not suitable for all countries or all communities but are intended as a conceptual model for how services may be expanded . First, rigorous evaluations must be implemented to assess efficacy of such programs, with an emphasis on community members' involvement in assessing program effectiveness . Important research questions include: what are the best methods for preparing chws for their role? Are such programs acceptable to the community (including women and those who may have power over their decisions and actions)? For information on longer - term programmatic effects, follow - up studies of women participating in the programs could measure women's attitudes, knowledge, and practices over time as well as track changes in external determinants of health that influence the feasibility of women participating in breast health care . Depending on the variability of conditions that could influence chw programs, randomized controlled trials may be necessary . A key resource in ongoing research and implementation is the breast health global initiative (bhgi), which recently opened the bhgi learning laboratory in kumasi, ghana and is training a new set of breast cancer health practitioners . The magnitude of lives lost to breast cancer in south africa and throughout low- and middle - income countries is unacceptable, and in large part, preventable . By building on existing infrastructure, utilizing lower - cost health service options, such as chws, and engaging in partnerships with affected communities,
Rolled - up nanotech has become a powerful technology with applications in a broad range of research fields, including optofluidics, micromachinery [4 - 6], magnetofluidics, biophysics, nanomechanics, and waveguiding for different spectral ranges and applications [10 - 12]. Large arrays of periodically ordered microtubes can be fabricated by a combination of lithography and deliberate self - rolling of strained layers upon selective underetching [1,13 - 15]. However, in some cases, it might be important to stimulate tube formation in certain areas and suppress tube formation outside those areas . Precise control over the lateral positioning and the number of windings is fundamental for the use of the tubes in optical resonators for sensors [16 - 20], hyperlenses, or electrical transport (curved electron gases) [23 - 27]. Numerous existing studies offer highly sophisticated methods for precise lateral positioning of rolled - up semiconductor nanostructures by controlling the starting edges [13 - 15,28,29], but difficulties arise for thin layers due to the extensive, successive lithography steps involved, and ways to control the stopping point of rolling remain largely lacking without modifying the structure . Here, we demonstrate illumination - controlled hydrofluoric acid (hf) etching of a buried thin alas sacrificial layer . Sufficiently intense light exposure of a certain substrate area leads to a complete suppression of the underetching effect, and as a result, the formation of rolled - up ingaas / gaas tubes can be easily controlled spatially . This method allows for control over the roll - up stopping point and is suitable for very thin sacrificial layers . The aim of this study was to quantify the observed etch - suppression effect (ese) and precisely control the release of ingaas / gaas bilayers with an illumination technique . V rolled - up nanotube (runt): when immersed in hf solution, the compressively strained double layer (a gaas layer on top of a larger - lattice constant inxga1x as layer deposited pseudomorphically on a gaas substrate with an alas sacrificial layer) releases from the substrate as the sacrificial layer is preferentially removed by the hf at a previously defined starting edge and begins to roll up in order to relax the built - in strain gradient . Finally, the bent bilayer forms a tube or scroll after performing a full rotation . The starting edge, which for ingaas / gaas layers should be oriented in the direction for optimal structural integrity of the resulting runts, can be defined either by scratching the surface, thereby laterally exposing the sacrificial layer, or by opening a window through the entire layer structure by photolithography and subsequent wet chemical etching . The tube diameter, i.e., the size of the cross section of the structure obtained after the etching step has been completed, depends on the thickness and inherent strain of the layers, and can be accurately varied in the nano- to micrometer range [1,30 - 32]. For precise positioning of runts on the surface, the important parameters in the roll - up process are the sacrificial layer thickness, etching time, and tube diameter . The ratio of inner - to - outer diameter can also be controlled over a wide range, based on the number of rotations performed by the tubes on the surface (or rolling distance, measured from the initial starting edge see schematic in fig . Bilayer rolling due to sacrificial layer removal in hf,3 . Rolled - up structure after one full rotation).bschematic of the etching experiment during in situ observation by optical microscopy and result for strained bilayers (relative ratio of mesa diameter to layer thickness not to size).coptical image of an alas / ingaas / gaas mesa structure etched in a 2.5% hf solution, with 20 microscope objective, for 6 min; the etch - suppression spot radius is 700 m.doptical images of adjacent mesas from (b) (black box), showing an increasing degree of etch suppression toward the center of the illumination here, the experiments were performed using 20 nm in0.33ga0.66as/20 nm gaas structures on alas grown on (001) gaas substrates by molecular beam epitaxy, where the thickness of the alas was varied from 4 to 20 nm . For experiments pertaining to the measurement of the onset of the etch suppression and rolling distance, the starting edges were defined by mechanical scratching, whereas photolithography and wet chemical etching (by h3po4:h2o2:h2o solution) were used for controlling the etch suppression . Our specially designed setup involved an hf - resistant trough for etching with hf under light focused by a zeiss optical microscope using different objective lenses of 5 through 50 magnification (for sample evolution during this process, see fig . Other measurements were performed using illumination from a hene laser with maximum output power of 2 mw, as well as a laser with adjustable power (up to 20 mw) and wavelength (535825 nm) with focusing optics or an optical fiber to illuminate the surface of the sample . For all measurements, the intensity of illumination was measured beforehand using a calibrated power meter . For samples that were etched, illuminated, and observed under the microscope, the settings on the illumination dial were used and the focused spot sizes for each magnification were assumed to be the same . For the quantitative measurements of tube rolling distance, optical images of the sample outside of solution once the etching was terminated were taken . From these images, the rolling distance could then be measured, which we define as distance from the starting edge to the center of the tube width, assumed to correspond to the stopping point of the underetching . The resolution of the es method was studied by precisely positioning the illumination spot on wet - chemically etched mesa structures of different shapes and sizes (on the order of 10100 m) during hf etching . The degree of etching suppression (given by the coverage area of unetched structures remaining on the sample) was measured versus distance from the center of illumination . Figure 1b shows an optical microscopy image of a structure composed of a 4 nm alas layer and a 40 nm symmetrical strained in0.33ga0.66as / gaas bilayer . The larger circular shape observed on the sample at the end of the hf experiment is formed by original mesa structures that were left unetched, while on the rest of the sample the bilayer mesas are underetched, leading to rolling up as expected . Figure 2, which plots the average rolling distance as a function of intensity for structures with varying alas thickness, illustrates the sudden onset of the ese . The error due to variations in experimental parameters such as quantity of hf solution, scattering of light and effective illumination intensity at the sample, as well as the method for evaluating rolling distances, is estimated to be within 15% . It can be seen that the maximum rolling distance increases as the alas thickness increases, while the ese is only present for sacrificial layers below 10 nm . Moreover, the onset of the ese, or the intensity of illumination necessary to suppress the alas etching, is higher for larger thicknesses . Onset of illumination effect for 20 nm ingaas/20 nm gaas samples with 4, 7, 10, 12, 13, 15, and 20 nm alas sacrificial layers, etched with 2.5% hf solution using 20 microscope objective, for 8 min; the starting edges were produced by mechanical scratching . The diamond data point shows a maximum rolling distance previously reported figure 3 shows the maximum rolling distance as a function of the sacrificial layer thickness . In a regime from 5 nm up to a thickness of 15 nm of alas the relationship between the maximum rolling distance and the sacrificial layer thickness is approximately linear, and then saturates for thicker alas layers . Although in previous studies ingaas - gaas runts (rolled - up nanotubes) have been noticed to exhibit a self - limiting rolling behavior for longer etching times, this limiting was not investigated as function of the sacrificial layer thickness . The fit through the data in the linear increase regime (r = 0.936) yields a unitless slope of 14.2e for the scaling of maximum rolling distance with talas . This indicates that in this first regime the maximum rolling distance is not dominated by intrinsic effects of the rolled - up layer and does not occur due to a fundamental limiting process (as, e.g., described by cendula et . Therefore, we ascribe the saturation in this regime to dynamic effects either between the rolled - up layer and the substrate or inside the etching solution caused by the process in the gap between the substrate and the rolled - up film . The second regime shows a clear saturation behavior for alas thickness larger than 15 nm . In this regime, the saturation should be dominated by intrinsic effects either from the layer system itself or possibly by some kind of fundamental process yet to be found . The linear scaling of the maximum rolling distance offers the possibility for lateral positioning of the rolled - up tube relative to a defined starting edge . 1a, indicating rolling distancedrollmeasured from the starting edge, alas thicknesstalas, ingaas / gaas bilayer thicknesstbil ., and radius of curvaturerof the rolled - up bilayer furthermore, as shown in fig . 4, the onset of ese changes for different etching speeds: the higher the hf concentration, the higher the intensity needed for suppression, while the thicker the alas layer, the more abrupt the etching behavior transition from the suppression regime to the illumination independent regime . For eachtalas, up to roughly 15 nm the onset of the ese increases approximately linearly with concentration, and scaling slopes are comparable for different thicknesses (12 mw / mm). From this, as well as fig . 2, it can be concluded that the onset of ese occurs in a different regime from that of intrinsic saturation of maximum rolling distance . The onset of the etch - suppression effect (ese) versus hf concentration for 20 nm ingaas/20 nm gaas structures with 4 nm and 10 nm alas sacrificial layers, with 20 microscope objective, for 8 min; the starting edges were produced by mechanical scratching . Thelinesthrough the data are guides for the eye we also found that the ese is time dependent, i.e., the etching time under illumination determines whether further etching will continue once the illumination is removed . In our experiments, we found that for samples where the high intensity light exposure was kept short, the layers were able to start rolling after being removed from the light source . This effect was found to not only depend on the light intensity but also on the thickness of the alas layer, which suggests that the etch suppression is a dynamic process that depends on the access of hf to the alas layer and the exchange of products and reactants in this region . This conclusion is further corroborated by the fact that with increasing alas thickness the rolling distance of the strained bilayer becomes independent of the alas gap size (fig . The ese was used to position and control the roll up of tubes from strained ingaas / gaas bilayers . The patterning allowed us to create well - defined starting edges suitable for the illumination experiment (as in fig ., one can obtain both etched regions with rolled - up layers and unetched regions within a small area on the sample . Furthermore, this technique is useful for applications since, during the es, the structures remain unchanged other than the blocking at the starting edge . 5, which is a plot of the degree of etching / suppression versus the distance from the center of the illumination for the 80-m diameter pattern shown in fig . The degree of etch suppression is the percentage of the circular mesa surface, which appears intact in optical images of the etched structures (see fig . As can be seen from this plot, the length scale on which the ese transitions from no significant suppression to full suppression is 100 m . Further refinement is possible either using smaller illumination areas, restricting light access close to the surface of the samples (for instance, by a shadow mask placed directly above the sample in the solution) or developing methods to reduce stray light and diffraction in the solution . (% of area) versus distance from the center of the illumination for the sample in fig . 1 . Thegreyareas indicate a fit based on the average degree of suppression and standard deviation values, for all points below and above a distance of 850 m, differentiating the regimes with ese and no significant ese, respectively figure 1b shows an optical microscopy image of a structure composed of a 4 nm alas layer and a 40 nm symmetrical strained in0.33ga0.66as / gaas bilayer . The larger circular shape observed on the sample at the end of the hf experiment is formed by original mesa structures that were left unetched, while on the rest of the sample the bilayer mesas are underetched, leading to rolling up as expected . Figure 2, which plots the average rolling distance as a function of intensity for structures with varying alas thickness, illustrates the sudden onset of the ese . The error due to variations in experimental parameters such as quantity of hf solution, scattering of light and effective illumination intensity at the sample, as well as the method for evaluating rolling distances, is estimated to be within 15% . It can be seen that the maximum rolling distance increases as the alas thickness increases, while the ese is only present for sacrificial layers below 10 nm . Moreover, the onset of the ese, or the intensity of illumination necessary to suppress the alas etching, is higher for larger thicknesses . Onset of illumination effect for 20 nm ingaas/20 nm gaas samples with 4, 7, 10, 12, 13, 15, and 20 nm alas sacrificial layers, etched with 2.5% hf solution using 20 microscope objective, for 8 min; the starting edges were produced by mechanical scratching . The diamond data point shows a maximum rolling distance previously reported figure 3 shows the maximum rolling distance as a function of the sacrificial layer thickness . In a regime from 5 nm up to a thickness of 15 nm of alas the relationship between the maximum rolling distance and the sacrificial layer thickness is approximately linear, and then saturates for thicker alas layers . Although in previous studies ingaas - gaas runts (rolled - up nanotubes) have been noticed to exhibit a self - limiting rolling behavior for longer etching times, this limiting was not investigated as function of the sacrificial layer thickness . The fit through the data in the linear increase regime (r = 0.936) yields a unitless slope of 14.2e for the scaling of maximum rolling distance with talas . This indicates that in this first regime the maximum rolling distance is not dominated by intrinsic effects of the rolled - up layer and does not occur due to a fundamental limiting process (as, e.g., described by cendula et . Therefore, we ascribe the saturation in this regime to dynamic effects either between the rolled - up layer and the substrate or inside the etching solution caused by the process in the gap between the substrate and the rolled - up film . The second regime shows a clear saturation behavior for alas thickness larger than 15 nm . In this regime, the saturation should be dominated by intrinsic effects either from the layer system itself or possibly by some kind of fundamental process yet to be found . The linear scaling of the maximum rolling distance offers the possibility for lateral positioning of the rolled - up tube relative to a defined starting edge . 1a, indicating rolling distancedrollmeasured from the starting edge, alas thicknesstalas, ingaas / gaas bilayer thicknesstbil ., and radius of curvaturerof the rolled - up bilayer furthermore, as shown in fig . 4, the onset of ese changes for different etching speeds: the higher the hf concentration, the higher the intensity needed for suppression, while the thicker the alas layer, the more abrupt the etching behavior transition from the suppression regime to the illumination independent regime . For eachtalas, up to roughly 15 nm the onset of the ese increases approximately linearly with concentration, and scaling slopes are comparable for different thicknesses (12 mw / mm). From this, as well as fig . 2, it can be concluded that the onset of ese occurs in a different regime from that of intrinsic saturation of maximum rolling distance . The onset of the etch - suppression effect (ese) versus hf concentration for 20 nm ingaas/20 nm gaas structures with 4 nm and 10 nm alas sacrificial layers, with 20 microscope objective, for 8 min; the starting edges were produced by mechanical scratching . Thelinesthrough the data are guides for the eye we also found that the ese is time dependent, i.e., the etching time under illumination determines whether further etching will continue once the illumination is removed . In our experiments, we found that for samples where the high intensity light exposure was kept short, the layers were able to start rolling after being removed from the light source . This effect was found to not only depend on the light intensity but also on the thickness of the alas layer, which suggests that the etch suppression is a dynamic process that depends on the access of hf to the alas layer and the exchange of products and reactants in this region . This conclusion is further corroborated by the fact that with increasing alas thickness the rolling distance of the strained bilayer becomes independent of the alas gap size (fig . The ese was used to position and control the roll up of tubes from strained ingaas / gaas bilayers . The patterning allowed us to create well - defined starting edges suitable for the illumination experiment (as in fig ., one can obtain both etched regions with rolled - up layers and unetched regions within a small area on the sample . Furthermore, this technique is useful for applications since, during the es, the structures remain unchanged other than the blocking at the starting edge . The resolution of this method is indicated by fig . 5, which is a plot of the degree of etching / suppression versus the distance from the center of the illumination for the 80-m diameter pattern shown in fig . The degree of etch suppression is the percentage of the circular mesa surface, which appears intact in optical images of the etched structures (see fig . As can be seen from this plot, the length scale on which the ese transitions from no significant suppression to full suppression is 100 m . Further refinement is possible either using smaller illumination areas, restricting light access close to the surface of the samples (for instance, by a shadow mask placed directly above the sample in the solution) or developing methods to reduce stray light and diffraction in the solution . Degree of suppression (% of area) versus distance from the center of the illumination for the sample in fig . 1 . Thegreyareas indicate a fit based on the average degree of suppression and standard deviation values, for all points below and above a distance of 850 m, differentiating the regimes with ese and no significant ese, respectively we investigated the rolling distance as a function of illumination intensity used during the hf etching of alas / ingaas / gaas structures on gaas for different sacrificial layer thicknesses . For alas layers thinner than 10 nm, total suppression of the etching process occurs beyond a threshold intensity that increases with increasing alas thickness . The ese is clearly influenced by the alas - hf reaction rate as well as the physical characteristics of the structure: the larger the sacrificial layer gap and faster the reaction rate, the higher the intensity of illumination needed to suppress the etching . This trend continues up to a point where the reaction is no longer illumination dependent and suppression is no longer possible . A further series of experiments involving etching with filtered light and a focused laser beam of varying wavelengths (not presented here) suggests that the ese is preserved for lower energies than the alas band gap and therefore any photochemical effect in hf cannot depend on light excitation of the alas material . We have also eliminated the possibility that heating plays a significant role in the ese, since experiments that involve heating samples in hf solution past the boiling point show an enhancement rather than suppression of the etching and hence would counteract this effect . We believe that for the ese found in samples etched with hf for shorter times under illumination levels close to the threshold value from fig . 2, the strained bilayer remains intact because the sacrificial layer was not underetched at the starting edge, which may happen through the accumulation of solid as and as oxide following from a photochemical interaction at the gaas surface in the presence of hf . The process through which alas sacrificial layers are wet - etched with hydrofluoric acid (hf) [34 - 43] as well as the laser - induced etching of semiconductors by a dilute acid solution [44 - 46] have been investigated previously in some depth . In hf - alas reactions, any passivating as formed at the surface is usually oxidized and then dissolved, thus allowing new access to the etch front and sustaining etching . But if the rate of as production exceeds that of oxide formation, or if the type of oxide is stable in the hf, then the etching will be inhibited . From laser - induced etching studies, it is clear that porous stable oxide films formed at the semiconductor surface give a time dependence for the process . During the redox reaction, as(iii) must be complexed by water and dissolved as haso2 and as2o3, but when the concentration of as(iii) surpasses the solubility limit, there is precipitation of as at the surface, slowing down the reaction . Our results match well with this type of process, and we propose that the ese occurs in a similar fashion: the formation of very small amounts of as or as2o3 at the alas gap, which would nonetheless be enough to block access of hf to films under 10 nm, can effectively suppress the underetching of the alas layer . In accordance with our findings, the thicker the sacrificial layer gap and the faster the hf etching rate, the less likely the ese takes place . In the suppression regime for thinner alas layers, for higher intensities of illumination the photogeneration of holes is more pronounced leading to a faster subsequent passivation (and reaching of the ese limit with time). For thicker alas layers, higher illumination can still lead to changes at the gaas substrate interface but does not hinder the etching of the alas . While the self - limitation of the maximum rolling distance of runts allows for the precise tuning of the number of rotations as a function of the sacrificial layer, the illumination permits the exact positioning of the tube in combination with common lithographic technology . Fine - tuning the etch suppression with patterned samples can yield a useful way of precisely controlling the roll up of strained ingaas / gaas bilayers and the entire tube fabrication process, as well as other more general laser - assisted microfabrication applications, with a convenient, customizable method . In this way, it is complementary to pre - pattering of the sample by lithographic means and allows for a full control over the position of the produced runts.
The current study utilizes data from the administrative records of a large workers compensation insurance company in the united states that included claims from a variety of different states, industries, organizations, and company sizes . All workers compensation claims with complete data for individuals who had an injury date between january 1, 2002, and december 31, 2008, were assessed for inclusion . We included all claims with at least 1 day of paid lost work time within 1 year of the injury date . Lost work time comprised both days of temporary total disability (ttd) and days of temporary partial disability (tpd). Two sets of criteria were used to identify lbp claims on the basis of the icd-9 diagnosis codes that were reported in the claimant's medical service bills for the first 15 days of medical treatment . Medical treatment typically began within 2 weeks of the injury date (90% of claims), although all claimants who received medical treatment within 1 year of the injury date and who had lost work time were included in the database . First, 99,127 claims were classified as a possible lbp claim based on having at least one icd-9 diagnosis code within the first 15 days of medical treatment relating to specific low back injuries or disorders, which are listed in supplemental table 1 . Second, from those identified as possible lbp claims, we required that over two - thirds of the claimant's icd-9 diagnosis codes in the diseases of the nervous system and sense organs (320389), diseases of the musculoskeletal system and connective tissue (710739), and injury and poisoning (800777) chapters within the first 15 days of medical treatment be for specific low back injuries or disorders (supplemental table 1 or nonspecific back injuries or disorders (supplemental table 2). In total, 76,955 claims met these criteria and approximately 70% of these claims had 100% of their icd-9 diagnosis codes within the first 15 days coming from the codes listed in supplemental tables 1 or 2 . Our sample was further restricted to claimants who had only one claim within a single calendar year . For some claimants with multiple claims in a single year, the claims were for different injuries . However, in certain cases, there were multiple episodes of disability for a single claim for individuals resulting from the same injury . This occurred in cases wherein an individual went back to work after an injury but after having returned to work went back out on ttd or tpd . In this study, we only included claims for individuals in whom multiple episodes of disability within the same claim had less than 14 days between episodes, in which case we considered this a single episode . If the duration between episodes was 14 days or longer, these claims were excluded . We also excluded claims for individuals who received a lump sum payment within 1 year of the injury date . Claims were excluded in cases wherein the first date of lost work time was more than 1 year after the injury date . Finally, only claims for individuals 18 to 80 years of age were included in this study . In total the first lag time is referred to as the reporting lag time and represents the number of days from the date of injury to the date at which the injury was first reported to the workers compensation insurer . The second lag time, which we refer to as the medical services lag time, is the number of days from the date of injury to the date at which a claimant first sought medical care for that injury . The third lag time is referred to as the work disability lag time and represents the number of days from the date of injury to the date at which a claimant first took tpd or ttd . Each of the three lag times were categorized into seven categories: 0 days lag (reference group), 1 to 3 days lag, 4 to 6 days lag, 1 week up to 2 weeks lag, 2 weeks up to 30 days lag, 30 days up to 60 days lag, and 60 days up to 1 year lag . The outcome variable was the length of disability, which was calculated from the date that a claimant first took tpd or ttd until the date at which tpd or ttd ended . Tpd or ttd was considered to have ended when no disability days were taken for at least 14 days consecutively . For claims in which the length of disability exceeded 1 year the following covariates were used: gender, annual income, industry, litigation status, injury severity, year of injury, age, and tenure . Sixteen ordered categories were used to assess annual income: $0 to $9999, $10000 to $19,999, $20,000 to $29,999, $30,000 to $39,999, $40,000 to $49,999, $50,000 to $59,999, $60,000 to $69,999, $70,000 to $79,999, $80,000 to $89,999, $90,000 to $99,999, $100,000 to $109,999, $110,000 to $119,999, $120,000 to $129,999, $130,000 to $139,999, $140,000 to $149,999, and $150,000 or more . Industry was categorized into 10 groups, including agriculture, forestry and fishing, construction, finance and insurance, manufacturing, mining, retail trade, services, transportation, public administration, and wholesale trade . Litigation status was coded 1 if the workers compensation insurer assigned an attorney to the claim and 0 if not . The codes in the more severe and less severe categories can be found in supplemental tables 1 and 2 . Injury severity was coded 1 for having at least one more severe diagnosis within the first 15 days of medical treatment and 0 for having only less severe diagnoses within the first 15 days of medical treatment . The more severe codes generally included diagnoses related to herniated disc, lumbar radiculopathy or neuropathy, spinal stenosis, sciatica, or possible instability . The less severe codes generally referred to diagnoses associated with degenerative changes, nonspecific back pain, or miscellaneous diagnoses . In addition, efforts were made to exclude cases of complicated back pain, such as those with diagnoses that were consistent with experiencing multiple work - related injuries or disorders, very severe injuries, or back pain due to cancer, infection, severe trauma, or an autoimmune disorder . Since the database included claims from 2002 to 2008, the analyses were controlled for the year of the injury . Tenure was also measured continuously on the basis of a claimant's organizational tenure at the time of injury . The first lag time is referred to as the reporting lag time and represents the number of days from the date of injury to the date at which the injury was first reported to the workers compensation insurer . The second lag time, which we refer to as the medical services lag time, is the number of days from the date of injury to the date at which a claimant first sought medical care for that injury . The third lag time is referred to as the work disability lag time and represents the number of days from the date of injury to the date at which a claimant first took tpd or ttd . Each of the three lag times were categorized into seven categories: 0 days lag (reference group), 1 to 3 days lag, 4 to 6 days lag, 1 week up to 2 weeks lag, 2 weeks up to 30 days lag, 30 days up to 60 days lag, and 60 days up to 1 year lag . The outcome variable was the length of disability, which was calculated from the date that a claimant first took tpd or ttd until the date at which tpd or ttd ended . Tpd or ttd was considered to have ended when no disability days were taken for at least 14 days consecutively . For claims in which the length of disability exceeded 1 year, the following covariates were used: gender, annual income, industry, litigation status, injury severity, year of injury, age, and tenure . Sixteen ordered categories were used to assess annual income: $0 to $9999, $10000 to $19,999, $20,000 to $29,999, $30,000 to $39,999, $40,000 to $49,999, $50,000 to $59,999, $60,000 to $69,999, $70,000 to $79,999, $80,000 to $89,999, $90,000 to $99,999, $100,000 to $109,999, $110,000 to $119,999, $120,000 to $129,999, $130,000 to $139,999, $140,000 to $149,999, and $150,000 or more . Industry was categorized into 10 groups, including agriculture, forestry and fishing, construction, finance and insurance, manufacturing, mining, retail trade, services, transportation, public administration, and wholesale trade . Litigation status was coded 1 if the workers compensation insurer assigned an attorney to the claim and 0 if not . The codes in the more severe and less severe categories can be found in supplemental tables 1 and 2 . Injury severity was coded 1 for having at least one more severe diagnosis within the first 15 days of medical treatment and 0 for having only less severe diagnoses within the first 15 days of medical treatment . The more severe codes generally included diagnoses related to herniated disc, lumbar radiculopathy or neuropathy, spinal stenosis, sciatica, or possible instability . The less severe codes generally referred to diagnoses associated with degenerative changes, nonspecific back pain, or miscellaneous diagnoses . In addition, efforts were made to exclude cases of complicated back pain, such as those with diagnoses that were consistent with experiencing multiple work - related injuries or disorders, very severe injuries, or back pain due to cancer, infection, severe trauma, or an autoimmune disorder . Since the database included claims from 2002 to 2008, the analyses were controlled for the year of the injury . Tenure was also measured continuously on the basis of a claimant's organizational tenure at the time of injury . The relationships between the three lag times and length of disability were estimated using random effects tobit models . We were only interested in following claimants for 1 year from the time of disability onset; however, for some claimants, disability may not have ended by 365 days and it is necessary for our models to take this into account . Tobit models or censored regression models, as they are sometimes referred to, were chosen to deal with the nature of the data used in the current study wherein length of disability was restricted to a range between 3 and 365 days . Similar to ordinary least squared regression models (ols), tobit models are generally used when estimating linear relationships between variables; however, ols generally assumes a continuous distribution without censored values, whereas the tobit model is able to accommodate left- and/or right - censoring of values . Ols produces biased estimates when values are censored, while tobit models eliminate that bias . In order to take into account the non - independence of observations across industry groupings, participants drawn from different industry groups may be thought of as representing subsamples within the larger sample . Participants disability durations may be clustered within these different industry groups such that the residuals are dependent on one another within groups . Random effects models take this clustering into account by allowing the constant to vary across groups, in this case industries, while at the same time, keeping the other estimates fixed . For all lag times, the 0 days lag category was used as the reference group . Separate analyses were conducted for each of the three lag times due to concerns about collinearity among the lag times . In addition to the main analyses, differences in the coefficients for the non - reference group lag time categories were assessed using wald tests . Analyses were adjusted for gender, annual income, industry, litigation status, injury severity, year of injury, age, and tenure . To reduce issues with multicollinearity, all continuous variables in the models were mean - centered . Stata 13 was used to perform the analyses (stata corporation, college station, tx). In the analyses, on the basis of this, we used a more conservative p value of less than 0.001 to represent statistical significance . The age of claimants at the time of injury ranged from 18 to 80 years, with an average age of 40 years (sd 11.2 years). The length of tenure at the time of injury ranged from 0 to 53 years, with an average tenure of 6 years (sd 7.7 years). Over half of the claimants had an annual income of $20,000 to $50,000 and only 1% of claimants had an annual income of over $100,000 . The large majority of claims were for less severe low back injuries (81%) and less than 30% (28%) of claims were involved in litigation . The length of disability ranged from 3 to 365 days, with an average length of disability of 96 days (sd 118.5 days). The breakdown of claims in each of the lag time categories is presented in table 1 . For all of the lag times, the greatest percentage of claims had a 1 to 3-day lag time (36% reporting lag time; 36% medical services lag time; 57% work disability lag time). Results of the random effects tobit model are presented in table 2 . For the reporting lag time, in comparison to the 0 days lag time category, having a 1 to 3 days lag time was related to a shorter length of disability, whereas having a lag time of 2 weeks up to 30 days, 30 days up to 60 days, or 60 days up to a year was associated with a longer length of disability . For the medical services lag time, having a lag time of 4 to 6 days, 1 week up to 2 weeks, 2 weeks up to 30 days, 30 days up to 60 days, or 60 days up to a year were all related to a longer duration of disability than having 0 days of lag time . Finally, for the work disability lag time, in comparison to all other lag time categories, having 0 days of lag time was associated with a shorter length of disability . To further compare the differences in the length of disability across the different lag times, in figure 3, we plotted the predicted values for the length of disability for each of the lag time categories . We also assessed significant differences across the lag time categories for each of the respective lag times using wald tests . Note: this figure is based on the predicted values for the length of disability at each of the respective lag times . Although not all of the differences among the lag time groups were statistically significant, across all three of the lag times, the trend was generally positive with the predicted length of disability increasing as the length of the lag increased . There were a few exceptions to this positive trend, specifically, for the 0 days and 1 to 3 days categories in the reporting lag time and the medical services lag time, the predicted length of disability actually decreased slightly as the length of the lag increased (note: the decrease was only statistically significant for the reporting lag time). In addition, the predicted length of disability again decreased slightly going from a lag time of 30 days up to 60 days to a lag time of 60 days up to a year in the medical services and work disability lag times (note: these decreases were not statistically significant). When examining the reporting lag time, there was over a 20-day difference in the predicted length of disability going from a lag time of 1 to 3 days to a lag time of 60 days up to a year . Overall, there was roughly a 10-day difference or slightly more for having less than 2 weeks of lag time in reporting the injury compared with having 2 weeks or longer for the lag time . For the medical services lag time, the greatest difference was between having 1 and 3 days of lag time where the predicted length of disability was 43.6 days and having a lag time of 30 days up to 60 days where the predicted length of disability was 66.9 days . In general, having a reporting lag time of 2 weeks or longer was related to a significantly longer predicted length of disability than having a lag time of less than 2 weeks . The largest difference in the predicted length of disability across the lag time categories was for the work disability lag time . The predicted length of disability was close to 40 days less for having a work disability lag time of 0 days compared with having a lag time of 30 days or longer . In addition, the predicted length of disability increased by approximately a week to a week and a half across each of the work disability lag time categories going from the 0 days category up to the 30 days up to 60 days category . In a sample of workers who experienced work - related low back injuries, the current study examined the associations between the length of work disability and three potential delays in the work disability process including the lag times from the work injury to (1) reporting the injury to an employer, (2) receiving medical treatment, and (3) taking time off of work or initiating light duty work . Understanding how these lag times are related to the length of disability can help guide health practitioners and employers in work disability case management . For the reporting lag time, our results suggested that the length of disability may be shorter when there is earlier reporting of the injury to the workers compensation insurer, as there was approximately a 10-day difference or more in the predicted length of disability when comparing having a lag time of less than 2 weeks to having a lag time of 2 weeks up to a year . However, within the first 2 weeks of the injury, the predicted length of disability was slightly higher when reporting the injury on the same day it occurred, in comparison to reporting it 1 to 3 days after it occurred, but reporting the injury in the first week was associated with a shorter predicted length of disability than reporting the injury after the first week . One possible explanation for this finding is that workers may attempt to wait to report an injury in hopes that the injury will get better on its own; however, some workers with more severe injuries may find that the injury does not resolve and ultimately have to report the injury in order to start the workers compensation process . This would result in those workers who delay reporting an injury having more severe injuries that do not get better on their own . Similarly, some workers may feel pressure from their supervisors to delay reporting an injury until they are sure that the injury is serious enough to require medical attention and time off of work, which again would result in a greater length of disability for those who delay the report of the injury . These findings for the reporting lag time are somewhat in line with a previous study of workers with low back injury, which reported that earlier injury reporting was related to a greater likelihood of having returned to work at 1 month following the lbp onset . In this previous study, all workers in the sample had reported the injury within 14 days of the pain onset and thus our findings for claimants reporting the injury after 14 days may not be comparable . Our results differ from the previous findings as we found an initially higher length of disability, but that was only for a zero - day lag time we measured length of disability and the previous study measured the likelihood of work disability at 1 month after the injury . It is also possible that the differences in findings resulted from differences in the lag time categories used in the current study compared with the study by shaw et al, which measured the lag time continuously . Our findings for the lag time between injury and receiving medical treatment were fairly similar to those for the reporting lag time with shorter lag times relating to a shorter length of disability . For the medical services lag time, we found a two and half week increase or more in the predicted length of disability when waiting 2 weeks or longer to receive medical treatment for a low back injury compared with receiving medical treatment within the first week of injury . Also of note, the predicted length of disability was about a week shorter when receiving medical treatment in the first 3 days of injury than receiving treatment 1 week to up to 2 weeks after the injury occurred . These findings could reflect that earlier treatment for low back injuries may help to prevent acute lbp from transitioning to chronic lbp . Seeking medical treatment for low back injuries in the first 2 weeks can provide workers with assurance about the course of their condition, as well as help to avoid further reinjury . It is possible that those workers who delayed seeking medical treatment for low back injuries did not take proper measures to stop further aggravation to the back injury, which may have ultimately resulted in a greater length of disability . For the medical services lag time, the findings are generally in line with previous studies of medical services lag times, which found that receiving medical treatment within 30 days of injury was associated with improved rtw outcomes . However, another previous study did not find a significant association between rtw and the lag time in receiving medical services when comparing a lag time of zero days with a lag time of 1 to 3 days or 4 days or more . Although our findings did not show a difference in the predicted length of disability for a lag time of 1 to 3 days when compared with zero days, we did find differences between a zero days lag time and a lag time of 4 days or more . It is possible that the differences in findings with this previous study could result from the difference in injury types examined . Our study focused specifically on low back injury, while the other study included all work - related injuries . In addition, the outcome variable used in the previous study was a dichotomous survey item indicating whether the worker was currently working, whereas we measured the length of disability continuously using retrospective claims data . For the work disability lag time, the predicted length of disability was found to increase by approximately a week to a week and half going across the different lag time categories . For example, the predicted length of disability was approximately a week longer for claimants with a 1 to 3 days lag time than those with a zero days lag time . The predicted length of disability seemed to plateau at 30 days lags time, with a little difference observed between the 30 days up to 60 days lag time group and the 60 days up to 1 year lag time group . It is possible that these findings reflect workers trying to tough it out in hopes of not having to take time off of work due to injury . However, after several days or weeks, workers may then find themselves in a situation in which the injury is too severe to remain at work . As a result, workers in this delayed group may have more severe injuries that lead to a greater length of disability . To our knowledge, no studies exist that examine the association between lag times in initiating work disability and the length of disability . Our findings provide a critical first examination of this relationship and indicate that, in general, workers who initiated work disability earlier had a shorter predicted length of disability . The results of the current study suggest that occupational low back injuries should be reported to the workers compensation insurer within the first week the injury occurs . Workers who reported the injury within the first 2 weeks after injury had on average a predicted length of disability over 14 days less compared with those whose injuries were reported 30 days or more after injury . With this in mind, interested parties, such as employers or insurers, may consider creating benchmarks for reporting a work - related injury no later than 2 weeks after the injury has occurred . Future research may also seek to investigate how to further mitigate barriers to reporting and facilitate trouble - free reporting, as this may help to shorten the lag time in reporting an injury . For the medical services lag time, this study demonstrated that the predicted length of disability was the shortest for individuals who received medical treatment within the first 3 days of injury . Approximately 10% of the sample did not receive any medical treatment during the first 2 weeks following the injury . For these individuals, the predicted length of disability was more than 2 weeks longer than those who received medical treatment within the first 3 days after injury . Stakeholders might consider systems to ensure that medical care is rendered within the first 3 days after injury, and to investigate the circumstances that lead to delays in seeking treatment . On the contrary, encouraging workers to seek immediate medical care may have unintended consequences such as increasing the quantity of unnecessary tests and procedures, and ultimately delaying returning to work . As our study was correlational, we are unable to assess whether early seeking of medical treatment directly causes shorter disability duration . In addition, the medical services gap in this study focused specifically on seeking any medical treatment . For some claimants, this may have been intensive medical care, which included a large number of tests and procedures, while for others, it may have been more passive where a doctor recommended waiting a few days to see how the injury progressed . Our implications must be interpreted cautiously with this in mind . With regard to the work disability lag time, surprisingly, there were approximately 14% of workers who worked for 1 month or more after injury before initiating work disability . For these workers, the predicted length of disability is more than doubled compared with those who initiated work disability the same day as the injury . An explanation for this doubling may be that a more severe reinjury or aggravation of the initial back injury could have occurred as a result of remaining at work after the injury, ultimately leading to an increase in the length of disability . Alternatively, some workers who delayed taking time off of work may have tried to self - manage their lbp, but eventually left work as their symptoms failed to resolve . In this case, these workers would represent a group of cases with likely greater severity of lbp . In either case, had workers more promptly taken time off of work or started light duty after the initial injury, then the length of disability may have been shorter . Taking into consideration that this study does not account for workers who received medical care but never lost a day of work, for those who are likely to go out on work disability, or who have persistent symptoms that significantly interfere with their function, they should be encouraged to not delay a transition to temporary light duty work, seeking medical care, or taking off work altogether . For disability case managers and employers, more research is needed to understand the reasons for delayed work disability for 30 days or more . It is possible that these individuals may represent unique cases in need of additional resources to fully rtw after this long initial delay . There were a number of strengths to this study, including the use of a wide range of workers compensation claims representing a variety of different industries, companies, and states . However, there are also several important limitations to note . As this study focused on the administrative data from a large, workers compensation insurer, this study does not provide any explanations as to why we observed differences in the lag times . For example, it may be assumed that individuals would seek treatment immediately after a work - related injury, but this study's findings suggest this is not always the case, although in this study we have little information as to why there was a delay . Gaining a better understanding of why there are lag times in the work disability process may help to shorten the length of disability following a work - related low back injury . Along these lines, there are several other variables of interest that may confound the relationship between lag times and the length of disability that were not available in the administrative data . Some of these factors include the availability of workplace accommodations, the quality of the relationship with the supervisor, the level of physical demands associated with a claimant's occupation, and a claimant's motivation to return to work . It is possible that if these variables were available, they may help to explain the relationship between lag times and the length of disability or possibility reduce the strength of the relationship . In addition, our measure of injury severity may be problematic for certain claims, as it could be an indicator of comorbidity . We focused only on the primary diagnosis relating to the main injury with no information about comorbid diagnoses that might impact recovery from the main injury . Another limitation associated with using administrative data is that we lacked a true measure of rtw . For analytic purposes, the length of disability was calculated on the basis of indemnity payments, with the assumption that the end of indemnity payments coincided with rtw . Although for many the end of indemnity payments is the result of returning to work, it is possible that in some cases, indemnity payments may have ended for a reason other than rtw . A major limitation in this study was that the analyses were limited to workers who at some point had lost work time compensated through the workers compensation system . This was done for practical reasons, as our analyses focused on the length of disability as an outcome, but it is well known that a large majority of workers compensation claims are for medical treatment only . As such, our findings must be interpreted with this in mind as they may only apply to workers who receive wage replacement for time off of work . Accordingly, the implications of our findings may be biased, as we only have the length of disability for individuals who did actually experience lost work time and our recommendation of earlier reporting and seeking of medical treatment may be detrimental for individuals who do not ultimately take time off of work, as early medical treatment may result in a greater number of unnecessary services . In addition, it is possible that some workers may have used vacation days to take off work for recovery purposes in order to avoid receiving lower wages in the workers compensation system, and thus, these cases were not included in our sample . Along these lines, our sample may suffer from the immortal time bias . In the administrative data, we only have information on individuals who did ultimately have a claim accepted for a given injury . We do not have information on individuals who may have had an injury but never filed a claim . It is possible that some of these individuals that never filed a claim did not do so because they recovered and returned to work relatively quickly after injury and felt that they did not need to file a claim . If this were the case, following our recommendation in all cases of encouraging early reporting of a claim and early commencement of work disability would be problematic, as it would result in more claims and more time away from work cases . It is therefore important to interpret our results with this in mind as our sample is selective to those who did file claims . Thus, these recommendations may make sense only for those persons who are likely to file a disability claim . One of the major findings of our study was that the amount of time individuals waited before taking time off of work was related to the length of disability . Although this was a major focus of the study, this may also present a potential limitation of our findings, as we are treating the length of disability as independent of when an injury actually occurred in relation to work disability onset . When calculating the length of disability, we utilize the date that lost work time began and the date that lost work time ended, without considering how many days there were between when the injury occurred and when lost work time started . The work disability lag time does include this information directly, but this variable could not be included in the models for the other two lag times because of collinearity concerns . Moreover, we have no information about what claimants with lag times higher than zero days were doing in those days before time off work began . Some of these individuals may have already been off work but not collecting benefits, others may have been using sick time, while others could have still been at work . Our study focused solely on the length of disability on the basis of receiving indemnity payments for lost wages, despite the fact that individuals may have lost wages for which indemnity payments were not received . First, due to the highly skewed distributions, for analytic purposes, the three lag time variables were measured as categorical variables . It would have been more ideal to run models with these variables coded continuously in order to be more informative about how a day or a week of difference in the lag times was related to the length of disability . We attempted to select meaningful categories for our analyses, but it is possible that valuable information was lost in the process . The second issue was that the three lag times were highly collinear and needed to be analyzed in separate models . As a result, in our analyses, we did not consider the relative strength of the different lag time relationships with the length of disability . Moreover, there may be combinations among the lag time categories that may further contribute to the relationship with the length of disability . Overall, the current study showed that across all three of the lag times, shorter lag times are related to shorter lengths of disability . These findings suggest that in workers with occupational low back injuries that are expected to go out on work disability, the length of disability may be decreased by earlier injury reporting, earlier receipt of medical care, and earlier initiation of light duty work and/or time off work.
Patients with neck pain often have impaired proprioception of the neck and reduced rom, for which proprioception training and rom training are usually prescribed in rehabilitation protocols . More and more researchers and clinicians are interested in the assessment1 and treatment of strength2, endurance, range of motion and proprioception3 of the cervical spine . Cervical rom has been used to evaluate the severity of impairment or disability of patients as it is related to cervical disorders and injuries . It has also been used as part of the clinical criteria for classification of disease as well as the evaluation of the efficacy of a rehabilitation program4 . The joint position error (jpe) test is considered the primary measure of neck proprioception and has been widely used as an outcome measure for patients with chronic neck pain5 . Abnormal jpe has been detected in patients with neck pain using either tests of ability to relocate the neutral head posture after an active movement or to actively relocate a position within a movement plane6 . Neuromuscular joint facilitation (njf) is a new therapeutic exercise based on kinesiology, and it integrates with the facilitation element of proprioceptive neuromuscular facilitation (pnf) with the joint composition movement, aiming to improve the movement of the joint through passive exercise, active exercise and resistance exercise . The njf technique uses the same motion pattern as pnf, but njf changes the resistance part . The purpose of this study was to examine the immediate effects of njf training and njf distal resistance training on cervical rom and position sense . Ten healthy people with an average age of (35.014.5) years, and an average height of (172.214.0) cm, an average body weight of (76.030.0) kg were recruited for this study . The characteristics of the subjects are shown in table 1table 1.subject characteristics (n = 10)age (yrs) 35.014.5height (cm) 172.1514weight (kg) 76.030.0values are mean sd . All the subjects were able to independently perform activities of daily living . They all agreed to participate in the study, after the researchers had given them a verbal explanation of the study method and aims . This study was approved by the research ethics committee of the international university of health and welfare . The subjects were divided into three groups: 10 in the control group, 10 in the njf distal resistance training group, and 10 in the njf training group . A miniature wireless motion recorder was used to record the maximum cervical rom and jpe before and after the interventions . Two miniature wireless motion recorders (mvp - rf8-gc, microston corperation, japan) were used in this study to measure the angle change of the neck . One was placed on the middle of the forehead, and the other one was placed on the manubrium sterni . The tests and performance were based on similar tests used in previous studies which investigated the neck proprioception8 . Subjects were comfortably positioned by sitting with their feet flat on the ground with their head in the neutral resting position . Subjects were familiarized with the tasks by performing a few practices in each test direction: extension, left rotation, and right rotation . For the formal tests, subjects were blindfolded, and their neutral head position was automatically set to zero by the sensor . In the three testing directions, the neck and head moved to the end of their range and returned as accurately as possible to the starting position . Three trials were performed of left and right neck rotation, and extension . Before each trial, subjects were allowed to remove the blindfold, open their eyes and face a big mirror to relearn the neutral position . Then, they were blinded again, and the head was repositioned back to the starting position the subjects by themselves . Prior to each new direction, the subject was able to recenter his starting position with the help of the mirror before being blindfolded again . Neutral position, the positions of maximum rotation (and extension), and the end position after the return to the neutral position . The differences in angle between the starting and the maximum range positions were calculated to deduce the rom of each movement at each sensor site . The differences in angle between the starting and the end range position were calculated to deduce the neck joint position error of each movement at each sensor site . Jpe and rom were calculated as the average data for the three trials in each direction (extension, right rotation, and left rotation). It is considered that left and right lateral flexion allows the trunk muscle to affect the neck movement . It interferes too much with the proprioceptive sense of the neck, so it was not used in the present study . The focus of the present study was proprioception sense . In the cervical region, most of proprioceptors are distributed in the deep flexion muscle groups of the neck9 . Test of neck position sense after the neck extension then back to the neutral position, involving neck flexion muscle group s activation, would be more persuasive for neck position ability in the present study . The interventions for control group was two minutes rest, for njf distal resistant training group was njf distal resistance training and for the njf group was njf neck flexion pattern training . Two - way repeated measures anova was used to compare joint position error between before and after the intervention . The two factors were group and intervention . If a significant interaction among groups was found, the two - way repeated measures anova with the bonferroni was performed to investigate the differences between before and after intervention . No difference in rom was observed among the three groups (table 2table 2.comparison of rom before and after intervention ()beforeafterextension njf 63.4212.4467.2015.31njf - d 64.5311.3062.8815.04control 67.5712.2563.8513.23 left rotationnjf 74.9413.8377.2915.81njf - d 71.2514.6272.9215.30control 72.4514.9271.1013.88right rotation njf 74.2517.1372.6114.09njf - d 72.9812.5475.2115.01control 76.3612.5274.3915.36values are mean sd . Comparison with before intervention: : p<0.05; : p<0.01 . Njf: neuromuscular joint facilitation group, njf - d: njf distal resistance group, control: control group, rom: range of motion). Two - way anova showed that there were significant interactions of jpe from extension to the starting neutral position among the groups (p<0.05), indicating that the changes were different among the groups . A significant pre- to post - intervention decrease in jpe from extension to the starting no other significant differences were observed among the three groups (table 3table 3.comparison of jpe before and after intervention ()beforeafterextension njf 10.164.26.464.37njf - d 5.954.043.402.79control 2.651.143.051.65left rotationnjf 3.012.013.651.42njf - d njf: neuromuscular joint facilitation group, njf - d: njf distal resistance group, control: control group, jpe: joint position error). Values are mean sd . Comparison with before intervention: : p<0.05; * : p<0.01 . Njf: neuromuscular joint facilitation group, njf - d: njf distal resistance group, control: control group, rom: range of motion values are mean standard deviation . Comparison with before intervention: : p<0.05; * *: p<0.01 . Njf: neuromuscular joint facilitation group, njf - d: njf distal resistance group, control: control group, jpe: joint position error in the current study, no difference was found in the neck range of motion of the different study groups . This finding may be due to the subjects being healthy individuals without any neck problems, who do not have any abnormal limited range motion of the cervical joint . Cervical spine studies have suggested that warm - up exercises which simulate the actual testing procedure can increase the compliance of neck soft tissues and minimize the process of creep related to repetitive measurements10, 11 . Before the test and the intervention, therefore, subjects were allowed to practice the task and perform movements in each direction, to warm up the neck muscle to avoid tight muscles . Therefore, it is reasonable to assume that healthy people would have the similar normal cervical joint range of motion at both pre and post intervention . If a method improves the proprioceptive function of the neck, the positioning ability of the neck should show more acuity . The result of this study indicate that njf training provides proprioceptive acuity that is superior to njf distal resistance training . This is because, in njf, proximal resistance is exerted, promoting the contraction of the neck multifidus thereby activating more proprioceptors . There was a pre- to post - intervention decrease in jpe of extension, possibly due to the patterns of njf training, with focus much more on the directions of extension and flexion of the neck, and less on the left and right rotation . According to the results of our study, it is evident that njf training can promote the proprioceptive function of the neck . Njf is suitable for neck dysfunction rehabilitation training and neck proprioceptive function disorder rehabilitation training . . Nevertheless, further investigations are necessary to find out the best approaches for improving proprioceptive function, and to investigate the different effects of different training programs after long - term interventions for healthy people and neck disorder patients . This result suggests that the better way to improve proprioceptive acuity is an intervention together with proximal resistance training, such as njf training.
Compound 1 was obtained as white needles (ch3oh) and had a molecular formula of c14h14o5, which was determined on the basis of hresims, showing eight degrees of unsaturation, suggesting the presence of two aromatic rings . The c nmr data (table 2) showed five aromatic methines [114.5 (c-5), 116.1 (c-5), 116.8 (c-6), and 117.8 (c-3 and c-3)], in addition to three quaternary and four oxygenated tertiary carbons [126.9 (c-1), 128.4 (c-6), 129.5 (c-4), 148.1 (c-1), 148.3 (c-2), 148.8 (c-4), and 150.9 (c-2)]. Four of them are deshielded, suggesting their attachment to electron - withdrawing groups . The h nmr spectrum (table 2) showed resonance for a diphenylmethylene group (3.73, s) as well as an oxygenated methylene group (4.53, s). The presence of two aromatic rings connected by a methylene group was further confirmed by the j hmbc correlations of h-8 (3.73, s) with c-3 (117.8), c-6 (128.4), and c-2 (148.3). The full assignments of the protons and carbons were supported by the j and j hmbc correlations (si). (4.53, s) showed j hmbc correlations with c-5 (116.1) and c-1 (126.9); h-5 (6.72, br s) showed j hmbc correlation with c-7 (60.8) and j hmbc correlations with c-4 (148.8) and c-6 (128.4); h-3 (6.51, br s) showed j hmbc correlations with c-5 (116.1) and c-1 (126.9); h-3 (6.50, br s) showed j hmbc correlations with c-8 (30.7), c-5 (114.5), and c-1 (148.1); h-5 (6.45, dd) showed j hmbc correlations with c-3 (117.8) and c-1 (148.1); h-6 (6.59, d) showed j hmbc correlations with c-4 (129.5) and c-2 (150.9). Data for 1 and 2 are in cd3od and pyridine, respectively (400 mhz for h, 100 mhz for c, in ppm). Compound 2, with the molecular formula c15h16o6, revealed by hresims, was obtained as colorless needles (ch3oh). The h and c nmr data (table 2) showed three oxymethines at 6.23 (d, j = 11.6 hz)/ 80.1, 5.00 (d, j = 8 hz)/ 72.8 and 4.59 (dd, j = 8, 11.2 hz)/ 77.5 and one sp methine at 7.04 (br s)/ 150.0; this was in addition to a carbonyl group at 194.4 and one methyl group at 1.84 (s)/ 15.7 . These signals were similar to those of 3 except for the downfield shifts of h-4/c-4 (from 4.52/62.8 in 3 to 5.00/72.8 in 2), h-5/c-5 (from 3.68/57.7 in 3 to 4.59/77.5 in 2), and h-6/c-6 (from 3.36/53.2 in 3 to 6.23/80.1 in 2). This in turn suggested the opening of the epoxide ring in 3, thus forming two hydroxy units in 2 . In addition, the h and c nmr spectra of 2 showed signals similar to those of 6-methylsalicylic acid (6), suggesting the presence of a similar moiety in the form of an ester . This was confirmed by the j hmbc correlation of h-6 (6.23, d) with the ester carbonyl carbon (169.9) of the 6-methylsalicylic acid unit (si), in addition to the downfield shifts of h-6/c-6 (6.23/80.1). The structure was further confirmed by the j and j hmbc correlations (si). H-3 (7.04, br s) showed j hmbc correlations with c-5 (77.5) and c-7 (15.7); h-4 (5.00, d) showed j hmbc correlation with c-2 (134.1) and j hmbc correlations with c-5 (77.5) and c-3 (150.0); h-5 (4.59, dd) showed j hmbc correlations with c-4 (72.8) and c-6 (80.1); h-6 (6.23, d) showed j hmbc correlation with c-4 (72.8) and j hmbc correlations with c-1 (194.4) and c-5 (77.5); h-7 (1.84, s) showed j hmbc correlations with c-1 (194.4) and c-3 (150.0) and j hmbc correlation with c-2 (134.1); h-3 (7.03, d) showed j hmbc correlations with c-1 (116.1) and c-5 (122.9) and j hmbc correlation with c-2 (160.0); h-4 (7.27, t) showed j correlations with c-2 (160.0) and c-6 (140.6); h-5 (6.76, d) showed j hmbc correlations with c-8 (22.4), c-3 (115.6), and c-1 (116.1); and h-8 (2.68, s) showed j hmbc correlations with c-1 (116.1) and c-5 (122.9) and j hmbc correlation of c-6 (140.6). By comparing these spectral data with the absolute configuration of compound 2 was determined as r, using the modified mosher esterification method, by preparing the diastereoisomeric esters separately with (r)- and (s)--methoxy--trifluoromethylphenylacetyl chloride (mtpa - cl), followed by analyzing their h nmr chemical shift differences []. Utilizing the acid chloride of mosher s reagent rather than the free acid commonly used in the typical mosher s method enabled running the reactions in the nmr tubes and rapidly acquiring the data . The h nmr of mtpa esters of the compound (pyridine - d5, 400 mhz) were assigned for the following protons: h-3 (34 hz), h-5 (2.4 hz), and h-6 (24.4 hz). Analysis of the above values established that the c-5-oh group has an r - configuration . The absolute configurations of c-4-oh and c-6-oco have been established to be r based on the noesy correlations between h-3, h-4, and h-5 . Compound 3 displayed a deprotonated molecular ion [m h] at m / z 139.040 99 (calcd 139.03951) in the negative mode hresims, corresponding to a molecular formula of c7h8o3 . The h and c nmr spectra showed the presence of three oxymethines; two are upfield shifted at 3.36 (d, j = 3.6 hz)/ 53.2 and 3.68 (dd, j = 0.8, 3.6 hz)/ 57.7, characterized for the epoxide, while the third oxymethine is at 4.52 (dd, j = 0.8, 4.8 hz)/ 62.8 . Also, the spectra revealed the presence of one carbonyl carbon at 195.1, suggesting the presence of a cyclohexenone, which was confirmed by the presence of an sp methine at 6.37 (d, j = 1.2 hz)/c 139.8, a singlet corresponding to a methyl group at 1.70 (3h, s)/c 15.7, and one quaternary carbon at 133.7 . The interpretation of the hmqc and hmbc correlations led us to the complete assignment of the structure . The absolute configuration of c-4 of compound 3 was determined as s, using the mosher esterification method . The h nmr of the mtpa esters of the compound (pyridine - d5, 400 mhz) were assigned for the following protons: h-5 (0 hz) and h-4 (+ 48 hz). Analysis of the above values established that the c-4 has an s - configuration . By comparing these data with those of closely related published compounds, we concluded that compound 3 is (2r,3r,4s)-2,3-epoxy-4-hydroxy-6-methylcyclohex-5-enone [(+) -epiepoformin]. Nine known compounds had been identified: ()-dihydroepiepoformin (4), (4s,5s)-4,5-dihydroxy-2-methylcyclohex-2-enone (5), 6-methylsalicylic acid (6), gentisylquinone (7), 3,4-dihydroxytoluene (8), 2,5-dihydroxybenzaldehyde (9), 3-hydroxybenzyl alcohol (10), 2,5-dihydroxybenzyl alcohol (11), and 3-hydroxytoluene (12). (+) -epiepoformin, 2,5-dihydroxybenzaldehyde, 2,5-dihydroxybenzyl alcohol, and 3-hydroxytoluene were all found to have activities against l. donovani, with ic50 values of 6.9, 3.3, 8.5, and 9.2 m, respectively, while compounds 1, (4s,5s)-4,5-dihydroxy-2-methylcyclohex-2-enone, and 10, 3-hydroxybenzyl alcohol have moderate activities against l. donovani, with ic50 values of 13.0, 47.3, and 34.0 m, respectively (table 1). All of the compounds were tested for antimicrobial activity, and only 3,4-dihydroxytoluene showed moderate antibacterial activity against methicillin - resistant staphylococcus aureus (mrsa) and staph . Aureus, with ic50 values of 72.9 and 147.8 m, respectively, [ciprofloxacin showed ic50 of 0.1 g / ml against mrsa and staph . Aureus; ic50 = the concentration that affords 50% inhibition of growth (g / ml)]. High - resolution mass spectra were measured using a bruker bioapex spectrometer . 1d and incubator shakers (new brunswick scientific, innova 4430) were used for incubating fungi . Sephadex lh-20 (mitsubishi kagaku, tokyo, japan) and silica gel (60120 mesh, merck) were used for column chromatography (cc). Solid - phase extraction (spe) cartridges (supelco, silica, 2, 5, and 10 g) were used under vacuum . Fractions from cc were monitored using precoated aluminum sheets [silica 60 f254, 0.25 mm (merck, darmstadt, germany)], with detection provided by uv light (254 and 366 nm) and by spraying with 1% vanillin diaion hp-20 (250 m, sigma - aldrich) was used for the separation of metabolites from the liquid media . Geosmithia langdonii (ascomycota: hypocreales) was provided by assiut university mycological center, assiut, egypt . A. m. moharram, at assiut university mycological center based on its morphological characters and comparison with the literature (accession no . The textiles were rinsed with water followed by surface sterilization in 70% etoh for 1 min, rinsed with sterilized water, cut into small pieces (2 cm in length and width), deposited on a petri dish containing modified pda medium (200 g of potato, 20 g of glucose, and 15 g of agar in 1 l of distilled water, supplemented with 100 mg / l chloramphenicol), and cultivated at 28 c for 3 days . The hypha tips were observed and transferred to new pda plates and subcultured until pure culture was obtained . Fungi were grown on modified potato dextrose agar (pda) plates at 28 c for 14 days . The fungus was grown on tryptic soy, malt extract, and pdb media . For small - scale extractions, the fungi were grown on a small scale using tryptic soy broth, malt extract broth, and pdb media . Fifty milliliters of each media were placed in 125 ml conical flasks and incubated with small pieces of actively growing mycelium . The cultures were incubated at 30 c under orbital agitation (160 rpm) for 14 days . After incubation, the contents of the flasks were filtered through sterile cotton using vacuum filtration, and then the filtrates were extracted exhaustively with ethyl acetate . The organic phase was vacuum concentrated to afford the extracts, which were then submitted for biological assays . G. langdonii was grown in 2800 ml erlenmeyer flasks containing 1 l of pdb medium (36 flasks), which had been prepared by dissolving 24 g of pdb in 1 l of distilled water and then autoclaved . Each flask was seeded by small fragments (25 mm) of the mycelium . The fungi were incubated at 30 c, using shakers (160 rpm), for 2 weeks . After the incubation period, the mycelia were filtered through sterile cotton using vacuum filtration, and the filtrates (40 l) were extracted with activated ion - exchange resin (diaion hp-20) by adding 100 g of resin to each 1 l of filtrate before being returned to the shakers and left overnight . The contents of the flasks were then filtered, and the hp-20 was washed with distilled water to remove salts and sugars . The meoh and acetone eluates were combined and dried under vacuum to yield a viscous residue, which was dissolved in water and successively extracted with n - hexane, dcm, and etoac (each 4 1 l). Each solvent was separately concentrated under vacuum to afford 2 g (n - hexane), 10 g (dcm), and 2 g (etoac). The crude dcm extract (10 g) was subjected to vacuum liquid chromatography (vlc) on flash silica gel (200 g, 15 15 cm) and eluted with gradients of n - hexane etoac (80:20100:0) and then with etoac meoh gradients (90:1050:50) to afford nine fractions [a i]. Fraction b (eluted with n - hexane etoac, 4:1, 385 mg) was chromatographed over silica gel cc (9 g, 50 3.5 cm) and eluted with n - hexane etoac gradients (100:080:20, 100 ml / fraction) to give five subfractions (b1b5). Mg) was further purified on sephadex lh-20 cc (10 g, 65 1.5 cm) with meoh etoac, 3:1, 460 mg) was subjected to solid - phase separation using an spe cartridge (silica, 10 g) under vacuum and eluted with n - hexane etoac with increasing polarities (95:550:50, 150 ml / fraction) to afford six subfractions (c1c6). Etoac, 17:3, 240 mg) was chromatographed over sephadex lh-20 cc (15 g, 65 1.5 cm), using meoh chcl3 (50:50) as the eluent, to yield subfraction c3 - 1 (170 mg) and 8 (35 mg). Subfraction c3 - 1 (170 mg) was further purified by cc over silica gel (5 g, 25 1.5 cm) using n - hexane etoac (95:585:15, 50 ml / fraction) to yield 3 (30 mg), 4 (1.5 mg), and 2 (5 mg). Fraction h (eluted with etoac meoh, 3:1, 2 g) was subjected to silica gel cc (50 g, 65 5 cm) using stepwise gradient elution of etoac and meoh (90:1070:30, 2 l / fraction) to yield three subfractions [h1h3]. Subfraction h1 (eluted with etoac meoh, 9:1, 200 mg) was further purified by sephadex lh-20 cc (15 g, 65 1.5 cm) and eluted with meoh the etoac crude extract (2 g) was subjected to silica gel cc (50 g, 65 5 cm) and eluted with n - hexane etoac in a manner of increasing polarities (100:00:100, 2 l / fraction) to afford 10 fractions [a j]. Fractions c, d, e, and f were further purified by sephadex lh-20 cc (15 g, 65 1.5 cm) and eluted with meoh chcl3 (50:50), yielding 7 (3 mg), 11 (53 mg), 5 (2.5 mg), and 1 (3.5 mg), respectively . The crude n - hexane extract (2 g) was subjected to silica gel cc (50 g, 65 5 cm) and eluted with n - hexane etoac with increasing polarities (100:070:30, 1 l each) to afford five fractions [a e]. White needles (ch3oh); uv (meoh) max (log) 298 (3.01) and 315 (3.24) nm; h nmr (cd3od, 400 mhz) see table 2; c nmr (cd3od, 400 mhz) see table 2; hresims m / z 261.06980 [m h] (calcd for c14h14o5, 261.07629). Colorless needles (ch3oh); []d 124 (c 0.06, meoh); uv (meoh) max(log) 296 (3.47), 308 (3.51), and 318 (3.45) nm; h nmr (pyridine - d5, 400 mhz) see table 2; c nmr (pyridine - d5, 400 mhz) see table 2; hresims m / z 315.0823 [m + na] and 607.1724 [2 m + na] (calcd for c15h16o6na 315.08448 and c30h32o12na 607.17916, respectively). In order to determine the absolute configuration, a modified mosher esterification method was used, where the diastereomeric (r)- and (s)--methoxy--trifluoromethylphenylacetyl chloride (mtpa - cl) esters of the compound were prepared in nmr tubes . First, the compound was mixed with (r)- and (s)-mtpa - cl separately in two different nmr tubes, in pyridine - d5 with a molar ratio of 1:1, respectively . The mixtures in the nmr tubes were warmed to 6070 c for 23 min, followed by acquiring the h nmr spectra for both the diastereoisomeric mtpa esters, and then their chemical shift differences [i.e., = (s - mtpa ester the antileishmanial activity was evaluated against a culture of l. donovani promastigotes grown in rpmi 1640 medium supplemented with 10% gibco fetal calf serum at 26 c . Growth of leishmanial promastigotes was determined by the alamar blue assay (biosource international, camarillo, ca, usa). Standard fluorescence was measured by a fluostar galaxy plate reader (excitation wavelength, 544 nm; emission wavelength, 590 nm). Pentamidine (ic50 3.2 m and ic90 5.9 m) and amphotericin b (ic50 0.2 m and ic90 0.4 pure compounds were tested for antimicrobial activity against the fungi candida albicans atcc 90028, c. glabrata atcc 90030, c. krusei atcc 6258, and aspergillus fumigates atcc 90906, as well as the bacteria methicillin - resistant staphylococcus aureus atcc 33591, cryptococcus neoformans attc 90113, staphylococcus aureus attc 29213, escherichia coli atcc 35218, pseudomonas aeruginosa atcc 27853, and mycobacterium intracellulare atcc 23068 . Amphotericin b (icn biomedicals, aurora, ohio) for fungal and ciprofloxacin (icn biomedicals) for bacterial bioassays were used as positive controls, respectively . White needles (ch3oh); uv (meoh) max (log) 298 (3.01) and 315 (3.24) nm; h nmr (cd3od, 400 mhz) see table 2; c nmr (cd3od, 400 mhz) see table 2; hresims m / z 261.06980 [m h] (calcd for c14h14o5, 261.07629). Colorless needles (ch3oh); []d 124 (c 0.06, meoh); uv (meoh) max(log) 296 (3.47), 308 (3.51), and 318 (3.45) nm; h nmr (pyridine - d5, 400 mhz) see table 2; c nmr (pyridine - d5, 400 mhz) see table 2; hresims m / z 315.0823 [m + na] and 607.1724 [2 m + na] (calcd for c15h16o6na 315.08448 and c30h32o12na 607.17916, respectively).
There is growing evidence that non - communicable diseases such as cardiovascular disease (cvd), stroke, hypertension and type 2 diabetes may originate in early life, a paradigm known as the developmental origins of health and disease (dohad). Investigations into mechanisms underpinning dohad using animal models and human specimens have suggested the involvement of epigenetics: mitotically heritable changes in gene expression controlled by chemical modifications to chromosomes without altering the dna sequence . One striking example implicates a role for the epigenetic mark of dna methylation in the long - term effects of the dutch famine during the second world war . By middle age, offspring previously exposed to maternal malnutrition during early gestation had a higher incidence of cvd than their unexposed siblings, together with differences in dna methylation in metabolic and cvd - related genes . Animal studies indicate that epigenetic modifications may be reversible by pharmacological or dietary interventions, suggesting approaches for future targeted interventions in humans . To accelerate progress towards this goal, these studies use techniques varying in sensitivity, coverage, sequence bias and amounts of dna required . A handful of ewas an important question raised by these studies is that of causality: when disease - associated epigenetic differences are identified, do they reflect causal pathological pathways of disease or a subsequent effect of disease? Furthermore, false positives can be captured in such screens ., recent ewas have found common epigenetic changes in pre- and post - symptomatic children with type 1 diabetes (t1d), and differences in fresh cord blood at gene loci whose expression was associated with body mass index in late childhood . Around the world, newborn babies are routinely screened for inborn errors of metabolism and other congenital disorders through testing of neonatal blood spot cards, a technique pioneered in the early 1960s by robert guthrie, after whom the cards are named . Collected within a few days of birth from heel pricks, the duration of guthrie card archiving varies between and within countries, ranging from a few months to indefinitely . In addition to serum analytes, dna has been extracted from guthries and has been used for such purposes as carrier screening for cystic fibrosis, detection of hiv and, more recently, genome - wide association studies . Modest degradation of dna occurs during storage and extraction that could affect data quality, although the quality appears sufficient for genomic assays . Remarkably, gene expression has also been analyzed in rna from 20-year - old guthries . As for the epigenetic regulators of gene expression, it has been shown that dna methylation can be analyzed at individual genes using the widely used technique of bisulfite conversion, which converts methylation differences to sequence differences; this has been applied to guthrie - based methylation screening of the fmr1 (fragile mental retardation 1) gene to predict cognitive impairment in individuals with fragile syndrome . A recent paper by beyan and colleagues has now taken this one stage further, conducting a proof - of - principle epigenome - wide pilot study using guthrie card methylomics . On average, 200 ng of dna from each 6 mm - diameter guthrie spot was extracted and used in two methods of genome - scale methylation profiling: one array - based, the other based on immunoprecipitation of methylated dna followed by high - throughput sequencing . For the array - based method, dna was extracted from 10-year - old guthries and compared with fresh blood and sperm from unrelated individuals . There was an excellent genome - wide correlation between archived guthrie dna and fresh blood, but a weaker correlation for the subset of regions showing small (<20%) differences in methylation between the two tissues . No comparisons were reported between fresh and aged dna from the same individual, which would have been an ideal control for the effect of storage of guthries on measurement of dna methylation . This represents an important caveat of the present study . For the immunoprecipitation - based method, which usually requires 2 g of dna the team then attempted to define the regions of the genome that differed between individuals but remained constant from birth to 3 years of age . This was an important comparison because it has been proposed that such' metastable epialleles' are influenced by environmental and stochastic factors in utero, remain constant thereafter, and can act as stable biomarkers for disease risk . For this, the team was careful to exclude genomic regions for which genetic heterogeneity could influence epigenetic variation and focused on clusters of variable, stable regions . Unfortunately, due to the low dna yields, the longitudinal comparisons were limited to the array - based technique at birth and the immunoprecipitation - based technique at 3 years of age . Nevertheless, up to a dozen metastable epialleles were identified, two of which had previously been associated with human disease . Highlights the utility of guthries for longitudinal ewas in which retrospective case - control studies can produce data more quickly and cheaply than birth cohort studies . However, the latter are designed to collect data on maternal exposures with minimal recall bias, which is not possible in retrospective studies . Furthermore, it is likely that a portion of the epigenome is still susceptible to environmental and stochastic influences in early postnatal life, making a case for repeat sampling . One disadvantage of using whole blood is that methylation levels represent an average of the levels in each of its component cell types, the proportion of which may change over time . However, if the early environment leaves an epigenetic legacy in multiple tissues, this will be a minor issue . Future longitudinal ewas (figure 1) will need to be sufficiently powered to detect disease - associated' epialleles' in contrast to the pilot study of beyan and colleagues, which was designed to detect large methylation differences (> 20%) in a small number of comparisons (n = 3 individuals). The group previously compared methylation profiles of 15 monozygotic twin (mz) pairs discordant for t1d, identifying 132 t1d - associated methylation variants with within - pair methylation discordance of 0.1 to 6.6% . Cross - cohort validation was performed with four additional t1d - discordant mz twins and the temporal origin of t1d - associated methylation differences were assessed with blood sampled from seven children with t1d before and after presentation, the latter using profiles from the same individuals positive for diabetes - associated auto - antibodies but negative for typical symptoms of t1d . Another recent study looked for methylation events that co - varied with body mass index at two time points in 74 individuals . Although different techniques were used, importantly, both of these studies used the same array platform at each time point . Epigenome - wide comparisons comparing blood from cases and controls at birth (guthries) and after disease presentation (blood in any form). After data filtering, differentially methylated regions will be compared to provide stable, disease - associated methylated events . It is also worth noting that there may be ethical barriers to longitudinal ewas, as the use of guthries without consent has been a major issue in some locations . Currently, cards can be used for limited forensic purposes and de - identified in research . But should consent always be sought for use of guthries in epigenetic research? Research studies using small numbers of samples are not generally problematic because it is easy to get consent from the individuals or parents . However, studies requiring large numbers of samples, such as well - powered ewas, are a problem because it may not be practical to obtain consent from all the individuals involved . Newborn infant screening programs have recognized these ethical issues and parents are presently better informed about potential uses of stored guthries, with some programs having introduced a consent process for future de - identified research . Beyan and colleagues have shown that it is possible to perform longitudinal ewas starting with blood samples from cases and controls after disease presentation and adding in blood samples obtained from guthries at birth (figure 1), a feat only previously achievable (in reverse order) through large birth cohort studies . Limitations that still need to be overcome include optimization of the amount and quality of dna extracted from guthries, identification of any technical artifacts associated with long term storage, an increase in study power and overcoming ethical barriers . In addition, longitudinal birth cohort studies should aim to sample at multiple time points to determine which disease - related epigenetic changes are present at birth and which develop after birth in response to postnatal environmental exposures . Nevertheless, the central message of the paper by beyan and colleagues is that we now have another arrow in our quiver with which to reach the ultimate target of ewas: to discover early, reversible biomarkers for human disease . Cvd: cardiovascular disease; dohad: developmental origins of health and disease; ewas: epigenome - wide association study; mz: monozygotic twins . We thank dr david godler for providing access to his manuscript in press, dr rony duncan and ms leah morenos for critical review of the manuscript, the murdoch childrens research institute for funding and the victorian government's operational infrastructure support program.
Harlequin ichthyosis (hi) is a rare genetic disorder of skin keratinization and is the most severe form of congenital ichthyosis with an incidence of about 1 in 300,000 births (1,2). Affected newborns have a thickened, grooved, hard (armor like) and hyperkeratotik skin with deep fissures that are most prominent over areas of flexion and waxy, plate like scaling over the entire of the body . It is associated with specific facial appearances including ectropion, everted and gapping lips (fish mouth), and ear and nose hypoplasia . Flexion deformities of all joints due to extreme inelasticity of the skin and hypoplastic digits are other signs of hi (4,5). In this paper we report a case of hi who was born by assisted reproductive technology (art). To the base of our search in scholar articles the aim of this report was to reinforce the importance of prenatal diagnosis, especially pre - implantation genetic diagnosis for hi in screening subsequent pregnancy . A 15 day - old boy was transferred to our hospital because of difficultly in feeding and dysmorphic appearance . He was the first child from first degree related healthy parents after 8 years of being infertile who born by ivf (in vitro fertilization) technique . His weight was 2,500 grams, 47 cm length and head circumference of 33 cm . Skin examination revealed thick horny shape 4 - 5 cm plaques with vertically and horizontally fissures . Ectropion, wide mouth and circum - oral cracking of skin were observed and ears and nose were crumpled and flattened . Characteristic features of harlequin ichthyosis in the baby the patient was admitted into the neonatology ward in an incubator with optimum temperature and humidity . Systemic antibiotics and fluids were started via an umbilical cut down and baby fed with feeding gavage . Topical antibiotics and emollients on the skin lesions and artificial tear drop for coverage of conjunctiva were prescribed . Pathological exam of skin biopsy revealed hyperkeratosis of epidermis layer that consisted with diagnosis of ichthyosis . On day 22 of his life while the neonate was in a good general condition and tolerated 150cc / kg / day of formula, he was transferred to another baby care center and did not return for follow up . In this article we reported a case of hi who was conceived by ivf technique . The hi is an extremely severe congenital form of ichthyosis characterized by a profound thickening of the keratin layer in fetal skin that is thought to primarily result from abnormal lipid metabolism in the epidermis (6,7). This disorder is caused by lack of function induced by mutations of the abca12 (adenosine - triphosphate - binding cassette a12) gene (1,3). Hi is a rare disorder with the incidence of 1:300,000 births and more than 100 cases have been reported in the literature (1,3,5). Most cases of hi have been recognized as having autosomal recessive inheritance although a dominant form may exist and sporadic cases occur frequently (5,8). The current case probably has an autosomal recessive pattern given the fact that his parents showed consanguinity . Although the histological findings of the variation in subtypes were varied, their clinical features were indistinguishable (2,5). Affected neonates die within few days because of feeding problems, infection, and respiratory failure . Recently with the better availability of neonatal intensive care facilities and benefits from oral retinoid some of affected newborns are surviving beyond neonatal periods (8). In 2007 reported a full term neonate with hi born in iran (gorgan) who died on the third day of hospitalization due to respiratory failure and sepsis . Reported another case of hi in a female preterm newborn in iran (mashhad) in 2009 . She also died on the third day of her life (4).diagnosisin both cases, was based on the clinical findings, but in our case, diagnosis was supplemented by skin biopsy . On the other hand the patient remained alive 22 days after birth during our follow up period . Rajpopat assessed clinical outcomes of 45 cases of hi and reported an overall survival rate of 56% ranged from 10 months to 25 years, that is probably the result of early introduction of oral retinoid and improvement of neonatal care (9). Noticeable point in our reported case is birth of affected newborn by ivf . To the best of our knowledge although ivf and other art techniques are generally considered safe, some studies have demonstrated an increased occurrence of major congenital malformations particularly congenital heart disease (10,11).on the other hand recent reports have suggested association between ivf and imprinting gene disorders such as angelman and beckwith - wiedmann syndrome (11). In a survey suggested that absolute risk of imprinting disorders in children born by art is small (<1%) and precise risk estimates are difficult to determine because of rarity of the conditions, hence recommended further investigation (12). Indeed in spite of low incidence of abnormalities in children conceived with arts, continuing surveillance of them including monitoring of their birth defects is essential (13). Although it seems that hi in our presented case was due to consanguinity of the parents, it is possible that this condition may occurred randomly following art pregnancy . The most important point and the main aim of this report was to emphasize on the importance of prenatal genetic diagnosis of hi in next offspring . Although prenatal genetic diagnosis is too expensive for low income family but it is very neccessary in high risk pregnancies such as our case . The diagnosis was done by fetal skin biopsy at 19 - 23 weeks of gestation using 3d or 4d ultrasonography (14,15). Akiyama et al reported the first dna - based prenatal diagnosis of hi by direct sequence analysis of abca12 mutation from amniotic fluid cells and demonstrated the efficiency of early dna - based prenatal diagnosis of hi . This new advancement created new horizons for earlier and accurate diagnosis of genodermatosis such as hi . We present a baby born with ivf suffering from hi that is a rare genodermatosis . In similar cases with improvement in dna - based prenatal diagnosis of hi new diagnostic procedures such as preimplantation diagnosis of hi especially for parents who had an affected child and can only be fertile by art is very critical and important for reduction of their emotional stress.
The lifetime risk of end - stage kidney disease (eskd) among african americans is threefold higher than that among whites . Differences in access to care and other socioeconomic factors do not entirely account for the markedly higher rates of eskd among african americans which suggests a possible role for genetic factors [16]. Recently, a region of chromosome 22, which includes apol1 and myh9 genes, was identified using mapping by admixture disequilibrium as a risk locus for non - diabetic forms of kidney disease, including idiopathic and hiv - associated focal segmental glomerular sclerosis (fsgs) and kidney disease clinically attributed to hypertension [79]. Genetic variants in the region show very strong associations with non - diabetic kidney disease . Specifically, a two - allele haplotype termed g1 consisting of two non - synonymous coding variants rs73885319 (s342 g) and rs60910145 (i384 m) along with rs71785313a 6 base pair deletion termed g2 and close to g1 in exon 5 of apol1 [10, 11]is likely to account for the majority of risk of non - diabetic kidney disease associated with the variants in this region, but the role of myh9 variants in non - diabetic kidney disease risk remains controversial . Regardless, variants in the region are more common in individuals of african descent compared with those of european descent [10, 11]. The fact that these variants are absent or less common in other populations has been used to explain the non - replication of these associations in non - african descent populations . The purposes of the current study were, therefore, to examine the frequency of these variants and to determine whether they are associated with ckd among native africans . The source population for both cases and controls was participants in the genetics of hypertension in blacks study, an ongoing project examining genetic variants for blood pressure among adults from the yoruba tribe . Participants in the current analyses included 88 and 81 adults with and without non - diabetic kidney disease, respectively . Participants were recruited from the university college hospital general medicine and nephrology clinics, university of ibadan, nigeria . The project was reviewed and approved by the institutional review board at the loyola university chicago stritch school of medicine, maywood, il and the joint ethical committee of the university of ibadan / university college hospital, ibadan, nigeria . The consent process was presented in english or yoruba, and written informed consent was obtained from all participants . Cases were defined as individuals of the yoruba tribe aged 1670 years with all stages of chronic kidney disease (ckd) of at least 3 months duration who met criteria for non - diabetic ckd clinically associated with long - standing hypertension, or ckd in the presence of proteinuria (proteinuric ckd) or ckd associated with hiv . Proteinuric ckd was defined as presence of spot urine protein / creatinine 2 g / g in the absence of red blood cell (rbc) casts or hematuria on urine microscopy or urinalysis and absence of known causes for ckd such as parasitic or other infections or diabetes . Clinically diagnosed hypertension - associated ckd was based on history or clinical evidence of long - standing hypertension (e.g., electrocardiogram evidence of left ventricular hypertrophy) and a spot urine protein / creatinine ratio <2 g / g, in the absence of other known causes for ckd, consistent with the african american study of kidney disease criteria . Ckd associated with hiv was defined as a positive hiv test and absence of other known causes for ckd including diabetes and parasitic diseases . Women who were pregnant, persons with diabetes mellitus, sickle cell disease, hepatitis b or c, acute kidney injury, or other terminal illnesses such as cancer were excluded . The clinical exam and laboratory studies included three serial blood pressure measurements using an automated device (omron hem-412c) previously evaluated in our field settings, anthropometric measures, a complete blood count and metabolic panel, electrocardiogram (ecg), testing for hiv and hepatitis b, urinalysis with microscopy and a bilateral kidney ultrasound when possible . All laboratory analyses in cases were completed at the university college hospital laboratory as part of routine clinical workup . Controls consisted of normotensive individuals (bp <140/90 in the absence of anti - hypertensive medication use) of the yoruba tribe without evidence of kidney disease (serum creatinine <1.4 and <1.2 mg / dl in men and women, respectively, and spot urine albumin / creatinine ratio <30 mg / g). All controls had standardized physical exams including three serial blood pressure measurements using the same device as used for the cases; fasting blood samples and a spot urine specimen were also obtained . Serum creatinine was measured in the control specimens at fairview laboratory in minnesota by rate reflectance spectrophotometry using thin film adaptation of the creatinine amidinohydrolase method on the vitros analyzer (johnson and johnson clinical diagnostics, rochester, ny). Genotyping was carried out on genomic dna from 88 non - diabetic ckd subjects and 81 non - ckd subjects randomly selected from among the controls described above . The genotyping was performed at the charles r. bronfman institute for personalized medicine (mount sinai school of medicine, new york, ny), using a custom fluidigm 96.96 array platform and abi taqman snp genotyping assays . The assays were originally selected for 96 published disease - associated single nucleotide polymorphisms (snps) and included 4 snps on apol1 and 6 snps on myh9 genes which have been reported to be associated with kidney disease . The assays also included variants associated with liver disease, type 2 diabetes or drug response . Standard quality control procedures were applied to the genotype data using all snps on the chip . As part of the procedures, 3 samples (1 case and 2 controls) and 2 snps with proportion of missing genotypes greater than 0.1 were filtered out . We also filtered out snps with minor allele frequency less than 0.05 (n = 23) or failing hardy weinberg equilibrium test at 0.01 (n = 3). The final 68 snps that passed quality control included 9 of the 10 apol1/myh9 variants . Since the objective of the current analysis was to examine the association between previously reported kidney disease associated snps and ckd in a sample of nigerians, subsequent screening for associations with ckd was therefore restricted to just the variants on apol1 (rs9622363, rs73885319, rs60910145 and rs71785313) and myh9 (rs11912763, rs2032487, rs4821481, rs5750248 and rs5750250) genes . To test for associations between ckd status and each single snp, we fitted logistic regression models in which each snp was presented as a predictor variable whose values were equal to the number of copies of the minor allele (0, 1, 2) (i.e., additive mode), or presence of at least one copy of the minor allele (0, 1) (i.e., dominant mode) or presence of two copies of the minor allele (0, 1) (i.e., recessive mode). The fitted model, which included sex and age as covariates, can be represented as: logit[pr(d = 1)] = + 1 g + 2sex + 3age, where d denotes ckd affection status; g denotes snp coded as additive, dominant or recessive; denotes the corresponding coefficient for each variable in the model (snp, sex or age), and its exponential is the corresponding odds ratio . To account for multiple comparisons, the statistical significance of each association test was empirically derived by permuting the data set 10,000 times . To explore the possible associations between ckd status and various joint allelic configurations of multiple snps, we performed haplotype association analysis of the apol1 and myh9 snps . We used the software haploview to compute the estimates of linkage disequilibrium (ld) for each pair of snps by the standard d - prime method and to determine the haplotype blocks regions with no evidence of historical recombination events, but significant level of ld . The haplotype blocks were defined by the four - gamete test [16, 17] as implemented in haploview . Analysis included all haplotypes with frequencies of at least 10% within the constructed blocks and also the two - allele haplotypes consisting of rs73885319 and rs60910145 that included g1 . All the haplotypes were individually tested for association with ckd by using logistic regression models as described above for tests of genotype association . The source population for both cases and controls was participants in the genetics of hypertension in blacks study, an ongoing project examining genetic variants for blood pressure among adults from the yoruba tribe . Participants in the current analyses included 88 and 81 adults with and without non - diabetic kidney disease, respectively . Participants were recruited from the university college hospital general medicine and nephrology clinics, university of ibadan, nigeria . The project was reviewed and approved by the institutional review board at the loyola university chicago stritch school of medicine, maywood, il and the joint ethical committee of the university of ibadan / university college hospital, ibadan, nigeria . The consent process was presented in english or yoruba, and written informed consent was obtained from all participants . Cases were defined as individuals of the yoruba tribe aged 1670 years with all stages of chronic kidney disease (ckd) of at least 3 months duration who met criteria for non - diabetic ckd clinically associated with long - standing hypertension, or ckd in the presence of proteinuria (proteinuric ckd) or ckd associated with hiv . Proteinuric ckd was defined as presence of spot urine protein / creatinine 2 g / g in the absence of red blood cell (rbc) casts or hematuria on urine microscopy or urinalysis and absence of known causes for ckd such as parasitic or other infections or diabetes . Clinically diagnosed hypertension - associated ckd was based on history or clinical evidence of long - standing hypertension (e.g., electrocardiogram evidence of left ventricular hypertrophy) and a spot urine protein / creatinine ratio <2 g / g, in the absence of other known causes for ckd, consistent with the african american study of kidney disease criteria . Ckd associated with hiv was defined as a positive hiv test and absence of other known causes for ckd including diabetes and parasitic diseases . Women who were pregnant, persons with diabetes mellitus, sickle cell disease, hepatitis b or c, acute kidney injury, or other terminal illnesses such as cancer were excluded . The clinical exam and laboratory studies included three serial blood pressure measurements using an automated device (omron hem-412c) previously evaluated in our field settings, anthropometric measures, a complete blood count and metabolic panel, electrocardiogram (ecg), testing for hiv and hepatitis b, urinalysis with microscopy and a bilateral kidney ultrasound when possible . All laboratory analyses in cases were completed at the university college hospital laboratory as part of routine clinical workup . Controls consisted of normotensive individuals (bp <140/90 in the absence of anti - hypertensive medication use) of the yoruba tribe without evidence of kidney disease (serum creatinine <1.4 and <1.2 mg / dl in men and women, respectively, and spot urine albumin / creatinine ratio <30 mg / g). All controls had standardized physical exams including three serial blood pressure measurements using the same device as used for the cases; fasting blood samples and a spot urine specimen were also obtained . Serum creatinine was measured in the control specimens at fairview laboratory in minnesota by rate reflectance spectrophotometry using thin film adaptation of the creatinine amidinohydrolase method on the vitros analyzer (johnson and johnson clinical diagnostics, rochester, ny). Genotyping was carried out on genomic dna from 88 non - diabetic ckd subjects and 81 non - ckd subjects randomly selected from among the controls described above . The genotyping was performed at the charles r. bronfman institute for personalized medicine (mount sinai school of medicine, new york, ny), using a custom fluidigm 96.96 array platform and abi taqman snp genotyping assays . The assays were originally selected for 96 published disease - associated single nucleotide polymorphisms (snps) and included 4 snps on apol1 and 6 snps on myh9 genes which have been reported to be associated with kidney disease . The assays also included variants associated with liver disease, type 2 diabetes or drug response . Standard quality control procedures were applied to the genotype data using all snps on the chip . As part of the procedures, 3 samples (1 case and 2 controls) and 2 snps with proportion of missing genotypes greater than 0.1 were filtered out . We also filtered out snps with minor allele frequency less than 0.05 (n = 23) or failing hardy weinberg equilibrium test at 0.01 (n = 3). The final 68 snps that passed quality control included 9 of the 10 apol1/myh9 variants . Since the objective of the current analysis was to examine the association between previously reported kidney disease associated snps and ckd in a sample of nigerians, subsequent screening for associations with ckd was therefore restricted to just the variants on apol1 (rs9622363, rs73885319, rs60910145 and rs71785313) and myh9 (rs11912763, rs2032487, rs4821481, rs5750248 and rs5750250) genes . To test for associations between ckd status and each single snp, we fitted logistic regression models in which each snp was presented as a predictor variable whose values were equal to the number of copies of the minor allele (0, 1, 2) (i.e., additive mode), or presence of at least one copy of the minor allele (0, 1) (i.e., dominant mode) or presence of two copies of the minor allele (0, 1) (i.e., recessive mode). The fitted model, which included sex and age as covariates, can be represented as: logit[pr(d = 1)] = + 1 g + 2sex + 3age, where d denotes ckd affection status; g denotes snp coded as additive, dominant or recessive; denotes the corresponding coefficient for each variable in the model (snp, sex or age), and its exponential is the corresponding odds ratio . To account for multiple comparisons, the statistical significance of each association test was empirically derived by permuting the data set 10,000 times . To explore the possible associations between ckd status and various joint allelic configurations of multiple snps, we performed haplotype association analysis of the apol1 and myh9 snps . We used the software haploview to compute the estimates of linkage disequilibrium (ld) for each pair of snps by the standard d - prime method and to determine the haplotype blocks regions with no evidence of historical recombination events, but significant level of ld . The haplotype blocks were defined by the four - gamete test [16, 17] as implemented in haploview . Analysis included all haplotypes with frequencies of at least 10% within the constructed blocks and also the two - allele haplotypes consisting of rs73885319 and rs60910145 that included g1 . All the haplotypes were individually tested for association with ckd by using logistic regression models as described above for tests of genotype association . After excluding participants whose genotype data did not pass quality control, the study sample consisted of a total of 166 subjects (87 cases and 79 controls). The descriptive characteristics of the cases and controls are presented in table 1 . Among both cases and controls,, the ckd subjects tended to be older (42.1 vs. 35.2 years), heavier (23.2 vs. 21.9 kg / m) and have higher blood pressure (systolic: 136.6 vs. 111.6 mm hg) when compared with the non - ckd controls . Among the 87 ckd subjects, the physician - reported diagnosis of kidney disease was proteinuric ckd in 35 (40.2%), hiv nephropathy in 8 (9.2%) and clinically attributed hypertensive ckd in 44 (50.5%). All the ckd subjects had a negative hepatitis b surface antigen test within 30 days of enrollment in the study . All but 3 cases had stage 5 ckd (1 with proteinuric ckd and 2 with ckd clinically attributed to hypertension.table 1descriptive characteristics of study subjectsnon - diabetic ckd cases (n = 87)controls (n = 79)all (n = 166)no . Of females (%) 41 (47%) 39 (49%) 80 (48%) age (years) 42.1 16.935.2 8.238.8 13.9body weight (kg)61.6 11.159.8 10.860.8 11.0height (m)1.6 0.11.7 0.11.6 0.1body mass index (kg / m)23.2 4.621.9 4.222.6 4.4systolic blood pressure (mm hg) 136.6 31.0111.6 10.0124.7 26.6diastolic blood pressure (mm hg) 88.1 20.766.9 6.178.0 18.8 ckd chronic kidney diseasedata presented as mean standard deviation values are significantly different (p <0.05) between cases and controls descriptive characteristics of study subjects ckd chronic kidney disease data presented as mean standard deviation values are significantly different (p <0.05) between cases and controls the frequencies of the risk allele of the 9 apol1/myh9 variants among the cases and controls are listed in table 2 . In the current analyses, the coded alleles are the same as the minor alleles of the variants . For any variant with reported odds ratio (or) greater than 1.0, the coded minor allele is also the risk or disease susceptibility allele . As would be expected, coded risk alleles tended to be more frequent in cases than in controls, for example, 0.442 versus 0.266 and 0.500 versus 0.301 for rs73885319 and rs60910145, respectively . The variant rs71785313, which is an apol1 insertion / deletion, had the least frequent minor allele (0.105) in both cases and controls . Results of the covariate - adjusted case / control analyses are also presented in table 2 for additive, dominant and recessive genetic modes of association . We observed significant associations for two apol1 snps rs73885319 and rs60910145 under all three genetic modes of association . Both variants have larger effects under the recessive mode (odds ratios: 3.85 and 3.12 for rs73885319 and rs60910145, respectively) when compared with the additive or dominant mode . The two variants are in almost perfect linkage disequilibrium (d - prime = 1.00, r = 0.82) and as such, when adjusted for either one, the association for the other disappeared.table 2snp associations with non - diabetic chronic kidney disease in 87 cases and 79 controlssnpgeneallelescoded (minor) allele (frequency,%) hwe p valuecoded allele frequency (%) association modeor (95% ci) p valuecasescontrolsunadjustedcorrected rs9622363 apol1 a / ga (27.71)0.78825.8629.75additive0.76 (0.451.31)0.3260.875dominant0.88 (0.471.66)0.6950.999recessive0.24 (0.051.29)0.0970.377rs73885319 apol1 a / ga (35.76)1.00044.1926.58additive2.29 (1.393.77)0.0010.005dominant2.59 (1.345.00)0.0050.025recessive3.85 (1.3111.36)0.0150.038rs60910145 apol1 g / tg (40.61)0.11450.0030.13additive2.04 (1.323.17)0.0010.006dominant2.54 (1.314.92)0.0060.034recessive3.12 (1.357.20)0.0080.015 g2: rs71785313 apol1 d / id (10.54)1.0008.6212.66additive0.61 (0.291.31)0.2070.701dominant0.64 (0.291.40)0.2630.816recessiveneneners11912763 myh9 a / ga (33.13)1.00038.5127.22additive1.68 (1.022.76)0.0400.197dominant2.03 (1.063.87)0.0320.183recessive1.70 (0.584.94)0.3340.872rs2032487 myh9 t / ct (22.12)0.77018.3926.28additive0.68 (0.401.16)0.1570.580dominant0.64 (0.331.23)0.1770.645recessive0.55 (0.142.22)0.4000.934rs4821481 myh9 t / ct (22.29)0.77718.3926.58additive0.66 (0.391.13)0.1320.532dominant0.61 (0.321.18)0.1430.583recessive0.55 (0.142.24)0.4070.940rs5750248 myh9 c / tc (30.72)1.00025.8636.08additive0.61 (0.370.99)0.0470.225dominant0.56 (0.291.05)0.0710.354recessive0.46 (0.151.41)0.1760.627rs5750250 myh9 a / ga (31.82)0.63526.1637.97additive0.56 (0.340.94)0.0270.141dominant0.51 (0.270.97)0.0400.208recessive0.44 (0.141.38)0.1570.576adjusted for age and gender snps single nucleotide polymorphisms, hwe hardy weinberg equilibrium, or odds ratio, ci confidence interval, ne not estimated p values corrected for multiple comparisons snp associations with non - diabetic chronic kidney disease in 87 cases and 79 controls adjusted for age and gender snps single nucleotide polymorphisms, hwe hardy weinberg equilibrium, or odds ratio, ci confidence interval, ne not estimated p values corrected for multiple comparisons the haplotype blocks formed by the apol1/myh9 variants are displayed in fig . 1 . There were two blocks consisting of three apol1 snps and four myh9 snps, respectively . Two snps, rs71785313 on apol1 and rs11912763 on myh9, were not included in any of the blocks . The apol1 block thus included rs9622363, rs73885319 and rs60910145, and the myh9 block included rs2032487, rs4821481, rs5750248 and rs5750250 . There were a total of five haplotypes each with a frequency of at least 0.10 (fig . 1). The results of the covariate - adjusted haplotype associations are presented in table 3 . We also present the results for the two - allele haplotypes of rs73885319 and rs60910145 that included the previously reported g1 haplotype . G haplotype of the apol1 snps (rs9622363rs73885319rs60910145) is a significant risk factor for ckd under any mode of association . The ors are 2.26 (p = 0.005), 2.54 (p = 0.023) and 3.79 (p = 0.041) for the additive, dominant and recessive modes of association, respectively, after correction for multiple comparisons . Similarly, the two - allele haplotypes of rs73885319 and rs60910145 otherwise termed g1, demonstrated strong levels of association with ckd . The ors for the g1:rs73885319rs60910145 (a g) haplotype are 2.25 (p = 0.006), 2.52 (p = 0.025) and 3.80 (p = 0.041) for the additive, dominant and recessive modes of association, respectively . The crude association [or, 2.67(95% ci, 0.798.97)] with the compound risk heterozygote state among subjects heterozygous (10 cases and 5 controls) for both the g1:a g and g2:d risk alleles and subjects (21 cases and 28 controls) without any of the risk alleles was not significant (p = 0.143). T haplotype of g1:rs73885319rs60910145 indicated significant protective association with ckd under the additive (or = 0.49, p = 0.005), dominant (or = 0.40, p = 0.025) and recessive (or = 0.32, p = 0.014) modes . The implication of this is a significantly high ckd risk for those carrying zero copies of the g1:g t haplotype with an or as high as 3.13 (p = 0.014). We did not observe significant evidence of association between any of the myh9 haplotypes and ckd after accounting for multiple testing (table 3).fig . 1plot of linkage disequilibrium between snps in apol1/myh9 region (top) and their haplotypes (bottom)table 3haplotype associations with non - diabetic chronic kidney disease in 87 cases and 79 controlssnp combinationhaplotypehaplotype frequencies (%) association modeor (95% ci) p valuecasescontrolsallunadjustedcorrected apol1 snps rs9622363 \| rs73885319 \| rs60910145g a g44.2526.9235.81additive2.26 (1.373.73)0.0010.005dominant2.54 (1.314.92)0.0060.052recessive3.79 (1.2811.20)0.0160.024 rs9622363 \| rs73885319 \| rs60910145a g t25.2930.1327.41additive0.72 (0.421.23)0.2310.641dominant0.81 (0.431.53)0.5240.392recessive0.24 (0.051.27)0.0930.983 rs9622363 \| rs73885319 \| rs60910145g g t24.7139.7432.16additive0.58 (0.370.90)0.0150.063dominant0.49 (0.260.93)0.0280.215recessive0.41 (0.161.03)0.0570.134 g1: rs73885319 \| rs60910145a g44.1926.9235.98additive2.25 (1.363.71)0.0020.005dominant2.52 (1.304.88)0.0060.051recessive3.80 (1.2911.22)0.0160.026 g1: rs73885319 \| rs60910145g t50.0069.8759.45additive0.49 (0.320.76)0.0010.005dominant0.32 (0.140.73)0.0070.018recessive0.40 (0.210.77)0.0060.031 myh9 snps rs2032487 \| rs4821481 \| rs5750248 \| rs5750250t t c a17.2426.5821.67additive0.62 (0.361.07)0.0830.302dominant0.57 (0.291.09)0.0910.899recessive0.50 (0.112.16)0.3520.373 rs2032487 \| rs4821481 \| rs5750248 \| rs5750250c c t g72.4162.0367.45additive1.66 (1.012.74)0.0460.184dominant2.16 (0.716.60)0.1760.299recessive1.80 (0.953.42)0.0730.609adjusted for age and gender or odds ratio, snps single nucleotide polymorphisms, ci confidence interval p values corrected for multiple comparisons plot of linkage disequilibrium between snps in apol1/myh9 region (top) and their haplotypes (bottom) haplotype associations with non - diabetic chronic kidney disease in 87 cases and 79 controls adjusted for age and gender or odds ratio, snps single nucleotide polymorphisms, ci confidence interval p values corrected for multiple comparisons in this study, we report the findings from a case / control association analysis of non - diabetic chronic kidney disease and variants in apol1 and myh9 genes in an african sample from southwest nigeria . Apol1 and myh9 variants are associated with non - diabetic ckd among african americans [79, 11, 18, 19] and hispanic americans . The purpose of our study was to investigate the possible evidence of association between these variants and non - diabetic ckd in a sample of africans of the yoruba tribe without history of european admixture . We replicated association with apol1 gene variants previously reported among african americans and hispanic americans [10, 11]. The strength of the association between the two - allele haplotype of apol1 variants rs60910145 and rs73885319 (g1) and non - diabetic ckd in the current study is about half of the sevenfold - increased odds of hypertensive kidney disease reported among african americans carrying multiple copies of apol1 risk alleles . Stronger associations have been reported with hiv nephropathy [11, 19]. Since sample size only affects statistical significance of estimates and not the strength of the estimates, the apparent attenuation of the observed association when compared to previous findings among african americans cannot be attributed to sample size . To confirm this, we investigated how much power the current study had to detect significant association between a variant with risk allele frequency at least similar to that observed for g1:a g (i.e., 0.360), a genetic effect of at least 3.0 under a population risk of 0.00001 . Using the software quanto, we estimated that the current study has at least 80% power to detect association under recessive mode and over 90% power under dominant or additive mode . We noted that the risk allele in the current sample is different for rs73885319 than previously reported . It should be noted that risk allele frequencies for this variant have been observed to differ substantially across african populations . In an african population sample set consisting of 676 samples from 12 african populations that included cameroon (2 ethnic groups), congo, ethiopia (4 ethnic groups), ghana (2 ethnic groups), malawi, mozambique and sudan, tzur et al . Observed that risk allele frequencies differ between groups from same country and also across the populations . In the bulsa and asante populations of ghana, the frequencies are respectively 0.11 and 0.41, whereas for the ethiopian groups the frequencies were zero . It is possible that non - diabetic kidney disease risk is not mediated by this variant but rather by other alleles in linkage disequilibrium with the variant or the haplotype g1 . As for myh9, we did not find significant association between non - diabetic ckd and any variant or haplotype after correction for multiple testing . A previous study indicated that g1 and g2 are in strong ld with variants in myh9, and most of the association previously attributed to myh9 variants or haplotypes with ckd is explained by ld with apol1 variant rs73885319 [10, 11]. In the present study, we observed reduction in the strength of ckd association with myh9 variants when we accounted for the apol1 variants by conditioning on either rs73885319 or rs60910145 in the regression models fitted for each of the myh9 variants . We note that the observed low ld (r 0.12) between apol1 and myh9 variants in our study sample may have contributed to the observed non - significant association of myh9 variants with ckd . Among african americans, the frequency of the apol1 risk allele is around 0.33 with the high - risk genotype frequency being about 0.11 . Previous studies have reported strong associations with apol1 risk alleles g1 (rs73885319 and rs60910145) and g2 (rs71785313) in an autosomal recessive fashion . Inheritance of 2 apol1 risk alleles (g1 and g1, g1 and g2, or g2 and g2) increases the risk of non - diabetic kidney disease by over sevenfold . For hiv nephropathy, associations may exceed 30-fold . In this study, we noted a fairly strong association between the g1 risk allele in a recessive model but no association was noted between g2 and non - diabetic kidney disease . This is likely due, in part, to the effect of sample size and the observed low minor allele frequency for g2 . It is also possible that differences in the association between apol1 variants and ckd in this study could, in part, be due to misclassification of cases or gene environment interactions but these interpretations remain speculative . In this study, the strength of the associations between 2 snps included in the myh9 e1 risk haplotype and non - diabetic kidney disease, though not significant after adjusting for multiple comparisons, was similar to associations reported in cases with end - stage kidney disease clinically attributed to hypertension . The mhy9 e1 risk haplotype consists of 4 snps (rs4821480, rs2032487, rs4821481 and rs3752462); robust associations have been documented in several studies between the e1 haplotype and non - diabetic kidney disease including idiopathic and hiv - associated fsgs and ckd clinically attributed to hypertension [8, 9, 18, 20]. Apol1 variants are in strong linkage disequilibrium with myh9 variants, and it remains controversial whether myh9 variants play a direct role in kidney disease risk [23, 24]. Due to limited resources and poor access to healthcare for non - urban areas of nigeria, many patients who present for diagnostic workup and treatment of kidney disease do not undergo kidney biopsy in ibadan . However, diabetes prevalence is very low in nigeria [25, 26], and patients with evidence of diabetic kidney disease as determined by the treating physician were excluded from participation in study . The small sample size limited the ability to determine associations with specific types of ckd such as hypertensive ckd or focal segmental glomerulosclerosis . However, even with the small sample, fairly strong associations were noted with apol1 variants and the haplotype g1 . The associations between apol1 variants and non - diabetic ckd among nigerians of the yoruba tribe demonstrate that the impact of these genetic factors on ckd risk appear to be independent of the environment . Diet, lifestyle and social structure are dramatically different in nigeria compared to the united states and other industrialized countries . Hypertension, diabetes and obesity prevalence are markedly lower in nigeria compared to the african american population [2529]. Despite these differences, apol1 variants are associated with non - diabetic ckd in this population . The discovery of the apol1/myh9 chromosomal 22 region as a region harboring genetic variants for non - diabetic ckd risk may be very important . It is possible that further delineation of the role of myh9 and apol1 variants may lead, in the future, to improved screening programs, prevention strategies and clinical interventions for ckd, one of the most common end - organ causes of morbidity and mortality worldwide . The perceived public health importance of these genetic variants is demonstrated by a patent application by the national institutes of health for these variants . This study demonstrates that these variants are also operative for non - diabetic kidney disease risk among nigerians of the yoruba tribe . Data suggest that the g1 and g2 variants in the apol1 region emerged in this population several thousand years ago as a result of conferring protection from trypanosoma brucei rhodesiense [10, 31], a story very similar to the rise in frequency of the sickle cell trait as a result of resistance to certain forms of malaria . In conclusion, apol1 risk variants are associated with non - diabetic forms of ckd among nigerians of yoruba ethnicity . Further information on apol1/myh9 variants may lead to screening programs which could lead to earlier detection and interventions for non - diabetic kidney disease . This article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
The ability of a newborn baby, fresh out of the womb, to attach to the maternal nipple and begin sucking leads many to label the behavior as innate . Some extend this concept of innate behavior to include reflexes, denoting fixed action patterns organized as sensory - motor circuits in the brain stem or spinal cord [1, 2], as well as the rhythmic firing of central pattern generators (cpgs), also located in the central nervous system, that produce correlated neuromuscular sucking rhythms . The concept of innate behavior is controversial, to say the least [4, 5]. Criticisms abound because such behaviors, when carefully observed and measured are not fixed but are highly variable [6, 7]. Moreover, so - called instinctive behaviors, under the scrutiny of experimental analysis, prove to be based on various forms of prior experience and learning [8, 9]. Similarly, while the firing of cpgs may correlate with sucking rhythms, it has yet to be shown that such isolated elements actually combine with other discrete components to create the real behavior of the suckling newborn, behavior that adapts to the unpredictable, dynamic geometry of the mother's body, and behavior in real time and in real contexts . Congenital is a more accurate and defensible term with which to denote a capability in behavior that is present at birth . In contrast to innate and instinctive, congenital is more obviously a description of status at birth than it is an explanation of its basis or origins [10, 11]. Explaining the developmental origins of a congenital capability such as nipple attachment and sucking by a newborn upon confronting for the first time its mother's external body in highly novel environment is a formidable challenge of clinical significance . The experimental literature, based on studies with non - human animals, contains a wealth of information pertinent to a better understanding of the onset and development of oral feeding skills . This literature includes some impressive findings concerning the onset of nipple attachment and sucking by newborn rat pups . A brief description of the postnatal onset of sucking by rat pups will help frame the forthcoming presentation and analyses: rat pups are born after a gestation period of about three weeks . They are born as litter, averaging 10 pups (mothers have 12 nipples), blind, deaf, and furless, with limited strength and coordination . For about 6 hrs prior to parturition, labor contractions can be seen rippling vertically on the mother's abdomen or indenting her sides as she stretches her body . More than 100 labor contractions can be observed during a rat's labor . As each pup emerges from the birth canal, encased in an amniotic sac with the umbilical cord and placenta trailing, the dam assists with licking and gentle tugging . The mother removes the sac by licking and nibbling, consumes the placenta and membranes, licks the pup some more, licks herself, and then repeats the sequence as the next pup emerges . Only after all the pups are thus delivered and the placentas are all consumed, does the rat dam turn her attention to the newborns, which she gathers into a clump in the nest and settles over them . We have described and quantified the labor and delivery process in rats, noting the stimulation received by the pups during labor and throughout the birth process . In the nest, with the dam hovering above the pups, the infants are active . They orient to the dam's ventrum, probe against her body and root along the ventrum until they orally grasp a nipple and suck . It is difficult to observe directly the natural sequence of events that lead to the rat pups' initial nipple attachments and sucking . When the dam settles on her newly born litter she is typically crouched above the pups within a nest that affords poor visibility . Fortunately, the newborn rat's behavior is robust and orderly; when placed under suspended artificial surfaces simulating various properties of the dam's ventrum, newborn rats show an organized repertoire of behaviors . They travel, wriggle, turn on their sides and upside down, ventroflex, probe the surface, and audibly bark, all in a state of heighted behavioral arousal . Other studies have been conducted with the dam anesthetized and the pup's behavior thus isolated for analysis . Thus, it is known that olfactory cues present on the rat mother's nipples and ventrum are necessary and sufficient for newborn pups to locate and orally apprehend a nipple to suck . These odor cues can be removed by washing the nipples and surrounding body surfaces that eliminates suckling [14, 15]. Nipple attachment and suckling by newborns can be reinstated, however, by painting onto the dam's ventrum a distillate of the wash taken from the dam's body or by painting nipples with amniotic fluid or maternal saliva . Other substances, both natural and atypical were tried, but no others were effective in reinstating nipple attachment . Knowledge that amniotic fluid is a sufficient stimulus to elicit the newborn's first nipple attachment led to preliminary considerations of two, mutually exclusive possibilities . One was that the key olfactory stimulus is somehow predetermined and that the newborn is correspondingly and inherently prepared (hard - wired the second possibility was that the perinate responds with nipple attachment and sucking to the amniotic odor stimulus because of its previous experience with amniotic fluid . They reasoned that if amniotic fluid is a behaviorally potent stimulus because the fetus experienced it previously, then if some other odor was similarly experienced, it should have the same behavioral potency as amniotic fluid . They tested this bold hypothesis by adding a novel, lemon - like substance to the amniotic fluid, and then testing whether this chemical would rescue the newborn's ability to make its first attachment to the washed nipples of a mother rat . The previously validated experimental procedure involved externalizing the uterine horns of a gestational day (gd) 20 dam and injecting a small quantity (0.2 ml) of a citral and saline solution through the transparent wall of the uterus into the amniotic fluid . The uteri were replaced in the dam's peritoneum, the laparotomy incision was closed, and gestation was completed without complications . Then, on gd 22, pups were delivered by caesarean section and placed immediately for one hr in a warmed nest where they were stroked with a soft artist's brush for 1 hr in the presence of the citral odor . The test procedure involved presenting the caesarean - delivered pups with an anesthetized parturient rat dam (not the subject pups' mother). If the dam's nipples were washed, pups did not attach to nipples, but when citral was on the dam, the treated newborns attached to a nipple and sucked! Natural odors of an unwashed dam were not effective for the citral - treated perinates . The new odor had replaced the natural stimulus . They performed an additional experiment in which pups were exposed to citral (a) in utero, (b) immediately after birth with stroking, or (c) both in utero and with postnatal stroking . Only pups with the combined experiences attached to the washed, citral - scented nipples and not to the unwashed, normal nipples . Pedersen and blass' study provided important new insights into the initial plasticity of the newborn rats' sucking, especially the specification of the cues that can activate and direct the behavior . It seems clear that the establishment of the olfactory control of sucking is determined by the experiences of the perinatal rat pup . But, what are the essential experiences for establishing the newborn's sucking responses to maternal cues? We adapted elements of our previous investigations of the perinatal rats' sensory experiences in the uterine environment and of the birth process [12, 1820] to demonstrate that specific components of maternal stimulation are sufficient conditions for the odor learning that establishes the newborn's sucking responses to maternal cues . The present paper is a review of some of this past research as well as a report of additional, previously unpublished data that, together, provide a new view of how the experience of being born creates a context for learning . That is, embedded in flow of events that constitute the birth process are forms and levels of stimulation that, together, create the contingencies for early, rapid learning in the fetus, as it becomes a newborn . We will show that this learning, though general in initial form, is expressed in the natural context of the mother's body as organized, adaptive, seemingly goal - directed behavior . We will first review an analysis of rat maternal behavior during labor and delivery from which we derived a set of novel tools that enabled us to simulate the major components of vaginal birth . We will also review some of our evidence that fetal and neonatal rats (perinates) have sensory capabilities sufficient to experience the birth process, at least the elements that are needed for basic associative learning . Then, we will present data showing that the perinates' responses to simulations of the birth process (a) augment nipple attachment and sucking, (b) establish odor - guided responses to the mother, and (c) induce neural conditions that mediate state transitions between fetal and neonatal behavioral systems which can account for the activation and expression of the newborn's initial sucking behavior . Under laboratory conditions, norway rats typically give birth on the 22nd day of gestation; by breeding our animals on a known day, we were able to be present with appropriate video arrangements to view and record the dams' labor and delivery . From these videorecordings, we quantified an average of 144.6 labor contractions during the six hours prior to the birth of the first pup from eight dams . Figure 1 illustrates the three types of visible labor contractions in rat dams and shows the average frequency of each during the 6 hrs of labor . Behavioral expressions of labor in the rat progress from uterine peristalsis to lordosis contractions followed by vertical contractions that occur in close association with birth of each pup . The brisk, linear decline in intercontraction intervals shown in figure 2 indicates how the contractions quicken as parturition approaches . From our systematic observations, we are able to describe the labor and delivery in the rat . Duration of the delivery phase of parturition (first to last birth) ranged from 40 to 136 min, with dams delivering and average of 10.1 (1.1) pups . Delivery duration and litter size were positively related (r = .73, p <as each pup begins to emerge from the birth canal, the dam typically adopts a head - between - heels posture, which facilitates delivery by enabling the mother to use her teeth to grasp the newborn and extract it from the vagina . Mothers lick and handle each pup, removing and consuming the embryonic membranes, activities that produce cutaneous stimulation and augmented evaporative cooling of the newborn's body . Dams provided about 2 min of continuous stimulation to each newborn while participating in its delivery . Each pup also received bursts of vestibular stimulation as the dam rotated its body while systematically consuming the products of gestation . During the initial phases of intense tactile and vestibular stimulation, as birth membranes were removed, especially from the head, pups began to emit the robust gasps that are characteristic of the onset of independent respiration, and thereafter they displayed gross movements and audible vocalizations . After the immediate postpartum licking and handling of each newborn, the dam often refocused her attention on previous newborns, providing each one with about 2.5 min of additional licking and handling . Overall, licking by the dams was distributed relatively evenly across the pups' bodies: head (39%), body (24%), anogenital area (32%), (see for additional details). Such observations help to specify events to which the newborn rat is exposed during birth . Each pup received a protracted bout of repetitive tactile and vestibular stimulation associated with the dam's handling and licking . During parturition, offspring are exposed to seemingly harsh forms of stimulation related to cooling and with compressions under the weight of the dam's body . After systematically describing and analyzing the kinds and amounts of stimulation sustained by rat fetuses as they were being delivered vaginally, we endeavored to assess quantitatively some of the most prominent forms of stimulation . Among the forms of birth stimuli that we analyzed were uterine compressions, cooling and rewarming, and maternal licking . From these analyses, we created a set of procedures and tools to mimic the biological stimuli that represent the physical bases of the pups' experience of being born . Uterine contractions, for example, were measured by surgically removing a single fetus from one of the paired uterine horns of a g18 rat installing a small balloon in its place in utero . The balloon was connected to a thin polyethylene tube that ran subcutaneously to the dam's back and was externalized at the nape of the neck . The tubing could be connected to a pressure transducer with which we measured the forces exerted on the fetuses by the mother's behavior and by uterine contractions . The dams' contractions ranged from 2 to 30 mm hg . By attaching an inflated balloon to a small, spring - based calibrated scale, we could apply with the balloon surface a reliable force of 15 mm hg to a single fetal rat or to a newborn (see figure 3). In this way, we established a protocol for simulating a vaginal birth for rat fetuses: 15, 20 sec - long compressions delivered at a rate of 1 per min, cooling (22c), stroking with a soft brush (2 min), and rewarming (33c). Several of our analyses have focused on the how the birth process, beginning with the mother's labor contractions, helps organize the fetal - to - neonatal transition . Breathing and suckling are two vital behavioral adaptations of the newborn . In one set of studies, we applied our tools to study the components of birth that are important in the onset of pulmonary respiration, perhaps the most essential and immediate requirement of the newborn . The respiratory movements present in utero are episodic and unrelated to gas exchange [22, 23]. At birth, however, breathing becomes continuous and regulated to meet the newborn's oxygen requirements . We found that compressions simulating uterine contractions were necessary for initiating breathing in late gestation rat fetuses . The effectiveness of simulated labor contractions could arise from some mechanical (nonsensory) effect of the compression, or cutaneous (sensory) effects on the offspring . In a study of gentle stroking of cesarean delivered pups (without simulated labor contractions), only 25% nonstroked pups survived for 1 hr postpartum compared to 100% stroked pups supporting a role for sensory stimulation . These observations fit well with reports of adaptive neuroendocrine changes and neurobehavioral advantages in neonates, both term and preterm, exposed to tactile and kinesthetic stimulation [25, 26]. Paradigm to examine more complex behavioral patterns in newborn rats in a study of how suckling becomes established . Fetal rats were either exposed to labor contractions or not then cesarean delivered as described earlier, except that we manipulated postpartum ambient temperature using one of three biologically relevant temperatures . Newborns were exposed to the cool room - temperature environment (22c) or to a warmer temperature maintained at nest (33c) or intrauterine (36c) temperature . After 1 hr postpartum exposure to one of the three temperature regimens, pups from all groups were placed at nest temperature then tested for nipple attachment . The 22c condition contained the sequence of thermal exposures experienced by a vaginally born rat pup under typical thermal conditions . The treatment regime, then, was designed to represent the sequence and duration of stimulation that normally occurs prior to and immediately after vaginal birth, leading to the onset of suckling . At 2 hr postpartum, we found that 90% of vaginally delivered pups attached to a nipple (table 1). The most dramatic effects of prenatal compression were seen between pups that experienced thermal conditions similar to those of normal, vaginally delivered pups (i.e., the room temperature condition), whereas thermal effects were most evident in pups exposed to atypically warm temperatures (i.e., the intrauterine temperature condition). These studies link the major postnatal milestones of pulmonary ventilation and suckling to birth experience . We sought to determine the mechanisms underlying the effectiveness of birth stimuli in facilitating the fetus - to - newborn transition . Human babies show a surge of plasma catecholamines associated with the stress of being born, a physiological response to labor and squeezing through the birth canal [27, 28]. Vaginally delivered infants show exhibit both enhanced respiratory performance and increased alertness compared to cesarean - delivered infants whose mothers did not undergo full labor [2931]. Catecholamine concentrations are higher in vaginally delivered human infants as compared to cesarean - delivered infants . We analyzed plasma catecholamines at 0 to 2 hr - old following either: (a) vaginal birth, (b) cesarean section with simulated labor contractions, or (c) cesarean section without labor contractions (mimicking planned cesarean delivery). Pups were exposed to the major elements of the rat's natural birth process, as we have described (i.e., umbilical cord occlusion, tactile stimulation and cooling). Only pups exposed to actual or simulated labor showed an immediate and profound rise in norepinephrine and epinephrine, to levels up to 35% greater than those of noncompressed pups . Our results, the first reported in the perinatal rat, closely parallel those reported in human studies and studies using the precocial sheep model [27, 33]. Labor contractions do more than move the fetus through the birth canal . Whether by design (natural selection) or by incidental effect, contractions provide a form of stimulation that serves to facilitate two neonatal achievements: pulmonary respiration and suckling . Birth stimuli, that is, the range, levels, and patterns of stimulation that comprise the birth process, might have multiple roles in the successful transition from fetal to postnatal life . Our simulated birth model incorporates actual forms and levels of sensory and physiological stimuli to which the rat is exposed during natural vaginal birth and allows us to specifically parcel out the effects of labor on postpartum functions . The experience of labor is associated with a number of positive neonatal outcomes, including lung compliance, respiratory integrity [3537], blood flow, resistance to oxidative stress, neonatal neurological condition, and complex global eeg patterns . Human infants are particularly responsive to odors emanating from their mother's nipple / areola region and can identify the nipple by smell [42, 43]. Amniotic fluid and breast odors are regulators of infant sucking behavior, comfort, and distress reactions [4446]. Learning about natural breast odors is enhanced in neonates that experience labor contractions, possibly mediated by ne . Together with the results reported herein, these studies support the view that prenatal events associated with labor initiate a cascade of neural, physiological and behavioral changes that assist the neonate's successful transition to postnatal life events that assist the newborn infant's adaptation to the extrauterine world . We now describe an original experiment conducted in our laboratory by abel in which individual, externalized, near - term rat fetuses received a combination of the simulated birth stimuli described earlier (see figure 3) while in the presence of the odor citral and then tested for their responses to a rat dam with natural odors, washed of natural odors and with citral added . Specifically, while still residing in their amniotic sac and uterine horn that had been gently brought outside the dam's abdomen, each pup received a series of simulated labor contractions . Pups were next removed from the uterus, at which time there occurred a bout of tactile stimulation associated with removal of the birth membranes . Following this birth, each pup was stroked with a soft brush, mimicking the normal maternal licking and it also experienced cooling as it would after a natural birth and then rewarming as it would, had it been brought into the nest for maternal brooding . In effect, we created a simulated birth sequence . One set of pups experienced their birth in the presence of citral that was injected into the amniotic fluid prior to the intrauterine compressions and that was in the air around the pup while it was stroked and cooled and rewarmed . Alternatively, saline was used instead of an odorant for the littermate control subjects, that otherwise experienced the same birth sequence . The goal was to test the hypothesis that an arbitrary odor, paired with the experience of birth stimuli, would become a conditioned odor capable of evoking nipple attachment behavior from a newborn . Our regime of stimulation was a controlled, 135 min analog of pedersen and blass' 50-hr - long process used to induce a newborn rat's nipple attachment to novel odor . In contrast to their approach, we were able to specify and control the kinds, quantities, and timing of a specific stimulation sequence, and to observe the perinate at each stage of experimental manipulation . We predicted that a perinatal sequence of experiences in association with an otherwise neutral olfactory cue would lead to rates of nipple attachment to that cue, similar to those of vaginally delivered newborn rat pups to the odor of amniotic fluid . If the outcome of the simulated birth experience was equivalent to a natural delivery, we would consider this a successful empirical demonstration of sufficiency . We will have demonstrated that the experience of a simulated birth, quantitatively comparable to a natural, vaginal birth, is sufficient to establish a conditioned response to an odor that is expressed as nipple attachment and the onset of sucking in an, otherwise, nave newborn . Animal experimentation was conducted in accordance with the guidelines of the indiana university institutional animal care and use committee and the nrc guide for the care and use of laboratory animals (copyright 1996, national academy of science). One hundred twenty - six fetal rats, derived from 24, time - mated sprague - dawley rat dams (rattus norvegicus) were used as subjects . All breeding and maintenance was conducted in the animal behavior laboratory at indiana university . The first day that sperm was detected in a vaginal lavage was recorded as the day of conception (gestational day [g]0), with birth expected on g22 . On g21, pregnant dams were briefly anesthetized with isoflurane (aerrane, ohmed ppd inc ., an area overlying the lumbar region was shaved and a small (3 cm) dermal incision exposed the vertebral column . To eliminate movement and sensation below the ribcage, 100% ethyl alcohol (0.1 ml) was administered via intrathecal injection between the t12 and l1 vertebrae . After confirming loss of sensation, the female was placed in a plexiglas holding apparatus and her lower body immersed into a heated (37.5c 5c) saline bath . A midline laparotomy was performed and the dams' paired uterine horns were gently externalized into the bath . Figure 4 depicts the prenatal and postnatal manipulations . For each dam, either citral (sigma chemical co., st . Louis, 50 l in 4 ml / l isotonic saline) or vehicle alone was injected into the amniotic fluid surrounding target fetuses . Beginning with the fetus in the second ovarian position, amniotic sacs of three - to - four adjacent fetuses were injected . A 30 ga hypodermic needle was inserted through the transparent uterine wall and into the amniotic sac near each fetus' snout . Immediately following either citral or saline injections, compressions of 1015 mm hg pressure were administered to fetuses in one uterine horn using a small latex balloon . Such pressures are within the range of pressures typically experienced by rat fetuses during labor contractions [4951]. Compressions were delivered at the rate of one, 15 sec compression per min for 15 min . Upon completion of the compressions, fetuses were removed individually from the uterus and delivered onto gauze pads moistened with either citral (1 ml of 4 ml citral / l isotonic saline) or isotonic saline - moistened (1 ml) gauze pads (figure 4). Immediately following delivery from the uterus, two cotton - tipped swabs were used to remove the birth membranes from the newborns, umbilical cords tied with surgical silk, and placentas removed . Each neonate was stroked with a soft - bristled artist's brush until respiratory activity was established (approximately 2 - 3 min per litter). Next, the newborns experienced temperature fluxes similar to those observed after a natural birth sequence in the laboratory . They were placed onto saline - moistened gauze pads in individual glass dishes (pyrex 80 40) at room temperature (22c 0.5c) for 60 min, then moved to an incubator maintained at nest temperature (33c 1c) for an additional 60 min . At 60 min postpartum, pups that had received pre- and postnatal exposure to citral were placed individually in glass dishes on citral - moistened gauze pads (1 ml citral solution / pad). These pups remained in the warm citral ambience for 5 min then transferred back to the original dishes containing saline pads in a noncitral incubator for the remainder of the second postnatal hr . Pups in both conditions were handled identically throughout the experiment except for the presence of citral . To ensure olfactory isolation, care was taken to maintain separate citral and noncitral incubators during the postnatal exposure and testing periods . Physical stimulation in the form of compression or stroking is necessary for the establishment of respiration in newborn rat pups [19, 20]. In the present experiments, postnatal stroking was required to elicit respiratory activity in the noncompressed subjects; equivalent durations of stroking were provided to all groups . To verify that alterations in frequency of suckling onset between compressed and noncompressed newborns were not related to deficits in respiration, respiratory movements were sampled (1 min) at 3 postpartum time points: 10 min postpartum; 1 hr postpartum (while at 22c); 2 hr postpartum (while at 33c). Approximately 20 min prior to testing nipple attachment, a recently parturient (1 - 2 day postpartum) dam was anesthetized with a ketamine / xylazine mix (ip; 100 mg / ml; 0.9 ml / kg, 20 mg / ml; 0.5 ml / kg) and placed within a 33c test incubator in the supine position . At 120 min post - delivery, each pup was gently grasped and held for up to 120 sec with its snout in contact with a nipple of the test dam . Successful attachment to a nipple was verified visually and then by testing if the pup maintained oral grasp of the nipple while gently retracted from the dam . Following the first attachment trial, the dam was moved to a second heated (33c) incubator and citral - scented gauze pads (5 pads; 1 ml citral solution / pad) were rubbed across the ventrum, thus infusing the fur with citral odor . The scented pads were then placed alongside the dam, further contributing to the citral odor within the incubator . Then, to further verify the effectiveness of perinatal exposure to citral in promoting nipple attachment to a citral - scented dam, one group of compressed but citral - nave newborns was tested first on a citral - scented dam and then on a normal dam . Mcnemar chi - square for dependent measures was used to analyze frequency of nipple attachment . Posthoc comparisons were made with newman - keuls with a cutoff of p <0.05 . The nipple attachment test used in the present experiment reveals robust and reliable behavior in newborn rats . The leftmost histogram bar in figure 5 shows that 90% of the vaginally - delivered newborns held before the ventrum of a natural (unwashed) anesthetized dam attached to a nipple and suckled . Note that this was the first attachment for each pup . Thus, this testing method enables rapid and reliable expression of the onset of postnatal ingestion, and the 90% attachment rate following vaginal birth can be used as standard against which we can evaluate the results of the simulated birth experiences . Newborn pups that experienced a simulated vaginal birth in the presence of natural amniotic odors, including the regime of in utero compressions caesarean delivery membrane stripping cooling stroking rewarming (see figure 3) attached to a nipple in 89% of the tests, as shown by the first hatched bar in figure 5 . The legend under that bar, amniotic fluid / natural, indicates that these pups experienced unadulterated amniotic fluid odors and were with a natural, unwashed dam . Newborns that experienced the a simulated vaginal birth absent compressions in the presence of natural amniotic odors, attached in only about 44% of the trials, which was a significant decrement in relation to littermates treated identically but with the compressions . The contrasting result is seen in the open bar next to the hatched bar in figure 5 . Thus, the complete simulated birth sequence (including compressions), produced rates of nipple attachment in newborns that were fully comparable to those in vaginally delivered pups . Newborn pups that experienced a simulated vaginal birth in the presence of citral in their amniotic fluid and in the atmosphere during stroking (figure 4) attached to a nipple in 89% of the tests with a citral - scented dam, as shown by the stippled bar in figure 5 . Newborns that experienced the simulated vaginal birth absent compressions in the presence of natural amniotic odors, attached in only about 20% of the trials with the citral - scented dam, a significant decrement in relation to littermates treated identically but with compressions . The open bar, adjacent to the stippled bar in figure 5, shows the contrasting outcome . Thus, the simulated birth sequence in the presence of citral, including compressions, produced rates of nipple attachment to a citral - scented dam that were fully comparable to those seen in vaginally - delivered pups and to pups that experienced the full simulated birth in the presence of amniotic odors when tested with a correspondingly natural - scented dam . The rightmost pair of histograms show that newborns that experienced simulated birth stimuli in the presence of citral in their amniotic fluid and in the atmosphere during stroking (figure 4) when tested with a natural scent dam (no citral during the test) attached to a nipple in only 20% and 4% of the trials, for the compressed and noncompressed subjects . Clearly, newborns that experienced birth in a citral environment were not prepared to attach to nipples on the body of a dam with only the natural scent of the species . But, we know that these newborns are capable of attaching to a nipple, as evidenced by the performance of the simulated birth group depicted by the stippled bar . For the citral - birthed pups, citral had become a necessary stimulus for the initial attachment . Respiratory rates at the three, sampled time - points (10, 60, and 120 min) were unaffected by either citral or compression (p>.10). As expected, however, there was a significant increase in respiration within all groups during the final hour at the warmer temperature (f(2,120) = 320.8, p <.01). The absence of in utero compressions of the fetus was associated with poor performance in the onset of nipple attachment . It might be tempting to conclude that compressions mimicking labor contractions are necessary for efficient initiation of nipple attachment in the newborn, but the present experiment was not designed to allow such a conclusion . Although we categorized each operation as a separate form of stimulation, the perinate might be less discriminating in its responsiveness and all forms of stimulation might simply be additive and incrementally increase the level of arousal in the pup . Thus, compressions might just add to the experience of general stimulation in the pup and nipple attachment rates might reflect levels of general arousal . Even if true, such an effect would not account for the second broad finding, that odors paired with birth stimuli become conditioned stimuli for nipple attachment . The experience of a vaginal birth, real or simulated, appears to give behavioral meaning to the odors experienced in association with the birth stimuli . Schaal and colleagues have suggested that amniotic odors provide a bridge from the fetus' prenatal world to its postnatal environment, and the present results suggest that this bridge is constructed by the experiences embedded in parturition and that they result in a newborn behavior that has been rapidly assembled to follow the bridge to a nipple and the onset of suckling . Stimulation associated with labor and delivery plays a key role in assisting the fetus' transition to postnatal life by inducing and canalizing specific behaviors, and thereby operating as a critical link in the chain of behavioral adjustments required for adaptation to the postnatal habitat . Fetal sensory experience appears to set into motion physiological processes that permit the onset of postpartum behavior and the expression of early learning . It makes sense, both logically and scientifically, to discard the idea that suckling is an innate or instinctive or hard - wired behavior in a newborn baby . Nevertheless, it is also sensible to revel in the readiness and competence of a newborn mammal to adjust immediately to severance of its umbilical connection to the uterine world and to make an oral connection to the mother's body and begin suckling . We recognized the ability of a newborn mammal to suckle by designating it as a congenital behavior, that is, present at birth . Clearly, suckling is an important congenital behavior worth understanding for it is one of the primary adaptations to newborn life for all infant mammals, serving not only nutritive, immunological, and general physiological functions, but it is also a powerful component of bonding with the mother and creating a social context which supports sensory, motor, and cognitive development . Much of what we explored in the present paper are lines of research that have been important in demystifying the kinds of basic processes that can explain the newborn's congenital abilities to orient to novel features on the mother's body surface and to initiate the complex, but vital behavior of suckling . From the findings that we reviewed, it can be concluded that the combination of sensory and motor processes that constitute successful suckling are rapidly assembled during the course of perinatal events . We focused on the roles of birth stimuli and specifically on the experience of being born . In the experiments we described, rat pups that did not experience the mechanical and thermal forces associated with vaginal birth failed to make the fetus - to - newborn transition . Moreover, by providing individual perinates with a simulated birth experience, it was possible to induce in them the dramatic developmental changes that serve the transition from fetus to newborn . There is now abundant evidence that learning is an important component of the birth transition in rats and in humans . Thus, this perinatal learning takes place in the context of the experience of being born . It appears that the set of sensory, endocrine, and neural events that comprise the physiological transition of birth also serve as factors in the perinate's learning about the odor cues that are present in utero and carry over into the ex utero world . These are the same cues that the newborn then uses to orient to the mother's body and that stimulate nipple attachment and suckling . We are impressed by the multi - leveled functions of sensory and physiological events of birth, but much more remains to be understood about them and how they operate during parturition . Here, we can speculate on some of the implications we see when considering the experimental findings from rats in the context of human births and the onset of suckling . As students of mammalian development, indeed, our past initial analyses of rat parturition (e.g., [12, 19, 20, 34, 51]) were shaped by lagercrantz and slotkin's perspective on the stress of human vaginal birth . The results of our experiments with rats resemble their observations with human birth, and we have been able to take advantage of opportunities to control and manipulate the birth stimuli to gain insights into the embedded and embodied learning processes . We are particularly struck by the contrasting picture presented by prematurely - born infants who enter the postnatal world at a stage of development when their sensorimotor function is not yet prepared for suckling . While it may be beautiful and exciting to witness the eventual onset of sucking in a baby born at less than 30 weeks of age, it is sobering to contemplate the dramatically different factors and unnatural schedule of experiences that direct the prematurely - born baby to suck: a premie's early postnatal development may be supported by intravenous nutrition and then gastric intubation until the baby presents signs of readiness for oral feeding . Nurses, therapists and parents may then use a variety of techniques to gently and gradually facilitate the transition to sucking and ingestion . How different it is for the baby born at term, for whom the process is short, and in many regards, even intense and abrupt . We see great potential in understanding the necessary and sufficient developmental steps as precursors to improved management and guidance of early ingestion . Other researchers have compared the development of feeding skills of term babies and prematurely born infants . Schaal and colleagues (cf .,), for example, have focused on the absence of pairings of chemosensory cues and nutritive intake when babies are fed by gavage . Their perspective extends to many aspects of experience that normally contribute to the integration of breathing, suckling, and swallowing . Recognition of such differences may be an important step towards identifying factors that contribute to the problems experienced by some babies and that lead to the higher incidence of feeding disorders in children born prematurely (cf .,).
Although behcet's disease can affect any organ system, pulmonary involvement is uncommon and typically manifests as pulmonary vascular lesions . Pulmonary artery aneurysms involving the large proximal branches of the pulmonary arteries are the most common finding on chest computed tomography and hemoptysis is a common presenting symptom . Pulmonary small vessel vasculitis and lung parenchymal involvement is rarely reported, although the true incidence is uncertain given that histopathologic correlation is seldom pursued . This is an unusual case of behcet's disease complicated by hypoxemic respiratory failure and acute respiratory distress syndrome . To our knowledge, this is the first case of adult respiratory distress syndrome associated with behcet's disease with diffuse alveolar damage (dad) and vasculitis on lung biopsy . A 34 year - old, non - turkish, caucasian female with a history of behcet's disease refractory to multiple immunosuppressive regimens presents with severe diffuse abdominal pain . She also reported a recent exacerbation of her behcet's disease with skin ulcerations and orogenital aphthae ., she had failed single - agent therapies, and therefore had required treatment with various combinations of immunosuppressants . Her current regimen included concomitant use of mycophenolate mofetil, methotrexate, azathioprine, infliximab, and prednisone at a dose of 50 mg daily . She self - discontinued methotrexate 2 weeks prior to her presentation due to gastrointestinal upset and the prednisone dose was increased to 80 mg given her current symptoms . The prednisone dose was then reduced to 50 mg several days later to avoid side effects from high - dose steroids . On day 2 of admission, she experienced rapidly progressive shortness of breath and hypoxemia requiring intubation and mechanical ventilation . Physical examination revealed a cushingoid - appearing female with a left mandibular healing ulcer, multiple punched out mucocutaneous ulcerations, and bilateral diffuse wheeze on auscultation . Computed tomography of the chest revealed numerous small nodules and patchy areas of ground glass opacity [figure 1b]. Intravenous methylprednisolone 60 mg every 6 h and empiric broad - spectrum antimicrobials were initiated . Bacterial, fungal, and viral cultures, including studies for aspergillus and pneumocystis jirovecii were unremarkable . Serology and immunology including polymerase chain reaction (pcr) for cytomegalovirus, flu antigens a and b, rapid human immunodeficiency virus (hiv) antibody, mycoplasma pneumoniae antibody immunoglobulin (ig) g and igm, legionella, and cryptococcal antigens were negative . Vasculitis panel, which includes serum myeloperoxidase antibodies (cytoplasmic antineutrophil cytoplasmic antibodies (c - anca)), serum proteinase 3 antibody (perinuclear - anca (p - anca)), rheumatoid factor, serum antinuclear antibodies (ana) titers, creatinine kinase, and aldolase levels were all unremarkable . Antibiotics were subsequently discontinued several days later . On day 5, a surgical lung biopsy was performed that revealed areas of necrosis and dad mixed with lymphocytic and necrotizing vasculitis with multiple small infarcts and thrombi [figures 1c and d]. Steroids were slowly tapered and she was discharged home on combination therapy with cyclophosphamide and prednisone . (c) diffuse alveolar damage characterized by intra - alveolar fibrin and hyaline membranes (hematoxylin and eosin stain (h and e)). This is a case of behcet's disease complicated by an acute fulminant pulmonary syndrome . The findings of necrotizing vasculitis with multiple infarcts and thrombi are consistent with previously described pulmonary manifestations of behcet's disease . The literature pertaining to pulmonary involvement in behcet's disease is mostly limited to case reports and case series describing large vessel vasculitis such as pulmonary artery aneurysms and their sequelae . The various pathologic findings reported include pulmonary infarction, hemorrhage, interstitial lung disease, and small vessel vasculitis . Dad in association with behcet's disease has rarely been described . As surgical lung biopsy in behcet's disease is seldom pursued, true incidence of pulmonary behcet's disease is likely underdiagnosed . To our knowledge, this is the first case of behcet's disease with pulmonary involvement manifesting as dad mixed with necrotizing vasculitis on lung biopsy . Dad is a well - described lung injury pattern which can occur in many settings including infection, collagen vascular disease, drug toxicity, inhalational injury, and uremia . The clinical diagnosis of acute interstitial pneumonia is appropriate . In this case, the coexistent vasculitis with multiple small infarcts consistent with lung involvement by behcet's disease may have triggered the onset of dad, and it provides an explanation for the clinical presentation . It is possible the abrupt tapering of steroids prior to her presentation led to an exacerbation of behcet's disease . Diffuse alveolar hemorrhage (dah), another diagnosis to consider in the setting of vasculitis, is a syndrome characterized by injury or inflammation of the pulmonary arterioles, venules, or alveolar septal capillaries, and is associated with three different histologic patterns, including pulmonary capillaritis, bland alveolar hemorrhage, and dad . Dah can result from a variety of conditions, such as coagulation disorders, drugs, toxins, collagen vascular diseases, and mitral stenosis . Dah is also commonly associated with anca positive vasculitis presenting as a pulmonary renal syndrome . Alveolar hemorrhage in behcet's vasculitis is rare but may occur in the setting of dad . Immunosuppressive therapy is the mainstay of treatment for behcet's disease, although evidence supporting specific agents is limited . Several immunomodulators and biologic agents, some of which our patient was using, have been reported to cause lung injury . Methotrexate, azathioprine, mycophenolate mofetil, as well as the tumor necrosis factor alpha inhibitor infliximab, have all been associated with varying degrees of pulmonary toxicity, including dad . A cohort study of 514 patients with acute lung injury reported an incidence of drug - associated lung injury of 9.5% . Pulmonary involvement in behcet's disease, although uncommon, is a real entity and can be life - threatening . Physicians who treat patients with behcet's disease, especially those with unexplained respiratory complaints, must keep this in their differential . Patients with behcet's disease, especially those on immunosuppressant therapy should be frequently evaluated for any such evolving respiratory complications as well as drug - related pulmonary toxicity . For those on chronic steroid therapy, abrupt withdrawal or rapid tapering of steroids high - dose corticosteroids appeared to have benefited our patient who had a complete recovery . Future lung biopsy - confirmed cases of dad in association with behcet's disease may help elucidate pathophysiologic mechanisms and optimize treatment . In conclusion, this is a case of pulmonary behcet's disease presenting as adult respiratory distress syndrome with dad and vasculitis on lung biopsy . Physicians must maintain a high index of suspicion and early lung biopsy may help in the diagnosis and management of patients with behcet's disease who present with unexplained respiratory failure.
Kohn sham density - functional theory (ks dft) is the method most widely used in electronic structure calculations, due to its modest computational cost combined with an accuracy that is often competitive with much more expensive ab initio methods . The accuracy of the method is limited by the quality of approximations required to describe the quantum mechanical exchange and correlation (xc) interactions of electrons . A large number of density - functional approximations (dfas) for the xc energy have been developed in recent decades . The simplest dfas are based on the local density approximation (lda), as proposed by ks in their 1965 paper, in which the xc energy is approximated as a functional of the density at a given point in space . The generalized gradient approximations (ggas) go beyond the lda by modeling the xc energy as a functional of the local density and its first derivative . The meta - ggas are closely related but their functional forms are also dependent on the ks kinetic energy density and/or, less commonly, the laplacian of the electron density . Further developments led to the introduction of the occupied ks orbitals as ingredients for the xc energy (hybrid functionals and self - interaction corrections,), and more recently also the virtual ks orbitals (double - hybrid functionals and random - phase approximations). Local hybrid functionals are also an interesting alternative approach to construct hybrid methods that are pertinent to the context of this work . The inclusion of additional dependencies in xc functionals has often resulted in significant improvements in their accuracy for general calculations . However, these improvements cannot be described as systematic in the same way that the accuracy of an ab initio calculation may be systematically improved by considering a larger number of excited determinants; some dfas give excellent results for particular systems but perform very poorly otherwise, and vice versa . There also remain many problems that none of the currently available dfas can accurately model . An important example of this, which is pertinent to this work, are strong correlation effects, commonly found in systems with near - degenerate orbitals such as the d- and f - block elements, but also in systems where chemical bonds are being broken or formed . In the present work, the problem of constructing dfas accurately for systems with and without strong correlation is examined by considering the adiabatic connection (ac) at the local level, i.e., at each point of space . The ac, discussed in subsection 2.1, provides an exact expression for the exchange and correlation energies by considering the changes that occur as the strength of electron interaction is smoothly increased from zero . This formalism has provided the basis for the development of several dfas, which attempt to interpolate the ac between the non - interacting and physical systems in order to estimate the xc energy . An advantage of the ac formalism crucial to our construction is that it allows the problem of strong correlation to be addressed in a more direct way, by creating interpolation models that are explicitly dependent on the strongly interacting limit, in addition to the non - interacting limit, of the ac . The strongly interacting limit of the ac has recently become the subject of much interest . The properties of the ac integrand in this limit reveal a highly nonlocal density dependence of correlation effects that cannot be obtained from the standard (semi)local or hybrid functionals . Study of the strongly interacting limit in dft has focused on the strictly correlated electrons (sce) functional, where the electrons have an infinite interaction strength . This limit is of particular interest from a theoretical point of view as it can be studied exactly for one - dimensional systems and may be closely approximated in systems with spherical or cylindrical symmetry . These studies show that in the limit of infinite interaction strength certain integrals of the density appear in the exchange correlation functionals, revealing a mathematical structure very different from the one of the usual semilocal or orbital - dependent approximations . The nonlocal radius (nlr) functional proposed in ref (43) approximates the sce functional with a model that retains some of the sce nonlocality, introducing integrals of the spherically averaged density around a reference electron . The inclusion of the nlr functional into global and local interpolations along the adiabatic connection has been very recently explored by zhou et al . In another recent work, kong and proynov constructed a functional combining the information from the becke13 model and approximating local quantities along the ac . The aim of the present work is to start a systematic study of local interpolation models along the adiabatic connection, using at a first stage exact input ingredients, thus disentangling the errors due to the interpolation models from those due to the approximate ingredients . The local ac for several closed - shell atoms has been recently computed to high accuracy between the non - interacting and physical systems using the legendre fenchel formulation of dft due to lieb, and the lieb maximization method of refs (4952). Local information pertaining to the strongly interacting limit is calculated using the sce functional, and together these quantities are used to calculate local analogues of some established global ac interpolation functionals . We also discuss how to approximate crucial local ingredients such as the initial slope of the local adiabatic curve . In section 2, relevant theoretical background is given including an overview of the ac formalism and the construction of dfas from both global and local variants of the ac . Techniques for computing quantities along the ac are discussed, including the determination of the local ac as introduced in ref (47). In section 2.3 the construction of a local model for the ac is discussed, considering the non - interacting and strong - interaction limits carefully in this context . Finally the forms of some local interpolation models, taken from successful existing global models, are introduced . In section 4 the performance of these models is assessed for the helium and beryllium isoelectronic series and for dissociation of the h2 molecule, a system that typifies the failure of present dfas to properly account for strong correlation . The ac was proposed in a series of reports, which suggested that further insight into electronic correlations in dft may be gained by considering a system at constant electron density as the interaction strength is smoothly scaled between zero, i.e., the ks auxiliary system, and the full physical interaction strength . This scaling of the interaction strength is achieved by the introduction of a simple coupling - constant coefficient,, such that the hamiltonian for any given is written as1where t is the kinetic energy operator, is the physical electron - interaction operator, and v is the operator representing an external potential v that binds the electron density at, such that it is always equal to the density of the physically interacting system (= 1,). As the value of is smoothly increased from zero to one, the system evolves adiabatically through a family of -dependent wave functions to the physical system described by 1 . Given a hamiltonian, one can define the corresponding -dependent universal density functional aswhere eq 2b follows from the application of the hellmann this allows the well - known ac formula to be derived, yielding the following exact expression for the xc energy of an electronic system,3where is the (global) ac integrand, given by4[] is the ground - state wave function of in eq 1, and u[] is the hartree (coulomb) energy . The ac integrand may be characterized by several features that can be exactly defined: the expansion of in the non - interacting limit is given by5while its expansion in the strongly interacting limit can be expressed as6here, the non - interacting terms and are the exchange energy and twice the second - order correlation energy given by grling their analogues at the strongly interacting limit, and, have been studied in refs (33, 34, and 38) and will be discussed further in section 3.2 . In addition to these asymptotic limits, the behavior of under uniform coordinate scaling is also well - defined, as discussed in ref (57). To construct practical dfas one could consider modeling the integrand of eq 4 using a function that interpolates between the limits of eq 5 and eq 6 . The sce limit is of particular importance in the present work; however, one could also consider models that intercept any other known point on the adiabatic connection for> 0 . Several attempts to develop dfas based on these ideas have been put forward in the literature; see, e.g., refs (30, 31, 33, 34, and 5862). Each form makes a choice of a simple model function and the parameters on which to base the model . These parameters often include the known exact expressions for the parameters in eq 5, and, since these may be computed from the set of kohn sham orbitals ({p}) and orbital energies ({p}). The calculation of requires the gl2 correlation energy, which leads to a computational cost similar to the second - order mller plesset (mp2) model used in ab initio quantum chemistry . The parameters in the sce limit entering eq 6 are clearly also of special interest in this context, and they can be computed numerically for atomic systems and molecules with cylindrical symmetry . More frequently dfas are derived for points along the ac with> 0, often by employing scaling relations to derive forms from existing dfas . A similar strategy can also be used to approximate by a dfa; see for example ref (60). In tandem with choosing a set of exact or approximate values to parametrize a model for the ac one must also choose an appropriate model function for the ac integrand . A number of these have been suggested and many have been benchmarked in practical applications . One of the simplest (and most often used) is that of a [1/1] pad, as suggested by ernzerhof . A range of forms were suggested by cohen et al . And tested using approximate parametrizations, leading to the mcy1 functional in which a [1/1] pad model is employed . Peach et al . Attempted to disentangle approximations in the choice of parameters from those in the choice of model ac function by utilizing nearly exact ks orbitals and orbital energies derived from full configuration - interaction data to calculate and and the corresponding interacting wave functions to evaluate via eq 4 . Our present study follows a similar philosophy, but applied to local, rather than global, interpolations . Seidl and co - workers were the first to make use of the strong - interaction limit (although approximated at a semilocal level, using the so - called point - charge - plus - continuum, or pc, functional) in constructing a global ac model, known as the interaction strength interpolation (isi) functional . The revisi model and the models of liu and burke were later constructed to take account of the dependence of the second term of eq 6, which was not correctly described by the isi approach . Teale et al . Also proposed forms for the ac integrand based on the structure of traditional ab initio methodologies and parametrized these forms to intercept values of the ac at any> 0 . The majority of these models for the global ac suffer from the fact they are not size consistent in practice . This deficiency arises from a nonlinear dependence on the parameters,, and a chosen approximation to . When these global parameters enter in a nonlinear fashion (often as ratios), then size consistency is difficult to achieve . One route forward is to construct local ac models, which can replace these global parameters with local values defined at each point in space and may be more amenable to the construction of models that recover size consistency (at least in the usual density - functional sense). The ac expression for the xc energy of a system as given in eq 3 describes a global quantity, integrated over the coupling constant . However, it may equally be written as the spatial integral of a local quantity analogous to the local value of an xc functional . To this effect, eq 3 may be rewritten as7where w(r) is the energy density at a given . It is well - known that the energy density cannot be uniquely defined; an arbitrary number of terms may be added to w(r), yet an identical recovered if their spatial integral is zero . Thus, any such energy densities are only defined within a particular gauge, and only energy densities defined in the same gauge may be meaningfully compared . In the context of the present work, it is both convenient and physically meaningful to define wxc,(r) in the gauge of the electrostatic potential of the exchange correlation hole,9and p2(r, r) is the pair density obtained from the wave function []10the definition of energy densities in the gauge of the xc hole is well - established in the literature, and further discussion may be found in refs (29, 72, and 73). The coupling - constant - averaged (-averaged) xc energy density is defined as11since the spatial integral of the product of this quantity and the density yields the xc energy, the same quantity may be considered as a target to be modeled by xc functionals, although ggas and metaggas often aim at energy densities within different definitions . Given the invariance of the exchange energy density to electron - interaction strength, eq 11 may be trivially resolved into separate exchange and correlation terms as12the aim of the local interpolation schemes examined in this work is to approximate wxc(r) and wc(r) through interpolating the local ac . In principle, this approach is analogous to that of the global ac interpolation schemes previously discussed, but rather than depending on quantities pertaining to the global ac, they are instead constructed from their local equivalents, w(r). Obviously, a local interpolation will only be meaningful if all of the local terms are defined in the same gauge . It is again both convenient and logical to define all local quantities in the gauge of eq 8, as in which highly accurate energy densities w(r) in the range 0 1 have previously been calculated, and additionally can be computed for small systems in the limit . At = 0, the energy density in the gauge of eq 8 is the exchange energy density w0(r) = wx(r), often denoted x(r) in the literature (also equal to 1/2 the nonlocal slater potential), which is the crucial ingredient of local hybrid functionals . Accurate and efficient computational schemes for this quantity have become available in recent years . In a way, local interpolation models can be viewed as local hybrids that carefully address the gauge problem . The local equivalent of is not as simple to define, yet is an essential component of ac interpolation schemes as it provides a measure of the departure from exchange - only behavior, in other words provides the information from which the correlation energy is approximated . While many global models use gl2 theory for this purpose, its dependence on global quantities makes it unclear how it could be applied to a local interpolation scheme . This is discussed in detail in section 3.1.2 . In order to assess the quality of our local interpolation functionals, it is necessary to have accurate data of energy densities, defined in the gauge of the xc hole . These may be acquired by the method of lieb maximization, described in refs (5052). The lieb maximization is an optimization algorithm developed using the convex conjugate functional defined by lieb in ref (48) as the legendre fenchel transform to the energy, in which the density and potential v are conjugate variables, belonging to the dual vector spaces14and e[v] is the energy yielded by a given electronic structure calculation at potential v(r). This convex conjugate formulation follows from the concavity of variationally determined energy e[v] in v, from which lieb showed that f[] must be convex in . Furthermore, the conjugate functional to a nonconcave energy, such as that which may result from a nonvariational calculation, remains well - defined as it is necessarily convex . A subsequent legendre fenchel transform of f[] yields the concave envelope (least concave upper bound) to e[v]; hence unique solutions to f[] can always be obtained . In the lieb maximization, the optimized density (r) is obtained by maximizing f[] with respect to variations in the potential v(r), rather than by minimizing e[v] with respect to (r) as is usually the case . Therefore, at convergence, the potential v(r) in eq 13b is that which yields (r). In the present work, lieb maximizations have been carried out at a number of points along the ac in the range 0 1; hence the density is constrained such that = =1, resulting in a -dependent optimizing potential . In order to effectively optimize with respect to the potential, we parametrize it by using the method of wu and yang (wy) as15where vext(r) is the external potential due to nuclei, vref(r) is a reference potential chosen to ensure that v(r) has the correct asymptotic behavior, and {gt} are a set of gaussian functions with coefficients {bt}. In all calculations in this work we choose the potential expansion basis set to be identical to the primary orbital basis set . Amaldi potential, and f[] is optimized with respect to the coefficients of the potential basis {bt}. Additionally, convergence is accelerated through the use of the newton method described in refs (5052). The relaxed - lagrangian formulation of helgaker and jrgensen is used to obtain relaxed densities for nonvariational wave functions, which serve as input to the lieb functional and are used in the determination of the derivatives required for its optimization . In this work, lieb maximization calculations are performed using the implementation of refs (5052) in a development version of the dalton quantum chemistry software package, in which e[v] is computed by using coupled - cluster singles and doubles (ccsd) and full configuration - interaction (fci) wave functions . At convergence, where the optimizing potential is such that = =1, the relaxed -interacting one- and two - particle density matrices are computed, with which the -dependent xc energy densities may be obtained as16 as described in section 2.3, the initial slope of the ac is an important part of many global ac models, in which it may be calculated directly by gl2 perturbation theory; however there is no analogous expression that yields the local equivalent, and we give such an expression in section 3.1.2 . Here, the local initial slope of the xc energy density that is given in eq 8 is defined as17and is related to the global slope, hence the gl2 correlation energy, by18 in this study w0(r) is numerically approximated by the method of finite difference, with a series of w(r) for 1 . The resulting local slopes in the h2 molecule with bond lengths of 1.4 and 6.0 au are plotted along the h h bond in figure 1, along with the densities from which they are calculated, at the fci level of theory and in the uncontracted aug - cc - pcvtz basis set . In both cases, the local slope is greatest in magnitude at the nuclei, as has been seen previously in atoms . It can be seen that the magnitude of the local slope is significantly larger in the stretched h2 molecule, mirroring observations previously made of the global ac in the dissociating hydrogen molecule . Plots comparing the values of (r) and w0(r), with respect to the distance from the bond midpoint, z / a.u ., along the principal axis of the h2 molecule with bond lengths of 1.4 au (upper panel) and 6.0 au (lower panel). The local slopes in the he and be isoelectronic series are plotted in figures 2 and 3, respectively . It is clear that, with increasing nuclear charge, the charge densities in both series become increaslingly contracted . The x - axis in both plots has been scaled by nuclear charge, highlighting a contrast in their behavior with respect to the uniform scaling condition,19which holds for nondegenerate ks systems . In figure 2, it can be seen that the slope of the ac for the he series becomes less negative with increasing z, tending to an asymptotic value as z, consistent with the scaling relation of eq 19 . However, the slope of the ac in the be isoelectronic series becomes more negative with increasing z, indicating that the scaling relation is not satisfied by this series . Plots of w0(r) for the helium isoelectronic series, with nuclear charges 1 z 10, and with radial distance from the nucleus r / a.u . Scaled by nuclear charge . Plots of w0(r) for the beryllium isoelectronic series, with nuclear charges 4 z 10, and with radial distance from the nucleus r / a.u . Scaled by nuclear charge . While it is useful to numerically approximate the local slope for the purposes of evaluating local interpolation schemes, such functionals would only be viable for mainstream use in dft calculations if they can be described by simple functional forms . In global models, the initial slope can be calculated directly from the occupied and virtual ks orbitals according to gl2 theory,20where the indices i, j and a, b pertain to occupied and virtual ks orbitals, respectively, vxks is the local ks potential, and vxhf the nonlocal hartree the first term in eq 20 is analogous to the correlation energy given by mp2 theory, in which p and p are canonical hf orbitals and eigenvalues rather than ks ones . The second term accounts for the difference between the ks and hf exchange potentials and has a form similar to a singles term in many - body perturbation theory . Previous studies of gl2 theory have found that the second term, although non - negligible, is small in magnitude relative to the mp2-like term evaluated with the ks orbitals . Therefore, it follows that an approximation to the gl2 correlation energy may be obtained by evaluating the mp2 correlation energy with the ks orbitals and eigenvalues, ecgl2 ecmp2 . Given that an approximation to the global ac slope may be obtained from an mp2-like calculation, it follows that an approximation to the local ac slope may be obtained by deriving a local form of this expression . While mp2 theory treats perturbations of the wave function, the analysis may be extended to energy densities in the gauge of the xc hole by means of eq 10, as the substitution of eq 16 into eq 17 yields the following,21where p2(r, r) is the derivative of the pair density at = 0,22notice that eq 21 ensures that w0(r) is in the gauge of the electrostatic potential of the xc hole . Given a non - interacting ground - state wave function, the perturbed wave function for \|\| \| \| can be appproximated by the series expansion23if one assumes that is nondegenerate and has the form of a single slater determinant, the first - order correction to the wave function is given by24restricting the space of k(0) to doubly - excited determinants reduces this expression to25 in mp2 theory, contributions to the correlation energy from singly - excited determinants are necessarily zero due to brillouin s theorem . However, this is not strictly true in gl2 theory as the singles term in eq 20 makes a small, but nonzero, contribution to the gl2 correlation energy . As such, considering only double excitations in the model for the local slope can only yield approximations to the local slope; spatial integration of this quantity will not return the exact gl2 correlation energy . Condon rules to eq 25 allows it to be rewritten as26where the coefficient to ijab may be identified as an mp2 doubles amplitude tijab,27to obtain p2(r, r), it is necessary to take the derivative of the pair density corresponding to the perturbed wave function, + . Substituting this into eq 10 and rearranging the resulting expressions yields the following,28where we assume that and are real and p2(r, r) = n(n 1) ij(r ri) (r rj) is the pair - density operator . Substituting eq 26 into this expression gives29which may then be resolved into the following orbital - explicit expression,30where i a is the kronecker over two spin functions: i*(ms) a(ms) dms = ia . Substituting eq 30 into eq 21 finally results in an expression for the local slope,31where vabij(r) is the antisymmetrized orbital potential,32multiplying the right - hand side of eq 31 by the density and integrating over all space, we recover twice the mp2-like expression . This is not an exact expression for the local slope, as the second term of eq 20 is not accounted for . However, the omitted term is generally small relative to the mp2-like term and vanishes entirely for two - electron systems; hence the expression for the local slope in eq 31 should, in principle, be a fair approximation of the exact local slope . In future work we will implement and test eq 31 against the numerical results in section 3.1.1 . The doubles amplitudes tijab are readily obtainable from standard quantum chemical packages, and the potential vabij(r) can also be readily calculated; however, it would likely be computationally expensive to evaluate on a numerical grid . To reduce this cost, a range of techniques, commonly used to accelerate the calculation of integrals in linear - scaling software packages, may be employed . We note that the behavior of the local slopes presented in figures 1, 2, and 3 may be rationalized in a manner similar to that commonly discussed for global models in terms of eq 20 . This is because of the key role of the doubles amplitude tijab in eq 31 . The doubles amplitude has a dependence on the orbitals and orbital energies that is similar to that of the gl2 energy in eq 20 . We see in figure 1 that the local slope of the hydrogen molecule displays the minima at the nuclei . Equation 31, which is exact for two - electron systems, can be used to rationalize this observation . For closed shell two - electron systems with only one virtual orbital, eq 31 is simplified as follows:33even if we used a minimal orbital basis for the evaluation of the expression given in eq 33 for the hydrogen molecule, we would see that the local slope is most negative at the two nuclei, for any bond length . While this effect is captured with the minimal basis, the same minimal basis model would incorrectly describe the slope at the bond midpoint . For example, in the top panel of figure 1 we see that w0(r) is less than 0 at the bond midpoint of h2 at r = 1.4 . Within the minimal basis, the local slope would be exactly 0 for any r, as the antibonding 2(r) orbital which enters eq 33 has a node at the bond midpoint . We also see in figure 2 that the correlation energy density for the he isoelectronic series scales quickly toward an asymptotic constant as z increases . Furthermore, the local slope decays smoothly with distance from the nucleus, reflecting the behavior of vabij(r). The ks homo lumo gap is known to increase as z increases from 4 to 10, from which one would expect the correlation energy to become less negative according to the behavior of tijab . In the core region this behavior holds; however in the valence region the trend is opposite, with the correlation energy density becoming more negative with increasing z. this suggests that the numerator of tijab and the spatial dependence of vabij(r) due to the form of the ks orbitals are dominant in this region, provided that eq 31 is sufficiently accurate for the be isoelectronic series . In recent years, the exact strong - coupling limit of the ac has been intensively studied . This limit reveals a new structure for the xc functional: instead of the traditional ingredients of dfas (local density, density gradients, ks kinetic energy density, and occupied and unoccupied ks orbitals), it is observed that certain integrals of the density appear in this limit, encoding highly nonlocal information . Tests on model physical and chemical systems (electrons confined in low - dimensional geometries and low - density, ultracold dipolar systems, simple stretched bonds and anions) have shown that taking into account this exact behavior can pave the way for the solution of the strong correlation problem in dft . However, the exact information encoded in the infinite coupling limit, described by the sce functional, does not come for free: the sce problem is ultra - nonlocal, and, although sparse in principle, its nonlinearity makes its exact evaluation for general three - dimensional geometry a complex task . A possible route to find suitable algorithms relies on the fact that constructing the exact sce functional for a given density is equivalent to solving an optimal transport (or mass transportation theory) problem with a cost function given by the coulomb interaction . This equivalence has triggered interest from the applied mathematics community working on optimal transport problems, which has led to the suggestion of several algorithms, together with very interesting exact results . So far, the sce solution is known exactly for one - dimensional systems . For spherically symmetric systems, a conjectured solution that is very close to the exact one (and it is in many cases, but not always, exact) has been proposed and used to address interesting physical problems . Using algorithms and ideas from optimal transport, the sce problem for the hydrogen molecule along the dissociation curve has just recently been solved and both the global and local sce quantities have been computed . A more practical way to proceed is to build approximations for the sce functional inspired by its exact form, as it was done in the construction of the nlr functional . It corresponds to the wave function that minimizes the hamiltonian of eq 1 when . One can argue that the sce system is a better starting point than the kohn sham system for the description of very strongly correlated systems . The sce functional is defined as34the xc part can be easily extracted from, as . The ks sce approximation, proposed in ref (35), uses the sce functional to approximate the hartree and exchange correlation energy, and it is equivalent to setting for all . It has been shown that ks sce yields good energies for systems where correlation plays a dominant role, such as electrons confined in low - density nanodevices or extremely stretched bonds . On the other hand, ks sce treats moderately and weakly correlated systems very poorly, giving energies that are unacceptably too low . A less drastic approximation is to construct a model, in such a way that its limit is given by the exact or approximate value of, as done in the pioneering work of seidl et al . Analogously, one can also model w(r), imposing that its limit is given by w(r). This latter approach is the main object of the following sections . In the sce limit, the electrons are infinitely or perfectly correlated and their positions are given by an infinite superposition of classical configurations . The basic idea is that the electronic positions are all determined by a collective variable r, a feature that is captured by the so - called comotion functionsfi(r). If a reference electron is at r, then the position of all of the other electrons in the system will be given by ri = fi(r). Since the electrons are perfectly correlated, the probability of finding the reference electron at r has to be the same as the probability of finding the ith electron at fi(r). Therefore, the comotion functions have to satisfy the following differential equation:35for more details on the comotion functions, including their group properties, see refs (29, 91, 99), and (33). In terms of the comotion functions, the sce functional is given by36despite the high nonlocality of the sce functional, evident from eq 35, we can easily compute its functional derivative from the following expression37equation 36 suggests the following energy density in the sce limit:38where vh(r) is the hartree potential . This expression is indeed in the gauge of the xc hole potential of eq 8, as proven in ref (29). Being derived from a wave function, the w(r) energy density decays like, similar to the physical (= 1) and the exchange (= 0) energy densities of eq 16 . Its functional derivative, eq 37, has also the correct asymptotic behavior . To solve the sce problem for spherically symmetric systems (the he and be isoelectronic series considered in this work), we have used the approach presented in ref (33), which is exact if n = 2 . For atomic densities with n> 2 it could be either a very good approximation for the true minimum of eq 34 or, again, the exact result . For the h2 molecule we have used the results of ref (37), where the sce energy density has been computed by obtaining the comotion function from the dual kantorovich formulation of the sce problem . The local interpolation models tested in this work are largely simple translations of the well - established global interpolation models into a local form . This was done for the model of seidl, perdew, and levy (spl), the simplified model of liu and burke, which will be referred to here as the lb model, and the pad[1/1] model . Each of the energy densities resulting from the three mentioned models is constructed from three local parameters, a, b, and c, which are defined in the gauge of the xc hole . In addition to these, we constructed a local form of the two - legged representation which, given some value of w1(r), takes the form whenever we used the two - legged representation to model the local ac in this work, we did it by incorporating the interpolated w1(r) of the lb model: w1(r) w1lb(r). By doing the local interpolation this way, we use the following three input quantities: w0(r), w0(r), and w(r) and circumvent the direct utilization of the full interacting energy density, w1(r). In each of these four models, integration of w(r) with respect to coupling constant gives the -averaged energy density wxc(r), which, if spatially integrated according to eq 7, yields the xc energy exc[]. An important observation in the translation of global to local models is that while the following global inequalities are always satisfied,40their local counterparts do not necessarily satisfy these same inequalities . It has previously been observed for the hooke s atom series that, in the tail regions of the density, w(r) can be less negative than w1(r). In this work, the crossing of w(r) with wxc(r), w1(r), and w0(r) has only been observed in the tail regions of the density and is thought to be an artifact of the numerical instability that occurs where the density is very small . Although the helium isoelectronic series is a set of only two - electron systems, it is a useful series to consider in evaluating the local interpolation models as most standard dfas incorrectly characterize the hydride ion (h), failing to predict its existence as a bound electronic system . Here, local interpolation models are constructed from energy densities acquired by the lieb maximization at the fci level, as described in section 2.4, in the range 0 1 and at = by evaluating the sce functional on the = 1 density, also at the fci level of theory . In table 2, the correlation energies given by local forms of the spl, lb, two - legged representation (the column labeled two - leg) and pad[1/1] models (the latter parametrized using the accurate values for w1(r), in order to compare with models that, instead, use the information) are given, along with that given by the global spl model and the fci correlation energy for comparison . These data show that the local interpolation correlation energies are in close agreement with the fci reference values; the mean absolute errors (mae) of the local interpolation models are 2.0, 1.5, 0.5, and 0.1 mh, for the two - legged representation, spl, lb, and pad[1/1] models, respectively . As would be expected, the local pad[1/1] is the most accurate of the models, given that it is derived from the full interacting energy density . These data further suggest that the local lb model is marginally superior to the local spl and the two - legged representation . However, comparing the global and local models shows a slightly lower error for the global model; the local spl model has an mae of 1.5 mh, compared to 1.3 mh for the global model . Figure 4 compares the fci wc(r) with that of the local lb and spl models, for the helium atom . This reflects the numerical data in table 2, both being very close to the fci energy density but with slightly lower error in the lb model . Plots comparing the fci, local lb, and local spl -averaged correlation energy density in the helium atom . The changes in correlation energy across the beryllium isoelectronic series are somewhat more complicated than those in the helium isoelectronic series, and its explanation involves the interplay of several effects . With increasing nuclear charge, the density becomes increasingly contracted, suggesting that the correlation energy should approach the high - density limit for very large z. however, this is accompanied by a changing ks homo lumo gap, here the energy difference between 2s and 2p orbitals, which increases from z = 4 13 before decreasing with higher z values . Table 3 shows the reference and interpolated ec results for the be isoelectronic series, with z in the range 410 . The wave function for values between 0 and 1 has been computed in the same way as that for the he isoelectronic series, however at the ccsd level of theory rather than fci . As for the helium series, the local pad[1/1] that uses w1(r) however, in contrast to the findings for he isoelectronic series, the local interpolation models are much more accurate than the global models . For example, in the case of f the global spl model has an mae of 47.4 mh, whereas the error for the local spl model is 3.5 mh . The local two - legged interpolation underestimates the correlation energies of the elements of the given series . The local spl and lb interpolation models appear to qualitatively capture the shell structure of wc(r); however, in some regions it overestimates the reference value while in other regions the converse is the case . The error cancellation that results from this is the most likely explanation for the superior accuracy of the local models in comparison to the global models . Plots comparing the ccsd, local lb, and local spl -averaged correlation energy density in the beryllium atom . Despite the development of dft into the most widely applied electronic structure method, and the wealth of xc dfas that have been developed, there are some systems for which no combination of dfas provide an accurate description . Standard dfas become increasingly inaccurate with greater h h bond length, reflecting a fundamental flaw of dfas in their inability to properly treat strong correlation . It has been seen previously that ks sce correctly predicts the dissociation of h2 in a spin - restricted formalism; however at equilibrium geometry the energies it predicts are extremely low and the bond lengths predicted are overly short . The overall accuracy of ks - sce for h2 dissociation can be substantially improved by the addition of nonlocal corrections . Figure 6 shows the dissociation curves for h2 given by the local interpolation models, along with those given by hf, fci, and the pbe functional for comparison . The computational details are the same as those of the he isoelectronic series, and the pbe, hf, and fci curves have been obtained from the dalton quantum chemistry package all within the uncontracted aug - cc - pcvtz basis set . Burke, two - legged representation combined with the liu burke model, pad[1/1] with w1(r). It can be seen in figure 6 that all of the interpolation models correctly predict the dissociation of h2, which follows from their inclusion of w(r). In global ac models, at infinite separation the initial slope diverges as a result of the vanishing homo lumo gap and the spl and lb models reduce to, yielding the exact energies . However, the dissociation curves produced by the local models approach the fci curve slowly, resulting in an unphysical bump-like feature . This is a well - known failing of dft, having been observed with other functionals, such as the random - phase approximation and even the global pad[1/1] model with . It can be seen in figure 6 that this is not remedied by the local interpolation approach, as the curve obtained by the local pad[1/1] also exhibits this unphysical bump, as does that given by the local spl model and, to a lesser extent, the local lb model . To analyze why the intermediate region is less accurately described by the local interpolation methods than the equilibrium and stretched region, we show in figure 7 the correlation component of the local ac at one of the nuclei of the hydrogen molecule at different bond lengths: r = 1.4 au (at equilibrium), r = 5.0 au (the intermediate region), and r = 13.0 au (stretched bond). The structure of the three local ac curves at one of the nuclei is very similar to the structure of the corresponding global ac curves . From the given figure we see that at equilibrium the local ac is almost linear, so we can expect that even a single line segment approximation to the local ac: w(r) w0(r) + w0(r) would properly capture the shape of the given local ac curve . The local ac curve at the nuclei of the stretched h2 exhibits the characteristic l - shape, which was also observed in the case of the corresponding global ac curve . We would expect that the two - legged representation would capture the given local ac very well, but even a single line segment approximation, w(r) w(r), this time coming from the strong coupling limit, would be highly accurate for the stretched h2 . In contrast to the local ac curves of the stretched and h2 at equilibrium, the curvature of the local ac curve at the intermediate bond length is highly pronounced . The shapes of the local ac curves at the nuclei mirror the difference in correlation regimes present in the hydrogen molecule at different bond lengths . While in h2 at equilibrium and at very stretched bond length, correlation is almost purely dynamical and almost purely static, respectively, in the intermediate dissociation region there is a subtle interplay between the dynamical and static correlation . Fci local correlation ac curves at one of the nuclei of h2 for different internuclear separations, r. in the intermediate region of the dissociation curve, where the unphysical bump is present, the local two - legged representation model is more accurate than the local pad[1/1] which we always use here with w1(r). This may be understood by comparing the exact local ac data with the interpolated quantities . The top panel of figure 8 shows the difference between wfci(r) and that of each of the local interpolation models, along the h h bond at the 5.0 au geometry, as a function of the distance from the bond midpoint z. this difference w(r) = wfci(r) wmodel(r), is multiplied by the density to represent an energy per volume element . It shows that the local spl energy density is the one that most overestimates w(r). The error is smaller for the lb model and even more so for the local pad[1/1] model . The error is smallest in the two - legged model, obtained using the w1(r) of the local lb . Furthermore, there is the error cancellation in the two - legged model, as there are regions where the w(r) of this model underestimates wfci(r). Plots of the difference between fci and interpolated -averaged energy densities, w(z) = wfci(z) wmodel(z), with respect to the distance from the bond midpoint, z / a.u . (upper panel), and the local ac curves at one of the nuclei of the fci and local interpolation models (lower panel), both in h2 with a 5.0 au bond length . It can also be seen that the curves shown in the top panel of figure 8 have a maximum at the nucleus (z = 2.5 a.u . ). Focusing on this region, it appears that the fci curve meets that of the pad[1/1] at = 1 and that the two - legged representation curve meets that of the lb model also at = 1 . This follows from the construction of the pad[1/1] and two - legged curves from w1fci(r) and w1lb(r), respectively . All curves, except for that of the two - legged model, lie above the fci curve . In the case of the two - legged interpolation model, the first line segment is below the fci curve, as a result of eq 39a and the convexity of the given local ac curve . The second line segment that starts at x 0.1 the resulting error cancellation makes it clear why the two - legged representation appears more accurate than the other models . In this work we have studied local interpolations along the adiabatic connection for the he and be isoelectronic series and the hydrogen molecule, by using accurate input local quantities computed in the gauge of the electrostatic potential of the xc hole and comparing the results with nearly exact energy densities defined in the same way . In order to obtain approximations to the local ac over the physical regime (0 1), we constructed interpolation models between the weak and strong coupling limits of dft . The weak coupling energy densities were obtained using the lieb variation principle, while the strong coupling limit energy densities were obtained using the strictly correlated electrons (sce) approach . The inclusion of the sce information in density - functional approximations helps to ensure their ability to capture the strong correlation effects . Unlike previous attempts in this direction that used global (integrated over all space) input quantities to model the ac, the local approach is more amenable to the construction of approximations that do not violate size consistency, at least in the usual dft sense . Since the aim here is to work in a restricted formalism, avoiding mimicking strong correlation with symmetry breaking, some care must be taken when discussing size consistency . In fact, strictly speaking, in a restricted framework the energy densities of the second - order perturbation theory and exact exchange are not intensive quantities in the presence of near degeneracy, which is the main challenge of capturing strong correlation within dft . In future work we will test different approximations for the sce energy densities and the local slope . The development of algorithms for solution of the sce problem is a very active research field . In spite of the recent improvements, we still lack an algorithm that will solve the sce problem for general 3d molecular geometries at low computational cost . However, a good candidate to approximate the sce energy density in the gauge of the xc hole potential is the nonlocal radius functional (nlr), which has been already implemented and used in ref (44). In addition to numerically exploring the local ac we have also reported the local weak - coupling slope of the adiabatic connection and derived an approximate expression for it in terms of occupied and unoccupied orbitals . This quantity is very important to signal the amount of correlation at each point of space . In our future work
The physician patient relationship has received extensive philosophical, legal, and literary attention since the time of hippocrates and is one of the key subjects in the modern medical literature . Patient trust in physicians is widely recognized as being central to the physician patient relationship . Furthermore, this trust, which can be considered a collective good, is necessary for an effective health care system . However, there is a widespread concern that patient trust in physicians is declining under various threats to the physician patient relationship worldwide . In china, patient violence against physicians has become a common occurrence . Thus, improving patient trust in physicians may be much more urgent in china than in other countries . Several researchers have confirmed that measuring patient trust in physicians is vital for improving patient trust . A systematic review of patient trust in physicians revealed that most studies on this topic have been conducted in the unites states and published in english . In china, several studies have discussed patient satisfaction with physicians in chinese, but none of them has discussed patient trust in physicians in chinese or in other languages . However, several researchers have confirmed that patient trust in physicians is distinct from patient satisfaction with physicians . The objective of this study was to assess patient trust in physicians through a quantitative study in shanghai, china . Furthermore, this paper aimed to identify the determinants of patient trust in physicians to provide appropriate suggestions for improving this trust in china, and to enrich the theories on the issue of trust in physicians . The data from a survey conducted in zhongshan hospital and shanghai tenth people's hospital, which are two tertiary public hospitals in shanghai, were used in this study . During the first eight workdays in july 2015, 100 outpatients were randomly selected using the outpatient registration numbers for each day in each hospital . We excluded patients with missing data and thereby obtained a total of 1210 respondents (a valid rate of 75.6%). Description of dependent and demographic variables (%) uebhis: urban employee basic health insurance scheme; ucbhis: urban citizen basic health insurance scheme; nrcms: new rural cooperative medical system; chi: commercial health insurance; fhi: free health insurance . In this study, this variable was measured using the following question: please indicate to what extent you trust the physicians on a scale from 1 (strongly disagree) to 5 (strongly agree). Furthermore, a 10-item scale was used to precisely describe the dependent variable . This scale is basically consistent with the 10- or 11-item scales employed in the unites states, which were constructed using psychometric analyses that focused on feasibility, factor structure, validity, and reliability . Responses and coding of each item were indicated on a typical 5-point likert scale: strongly agree (5), agree (4), neutral (3), disagree (2), and strongly disagree (1). Description of the scores of patient trust in physicians by the 10-item scale * the negatively worded item has been reverse coded . The demographic characteristics (gender, age, education level, marital status, household registration, income, and health insurance) were independent variables of trust in physicians . Dichotomous variables were scored as either 1 (male and household registration in shanghai) or 2; age was measured on a scale from 1 (1829 years) to 6 (70 years and above). Education level was measured from 1 (primary school and below) to 6 (master's degree and above). The annual income was assessed from 1 (rmb 0) to 6 (rmb 120,000 and above or approximately usd 19,000). Furthermore, health insurance coverage was measured from 1 (no health insurance) to 7 (urban employee basic health insurance scheme [uebhis] and commercial health insurance [chi]). Chicago, il, usa), and a p <0.05 was considered statistically significant . Binomial logistic regression was employed to analyze the factors associated with the dependent variable, which was divided into two categories on the basis of the responses (1: strongly agree or agree and 0: strongly disagree, disagree, or neutral). In addition, logistic regression results are presented as odds ratios (ors), 95% confidence interval (ci), and p values . The data from a survey conducted in zhongshan hospital and shanghai tenth people's hospital, which are two tertiary public hospitals in shanghai, were used in this study . During the first eight workdays in july 2015, 100 outpatients were randomly selected using the outpatient registration numbers for each day in each hospital . We excluded patients with missing data and thereby obtained a total of 1210 respondents (a valid rate of 75.6%). Description of dependent and demographic variables (%) uebhis: urban employee basic health insurance scheme; ucbhis: urban citizen basic health insurance scheme; nrcms: new rural cooperative medical system; chi: commercial health insurance; fhi: free health insurance . This variable was measured using the following question: please indicate to what extent you trust the physicians on a scale from 1 (strongly disagree) to 5 (strongly agree). Furthermore, a 10-item scale was used to precisely describe the dependent variable . This scale is basically consistent with the 10- or 11-item scales employed in the unites states, which were constructed using psychometric analyses that focused on feasibility, factor structure, validity, and reliability . Responses and coding of each item were indicated on a typical 5-point likert scale: strongly agree (5), agree (4), neutral (3), disagree (2), and strongly disagree (1). Description of the scores of patient trust in physicians by the 10-item scale * the negatively worded item has been reverse coded . The demographic characteristics (gender, age, education level, marital status, household registration, income, and health insurance) were independent variables of trust in physicians . Dichotomous variables were scored as either 1 (male and household registration in shanghai) or 2; age was measured on a scale from 1 (1829 years) to 6 (70 years and above). Education level was measured from 1 (primary school and below) to 6 (master's degree and above). The annual income was assessed from 1 (rmb 0) to 6 (rmb 120,000 and above or approximately usd 19,000). Furthermore, health insurance coverage was measured from 1 (no health insurance) to 7 (urban employee basic health insurance scheme [uebhis] and commercial health insurance [chi]). Chicago, il, usa), and a p <0.05 was considered statistically significant . Binomial logistic regression was employed to analyze the factors associated with the dependent variable, which was divided into two categories on the basis of the responses (1: strongly agree or agree and 0: strongly disagree, disagree, or neutral). In addition, logistic regression results are presented as odds ratios (ors), 95% confidence interval (ci), and p values . Among all sampled patients, 47.4% were male, 26.3% were 60 years and above, and 48.4% had high school or lower education . Furthermore, 58.3% of patients had household registration in shanghai, and 45% were covered by the uebhis . Patient trust in physicians was scored using the 10-item scale; the mean score of this trust was 35.4 from a total score of 50 . Table 2 indicates that the highest score was obtained for the item i can tell doctors anything about my disease among the ten items . In addition, the lowest scores were obtained for the items doctors sometimes pretend to know things when they are really not sure and if a mistake were made in my treatment, then doctors would try to hide it from me . According to the logistic regression results, patient trust in physicians was significantly correlated with the age of patients . Compared with patients aged 1829 years, those who were any other age except for 70 years and above were more likely to trust physicians . For example, patients aged 6069 years were more likely to trust physicians (or 3.36; 95% ci 2.005.67). Furthermore, table 3 shows that patient trust in physicians was also significantly correlated with the annual income of patients . Compared with patients without any income, those with any other income level except for rmb 40,00080,000 were more likely to trust physicians . For example, patients with an income of rmb 80,000120,000 were more likely to trust physicians (or 7.56; 95% ci 4.0714.06). Table 3 also illustrates that trust in physicians was significantly correlated with the education level of patients . Compared with patients with primary school education and below for example, patients who received a bachelor's degree were more likely to trust physicians (or 5.27; 95% ci 2.4811.20). Factors associated with trust in physicians by the logistic regression * p<0.05; p<0.01; p<0.001 . Uebhis: urban employee basic health insurance scheme; ucbhis: urban citizen basic health insurance scheme; nrcms: new rural cooperative medical system; chi: commercial health insurance; fhi: free health insurance; or: odds ratio; ci: confidence interval . In addition, table 3 displays that patient trust in physicians was significantly correlated with the type of health insurance coverage . Compared with patients without health insurance, those covered by chi were more likely to trust physicians (or 0.36; 95% ci 0.150.86), and those covered by free health insurance were less likely to trust physicians (or 0.08; 95% ci 0.030.20). Patients covered by both the uebhis and chi were also less likely to trust physicians (or 0.39; 95% ci 0.180.87). Furthermore, this study found that trust in physicians was not significantly correlated with patient gender, marital status, or household registration . Among all sampled patients, 47.4% were male, 26.3% were 60 years and above, and 48.4% had high school or lower education . Furthermore, 58.3% of patients had household registration in shanghai, and 45% were covered by the uebhis . Table 1 shows that 67% of patients trusted or strongly trusted physicians . In addition, 4.2% of patients distrusted or strongly distrusted physicians . Patient trust in physicians was scored using the 10-item scale; the mean score of this trust was 35.4 from a total score of 50 . Table 2 indicates that the highest score was obtained for the item i can tell doctors anything about my disease among the ten items . In addition, the lowest scores were obtained for the items doctors sometimes pretend to know things when they are really not sure and if a mistake were made in my treatment, then doctors would try to hide it from me . According to the logistic regression results, patient trust in physicians was significantly correlated with the age of patients . Compared with patients aged 1829 years, those who were any other age except for 70 years and above were more likely to trust physicians . For example, patients aged 6069 years were more likely to trust physicians (or 3.36; 95% ci 2.005.67). Furthermore, table 3 shows that patient trust in physicians was also significantly correlated with the annual income of patients . Compared with patients without any income, those with any other income level except for rmb 40,00080,000 were more likely to trust physicians . For example, patients with an income of rmb 80,000120,000 were more likely to trust physicians (or 7.56; 95% ci 4.0714.06). Table 3 also illustrates that trust in physicians was significantly correlated with the education level of patients . Compared with patients with primary school education and below, those with any other education level for example, patients who received a bachelor's degree were more likely to trust physicians (or 5.27; 95% ci 2.4811.20). Factors associated with trust in physicians by the logistic regression * p<0.05; p<0.01; p<0.001 . Uebhis: urban employee basic health insurance scheme; ucbhis: urban citizen basic health insurance scheme; nrcms: new rural cooperative medical system; chi: commercial health insurance; fhi: free health insurance; or: odds ratio; ci: confidence interval . In addition, table 3 displays that patient trust in physicians was significantly correlated with the type of health insurance coverage . Compared with patients without health insurance, those covered by chi were more likely to trust physicians (or 0.36; 95% ci 0.150.86), and those covered by free health insurance were less likely to trust physicians (or 0.08; 95% ci 0.030.20). Patients covered by both the uebhis and chi were also less likely to trust physicians (or 0.39; 95% ci 0.180.87). Furthermore, this study found that trust in physicians was not significantly correlated with patient gender, marital status, or household registration . A global survey used the same questionnaire to compare the differences in public trust in physicians among different countries . One survey in the unites states showed that 70% of patients completely trusted their physicians, and another survey of cancer patients in the united kingdom revealed that 94% of patients completely trusted the hospital physicians . Although no research has evaluated patient trust in physicians in china, several news media reports have shown that patients distrust physicians in china . For example, a survey by the china youth newspapers in 2013 found that approximately 70% of patients did not trust physicians in china . This result is consistent with the score determined using the 10-item scale, with a mean score of 35 from a total score of 50 . The percentage and score of patient trust in physicians are similar to those in the aforementioned studies in the united states and are much higher than those in previous reports . Therefore, this study demonstrated that patient trust in physicians in shanghai is much higher than in the locations examined in previous reports in china . Previous studies have shown that the for - profit characteristic of physicians in china is the most crucial reason for the poor physician furthermore, this for - profit characteristic is evidenced by the connection between physicians prescription provision and their salaries . The results of this study illustrated that the item doctors make decisions on my treatment regardless of their salaries was ranked in the last fourth among the ten items of the scale used to assess patient trust in physicians . Therefore, we believe that the so - called for - profit characteristic of physicians is not the most crucial reason for patient distrust in physicians . More importantly, analyses of patient trust in physicians according to the 10-item scale revealed that the information asymmetry theory plays a crucial role in patient trust in physicians . The item i can tell doctors anything about my disease ranked first, and the item if a mistake were made in my treatment, then doctors would try to hide it from me ranked last among the ten items of the scale used to assess patient trust in physicians . The considerable differences in the scores between the two items showed that patients possessed full information about themselves but did not possess full information about the physicians diagnosis and treatment because of the obstacles in acquiring professional medical knowledge . Because it is nearly impossible for patients to possess the same amount of information that physicians do, altering the information asymmetry between patients and physicians is difficult . Therefore, we believe that being pessimistic about patient distrust in the honesty of physicians is unnecessary . In addition, hospitals and physicians should establish suitable mechanisms and channels for improving communication with patients, such as increasing the transparency of the protocol of diagnosis and treatment . Previous studies have illustrated that critical citizens who are younger and highly paid as well as have a higher education level are considered to be a vital reason for the lower trust in local governments and their provisions, such as public services, in developed countries . Furthermore, critical citizens who have a lower trust in local governments and public services have also been found in urban china . For this reason and because of the common occurrence of patient violence against physicians, several researchers attribute the poor physician although we found that younger patients were less likely to trust physicians, those with a higher education level or a higher income were more likely to trust physicians . Therefore, this study found no strong evidence to confirm the existence of critical citizens among patients in shanghai . In conclusion, according to empirical evidence from two public hospitals in shanghai, patient trust in physicians in china is higher than previously reported . Furthermore, the most crucial reason for patient distrust in physicians is the information asymmetry between patients and physicians, which is a natural property of the physician patient relationship, rather than the so - called for - profit characteristic of physicians or patients excessive expectations . In addition, because this study was a cross - sectional survey, deducing a continuous trend in patient trust in physicians over a long period was impossible . Regarding the patients sampled from shanghai's hospitals, extrapolating the results of this study to the entire population of china is difficult . Therefore, conducting a continuous and comprehensive survey of patient trust in physicians in multiple regions of china has been planned in order to provide more appropriate suggestions for improving the physician this work was supported by grants from national social science foundation of china (no . 13cgl135), and the humanities and sciences cross - disciplinary foundation of shanghai jiao tong university (no . This work was supported by grants from national social science foundation of china (no . 13cgl135), and the humanities and sciences cross - disciplinary foundation of shanghai jiao tong university (no.
Oral contraceptive pills (ocps) are commonly used for contraception, polycystic ovarian syndrome, amenorrhea, menorrhagia, dysmenorrhea, endometriosis, etc ., common side effects of ocps include nausea, vomiting, headache, bloating, breast tenderness, swelling of the feet, weight gain, breakthrough bleeding, and venous thromboembolism . Neurological adverse effects associated with ocps are migraine, depression, psychosis, and cerebral infarction . Chorea caused by ocps is extremely rare and only very few cases are reported in the literature . The onset of chorea usually varies from few weeks to several years after starting therapy . Here, we report a case of chorea developing within 1 week of initiating ocps in an adolescent girl with polycystic ovarian disease . A 17-year - old non - obese, unmarried girl presented to our hospital with irregular cycles and hirsutism for 2 years duration . Investigations revealed raised lh: fsh ratio (3:1) and a slightly raised serum testosterone level . She was diagnosed to have polycystic ovarian disease and was prescribed ocps for treatment of irregular cycles . The ocps contained a combination of ethinyl estradiol 0.035 mg and cyproterone acetate 2 mg, which was prescribed once daily for 21 days . The patient presented with complaints of abnormal movements of right extremities within 1 week of starting therapy . There was no previous history suggestive of streptococcal infection, rheumatic fever or drug therapy . On examination, investigations revealed normal blood counts, erythrocyte sedimentation rate, serum electrolytes, renal, liver, and thyroid function tests . She was diagnosed to have ocps induced hemichorea after ruling out other common causes of chorea . Neurological side effects of ocps have been linked to alterations in coagulation pathways and secondary vascular complications . Chorea is an abnormal involuntary movement disorder characterized by brief, semi - directed, irregular movements that are not repetitive or rhythmic, but appear to flow from one muscle to the next . The first description of chorea associated with ocps was reported by fernando in 1966 and subsequently few similar published reports established ocps as a cause of chorea . No particular oral contraceptive preparation was consistently associated with chorea, and both low and high - dose preparations can cause this movement disorder . It has been well - established that estrogens have a definite but complex modulatory action in dopaminergic systems . Hence, the estrogenic component of ocps is most likely the causative factor in the chorea induced by these agents . Some cases of ocps induced chorea are caused by reactivation of sydenham's chorea seen in rheumatic fever . However, ocps induced chorea have no clear relationship with sydenham's chorea, or streptococcal infection have also been reported, similar to our case . The onset of chorea varies widely from few weeks to several years (average duration: 9 weeks). The shortest time interval between ocp intake and development of chorea reported in the literature is eight weeks in the patient with no significant medical history . In most cases, symptoms subside promptly after withdrawing the offending drug, usually within a period of 23 months . In this index case, causality assessment by naranjo adverse drug reaction probability scale got a score of 6, suggesting a probable relationship between the reaction and the drug . Prior knowledge of this rare adverse effect and high index of suspicion can help in early diagnosis and discontinuation of therapy.
Amyloidosis is a heterogeneous group of diseases in which misfolding of extracellular protein is the pathogenic factor . This process produces insoluble, toxic protein aggregates that are deposited in tissues in bundles of -sheet fibrillar protein . The most common cause of amyloidosis is clonal plasma cells in the bone marrow producing immunoglobulins that are amyloidogenic (light chain amyloidosis or al). Hemorrhagic events, ranging from mild subcutaneous hemorrhage to life - threatening bleeding, account for a significant proportion of morbidities and mortality in al . Bleeding tendency is frequently encountered in al, and while mild subcutaneous hemorrhage is the most common manifestation, life - threatening bleeding has also been reported [3 - 6]. Acquired hemostatic abnormalities, including coagulation factor deficiencies, hyperfibrinolysis, and platelet dysfunction are the background of bleeding tendency [5, 6]. In particular, acquired deficiency of factor x is the most common coagulation factor deficiency in patients with al, and it is postulated to occur via the adsorption of factor x to amyloid fibrils [3, 5 - 8]. We herein report 2 korean patients with acquired factor x deficiency in association with al . A 55-yr - old woman with generalized edema was diagnosed with al on the basis of a renal biopsy . Serum and urine electrophoresis combined with immunofixation revealed m - protein of igg / lambda type, and bone marrow analysis showed monoclonal proliferation of plasma cells . Coagulation tests revealed a prolonged prothrombin time (pt) of 2.51 international normalized ratio (inr) and an activated partial thromboplastin time (aptt) of 75.1 sec (29.1 - 41.9 sec). Thrombin time was within the normal range (18.6 sec; reference interval, 15.6 - 20.0 sec). Complete correction of the prolonged pt and aptt on mixing with normal plasma prompted us to proceed with factor assays, which revealed markedly decreased factor x activity at 5% (69 - 126%) and mildly decreased factor v activity 63% (81 - 160%). Follow - up coagulation tests revealed improvement of prolonged pt / aptt (pt, 1.77 inr and aptt, 52.5 sec) and normalization of factor v activity (136%). However, factor x activity was still decreased at 12% . Despite the improvement of coagulopathy, monoclonal immunoglobulins of igg / kappa type were detected in the serum and urine, and bone marrow analysis showed an increase of monoclonal plasma cells . Coagulation tests showed prolongation of both pt (1.63 inr) and aptt (50.3 sec), which were corrected on mixing with normal plasma . He received chemotherapy, and follow - up coagulation tests showed a normalized aptt, but the pt was still prolonged (1.64 inr). A 55-yr - old woman with generalized edema was diagnosed with al on the basis of a renal biopsy . Serum and urine electrophoresis combined with immunofixation revealed m - protein of igg / lambda type, and bone marrow analysis showed monoclonal proliferation of plasma cells . Coagulation tests revealed a prolonged prothrombin time (pt) of 2.51 international normalized ratio (inr) and an activated partial thromboplastin time (aptt) of 75.1 sec (29.1 - 41.9 sec). Thrombin time was within the normal range (18.6 sec; reference interval, 15.6 - 20.0 sec). Complete correction of the prolonged pt and aptt on mixing with normal plasma prompted us to proceed with factor assays, which revealed markedly decreased factor x activity at 5% (69 - 126%) and mildly decreased factor v activity 63% (81 - 160%). Follow - up coagulation tests revealed improvement of prolonged pt / aptt (pt, 1.77 inr and aptt, 52.5 sec) and normalization of factor v activity (136%). However, factor x activity was still decreased at 12% . Despite the improvement of coagulopathy, monoclonal immunoglobulins of igg / kappa type were detected in the serum and urine, and bone marrow analysis showed an increase of monoclonal plasma cells . Coagulation tests showed prolongation of both pt (1.63 inr) and aptt (50.3 sec), which were corrected on mixing with normal plasma . He received chemotherapy, and follow - up coagulation tests showed a normalized aptt, but the pt was still prolonged (1.64 inr). Acquired factor deficiency with or without bleeding symptoms is not infrequent in al, whereas it is rare in other types of amyloidosis . Specific coagulation factor deficiencies in al have long been recognized and have been explained by the adsorption of coagulation factors to amyloid fibrils . Evidence showing the interaction of amyloidogenic light chains with coagulation factors and anecdotal reports of improved hemostasis after removal of amyloidotic spleen support the fact that coagulation factors bind to the amyloid fibrils . Both isolated coagulation factor deficiency and combined deficiencies have been reported . Factor x deficiency has been described as the most common acquired coagulation factor deficiency, affecting up to one - third of patients with al . Deficiencies of other coagulation factors such as ii, v, vii, and ix have also been reported [5 - 7]. According to a previous study that evaluated a total of 368 patients with al, 32 patients had a factor x activity below 50% of normal . Eighteen of these patients (56%) had bleeding complications, which were more severe in the 12 patients with factor x activity below 25% of normal . The 2 patients with acquired factor x deficiency from al described in the present report had no bleeding symptoms, even with the very low level of factor x activity (5%) in patient 1 . A review of the literature revealed a single case report on acquired factor x deficiency in al patients in korea . Kim et al . Reported factor x deficiency in a patient with al and nephrotic syndrome . The patient had mildly decreased factor x activity of 49% (70 - 120%), prolonged pt of 1.32 inr and upper normal aptt of 39.4 sec (32 - 41.2 sec). Despite normal aptt, mildly prolonged pt, and slightly decreased factor x activity, he had bleeding tendency and symptoms such as petechiae . Previous studies reported that high - dose chemotherapy followed by autologous stem cell transplantation led to complete remission and normalization of the factor x level [7, 10, 11]. The patient showed improvement of prolonged pt and aptt (2.51 to 1.77 inr and 75.1 to 52.5 sec, respectively). However, factor x activity was still significantly decreased (12%), and the patient developed bleeding diathesis during the course of treatment . Indeed, it was reported that the baseline factor x level was not predictive of bleeding risk, and optimal management of factor x deficiency in al is still elusive . The findings in the previously reported patient and the 2 patients described in the present report demonstrate the heterogeneous clinical and laboratory manifestations of factor x deficiency in korean patients with al . To the best of our knowledge, this is only the second report of factor x deficiency in association with al in korea . Further prospective studies involving multiple institutions are needed to investigate the epidemiology and clinical implications of acquired factor x deficiency in korean patients with al . Appropriate coagulation workup should be conducted in patients with al with prolonged coagulation times, and further studies are needed to obtain laboratory and clinical data to delineate the frequency and clinical implications of acquired coagulopathy in korean patients with al.
The reverse trial was a prospective, randomized, double blind, parallel - controlled multinational study designed to determine whether crt limited the progression of hf compared with optimal medical therapy alone . The study included 610 patients with acc / aha stage c, nyha class i or ii hf patients with qrs more than 120 msec, and lvef less than 40% on optimal medical therapy . Patients were implanted with a crt - device with (crt - d) or without (crt - p) defibrillator and randomized 2:1 to crt on (n = 419) or crt off (n = 191). Patients were then programmed as randomized through 12 months in north america and through 24 months in europe . By the study design, all patients were programmed to crt on after the randomization period and followed for 5 years from implantation . First enrollment occurred in september 2004, and enrollment was completed in september 2006.the final 5-year follow - up was in november 2011.the primary end point was hf clinical composite response, which scores patients as improved, unchanged, or worsened . The initial results were published in 2009 in the journal of american college of cardiology . They showed the hf clinical composite response end point, which compared only the percent worsened, indicated 16% worsened in crt - on compared with 21% in crt - off (p 0.10). Patients assigned to crt - on experienced a greater improvement in lv end - systolic volume index (18.4 29.5 ml / m 2 vs. 1.3 23.4 ml / m 2, p 0.0001) and other measures of lv remodeling . Time - to - first hf hospitalization was significantly delayed in crt - on (hazard ratio: 0.47, p 0.03). The five - year follow up analysis was confined to 419 subjects randomized to crt on, who received up to 5 years of crt therapy . The 6-min hall walk increased by 18.8 102.3 minutes and the minnesota and kansas city scores improved by 8.2 17.8 and 8.2 17.2 unites, respectively . The mean decrease in left ventricular end - systolic volume index and left ventricular end - diastolic volume index was 23.5 34.1 ml / m and 25.4 37.0 ml / m and the mean increase in lvef 6.0 10.8% with sustained improvement thereafter (figure 1). The annualized and 5-year mortality was 2.9 and 13.5% and the annualized and 5-year rate of death or first hf hospitalization 6.4, and 28.1% . The 5-year results of reverse confirmed that crt in mildly symptomatic hf and wide qrs reverses remodeling and is associated with low rates of hf hospitalization and all - cause mortality over the entire follow - up . Crt effect in mildly symptomatic hf patients was first tested on 186 patients (101 control and 85 crt) in the miracle icd ii trial, which was published in 2004 . The results from the study demonstrated that crt significantly improved the cardiac structure and function over a 6-month period of follow up in optimally treated patients . In the larger madit - crt, published in 2009, the use of crt combined with an icd in asymptomatic or mildly symptomatic patients with heart failure and a reduced ejection fraction and wide qrs complex was associated with 34% reduction in the risk of death or heart failure events, as compared with the use of an icd alone . This benefit was driven by 41% reduction in the risk of heart failure events, a finding that was evident primarily in subgroup of patients with a qrs duration of 150 msec or more . Similarly, the raft trial, published in 2010, found the addition of crt to an icd and optimal medical therapy reduced rates of death and hospitalization for hf among patients with mild - to - moderate hf, a reduced lv ef, and a wide qrs complex over a period of 40 months of follow - up . The echocardiographic studies from reverse, madit - crt, and care - hf showed substantial improvements in lv size and function, lvef, rv function, left atrial size and mitral regurgitation severity in patients treated with crt compared with icd only . These findings were strongly concordant with and predictive of the primary outcome of death or a hf event and suggest a compelling cardiac structural and functional mechanism by which crt therapy improves outcomes . These results suggest that in the long term, crt lowers the risk of hf - related adverse clinical events and prevents or reduces the progression of disease by reverse lv remodelling . Four trials have studied the effect of crt on rates of death among patients with hf . Care - hf and companion included patients with moderate to - severe nyha class iii and ambulatory class iv hf . Raft and madit - crt included less symptomatic patients with nyha class ii and iii in the raft and nyha class i or ii in the madit - crt . The raft trial found a significant reduction in the rate of death from any cause associated with the use of crt in addition to icd and optimal medical therapy . The relative risk of death was reduced by 25%, resulting in an absolute mortality reduction of 6 percentage points at 5 years . Possible reasons for the differences in mortality rates between raft and madit crt are that raft had longer follow up period and that patients had slightly more advanced disease with a slightly lower ef and a higher proportion of the raft patients and ischemic heart disease . Although reverse was not designed as a morbidity and mortality trial, its 5-year follow - up confirmed the raft findings that crt in mild hf patients over a longer treatment period provided benefits in morbidity and mortality . This positive impact on mortality was in fact predicted by the reverse authors in their 2009 publication as they observed a delay in time to first hf hospitalization in the active crt group in the 24 months follow - up . However, the investigators were not blinded to the follow - up cohort and this could represent a potential bias to the results . Like the madit - crt, the positive reverse findings were greatest in patients with a qrs width of more than 150 msecs, left bundle branch block (lbbb), and of non - ischemic aetiology . In fact, this finding was confirmed in a meta - analysis of five randomized controlled trials . A detailed non - invasive 3-dimensional electrocardiographic mapping, in 33 consecutive crt candidates, demonstrated that patients with lbbb had uniform patterns of activation, whereas in patients with nonspecific interventricular conduction delay, conduction patterns were highly variable . Left ventricular electrical uncoupling was consistently elevated in all lbbb patients, and properly identified clinical crt responders . Among the 419 patients, 51 (12.2%) experienced 63 lv lead - related complications . Most events occurred within the first 3 months of implant with a rate of 7.9% (95% ci: 5.710.9%), and a rate of 12.5% (95% ci: 9.616.2%) at 5 years . The majority were due to lv lead dislodgment (n = 33) and/or diaphragmatic stimulation (n = 10). Over the course of the 5-year follow - up, 132 of the 419 patients (31.5%) underwent device replacement . These findings were relatively fewer than the 16% complication rate (lv 11%, rv 5%) reported in nyhaclass iii and iv patients with 6-month follow - up and comparable to the reported european crt survey with 1-year follow - up (10.3%). Despite the low mortality rates, the cost effective analysis from reverse showed that compared with crt - off, 0.94 life years or 0.80 qalys were gained in the crt on group at an additional cost of 11,455 - yielding an incremental cost - effectiveness ratio of 14,278 per quality - adjusted life year (qaly) gained . At a threshold of 33,000 (30,000) per qaly in the usa 5 million individuals - from a total population of nearly 294 million - have hf . In europe asymptomatic left ventricular dysfunction is estimated to have at least the same prevalence as congestive hf . Overt hf symptoms generally follow the asymptomatic phase and are linked to increased morbidity and mortality . The clinical efficacy of crt in patients with moderate to severe hf has been established in a number of trials . In contrast, for patients with asymptomatic or mildly symptomatic hf, crt represents a relatively novel treatment option . Evidence on the efficacy of crt in this patient group has recently been demonstrated with the publication of the reverse and madit - crt trials . The long term follow up confirmed the initial findings of reverse by showing that cardiac reverse remodelling lowers hf related events and mortality rates with longer follow up time . The possibility of long - lasting benefit of crt on hf progression outweighs the risk, as the complication rate related to crt remained acceptable after 5 years . However, these results are exclusive to patients with lbbb and wide qrs complex more than 150 milliseconds, emphasizing a specific electrophysiological process that takes place in crt responders.
Paranasal sinus osteoma is a slow growing benign encapsulated tumor occurring most commonly in frontal, ethmoid and maxillary sinuses . Osteoma size generally range in 2 mm to 30 mm diameter, osteoma having diameter> 30 mm or weight more than 110 g is typically considered a giant osteoma of the paranasal sinuses are rare but readily extend into the intraorbital or intracranial cavity, causing serious complications . However, secondary invasion of the orbits by osteoma is relatively uncommon, with an incidence of 0.95.15% of all orbital tumors . Giant osteoma of the paranasal sinuses are infrequent lesions, especially in the pediatric age group and those of the spheno - ethmoid sinuses are much rarer . We report a rare case of a giant spheno - ethmoid osteoma in a 14-year - old boy with extension into the orbit and optic canal and compression of the optic nerve, causing visual impairment . A 14-year - old boy presented with a history of progressive proptosis with diminution of vision of left eye for 1 year . There was no antecedent history of trauma, chronic sinusitis or nasal surgery . Examination revealed extra - axial proptosis of the left eye in outward and forward direction . Visual acuity was 6/60 in the left eye and 6/6 on the right side, and fundus showed mild primary optic atrophy on the left side . Plain computed tomography (ct) scan of the head and orbit showed a bone density mass arising from the sphenoid and left posterior ethmoidal cells, which was extending to the left orbital apex and roof of the optic canal and anterior clinoid process, which was causing compression of the left optic nerve and anterolateral displacement of the optic globe [figures 1 and 2]. Computed tomography scan head and orbit, coronal section showed a mass of bone density over sphenoid and left posterior ethmoidal cells with extension to the left orbital apex and optic canal computed tomography scan head and orbit, axial section showing mass over sphenoid and left posterior ethmoidal cells with extension to the left orbit he underwent left frontotemporal craniotomy with combined orbitozygomatic osteotomy using transcranial extradural approach with total excision of a giant osteoma, measuring about 6 5.5 5 cm [figure 3]. The excision was accomplished using a high speed drill and osteoma was gradually mobilized from the surrounding thin remodeled bones . At the end of surgical procedure, optic nerve in the optic canal was visualized, with completely thinned out bony canal . He regained visual acuity of 6/12 on the left side on follow - up at 3 months following surgery . Histopathology was consistent with benign cancellous osteoma, which was consisting of trabeculae and fatty bone marrow . The distribution of osteoma in the paranasal sinus, according to mansour et al . Includes frontal (71.8%), ethmoidal (16.9%), maxillary (6.4%) and sphenoid (4.9%) sinuses . Osteomas are commonly observed in third to fourth decades of life, with a mean age of 37.8 years . Giant osteomas are rare however, much rarer to occur in the pediatric age group, and only nine cases of giant osteoma originating in ethmoid sinus with extension to the orbit are reported [table 1]. Frontal osteoma is slow growing and lobulated and can acquire cauliflower or dumbbell shape causing proptosis in downward and outward direction with rarer intracranial extension . However, ethmoid osteoma is pedunculated, have a propensity to relatively rapid growth and causes proptosis in the outward direction . Sphenoid osteoma is characterized by variable growth rate and shape, causes proptosis in an outward direction and carries high predilection for optic nerve compression and it may cause complete blindness . Common presenting symptom of paranasal osteoma is localized headache besides nonocular manifestations and sinusitis, frontal mucocele, anosmia, cerebrospinal fluid rhinorrhea and rarely meningitis, brain abscess, and seizures . Ocular manifestations include proptosis, extra ocular muscle displacement, optic disc edema, choroidal folds and orbital infection . Visual loss results from direct pressure on the globe or the optic nerve, or interference with the ocular blood supply, venous drainage, or infection . In long standing cases optic atrophy develops . However, early visual compromise results only with sphenoid osteoma and it even causes primary bilateral optic atrophy . Osteoma is composed of mature lamellar bones, originate in the bones formed by intramembranous ossification . It can be compact type, which is most common being composed of mature lamellar bone with haversian systems and containing minimal bone marrow space and x - ray shows rounded sclerotic lesion, while another type called cancellous or spongy type have trabecular architecture with peripheral cortical bone margin . Ct scan is the investigation of choice and shows well demarcated hyperdense mass homogenous consistency, may be lobulated, lying on the floor of the sinus causing expansion and distortion of the involved sinus . Surgery is indicated if osteoma filling of more than 50% of the volume of the frontal sinus, or located near the frontal sinus ostium, extension beyond the confines of sinus or showing enlargement on serial imaging other indication for surgical intervention include large osteoma, symptomatic causing headache, recurrent sinusitis, cosmetic deformity, ocular or central nervous system symptoms . The surgical approach depends on the size, location and extension, minimally invasive surgery including endoscopic method is a choice . However, small osteoma of the ethmoid sinus can be approached through a small external incision around the medial canthus while those of the sphenoid sinus through transseptal, subnasal midline, transpalatal or transorbital transpalate routes . However, giant osteoma as the current case can be removed, and optic canal decompression can be successfully accomplished by the transcranial extradural approach frontotemporal craniotomy with orbitozygomatic osteomy . While selecting transcranial approach, consideration of protection of vital structures, with minimal cosmetic deformity prevails . They can also be approached via the endonasal route with the help of microscope or endoscope . Sphenoorbital osteomas are rare, but important cause of visual impairment due to optic nerve compression in the optic canal.
Carcinogenesis is a long and multi - step process that includes initiation, promotion and progression as a consequence of an imbalance between cell proliferation and cell death, during which complex interplay concerning inflammation between epithelial layer and mesenchymal tissues occurred, after which more genetic and epigenetic events are required to drive from initiated cells to malignant tumors, each conferring one and another type of growth advantage, and leads to the progressive conversion of normal cells into cancer cells [13]. In fact, a wide array of chronic inflammatory conditions predisposes susceptible cells to neoplastic transformations during this journey to carcinogenesis [45]. In general, the longer the inflammation persists, the higher the risk of cancer, suggesting that though inflammation is a step - by - step process that includes injury, repair and resolution, all inflammatory cells, for instance, neutrophils, monocytes, macrophages, eosinophils, dendritic cells, mast cells, and lymphocytes, are recruited after a damage or an infection, and may contribute to the onset and progression of cancer . Moreover, once tumor develops, tumor cells educated macrophages or inflammatory cells to promote tumor progression . With the continuing medical education that inflammatory cells including macrophages are continuously educated by the tumor microenvironment, they adopt a trophic role that facilitates angiogenesis, matrix breakdown and tumor cell motility, all of which are elements of the metastatic process . During an inflammatory response, these inflammatory cells also produce many compounds, ranging from mutagenic oxygen and nitrogen radicals to angiogenic factors that further contribute to cancer initiation and promotion . Therefore, crudely saying, this is why inflammation represents an important drug target for cancer prevention and cure . It has been reported that approximately 1520% of all malignancies are initiated or exacerbated by inflammation . Furthermore, the recruitment and infiltration of inflammatory cells in the tumor microenvironment activates them to spurt the malignant progression of cancer cells . Almost 150 years, rudolf virchow first indicated the concept that lymphoreticular infiltration reflects the origin of cancer at sites of chronic inflammation, suggesting a close connection between inflammation and the development of cancer, of which theory was further extended to implicate the stromal interactions between malignant cells and inflammatory cells associated with angiogenesis . Inflammatory stimuli include chemicals and foreign bodies (e.g. Asbestosis, fiber, silica particles, catheters, alcohol, bile acids, gastric acids, gall bladder stones, and ultraviolet light) and infectious organisms (e.g. Helicobacter pylori (h. pylori), hepatitis b and c viruses, epstein - barr virus, herpes virus, papilloma virus, hiv) [910]. Irrespecive of involved organs and etiologies of inflammations, the common features essential to inflammation - associated carcinogenesis were as follows; proinflammatory cytokines including tumor necrosis factor- (tnf-), interleukin (il)-1, il-6, il-8, interferon-, mdm2, p53 etc, prevail in inflamed sites, imbalance between reactive oxygen species (ros)-generating enzymes and anti - oxidant defense mechanisms, so called, oxidative stress, activation of nf-b, inducible nitric oxide synthase (inos), cyclooxygenase-2 (cox-2), proteinase, and activation of oncogenes . Based on these implications, various chemopreventive agents targeted for attenuating inflammation have been studies, including curcumin, retinoids, vitamin e, silimarin, aspirin, celecoxib, some plant polyphenols, proton pump inhibitor and others [1113]. Though it shows the tendency of decrease in gastric cancer for last 80 years, it is still second highest cause of death following lung cancer and in korea . 2530 people out of 100,000 catch gastric cancer regardless of their gender and its motility rate is number one . However, the discovery of causative pathogen of gastric cancer, h. pylori, has shown the light to inhibit gastric cancer because international agency for research on carcinogenesis (iarc, lyon) in 1994 defined h. pylori as an imminent carcinogenic pathogen based on many studies, epidemiological research and animal studies . The definition has the historical background based on the theological evidence and several animal experiments results showing infection of h. pylori causes infiltration of inflammatory, oxidative damage and mutations . In a resected specimen due to gastric cancer, the intestinal metaplstic lesions were easily identified around cancer lesions, for which h. pylori infection is closely associated . The reason why gastric cancer is not controlled even after killing h. pylori, is that under the equation: chronic atrophic gastritis intestinal metaplasia - gastric cancer, the patients who did not stepping into malignant process could be benefited from h. pylori - associated carcinogenesis [1415]. In other words, even though h. pylori, defined as class i carcinogen, are eradicated, the inflammation itself remains in the stomach, thus it seems that the control and the amelioration of gastric inflammation itself is far more essential in cancer prevention rather than the eradication of the pathogen itself . This inversely emphasizes that chronically continuation of gastritis has greater chance of onset of gastric cancer than infection of h. pylori itself . Pylori - induced gastric cancer is based on chronic atrophic gastritis as well as intestinal metaplasia, and gone into the stage of dysplasia, resulting in intestinal gastric cancer and accompanied by genetic or environmental change synergistically to accelerate the formation of cancer . With this background, genetic factors, toxicity of the pathogen and environmental factors are orchestrated to develop h. pylori - induced gastric cancer from inflammation, thus with broad category, alleviation or treatment of inflammation would be the fundamentals to the prevention of gastric cancer . Then, we have another question whether atrophic gastritis can be restored as before infection with either the removal of bacteria or clearing gastric inflammation . In spite two recent large - scale, prospective studies, both performed in high risk population, have reported that h. pylori infection as a definite risk factor for the development of gastric cancer, the opposite premise that eradication of h. pylori infection is an appropriate target for the prevention of gastric cancer is still uncertain . Tree randomized, placebo - controlled trials performed in china and columbia demonstrated no significant protective effect by h. pylori eradication [1618], whereas contradictory results have emerged out of three japanese studies published recently [1921], indicating that h. pylori eradication may prevent the development of gastric cancer significantly, even in patients with precancerous gastric lesions . These reciprocal results can be explained by the fact that, unlike the studies from china and columbia, protective studies from japan were neither randomized nor placebo - controlled . However, the common evidence of each japanese study was that no gastric cancers developed after eradication treatment in patients without precancerous gastric lesions at entry . Stated the other way around, all gastric cancer cases appeared in patients who had chronic atrophic gastritis / intestinal metaplasia at trial entry before h. pylori eradication . This observation ascertains us that earlier eradication therapy or other kinds of efforts to lessen gastric inflammation could be beneficial in high - risk populations to completely abolish overall gastric cancer risk arising from chronic atrophic gastritis . Another key issue regarding the influence of h. pylori eradication on gastric cancer prevention is the fact that atrophic gastritis is reversible lesion after h. pylori eradication, leading to hypothesis that h. pylori eradication could retard or reverse gastric carcinogenesis before it reaches the stage of h. pylori - associated chronic atrophic gastritis and/or dysplasia . As clear evidence of merit of h. pylori eradication, fukase et al . Have published important results from a study where, following endoscopic resection of early gastric cancer, a group of patients in a randomized control trial were subjected to h. pylori eradication treatment and monitored at different time intervals . At 3 years, metachronous gastric cancer had developed in only 9 of 255 patients in the eradication group compared with 24 of 250 patients in the control group, a significant difference with indicates that prophylactic eradication of h. pylori in atrophic gastritis can substantially reduce gastric cancer rates . However, similar to afore - mentioned implication of h. pylori eradication on gastric cancer prevention, studies examining the reversibility of precancerous lesions following eradication of h. pylori also have provided conflicting results . Among earlier studies, several reported that the severity of gastric atrophy and intestinal metaplasia does not change after treatment . In contrast, another study has suggested that gastric atrophy and some of intestinal metaplasia can improve after h. pylori eradication . The contradictory results can be explained by a fact that significant proportions among the eradicated patients have progression of premalignant lesions, already stepping to pass a point - of - no - return . Therefore, the eradication of h. pylori infection might not be beneficial if therapy is given passing the point - of - no - return . However, it is very difficult to discriminate whether the gastric premalignant lesion is passed such a point - of - no - return or not by conventional endoscopy or histopathology [2627]. Many efforts had been made to find biomarkers or clinical findings that suggest reversibility of gastric atrophy or intestinal metaplasia; ideally, these might adopt high throughput analytical techniques, including microarray or proteomics . Based on these observations, we hypothesize that supplementary or continuing suppressive way for gastric inflammation to mitigate chronic gastric gastritis will be the next step for gastric cancer prevention and we strongly send confidence that korea red ginseng can play tackling in the progression of atrophic gastritis and tangle to revert to non - atrophic gastritis . Before entering the description about the beneficial effects of korea red ginseng on h. pylori - associated atrophic gastritis, we will briefly touch the cancer preventive action of panax ginseng ., based on the humanoid shape of the root or rhizome of the plant, which is the part of the plant most commonly consumed . The name panax means all healing (panacea), which describes the traditional belief that ginseng has properties to heal all aspects of the body . Even though there are several different species of ginseng, two of the most commonly used are p. ginseng (chinese ginseng) and p. quinquefolius (american ginseng), among which p. ginseng c.a . Meyer (araliaceae) has been used as a medicine by the people of eastern asia for at least 2,000 years [2931]. Native to korea, this red - berried plant, commonly called korean ginseng, is now open employed for diabetes, cardiovascular diseases, neurodegenerative diseases, urological diseases including sexual improvement, several kinds of viral diseases, including various kinds of cancer . A research of pubmed for cancer and p. ginseng yields over 400 articles, signaling that p. ginseng apparently mitigates cancer through their anti - inflammatory, antioxidant, and apoptotic mechanisms to influence carcinogenic process . Briefly stated, 1) inducing differentiation; ginseng s induction of repair or reverse transformation of cells into more differentiated cells has been noted in hepatoma, melanoma, and teratocarcinoma cells, 2) reduced effects from chemical carcinogens; inhibitory effects on the development of rat mammary adenocarcinoma induced by methyl - n - nitrosourea and n - ethyl - n - nitrosourea administration, 3) mitigating inflammatory carcinogenesis; ginseng reduced cox-2, inos, and nk-b, 4) antioxidant chemoprevention; ginseng extracts have been shown to scavenge reactive oxygen species (ros) as well as lipid peroxidation . As shown in fig . 1a, electron spin resonance (esr) measurement showed direct antioxidative action of korea red ginseng extracts on fenton reaction - induced hydroxyl radicals, 5) induction of apoptosis; active components of ginsenoside induced direct executions of apoptosis, 6) inhibition of proliferation; inhibition of cell cycle progression has been implicated as a chemopreventive mechanisms of ginsenosidesm rh2 rg3, etc, halting the metastasis in addition to tumorigenesis except one study showing increased metastatic potential of p. ginseng, 7) immnuomodulation; mostly studied postoperatively to encourage the recovery from surgery or illness, 8) the relief of multi - drug resistance (mdr); chemosensitizing effect and reduced efflux pump activity related to mdr mechanism, 9) anti - angiogenic or angiogenic effect; dual effects on angiogenesis imposing anti - angiogenic action against carcinogenesis, but angiogenic effects for healing . As for effective gradients of ginseng, ginsenosides or ginseng saponins are the principal active ingredients in ginseng and more than 40 different ginsenosides have been identified . Based on their structural differences, they can be classified into three categories; the panaxdiol group (e.g. Rb1, rb2, rc, rd, rg3, rh2), the panaxatriol group (e.g. Re, rf, rg1, rg2, rh1) and the oleanolic group (e.g. Ro). Yun issued human epidemiology and well organized animal study in the lancet oncology (2001). He investigated the effects of ginseng consumption on the risk of cancers by interviewing 905 pairs of cases and controls matched by age, sex, and date of admission to the korea cancer center hospital, seoul . As results, a trend test showed a significantly decrease in the frequency of cancer cases among those with the highest ginseng intake for men (p<0.0001) and for women (p<0.05), strongly support the hypothesis that ginseng has cancer preventive effects . Several studies in the filed of microbiology and immunology have demonstrated that ginseng exerts antioxidant, anti - bacterial growth, and anti - inflammatory effects . Ginsenoside rb1 has been known to inhibit lipopolysaccharide (lps)-induced expression of the proinflammatory cytokine, tnf-, and acidic polysaccharide from ginseng have been reported to inhibit the adherence of h. pylori to human gastric epithelial cells . Also a growth inhibiting effect of the ginseng fraction, panaxdiol, on h. pylori was suggested in vitro with an mic of 50 g / ml [4243]. In addition to these anti - microbial actions of ginseng ingredients, it has been documented that ginseng has distinctive actions on cell growth in various carcinoma cells, through growth inhibition or cytotoxic effects in some cell lines, such as human melanoma cells a375-s2, hepatoma cells sk - hep-1, and prostate cells lncap, but growth stimulating or protective cells in other cell lines, such as neuroblastoma shsy5y and keratinocyte hacat, have been reported, suggesting that ginseng might provide diverse biological actions depending on cell context [4445]. First, we documented that korea red ginseng could rescue h. pylori - induced cytotoxicity . In addition to rescuing actions of korea red ginseng, they mitigate the oxidative stress - induced dna mutation . Red ginseng extracts significantly attenuated both h. pylori - induced dna damage assessed by comet assay, apoptosis measured by dna fragmentation, and expressions of 8-oh - dg (fig . 1b) for which inactivation of erk1/2 signal transduction and attenuation of caspase-3 activation were operated . Simutaneously, korea red ginseng decreased the h. pylori - stimulated il-8 expression, suggesting that based on these gastroprotective effects of ginseng against h. pylori - associated gastric mucosal damages, red ginseng could be used as a medicinal phytonutrient against h. pylori infection (fig . Then, we extended our experiment to prove whether red ginseng extract influences 5-lipoxygenase (5-lox) pathway, thereby suppressing the biosynthesis of 5(s)-hete . H. pylori infection increased exclusively 5-lox enzyme activity, which is strongly associated with carcinogenic pathway . Red ginseng extracts inhibited these increments of h. pylori - stimulated 5-lox through inactivation of c - jun phosphorylation and redox - sensitive transcriptional activation together, finally led to reduced levels of il-8 and 5-lox in gastric mucosal cells (fig . The lox inhibiting capacity of red ginseng was equivalent to 200 m of geraniin or egta . All of our study described above for the first time, revealed the h. pylori infection induced significant levels of 5-lox activity rather than its expression and documented the apparent biosynthesis of its metabolite, 5-hete, which eventually contributes to inflammatory activities through ltb4 formation . Red ginseng treatment was efficient in decreasing h. pylori - induced 5-lox activities and attenuating 5-lox expression through inactivating c - jun pathway . Several studies showed nsaid played significant chemopreventive effects against h. pylori - induced gastric carcinogenesis through attenuating cox activities, but our study opens the possibility that lox activity as much as cox activity might influence the inflammatory or carcinogenic activities related to its disasters associated with h. pylori infection, suggesting that modulation of activities of both cox and lox will be quite beneficial in avoiding the deleterious outcomes of chronic h. pylori infection . As korea red ginseng has been reported to show a significant protective effect against h. pylori - induced cytotoxicity and dna damage in vitro, we designed a clinical study to assess the efficacy of red ginseng treatment in patients with h. pylori - associated chronic gastritis . A total of 84 patients with h. pylori - associated chronic gastritis were recruited and randomly divided into two groups . During the trial, 34 patients out of 42 patients in the placebo control group and 36 patients out of 42 patients in the red ginseng group completed the protocol . The patients received a one week triple therapy for the eradication of h. pylori and then received either placebo capsules that were composed of flour for the placebo group or red ginseng capsules for the treatment group, which were administered for 10 weeks . An endoscopic examination of gastritis with a visual analogue scale, a test for detection of h. pylori, immunohistochemistry of 8-ohdg, the 8-ohdg immunohistochemical staining for assessing oxidative dna damage and tunel staining for apoptosis were performed, respectively . As results, h. pylori eradication rates were augmented in the red ginseng group as compared to the placebo group (91.7% in the red ginseng group and 79.4% in the placebo group), but there was no statistical significance (p = 0.147). For an analysis of gastritis based on updated sydney system (fig . 3a), the red ginseng group showed significant improvement in neutrophil infiltrations (p = 0.008), chronic atrophic gastritis (fig . 3b, p<0.05), and even intestinal metaplasia (p = 0.005). An attenuation of 8-ohdg immunohistostaining after treatment was seen more frequently in the red ginseng group (p<0.001) (fig . 4). An attenuation of dna damage and apoptosis was seen for the red ginseng group as compared to the placebo group (p<0.001). Therefore, supplementary administration of red ginseng augmented eradication rates of h. pylori, attenuated gastric inflammation, and reduced oxidative dna damage and apoptosis, suggesting the clinical usefulness of red ginseng . Recently we could add more evidence that korea red ginseng was very efficient in eliminating troublesome halitosis . Since halitosis is closely associated with erosive changes in the stomach, the halitosis improving actions of korea red ginseng were dependent on either direct suppressive action of korea red ginseng on enzyme responsible for generating volatile sulfure compounds, that is, cystathionine -lyase and cystathionine -synthase, or cyto - restorative actions . Ginseng has been reported to reduce the human cancer risk of lip, oral cavity, pharynx, larynx, esophagus, lung, liver, pancreas, ovary, colon, rectum, and stomach documented by clinical and epidemiological studies, rendering panax ginseng as non - organ specific cancer preventive . However, still many people have questioned the use of traditional medicinal herbs and this has been a prime issue in complementary and alternative medicine . Even though we issued several in vitro and in vivo results of feasibility of korea red ginseng administration for mitigating h. pylori - associated atrophic changes and carcinogenesis, science and evidence based medicine must develop through verification of observations, necessitating that the need for clinical studies should be strongly recommended, especially more to put korea red ginseng as h. pylori - associated gastric cancer preventive based on our publications [4648, 51]. Scientific clinical trials of korea red ginseng are now warranted to reach to the conclusion that rejuvenation of atrophic gastritis could be coming true with supplementation of korea red ginseng after the eradication of h. pylori infection . Overall, ginseng is a good example of a natural herb that has ubiquitous properties that are conductive to stopping inflammatory carcinogenesis.
Reversible cerebral vasoconstriction syndrome (rcvs) is characterized by severe headaches, with or without other acute neurological symptoms, and by the diffuse segmental constriction of the cerebral arteries that resolves spontaneously within three months (1). In addition to the simple vasoconstriction demonstrated by magnetic resonance angiography (mra), rcvs - associated abnormalities, including posterior reversible encephalopathy syndrome (pres) and/or ischaemic / haemorrhagic strokes (accompanied by rcvs), have also been demonstrated by magnetic resonance imaging (mri). However, cases of rcvs with transient splenial lesions (tsl) detected by mri are rare; only two such cases have been reported previously (2,3). A 28-year - old woman with no history of migraine or any other disease developed a thunderclap headache immediately after an uncomplicated pregnancy and spontaneous vaginal delivery at 40 weeks of gestational age . The patient had a normal blood pressure during pregnancy, indicating that she did not have preeclampsia . She had a history of normal uncomplicated vaginal delivery at 21 and 25 years of age; this was her third pregnancy . She had not undergone epidural anaesthesia or been given any vasoactive drugs . Although the patient's headache improved the day after disease onset, it re - emerged 17 days after onset, and again improved 18 days after onset . Brain diffusion - weighted imaging showed an intensified signal in the splenium of the corpus callosum (figure a). The apparent diffusion coefficient (adc) indicated a low signal in the same area (figure b). Mra showed segmental constriction of the left middle cerebral artery and bilateral posterior cerebral arteries (figure d). A cerebrospinal fluid analysis revealed an opening pressure of 100 mm h2o, no red blood cells, 1 white blood cell / mm, a glucose level of 62 mg / dl and a protein level of 17 mg / dl . Her symptoms did not worsen or relapse, despite not using either calcium - channel blockers or any other agents . Mri and mra 20 days after the first examination were normal with a complete resolution of the tsl and all other vascular abnormalities (figure c and f). (a) brain axial diffusion - weighted images (dwi) at first examination (19 days after onset) showing signal hyperintensity in the splenium of the corpus callosum . (b) the apparent diffusion coefficient indicating a low signal in the same area . (c) dwi at 9 days after the first examination showing splenial lesion recovery . (d - f) serial magnetic resonance angiography showing focal constriction of the bilateral posterior cerebral arteries and the middle cerebral arteries (arrow head) and serial recovery (d, at first examination; e, 9 days after the first examination; and f, 20 days after the first examination). Based on the typical and main symptoms of rcvs, our patient experienced a thunderclap headache immediately after delivery . The mra abnormalities of rcvs and mri abnormalities of tsl with serial recovery in our patient indicated an association between delivery and the development of both rcvs and tsl . Rcvs - associated mri abnormalities include three types of stroke (cerebral infarction, convexity subarachnoid haemorrhage, and intracerebral haemorrhage) and reversible brain edema such as pres (1). In our case, the decreased adc indicated cytotoxic oedema clearly distinct from pres, as the adc levels do not decrease in pres . Only two cases of rcvs with tsls have been reported to date, and both cases, like the present case, occurred postpartum (table) (2,3). Although the timing and clinical features, such as signs, symptoms and serial mri findings varied in these three cases, they indicated that delivery may be strongly associated with the development of both tsls and rcvs . The important difference between the previously reported two cases and our case was the presence of neurological symptoms / sings . Our patient did not develop either seizures or neurological deficits and also had a normal blood pressure . Postpartum / peripartum patients with headaches, seizure, dysarthria, or eclampsia are immediately suspected of having neuroimaging abnormalities and require an urgent diagnostic work - up to identify the cause . Meanwhile, rcvs cases presenting with headache only, like in our case, might be overlooked or underestimated . Postpartum tsls are also rare; however, a few cases of tsls (without rcvs) after pregnancy have been reported (5 - 8). Tsls are associated with a variety of disorders, including infection, antiepileptic drug withdrawal, high - altitude cerebral oedema, metabolic disturbance and others (9). Despite the large number of aetiologies, it remains uncertain as to why tsls selectively occur in the splenium; moreover, the exact pathogenesis of tsls remains elusive . The splenium differs from other parts of the corpus callosum with respect to its vascular supply; the rostrum, genu and trunk, which constitute the anterior part of the corpus callosum, are supplied by branches of the artery originating from the anterior circulation, whereas the splenium is supplied by both the anterior and posterior branches of the artery (10). However, because of the reversibility of the lesions and the absence of any other lesions in vascular distributions, it is unlikely that tsls are the result of ischaemia (11). It has been postulated that the splenium has a specific vulnerability to excitotoxic injury in metabolic diseases, making it selectively involved in differential pathological events (12). Another possible explanation for such tsl development is that it may be related to a relative lack of adrenergic tone, making it susceptible to hypoxic vasodilation and autoregulation failure with resultant hyperperfusion (9,13). On the other hand, transient failure of regulation of cerebral arterial tone with sympathetic hyperactivity seems to play a role in rcvs development (1). Although hormonal and metabolic changes during the peripartum period can contribute to the pathogenesis, of rcvs and tsl, a transient failure of regulation of arterial tone after delivery might be the common mechanism underlying both tsl and rcvs development . Previously reported and present case(s) of reversible cerebral vasoconstriction syndrome with transient splenial lesion . Gtc: generalized tonic - clonic seizure, sd: speech disturbance, pres: posterior reversible encephalopathy syndrome, sv: segmental vasoconstriction demonstrated by mra, tsl: transient splenial lesion, nd: not described, mri: magnetic resonance imaging . Patients with tsls are often asymptomatic, and these lesions are incidentally noticed during mris performed for other reasons . Moreover, rcvs presenting only with a headache is also common, and a diagnosis of rcvs depends on the mra findings . The actual incidence of rcvs with tsls or postpartum tsls is therefore unknown; rcvs with tsls may be overlooked and might therefore be a more common condition than is currently thought.
There is a consensus that artemisinin combination therapies (acts) are efficacious in the treatment of malaria (1). Kenya adopted artemisinin lumefantrine (al) as its first - line act malaria treatment in april 2004 . The same year, the government of kenya also began distributing free al branded coartem in the public formal sector (5). What is required is optimal malaria treatment as even the most efficacious antimalarial drug if not used correctly may fail to cure malaria . There are a number of factors that inhibit access and use of antimalarials in real life settings . Complicated drug schedules and limited understanding of how or why to adhere to recommended antimalarial drug is a major hindrance to malaria therapy (69). Adverse effects, poor instructions, poor provider - patient relationship, loss of drugs, forgetting to take a drug, patient s disagreement with the need for treatment, perceived ineffectiveness of the medicine, the inability to pay for treatment (10, 11) and perceived feeling of recovery from an illness (12) are just but a few documented reasons for non - adherence . The need of a caretaker most often a parent compounds the issue of adherence in children (1). With a view of scaling down malaria, the kenyan government permitted the sale of acts over the counter in 2011 (5). However, there are limited adherence to act s studies in particular coartem which is widely used in the public formal sector . This study therefore, sought to determine the level of adherence to coartem in the routine treatment of uncomplicated malaria among children under the age of five years in nyando district of kenya . In this cross sectional survey, nyando district hospital, in nyanza province was chosen for this evaluation because of its central location and high usage . Malaria is a major health problem accounting for approximately two - thirds of all out - patient department (opd) consultations in the health facility . Plasmodium falciparum is present in more than 95% of all malaria infections (5). Each day over 100 new patients flock the hospital, yet just one doctor, 15 nurses and four clinical officers are on hand to cover all shifts . The lean workforce means that patients often have to wait for long periods to get attention . Old, faulty, and limited technology and poor facilities compound the shortage of the health care staff . Children below the age of five years with microscopically confirmed uncomplicated p. falciparum malaria and prescribed coartem during the normal opd hours on 27 of april to 15 of may 2009 comprised the study subjects . Assuming an adherence of 80%, a precision of 10%, a type 1 error of 5% and a 20% loss to follow - up, 73 patients were randomly recruited into the study . Patients included in the study were residence of nyando district aged 1259 months, no household member participating in the study, reported fever within the last 48 hours or axillary temperature 37.5c on presentation at the clinic and weighed 10 kg . Patients who had signs of complicated malaria manifested by microscopically confirmed high parasitemia levels (> 100,000 parasites/l), inability to sit or stand, altered consciousness lethargy or coma, breathing difficulties, severe anemia (hemoglobin concentration 5 g / dl), recent history of convulsions, inability to drink or breastfeed or persistent vomiting were excluded from the study . The blood was stained with giemsa and read for species and parasitemia by an experienced technician . Parasitemia were calculated against 200500 leukocytes according to the formula: parasitemia (/l) = number of parasites x 8,000/number of leukocytes (13). Patients were classified as adherent, probably non - adherent and definitely non - adherent based on the findings from the blister pack check and questionnaire . Adherent if their caretakers followed exactly the medication instructions when administering the standard 3-day course of coartem (10.0 14.9 kg: 1 tablet per dose; 15.0 24.9 kg: 2 tablets; novartis pharma ag, basel, switzerland). Patients who had tablets remaining in the blister pack were classified as definitely non - adherent irrespective of whether they gave correct or incorrect account on how they administered the medicine . Those who had blister pack missing or empty and the caretaker did not report administering all the doses at the correct time and amount were considered as probably non - adherent or as probably adherent when the caretaker reported administering all doses at the correct time and amount . Adherence was measured on day 3 through a semi - structured questionnaire, a pill count and a blister pack recovery where possible . Secondary outcomes were the number of doses taken correctly and number of tablets remaining in the blister pack (where applicable). All the variables that were significant at 95% ci (p 0.05) were entered into logistic regression model to test for linear relationship . The study was given ethical approval by institutional research and ethics committee (irec) of moi university and moi teaching and referral hospital (mtrh). In this cross sectional survey, nyando district hospital, in nyanza province was chosen for this evaluation because of its central location and high usage . Malaria is a major health problem accounting for approximately two - thirds of all out - patient department (opd) consultations in the health facility . Plasmodium falciparum is present in more than 95% of all malaria infections (5). Each day over 100 new patients flock the hospital, yet just one doctor, 15 nurses and four clinical officers are on hand to cover all shifts . The lean workforce means that patients often have to wait for long periods to get attention . Old, faulty, and limited technology and poor facilities compound the shortage of the health care staff . Children below the age of five years with microscopically confirmed uncomplicated p. falciparum malaria and prescribed coartem during the normal opd hours on 27 of april to 15 of may 2009 comprised the study subjects . Assuming an adherence of 80%, a precision of 10%, a type 1 error of 5% and a 20% loss to follow - up, 73 patients were randomly recruited into the study . Patients included in the study were residence of nyando district aged 1259 months, no household member participating in the study, reported fever within the last 48 hours or axillary temperature 37.5c on presentation at the clinic and weighed 10 kg . Patients who had signs of complicated malaria manifested by microscopically confirmed high parasitemia levels (> 100,000 parasites/l), inability to sit or stand, altered consciousness lethargy or coma, breathing difficulties, severe anemia (hemoglobin concentration 5 g / dl), recent history of convulsions, inability to drink or breastfeed or persistent vomiting were excluded from the study . The blood was stained with giemsa and read for species and parasitemia by an experienced technician . Parasitemia were calculated against 200500 leukocytes according to the formula: parasitemia (/l) = number of parasites x 8,000/number of leukocytes (13). Patients were classified as adherent, probably non - adherent and definitely non - adherent based on the findings from the blister pack check and questionnaire . Adherent if their caretakers followed exactly the medication instructions when administering the standard 3-day course of coartem (10.0 14.9 kg: 1 tablet per dose; 15.0 24.9 kg: 2 tablets; novartis pharma ag, basel, switzerland). Patients who had tablets remaining in the blister pack were classified as definitely non - adherent irrespective of whether they gave correct or incorrect account on how they administered the medicine . Those who had blister pack missing or empty and the caretaker did not report administering all the doses at the correct time and amount were considered as probably non - adherent or as probably adherent when the caretaker reported administering all doses at the correct time and amount . Adherence was measured on day 3 through a semi - structured questionnaire, a pill count and a blister pack recovery where possible . Secondary outcomes were the number of doses taken correctly and number of tablets remaining in the blister pack (where applicable). All the variables that were significant at 95% ci (p 0.05) were entered into logistic regression model to test for linear relationship . The study was given ethical approval by institutional research and ethics committee (irec) of moi university and moi teaching and referral hospital (mtrh). A total of 73 (15.6%) patients out of the 469 children screened were recruited into the study . The vast majority of these patients had pure p. falciparum (90.3%), fever on presentation at the health facility (85.5%), low parasitemia (<500 parasites/l) levels (69.4%) and body weights of 10.0 14.9 kg (75.8%). (table 1) of the 73 patients recruited, eight (11.0%) could not be traced for home visit leaving data for 65 home visit interviews . Among the 65 patients who completed the follow - up visit, three had incomplete data for analysis of adherence due to failure of the caretakers to give analyzable information on the timing of the doses or the number of tablets taken per dose and were thus excluded from the analysis . Of the 62 children, nine (14.5%) had tablets remaining in the blister pack, and were thus classified as definitely non - adherent; six (9.7%) reported taking the regimen in a non - adherent manner and were thus classified as probably non - adherent; 47 (75.8%) reported taking the regimen in an adherent manner and were therefore classified as probably adherent (table 2). The caretakers of patients who had tablets remaining in the blister packs were asked to specify why they did not administer the full treatment course . These reasons were significantly varied depending on the number of the missed dose(s) (p = 0.029). One caretaker (11.1%) did not give one dose because the child did not like the medicine, five (55.6%) discontinued the treatment prematurely by a dose because the patients condition had improved, two (22.2%) gave the patient few tablets with two doses and one (11.1%) could not give any reason for not administering a dose (table 3). None of these reasons however were influenced by the caretakers level of education (p = 0.825), caretaker relation to the child (p = 0.145), number of children cared for by the caretaker (p = 0.549), household size (p = 0.825) or the sex of the patient (p = 0.687). Apparently, only one caretaker who did not give a reason for not administering a full dose admitted to have shared the medicine meant for the study participant with a sick member of the family (p = 0.021). A total of 73 (15.6%) patients out of the 469 children screened were recruited into the study . The vast majority of these patients had pure p. falciparum (90.3%), fever on presentation at the health facility (85.5%), low parasitemia (<500 parasites/l) levels (69.4%) and body weights of 10.0 14.9 kg (75.8%). (table 1) of the 73 patients recruited, eight (11.0%) could not be traced for home visit leaving data for 65 home visit interviews . Among the 65 patients who completed the follow - up visit, three had incomplete data for analysis of adherence due to failure of the caretakers to give analyzable information on the timing of the doses or the number of tablets taken per dose and were thus excluded from the analysis . Of the 62 children, nine (14.5%) had tablets remaining in the blister pack, and were thus classified as definitely non - adherent; six (9.7%) reported taking the regimen in a non - adherent manner and were thus classified as probably non - adherent; 47 (75.8%) reported taking the regimen in an adherent manner and were therefore classified as probably adherent (table 2). The caretakers of patients who had tablets remaining in the blister packs were asked to specify why they did not administer the full treatment course . These reasons were significantly varied depending on the number of the missed dose(s) (p = 0.029). One caretaker (11.1%) did not give one dose because the child did not like the medicine, five (55.6%) discontinued the treatment prematurely by a dose because the patients condition had improved, two (22.2%) gave the patient few tablets with two doses and one (11.1%) could not give any reason for not administering a dose (table 3). None of these reasons however were influenced by the caretakers level of education (p = 0.825), caretaker relation to the child (p = 0.145), number of children cared for by the caretaker (p = 0.549), household size (p = 0.825) or the sex of the patient (p = 0.687). Apparently, only one caretaker who did not give a reason for not administering a full dose admitted to have shared the medicine meant for the study participant with a sick member of the family (p = 0.021). Adherence to antimalarial medications is an important component of malaria control although measuring it is difficult as the available measurement techniques have drawbacks (14). In this study, adherence was assessed using self - report measurements that had been standardized, validated, and well accepted in the adherence literature (15). The study reported encouraging high levels of adherence than in zambia (16); southern sudan (17) and uganda (13) but lower levels than that in uganda (12). In zambia and southern sudan, the studies were conducted in complex settings in refugee and internally displaced populations, whereas, in a stable semi - urban area with high educational level in uganda (13). These were not easily comparable to this study site in an unstable typical african rural setting with low level of education . Many patients were reported to be definitely non - adherent, an indication that the prevalent form of non - adherence was not the way in which coartem was administered with regard to time and number of tablets but rather the failure to complete tablets (18, 19). This calls for improved caretakers counseling on the importance of giving the patient full dose when administering the drug . Admittedly, self - report and tablet counts alone are imperfect measures of adherence . These methods are vulnerable to overestimates of adherence and under - estimates of non - adherence . Interviews had been shown to identify 80% of the true non - adherence as assed by pill count (14). However, interviews are not equally sensitive for all subgroups of patients (20). Therefore, a pharmacological assessment of lumefantrine - plasma concentrations would have been more robust in this study . Nevertheless, there is a significant difference in the concentration of lumefantrine levels between the adherent and non - adherent groups (13). However, there is a need for study to be conducted outside a health facility to ascertain whether the same adherence levels could be obtained . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors.
This randomized controlled trial was conducted in five primary care clinics affiliated with the edmonton south side primary care network in edmonton, canada . These primary care teams were akin to the patient - centered medical home (15) and consisted of physicians and nurses who had support from dietitians, physiotherapists, and social workers as needed . Patients were eligible if they had type 2 diabetes, were regularly seen by the primary care team, and did not qualify for urgent specialist referral and assessment (according to protocol, a fasting blood glucose 17 mmol / l, blood pressure 220/120 mmhg, or triglycerides 15 mmol / l). We excluded patients who were followed in specialty clinics for diabetes, hypertension, or dyslipidemia; who were cognitively impaired; who were not responsible for their own medication administration; or who were unable to communicate in english . Blood pressure screening was not conducted during patient recruitment to minimize contamination of controls . Because type 2 diabetic patients have higher blood pressure levels than the general population (1) eligible patients were identified from the clinic roster, and a clinic staff member made initial contact to tell patients about the study . Patients were told that the study was designed to help improve medication therapy for heart disease risk in patients with type 2 diabetes . The specific focus of the study, hypertension, was listed among other risk factors for heart disease . The university of alberta health research ethics board approved the study protocol, and all participants gave written informed consent . Pharmacists were given access to the patient's clinical chart after consent to participate in the study was obtained . A central randomization service (www.epicore.ualberta.ca) provided computer - generated random sequences stratified by the primary care clinic for treatment allocation . Pharmacists, analysts, and investigators were unaware of the block size and allocation sequence to preserve allocation concealment . Control patients received usual care by the primary care team without contributions from study pharmacists, except for standardized blood pressure measurements at the end of the follow - up period (see below). Two pharmacists providing the intervention program held a bachelor's degree in pharmacy, were certified diabetes educators, and had practiced in community pharmacies for over 5 years . Both pharmacists completed structured online training courses for hypertension and diabetes management (www.pharmalearn.com) and reviewed the canadian hypertension education program and canadian diabetes association guideline recommendations prior to starting the study (16,17). The intervention program began with an in - person visit with a study pharmacist to identify all prescription, nonprescription, complementary, and alternative medications . Pharmacists also measured the patient's height, weight, heart rate, and blood pressure . Blood pressure was measured according to the canadian hypertension education program recommendations using the bptru bpm-100 (vsm med tech, coquitlam, bc) automated machine set to report the average of five measurements at 1-min intervals (16). Pharmacists then formulated guideline - concordant recommendations to optimize medication management of blood pressure and other cardiovascular risk factors . These recommendations were discussed with the primary care physician who was responsible for authorizing medication changes . Interim contact with intervention patients was made at the discretion of the pharmacist, physician, or patient and could be conducted via telephone or in person . Interim contacts were used to determine whether medication changes were implemented and to address questions or concerns since the previous encounter (e.g., side effects, adverse events, or adherence issues). Pharmacists recorded the date, duration, and nature of each contact with a study patient . After 1 year, all patients were seen in the primary care clinic to review medications, measure blood pressure with the automated machine, and obtain a fasting blood sample to measure blood glucose, a1c, and cholesterol profile . Patients also reported the number of encounters with specialists, other health care professionals, regional health care resources, emergency - room visits, and hospitalizations during the previous year . The primary outcome was achievement of a clinically important reduction in blood pressure, defined as a 10% decrease in systolic blood pressure at 1 year (18). Secondary outcomes included the absolute change in systolic blood pressure from baseline to 1 year, achievement of recommended blood pressure targets (<130/80 mmhg) (8), and antihypertensive medication changes . We also measured the change in predicted 10-year risk of cardiovascular disease using the uk prospective diabetes study (ukpds) risk engine (19). Baseline and follow - up values for a1c, systolic blood pressure, total cholesterol, and hdl cholesterol were used to calculate the change in the ukpds risk engine score . Our sample size was based on observations from a previous study (20) examining the effect of a diabetes intervention program aimed at physicians . In that study, 40% of intervention patients and 25% of control patients achieved a 10% decrease in blood pressure at 6 months . Although the current study follow - up was twice as long and the intervention was directed at patients, we estimated that the event rates would be similar . With a two - sided of 0.05 and 80% power, we estimated the total sample size would be 300 patients . We used statistics to test for between - group differences in the primary outcome . The association between treatment group and achievement of the primary outcome was also examined using a logistic regression model to calculate an odds ratio (or) and 95% ci . Based on the results of related trials in our region (13,20,21), we assumed that patient - related outcomes were statistically independent of one another, with intracluster correlation coefficients <0.01 . All patients were evaluated in the groups to which they were randomly allocated according to the intention - to - treat principle . Missing data were replaced by carrying the last observation forward . To test the robustness of our observations, we restricted our analyses to patients who completed the full study protocol and patients with inadequately controlled hypertension at baseline . First, we used indicator variables to directly adjust for all 18 family physicians involved in the study in a multivariate logistic regression model . Second, we used generalized estimating equation methodology to account for the potential correlations of outcomes among patients treated by the same physician . None of the sensitivity analyses changed the direction, magnitude, or statistical significance of our findings; therefore, we report only our prespecified analyses . A p value of <0.05 was considered statistically significant, and pasw statistics version 18.0 (spss, chicago, il) was used for all analyses . A central randomization service (www.epicore.ualberta.ca) provided computer - generated random sequences stratified by the primary care clinic for treatment allocation . Pharmacists, analysts, and investigators were unaware of the block size and allocation sequence to preserve allocation concealment . Control patients received usual care by the primary care team without contributions from study pharmacists, except for standardized blood pressure measurements at the end of the follow - up period (see below). Two pharmacists providing the intervention program held a bachelor's degree in pharmacy, were certified diabetes educators, and had practiced in community pharmacies for over 5 years . Both pharmacists completed structured online training courses for hypertension and diabetes management (www.pharmalearn.com) and reviewed the canadian hypertension education program and canadian diabetes association guideline recommendations prior to starting the study (16,17). The intervention program began with an in - person visit with a study pharmacist to identify all prescription, nonprescription, complementary, and alternative medications . Pharmacists also measured the patient's height, weight, heart rate, and blood pressure . Blood pressure was measured according to the canadian hypertension education program recommendations using the bptru bpm-100 (vsm med tech, coquitlam, bc) automated machine set to report the average of five measurements at 1-min intervals (16). Pharmacists then formulated guideline - concordant recommendations to optimize medication management of blood pressure and other cardiovascular risk factors . These recommendations were discussed with the primary care physician who was responsible for authorizing medication changes . Baseline characteristics were obtained from the clinic chart . Interim contact with intervention patients was made at the discretion of the pharmacist, physician, or patient and could be conducted via telephone or in person . Interim contacts were used to determine whether medication changes were implemented and to address questions or concerns since the previous encounter (e.g., side effects, adverse events, or adherence issues). Pharmacists recorded the date, duration, and nature of each contact with a study patient . After 1 year, all patients were seen in the primary care clinic to review medications, measure blood pressure with the automated machine, and obtain a fasting blood sample to measure blood glucose, a1c, and cholesterol profile . Patients also reported the number of encounters with specialists, other health care professionals, regional health care resources, emergency - room visits, and hospitalizations during the previous year . The primary outcome was achievement of a clinically important reduction in blood pressure, defined as a 10% decrease in systolic blood pressure at 1 year (18). Secondary outcomes included the absolute change in systolic blood pressure from baseline to 1 year, achievement of recommended blood pressure targets (<130/80 mmhg) (8), and antihypertensive medication changes . We also measured the change in predicted 10-year risk of cardiovascular disease using the uk prospective diabetes study (ukpds) risk engine (19). Baseline and follow - up values for a1c, systolic blood pressure, total cholesterol, and hdl cholesterol were used to calculate the change in the ukpds risk engine score . Our sample size was based on observations from a previous study (20) examining the effect of a diabetes intervention program aimed at physicians . In that study, 40% of intervention patients and 25% of control patients achieved a 10% decrease in blood pressure at 6 months . Although the current study follow - up was twice as long and the intervention was directed at patients, we estimated that the event rates would be similar . With a two - sided of 0.05 and 80% power, we estimated the total sample size would be 300 patients . We used statistics to test for between - group differences in the primary outcome . The association between treatment group and achievement of the primary outcome was also examined using a logistic regression model to calculate an odds ratio (or) and 95% ci . Based on the results of related trials in our region (13,20,21), we assumed that patient - related outcomes were statistically independent of one another, with intracluster correlation coefficients <0.01 . All patients were evaluated in the groups to which they were randomly allocated according to the intention - to - treat principle . Missing data were replaced by carrying the last observation forward . To test the robustness of our observations, we restricted our analyses to patients who completed the full study protocol and patients with inadequately controlled hypertension at baseline . First, we used indicator variables to directly adjust for all 18 family physicians involved in the study in a multivariate logistic regression model . Second, we used generalized estimating equation methodology to account for the potential correlations of outcomes among patients treated by the same physician . None of the sensitivity analyses changed the direction, magnitude, or statistical significance of our findings; therefore, we report only our prespecified analyses . A p value of <0.05 was considered statistically significant, and pasw statistics version 18.0 (spss, chicago, il) was used for all analyses . We enrolled 260 patients between 28 february 2006 and 6 december 2007 and randomly allocated 131 to the intervention and 129 to the control group (fig . The main reasons for exclusion were that the pharmacist could not contact eligible patients (700 of 1,183 [59%]) and patient refusal (211 of 1,183 [18%]). There were 21 intervention patients (14 withdrew, 6 were lost to follow - up, and 1 died) and 16 control patients (10 withdrew and 6 were lost to follow - up) who did not complete the study (p> 0.05 for all comparisons). There were no differences in age, sex, diabetes duration, or baseline blood pressure between the patients who did or did not complete the study . Icd-9, international classification of diseases, 9th revision; bp, blood pressure; itt, intention to treat . There were 149 (57.3%) women, mean (sd) age was 59.1 11.6 years, bmi was 32.5 6.5 kg / m, diabetes duration was 5.5 6.5 years, and a1c was 7.4 1.4% . The mean blood pressure at baseline was 129.4 15.3/74.1 10.4 mmhg, with 82 of 131 (63%) intervention patients and 71 of 129 (55%) control patients having inadequately controlled hypertension (130/80 mmhg; p = 0.22 for difference). Of 107 patients with a blood pressure <130/80 mmhg, 76 were taking one or more antihypertensive medications at baseline . Baseline characteristics data are means sd for continuous variables and n (%) for categorical variables . Over 1 year, there was a statistically significant reduction in systolic blood pressure for intervention patients (mean decrease 7.4 mmhg [95% ci 4.610.2]; p <0.001) but not for control patients (2.5 mmhg [0.1 to 5.2]; p = 0.06) (supplementary fig . The between - group difference in systolic blood pressure change at 1 year was 4.9 mmhg (95% ci 1.08.7; p = 0.01) (supplementary table 2) in favor of the intervention . The primary outcome was achieved by 48 of 131 (37%) intervention patients and 30 of 129 (23%) control patients (or 1.91 [95% ci 1.113.28]; p = 0.02) (fig . The absolute difference of 14% translates to a number needed to treat (nnt) of seven patients followed for 1 year by a pharmacist to achieve one additional patient with better blood pressure control compared with usual care . Proportion of patients achieving a 10% decrease in systolic blood pressure at 1 year (primary outcome). Limiting our analyses to 223 patients who completed the mean decrease in systolic blood pressure was 7.7 mmhg (95% ci 4.510.9; p <0.001) for intervention patients and 2.8 mmhg (0.2 to 5.8; p = 0.07) for control patients (p = 0.03 for between - group differences). More intervention patients achieved the primary outcome (41 of 110 [37%]) compared with control patients (30 of 113 [27%]); however, this difference was not statistically significant (p = 0.09). We observed a larger treatment effect when blood pressure changes were examined in 153 patients with inadequately controlled hypertension at baseline . Mean blood pressure at baseline was 138.7 11.4/78.8 9.4 mmhg for intervention patients and 137.9 14.1/78.1 11.4 mmhg for control patients (p> 0.05). All other baseline characteristics for these 153 patients were well balanced between the two groups (p> 0.05 for all comparisons). Systolic blood pressure decreased a mean of 13.9 mmhg (95% ci 10.617.1; p <0.001) for intervention patients and 6.7 mmhg (3.210.1; p <0.001) for control patients (p = 0.002 for between - group differences) (supplementary fig . The primary outcome was achieved by 41 of 82 (50%) intervention patients and 20 of 71 (28%) control patients (or 2.55 [95% ci 1.305.01]; p = 0.007) (fig . Moreover, among these 153 patients, 44 of 82 (54%) intervention patients and 21 of 71 (30%) control patients achieved recommended blood pressure targets at 1 year (2.76 [1.415.39]; p = 0.003; nnt = 4). Fifty - five (42%) intervention patients had 85 changes and 32 (25%) control patients had 44 changes to their antihypertensive medication regimen (or 2.19 [95% ci 1.303.71]; p = 0.003) (supplementary fig . 2). Among those with uncontrolled hypertension at baseline, only 61 of 153 (40%) had changes . The most common antihypertensive medications added to a patient's regimen were ramipril (10 patients), hydrochlorothiazide (9 patients), and irbesartan (8 patients). Although changes in glycemic control and lipid parameters all favored the intervention, other than blood pressure control, none achieved statistical significance (supplementary table 2). Using the ukpds risk engine (19), there was a statistically significant reduction in predicted 10-year risk of cardiovascular events for intervention patients (mean decrease 2.7% [95% ci 1.53.9]; p <0.001) but not for control patients (1.2% [0.1 to 2.4]; p = 0.06). The between - group difference was 1.5% (95% ci 0.2 to 3.3; p = 0.005) in favor of the intervention (supplementary table 2). The total number of health care related contacts was 1,439 for intervention patients and 420 for control patients (p <0.01; supplementary table 3). However, 1,442 (77.6%) of these contacts were either protocol driven (baseline and 1-year follow - up) visits or interim contacts between intervention patients and study pharmacists . There were no differences in emergency - room visits (11 [8.4%] vs. 11 [8.5%]), hospitalizations (4 [3.1%] vs. five [3.9%]), or all - cause mortality (1 [0.8%] vs. 0 [0%]) between groups during the study . There were 149 (57.3%) women, mean (sd) age was 59.1 11.6 years, bmi was 32.5 6.5 kg / m, diabetes duration was 5.5 6.5 years, and a1c was 7.4 1.4% . The mean blood pressure at baseline was 129.4 15.3/74.1 10.4 mmhg, with 82 of 131 (63%) intervention patients and 71 of 129 (55%) control patients having inadequately controlled hypertension (130/80 mmhg; p = 0.22 for difference). Of 107 patients with a blood pressure <130/80 mmhg, 76 were taking one or more antihypertensive medications at baseline . Baseline characteristics data are means sd for continuous variables and n (%) for categorical variables . Over 1 year, there was a statistically significant reduction in systolic blood pressure for intervention patients (mean decrease 7.4 mmhg [95% ci 4.610.2]; p <0.001) but not for control patients (2.5 mmhg [0.1 to 5.2]; p = 0.06) (supplementary fig . The between - group difference in systolic blood pressure change at 1 year was 4.9 mmhg (95% ci 1.08.7; p = 0.01) (supplementary table 2) in favor of the intervention . The primary outcome was achieved by 48 of 131 (37%) intervention patients and 30 of 129 (23%) control patients (or 1.91 [95% ci 1.113.28]; p = 0.02) (fig . The absolute difference of 14% translates to a number needed to treat (nnt) of seven patients followed for 1 year by a pharmacist to achieve one additional patient with better blood pressure control compared with usual care . Proportion of patients achieving a 10% decrease in systolic blood pressure at 1 year (primary outcome). Limiting our analyses to 223 patients who completed the mean decrease in systolic blood pressure was 7.7 mmhg (95% ci 4.510.9; p <0.001) for intervention patients and 2.8 mmhg (0.2 to 5.8; p = 0.07) for control patients (p = 0.03 for between - group differences). More intervention patients achieved the primary outcome (41 of 110 [37%]) compared with control patients (30 of 113 [27%]); however, this difference was not statistically significant (p = 0.09). We observed a larger treatment effect when blood pressure changes were examined in 153 patients with inadequately controlled hypertension at baseline . Mean blood pressure at baseline was 138.7 11.4/78.8 9.4 mmhg for intervention patients and 137.9 14.1/78.1 11.4 mmhg for control patients (p> 0.05). All other baseline characteristics for these 153 patients were well balanced between the two groups (p> 0.05 for all comparisons). Systolic blood pressure decreased a mean of 13.9 mmhg (95% ci 10.617.1; p <0.001) for intervention patients and 6.7 mmhg (3.210.1; p <0.001) for control patients (p = 0.002 for between - group differences) (supplementary fig . The primary outcome was achieved by 41 of 82 (50%) intervention patients and 20 of 71 (28%) control patients (or 2.55 [95% ci 1.305.01]; p = 0.007) (fig . Moreover, among these 153 patients, 44 of 82 (54%) intervention patients and 21 of 71 (30%) control patients achieved recommended blood pressure targets at 1 year (2.76 [1.415.39]; p = 0.003; nnt = 4). Fifty - five (42%) intervention patients had 85 changes and 32 (25%) control patients had 44 changes to their antihypertensive medication regimen (or 2.19 [95% ci 1.303.71]; p = 0.003) (supplementary fig . 2). Among those with uncontrolled hypertension at baseline, only 61 of 153 (40%) had changes . The most common antihypertensive medications added to a patient's regimen were ramipril (10 patients), hydrochlorothiazide (9 patients), and irbesartan (8 patients). Although changes in glycemic control and lipid parameters all favored the intervention, other than blood pressure control, none achieved statistical significance (supplementary table 2). Using the ukpds risk engine (19), there was a statistically significant reduction in predicted 10-year risk of cardiovascular events for intervention patients (mean decrease 2.7% [95% ci 1.53.9]; p <0.001) but not for control patients (1.2% [0.1 to 2.4]; the between - group difference was 1.5% (95% ci 0.2 to 3.3; p = 0.005) in favor of the intervention (supplementary table 2). The total number of health care related contacts was 1,439 for intervention patients and 420 for control patients (p <0.01; supplementary table 3). However, 1,442 (77.6%) of these contacts were either protocol driven (baseline and 1-year follow - up) visits or interim contacts between intervention patients and study pharmacists . There were no differences in emergency - room visits (11 [8.4%] vs. 11 [8.5%]), hospitalizations (4 [3.1%] vs. five [3.9%]), or all - cause mortality (1 [0.8%] vs. 0 [0%]) between groups during the study . To our knowledge, this is the largest randomized controlled trial reporting the effect of adding pharmacists to primary care teams on blood pressure control in type 2 diabetic patients . On average, most patients were relatively well controlled in terms of a1c, blood pressure, and other cardiovascular risk factors; a reflection of both the quality of usual care in this primary care network and the fact that study participants tend to be healthier than nonparticipants . Nevertheless, adding pharmacists to primary care teams resulted in more intervention patients achieving a clinically important reduction in systolic blood pressure at 1 year compared with control patients . The absolute difference of 14% translates to an nnt of seven, and absolute benefits were even greater among those who had inadequately controlled hypertension (i.e., 22% improvement, nnt of five). Glycemic control, cholesterol management, and predicted 10-year risk of cardiovascular disease all showed a trend toward improvement with the pharmacist intervention . A 10% reduction in systolic blood pressure is considered clinically worthwhile, (18) and, if sustained for another 4 years, would be associated with an 25% reduction in cardiovascular events (22). Our observations are broadly consistent with three previous studies (1214) that examined pharmacist contributions to diabetic hypertension management . All three studies reported a significant difference in systolic blood pressure change between groups and favored pharmacist intervention (supplementary fig ., we might have seen a greater difference in systolic blood pressure change between groups if we had excluded people with normal blood pressure or well - controlled hypertension (n = 107 [41%]) or included those with elevated hypertension (220/120 mmhg). We believe that the success of this study can be attributed to three critical components of the pharmacist intervention . These evidence - based resources provided a validated, external benchmark to identify treatment options . Second, pharmacists discussed their recommendations directly with primary care physicians and other health care professionals, which is considered an essential component of successful management programs (23). Discussions facilitated a richer exchange of patient - specific ideas compared with more impersonal e - mails or faxes that are commonly used by pharmacists in the community . Third, the frequency of follow - up contact was tailored to the patient's needs . We have found that the effects of an intervention decay over time without direct, continuous involvement and individualized support of clinicians (20,21,24). First, we examined relatively short - term changes in surrogate measures rather than harder longer - term clinical end points such as myocardial infarction, stroke, or death . This limitation may be ameliorated somewhat by the significant changes in the ukpds risk score observed with the intervention . Second, there was the possibility of contamination or cointervention because both intervention and control patients were drawn from the same primary care team . Although we considered a cluster - randomized trial, we estimated that there were not enough primary care teams to carry out such a study . Contamination would only tend to bias to the null, and without the pharmacist's active intervention it is unlikely that the primary care team would pay greater attention than usual to blood pressure control in those with diabetes . Third, our 14% drop - out rate was high but consistent with other randomized controlled trials of pharmacist involvement in diabetic hypertension management (1114). Withdrawal rates were similar between groups, and there were no significantly different characteristics between patients who withdrew or completed the study . Fourth, our intervention was conducted in a jurisdiction with universal health care coverage and set within established primary care teams or patient - centered medical homes, so usual care was already much better than reported in the previous literature . Nonetheless, there was still room for improvement, and adding pharmacists to this team did improve care . It is likely, therefore, that the intervention would have an even greater effect when implemented in settings with a lower baseline quality of care . Last, the multifaceted nature of our intervention program makes it difficult to attribute the observed differences to a specific component . We believe the next stage in this line of research could be an active comparator study examining the effects of pharmacists, perhaps with prescriptive autonomy, relative to other case managers . Working in collaboration with the patient, primary care physician, and other health care professionals, pharmacists can have a significant, positive impact on blood pressure management in type 2 diabetes . We believe our results are applicable to a broad range of patients with type 2 diabetes managed in primary care settings and can be extended to nondiabetic patients with inadequately controlled hypertension . First, we examined relatively short - term changes in surrogate measures rather than harder longer - term clinical end points such as myocardial infarction, stroke, or death . This limitation may be ameliorated somewhat by the significant changes in the ukpds risk score observed with the intervention . Second, there was the possibility of contamination or cointervention because both intervention and control patients were drawn from the same primary care team . Although we considered a cluster - randomized trial, we estimated that there were not enough primary care teams to carry out such a study . Contamination would only tend to bias to the null, and without the pharmacist's active intervention it is unlikely that the primary care team would pay greater attention than usual to blood pressure control in those with diabetes . Third, our 14% drop - out rate was high but consistent with other randomized controlled trials of pharmacist involvement in diabetic hypertension management (1114). Withdrawal rates were similar between groups, and there were no significantly different characteristics between patients who withdrew or completed the study . Fourth, our intervention was conducted in a jurisdiction with universal health care coverage and set within established primary care teams or patient - centered medical homes, so usual care was already much better than reported in the previous literature . Nonetheless, there was still room for improvement, and adding pharmacists to this team did improve care . It is likely, therefore, that the intervention would have an even greater effect when implemented in settings with a lower baseline quality of care . Last, the multifaceted nature of our intervention program makes it difficult to attribute the observed differences to a specific component . We believe the next stage in this line of research could be an active comparator study examining the effects of pharmacists, perhaps with prescriptive autonomy, relative to other case managers . Our observations support the addition of pharmacists to primary care teams . Working in collaboration with the patient, primary care physician, and other health care professionals, pharmacists can have a significant, positive impact on blood pressure management in type 2 diabetes . We believe our results are applicable to a broad range of patients with type 2 diabetes managed in primary care settings and can be extended to nondiabetic patients with inadequately controlled hypertension.
Carbapenems are considered to be one of the few drugs that are useful for the treatment of infections caused by multiresistant gram - negative bacteria . The emergence of carbapenem - resistant enterobacteriaceae is a serious public health concern due to the large spectrum of resistant genes and the lack of therapeutic options (1, 2). Therefore, there is an ongoing effort in the development of earlier and more sensitive detection of carbapenemase producers . With regard to carbapenem resistance, it must be taken into account that carbapenemase - producing bacteria may sometimes exhibit only a slight increase of minimal inhibitory concentration (mic) values for carbapenems, which reflects the importance of molecular approaches to phenotypic tests (3). Commonly cause nosocomial pneumonias and infections in the bloodstream, urinary tract and intra - abdominal region (4, 5). Carbapenem resistance mechanisms in enterobacteriaceae include the following: (i) enzymatic inactivation by -lactamases; (ii) modification of outer - membrane proteins (porins) and penicillin - binding proteins; and (iii) efflux pumps (6). Carbapenem - hydrolyzing -lactamases belonging to molecular class a (e.g., kpc, ges, imi, sme), class b (e.g. Imp, vim, ndm, gim) and class d (e.g. Oxa-23 and oxa-48), are the main source of antibiotic resistance in enterobacteriaceae . Genes encoding these types of carbapenemases are extensively reported among e. coli and k. pneumoniae isolates from many european countries (1). The carbapenem - hydrolyzing class d -lactamase oxa-48 has been identified in a k. pneumoniae isolate from europe (7, 8), oxa-23 in proteus mirabilis from france (9), oxa-162 in k. pneumoniae isolates from hungary (10), oxa-181 in k. pneumoniae from romania (11) and citrobacter freundii from france (12), oxa-232 in k. pneumoniae from france (13) and oxa-244 and 245, both in k. pneumoniae from spain (14). Recently, blaoxa-51-like, blaoxa-58 and bladim-1 carbapenemase genes have been found in a large variety of enterobacterial species (16). Further, the presence of carbapenemases and extended - spectrum -lactamases (esbls) were also described: (i) blatem-1, blashv-11, blactx - m-15 and blaoxa-9 genes were present in the k. pneumoniae isolates harboring blaoxa-48; (ii) blatem-1 and blaoxa-1 genes were found in e. coli harboring blaoxa-162 and blaoxa-48; (iii) blashv-5 in c. freundii harboring blaoxa-162; and (iv) blatem-1 and blactx - m-15 in enterobacter cloacae carrying blaoxa-48 (17). Moreover, e. coli and k. pneumoniae clinical isolates producing ctx - m-2 and ctx - m-92 and a k. pneumoniae isolate with ctx - m-3 were detected, and each had the oxa-2 type beta - lactamase (18). In addition, kpc-2 and tem-1 enzymes were identified in k. pneumoniae isolates, and vim-1 and tem-1 in p. mirabilis, both of which were positive for blaoxa-10 (19). In this context, a recent study has demonstrated that oxa-2 and oxa-10 are in fact carbapenem - hydrolyzing class d beta - lactamases (chdls), also called oxacillinases (oxa) (20). Interestingly, development of carbapenem resistance in ctx - m-1-producing enterobacteriaceae has been reported (21, 22). Furthermore, it has been reported that cmy-2 -lactamase plays a role in carbapenem resistance (" trapping " of meropenem) (23). However, genes encoding oxa overcome this deficiency by possessing efficient promoters, leading to their overexpression and to increased carbapenem resistance (24). Thus, rapid and useful detection methods are important for the implementation of appropriate infection control measures to prevent the further spread of esbls and carbapenemases . The aim of the present study was to develop the primers for single and/or multiplex pcr amplification assays for identification of all known genes that confer carbapenem resistance in enterobacteriaceae and evaluate the amplification efficiency of the primers . Fifteen carbapenemase - producing strains were used as positive controls for optimizing the multiplex pcr assay; six reference strains and nine clinical isolates . The reference strains used were: (i) imp - type (nctc 13476), producing e. coli; (ii) kpc-3 positive k. pneumoniae (nctc 13438); (iii) ndm-1 positive k. pneumoniae (nctc 13443); (iv) vim-10 positive pseudomonas aeruginosa (nctc 13437); (v) oxa-23 positive acinetobacter baumannii (nctc 13301); and (vi) oxa-48 positive k. pneumoniae (nctc 13442). The clinical isolates were: (i v) k. pneumoniae that produce vim (v602b), kpc-2 (v117), kpc-3 (v514) and kpc (v601, v646); (vi viii) p. aeruginosa - producing imp (v7424) and vim (v109, v7393); and (ix) ndm-1 positive a. baumannii (v509). Faculty of medicine and university hospital in plzen, charles university in prague, plzen, czech republic . Two multiplex pcrs were designed in this study: a blakpc / blaoxa-48-like / blavim multiplex pcr and a blandm-1/blaimp variants / blaoxa-23-like mutliplex pcr . The sequences of genes that encode carbapenemases (kpc, vim, imp, sme, imi, ges, ndm, oxa) so far described (http://www.lahey.org/studies/; last accessed february 2015) were downloaded from the genbank databases and were aligned using geneious pro 4.8.5 (biomatters ltd, newark, nj, usa) to identify highly homologous regions suitable for designing primers . Two sets of primers were tested against reference standard strains, as well as clinical isolates, in a single pcr reaction and then in a multiplex format . These reference strains included nctc 13476, nctc 13438, nctc 13443, nctc 13437, nctc 13301 and nctc 13442 . Representative v602b, v117, v514, v601, v646, v7424, v109, v7393 and v509 were used as clinical isolates . The primer sequences, concentrations and calculated lengths of the corresponding amplicons are listed in table 1 . For degenerate primers: r = a or g; s = g or c; y = c or t. designed primers but not tested . Dna preparation was performed by suspending a colony of each bacterial strain in 100 l of distilled water, boiling at 98c for 10 minutes and centrifuging the cell extract for five minutes at 13,000 rpm . A multiplex pcr assay was designed to detect and differentiate one family of class a carbapenemase (kpc), three families of class b carbapenemases (imp, ndm and vim) and two families of class d carbapenemases (oxa) in two reactions . Both multiplex pcrs were performed with six pairs of specific primers (table 1), which were used to amplify fragments (different in size) of 340 bp (kpc), 597 bp (oxa-48), 247 bp (vim), 439 bp (ndm), 183 bp (imp) and 736 bp (oxa-23). The pcr reaction mixture contained: 0.5 l dna (50 ng) in 24.5 l complete reaction buffer with mgcl2 (containing 100 mmol / l tris - hcl [ph 8.8], 500 mmol / l kcl, 1% triton x-100, 15 mmol / l mgcl2) (top - bio, czech republic; 4 l), dntp (10 mm, 0.5 l), 15 pmol of each primer (0.5 l) and taq dna polymerase (top - bio, czech republic; 0.2 l). The pcr conditions were as follows: initial denaturation at 95c for five minutes, 35 cycles at 95c for one minute, at different annealing temperatures for one minute and 72c for one minute, followed by a single, final elongation step at 72c for five minutes . The annealing temperature was optimal at 56c for amplification of the blakpc, blaoxa and blavim genes and optimal at 52c for amplification of the blandm, blaimp and blaoxa genes (table 1). Amplicons were visualized after running at 100 v for 90 minutes on a 1.5% agarose gel containing ethidium bromide (figure 1). A 200 1500 bp dna ladder (top - bio, czech republic) was used as a size marker . Notes, lane 1, control blakpc gene; lane 2, control blaoxa-48 gene; lane 3, control blavim gene; lane 4, control blandm-1 gene; lane 5, control blaimp gene; lane 6, control blaoxa-23 gene . Fifteen carbapenemase - producing strains were used as positive controls for optimizing the multiplex pcr assay; six reference strains and nine clinical isolates . The reference strains used were: (i) imp - type (nctc 13476), producing e. coli; (ii) kpc-3 positive k. pneumoniae (nctc 13438); (iii) ndm-1 positive k. pneumoniae (nctc 13443); (iv) vim-10 positive pseudomonas aeruginosa (nctc 13437); (v) oxa-23 positive acinetobacter baumannii (nctc 13301); and (vi) oxa-48 positive k. pneumoniae (nctc 13442). The clinical isolates were: (i v) k. pneumoniae that produce vim (v602b), kpc-2 (v117), kpc-3 (v514) and kpc (v601, v646); (vi viii) p. aeruginosa - producing imp (v7424) and vim (v109, v7393); and (ix) ndm-1 positive a. baumannii (v509). Faculty of medicine and university hospital in plzen, charles university in prague, plzen, czech republic . Two multiplex pcrs were designed in this study: a blakpc / blaoxa-48-like / blavim multiplex pcr and a blandm-1/blaimp variants / blaoxa-23-like mutliplex pcr . The sequences of genes that encode carbapenemases (kpc, vim, imp, sme, imi, ges, ndm, oxa) so far described (http://www.lahey.org/studies/; last accessed february 2015) were downloaded from the genbank databases and were aligned using geneious pro 4.8.5 (biomatters ltd, newark, nj, usa) to identify highly homologous regions suitable for designing primers . Two sets of primers were tested against reference standard strains, as well as clinical isolates, in a single pcr reaction and then in a multiplex format . These reference strains included nctc 13476, nctc 13438, nctc 13443, nctc 13437, nctc 13301 and nctc 13442 . Representative v602b, v117, v514, v601, v646, v7424, v109, v7393 and v509 were used as clinical isolates . The primer sequences, concentrations and calculated lengths of the corresponding amplicons are listed in table 1 . For degenerate primers: r = a or g; s = g or c; y = c or t. designed primers but not tested . Dna preparation was performed by suspending a colony of each bacterial strain in 100 l of distilled water, boiling at 98c for 10 minutes and centrifuging the cell extract for five minutes at 13,000 rpm . A multiplex pcr assay was designed to detect and differentiate one family of class a carbapenemase (kpc), three families of class b carbapenemases (imp, ndm and vim) and two families of class d carbapenemases (oxa) in two reactions . Both multiplex pcrs were performed with six pairs of specific primers (table 1), which were used to amplify fragments (different in size) of 340 bp (kpc), 597 bp (oxa-48), 247 bp (vim), 439 bp (ndm), 183 bp (imp) and 736 bp (oxa-23). The pcr reaction mixture contained: 0.5 l dna (50 ng) in 24.5 l complete reaction buffer with mgcl2 (containing 100 mmol / l tris - hcl [ph 8.8], 500 mmol / l kcl, 1% triton x-100, 15 mmol / l mgcl2) (top - bio, czech republic; 4 l), dntp (10 mm, 0.5 l), 15 pmol of each primer (0.5 l) and taq dna polymerase (top - bio, czech republic; 0.2 l). The pcr conditions were as follows: initial denaturation at 95c for five minutes, 35 cycles at 95c for one minute, at different annealing temperatures for one minute and 72c for one minute, followed by a single, final elongation step at 72c for five minutes . The annealing temperature was optimal at 56c for amplification of the blakpc, blaoxa and blavim genes and optimal at 52c for amplification of the blandm, blaimp and blaoxa genes (table 1). Amplicons were visualized after running at 100 v for 90 minutes on a 1.5% agarose gel containing ethidium bromide (figure 1). A 200 1500 bp dna ladder (top - bio, czech republic) notes, lane 1, control blakpc gene; lane 2, control blaoxa-48 gene; lane 3, control blavim gene; lane 4, control blandm-1 gene; lane 5, control blaimp gene; lane 6, control blaoxa-23 gene . After optimizing the pcr (amplification) conditions, all positive controls (reference strains and clinical isolates) yielded amplicons of the predicted sizes and confirmed the specificity of the primers used (figure 1). The primer paris were tested in mixed (table 1, figure 1) and individual reactions (data not shown). The two multiplex pcr assays were 6 validated with a panel of fifteen characterized gram - negative bacterial strains . This collection includes 5 class a carbapenemases (5 kpc), 8 class b carbapenemases (2 imp, 2 ndm and 4 vim) and 2 class d carbapenemases (2 oxa). The resistance genes of the control isolates were correctly determined by each multiplex pcr (accuracy 100%; figure 1). In addition, eleven pairs of primers have been proposed but not tested experimentally in pcr and/or multiplex reactions (table 1). The emergence of carbapenem resistance in enterobacteriaceae has become a substantial clinical problem, since carbapenemase production cannot be easily inferred from the antimicrobial resistance profiles, thus, dissemination of these enzymes among nosocomial pathogens (e.g., enterobacteriaceae) is a threat to public health and must be closely monitored (phenotypic and genotypic tests). In addition, delay in detection of multidrug - resistant enterobacteriaceae results in longer hospitalizations and increased healthcare costs (26). Recently, multiplex pcr assays for the detection of blaimp, blavim, blaoxa, blandm and blakpc have been described (25 - 30). Unfortunately, the primers used had lower detection ranges (many homologs are missed). (29) targeted vim and imp enzymes, among other carbapenemases, and allowed the detection of 22 variants of imp (imp-1 to imp-22) and 13 variants of vim (vim-1 to vim-13). However, according to the lahey clinic website, to this day over 41 imp and 39 vim derivatives have been documented . (30) targeted imp, oxa-48-like types (oxa-48, 162, 163, 181, 204, 232, 244, 245, 370) and other carbapenemase genes . The assay reported in this study allowed the detection of 33.3% of imp variants (14/42) and was not able to detect oxa-23 and oxa-247 . This study shows that primers in two multiplex pcrs are able to detect of kpc, vim, ndm-1, oxa (oxa-23-like and oxa-48-like enzymes) and imp variants (except imp-3, imp-16, imp-27, imp-31, imp-34 and imp-35) present in enterobacteriaceae clinical isolates . Due to the success of the first primer sets, the additional untested sets (table 1) are predicted to detect the remaining ndm- and imp - type enzymes and other carbapenemases . Therefore, the combination of primer sets presented in this study will cover all known carbapenemase genes found in enterobacteriaceae . The multiplex pcr assay described in this study is a fast, low - cost, efficient and accurate test for rapid screening of enterobacteriaceae isolates with respect to drug resistant genes for blakpc, blavim, blaimp, blandm and blaoxa and cover 100% (19/19), 100% (39/39), 85.7% (36/42), 9.1% (1/11) and 8.5% (25/293) of the genes described in the lahey database, respectively . The proposed untested primers may be also used for detection of the remaining carbapenemases in single and/or multiplex pcr reactions (table 1). The resulting assays could collectively cover 92.9% (39/42), 100% (11/11), 100% (5/5), 100% (4/4), 95.8% (23/24) and 52.6% (154/293) of the blaimp, blandm, blasme, blaimi, blages, blaoxa and other genes, respectively . We included here all known carbapenemase genes that have been identified in clinical isolates of enterobacteriaceae (blakpc, blaoxa, blavim, blandm, blaimp, blasme, blaimi, blages, blagim, bladim and blacmy). In conclusion, primers tested in silico and in vitro may be used in single and/or multiplex pcr for screening encountered carbapenemases in enterobacteriaceae, as well as for monitoring their emergence and spread (e.g., outbreaks).
Anti - retroviral therapy (art) has improved the quality of life of human immunodeficiency virus (hiv) patients worldwide . A reduction in hiv - related morbidity and mortality has been recognized in countries where art has been made widely available . To achieve optimal results from art, high levels of patient adherence to art is essential . High levels of adherence to art (at least 95%) is needed to ensure optimal benefits . Adherence is defined as a patient's ability to follow a treatment plan, take medications at prescribed times and frequencies, and follow restrictions regarding food and other medications . Adherence is a problem in any chronic disease and an average non - adherence rate of 24.8% have been reported . It is important to identify factors that lead to non - adherence and develop strategies to improve long - term adherence . This study was designed to identify the levels of adherence and the factors influencing adherence to art at a tertiary care institution in southern india . Ethical clearance was obtained from the institutional ethics committee of kasturba medical college, mangalore . A facility based cross - sectional study was carried out among 116 people living with hiv (plhiv) on art.the sample size was calculated based on expected proportion of adherence to art among plhiv based on previous studies, with a relative precision of 10% and a confidence interval (ci) of 95%.the study subjects were adult patients (aged 18 years or above) on first line anti - retroviral treatment (two nucleoside reverse transcriptase inhibitor and one non - nucleoside reverse transcriptase inhibitor) for more than 1 year and who gave written informed consent . During our study period of 2 months, 288 hiv positive patients on art visited the hiv clinic of our hospital . Out of 288 patients, 134 patients were on art for more than 1 year . Out of 134 patients, 116 consecutive patients who gave consent participated in the study . The study was carried out at kasturba medical college hospital (tertiary health care centre) in mangalore (south india). Patients who agreed to participate were interviewed by the investigators for 25 to 30 minutes . The interview was guided by a pretested semistructured questionnaire that covered sociodemographic characteristics of the study population, duration of use of art, distance traveled to procure art, and whether they got government sponsored free art . Adherence was assessed retrospectively based on a 4-day recall as used in adult aids clinical trials group (aactg) follow up questionnaire . Adherence index was calculated by the formula: patients with more than 95% of adherence were considered as having high adherence and those with less than 95% were considered as having low adherence . Investigators asked leading questions to assess stigma and family support and then it was categorized as present or absent . Chi - square test was done . A p value of <0.05 was considered statistically significant . A facility based cross - sectional study was carried out among 116 people living with hiv (plhiv) on art.the sample size was calculated based on expected proportion of adherence to art among plhiv based on previous studies, with a relative precision of 10% and a confidence interval (ci) of 95%.the study subjects were adult patients (aged 18 years or above) on first line anti - retroviral treatment (two nucleoside reverse transcriptase inhibitor and one non - nucleoside reverse transcriptase inhibitor) for more than 1 year and who gave written informed consent . During our study period of 2 months, 288 hiv positive patients on art visited the hiv clinic of our hospital . Out of 288 patients, 134 patients were on art for more than 1 year . Out of 134 patients, 116 consecutive patients who gave consent participated in the study . The study was carried out at kasturba medical college hospital (tertiary health care centre) in mangalore (south india). Patients who agreed to participate were interviewed by the investigators for 25 to 30 minutes . The interview was guided by a pretested semistructured questionnaire that covered sociodemographic characteristics of the study population, duration of use of art, distance traveled to procure art, and whether they got government sponsored free art . Adherence was assessed retrospectively based on a 4-day recall as used in adult aids clinical trials group (aactg) follow up questionnaire . Adherence index was calculated by the formula: patients with more than 95% of adherence were considered as having high adherence and those with less than 95% were considered as having low adherence . Investigators asked leading questions to assess stigma and family support and then it was categorized as present or absent . The collected data was analyzed using spss version 11.5 . Chi - square test was done . Of the 116 subjects, 80 (69%) were males and 36 (31%) were females . Majority (51.72%) of study population was in the age group of 21 - 40 years . 72.41% of the patients in our study received free art provided by the government, and 27.58% of the patients paid from their pocket [table 1]. Baseline characteristics of study population (n=116) among 116 participants, 74 (63.7%) reported adherence> 95% . Hundred percent adherence was reported by 54.3% of patients . Among patients who received free art, 64 (76.2%) of them had high adherence, whereas, only 10 (31.3%) patients who paid for art had high adherence . Among patients who were self - motivated to take art, 63 (70%) of them had high adherence . Among patients with low adherence, 14 (33%) simply forgot to take tablets and 13 (30%) patients reported that cost of treatment was responsible for low adherence . All patients who lacked family care, patients who had depression and patients who consumed alcohol had low adherence which was statistically significant . Social stigma and side effects to treatment also contributed to low adherence which was statistically significant [table 2]. Of the 116 participants in our study, 42 (36%) patients reported adherence of <95% . Financial constraints, forgetting to take medication, lack of family care, depression, alcohol use, social stigma, and side effects to art were associated with low adherence in our study . Adherence is a major issue in management of chronic diseases . In a meta - analysis of 569 studies, 24.8% of subjects were non - adherent . In hiv, studies indicate that high levels of adherence are necessary for viral suppression, prevention of resistance, and disease progression . A meta - analysis of studies on art adherence found that 77% of patients in africa achieved adequate adherence of 95% compared to just 55% of patients in north america . Studies from india have shown that financial problems, lack of family care, substance abuse, depression, social stigma, and side effects are barriers to adherence to art . Wanchu et al ., have shown that the major reasons for non - adherence was financial constraints, forgetting to take the medication, drug toxicity, lack of access to drug, fear of getting immune to the benefit of the drug, and to avoid side effects . Not having money to travel to art centre and forgetting to take the medication were the major reasons for non - adherence in the african setting . A meta - analysis a meta - analysis has shown that individuals who consumed alcohol had reduced adherence to art . There is concern that patients on art would become less adherent once they feel better . Education may impact adherence in several ways including facilitating communication with health care providers . In our study, self - reporting is the most commonly used measure of adherence in resource limited settings because it is easy to include in routine clinical practice . Medication event monitoring system (mems), pharmacy refill data, and directly observed therapy (dot) are other methods to monitor adherence . Adherence was assessed through a self - reporting adherence questionnaire and other objective tools such as electronic pill caps, viral load and cd4 count were not used . Our study group was predominantly urban so our results do not necessarily reflect practices in other settings . Finally, the cross sectional study design has its own limitations alcohol use, drug side effects, depression, stigma and lack of family support are factors associated with reduced adherence . Busy medical practitioners must find sufficient time for counseling, which is very essential for the success of art program in our country.
Cancer is defined as an abnormal growth of cells caused by multiple changes in gene expression leading to deregulated balance of cell proliferation and cell death . Cancer is those tumors that have developed the ability to invade the surrounding normal tissues . Cancers are caused by exogenous chemical, physical, or biological carcinogens in humans and the mechanisms of carcinogenesis are often multifactorial and complex . A cancerous cell is traveling throughout the body using the blood or lymph systems, destroying healthy tissue in a process called invasion, and that cell manages to make new blood vessels to feed itself in a process called angiogenesis . Tumors may activate angiogenic inhibitors (angiostatin and endostatin) that can modulate angiogenesis at both the primary site and downstream sites of metastasis [3, 4], when a tumor successfully spreads to other parts of the body using the blood or lymph systems known as metastasis . Cancer is a leading cause of death in the western world . In the united states and a number of european countries, cancer is the second leading destroyer after cardiovascular diseases . Cancer can occur at any age and the average age at the time of diagnosis for cancer is 67 years, and about 76% of all cancers are diagnosed at the age of 55 or older . Although cancer is relatively rare in children, it is the second leading cause of death in children ages of 114 . In this age the overall death rates due to cancer have almost tripled since 1930 for men and gone up over 50% for women . World health organization (who) estimates that some 84 million people will die of cancer between 2005 and 2015 around the world . In 2007, there were 7.9 million deaths from cancer, around 13 percent of all deaths . The national institute of cancer research and hospital (nicrh) started a cancer registry in 2005 for the first time in bangladesh along with the world health organization (who). Data were collected from 24,847 cancer patients who appeared in the nicrh for the first time . Among them, 10,847 (57.6%) were males . Lung cancer was the leading cancer (17.3%), followed by cancers of breast (12.3%), lymph nodes and lymphatics (8.4%), and cervix (8.4%) for sexes combined in all ages . In males' lung (25.5%) and in females in children aged 14 years or younger, lymphoma, retinoblastoma, osteosarcoma, leukemia, and kidney cancers were most prevalent . Lung cancer in males and cervical and breast cancer in females constitute 38% of all cancers in bangladesh . According to the who data published in april 2011, oral cancer deaths in bangladesh reached 11,562 or 1.21% of total deaths . The age adjusted death rate is 12.52 per 100,000 of population ranking bangladesh 4 in the world . There are more than one million (10 lakh) cancer patients in bangladesh while approximately 200,000 new patients, mostly women, are added every year creating a social burden on the country [8, 9]. Various plants have been used against cancer and tumor in traditional medicine system of bangladesh since many years . Traditional medicine is practiced in bangladesh by folk medicine practitioners, also known as kabirajes who utilize various formulations of medicinal plants in most of their preparations . We have observed that the kabirajes of various districts and areas use diverse varieties of plants for the treatment of schizophrenia and psychotic problems, cardiovascular problems, eye infections, snakebite, diabetes, gastrointestinal disorders [16, 17], hiv / aids related infections, rheumatoid arthritis, cattle diseases, and so on . It was objective of the present study to conduct a randomized ethnopharmacological survey to learn more about the medicinal plants used by folk medicine practitioners of bangladesh for the treatment of cancer and also to do comprehensive study on several published articles attributed to the in vivo or in vitro anticancer properties of these species . The anticipation was that the medicinal plants used by the kabirajes can prove to be a useful source for further scientific studies leading to discovering more efficacious antineoplastic drugs . The present randomized surveys were carried out between october 2013 and march 2014, among the kabirajes of three districts of bangladesh, namely, jessore, khulna, and narail . It is located at 23100 north, 89130 east, bordered by khulna and satkhira district to the south, india to the west, magura and narail district to the east, and jhenaidah district to the north . Khulna and narail district geographically coordinate at 22480 north, 89330 and 23100 north, 89300 east, respectively . The surveys were conducted with the help of a semistructured questionnaire and the guided field - walk method [21, 22]. Kabirajes were asked whether they know about cancer and whether they treat the cancer on a regular basis . The kabirajes mentioned the plants with which they treated cancer and took the interviewers to spots from where they collected the plants . Plant specimens were collected and dried in the field and later brought back to dhaka for complete identification at the bangladesh national herbarium . Nomenclature of the identified species was documented from the plant list database [http://www.theplantlist.org/]. A total of 20 plant species were obtained from the kabirajes of the three districts surveyed . The acanthaceae, cucurbitaceae, and fabaceae family contributed two plants each; the rest of the families contributed one plant each . Whole plant as well as plant parts like leaves, barks, roots, fruits, and seeds was used for preparing medicine . Roots, fruits, and seeds each constituted accordingly 15.6%, 12.5%, and 9.4% of total uses . The other plant parts (whole plant, stem, bark, flower, and tuber) mentioned constituted, respectively, 9.4 and 3.1% of total uses (figure 2). Among developed countries, the incidence and mortality rates for various cancers are almost the same . Lung cancer is the most common cancer among men in both developing and developed countries of the world and breast cancer is the most common cancer in women . Annually, the global death rate for cancer is estimated to be more than 6 million people and over 22 million individuals have been diagnosed with cancer worldwide . Many developing countries have intensified their efforts in documenting the ethnomedical data and scientific literature on medicinal plants . In 2000, natural product derivatives were involved in 14 of the top 35 drugs based on worldwide sales . Medicinal plants have been used for cancer treatment in many countries of the world from a prolonged period of time [26, 27] and the treatment or prevention is attributed to their safety, low cost, and oral bioavailability as well; natural plant derivatives claimed extensive scientific screening and clinical experiments for the development of anticancer drugs . Over 3000 plants species have been reported to have anticancer properties and about 35000 plant samples from 20 countries have been collected and around 114,000 extracts were screened against tumor systems used as a primary screen . Clinically active antineoplastic agents should be able to prolong the survival and decrease the leukocyte count of blood of tumor - bearing animals . Examples of some well - known plant - derived antineoplastic lead compounds along with their specific mechanism of actions are summarized in table 3 . Secondary metabolites are compounds belonging to varied chemical groups that exert biological activities both on human and animal cells . Products of secondary metabolites are the main phytochemical constituents with various pharmaceutical properties serving either as protective agents against various pathogens or growth regulatory molecules . Plant - derived commercial anticancer drugs (vinblastine and vincristine from catharanthus roseus) are still produced by isolation from growing plants . In table 4, we have listed some reported plant - derived chemical compounds from the antineoplastic plants used by the bangladeshi folk health practitioners in the treatment of cancer . Hydroalcoholic seed and leaf extracts of abelmoschus moschatus exhibited antiproliferative activity against colorectal adenocarcinoma and retinoblastoma human cancer cell lines . The ethanol leaves' extract of the plant was found to be cytotoxic towards lung fibroblast cells in mtt assay . Another study reported that the plant extract has been shown to prevent dna alterations in a transplantable ehrlich ascites carcinoma - bearing murine model and in enlargement of the survival of the animals against the proliferation of ascites tumor . Ethyl acetate extract of the whole plant of a. ilicifolius has a potential cytotoxic activity on hela cell and kb cell lines by comet assay . Active compounds of a. ilicifolius flower play a role in killing artemia salina nauplii and can be considered as potential cytotoxic agents as well as future candidate for cancer therapy . The cytotoxicity and antitumor activity of the chloroform extracts of aristolochia indica were assessed in human breast cancer cell line by mtt assay using taxol as standard and showed pronounced anticancer activity against ehrlich ascites carcinoma cell line [38, 39]. Aristolochic acid was reported to possess various biological activities including antiadenocarcinoma, antineoplastic, and antitumor activities . Dammarane triterpenoid 1, isolated from borassus flabellifer seed coat, inhibits tumor necrosis factor- and showed good antiproliferative activity against pancreatic cancer cell line . Apoptosis inducing activity was confirmed based on increased sub - g0 phase cell population in cell cycle analysis, loss of mitochondrial membrane potential, elevated levels of cytochrome c, nuclear morphological changes, and dna fragmentation in mia paca-2 pancreatic cancer cells . B. flabellifer seed coat extracts were screened in another study for their possible anticancer activity on growth of the hela cells and these preliminary studies indicated that even the lower concentrations of plant extract showed significant antiproliferative activity . There is an in vitro study that showed that blumea lacera exhibited broad spectrum antileukemic activity against k562, l1210, p3hr1, and u937 leukemia cells . Methanolic extract of b. lacera leaves has also showed cytotoxic activity against human gastric adenocarcinoma cell line, human colorectal adenocarcinoma cell line, and human breast ductal carcinoma cell line . The interest in anticarcinogenic properties of cannabinoids was renewed after the discovery of the endocannabinoid system . The administration of 9-thc, 8-thc, and cannabinol inhibited the growth of lewis lung adenocarcinoma cells in vitro as well as in vivo after oral administration in mice . Antitumorigenic mechanisms of cannabinoids are showing their ability to interfere with tumor neovascularization, cancer cell migration, adhesion, invasion, and metastasis . The mechanism of cannabinoids' anticancer action depends on the ability of their agents to stimulate autophagy - mediated apoptotic cancer cell death; thus, cannabinoid action helps in cancer cell death, impairs tumor angiogenesis, and blocks invasion and metastasis and cannabinoids are currently also being tested as anticancer agents in phase i / ii clinical studies . Methanol extract of cucurbita maxima aerial parts has been performed against ehrlich ascites carcinoma model in mice by saha et al . For the antitumor activity and the results revealed that c. maxima possesses significant anticancer activity which may be due to its cytotoxicity and antioxidant properties . L - asparaginase is an antineoplastic agent, identified from fruit of c. maxima, used for treatment of a type of cancer that is acute lymphoblastic leukemia and non - hodgkin's lymphoma as well as being experimentally used as an anticancer agent in human patients [54, 55]. The methanolic extract of d. indica has been found to have significant antileukemic activity in human leukemic cell lines u937, hl60, and k562 . Methanolic extracts of betulinic acid were prepared from the d. indica fruits inducing apoptosis in ht-29 cells via mitochondrial dependent pathway and proving to be a potential therapeutic agent for colon cancer . Petroleum ether fraction of the plant showed potential effects against hepa with microstructure abnormality of hepa cells surface . Immune system modulation might be related to antitumor effects of d. bulbifera rhizome, as reported in s180 and h22 tumor cells bearing mice . The aqueous and methanolic extracts of the leaves of emilia sonchifolia gradually exhibit antitumor activities . The n - hexane extract of e. sonchifolia has anticancer effect and is rich in terpenoids and terpenoids were evaluated for their potential antineoplastic activity in various human cancer cell lines such as gastric, pancreatic, and colon carcinomas . Steroid derived from the stem bark and the leaves of erythrina variegata showed anticancer activity against in vitro breast cancer cell t47d . Alkaloids (10,11-dioxoerythratidine and crystagallin a) extracted from the leaves and stem bark of e. variegata plant strongly stated in vitro anticancer activity against breast cancer t47d cell lines in vitro using the sulforhodamine b (srb) assay . The effect of h. auriculata on carbohydrate metabolizing enzymes in n - nitrosodiethylamine induced hepatocellular carcinoma in rats . The aqueous seed extract from h. auriculata displayed selective cancer cell cytotoxicity with an ic50 value of 0.22 mg ml against colon cancer cells . In vitro study of h. auriculata extracts ahmad et al . Reported antitumor activity from plant extract against chemically induced hepatocarcinogenesis in wister rats . Different leaf extracts of m. oleifera produced significant cytotoxic effects on human multiple myeloma cultured cell lines . A study showed that leaves extract of m. oleifera can significantly obstruct the growth of cultured human pancreatic carcinoma cells by inhibiting the nf-b signaling pathway . Most of the anticancer studies of m. oleifera have not focused on the molecular basis of the tumor - suppressive activity but strongly suggested that it could potentially be a supreme anticancer candidate specific to cancer cells [73, 74]. The methanolic extract of nymphaea nouchali roots has showed inhibitory activity towards tumor promoter in the raji cells . In vitro antiproliferative activity of polygonum hydropiper (synonymy) extracts was evaluated against cervix epithelial adenocarcinoma, skin epidermoid carcinoma, and breast epithelial adenocarcinoma cells and the results confirmed substantial cell growth inhibitory activity against one or more cell lines . A triterpenoid compound named cucurbitacin b isolated from trichosanthes kirilowii showed the potent inhibitory activity against hif-1 activation induced by hypoxia in various human cancer cell lines . In vivo studies confirmed the inhibitory effect of cucurbitacin b on the expression of hif-1 proteins, leading to a decrease growth of hela cells in a xenograft tumor model . Cucurbitacin d isolated from the plant has also been shown to suppress proliferation of ht-29 human colon cancer cells and the compound could be potent therapeutic agent for breast cancer by blocking tumor cell proliferation and inducing apoptosis through suppression of stat3 activity and it could also induce apoptosis in human hepatocellular carcinoma cells . Among twenty plant species, four of the species used by folk medicine practitioners have no strong published data regarding anticancer or cytotoxic activities . These 4 species are c. inerme, m. paniculata, s. sesban, and v. officinalis . From just a brief survey of the literature, it appears that the rest of the sixteen plants used by the kabirajes in three districts of bangladesh present considerable potential in the treatment of cancer . Further scientific studies need to be conducted on these plants towards discovery of lead compounds, which can lead to formulation of new drugs for the prevention and management of malignant neoplastic diseases with giving less or no side effects.
The gag reflex is a physiological response which safeguards the airway from foreign bodies . Gagging can become a conditioned response that makes dental treatment difficult or impossible for both the individual and the dentist . Clinicians successfully treat many patients with mild gagging problems using only minor procedural modifications or behaviour techniques . Generally, the ultimate goal for patients is to make routine dental care possible by helping them unlearn the behaviour that leads to gagging . Therefore, strategies to overcome the gag reflex in treatment have focused on behaviour modification using systemic desensitization and distraction methods . Some patients suffered from such severe gagging that behaviour techniques did not sufficiently reduce gagging in dentistry . In these patients pharmacological management (sedatives [propofol, midazolam, nitrous oxide e.g. ], antihistamine, or parasympathetic depressants) was thought to be the last alternative to eliminate the reflex [4 - 6]. We present three cases which describe the rehabilitation of a female and two paediatric patients who have exaggerated gag reflex requiring prosthetic and orthodontic procedures with the help of intranasal midazolam . A 50 year old, 65 kg female patient was referred to the prosthetic department of seluk university for rehabilitation of their oral function and aesthetic . She had malformed anterior teeth in the maxilla and left first molar present in the mandible . The treatment was planned as providing partial removable dentures retained with fixed partial dentures . Intranasal midazolam 2.5 mg (demizolam, dem medikal, istanbul) was employed to facilitate the taking of accurate dental impressions without gagging during the whole procedure by the anaesthesiologist . The patient was given verbal information about the procedure at an assessment visit prior to the impression appointment . The patient had no gag reflex and allowed the dentist to apply the impression tray accurately in the mouth . The patient was controlled after sedation for 45 minutes before she was discharged . In addition, planning for horse - shoe palatal connector reduced palatal coverage area, thereby, providing less interference for tongue and reduced gag reflex (figure 1 and 2). Initial intraoral photograph of patient 1 . A 15 year old, 56 kg child patient was referred to the prosthetic department of seluk university . Patient was suffering from chewing dysfunction resulting from maxillary tooth agenesis and deficiency . He did not allow the dentist to put the tray into his mouth and this encouraged us to administer midazolam intranasally for its inhibitory effect on gagging . Intranasal midazolam 2.5 mg (demizolam, dem medikal, istanbul) were applied incrementally in both nostrils . Patient was able to overcome his gagging reflex and completed the treatment by the help of midazolam . An 11 year old, 45 kg child patient was referred to the orthodontic department of seluk university . It was learnt from the medical history of the patient that he used regular medication for hyperactivity . In extraoral clinical examination, it was observed that the patient had a skeletal class iii malocclusion and concave profile . In the intraoral and radiographic examination, it was detected that the patient was in mixed dentition stage and angle class ii molar relationship on both sides . According to the facial midline, the maxillary midline was 1 mm on the left and the mandibular midline was 1 mm on the right . The orthodontic treatment plan included non - extraction treatment after using face mask . During obtaining initial records, taking impression procedure was elicited patient s hypersensitive gagging reflex . Therefore, it was decided to administer intranasal midazolam in advantage to take precise dental impressions without gagging during the whole procedure . After patient s parents were given verbal information about the procedure, 2.5 mg intranasal midazolam (demizolam, dem medikal, istanbul) were applied incrementally in both nostrils . Five minutes later, the patient felt himself relaxed and the level of sedation was sufficient to take dental impression . We experienced the benefit of intranasal midazolam to facilitate taking precise dental impressions in problematic gagging patients intolerable to dental therapy . All patients suffered moderate burning in the nostril after delivery of intranasal midazolam (figure 3 and 4). Retentive denture prosthesis is a major factor in achieving a successful result when providing partial removable dentures . The best results are obtained when the prosthesis bases contain all anatomic landmarks in the upper and lower arches . This is really complicated when the patients anamnesis tells us the difficulty with the impression phase of treatment due to a hypersensitive gag reflex . Intranasal midazolam may be safely used to facilitate the taking of dental impressions in these patients . In the aetiology of gagging, anatomical abnormalities or neural hypersensitivity in the oropharynx have been identified as somatogenic factors in the development of the gag reflex . In a previous study by sata et al ., it was reported that psychological factors were identified in all patients with strong anxiety indicated as the probable cause of the gag reflex . However same authors were stated that the gag reflex was caused by touching the oral mucosa in most of patients with some difference in degree of severity . In our patients, a pre - treatment oral examination was not performed to determine gagging severity but a hypersensitive gag reflex was developed during taking impression in all patients . However it may not be easy to distinguish between two aetiological factors because physical stimuli may still provoke gagging of psychogenic origin . A number of techniques for reduction of gagging have been suggested, including distraction of patient s attention from the dental procedure, relaxation, hypnosis, acupuncture, drugs and general anaesthesia (ga). Ga is also conducted in a patient with extremely problematic gagging who is intolerable to dental therapy under intravenous sedation . The advantage of ga is complete elimination of the reflex, but costs should be considered in comparison to the benefit of dental care . As another alternative, inhalation sedation using nitrous oxide is the most common method of delivery of conscious sedation in dentistry . Nitrous oxide inhalation sedation was shown to be useful for control of the gagging reflex . Nitrous oxide is a commonly used pharmacologic agent but has the main disadvantage in especially children of being a nasal inhalation agent . One of the popular medications available to the dentist for use in children is midazolam . Midazolam is a short - acting benzodiazepine with a short half - life, in children, of two hours compared to 18 hours for diazepam . Midazolam has been used for preoperative sedation by the intramuscular, rectal, oral and nasal routes . The injections are painful and injection procedure is the most common fear of the children . The anaesthetics used orally and rectally start to relieve symptoms in a longer time . This technique has advantages when compared with oral administration as the bioavailability of intranasal administered midazolam is approximately 55%, compared with 15% when administered orally . The rate of onset and recovery are more rapid, but intranasal burning is the main disadvantage of the nasal application of midazolam . The nasal anaesthesia is popular as a useful and safe way . The short absorption time and the fast relief process of anaesthetics transferred to the systemic circulatory system are important advantages of nasal anaesthesia . No serious adverse effects were observed and the patients remained cooperated in the impression phase as in the beginning of the treatment; they had no gagging during the entire procedure which was completed to the full satisfaction of all involved . These three cases showed that intranasal midazolam could be very effective treatments in exaggerated gag reflex in paediatric and adult patients . For some patients, however, severe gagging can be elicited by the dentist s fingers or instruments contacting the oral mucosa or even by nontactile stimuli, for example, patients seeing the dentist or remembering a previous dental experience . Some clinicians swab the sensitive mucosal areas with topical anaesthetics, whereas others suggest that impressions should be made under general anaesthesia or hypnosis . Anaesthetic sprays are difficult to control and their use may result in increased risk of toxicity . Intranasal midazolam has been found to be effective in doses ranging from 0.2 to 0.6 mg / kg (maximum 15 mg) when used for conscious sedation and as a premedicant for children . In our clinic; 2.5 mg or 5 mg intranasal midazolam is being used according to the anaesthesiologists preference in adults and 0.5 mg / kg (maximum 10 mg) in child . In this clinical report the reason of this quantity of dose preference for the child patients is their weights (56 and 45 kg) and no sedation effect is required for them . The beneficial effects of midazolam include sedation, anxiolysis, and reduction of postoperative vomiting . Our patients were given written consent before orthodontic and prosthetic treatment and also verbal information about the midazolam procedure at an assessment visit prior to the impression appointment . Premedication with oral midazolam has shown to be more effective than parental presence or placebo in reducing gagging anxiety and improving compliance at induction of anaesthesia . Intranasal drug administration is relatively quick, simple, and may have benefits over transmucosal routes or rectal administration, which requires more patient cooperation . And, intravenous way is not needed . The use of conscious sedation with inhalational, oral, or intravenous agents may temporarily eliminate gagging during dental treatment . In dental procedures; midazolam is accepted as one of the most effective agents to reduce gagging reflex due to its sedative effects . In a study by murphy et al . Which has investigated the effect of intravenously administered midazolam on the sensitivity of upper airway reflexes in volunteers, the authors concluded that midazolam produced significant depression of upper airway reflex sensitivity . And this conclusion may also explain our clinical results . Besides the sedative effect of midazolam, the intranasal midazolam was found to be very useful for taking impressions probably effecting on the depression of upper airway reflex sensitivity . Further investigations by using quantitative methods are needed to be done to clearly prove this effect of midazolam in dentistry.
A 57-year - old female patient came to the hospital with pain in both thighs, especially on the right side . The pain was exacerbated during weight bearing and walking and decreased while resting . In the physical examination, the range of motion of the hip joint was normal, and there were no abnormal neurological findings . On the simple radiographic inspection, the cortical bone of the right proximal femur was thinned and showed cystic change . Decreased bone density was also observed and the trabecular bone of the overall femur was thinned (fig . 1a). The magnetic resonance imaging (mri) was performed on the part showing cystic bone change to determine the bone tumor . 1c) were observed in the proximal femur on both sides and in the right pubis . An additional blood test was conducted and serum intact parathyroid hormone was 2,720 pg / ml (normal, 15 - 65 pg / ml), alkaline phosphatase was 2,417 u / l (normal, 104 - 338 u / l), calcium was 14.5 mg / dl (normal, 8.6 - 10.4 mg / dl), and phosphate was 2.0 mg / dl (normal, 2.6 - 4.4 mg / dl). To find out the cause of hyperparathyroidism, we did the ultrasonography of the thyroid . Since a nodule was observed in the left lobe of the parathyroid, fine needle aspiration was performed to obtain pathological results from the parathyroid . Furthermore as malignant change could not be excluded with the presence of a relatively large nodule, a parathyroidectomy was decided . On the 14th day from admission, the day for parathyroidectomy, the patient felt sudden pain in the right thigh while getting up from a bed to sit down in the morning, and radiographic inspection was performed . In the femur anteroposterior and lateral images, a pathological fracture was confirmed in a site where the brown tumor had formed (fig . 2). Closed reduction and internal fixation with the intramedullary nailing was performed in orthopaedic surgery department, and a biopsy was conducted using specimen tissue obtained during the medullary reaming . And the additional biopsy was performed along with the parathyroidectomy in the head and neck surgery department . The blood calcium concentration decreased to 10.0 mg / dl after the surgery, and the blood parathyroid hormone concentration became normalized after a month (table 1). In a biopsy on the femoral lesion, brown tumor was diagnosed . Four months after the surgery, the union of the fracture was confirmed (fig . The patient became capable of walking without a crutch or walkers by a follow - up ten months after the surgery . An increase of blood parathyroid hormone concentration due to hyperparathyroidism destroys the balance of osteoblasts and osteoclasts through activation of the osteoclast . As a result, bleeding and repetitive granulation of tissue partially develops in the bone, which is gradually filled with fibrous tissues which are rapidly proliferate . These changes of bone are observed in approximately 10% to 20% of patients with hyperparathyroidism and are referred as brown tumor35). A cyst formed by the osteoclast function has clinical significance, as it increases pain and the risk of fracture . In radiological aspects, the difference is that it does not invade adjacent tissues and does not induce changes around the periosteum124). In our case, an osteolytic lesion in the proximal part of the right femur was observed in simple x - ray . The cortical bone was thinned to approximately one - third the thickness of other parts of the bone . Multiple osteolytic lesions existed not only in the right proximal femur but also in the right acetabulum and left pubis . However, a periosteal reaction, which is frequently observed in cases of metastatic tumor, was not observed . With a high concentration of blood parathyroid hormone, as it was reported that osteolytic lesions caused by brown tumor are recovered when hyperparathyroidism is treated13), we decided to treat the hyperparathyroidism for the first, including a parathyroidectomy, instead of doing surgical therapy on the bone lesion . However, in this case, a fracture in the right femur occurred unfortunately without any trauma while the patient was bending her legs to sit up on a bed . Although she had mild pain, her bony lesion was located in a subtrochanteric site, and it showed severe cystic and osteolytic changes . The patient's mirels score, which indicates the possibility of a pathological fracture development, was ten (table 2)78). As we thought that recovery of bone lesions can be expected with therapy for hyperparathyroidism without prophylactic internal fixation so we did not undergo primary stabilization with intramedullary nail at first time . But through this case, it is assumed that it will still be necessary to actively do the preventive fixation by applying the mirels score, as brown tumor is in lower extremities which is the weight bearing portion and could easily induce pathological fracture . In our case, there were other bone lesions except the right femur lesion . Through the treatment for hyperparathyroidism, her pain on groin and left thigh region were relieved clinically and osteolytic bony changes in pubis and left proximal femur were recoverd by the follow up x - ray after ten months from the diagnosis (fig . The blood parathyroid hormone and calcium concentration were normalized within a few days after the parathyroidectomy . In conclusion, as brown tumor due to hyperparathyroidism has a characteristic of multiple osteolytic lesions, if there were multiple osteolytic lesions in plain x - ray film, hematological tests including blood calcium and parathyroid hormone concentration are required for differential diagnosis . When a brown tumor does not accompany the pathological fracture, conservative treatment should be considered first . However, we believe that it will be necessary to prophylactic internal fixation according to the mirels score in case of a high risk of fracture, as in our case.
Single and double, fluorescent and non - fluorescent, in situ hybridization and immunohistochemical stainings were performed using standard protocols . To analyze the ltbp3 loss of function phenotype, we injected anti - sense ltbp3 morpholinos into one - cell stage wt and transgenic embryos . For genetic lineage tracing, a transgenic driver strain expressing cre recombinase in ltbp3 cells and four cre - responsive color switching the driver strain was crossed individually to each of the reporter strains and their double transgenic progenies were analyzed for zsyellow protein fluorescence using confocal microscopy . To follow the migration of zebrafish shf cells, a tracking dye was injected into the zsyellow, amcyan region of tg(nkx2.5::zsyello); tg(cmlc2::csy) embryos at 24hpf, the embryos were imaged immediately, and then again at 48 and/or 72hpf . A transgenic strain carrying a cdna encoding a constitutively active human tgf type i receptor (caalk5) under control of the zebrafish heat shock promoter was generated and used to rescue the myocardial defect in ltbp3 morphant embryos . Full methods and any associated references are available in the online version of the paper at www.natre.com/nature
Transurethral resection of bladder tumor (turbt) is the treatment of choice for non - muscle - invasive urothelial carcinoma . Intraperitoneal perforation is a rare and serious complication of turbt, which can be complicated by increased leakage, systemic absorption of irrigation fluid and by bowel injury . Intraperitoneal bladder perforation, although infrequent, is considered so serious that it requires immediate treatment . Traditionally, intraperitoneal perforation has been managed with open surgical repair . Minimal invasive management such as percutaneous peritoneal drainage, or laparoscopic repair of the bladder defect[24] have been described; however, there are no large series or long - term follow - up studies . Laparoscopic bladder repair was safely performed in an old patient as the following case illustrates . She underwent staging turbt after 4 weeks which again showed t1 high grade urothelial carcinoma . Deep tumor resection was performed using a flat, equatorial loop to resect the posterior wall tangentially . During the resection which took 20 minutes this was recognized by cystoscopic visualization of the intraperitoneal cavity through a laceration in the middle of the resected area near the dome, while fulgurating the edges at the completion of the procedure . Immediately, exploratory laparoscopy was performed by a colleague from the department of general surgery experienced in laparoscopic techniques showing a 20-mm rupture at the posterior wall of the bladder . Three trocars were used, a 10 mm umbilical trocar and two 5 mm trocars, one in each iliac fossa . There was no injury to the bowel and the bladder was repaired with two absorbable 2 - 0 vicryl sutures using a one - layer full - thickness suture pattern . A percutaneous intraperitoneal 12f silicon drain was placed to monitor for possible urine extravasation post operation . For bladder drainage, five days after the operation a cystogram revealed no evidence of leak, and the catheter was removed . Intraperitoneal bladder perforation is a rare but serious complication that deserves immediate treatment to prevent complications such as peritonitis, uremia, acidosis, hypervolemia due to irrigant fluid absorption, and tumor cells seeding in the peritoneum . Bladder perforation is more likely to occur in elderly patients who have a thin bladder wall . The incidence of bladder perforation is difficult to assess because many perforations heal spontaneously and go unnoticed since they do not cause any perioperative and postoperative problems . Therefore, bladder perforations following transurethral surgery may occur much more often than observed or reported in the literature . Balbay et al . Carefully evaluated this by performing a cystogram before and after each turbt, and found that 58% of the cases had some extravasation although the surgeon did not believe there was a perforation . Some means to reduce the incidence of bladder perforation are to perform resections in elder patients with special care (especially for tumors at the dome), to avoid overdistension of the bladder, to use the loop in a manner that follows the bladder contour, and to use low power settings on the diathermy unit . Whereas in many patients with small extraperitoneal perforations, free bladder catheter drainage and careful observation is sufficient, such treatment is not enough in patients with significant intraperitoneal perforations . The traditional treatment has been a formal laparotomy with drainage of the intraperitoneal fluid, repair of the perforated area, exclusion of small bowel injury, and placement of intraperitoneal drains . As demonstrated in this case and by others,[24] advantages over open repair are obvious, including less hospitalization and avoidance of the morbidity associated with open surgery . In our opinion, simple bladder drainage might be insufficient, considering that it does not allow any control of the fluid extravasating into the peritoneal cavity during the postoperative period . Continuous or intermittent bladder irrigation can be necessary in these cases to deal with bleeding caused by incomplete tumor resection and insufficient hemostasis . On the other hand, percutaneous placement of a peritoneal drainage tube might put the patient at risk of bowel perforation or injury to abdominal wall vessels . Moreover, it avoids open surgery and the limits and risks of the standard conservative approaches . Although diagnostic laparoscopy can be performed with either local or regional anesthesia, therapeutic laparoscopic procedures are generally performed using inhalation anesthesia and controlled ventilation . Next, consideration must be given to the special equipment and adequate expertise needed for these procedures . As demonstrated in this case, the procedure can be performed in cooperation with any surgeon experienced in laparoscopic techniques . According to a consensus statement from 2004 on bladder injuries, ureters or bladder neck a further major concern of bladder perforation during turbt is the possibility of tumor cell dissemination . Unrecognized bladder cancer seeding after resection may alter the natural course of the disease process . Existing literature supports that bladder perforation during transurethral resection has a slight but possible chance of extravesical recurrence even for a low stage tumor . Noted that of the 11 patients who had bladder perforations during turbt, only 1 had extravesical recurrence . Indicated that open procedures to close the bladder were associated with an increased risk of extravesical recurrence and this negatively impacted patient prognosis . Their statement was based on an extensive review of more than 3,400 patients and 34 instances of bladder perforation . Of course, dissemination alone does not result in metastases, which require target tissue that are receptive and support angiogenesis . Laparoscopic repair should be considered in case of inadvertent intraperitoneal bladder perforation during transurethral surgery . It can be safely performed and offers an excellent modality for repair of this rare complication even in old patients.
Adhesive dentistry emphasizes on the development of materials to understand the interaction with the tooth tissues.1 the rapid advancement in adhesive technology has extensively influenced modern restorative dentistry . Despite numerous advances made in adhesive technology during the last 50 years, the bonded interface itself remains the achilles heel of an adhesive filling . Especially water sorption is thought to destabilize the adhesive - tooth bond, which include the heterogeneity of tooth structure and composition, the features of the dental surface exposed after cavity preparation, and the characteristics of the adhesive itself, such as its strategy of interaction with both substrates and its basic physicochemical properties.2 cementation is a vital step in the process of retention, marginal seal and durability of indirect restorations . From the past two decades, resin cements continue to evolve with advancements in adhesive dentistry.3 dual - polymerizing resin luting agents are widely used to bond esthetic indirect restorations because of their low solubility, low viscosity, clinically acceptable film thickness, better mechanical properties than conventional cements, ability to bond tooth to restorative material when used with bonding agents, and lower microleakage compared to other luting materials.4 effective adhesion to enamel has been achieved with relative ease and has reportedly proven to be a durable and clinical procedure for routine applications in modern adhesive dentistry.5 bonding to dentin has been considered more difficult and less predictable . The main obstacle is the heterogenous nature of dentin, with hydroxyapatite deposited on a mesh of collagen fibers with hydrophilicity definitely presenting one of the major challenges for the interaction of modern adhesives with dentin.6,7 different mechanical tests can be found to evaluate bond strength to tooth structures . The microtensile bond strength (-tbs) is more versatile, as there is better economic use of teeth, as multiple specimens obtained from a single tooth enable more inventive set - ups and better - controlled substrate variables . However, several micro - specimen preparation protocols are being used worldwide, one is being more technique - sensitive than the other . Non - trimmed micro - specimens are definitely most easy and fast to prepare . Trimming the micro specimens at the interface to so - called hourglass - shaped specimens better concentrates stresses at the interface, but involves a more invasive specimen procedure.2 the present in - vitro study was conducted in the department of conservative dentistry and endodontics, m.r . 40 recently extracted intact human molars were used in this study . Attached soft tissue and calculi teeth were cleaned with slurry of pumice and water and examined under a 30 stereomicroscope (lynx, shimadzu, tokyo, japan) to ensure that they were free of surface cracks, decalcification or any sign of previous grinding . Forty teeth were embedded in acrylic resin after sectioning of roots in a custom made alignment to enable standardization of the specimen during curing . The buccal enamel surface was then demarcated to outline the flat - test area for bonding . The occlusal third of the buccal and lingual surfaces was usually outside the bonding area due to its inclination . The enamel surfaces were manually ground with a 60 grit -silicon carbide paper under water cooling for 60 s. for dentinal exposure, the crowns of the teeth were sectioned using a low - speed diamond disc with water lubrication up to 1 mm below the dentino - enamel junction . An artificial smear layer was obtained by wet grinding with 600 grit silicon carbide paper . The commercial name, composition, and the manufacturer or the materials used in the study are listed in table 1 . Calibra resin cement was used with dual - cured prime and bond nt (two step etch - and - rinse bonding system). The substrates were etched for 15 s with 37% phosphoric acid, then rinsed for 30 s and dried . Mix equal drops of prime and bond nt and self - cure activator into the same mixing well for 1 - 2 s with a clean, unused brush tip . Using the disposable brush supplied, immediately apply mixed adhesive / activator to thoroughly wet all the tooth surfaces . . Equal parts of base and catalyst were mixed for 20 s, and then applied to the surface of the tooth . To standardize the amount of luting material placed on each specimen, templates was prepared, which was 3 0.2 mm for all specimens . The composite resin of dimensions 4 mm 4 mm was built incrementally over the cured resin cement . The cement was cured for 40 s in two different areas for a total of 80 s. paracem dual - cured resin cement was used with parabond adhesive system (two - step self - etch), which is chemical curing adhesive system . The tooth substrates were conditioned for 30 s with a non - rinse conditioner and gently air - dried for 2 s. after conditioning, equal amounts of adhesive a and adhesive b were mixed and applied to the dentin surface in two coats, the bonding agent was left in place undisturbed for 30 s before the excess was gently air dried . Variolink cement was used with dual - cured excite f dsc (two step etch - and - rinse). The substrates were etched for 15 s with 37% phosphoric acid, then rinsed for 30 s. apply it to the enamel and dentin and agitate the adhesive on the prepared surfaces for 10 s. light cure for 10 s. the procedure is same as mentioned above . This cement was used with adper single bond 2 (two step etch - and - rinse). Substrates were etched for 15 s with 37% phosphoric acid, then rinsed for 30 s and dried just enough to remove all the water from tooth surface, but without desiccating the tooth . Immediately after blotting, apply 2 - 3 consecutive coats of adper single bond 2 adhesive to etched enamel and dentin for 15 s with gentle agitation using a fully saturated applicator . Specimens were stored at 37c in 100% relative humidity for 24 h to ensure complete polymerization . Each tooth was sectioned perpendicular to the resin - substrate interface with a slow speed diamond saw under water cooling yielding sections of approximately 1 mm (figure 1). On an average, the beams obtained after sectioning were stressed to failure under tension in a custom made stainless steel forceps (figure 2) held in a universal testing machine (lloyd) at a crosshead speed of 1.0 mm / min (figure 3). Subsequent to -tbs, fracture modes were observed by stereomicroscopic observations of both sides of the failed bonds . The actual mode of failure was recorded according to the following criteria given by morais et al.4 the statistical data derived from the bond strength of four dual - cured resin cements to enamel and dentin was analyzed by two - way analysis of variance (anova), independent t - test and turkey s hsd (p <0.05) to determine the effect of the substrate on each material . Calibra resin cement was used with dual - cured prime and bond nt (two step etch - and - rinse bonding system). The substrates were etched for 15 s with 37% phosphoric acid, then rinsed for 30 s and dried . Mix equal drops of prime and bond nt and self - cure activator into the same mixing well for 1 - 2 s with a clean, unused brush tip . Using the disposable brush supplied, immediately apply mixed adhesive / activator to thoroughly wet all the tooth surfaces . . Equal parts of base and catalyst were mixed for 20 s, and then applied to the surface of the tooth . To standardize the amount of luting material placed on each specimen, templates was prepared, which was 3 0.2 mm for all specimens . The composite resin of dimensions 4 mm 4 mm was built incrementally over the cured resin cement . The cement was cured for 40 s in two different areas for a total of 80 s. paracem dual - cured resin cement was used with parabond adhesive system (two - step self - etch), which is chemical curing adhesive system . The tooth substrates were conditioned for 30 s with a non - rinse conditioner and gently air - dried for 2 s. after conditioning, equal amounts of adhesive a and adhesive b were mixed and applied to the dentin surface in two coats, the bonding agent was left in place undisturbed for 30 s before the excess was gently air dried . Variolink cement was used with dual - cured excite f dsc (two step etch - and - rinse). The substrates were etched for 15 s with 37% phosphoric acid, then rinsed for 30 s. apply it to the enamel and dentin and agitate the adhesive on the prepared surfaces for 10 s. light cure for 10 s. the procedure is same as mentioned above . This cement was used with adper single bond 2 (two step etch - and - rinse). Substrates were etched for 15 s with 37% phosphoric acid, then rinsed for 30 s and dried just enough to remove all the water from tooth surface, but without desiccating the tooth . Immediately after blotting, apply 2 - 3 consecutive coats of adper single bond 2 adhesive to etched enamel and dentin for 15 s with gentle agitation using a fully saturated applicator . Specimens were stored at 37c in 100% relative humidity for 24 h to ensure complete polymerization . Each tooth was sectioned perpendicular to the resin - substrate interface with a slow speed diamond saw under water cooling yielding sections of approximately 1 mm (figure 1). On an average, the beams obtained after sectioning were stressed to failure under tension in a custom made stainless steel forceps (figure 2) held in a universal testing machine (lloyd) at a crosshead speed of 1.0 mm / min (figure 3). Subsequent to -tbs, fracture modes were observed by stereomicroscopic observations of both sides of the failed bonds . The actual mode of failure was recorded according to the following criteria given by morais et al.4 the statistical data derived from the bond strength of four dual - cured resin cements to enamel and dentin was analyzed by two - way analysis of variance (anova), independent t - test and turkey s hsd (p <0.05) to determine the effect of the substrate on each material . When mean bond strengths of four resin cements (subgroups 1, 2, 3, 4) were compared, subgroup 4 (rely x arc) showed highest mean bond strength to both the substrates, which is significant (p <0.001) compared with other groups, whereas subgroup 2 (paracem) showed the lower bond strength to both the substrates when compared to other groups (table 1 and 2). When comparison of mean tbs to enamel and dentinal substrates was done using two - way anova, cements bonded to enamel substrates showed higher mean bond strength compared to dentin, which is statistically significant with p <0.001 (tables 3 and 4). Inter comparison of tbs between subgroups in group a done using tukey s hsd post - hoc multiple comparison test . Inter comparison of tbs between subgroups in group b done using tukey s hsd post - hoc multiple comparison test . It apparently places stress on the adhesive interface during specimen preparation and handling.8 very few studies were done comparing the -tbs of dual - cured resin cements to enamel and dentin.9 - 11 the present study compared the -tbs of dual - cured resin cements to enamel and dentin on recently extracted human molar teeth . Four resin cements were compared i.e., calibra (two - step etch - and - rinse adhesive system), paracem (two - step self - etch adhesive system), variolink ii (two - step etch - and - rinse adhesive) and rely x arc (two - step etch - and - rinse adhesive system). These cements were bonded to enamel (buccal surface of the tooth) and to the dentin (1 mm below the dej on the occlusal surface) of the tooth according to the manufacturer s instructions . Mota et al.12 and eick et al.13 claimed that bond strengths to enamel and dentin should be higher than 20 mpa to compensate adequately for the stresses caused by polymerization shrinkage . When substrates were compared, the study showed significant bond strength to enamel compared to dentin . This is in agreement with studies done by la fuente et al.9 and ritter et al.11 this can be explained on the basis that on enamel, acid - etching selectively dissolves the enamel rods, creating micro porosities, which are readily penetrated, even by ordinary hydrophobic bonding agents, creating micromechanical interlocking of resin tags with capillary attraction, whereas on dentine the main obstacle is the heterogeneous nature, with hydroxyapatite deposited on a mesh of collagen fibers . In addition, dentin is closely connected with pulp tissue through numerous fluid - filled tubules, which traverse through dentine from the pulp to the dentino - enamel junction . The hydrophilic property of dentin represents one of the major challenges for the interaction of current adhesives with dentin . The presence of smear layer and smear plugs that obstruct the dentinal tubules is also an another co - factor that may not be underestimated.6 on enamel, among the resin cements tested, significant differences in the mean bond strength was found . Rely x arc showed an average mean bond strength value of 29.36 mpa, which is statistically significant compared to other dual - cured resin cements . Acid - etching creates micro porosities on the inter - prismatic enamel through which the hydrophobic monomers of the bonding agent may penetrate creating high micromechanical retention.14 rely x arc used with adper single bond 2 has shown low viscosity, which could contribute for its best penetration into the surface to provide reliable bond strength preventing micro leakage.13 - 15 the low viscosity monomer allows better mobility and distribution of free radicals inside the resin material, which can increase the polymerization reaction and the monomer conversion . This cement also contains larger amount of chemical and physical initiators, which in turn resulted in higher degree of conversion under the three polymerization conditions.13 on dentine, among the resin cements tested, significant differences in the mean bond strength was found . Rely x arc showed an average mean bond strength value of 26.48 mpa, which is statistically significant compared with other dual - cured resin cements . This could be attributed to that on application of 37% phosphoric acid, the exposed collagen of superficial demineralized dentin may provide reactive groups that can chemically interact with bonding primers . The solvent present in single bond, due to its high vapor pressure, helps in forming a framework for the creation of a resin - demineralized dentin hybrid layer, resulting in a strong micromechanical interlocking between resin and the superficially demineralized dentin . This could probably account for the higher bond strength values showed by subgroup 4 when compared to all the other groups.8 it should be considered that adhesive systems containing acetone (prime and bond nt) used with (calibra) or ethanol (excite f dsc) used with variolink ii are unable to rehydrate a surface that was dried after applying acid to form an effective hybrid layer, whereas adper single bond 2 used with rely x arc is an ethanol based adhesive, which invariably contains small amount of water . This extrinsic water together with an increase in intrinsic moisture caused by the removal of the smear layer could have resulted in rehydration of the partially collapsed collagen matrix during the adhesive application enhancing the bonding . In this study, paracem cement showed the least mean bond strength to enamel and dentine (17.63 mpa and 18.66 mpa) when compared to other cements whereas in contrast to other cements, it showed higher bond strength to dentine compared to enamel though which is not statistically significant . 11 whereas conflicting results were reported by hannig et al.16 shimada et al.17 and hikita et al.10 who demonstrated that enamel bonding with self - etch adhesives is of the same magnitude as enamel bonding after phosphoric acid etching . The results of this study could be attributed to that paracem is used with two - step self - etch adhesive system, which showed minimal etching pattern with enamel resulting in shallow inter - crystallite infiltration of the resin and lack of inter - prismatic resin tag formation, which in turn results in poor penetration of adhesive monomer,1,15 whereas on dentine, the lower bond strength could be attributed to that, self - etching priming systems combine the etching and priming steps . This can be attributed to the calcium and phosphate ions being solubilized from the apatite crystals, which are suspended in alcohol and water solvents in the primer, which limits the ability of adhesives to penetrate the primed surface, due to the buffering capacity of dentin and due to the high ion concentrations of calcium and phosphate.8 in addition to it, chemical incompatibility between chemical / dual - cured resin cements and simplified adhesive systems can negatively influence the adhesive cementation of indirect restorations, which in turn result in incomplete polymerization of resin - based materials.19 a review by kramer et al . Showed that the newer luting materials exhibit excellent flow characteristics with mean film thickness ranging from 8 to 21 m . The thickness of the luting materials used in the study was not equal to the thickness usually obtained clinically beneath the indirect restorations.20 in this study, in order to produce specimens viable for testing with a standardized thickness, a 3 mm thick layer is used . Therefore, these materials may produce different results when compared to that applied in a thin layer . The loss of specimens during microtensile specimen preparation is not uncommon . In the present study several approaches have been applied to deal with the pretesting failures: (a) exclude all pretesting failures from further statistical analysis, which obviously overestimates the actual bond strength; (b) assign a bond strength value of for instance 0 mpa to each pretesting failure . This actually penalizes the product too severely, as there was a certain bond strength; and (c) a modification of the former approach by assigning a pre - determined value to each pretesting failure, as for example the lowest -tbs value measured within the respective group 2 however, in the present study third approach is followed i.e., the pretested samples were assigned a low -tbs value measured within the respective group . In the present study, observations of both sides of the failed bonds were evaluated and it was observed that bond strength values may be accountable for the modes of failures at the bonded interface as the cements with higher bond strength values have increased cohesive failures.21 failure modes of resin cements to enamel and dentin were given for rely x arc and variolink ii predominantly failures were cohesive in resin cement . This could be attributed to the presence of the suitable bond between the resin adhesive and tooth substrate as found in accordance with the studies done by la fuente et al.,9 thaine et al.18 whereas for paracem resin cement, more failures were adhesive between the resin cement and substrate interface . This could be attributed to the higher permeability of the simplified self - etch systems, which promote faster hydrolytic degradation . In addition to hydrolytic degradation, chemical incompatibility between dual - cured resin cements and simplified adhesive systems can negatively influence the adhesive cementation of indirect restorations resulting in incomplete polymerization of resin - based materials.18 therefore, the results of the present in - vitro study provide an insight for the clinicians about the interaction of the dual polymerizing materials with etch - and - rinse and self - etch adhesives systems to enamel and superficial dentin . It can be concluded that the -tbs value obtained for group a was significantly higher than group b. among the cements tested in group a and group b, rely x arc (subgroup 4) showed higher bond strength, followed by variolink (subgroup 3), calibra (subgroup 1) and paracem (subgroup 2). When failures modes were assessed most of the specimens in rely x arc showed cohesive failures in the resin cement whereas for paracem resin cement most of the specimens debonded in adhesive mode.
The term adenoid cystic carcinoma (acc) was first used by spies in 1930 . It is more common in the minor salivary glands (> 25%) rather than in the major salivary glands (about 5%). The tumor is characterized by slow and relentless growth with a tendency for extensive local invasion . A particular feature of acc is the tumor's propensity for perineural invasion that may result in the spread along the involved nerves . A few point mutations in known cancer genes, including kras, braf and tp53 have been reported in accs . Recently, recurrent translocation t (6;9)(q22 - 23;p23 - 24) was identified resulting in the fusion of the v - myb myeloblastosis viral oncogene homolog (myb) gene on chromosome 6 to the nuclear factor i / b gene on chromosome 9 in a major number of cases . However, the extent to which other genes contribute to the disease is not well understood . Most commonly followed the method of treatment for acc seems to be surgical resection combined with radiotherapy . A 64-year - old man presented to the department of oral medicine and radiology with a 3-month history of swelling in the right anterior floor of the mouth . The patient had noted reduced salivation for 2 months along with a little discomfort in speech and mastication due to the size of the tumor . Extra - oral examination revealed a palpable right submandibular lymph node measuring around 2 cm 2 cm in size, tender and soft in consistency suggestive of an inflammatory nature of the lesion . Intra - oral examination revealed a tender, solitary, soft, and sessile swelling in the right anterior floor of the mouth measuring about 3 cm 2 cm extending mesiodistally from central incisor to premolar region and superioinferiorly from the depth of the lingual vestibule to the level of occlusion . Examination also revealed an ulcer over the swelling [figure 1]. Considering the history of reduced salivation and discomfort in mastication, provisional diagnosis of submandibular salivary sailolith and ranula was considered . Figure 164-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . Intraoral photograph shows smooth surface with well - defined borders (solid arrow). 64-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . Acc may require ultrasonography (us), computed tomography (ct), and magnetic resonance imaging (mri) for evaluating perineural, vascular, or skull base infiltration . The limitation of us includes its inability to evaluate the deep lesions . Because ct is less accurate than mri for detecting the extent of disease, mri is preferred over ct . In case of bone pain, acc is best evaluated by using t1-weighted and fat - suppressed t2-weighted and gadolinium enhanced mri . The lesion exhibits homogenous hypointense signals with ill - defined margins on t1-weighted mri and after injecting gadolinium intravenously acc shows continuous rapid or gradual enhancement pattern . On t2-weighted mri, low signal intensity corresponds to the solid type with poor prognosis and high signal intensity corresponds to either the cribriform or tubular subtypes with better prognoses . Mandibular cross - sectional occlusal radiograph of the 64-year - old patient showed no identifiable sailoliths [figure 2]. The patient's socioeconomic status did not allow use of the more expensive diagnostic imaging modalities . With a provisional diagnosis of ranula, surgical excision of the swelling figure 264-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . 64-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . The swelling was found to begin from the sublingual duct and to obstruct the submandibular duct [figures 3 and 4]. Histological investigation of the excised tissue stained with hematoxylin and eosin revealed basaloid epithelial cells arranged in cyst like spaces (duct like appearance), with many of the spaces presenting with hyalinized and mucoid appearance . The cells appeared small and uniform with deeply basophilic nuclei and a predominently cribriform architecture suggestive of acc arising from the right sublingual gland [figure 5]. Considering the patient's age and socioeconomic status, no further investigations or radiotherapy was done . Patient was advised to return for a follow - up examination once in 3 months because of the relentless growth of acc which has a tendency for extensive local invasion . Figure 364-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . Figure 464-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . Figure 564-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . Photomicrograph of the hematoxylin and eosin stained tissue (10) shows multiple basaloid epithelial cells with isomorphic and basophilic nuclei arranged in cribriform architecture (solid arrow). 64-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . 64-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . 64-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . Photomicrograph of the hematoxylin and eosin stained tissue (10) shows multiple basaloid epithelial cells with isomorphic and basophilic nuclei arranged in cribriform architecture (solid arrow). Accs may show any one of the following patterns: solid, trabecular, tubular, and cribriform . Among these cribriform type is the most common variant . Cribriform form shows the presence of extensive sheets, uniform bands and is composed of small, darkly stained, slightly separated basal cells with duct like structures, which may contain secretory products . The present case showed the most common cribriform pattern of acc . According to many authors histological subtype influence the prognosis of the disease . Histological subtypes of tubular and cribriform acc have a better prognosis than lesions with trabecular and solid patterns . Acc of the sublingual salivary gland causing obstruction of the submandibular duct is exceedingly rare: only three cases have been reported in the literature . The peak incidence is seen during the fifth and sixth decades of life and shows a female predominance . Most of the patients with acc present with a mass, which has been there for months or years . Pain is felt by a few patients and is more often associated with advanced or recurrent tumors . In the present case, a 64-year - old - male patient presented with swelling of only 3-months duration and experienced mild pain, which may be associated with traumatic ulceration on the swelling rather than due to the tumor itself . Possible treatment modalities of acc include surgical therapy, radiotherapy, chemotherapy, and combined therapy . Post - operative radiotherapy is indicated for tumors associated with perineural spread, local recurrence, and distant metastases . According to avery et al . Alcedo et al ., opened up a new possibility in the pharmacological treatment of this tumor by demonstrating its response to imatinib mesylate, a potent inhibitor of kit tyrosine kinase, an enzyme involved in the pathogenesis of this tumor . Several genomic investigations have been carried out in accs to identify biological markers of therapeutic potential . These efforts, however, have been largely unrewarding and more investigations of new targets are needed . In the present case, only surgical therapy was preferred and further management was not done due to patient's socio - economic status . In spite of only surgical management, 9-month follow - up showed no recurrence . This may be due to better prognosis associated with the less aggressive cribriform type of the lesion . Further studies are required to validate treatment with only surgery in cases of tubular and cribriform forms of accs . Acc of sublingual glands is rare and tumors causing obstruction to submandibular duct are extremely rare . Review of the literature shows that until date, only three cases have been reported . Us, ct, and mri are helpful tools in the diagnosis of accs . The diagnosis can be confirmed and the subtype identified by histological investigations . Most common method of treatment for acc seems to be surgical resection combined with radiotherapy . The present case was managed only with surgical modality and 9-month follow - up showed no recurrence . Long - term follow - up and newer biological markers of therapeutic potential are necessary to validate use of only surgery to manage tubular and cribriform accs.
Ovarian cancer is the most common cause of death among women with gynecologic malignancies and the fifth leading cause of cancer death in women in the united states . Approximately 21 550 new cases and 14 600 deaths are estimated annually in the us . There are, however, large variations in the incidence of ovarian cancer in different areas of the world . The highest incidence areas are in europe (especially austria, germany and the united kingdom) and north america [24]. The estimated number of newly diagnosed cases in the european union was 42 700 in 2004 with a mortality of 12/100 000 women / year . Metastasis of ovarian serous carcinoma to the breast and axillary lymph nodes is very rare and usually occurs very late in the course of the disease [5, 6]. Therefore, metastatic cancer to the breast from an ovarian primary is associated with shortened survival, most of the patients dying a few months after the metastasis . Reported a retrospective analysis of 29 patients with epithelial ovarian cancer and breast / axillary mass from 1990 to 2007 . Ten of them were metastatic to breast, and 19 patients had secondary primary carcinoma of the breast . The mean disease - free survival (dfs) was 14.9 versus 77.4 months for patients with epithelial ovarian cancer metastatic to breast and for patients with secondary primary breast cancer respectively . A 52-year - old woman with complaints of inguinal tenderness and increased abdominal size for three months was admitted to our hospital, izmir ataturk training and research hospital, department of gynecology, in september 2006 . An irregular, fixed pelvic mass and ascites were detected in her physical and pelvic examination . She underwent an abdominal ultrasound examination, abdominopelvic computerized tomography (ct), and a plain chest radiography . These imaging studies demonstrated ascites, bilateral adnexal masses, and a right - sided minimal pleural effusion . After preoperative evaluation, the patient underwent laparotomy and an optimal cytoreduction procedure with total abdominal hysterectomy, bilateral salpingo - oophorectomy, omentectomy, and lymphadenectomy . An 8 cm diameter tumor on the right ovary and a 10 cm tumor on the left ovary and 2 liters of ascites were detected . The frozen and final pathology was compatible with well differentiated, malignant serous carcinoma (fig . 1), with invasion of the omentum, peritoneum, and uterine wall (stage iiic). Immediate postoperative ct scan showed a solid 4 - 5 sized mass in the right inguinal region, and multiple millimetric lymph nodes . Malignant serous tumor of the ovary she received a combination therapy of carboplatin (auc = 6, d1 of a 21-day cycle) and paclitaxel (175 mg / m, on day 1 of a 21-day cycle) which was initiated in december 2006 . After 6 chemotherapy cycles, in april 2007, serum ca 125 level was 860 u / ml without any radiological improvement . Then she was treated with a combination therapy of liposomal doxorubicin (30 mg / m, on day 1 of a 21-day cycle) and gemcitabine (1000 mg / m, on days 1 and 8 of a 21-day cycle). The chemotherapy was completed in july 2008 and serum ca125 level was 95.5 u / ml at that time . The solid size of the solid mass in the right inguinal region was same, but lymph nodes were not seen in the follow - up ct of the abdomen / pelvis . Surgery was not planned because it was considered as a postoperative benign lesion by her gynecologist . Five months later, in the thorax ct, a 2 cm - sized lymph node in right axillary fossa was seen . The mammography showed a 9 mm sized opacity without malignant features in the right breast . During the follow - up, for that reason, oral etoposide (100 mg / day, 14/28 day) was started . After 6 cycles, the ca125 level increased to 914 u / ml and the axillary lymph node and intramammary lesion showed progression . Surgical biopsy of the intramammary lymph node was compatible with metastasis of ovarian serous carcinoma (fig . 3). Following this diagnosis, a combination of carboplatin (auc = 6, on day 1 of a 21-day cycle) and cyclophosphamide (750 mg / m, on day 1 of a 21-day cycle) was started in december 2009 . After 6 cycles, the number and size of axillary and intramammary lymph nodes increased and newly appearing intra - abdominal lymph nodes were seen in the ct scan . After then, the patient was treated with topotecan (4 mg / m, weekly), however she progressed after 4 months, and paclitaxel (80 mg / m, weekly) was treated with . She has been still treated by weekly paclitaxel for 4 months and has a stable radiological and clinical response . Metastatic ovarian serous carcinoma in the mammary lymph node wt-1 positivity (seen as brown) in metastatic serous ovarian cancer cells approximately 21 550 women in the united states are diagnosed with ovarian cancer annually, and an estimated 15 520 women die of the disease . Metastasis of ovarian serous carcinoma to the breast and axillary lymph nodes is very rare [5, 6]. Moreira et al . Reported a 29-year - old woman who presented with an intramammary lymph node metastasis of a borderline papillary ovarian tumor which had been resected a year before diagnosis . Reported a series of 14 cases of ovarian serous carcinoma which had metastases to breast and axillary lymph nodes . One of them was stage i at the initial diagnosis while 10 of them were stage iii and 3 of them were stage iv at the time of the diagnosis . Only one patient had stage iv disease initially with both axillary lymph node metastasis and breast lesion . The breast and/or axillary lymph node metastases were discovered at an average of 30 months after presentation (in patients with stage iii). All of them had stage iii disease and presented with a mass in the breast . Metastatic cancer to the breast from an ovarian primary is associated with a poor prognosis, with a majority of patients dying within one year [6, 11]. After two years, she presented with bilateral metastatic breast disease derived from the primary ovarian cancer . Differentiation between metastatic and primary tumors of the breast is of great importance because treatment and prognosis differ significantly . Clinical history, the presence of papillary architecture, and immunohistochemical features are important in establishing the correct diagnosis . Wt-1, the wilms tumor gene product, can be expressed in some types of ovarian tumors, other solid tumors (such as stomach, prostate, biliary and urinary system tumors, malignant melanoma) and leukemias . Most of the studies showed that serous carcinomas are usually positive and endometrioid ovarian cancers are usually negative for this marker yamamoto et al . Analyzed wt-1 immunoreactivity in 119 patients with ovarian serous cancer and showed that wt-1 positivity is positively correlated with high grade, advanced stage and higher ki-67 labeling index, higher bcl-2 expression and poorer outcome in a study . Although wt-1 positivity can be seen in many solid tumors, it has been identified as a possible marker mostly for ovarian cancer . A study of 117 patients involving primary breast cancer, breast cancer metastatic to ovary, and primary ovarian cancer showed that 76% of primary ovarian cancer patients were positive for wt-1, but none of the patients with breast cancer showed wt-1 positivity . Gross cystic disease fluid protein (gcdfp-15) and mammaglobin are both widely used and accepted markers for epithelia of breast origin . Cytoplasmic expression of gcdfp-15 and mammaglobin was observed in respectively 73.3% and 72.1% of invasive breast carcinomas in a study . These two markers were studied in paraffin - embedded tissues of the patients with primary breast cancer, serous carcinoma (ovarian and uterine), and metastatic breast cancer to the ovary . Mammaglobin was negative in all serous carcinomas, while it was more frequently and diffusely expressed in breast carcinomas when compared to gcdfp-15 (14/21 patients vs. 8/21 patients for gcdfp-15). Chia et al . Compared the diagnostic utility of these two markers in confirming a breast origin for recurrent tumors in a recent study; they demonstrated that metastases to the breast from other organs and metastatic lesions from non - breast primaries were uniformly negative for both mammaglobin and gcdfp-15, and also mammaglobin was superior to gcdfp-15 in detecting a tumor of breast origin . As a result, in addition to papillary morphology, metastatic breast lesions from ovarian serous tumor origin are usually negative for gcdfp-15 and mammaglobin and positive for wt-1 . We established the diagnosis of metastasis from ovarian cancer by using wt-1 immunoreactivity and typical papillary architecture of serous cancer cells in this case . The present patient had a primary ovarian carcinoma and had been heavily treated with multiple chemotherapy regimens . The patient has been receiving a sixth - line chemotherapy protocol and she is still alive 29 months after intramammary lymph node metastasis was detected . Although she has an advanced disease, she has no visceral organ metastasis and she has in total a 56-month survival time since diagnosis.
Healthy aging is defined as aging without disease . With the current attempts to increase the life span, understanding the molecular interactions and mechanisms involved in normal brain aging continues to be a challenge . Cerebral aging is a complex and heterogenous process that is associated with a high degree of interindividual variability . The last 20 years have witnessed a great increase in our knowledge of its basic mechanisms . Functional analyses have identified signaling pathways acting as master regulators of aging and lifespan that are conserved in many animals, suggesting that the rate of aging is not inevitably fixed, but is plastic and open to modifications . Based on experimental evidence, the evolution of aging is probably the result of determinants of neuronal vulnerability, which include altered protein interaction networks, mitochondria, reactive oxidative species and intracellular calcium homeostasis, autophagy, signal transduction pathways, stem cell proliferation, and stress resistance mechanisms . Perturbations in the functional state of these processes may lead to a state of decreased homeostatic reserve, where the aged neurons could still maintain adequate function during normal activity, although they become vulnerable . Neurons have significant homeostatic control of essential physiological functions like synaptic excitability, gene expression, and metabolic regulation . Any deviation in these physiological events can have severe consequences, as observed in aging . A recent study in a large cohort of> 10 000 persons showed that a measurable decline in generalized cortical function is already present by 45 to 49 years of age, with evidence of faster decline in older people . Dementia due to alzheimer's disease (ad) is preceded by about 5 to 6 years of accelerated decline of multiple cognitive functions; by contrast, little decline is evident in persons who do not develop ad . Compromised brain energy metabolism / oxygen delivery to neurons and blood flow differences in the regions most vulnerable to neurodegeneration are possible mechanisms of progression from healthy to unhealthy the human brain is uniquely powerful with respect to cognitive abilities, yet many neuronal networks, in particular the hippocampal and neocortical circuits that mediate such complex functions, are highly vulnerable to aging . Loss of neurons, now recognized to be more modest than previously suggested, mainly involves these specific neuroanatomical areas . Cognition and its decline associated with brain aging also seems to be variable and possibly open to modifications . Studies in humans and animal models suggest that age - related cognitive decline is more likely to be associated with alterations in synaptic connectivity than with neuronal loss and white matter changes . According to recent studies, alterations of intracellular -secretase mediated signaling pathways may be involved in synaptic pathogenesis of ad, and apolipoprotein e is suggested to enhance the toxic effects of oligomeric amyloid beta (a), causing synapse loss, a major correlate of cognitive decline in ad . Although dementia - associated hallmarks of ad pathology (neuritic plaques and neurofibrillary tangles) become less prominent with increasing age, synaptic marker abnormalities in dementia remain constant and may represent an independent substrate of dementia spanning all ages . These and other changes induce functional network disruptions in degenerative dementia, suggesting that disease progress is transmitted by neural pathways . Postmortem and in vivo magnetic resonance imaging (mri) studies of healthy brains have reported different location, extent, and severity of these changes with aging, some brain regions with greater activation being linked to better cognitive performance . Besides hemispheric asymmetry reduction they indicated increased activity in (pre)frontal regions, suggesting posterior - anterior shift models of functional brain aging . There is a strong relationship between cognitive ability and cortical fine structure in the prefrontal cortex . Postmortem studies of human brains revealed more prominent age - related changes in the anterior and posterior white matter, but not in gray matter volumes, histology showing less severe changes than the imaging methods . While in previous studies postmortem mri of white matter lesions (wmls) was less sensitive than pathology, more recent ones showed that postmortem mri is a valid tool for the assessment of subcortical pathologies . Mri investigations showed widespread age - related changes in prefrontal cortex and white matter, somatosensory cortex, and, to a lesser degree, in motor cortex, the prefrontal white matter being most susceptible to the influence of age . In cognitively normal elderly subjects, wmls were inversely correlated with gray matter volume, with greatest volume loss in the frontal cortex . Both advancing age and hypertension predict higher wml load, which is itself associated with gray matter atrophy . Low white matter grade and ventricular grade on mri are powerful determinants of long - term survival among older individuals . Recent functional neuroimaging studies indicated reduced cortical activation in the default - mode network for mild cognitive impairment patients, compared with age - matched healthy elderly persons, mainly in the retrosplenial region / posterior cingulate cortex, left hippocampus, and bilateral inferior and middle frontal areas, while increased activation for patients was observed in the medial prefrontal and bilateral middle temporal/ angular cortex, probably as a compensatory mechanism . This region is of particular interest given its contribution to memory function, working memory decline being a common complaint in healthy aging and one of the earliest signs of ad . Impaired hippocampal synaptic function is an early detectable pathologic alteration, well before amyloid plaque accumulation and cell death . Positive relationships emerged consistently between the hippocampal formation, global cognition, and memory, and between frontal measures and executive function . The hippocampal formation and the papez circuit are targeted differentially by diseases of late life . Volumetric mri of temporal and parietal brain structures distinguishes ad patients from healthy subjects, volumetry of the left and right hippocampus providing the highest diagnostic accuracy in separating these groups . Recent advances in imaging techniques (diffusion tensor imaging [dti] and magnetization transfer imaging [mti]) indicate that age - related small - vessel disease is a diffuse process affecting the whole brain and that wmls are probably only the tip of the iceberg, while decreased gray matter diffusivity might be a potential new biomarker for early ad . Age - related neuronal dysfunction involves a host of subtle changes such as reduction in the complexity of dendritic arborization and length, decrease in spine numbers and related synaptic densities, changes involving receptors, neurotransmitters, cytology, electric transmission, vascular or alzheimer - related changes, and myelin dystrophy . Together, these multiple alterations in the brain may lead to age - related cognitive dysfunction . However, every lesion in the nervous system triggers an endogenous neuroprotective reaction, combining neuroplasticity and neurogenesis, which are initiated and regulated by neurotrophic factors in a multimodal way . Extrusion of misfolded and aggregated (toxic) proteins may be a protective strategy of aging neurons . It is increasingly recognized that the correlation between neuropathological lesions and cognition is modest and accounts for about a quarter of the variance in cognition of older adults . Concerning factors that modify or mediate the association between neuropathology and cognition, it was hypothesized that the concept of resilient aging can be useful to understand mechanisms that underlie healthy aging amidst disease - related pathology . Some individuals maintain normal cognitive function despite significant brain pathology, while others suffer varying degrees of cognitive and neurological deterioration . Many aged people do not exhibit cognitive impairment or other symptoms of disease and live normal lives, but nonetheless display pathological changes that are characteristic of ad, parkinson's disease (pd), cerebrovascular disease (cvd), or other disorders . Although the best morphologic correlates of cognitive impairment / dementia are; (i) the number of neocortical neurofibrillary tangles (nfts); and (ii) loss of synapses, between 8% and 45% of nondemented, often cognitively stable older adults were found to have ad - related pathologies . Many of them showed only minimal to mild neuritic changes corresponding to braak tau stages 0-iv, while 31% to 88% showed national institute for aging and reagan institute (nia - ri) criteria of no likelihood for ad criteria . The frequency of intermediate likelihood of ad criteria ranged from 11.9% to 35.8%, and only 1.5 to 3% were scored as having a high likelihood of ad . The presence of ad lesions in nondemented aged individuals may represent ad at a stage prior to clinical expression (presymptomatic or unrecognized early forms). This is supported by observations that the mechanisms responsible for these changes in nondemented elderly appear similar if not identical to those found in ad, and their distribution corresponds to the hierarchical topographical procession associated with symptomatic ad . The concept of preclinical ad pathology has been further solidified in biomarker studies using csf a-42 and more directly in vivo positron emission tomography (pet) amyloid scanning, demonstrating that 20% to 30% of healthy elderly subjects have elevated pib signals indicative of extensive amyloid deposition . These data suggest a high frequency of preclinical ad pathology in normal elderly similar to that seen in clinico - pathologic cohorts . They further suggest that preclinical changes are not static, but progress over time . Among 555 nondemented persons with false - positive pathological nia - ri high likelihood for ad, only 1.6% corresponded to braak stage v, 0.5% to stage vi, and 2.6% to stage v - vi, while in other studies between 35% and 88% were nia - ri negative; 18% to 25% met the consortium to establish a registry for alzheimer's disease (cerad) criteria for ad . Review of the data from national disease coordinating center (ndcc) database and the nun study emphasized that there may be no documented example of truly end - stage neurofibrillary pathology with intact cognition . Although in the adult changes in thought (act) and nun studies, nondemented seniors with severe ad pathology (mean age of 89.156.9 to 90.805.2 years) amounted to 8% and 12%, respectively, most of them showed neuritic braak stage v, and frontal nft counts were slightly lower than in a comparable dementia group . Moreover, review of clinical data from those studies revealed that most of the seniors classified as nondemented were indeed significantly memory - impaired . A recent study of nondemented elderly demonstrated 62% with low and 28% with high nft levels70; 87 nondemented elderly (mean age 87 5.9 years; mean mmse 28.3) showed mean braak stage 3.00.9, a total nft score of 4.52.5, and mean neuritic density of 1.31.1, whereas ad cases showed much higher cortical neuritic and striatal amyloid plaque scores . The 90 + study revealed significantly less severe a, -synuclein, and tpd-43 pathologies, and hippocampal sclerosis in nondemented subjects, while a distribution showed no essential differences; nondemented individuals had limited hippocampal tau and neocortical a pathology . A recent clinicopathologic study of 296 persons without cognitive impairment of the religious order study (ros) and the memory and aging project (map) showed a common presence of ad pathology and macroscopic infarctions . Amyloid load was related to global cognition (p<0.05), with only a trend for nfts (p = 0.08), while nfts and macroscopic infarctions were related to episodic memory (p = 0.03 and 0.02, respectively); ad pathology and a load to working memory (p = 0.02 and 0.03, respectively). Comparing the biochemistry of ad and nondemented nonagenerians revealed the lack of clear amyloid - related pathological/ biochemical determination between both groups . A personal retrospective study of 100 nondemented elderly (mean age 81.235.47 years, mean mini mental state examination (mmse) score 29) revealed negative khachaturian criteria and cerad stage 0 in 83% and 86%, respectively, only 13% with cerad stage a and 1% stage b. braak neuritic stages ranged from 0 to iv with an average score of 2.30.8 . 12% were scored nia - ri low, and only 2% intermediate likelihood for ad . Thus, mounting evidence from clinicopathologic studies support the view that ad is a continuous spectrum between asymptomatic lesions in cognitively normal elderly and dementia, with mild cognitive impairment (mci) as a transition phase between them . Although correlations between cognitive deficits and the severity and extension of senile plaques (sp) and nfts (see ref 42) have been found, at least in those brains without other pathologies, the distinction between physiological (in nondemented subjects) and pathological aging (pa) is difficult . A postmortem classification for individuals reported to be cognitively normal before death, their brains showing plaque pathology similar in extent to ad with only minimal cortical tau pathology, may also be difficult . Recent biochemical studies found extensive overlap with only subtle quantitative differencies between a levels, peptide profiles, solubility, and oligomeric assemblies in pa and ad brains, suggesting that pa represents an initial prodromal stage of ad and that these individuals would eventually develop clinical symptoms, if they lived long enough, or an inherent individual resistance to the toxic effects of a. recent studies suggest that two independent processes (synapse - mediated and apoe - mediated) may contribute to region - specific a accumulation in nondemented individuals, and may influence the mechanisms of the regional vulnerability to a accumulation, which is prevented by apoe . A coding mutation (a673 t) in the app gene that reduces the p - cleavage of app may protect against ad and also against cognitive decline in the elderly without ad . Older persons with overall normal cognitive function and preclinical ad changes by brain autopsy usually have lower scores on cognitive function tests, particularly episodic and working memory . A biomarker studies also confirmed the relations between preclinical ad and cognition, and a clinicopathologic study indicated that elders with ad changes but without overt dementia are more likely to have memory complaints . The definition of nondemented subjects with ad pathology raises important questions regarding the cognitive profile of these people who are relatively protected from the devastating effects of ad - related lesions . Normal aging process, such that plaques and tangles are secondary to aging or that the primary aging effect is on synapses and neurons independent of these morphological ad markers . Ad is indeed a disease that accompanies human aging, but it is not an inevitable consequence of it . However, the suggestion that plaques and tangles may cause this disorder is oversimplified or even wrong, since accumulating evidence suggests that ad pathology represents effect rather than cause or at least a host response to injury, equaling adaptive or neuroprotective reactions . Many studies emphasize multiple additional pathologies in nondemented elders, in particular cerebrovascular lesions (cvls), eg, small or large cerebral infarctions, lacunes, wmls, in 22 up to almost 100% . Evaluation of 336 cognitively normal (cn) seniors from four studies revealed moderately to frequent neuritic plaque density in 47%; of these 6% also had braak stages v or vi; medullary, nigral, and cortical lewy bodies in 15%, 8%, and 4%, respectively; cerebral microinfarcts in 33% and high - level cerebral microinfarcts in 10% . The burden of brain lesions and comorbidities varied widely within each study but was similar across studies . Among 418 nondemented participants of the religious order study (mean age 88.55.3 years), 35% showed macroscopical infarcts, 8% microinfarcts, 14.8% arteriosclerosis, 5.7% both, only 37.5% being free of cvls . Up to 75% of cn seniors had various degrees of cerebral amyloid angiopathy (caa), occasional hippocampal sclerosis, lewy body pathologies in up to 18%, argyrophilic grains in up to 23%, and mixed pathologies in 7% to 14.8% . In a small autopsy series of cn elders, among 100 nondemented seniors, mild, moderate and severe intracranial atherosclerosis was present in 31%, 17%, and 6%, respectively, lacunar state in basal ganglia and/or white matter in 73%, hippocampal sclerosis in 3%, whereas only 9% were free of cvls . Lewy bodies were observed in 5%, tau pathology in brain stem in 60%, and mixed cerebral pathologies (cvls and moderate neuritic braak stages) in 6% . A recent british nondemented sample (n = 53; mean age 81.57.4 years; mmse score 27 - 30) showed maximum score neuritic plaques in 32% to 49%, nfts in hippocampus and neocortex in 81% and 30.8%, respectively, white matter changes 55% to 83.7%, small vascular disease 45%, infarcts 13.7%, lacunes 6%, and hemorrhages 10% . Thus, clinically silent pathology is widespread in normal aging, and the term healthy aging is inappropriate at the cellular level, and is manifested by regional heterogeneity in the scenario of general volume loss in the human brain . Aging is associated with progressive loss in function across multiple systems, including sensation, cognition, memory, motor control, and affect . The traditional view has been that functional decline in aging is unavoidable because it is a direct consequence of brain machinery wearing down over time . In recent years, however, an alternative perspective has emerged that, based on extensive experimental work, argues that as people age, brain plasticity processes with negative consequences begin to dominate brain functioning . Four core factors reduced schedules of brain activity, noisy processing, weakened modulatory control, and negative learning interact to create a self - reinforcing downward spiral of degraded brain function . These interrelated functions promote plastic changes in the brain that result in substantial improvement in function and/or recovery from functional losses . Neuroplasticity can be defined as the ability of the nervous system to respond to intrinsic and extrinsic stimuli by reorganizing its structure, function, and connections . It is both a substrate of learning and memory and a mediator of responses to neuronal attrition and injury (compensatory plasticity). This continuous process in reaction to neuronal activity and injury involves modulation of structural and functional processes of dendrites, axons, and synapses . However, mechanisms of neuroplasticity may vary with age, and occur in many variations and in many contexts, while common areas of plasticity that emerge across diverse cns conditions include experience dependence and circuit training . Contrary to assumptions that changes in brain networks are possible only during crucial periods of development, recent research has supported the idea of a permanent plastic brain . Novel experience, altered afferent input due to environmental changes, and learning new skills are now recognized as modulators of brain function and underlying neuroanatomic circuitry . Results in animal experiments and discovery of increases in gray and white matter in the adult human brain as a result of learning and exercise have reinforced the old concept of cognitive reserve, that is, the ability to reinforce brain volume in certain areas and thus provide a greater threshold for age - dependent deficits, or the capacity of the brain to manage pathology or age - related changes, thereby minimizing clinical manifestation . The concept of cognitive reserve and a broader theory of brain reserve was originally proposed to help explain epidemiological data indicating that individuals who engaged higher levels of mental and physical activity via education, occupation, and recreation were associated with slower cognitive decline in healthy aging and are at lower risk of developing ad and other forms of dementia . The aging process that results in loss of synapses and possible neurons may be far more detrimental for those with little brain reserve as compared with those with a high one . The construct of cognitive reserve is a set of variables including intelligence, education, and mental stimulation which putatively allows the brain to adapt to underlying pathologies by maintaining cognitive function despite underlying neuronal changes . It also indicates a resilience to neuropathological damage, and could be defined as the ability to optimize or maximize performance through effective recruitment of brain networks and/or alternative cognitive strategies . Childhood cognition, educational attainment, and adult occupation all contribute to cognitive reserve independently . Enriched environment and physical activity influence the rate of neurogenesis in adult animal model hippocampi . In people with high reserve structural and functional brain imaging studies have revealed selective changes in aging brain that reflect neural decline as well as compensatory neural recruitment, representing possible neural substrates of cognitive reserve, but its neural basis is still a topic of ongoing research . While aging is associated with reductions in cortical thickness, white matter integrity, transmitter activity, and functional engagement in the hippocampus and occipital areas, there are compensatory increases in frontal functional engagement that correlate with better behavioral performance in the elderly . Those cortical regions most consistently shrinking in aging prefrontal and parietal cortices are the same regions showing increased regional activation in aging, suggesting that losses in regional brain integrity drive functional reorganization through changes in processing strategy . Cognitive reserve allows individuals greater neural efficacy, greater neural capacities, and the ability for compensation via the recruitment of additional brain regions . Frontal and supramarginal cortical activity has been suggested to compensate for an age - related decrease in inferior - frontal junction recruitment of verbal fluency processing . Larger brain and hippocampal values, and neuronal hypertrophy were associated with preserved cognitive function despite a high burden of ad pathology (asymptomatic ad). The structural and functional imaging correlates of cognitive and brain reserve hypothesis have recently been reviewed . A complementary hypothesis of metabolic reserve is characterized by neuronal circuits that respond adaptively to perturbations in cellular energy metabolism and thereby protect against declining function, mediated by neurotrophic factor signaling, and glucose metabolism . Increased basal forebrain metabolism in mci is an evidence for brain reserve in incipient dementia . Neuroprotective effects of noradrenaline both in vivo and in vitro suggest noradrenaline's key role in mediating cognitive reserve by disease compensation, modification, or a combination of both, a viable hypothesis . The structural elements that embody plasticity include synaptic efficacy and remodeling, synaptogenesis, neurite extension including axonal sprouting and dendritic remodeling, neurogenesis, and recruitment from neural progenitor cells . Phenomenological processes that manifest plasticity are: synapse, neurite, neuronal cell bodies, anterograde and retrograde transport, cell interactions (neuron - glia), neuronal networks, and related activities . They include intraneuronal, interneuronal, and intercellular signaling through glia, and involve extracellular matrix molecules, immunoglobulins, myelin - associated inhibitors, tyrosine kinase receptors, neurotrophic and growth factors, inflammatory cytokines, and neurotransmitters . These processes are regulated by cell - autonomous and intercellular programs that mediate responses of neuronal cells to environmental input . By generating energy and regulating subcellular ca and redox homeostasis, mitochondria may play important roles in controlling fundamental plasticity processes, including neuronal and synaptic differentiation, neurite outgrowth, neurotransmitter release, and dendritic remodeling . Receptor protein tyrosine phosphorylase (rptp) regulates synapse structure, function, and plasticity . Emerging data suggest that mitochondria emit molecular signals, eg, reactive oxygen species, proteins, and lipid mediators that can act locally or travel to distant targets . Disorders in mitochondrial functions and signalling may play roles in impaired neuroplasticity and neurodegeneration . Both aging and a that as a normal product of neuronal metabolism has an essential regulatory function at the synapse, independently decrease neuronal plasticity . The major growth of a burden occurs during a preclinical stage of ad, prior to the onset of ad - related symptoms . It is associated with lower cognitive performance both in ad patients and normal elderly, but the association is modified by cognitive reserve, suggesting that this may be protective against amyloid - related cognitive impairment . On the other hand, endogenous a is necessary for hippocampal plasticity and memory within the normal cns, due to regulation of transmitter release, activation of nicotinic acetylcholine receptors, and a-42 production . The basis of age - related toxicity partly resides in mitochondrial dysfunction and an oxidative shift in mitochondrial and cytoplasmic redox potential . In turn, signaling through phosphorylated extracellular signal - regulated protein kinases is affected along with an age - independent increase in phosphorylated cyclic adenosine monophosphate (camp) response element - binding protein . Furthermore, the production of inflammatory mediators (inflammatory cytokines, interleukins, neurotrophins), activation of glia and other immune cells disrupting the delicate balance needed for the physiological action of immune processes produces direct effects on neural plasticity and neurogenesis, facilitating many forms of neuropathology associated with normal aging as well as neurodegenerative diseases . Recent evidence shows that key regulations of communication between neuron and microglia disruption in the aged brain may be one of the factors that precedes and initiates the increase in chronic inflammatory states underlying age - related impairments of cognition and hippocampal neurogenesis . Effective treatments that dampen inflammatory activity are expected to have beneficial effects on cognitive performance and neural plasticity . Functional recovery of synaptic circuitry requires that reactive synaptogenesis not exacerbate dysfunction, since aberrant misconnection by innervating the wrong target may cause misguided synaptogenesis, and inhibition of sprouting may be protective by sequestering dysfunctional neurons . Aberrant, excessive, insufficient, or mistimed plasticity may represent the pathogenic cause of neurodevelopmental and neurodegenerative disorders . Neuroplasticity is impaired in patients with ad and pd as a result of diminished growth factor expression and failure of delayed nonsynaptic neural plasticity mechanisms . Understanding normative changes in brain structure that occur as a result of environmental changes is pivotal to understanding the ability of the brain to adapt . Studies in animals and humans revealed dramatic effects of environmental enrichment, increased physical exercise documenting positive effects of mental and physical exercise, mediating brain and cognitive reserve, thus showing no compromise in daily life despite higher a plaque load . Other studies in animal models showed preventive or therapeutic action of environmental enrichment counteracting a pathology by different molecular mechanisms and by mitigating alzheimer - like pathology, and increasing synaptic immunore activity due to reduction of cerebral oxidative stress . Examination of synaptic physiology revealed that environmental experience significantly enhanced axonal transport in hippocampal and cortical neurons after enrichment, enhanced hippocampus long - term potentiation, without notable alterations in synaptic transmission . These data suggest that environmental modulation can rescue the impaired phenotype of the ad brain and that induction of brain plasticity may represent therapeutic and preventive avenues in ad . Recent studies demonstrated that the magnitude of the contribution of education is greater than the negative impact of either neuropathological burden of ad or cvls with standardized regression weights of -0.14 for hyperintensities and -0.20 for hippocampal atrophy . However, a large clinicopathologic study at 27 ad centers found no evidence of larger education - related differences in cognitive function when ad pathology was more advanced, suggesting that the advances of cognitive reserve may ultimately be overwhelmed by ad pathology . Neurogenesis or the birth of new neural cells was thought to occur only in the developing nervous system, but recent studies have demonstrated that it does indeed continue into and throughout adult life . However, the age of olfactory bulb neurones, that are assumed to be derived from neuroblasts via the rostral migratory stream (rms), has been assessed recently by measuring the levels of nuclear bomb test - derived 14c in genomic dna . Data from this study suggest that there is very limited, if any, postnatal neurogenesis in the human olfactory bulb . Certain areas of the brain may retain pluripotent precursors with the capacity to self - renew and differentiate into new neural lineages in adult mammals, nonhuman primates, and humans . Physical activity causes a robust increase in neurogenesis in the dentate gyrus of the hippocampus, a process that would implement a form of network plasticity analogous to that at the synaptic level, but occurring at the cellular network level . Neurogenesis represents a key factor of adult brain to response to environmental stimuli, and abnormalities in neurogenesis have been detected in neurodegenerative disorders such as ad . It occurs in the subventricular zone and the subgranuiar layer of the hippocampus, and follows a multistep process probably in five stages, including proliferation, differentiation, migrating, targeting, and integration phases, respectively . Stimuli that entail an increase in neuronal activity have been shown to stimulate neurogenesis and enhance survival of new neurons in the adult mammalian hippocampus . The incorporation of functional adult - generated neurons into existing neural networks provides higher capacity for plasticity, while they favor the encoding and storage of certain types of memories . Although neurogenesis continues throughout life, its rate declines with increasing age, and the proportion of neuronal stem cells that survive to become mature neuronal ceils is reduced . This may be due to intrinsic decline in neuronal stem cell responsiveness to stimulating environmental cues, to a decrease in or disappearance of these environmental cues, or to accumulation of inhibitory factors . Intrinsic properties of neural progenitor cells such as gene transcription and telomere activity change with age, which may contribute to decline in neurogenesis . While most studies indicated a correlation between decreased hippocampal neurogenesis and impaired performance in hippocampus - dependent cognitive tasks in age mice, few have demonstrated that young and aged mice are equivalent in their cognitive ability . The lack of neuronal ability to divide may be overcome by replacing damaged neurons or by restoring their function . Thus, kittappa et al revisited the molecular mechanisms responsible for neuronal renewal from stem cells, which are present in specific niches within the adult brain . The authors provided the novel notion that even non - terminaliy differentiated neural stem cells play roles in the regeneration of neurons and their synaptic function by mechanisms beyond mere cell replacement . These cells signal specific survival pathways that are worth investigating in search for novel therapeutic strategies against neurodegeneration . According to this notion, noninvasive tools to follow up synaptic function in the living brain are therefore essential for our better understanding of neuronal regeneration . Although neuronal turnover is reduced in every neurogenic region of the aged brain, neuronal precursor cells clearly survive, remain responsive to growth factors and other physiological stimuli, and can increase their activity in response to damage . Exploration of the regulation of neuronal progenitor cells in the aging brain is critical not only for understanding age - related cognitive deficits, but also for progress toward the goal of using the brain's regenerative potential to restore functional loss . Dysregulated or impaired neurogenesis may compromise plasticity and neuronal function in the hippocampus and other neuronal systems, and exacerbate neuronal vulnerability . Interestingly, increasing evidence suggests that molecular players in ad, including presenilin1, amyloid precursor protein, and its metabolites, play a role in adult neurogenesis, while alterations in tau phosporylation may interfere with the potential role of tau proteins in neuronal maturation and differentiation . This indicates a crosstalk between signaling molecules involved in both neurogenesis and neurodegeneration, and the ways by which ad - linked dysfunction of these signaling molecules affect neurogenesis in the adult brain . In ad, both increased and decreased neurogenesis has been reported and cholinergic activity may be involved in neurogenesis . However, most of these new neurons die, and fibrillar a-42 seems to be involved in generating an inappropriate environment for those neurons to mature . These findings open up prospects for new strategies that can increase neurogenesis in pathologic processes in the aging brain . Recent studies confirming the assumption that cholinergic pathology has a detrimental influence on neurogenesis suggest an attenuation of stem cells together with compensatory increased proliferation that, however, does not result in an increased number of migratory neuroblasts and differentiated neurons in ad . There are indications that neurogenesis is impaired in pd, which might be due to a lack of dopamine in the subventricular zone, but recent studies did not find evidence that dopamine has a direct effect on human stem cell proliferation in vitro . Thus, it was concluded that the number of adult neural stem cells is probably not diminished, and the proliferative capacity of the subventricular zone is maintained in the parkinsonian brain . Neural stem cells have been identified also in areas where neurogenesis does not occur under physiological conditions, such as the midbrain and striatum, suggesting that they may have the potential to be used as a non - invasive cell replacement therapy in pd . Recent studies have shown that the deleterious effects of -synuclein on newly generated neurons, in particular on their dendritic outgrowth and spine development, thus having negative impact on adult neurogenesis and neuronal maturation . Further elucidation of the mechanisms regulating the synaptic integration of adult - born neurons is not only crucial for our understanding of the age- and disease - related neuroplasticity / brain plasticity, but also provides a framework for the manipulation and monitoring of endogenous adult neurogenesis as well as grafted cells potential therapeutic applications . A major problem in studying aging is how to separate the effects of aging from disease . Cerebral aging is a complex and heterogenous process that is associated with a high variety of molecular interactions, morphological, and functional changes, summarized in table i. the interrelations between them need further elucidation . Brain aging results in loss of synapses and possible neurons, which is associated with structural changes in cerebral areas and neural neworks that are essential for cognitive and memory function . Many cognitively unimpaired eldery subjects are involved by alzheimer - related or other pathologies of various severity and extent . Knowing the substrate of the resilience to cognitive decline in the presence of abundant ad and/or mixed pathology might be crucial not only for the understanding of the pathophysiology of nondemented aged people, but also to discover new prophylactic and/or therapeutic targets for aging processes . As expected from the significant clinicopathologic correlations of synaptic and neuronal loss in ad, high - pathology nondemented controls have preserved densities of synaptophysinlabeled presynaptic terminals and dendritic spines as compared with ad dementia patients with a similar burden of plaques and tangles . Greater amounts of specific presynaptic proteins and distinct protein - protein intreactions may be components of cognitive reserve that reduce the risk of dementia with aging . They may have no significant neuronal loss, not even in vulnerable regions, such as the entorhinal cortex and hippocampus, and have lower levels of neuroinflammatory markers than pathology - matched ad patients . This resistance to ad pathology has also been related to a nucleolar, nuclear, and cell body hypertrophy of the hippocampal and cortical neurons, suggestive of a compensatory metabolic activation to face the neurotoxic effects of ad lesions . Resilience to ad is also attributed to genetic factors, particularly apolipoprotein e2 and combinations of other genetic polymorphisms . Premorbid brain volume has been found to provide protection against clinical manifestation of dementia despite evidence of ad pathology, supporting the brain reserve hypothesis of resilience to ad . Although multiple factors and possible interventions may influence cognitive reserve and susceptibility to dementia, much work is required on the mechanisms of action in order to determine which, if any, may improve the clinical and epidemiological picture . On the other hand, the unique observation of a cognitively intact woman aged 115 years with only slight tau pathology corresponding to braak stage ii, almost no plaques or vascular changes, and normal neuron count in the locus ceruleus indicates that the limits of human cognitive function extend far beyond the range that is currently enjoyed by most individuals and that brain disease, even in supercentenarians, is not inevitable . The association between vulnerability and protective factors varies with age, since the effects of these factors on the risk for ad may differ in younger (age <80) versus older (age> 80) individuals . The understanding of the dynamic of these factors at different age periods will be essential for the implementation of primary prevention treatments for ad . The importance of understanding ageing and the complex interplay of multiple influences on successful cognitive ageing is clear . Understanding how brain reserve might be influenced to minimize this is a crucial prerequisite to meaningful research in dementia and illustrates how life - long intellectual engagement can mitigate the negative impact of brain pathology even on healthy ageing . The neuronal underpinning of the dynamic compensatory mechanism opens the possibility for strategic interventions based on environmental approaches . Future work should measure the contribution of more diverse influences on cognitive reserve that might operate in early and midlife, such as socioeconomic conditions and social relationships, which might be modified through public education in order to have a positive impact on the looming public health disaster that is dementia . Recent studies in a nondemented population have shown that intellectual and physical activity lifestyle factors were not assessed with ad biomarkers, while intellectual lifestyle factors explained the variability in the cognitive performance, providing evidence that lifestyle activities may delay the onset of dementia, but do not significantly influence the expression of ad pathophysiology . The neuropathological distinction between nondemented, cognitively intact, and cognitively impaired / demented subjects, elucidation of the relationship of additional pathologies with minor often clinically latent ad lesions observed in many but not all elderly persons without cognitive impairment is important, allowing further insights into the mechanisms of brain plasticity and the basic mechanisms of adult neurogenesis warrants further experimental and prospective, well documented clinico - pathological studies of elderly individuals . In this continuously growing field, new acquisitions, derived from basic research and clinical grounds, on cognitive reserve mechanisms, neuroplasticity, and the potential application of novel therapeutic targets in neurodegeneration and aging disorders are necessary . As a basis for potential prophylactic and therapeutic options for brain aging,
Acetaminophen gained widespread popularity in the 1960s as a less toxic, analgesic antipyretic agent than aspirin . Ironically, acetaminophen is now the second leading cause of toxic drug ingestions in the united states . Acetaminophen toxicity is thus a real burden on our health care system, and hepatotoxicity due to acetaminophen overdose has become an important problem . One is the' garden variety', wherein the patients ingested large amounts of acetaminophen with a suicidal intent . The other pattern is seen in chronic alcoholics who ingest smaller amounts of acetaminophen in an attempt to relieve pain . Some cases of acetaminophen overdose have been described as parasuicidal, where the attempted suicide is more of a gesture than an act of lethal intent . The aim of our study was to determine the epidemiology of various types of acetaminophen poisoning and analyze their outcome compared with their admission characteristics . This study was conducted at nassau university medical center (east meadow, ny, usa) which represents an urban county hospital . Computer associated search was undertaken of all admission records from january 1996 to april 1999 . The records of all the patients with a discharge diagnosis of acetaminophen overdose were analyzed . Patients who were not admitted to the hospital after being assessed in the emergency room were excluded from this study . We included patients who had a history of acetaminophen ingestion reported by either the patient or the family . We went on to confirm that the patient had substantial acetaminophen ingestion by history, blood acetaminophen level> 10 mg / l, or serum aminotransferase level> 1000 two out of three of these conditions had to be met for a case to be included in our study . Patients who had elevated serum aminotransferase levels but in whom substantial acetaminophen ingestion could not be adequately confirmed were excluded from the study . Chronic alcohol abuse was defined by the diagnostic and statistical manual of mental disorders 4th edition (dsm - iv) criteria . For the patients with accidental acetaminophen ingestion, the triage was based on their clinical condition . There was a standard management plan for all the patients, as suggested by the national poison control center . Most of the patients were observed for 24 hours in the intensive care unit before being transferred to the medical floor . The following information was recorded: age; sex; race; acetaminophen dose; reason for ingestion; history of alcohol abuse or concurrent intoxication; time to presentation to the emergency room since ingestion; and n - acetylcysteine therapy . The laboratory data included peak acetaminophen level, peak aminotransferase level, prothrombin time, serum bilirubin, and serum creatinine . Using the international classification of diseases ninth revision (icd-9) code, the total number of cases of poisonings admitted during the same time period was determined to calculate the prevalence of acetaminophen toxicity . Quantitative data was analyzed using the students unpaired t - test and the mann - whitney rank sum test . Analysis of qualitative data was done using the fisher exact test and the chi - square test . Quantitative data was analyzed using the students unpaired t - test and the mann - whitney rank sum test . Analysis of qualitative data was done using the fisher exact test and the chi - square test . Four patients had to be excluded, because acetaminophen did not appear to be the culprit for their clinical state . Three others were excluded because they presented with another comorbid condition that could have been responsible for their hepatic profile . Of the 93 patients, 80 were classified as suicidal based on psychiatric evaluation, and 13 had accidentally poisoned themselves in an attempt to relieve pain . The causes of chronic pain in the accidental overdose group were toothache, chronic backache, or headache . Eighty - eight of the 93 patients were admitted to the intensive care unit for first 2448 hours monitoring . Table 1 shows that the patients with suicidal ingestion tended to be younger than the accidental group . Female to male ratio was 2:1, with a preponderance of whites in both the groups . It is evident that, although the peak acetaminophen level was higher in the suicidal subgroup (mean 121.7 97.0 vs 64.5 61.8 mg / l, p <0.05), a peak aminotransferase level> 1000 iu / l was seen more frequently in the patients with accidental overdose (39% vs 12%, p <0.05) (table 2). The renal function was overall unaffected and not significantly different between the two groups (data not shown). There were only two deaths, both in the accidental group; one of whom had a history of chronic alcohol abuse . N - acetylcysteine therapy was given to 62% of the patients in the accidental group, compared to 73% in the suicidal overdose group . The hospital stay was higher in the accidental overdose group (mean 6.4 6.1 days vs 3.9 2.7 days, p <0.01). This study did not take the dose of acetaminophen ingested into consideration due to several reasons . Accurate dose estimations could not be made in the accidental group because many of these patients had inadvertent ingestion of acetaminophen in more than one form over several days . The other problem we faced was that some patients in the accidental group were unaware of the presence of acetaminophen in some over - the - counter drugs . About 40% of the patients in the suicidal group could not give an accurate history about the dose of acetaminophen ingested . A substantial number of patients in the suicidal group were drowsy and sedated due to other concomitant ingestions . Moreover, most of the studies in the literature fail to demonstrate any clear - cut, direct relationship between acetaminophen dose and hepatotoxicity . Low acetaminophen levels (<10 mg / l) were found in 38% patients in the accidental group, compared to 10% in the suicidal overdose group . This is probably because these patients presented late, and they had ingested small doses over a prolonged period . Surprisingly, the morbidity and mortality were higher in the accidental overdose group than the suicidal group (15% vs 0%, p <0.05). First, these patients present late and sometimes the diagnosis is delayed, both of which hinder optimal antidotal treatment . The second reason could be the increased chronic alcohol use among the accidental group (39% vs 12%, p <0.05). However, the data on the amount of alcohol, duration of intake, or interval between intake of alcohol and acetaminophen are subject to recall bias, withholding of information by the patient, or non - responsive state of the patient on presentation . The presumed basis for the potentiation of acetaminophen - induced hepatotoxicity by chronic ethanol ingestion has been amply discussed . Elegant studies performed in the early 1970s established that acetaminophen, taken in therapeutic doses, is metabolized by the liver through two pathways . Most of the drug (8090%) is conjugated with either glucuronic acid or sulphates, yielding the nontoxic conjugates that are excreted by the kidney . A small proportion (5%) this metabo - lite is rendered nontoxic by conjugation with glutathione to form mercapturic acid and related conjugates that are also excreted in the urine . If the drug is taken in excessive doses, an augmented amount is converted by cytochrome p-450 to the highly reactive, toxic intermediate metabolite . It then may reach a level that overwhelms the protective mechanism of glutathione conjugation and ultimately, through covalent binding to hepatocyte proteins, leads to hepatocellular necrosis . Therapeutic doses of acetaminophen have the potential of producing liver damage if they are associated with circumstances that enhance the activity of the p-450 system leading to increased production of toxic metabolite, or that interfere with the protective mechanism by depleting the available glu - tathione . Ethanol can potentiate damage due to both of these reasons . In the present study 6/9 (66%) patients in the accidental overdose group and 7/55 (13%) in the suicidal overdose group presented to the emergency room more than 24 hours after the overdose . N - acetylcysteine therapy was given to 62% of the patients in the accidental group, compared to 73% patients in the suicidal group . Treatment with n - acetylcysteine has been very successful in preventing or ameliorating hepatic injury after suicidal acetaminophen overdose . However, the benefit of n - acetylcysteine in the syndrome of acetaminophen injury as a therapeutic misadventure is not clearly defined . The final outcome in the case of accidental overdose is dependent on a multitude of factors, and thus a large number of cases will be required for multivariate analysis to identify the role of n - acetylcys - teine . As the efficacy of n - acetylcysteine as an antidote decreases after eight hours, the treatment must be started immediately following all potentially toxic doses of acetaminophen (> 10 g). However, a large retrospective trial indicated that n - acetylcysteine therapy decreased the incidence of hepatotoxicity when administered up to 24 hours post overdose . Patients, who have an increased susceptibility to acetaminophen toxicity, particularly alcoholics, should be considered for n - acetylcysteine therapy at plasma levels that are half of those indicated in the standard graph . In addition, patients with accidental overdose acquired over a number of hours should be still considered for treatment, because in these cases plasma levels are unreliable in predicting hepatotoxicity . Our study also showed that, although patients with suicidal overdose had higher peak acetaminophen levels than the accidental overdose group, the peak aminotransferase level (> 1000 iu / l) was more often seen in the accidental overdose group (39% vs 12%, p <0.05). The suicidal overdose patients with high levels of liver enzymes more often had a history of chronic alcohol abuse and they presented late . Even when acute hepatic failure develops because of suicidal overdose of acetaminophen, these patients have a good prognosis in terms of liver transplantation and death . . In fact, none of our patients in the suicidal overdose group died or had to be referred for transplantation . In contrast to this, in the accidental overdose group 2/13 patients developed fulminant hepatic failure leading to hepatic coma and ultimately to death . Thus peak acetaminophen levels correlate poorly with hepatic dysfunction, morbidity and mortality . There are numerous studies which have shown that the total cost of treating a patient in the intensive care unit in the us is approximately $25,00035,000 per day . It is a tradition in many hospitals in the country to admit patients with acetaminophen overdose to critical care units . In our study, out of the 80 patients in the suicidal group, 75 were admitted to the intensive care unit, whereas all the 13 patients in the accidental overdose group were admitted to this unit . Because of their benign clinical course, we recommend that patients with suicidal acetaminophen overdose can be safely managed on the medical floors unless there is a history of chronic alcohol abuse or other concomitant poisonings . In our hospital in summary, acetaminophen poisoning continues to remain a significant reason for hospital admissions among young adults . The admission of the patients to the intensive care unit or ward should be determined by the degree of derangement of their clinical parameters . Patients with suicidal overdose and no history of chronic alcohol use do not need be admitted to the intensive care unit . This excludes patients for whom there is any doubt about the history of chronic alcohol abuse and those who have accidentally poisoned themselves . These are the patients who have an unfavorable clinical course and should be monitored in a critical care setting . Characteristics of patients with acetaminophen overdose clinical variables seen in the suicidal and accidental overdose groups
The exocrine pancreas functions are the synthesis, storage, and secretion of digestive enzymes into the lumen of the gastrointestinal tract where the enzymes are responsible for converting foodstuffs ingested into aqueous soluble molecules that can be absorbed by the intestinal epithelium . The two key epithelial cell types of the exocrine pancreas involved in digestive enzyme secretion are the acinar cell and the duct cell . The acinar cell is the site of digestive enzyme synthesis, storage, and regulated secretion while the duct cell provides ions and water necessary for transporting the acinar cell secreted products in the pancreatic ductal system to the intestinal lumen . Ductal cell ion secretions are rich in nahco3 which is involved in neutralization of gastric acid emptied into the duodenum . The neutralization of gastric acid is necessary because the pancreatic enzymes have optimal activity at neutral ph . The acinar cell of the exocrine pancreas has the greatest rate of protein synthesis of any mammalian organ and thus is endowed by a highly developed endoplasmic reticulum (er) system (palade, 1975; case, 1978). In addition to its functions in performing protein synthesis and processing, the er is the major storage site for intracellular calcium which when released into the cytoplasm is the mediator of regulated secretion of stored digestive enzymes into the pancreatic ductal system (petersen and tepikin, 2008). Each protein synthesized in the er must undergo specific secondary modifications and folding before it can be transported to destination organelles such as golgi, zymogen granule (storage for the digestive enzymes), and lysosome; or membrane sites . Further, because of variation in the demand for protein synthesis as a function of diet; and because protein processing in the er could be adversely affected by environmental factors such as alcohol, smoking, altered metabolism, and xenobiotics, it is likely that there are regulatory systems within the er that allow it to adapt its functions to such stressors . Alcohol abuse and smoking are key risk factors in the epidemiology of both diseases (go et al ., 2005; pandol et al ., 2009; yadav and whitcomb, 2010). In the case of alcohol abuse the increased risk for pancreatic cancer occurs largely through the effect of alcohol abuse causing chronic forms of pancreatitis (lowenfels et al ., smoking also contributes to the development of pancreatitis and is a major risk factor for pancreatic cancer independent of pancreatitis (lowenfels et al . Recent epidemiologic studies demonstrate that smoking accelerates the development of pancreatitis in alcoholic patients and may have an additive or multiplicative effect when combined with alcohol to cause pancreatitis (maisonneuve et al ., 2005; yadav and whitcomb, 2010). The mechanisms underlying the effects of alcohol and smoking on the development of pancreatic diseases an important and unexplained observation is that only a small proportion of heavy drinkers / smokers develop pancreatic diseases (pandol et al ., 2007). Although the reason for lack of development of pathology in the majority of those who drink and smoke is unknown, we hypothesize that an adaptive unfolded protein response (upr) is sufficiently robust in most individuals to prevent pathology . Proteins enter the er as unfolded polypeptides that require further processing for activation and targeting to the appropriate organelle or membrane site . Further, the er is faced with several challenges in completing these functions with high fidelity . Figure 1 illustrates many of these challenges which are often referred to as er stress that we have applied to our hypothesis of er stress for the exocrine pancreas . At the bottom of the figure a key implication from the figure is that upr activation leads to an adaptive response in the er . In this figure we hypothesize potential er stressors in the pancreas from what is known about er stressors in general (ron and walter, 2007). For example, it is likely that increases in protein synthesis rates that occur to replenish digestive enzyme stores after a meal will increase the demand for protein folding resulting in increased need for synthesis and functionality of chaperones and foldases in the er . Because the rate of unfolded / misfolded protein formation would also increase with increased proteins synthesized, the er quality control system to degrade these unusable proteins called er - associated protein degradation (erad) is required to be present and functional to rid the cell from accumulation of permanently misfolded and unfolded proteins that could present toxicity to the cell . For example, an increased folding demand will result when there is a need for increased digestive enzyme synthesis . . Altered er calcium levels as occurs during forms of pancreatitis can also cause er stress . On the left hand side of the figure these include genetic mutations in digestive enzymes; alcohol abuse; smoking; metabolic disorders such as diabetes and hyperlipidemia; xenobiotics such as drugs and intestinal bacterial metabolites; and reactive oxygen species that are generated in many of these situations as well as during acute and chronic pancreatitis . These pancreatic er stressors lead to further accumulation of unfolded and misfolded proteins which, in turn, lead to further activation of the unfolded proteins response in an attempt to adapt the pancreas to function in the face of the er stressors . There are several genetic, environmental, and disease - related stressors illustrated in figure 1 that occur in the pancreas that are likely significantly increase er stress requiring the acinar cell to activate its adaptive upr or face the possibility of cellular pathologies . Mutations in key protease digestive enzymes are known to lead to chronic forms of pancreatitis (hereditary pancreatitis) and increased rate of pancreatic cancer (whitcomb, 2010). In fact, a recent report (kereszturi et al ., 2009) demonstrates that a mutation in human cationic trypsinogen causes er stress in pancreatic cells . These results support a hypothesis that hereditary pancreatitis results from er stress caused by a mutated protease . Another example is altered er calcium levels that occur in experimental models of pancreatitis (sutton et al ., 2008). Calcium is stored in the er by its resident chaperones and foldases which act as low affinity, high capacity ca - binding proteins (gorlach et al ., 2006). Importantly for er stress as discussed below, their chaperone and foldase functions are markedly altered with perturbations in er calcium homeostasis . Thus, alterations in er calcium stores in cells can lead to pathologic consequences at least in part because of the er stress induced . Further, as discussed in more detail below, manipulation of er calcium stores in pancreatic acinar cells has been shown to activate er stress signals . Other potential stressors listed in figure 1 include alcohol, smoking, metabolic disorders, and xenobiotics as well as reactive oxygen species (ros). Except for information on alcohol abuse recently published (lugea et al ., 2010) and reviewed below, there is little information on whether these factors affecting the pancreas indeed cause er stress and whether pathology results from an insufficiently robust upr . However, alcohol, smoking, diabetes, obesity, hyperlipidemia, and drugs account for large percentage of the burden of pancreatic diseases (pandol et al ., 2007); and ros participate in their pathogenesis . Thus, the potential for roles of er stress in pancreatic disease pathogenesis is great . The adaptive upr has three major types of outputs (marciniak et al ., 2006; ron and walter, 2007; rutkowski and kaufman, 2007; kaser et al ., 2008; kim et al ., 2008 these include: (1) upregulation of the expression and function of chaperones and foldases to augment the folding and export capacity of the er; (2) activation of the er - associated protein degradation (erad) system to rid the er of accumulated unfolded and misfolded proteins; and (3) a global reduction in translation of mrna to decrease the processing demand for newly synthesized proteins . The upr also activates cell death programs under conditions of severe and/or prolonged er stress when the adaptive upr responses are exceeded or when a dysfunctional upr is unable to correct the folding disorders presented to it . As illustrated in figure 2, the mammalian upr is initiated mainly by three er stress sensor transducers located in the membrane of the er (ron and walter, 2007; rutkowski and kaufman, 2007). These three are inositol - requiring protein-1 (ire1), activating transcription factor-6 (atf6), and protein kinase rna (pkr)-like er kinase (perk). In each case the transmembrane sensor transducer determines the folding status of proteins in the er lumen and transmits this information across the er membrane to the cell cytosol . In some cases, the transmembrane sensor transducer is silenced by binding of an er chaperone called immunoglobulin - binding protein (bip) to its luminal domain . Bip is also known as also known as 78 kda glucose - regulated protein (grp78). Er stress unfolded and misfolded proteins compete for binding bip resulting in removing its silencing effect resulting in activation of the sensor . Are inositol - requiring protein-1 (ire1), activating transcription factor-6 (atf6), and protein kinase rna (pkr)-like er kinase (perk). These sensor transducers is followed specific pathways resulting in transcriptional regulation of chaperones, foldases, and components of er - associated protein degradation (erad) system and lipid synthesis for expansion of the er mediated by the combined effects of ire1 and atf6; or translational attenuation and transcriptional upregulation pathways involved in antioxidant synthesis and cell death through the transcription factor c / ebp homologous protein (chop) as in the case of perk . The participants in the pathways involved in the downstream effects of ire1, atf6, and perk activation include x - box binding protein1 (xbp1), site-1 and site-2 proteases (sp1/sp2), and eukaryotic translation initiation factor-2a (eif-2a), and activating transcription factor-4 (atf4) as discussed in the text . Activation of the er transmembrane protein ire1 initiates a response to increase the expression of er chaperones and foldases in order to assist in protein folding and transport (figure 2). Er stress induces ire1 homodimerization and trans - autophosphorylation to activate its rnase activity toward the mrna for unspliced x - box binding protein1 (xbp1-u). Activated ire1 removes a 26-nucleotide intron from xbp1-u resulting in a mrna that translates into a potent transcription factor, spliced xbp1 (xbp1-s; yoshida et al ., 2001; calfon et al ., 2002; xbp1-s, in turn, binds to er stress element (erse) and the upr element (upre) dna binding sites to upregulate many upr target genes such as the chaperones bip / grp78 and grp94 and the gene encoding xbp1-u (yoshida et al ., 2001, 2003; calfon et al ., 2002; lee et al ., this ability to increase transcription of xbp1-u leads to more substrate for expression of the xbp1-s transcription factor thus augmenting this protective response . The ire1/xbp1 pathway also leads to increased expression of foldases such as protein disulfide isomerase (pdi), enzymes for lipid synthesis for expanding the er membrane and er capacity, components of the er - associated degradation (erad), all protective mechanism to lessen er stress (yoshida et al ., the importance of xbp1 for the function of the pancreatic acinar cell has been demonstrated in xbp1 deficient mice with a transgene expressing xbp1 in hepatocytes to prevent embryonic lethality (lee et al ., 2005). These mice show abnormalities limited to the secretory organs, the exocrine pancreas, and salivary gland . The er in the pancreatic acinar cells in these animals is poorly developed accompanied by a decreased in the expression of er chaperones and marked apoptosis of the cells . Activating transcription factor-6 represents a second er transmembrane protein that is sensitive to er stress (figure 2). The n - terminal cytoplasmic domain contains a dna transcription - activating domain while the c - terminal luminal domain is sensitive to er stress . Release from bip permits atf6 transport to the golgi compartment where it is cleaved by site-1 and site-2 proteases (sp1/sp2) to a 50- to 60-kda fragment that migrates to the nucleus to activate transcription of xbp1-u and other upr target genes (shen et al ., 2002). Thus, the ire1 and atf6 pathways work in concert to upregulate an er protective response utilizing xbp1 . As indicated, xbp1-s is a potent transcription activator for many upr target genes including the molecular chaperone bip . The increased expression allows more bip available to regulate the er sensors . As discussed here, bip has several functions including acting as a chaperone, a luminal sensor of unfolded proteins, a regulator of activation of upr pathways upr, and a regulator of its own expression . Recently, we found that genetic inhibition of bip leads to augmented severity of experimental pancreatitis showing the importance of bip in cellular protective responses (ye et al ., 2010). The perk plays a key role in adjusting the cell to er stress by causing a significant attenuation of general protein synthesis (figure 2). The activation of perk by autophosphorylation (thr) leads to its phosphorylation of the alpha subunit of the eukaryotic translation initiation factor-2a the non - phosphorylated form of eif-2a in its gtp - bound form is essential for translation initiation because it recruits the first trna (trna) to the ribosomal subunits to start translation of the attached mrna . Phosphorylation of eif2a at ser51 by perk blocks eif2a - mediated initiation resulting in a general inhibition of protein synthesis . Cells with genetic deletion of perk or cells containing eif2a with position 51 containing alanine instead of serine to prevent phosphorylation do not attenuate protein synthesis with er stress (harding et al ., 2000; scheuner et al ., 2001). As a consequence, cells are more sensitive to er stress pointing out a potential protective role of the perk signaling pathway in the er stress response . Of note, previous studies in acinar cells have demonstrated that concentrations of cholecystokinin peptides which cause pancreatitis also inhibit translational initiation; and that this block in translation is mediated by phosphorylation of eif2a (perkins et al ., 1997; sans et al ., 2002). Of interest, the phosphorylation of eif2a is augmented by depletion of calcium from er stores, showing that this manipulation of er calcium homeostasis can initiate er stress . Because cholecystokinin as well as acetylcholine can also deplete er calcium stores, the findings suggest that changes in er calcium provide an er stress signal in the pancreatic acinar cell . Persistent phosphorylation of eif2a leads to specific translational upregulation of atf4 that targets genes involved in antioxidant effects including synthesis of glutathione (harding et al ., atf4 also upregulates the expression of the transcription factor c / ebp homologous protein (chop) which induces apoptosis (oyadomari and mori, 2004). The mechanisms of the erad system to rid the er of accumulated unfolded and misfolded proteins is complex, debated, and only partly understood in any cell (brodsky and wojcikiewicz, 2009; yoshida and tanaka, 2010). The degradation of erad substrates requires the multi - catalytic 26s protease called the proteasome . The proteasomes are not located in the er but in the cytoplasm adjacent to the er . Proteasomes act complementarily to the lysosome / vacuole to mediate the disassembly of proteins for recycling of their amino acids . The mechanisms of delivery of proteins from the er to proteasomes are under active investigation and likely require a proteinaceous channel in the er membrane needed to transfer proteins to the cytoplasmic located proteasomes . The modification with ubiquitin both aids in transfer and targets a protein to the proteasome . Of importance, we recently reported (lugea et al ., 2010) that alcohol feeding in animals with genetic inhibition of xbp1 expression led to a marked decrease in expression of er degradation - enhancing alpha - mannosidase - like 1 (edem1), a key participant involved in erad and targeting unfolded / misfolded proteins to the proteasome (yoshida and tanaka, 2010). These findings suggest a key role for erad in the adaptive response of the exocrine pancreas to environmental stressors . Previous studies (kubisch et al ., 2006) demonstrated activation of all three er stress sensor transducers (i.e., ire1, atf6, and perk) and their downstream pathways during the development of pancreatitis in experimental models pancreatitis suggesting involvement of er stress responses in this disorder . However, there was no information on er stress signals and the upr in providing adaptation of environmental insults such as alcohol abuse . Previous reports (gukovsky et al ., 2008) have demonstrated that long - term ethanol feeding in animal models causes little evidence of damage of the pancreatic parenchyma as determined by histology and blood concentrations of pancreatic digestive enzymes . For these reasons we designed studies to determine if alcohol abuse causes er stress and activates the upr in the exocrine pancreas; and studies to investigate the role of upr signals in adapting the pancreas to the effects of alcohol abuse (lugea et al ., 2010). For these studies rats and mice were fed control diets or ethanol containing diets for 46 weeks using the tsukamoto french intra - gastric model (tsukamoto et al ., 1985) which provides continuous feeding of the diets to the animals . As reportedly previously (gukovsky et al ., 2008), light microscopic examination of the exocrine pancreatic tissue demonstrated no obvious injury . However, careful structural examination by electron microscopy demonstrated extensive distention of the er of acinar cells and altered distribution of zymogen granules . In addition, an investigation of the redox status of the er demonstrated that there was a significant decrease in reduced and an increase in oxidized glutathione indicating that the alcohol feeding has a significant oxidative effect in the er of the acinar cell . Taken together, these findings indicated that ethanol has the oxidative effect in the er of acinar cells associated with morphologic signs of er stress . We then evaluated the effect of the alcohol treatment on the sensors and signals of the upr . The major findings were significant effects of alcohol feeding on ire1 and xbp1-s expression with smaller effects on perk induction (lugea et al ., 2010). Also, we found that a key oxido - reductase, pdi, was significantly upregulated in the pancreas of the ethanol - fed animals . Further, we found that a much greater proportion of the pdi was in its oxidized state in the ethanol - fed animals compared to control fed . Pdi is critical for protein folding by catalyzing disulfide bond formation, a key step for maturation of proteins in the secretory pathway (ellgaard and ruddock, 2005). That is, pdi requires reduced glutathione to sustain its catalytic redox cycling (zhang and kaufman, 2008). The combination of these findings suggest that pdi activity and thus appropriate folding of proteins in the secretory pathway of the acinar cell are impaired due to the oxidizing effects of alcohol feeding . The findings that alcohol feeding increased ire1 and xbp1-s expression suggested to us the possibility that these sensors and signals are important for adapting the er of the acinar cell to the er stress caused by the alcohol feeding . In order to test this possibility we obtained and tested animals with heterozygous deficiency in xbp1 . Homozygous deletion of xbp1 is lethal and pancreatic tissue specific deletion leads to lack of development of the pancreas (lee et al ., 2005). We hypothesized that there would be an incomplete adaptive response to alcohol feeding in the heterozygous animals . In contrast to wild - type animals, there were marked morphological and biochemical effects of alcohol feeding in animals with heterozygous deletion of xbp1 . Of note, alcohol feeding increased levels of xbp1-s in the pancreas of heterozygous animals to the same level observed in wild - type animals receiving the control diet but not the augmented levels observed in the pancreas with alcohol feeding in the wild - type animals . Thus, the heterozygous deletion specifically prevented the ethanol feeding - induced augmentation in xbp1-s levels in the pancreas . The predominant morphologic abnormalities in the xbp1 deficient animals receiving alcohol were a disorganized ultrastructure with decreased number of zymogen granules with the remaining ones inappropriately scattered throughout the cell and not localized to their normal apical position . There was extensive dilation of the er with occasional dense luminal inclusions, a hallmark of er stress, and a significant accumulation of autophagic vacuoles . There was also a marked decrease in levels of the digestive enzyme amylase corresponding to the decrease number of digestive enzyme storing zymogen granules . These areas showed acinar cell necrosis, apoptosis, and inflammation with replacement by stromal cells and regenerating ductular structures indicative of a response to severe injury . An analysis of the upr revealed that xbp1 deficiency prevented alcohol's effect to increase pdi while it markedly promoted perk and eif2a phosphorylation and expression of atf4, all features of prolonged and severe er stress (harding et al ., 2003). Also of importance, xbp1 deficiency decreased levels of edem1, a key participant in the erad pathway for degradation of misfolded proteins . Of note, deficiencies in edam1 have been shown to result in increased degradation of misfolded proteins via autophagy (hetz et al ., 2009) which may explain the increased autophagy observed in our studies . The results of the in vivo studies described above showing the role of the upr in adapting the pancreas to alcohol treatments allow us to propose the model illustrated in figure 3 . We suggest that alcohol abuse usually does not cause pancreatic disease because of an adaptive upr predominated by ire1 and xbp1 . The oxidative stress caused by alcohol metabolism in the er activates ire1 leading to splicing of xbp1 mrna resulting in translation of the active transcription factor, xbp1-s . This transcription factor in turn mediates the expression of chaperones, pdi, erad proteins (for example edem1), and increased lipid synthesis to expand the capacity of the er . These should all result in an attenuation of the er stress . When there is an insufficient upr response as shown in our experiments using animals with deficient xbp1, there is unresolved and augmented er stress resulting in perk activation causing global protein synthesis inhibition and even atf4-mediated activation of cell death pathways via chop expression . These are only a few events that must be occurring in the cascade of pathobiologic events resulting in the significant tissue injury we observed . This figure illustrates the results of our experiments in alcohol fed animals showing er stress from ethanol and its metabolites, acetaldehyde, and fatty acid ethanol ester (faee), by inducing an oxidative state in the er mediating misfolding and unfolding of proteins and oxidation of lipids . The er stress leads to upregulation of the ire1 pathway leading to splicing of xbp1 mrna resulting in the translation of the transcription factor xbp1-s which is transported to the nucleus where it upregulates the expression of chaperones, oxido - reductases / foldases such as pdi, erad proteins, and lipid synthetic enzymes through dna binding elements, er stress element (erse) and the upr element (upre). The activation of this pathway leads to adaptation of the pancreas to the stress of ethanol abuse and prevents pancreatic injury and disease . The findings related to the oxidative state of glutathione and pdi provide important insights for er stress and the upr in the exocrine pancreas . Figure 4 illustrates the mechanism of oxidative folding of nascent proteins (ellgaard and ruddock, 2005; hatahet and ruddock, 2009; shimizu and hendershot, 2009). The ability of pdi to mediate folding of a nascent protein is a function of its ability to catalyze disulfide pdi recognizes and binds to nascent unfolded proteins, semi - folded proteins and misfolded proteins but not correctly folded proteins through mechanisms that are not entirely known . Through electron transfer, oxidized pdi catalyzes disulfide bridge formation of the substrate protein as shown in the center of figure 4 . Oxidative folding in the er . This figure illustrates the er oxido - reductases involved in oxidative folding of nascent proteins in the er . The oxido - reductases pdi and ero1 are coupled and mediate the disulfide bridge formation and protein folding of proteins such as digestive enzymes synthesized in the er . Another product of these coupled reactions is ros . Reduced glutathione is necessary to reduce incorrectly placed disulfide bonds so that the protein can be recycled for correct disulfide bridge formation and folding . The re - oxidation of pdi is catalyzed by another oxido - reductase called er oxidase (ero1) as shown on the left side of figure 4 . Ero1 is a flavo - enzyme that is induced by the upr and it is directly oxidized by molecular oxygen in a fad - dependent reaction (shimizu and hendershot, 2009; tavender and bulleid, 2010). Fad is synthesized in the cytoplasm and easily enters the er . In the process ero1 delivers the electrons that have been transferred from pdi to molecular oxygen with resultant ros formation . It is known that pdi function and oxidative folding are dependent on ero1 (chakravarthi and bulleid, 2004; tavender and bulleid, 2010). As illustrated on the right side of figure 4, the reduction of disulfides is important for the reduction of incorrect disulfide bonds that can form during folding (chakravarthi and bulleid, 2004). Illustrated at the top of figure 4 is the suggestion that glutathione may be involved in regeneration of the oxidized state of pdi in some systems . The activities and direction of the reaction catalyzed by oxido - reductases such and pdi and ero1 are dependent on the oxidative state of their catalytic sites as well as the local environments of the catalytic sites . The results from the experiments in the exocrine pancreas do not yet provide information on the detail of the oxidative state of the oxido - reductases or about their kinetics during the er stress produced by alcohol treatments . However, the initial findings of a marked increase in the ratio of the oxidized to the reduced form of glutathione in the er and the increase in the oxidized form of pdi suggest the likelihood of significant disturbances in oxido - reductase enzyme kinetics which would lead to the findings described . In closing, we hypothesize that alcohol abuse and many other causes of pancreatitis including genetic mutations in digestive enzymes, smoking, metabolic disorders such as diabetes and hypertriglyceridemia, and drugs may cause the disease at least in part by causing er stress . The key question related to why some individuals develop disease while others do not is not known . It is certainly possible that alterations in key components of the upr through mutation (or environmental factors) are present in the pancreas of those individuals who develop tissue injury in fact, genetic variants of xbp1 have been associated with the risk of development of inflammatory bowel disease (kaser et al ., 2008) supporting this possibility . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Granular cell tumors (gct) are uncommon soft tissue neoplasms that may be asymptomatic or may present as a slow growing nodule . We present a case of granular cell tumor in the right inguinal region misdiagnosed as a reactive lymph node on cytology . A 63-year - old male presented with swelling in the right inguinal region for the past 10 years with a sudden increase in size since two months . On examination, a single, firm three cm swelling was felt in the right inguinal region . No other lesion such as an ulcer or swelling was noted in the right leg or thigh . There was no history of fever, rigors and loss of weight or appetite in the recent past . An ultrasonogram of the right inguinal mass showed circumscribed hypoechoic mass suggestive of a lymph node shadow . Cytosmears examined were moderately cellular with dual cell population, composed predominantly of oval to polygonal cells with abundant cytoplasm and round bland nuclei resembling histiocytes [figure 1]. The background showed few discrete round cells with round nuclei and scanty cytoplasm suggestive of mature lymphocytes [figure 1]. No precursor lymphoid cells were seen on cytosmears . In correlation with clinical and image findings, an impression of reactive lymph node a clinical follow - up and excision was advised if clinically suspicious or lesion further increased in size . (a) moderately cellular cytosmear showing clusters of cells with histioid appearance, lymphoid cells and few bare nuclei (mgg, 100); (b) cytological smear with loose clusters of oval to polygonal cells having basophilic granular cytoplasm (mgg, 200) an excision biopsy was performed which showed skin with a neoplasm located in lower dermis and subcutis . Individual cells appeared round to oval with abundant granular eosinophilic cytoplasm and centrally placed bland nuclei [figure 2]. Immunohistochemistry showed s100 positivity in nucleus and cytoplasm ([figure 2] inset). Section show diffusely arranged tumor cells with granular eosinophilic cytoplasm intervening adnexal structures and presence of lymphoid aggregate (h and e, 100); inset show tumor cells showing cytoplasmic and nuclear immunopositivity for s100 protein (ihc, 100) granular cell tumor is a rare benign tumor arising predominantly in skin, subcutaneous tissues and tongue . They may occur at any age, but commonly affects third to fourth decades of life . Generally, these are isolated lesions, but can be multiple in about 15% of cases . Usually, these tumors behave in a benign fashion, but malignant variant can also occur de novo or from transformation of a benign one . Earlier gct was proposed to be derived from immature skeletal muscle cells (hence, the term granular cell myoblastoma was given). However, later ultrastructure and immunohistochemistry studies have proven it to be of schwannian differentiation . Granular cell tumors show non - specific features on mri and may only be useful in assessing precise location of tumors which is essential for preoperative evaluation . In our case study, only an ultrasonogram was done . In general, cytological smears of gct show moderate to high cellularity with uniform appearing large cells arranged in syncitium and as isolated cells . Cytoplasmic granules are positive for periodic acid schiff stain and resistant to diastase . Rarely, intranuclear inclusion has also been reported . The granularity in cytoplasm of tumor cells is due to accumulation of secondary lysosomes resulting in its typical appearance on microscopy and thus deriving its name . In our case, due to the presence of lymphoid cells, the lesion was mistaken for a lymph node and the granular cells were incorrectly identified as histiocytes on cytology . Since it was a long - standing inguinal swelling with mild increase in size for the last two months on reviewing the cytosmears, the larger cells appearing as histiocytes were definitely very granular and this is not a feature of sinus histiocytes . Misdiagnosis, in our case, was due to several reasons, foremost being the site of occurrence, clinical and radiological opinion of enlarged lymph node and finally misinterpretation of cytosmears . Cytological smears in our case showed mature lymphocytes and histopathology of the excised tissue showed features of granular cell tumor with many lymphoid aggregates . This finding suggests that lymphocytes can sometimes feature in cytological smears of gct and can confuse the pathologist . Another feature on cytology that can further confound is the occurrence of stripped nuclei from tumor cells of gct resembling nucleus of lymphocyte . Granular cell tumor can rarely occur within a lymph node and it is important to know this fact to avoid a mistaken diagnosis of a metastatic tumor . Cell block is a useful technique that provides additional information from cytological specimen as it displays microhistology which can be further utilized for immunohistochemical studies . In the present case, our case further emphasizes that cell blocks should be considered in image guided aspiration as the material obtained in such cases is through a specialized invasive technique and a repeat aspiration would be cumbersome task . We present this case to create awareness among the cytologists regarding the difficulties encountered in the cytodiagnosis of granular cell tumor . The possibility of granular cell tumor should be considered when viewing single and cluster of large cells with abundant eosinophilic granular cytoplasm and vesicular nuclei on cytology.
A large fraction of all dna damages are formed at 2-deoxyguanosines (dgs). Of the four common nucleosides in dna, oxidation takes place most easily at dg residues, giving rise to a variety of products including 7,8-dihydro-8-oxo-2-deoxyguanosine (8-oxo - dg) (figure 1). 8-oxo - dg is more susceptible to oxidation than dg, and it generates a number of secondary oxidation products . The pathway leading to 8-oxo - dg is believed to involve a c8-hydroxyl radical, which also forms fapydg (figure 1). Methylating and ethylating agents preferentially react at n7, but they also alkylate o of dg, and the fraction of alkylation at o increases with harder the n7-methyl - dg (n7-me - dg) adduct is unstable, which either depurinates to form an abasic site or undergoes ring opening to generate mefapydg (figure 1). Interestingly, a vast majority of the bulky adducts are formed at either n7 or the exocyclic n position of dg . The unstable dg - n7 adducts formed by the metabolically activated aromatic amines and nitro compounds rearrange to stable dg - c8 adducts, while minor adducts at the n position of dg have also been isolated . In contrast, a majority of the metabolically activated epoxides of polycyclic aromatic hydrocarbons (pahs) form the dg - n adducts as the major products . Metabolically activated aflatoxin b1, however, forms the primary dg - n7 adduct, which undergoes ring opening to a stable formamidopyrimidine (fapy) derivative . Like the pah epoxides, the antitumor agent mitomycin c (mc) containing an aziridine ring preferentially forms the dg - n adducts . The genotoxicity and mutagenicity of many of these adducts have been investigated in prokaryotic and eukaryotic cells for the last three decades . Replication of these dna lesions do not follow a unifying mechanism, and each lesion exhibits a characteristic mutational spectrum . However, increasingly it became clear that the mutational signature of a dna lesion is directly related to the identity of the dna polymerase(s) that bypass it and the mechanism of its nucleotide insertion and extension, though additional factors such as dna sequence context play a role as well . A human cell contains at least 17 different dna polymerases (pols) to perform different functions of the cell, which include dna replication of undamaged and damaged dna, replication as part of various dna repair pathways, recombination, telomere maintenance, and other tasks . On the basis of sequence homology, pols have been divided into seven families (a, b, c, d, x, y, and rt), of which c family pols were only found in prokaryotes . In eukaryotes, the b - family enzymes are important since pol and pol of this family carry out a large fraction of nuclear dna replication, whereas pol is involved in initiation and priming . These three pols are essential for dna replication in eukaryotes . In the current model of dna replication, pol carries out a majority of leading strand dna replication of the undamaged genome, whereas pol primarily replicates the lagging strand . However, this model has recently been challenged, and data supporting the involvement of pol in both leading and lagging strand replication have been presented . The discovery of translesion synthesis (tls) dna pols in the 1990s invigorated the area of replication of dna lesions, and since then, numerous articles have been published on the catalytic and noncatalytic roles of these pols in the context of damaged dna replication . Lesion bypass is carried out principally by the y - family pols, although x- and b - family pols are also frequently involved . Like the replicative pols, these pols possess right - handed topology with the active site located in the palm domain, except that the active site is much larger in order to accommodate the dna lesions . Unlike the replicative pols, in which the finger and thumb domains ensure correct pairing with the incoming nucleotide, they are shorter and make little interaction with the template and the incoming dntp, thereby reducing the pol s ability to discriminate the accuracy of nucleotide insertion . A little finger domain assists to stabilize the y - family pol on dna . An important aspect of the y - family pols and pol of the b - family is that they lack the 35 proofreading function, making them error - prone but letting them carry out tls . From the perspective of tls, dna lesions can be broadly divided between weak and strong replication blocks . Small dna lesions such as o - me - dg and 8-oxo - dg stall but do not completely stop dna synthesis, whereas most bulky dna lesions, such as the adducts formed by the pahs and aromatic amines, are much stronger replication blocks and require the assistance of tls pols to bypass . The current paradigm on tls is as follows . When a processive dna pol encounters a blocking lesion, the pol dissociates, and a tls pol binds to the dna and incorporates a dntp opposite the lesion . In many cases, the same pol continues elongation for a few more bases before dissociating, while in other occasions this tls pol is replaced by another tls pol for the elongation steps . Tls pols exhibit higher rate of errors on unmodified templates and are also highly error - prone when bypassing most dna lesions . Soon after bypassing the lesion, the processive pol returns to continue dna synthesis . However, the actual process of pol switching is still speculative, and many related questions remain unanswered at the present time . During cellular replication, the fork utilizes many proteins, including dna pol, helicase, and single strand binding proteins, to name a few . A prerequisite for tls is the rad6/rad18-mediated monoubiquitination of proliferating cell nuclear antigen (pcna) at the highly conserved lysine k164 . Y - family pols contain ubiquitin - binding domains that confer affinity to monoubiquitinated pcna . In mammalian cells, in addition, two human rad5-related proteins, snf2 histone - linker phd - finger ring - finger helicase (shprh) and helicase - like transcription factor (hltf), transform monoubiquitinated pcna into the polyubiquitinated form . Additional dna damage response pathways, including shprh / hltf - mediated template switching, also depend on pcna ubiquitination . So, when replication by pol or pol is blocked by a dna lesion, pcna is monoubiquitinated by the rad6-rad18 protein complex and promotes the switch to a tls pol at the damage site (figure 2). Evidently, the activity of the tls pols must be tightly regulated so that they only gain access to genomic dna when there is dna damage . For example, monoubiquitination of pol inhibits its interaction with pcna, thereby preventing its activity on undamaged dna, but monoubiquitination is downregulated by the dna damaging agents . This mechanism allows optimal availability of nonubiquitinated and active pol following dna damage . Post - translational regulation of these proteins is an area where much emphasis has recently been placed . Despite the predominant role of these bypass pols in tls, it is also worth noting that there is evidence that replicative pols (such as pol) may take part in some tls events . Abridged tls scheme using pol as an example of the tls polymerase . Oxidative stress generates many different dna lesions, but 8-oxo - dg is the most widely studied dna lesion formed by reactive oxygen species such as hydroxyl radicals (figure 1). 8-oxo - dg does not strongly block dna synthesis in eukaryotic cells, as reflected by the number of progeny derived from replication of singly adducted vectors . Crystallographic studies using a high fidelity pol indicated that 8-oxo - dg adopts syn conformation at the preinsertion stage and pairs preferentially with adenine via hoogsteen base pairing in the pol active site . However, in vitro experiments using yeast pol showed that only about 10% tls takes place in the absence of any accessory proteins . Even in the presence of pcna, steady - state reactions of calf - thymus dna pol were decreased by a factor of 12 for datp and dctp incorporation opposite 8-oxo - dg . The major dna pols in mammalian cells, pol, pol, and pol extend an 8-oxo - g: a pair more efficiently than the correct 8-oxo - g: c pair.in vitro experiments showed that pol is inefficient in nucleotide insertions opposite 8-oxo - dg, but it can efficiently extend from the nucleotides inserted opposite it by pol . Yet, in human cells tls of 8-oxo - dg is largely error - free (mutation frequency (mf) 1% in duplex dna and 420% in single stranded dna). Several repair systems, including base excision repair and mismatch repair, excise 8-oxo - dg from duplex dna, justifying low mf, but most repair systems are inefficient in 8-oxo - dg repair in single - stranded dna . One might wonder why the tls of 8-oxo - dg in single - stranded dna is mostly error - free . The answer came from in vitro and cellular experiments, which determined a crucial role of pol, an x - family enzyme, in 8-oxo - dg bypass . The preference for dctp incorporation over either datp or dgtp incorporation opposite 8-oxo - dg is 12-fold by pol . However, it is remarkable that in the presence of the accessory proteins, human pcna and replication protein a (rpa), correct incorporation of dctp over other dntps opposite 8-oxo - dg increased to 1200-fold by pol . In a similar vein, pcna and rpa increased the preference for dctp over datp or dgtp incorporation opposite 8-oxo - dg by pol from 2.5-fold to 68-fold . On the basis of these results and additional data from mouse embryonic fibroblasts and human cell lines, it was suggested that the switch from pol involved pol and not pol or pol since mutations by 8-oxo - dg increased considerably in pol knockout or knockdown cells . In yeast chromosome, however, the switch to pol, which replicates 8-oxo - dg with an accuracy of 94%, was reported . In the absence of pol, dna pol -interacting protein 2 (poldip2, also known as pdip38) physically interacts with pol and increases the efficiency of elongation past 8-oxo - dg by pol, suggesting an important role of this protein in pol switch and elongation steps during tls . If pol (in the presence of the accessory proteins) were the only pol that bypasses 8-oxo - dg, mf would have dropped to less than 1% . The 420% mf, which depends on the dna sequence context and the type of assay, in single - stranded dna indicates, however, that in addition to pol, other pols bypass the lesion . In human embryonic kidney (hek) 293 t cells, depending on the dna sequence context, we observed 3850% increase in mutations induced by 8-oxo - dg, upon knockdown of pol . It is interesting that g t mutations were not significantly increased in pol knockdown cells . The increase in mutations was primarily due to an increase in dinucleotide deletions, involving the lesion and one of its neighboring bases . Others have also reported targeted one - base or small deletions in the absence of pol . It appears, therefore, that pol prevents these deletions induced by 8-oxo - dg . In addition to the dna pols, an additional factor is the participation of a homologue of muty glycosylase . Muty human homologue (mutyh) shares 41% and 79% of sequence homology to its e. coli counterpart muty and mouse homologue mmyh, respectively . Muty removes adenine from the 8-oxo - g: a mispair, which allows another chance to incorporate c opposite 8-oxo - dg by a pol . In a study in human lymphoblastoid cells, replication of 8-oxo - dg generated 14% mutants, including 6% g t and 2% targeted single - base deletions . Overexpression of mutyh reduced the g t mutations, but the deletions remained unaffected, which also suggests the role of an unidentified dna pol in the 8-oxo - dg induced deletions . While the role of these deletions in human diseases is unknown, inherited variants of mutyh in a family affected by colon cancers show a pattern of high g: c t: a mutations implicating a role of unrepaired 8-oxo - dg lesions in human cancer . Is generated at comparable levels under many conditions to 8-oxo - dg, but only a limited number of biological studies have been conducted with this lesion . Bypass efficiency of purine - ring opened fapydg is slower than 8-oxo - dg . Like 8-oxo - dg, however, the mf is highly dependent on the dna sequence context . For example, the mf of fapydg in the tgt sequence is significantly higher than when it is located in the tga sequence in both simian (cos-7) and human embryonic (293 t) kidney cells . In human cells, in some sequence contexts fapydg is more mutagenic than 8-oxo - dg, while in others the opposite is true . The major difference between the two lesions, however, is that knockdown of pol reduced the level of g t mutations induced by fapydg, in contrast to an increase in mf for 8-oxo - dg . This suggests that pol is involved in a significant fraction of fapydg induced g t mutations, whereas it carries out error - free bypass of 8-oxo - dg . It is interesting, however, that the level of small deletions increases upon replication of either 8-oxo - dg or fapydg in human cells in which pol was knocked down . Unlike 8-oxo - dg, which adopts syn conformation to pair with adenine, a structural study of the carbocyclic analogue of fapydg by a high fidelity pol (bst pol i) showed that the lesion maintains its anti conformation of the glycosidic bond during both error - free and error - prone replication . Most biological assays indicate that n7-me - dg is not mutagenic but that its ring - opened derivative mefapydg (figure 1) is mutagenic.in vitro assays showed that the mefapydg is a strong block to the high fidelity replicative dna polymerases at both the insertion and the extension steps . However, hpol and hpol as well as hrev1 and ypol together can carry out facile tls . With hpol and hpol, the predominant replication product is the error - free extension product, whereas hrev1 and ypol together accomplish entirely error - free tls . Up to 29% mutagenic tls, including each of the targeted base changes and one - nucleotide deletion products, were identified from replication products generated by hpol and hpol . In cos-7 cells, mefapydg induces g t mutations and single and dinucleotide deletions as do 8-oxo - dg and fapydg . However, cellular experiments in human cells analogous to fapydg have not been performed with mefapydg, and it would certainly be interesting to compare the replicative properties of fapydg with mefapydg using the same approach . O - methyl - dg (o - me - dg) is one of the first mutagenic dna lesions identified as a result of dna methylation (figure 1). It is highly mutagenic but is quickly repaired in a cell by multiple repair systems . A great deal of circumstantial evidence indicates that it plays a role in the etiology of human cancer . Using an intrachromosomal probe, 19% g a mutations were detected after replication of a site - specific o - me - dg in chinese hamster ovary cells deficient in the repair enzyme o - alkylguanine - dna alkyltransferase, but in repair proficient cells, mutation frequency dropped to an 1% level . Like 8-oxo - dg, it allows partial bypass of several purified dna polymerases, but pol is only slightly inhibited in vitro and inserts dctp and dttp equally well opposite o - me - dg . However, pol is strongly blocked one base before o - me - dg.o - me - dg also is a strong but not absolute block of human pol, and even though hpol inserts dttp more efficiently than dctp opposite the lesion, it preferentially extends the correct o - me - g: c pair . In the absence of accessory proteins, the human tls pol and pol produce mainly one - base incorporation products opposite this lesion, but hpol is much more efficient . Steady - state kinetic analysis showed similar efficiencies of insertion of dctp and dttp opposite o - me - dg by hpol and hpol, whereas hpol showed a higher preference for dttp insertion . Genetic studies in yeast implicate both pol and pol in the tls of o - me - dg, even though biochemical studies suggest that hpol is more efficient than hpol . Similar to 8-oxo - dg, in yeast pol is very inefficient at inserting nucleotides opposite o - me - dg, but it can efficiently extend from the nucleotides inserted opposite it by pol . As a result, the most efficient bypass can be accomplished in vitro when both pol and pol were used for the tls of templates containing o - me - dg . Even though this perspective is focused on dg lesions, it may be pertinent to mention investigations that established a specialized role of pol in efficient and error - free bypass of uv light - induced cis - syn cyclobutane pyrimidine dimers (cpds). This is due to pol s unique ability to accommodate both pyrimidine residues of this bulky lesion in its active site and perform accurate and efficient tls . Pol and pol, on the other hand, provide an alternate, albeit highly error - prone, pathway of tls of cpds . In the absence of pol and pol, tls of cpds carried out by pol is error - free, and mutations decrease to the background level . Mutations in this gene (polh) result in xpv, a variant type of the genetic disease, xeroderma pigmentosum, which is characterized by extreme sensitivity to uv light . No other dna pol exhibits such a precise and dedicated role, but the main characteristics of the other bypass pols have been established . One example is the ability of pol in the error - free bypass of dg - n lesions (discussed later). The enlarged active site of pol allows it to accommodate even the cisplatin - derived large intrastrand n7pt crystal structure analysis showed that to allow the lesion to fit in its active site, pol goes through a backbone rearrangement to stabilize the lesion and incorporate dctp opposite the two guanines . However, it also shows that the rigid backbone of the ternary complex with pol does not allow extension, which necessitates another tls pol such as pol to extend it . The potent hepatocarcinogen aflatoxin b1 (afb1) forms two major dna adducts upon metabolic activation of afb1 to afb1 - 8,9-epoxide by the liver cytochrome p450 enzymes (figure 3). The primary dna adduct, afb1n7-dg, is formed at the n7 position of dg . This adduct is chemically unstable due to the positive charge at n7, which can undergo either spontaneous depurination to generate abasic sites or ring opening to form afb1fapy - dg (figure 3). Both these adducts are mutagenic in simian kidney (cos-7) cells when the adduct is located in a ttgaa sequence, but afb1fapy - dg induces 97% mutations compared to 45% mutations by the afb1n7-dg adduct . Interestingly, in vitro tls assays showed that pol bypasses afb1n7-dg in an error - free manner, whereas it is responsible for the erroneous bypass of afb1fapy - dg . Because of the importance of these adducts in human cancer, additional structural, genetic, and in vitro studies on the two dna adducts in the future would certainly be of significant interest . Pahs are ubiquitous in our environment, and many of them, notably those with a bay or fjord region, are highly mutagenic and carcinogenic . The most extensively studied pah is benzo[a]pyrene (bp), an extremely carcinogenic chemical, which upon metabolic activation binds to dna, predominantly at the n position of dg (figure 4). Bp is metabolized by the mammalian monooxygenase enzymes to form the diastereomeric anti- and syn - benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (bpde). The metabolically activated (+) -anti bpde is a potent mutagen and the most tumorigenic metabolite of bp . It is believed to be the ultimate carcinogenic form that leads to trans- and cis - dg - n adducts (figure 4). The principal mutation in mammalian cells induced by the major dg adducts of bp is g: c t: a transversion . Bp adducts are strong blocks of replication by replicative pols, but the tls pols can bypass them at varying efficiencies . In vitro studies using either hpol or hpol showed that the bpde dg adducts allow slow bypass, which results in a high frequency of nucleotide misincorporations . In yeast, however, ()-anti - bpde mutagenesis requires pol and partially involves pol, but pol mainly contributes to deletions and insertions of 13 nucleotides . In contrast, pol performs accurate and reasonably efficient replication of the bpde dg adducts . The extent of bypass drops, and mutagenesis increases significantly in human and murine cells lacking pol . Pol s catalytic site, unlike that of pol, can only accommodate one watson crick base pair . However, it is capable of tls of many dg - n adducts, including the dna adducts formed by bp . Specifically, for the (+) -trans - anti - dg - n - bpde adduct, genetic, in vitro kinetics, and structural studies show that pol performs efficient and accurate tls . For the mutagenic tls, genetic evidence suggests that a non - y family pol inserts a wrong nucleotide (datp or dttp) opposite the adduct but that extension is performed cooperatively by pol and rev1 . Crystal structure analyses of the (+) -trans - anti - dg - n - bpde adduct showed that the active site of pol is opened up at the minor groove side of the primer the amino acid residues of the protein in the minor groove side of dna stabilizes the hydrophobic bpde ring and maintains watson crick base pairing with an incoming dctp for accurate replication . Pol also bypasses many other dg - n adducts accurately and efficiently, which includes n-(1-carboxyethyl)-dg and n - furfuryl - dg as well as much bulkier adducts formed by iq and mitomycin c (discussed in the next section). We have recently studied the minor, albeit persistent, dg - n adduct (figure 5) formed by the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (iq), a heterocyclic aromatic amine formed during high temperature cooking of meat, and two dg - nadducts (figure 6) formed by the antitumor agent, mitomycin c (mc), and its metabolite, 27-diaminomitosene (2,7-dam). Structures of the dg - n adducts formed by mitomycin c and its metabolite 2,7-diaminomitosene . The dg - n - iq adduct was studied in the three different guanines of the nari restriction site (5-g1g2cg3cc-3). As shown in table 1, in hek293 t cells mf increases upon knockdown of only pol, whereas knockdown of pol, pol, pol, or rev1 results in a reduction in mf . The greatest reduction in mf occurred when pol, pol, and rev1 were concurrently knocked down . This suggests that pol is involved in error - free bypass of the dg - n adduct formed by iq, whereas pol, pol, and rev1 cooperatively carried out mutagenic tls . Similar results were obtained with the mitomycin c adducts (table 1), indicating that they also follow analogous mechanisms . It was also established that with the increasing bulk of the dg - n adducts, the misincorporation frequency of datp relative to dctp increases significantly . Taken together, there seems to be a predictable pattern of error - free and error - prone tls of dg - n adducts by the tls pols . Exceptions to this rule, however, are the minor groove adducts -hydroxy-1,n - propano - dg and trans-4-hydroxy-2-nonenal - dg, in which case pol is inefficient in nucleotide insertion opposite the lesion, but it efficiently acts as an extender . In both these cases, pol can insert dctp opposite the lesions but is inefficient in extending the g:c pair . In contrast, pol is unable to insert a nucleotide opposite these lesions, but it can extend the g:c pair . Thus, the sequential act of pol and pol promotes efficient and error - free tls of these lesions . It is noteworthy that these are cyclic adducts with a covalent bond with n1 in addition to n of dg, suggesting that pol one of the most extensively studied dna adduct is dg - c8-aaf (figure 7), the dg - c8 adduct formed by n - acetyl-2-aminofluorene (aaf), which induces frameshift mutation in bacteria and human cells, but in simian kidney (cos-7) cells, when the adduct is placed in a single stranded plasmid, it causes largely g t mutations . However, in a subsequent study, also in cos-7 cells but in duplex dna, at the third guanine of 5-ggg-3 and 5-ggcgcc-3 (nari site), 1 and 2 frameshift mutations, respectively, were detected . The frameshift mutations at the 5-ggg-3 sequence are dependent on pol but not pol or pol . Furthermore, this pol -mediated erroneous pathway requires rad18 and ubiquitination of pcna . On the other hand, tls is only partially dependent on pol and rad18 when the adduct is situated at the nari site this indicates that the same adduct may follow different mechanisms for mutagenesis in different sequence contexts . Structures of the dg - c8 adducts formed by n - acetyl-2-aminofluorene, 1-nitropyrene, and 3-nitrobenzanthrone . The mechanism of both 1 and 2 frameshifts was suggested to follow a slipped frameshift intermediate, and while most pols are inefficient in extending such an intermediate, pol can extend them, albeit slowly . In duplex dna, dg - c8-aaf is known to rotate the guanine base to syn conformation, in contrast to an overwhelming anti conformation of an unmodified dg . Biophysical and computational studies indicate that syn conformation in a base - displaced intercalated structure of the dg adduct allows formation of stable slipped intermediates . Such intermediates, upon elongation, would cause frameshift mutations, the major types of mutations detected in bacteria and occasionally in mammalian cells (or cell - free extracts). The role of pol in bypassing misaligned adducts has been explored, which showed that depending on the base sequence, a cytosine inserted opposite the dg - c8 lesion slips to generate a 1, 2, or 3 frameshift intermediate that pol can continue to replicate, in spite of a bulge . In a crystal structure study, however, pol was able to incorporate dctp opposite the dg - c8-aaf adduct, in which tls occured without rotation of the adduct into the anti conformation, and only one hydrogen bond was formed between the lesion and dctp . This structural investigation recognized pol s ability to perform error - free replication of dg - c8-aaf, in addition to its propensity to carry out frameshifts . Like the dg - n adducts, the roles of tls dna pols in bypassing the c8-dg adduct (dg - c8-iq) (figure 5) formed by iq were explored at the g1-, g2-, or g3-positions of the nari recognition sequence after replication in hek293 t cells . Mf was the highest (50%) when the adduct was placed at g3, compared to 18% and 24% mf when the adduct was located at g1 and g2, respectively, inducing mainly g t transversions at each site . Mf of dg - c8-iq was reduced in varying degrees upon sirna knockdown of pol, pol -, pol -, or rev1-knockdown cells (table 2), indicating that these pols are involved in error - prone synthesis of this adduct . In contrast, mf was increased by 826% in pol knockdown cells, suggesting that pol bypasses the lesion accurately . Largest% change was noted with pol and pol simultaneous knockdown, which gave 70% reduction in mf . Upon simultaneous knockdown of pol, pol, and rev 1, a synergy was observed in that mf was reduced by more than 90% in each case (table 2). In vitro experiments using yeast pol confirmed that it can extend the g3:a pair more efficiently than the g3*:c pair, although it is inefficient at nucleotide incorporation opposite dg - c8-iq . It is, therefore, conceivable that pol and pol cooperatively carry out the majority of the error - prone tls of dg - c8-iq, whereas rev1 may play a noncatalytic role in assembling the tls pols . By contrast, pol is involved mostly in its error - free bypass . Similar experiments have also been conducted with dg - c83-aba, the major dna adduct formed by the carcinogen 3-nitrobenzanthrone (3-nba) (figure 7). Like dg - c8-iq, dg - c83-aba induces g t as the major type of mutations in human cells . However, the polymerase knockdown results are different . Pol and pol were found to be the major contributors of the mutagenic tls of dg - c83-aba since mf dropped by 70%, when these pols were simultaneously knocked down, although mf actually increased upon knockdown of pol alone . In contrast, pol is involved in the error - free bypass of the lesion since mf increased by 60% in pol knockdown cells . A recent in vitro presteady state kinetic investigation showed that hpol and hpol efficiently bypassed a site - specifically placed dg - c83-aba, whereas hpol and hrev1 were severely stalled by the lesion . Crystal structure analysis of dg - c83-aba at the insertion stage of hpol showed that the adduct is wedged at the hydrophobic cleft in the active site in anti conformation stabilized by a hydrogen bond between the c8 amino group and the phosphate, while the 2-deoxyribose adopts c3-endo pucker . This structure provides a model for an accurate but slow bypass of the adduct by pol . The structure of an erroneous bypass of dg - c83-aba by a pol is yet to be solved . We postulate that both pol and pol conduct error - free tls of dg - c83-aba . However, pol also extends mispairs generated by incorporation of datp by pol opposite the adduct . It is noteworthy that single - nucleotide incorporation opposite a dg - c83-aba lesion catalyzed by hpol in vitro showed that at short reaction time frames incorporation of dctp is greater than datp but that with longer time incorporation of these two nucleotides becomes comparable . Rev1 likewise is important for mutagenesis, as reflected by 60% reduction in mf upon rev1 knockdown, but as with dg - c8-iq, it probably plays a noncatalytic role by physically interacting with the other two y - family pols . The noncatalytic role of rev1 was indicated by its inability to bypass the lesion in vitro . Therefore, the c8-dg adducts dg - c8-iq and dg - c83-aba do not behave the same way with different polymerases . As mentioned earlier on the mechanism of frameshift mutations induced by dg - c8-aaf, many bulky adducts formed at the c8 position of dg, such as dg - c8-iq and dg - c83-aba, rotate the base to syn conformation, which is believed to play a structural role in frameshift mutations observed in bacteria . More frequently in mammalian cells, however, these adducts induce base substitutions . Since these purine lesions rotate to syn conformation, one can anticipate a role of pol in bypassing them, as this enzyme uses hoogsteen base pairing to select the incoming nucleotide . Pol can bypass only small dg - n adducts since n is oriented toward the major groove, and rotation to syn is inhibited for bulky dg - n adducts . In contrast, bulky dg - c8 adducts can be accommodated in the pol active site more efficiently . An example of pol s potential involvement in dg - c8 adduct bypass is its interaction with dg - c8-ap, the major adduct formed by the environmental carcinogen, 1-nitropyrene (1-np) (figure 7). Dg - c8-ap, like the other dg - c8 adduct mentioned earlier, induces predominantly g t mutations in simian and human embryonic kidney cells . The adduct, as other bulky dg - c8 adducts, exists in syn conformation in a base - displaced intercalated solution structure . Replication of dg - c8-ap stalls when in vitro bypass is conducted by the tls pols . Of the human tls pols, hpol is most proficient in bypassing it in vitro, but hpol and hpol can incorporate a nucleotide opposite the lesion . Crystal structure analyses showed that dctp incorporation opposite dg - c8-ap forces the adduct to rotate to the anti conformation to avoid steric hindrance at the minor groove side . However, this structure inhibits further extension, due to a clash with the little finger domain of the enzyme . In contrast, the adduct can maintain syn conformation when datp is inserted, in which the adenine is stacked above the pyrene ring intercalated in the helix . This structure allows further extension . Therefore, error - prone replication of dg - c8-ap potentially may occur by two tls pols, with pol being involved in the insertion stage . Another dg - c8 adduct, (5s)-8,5-cyclo - dg, a cyclic dna adduct containing a covalent bond between c8 of guanine and 5 c of 2-deoxyribose, was investigated in human cells, which showed that pol, pol, and pol but not pol are involved in tls . Unlike the dg - n adducts, therefore, a pattern for tls of the dg - c8 adducts could not be determined . For example, pol is involved in extension of the correct pair of the dg - c83-aba, whereas it extends the wrong pair with dg - c8-iq . Studies on additional dg - c8 adducts might give us a clue as to why they fail to follow a unifying mechanism of tls . Genetic studies in repair and replication competent cells provide data on the outcome of the damage, and a comparison of these in genetically altered cells (including knockout or knockdown of specific genes) has been employed to investigate the role of each tls pol . In vitro experiments using purified pols and accessory proteins elucidate how each pol can deal with the dna damage, whereas structural and computational studies give a more intimate snapshot of the lesion bypass . Each approach has its limitations, and consequently, combined approaches are essential to comprehend the mechanism of tls of a dna lesion . Mechanistic information on replication of the dna lesions is critical to follow the underlying process for the development of cancer, aging, and various other diseases . These fundamental studies are now paving the way to application of the acquired knowledge toward therapeutic application, as inhibiting the activity of some of the tls pols may enhance the effect of an antitumor agent . As yet, more tls work has been done with the pols from prokaryotes and archaea than from eukaryotes . It is certain that this dynamic area of research is still in its early stage and will continue to enrich the field of toxicology with many novel findings.
Endometrial stromal sarcomas (ess) are uncommon mesenchymal tumors of the uterus which are composed of cells closely resembling normal proliferative endometrial stromal tissue . Traditionally, ess were classified into low - grade and high - grade ess based on mitotic rate . High - grade tumors however, have little resemblance to original endometrial stroma . Therefore, high - grade tumors are presently classified as undifferentiated endometrial or uterine sarcoma . In this classification the differentiation between low - grade and undifferentiated tumors is not made on mitotic count but on the presence of nuclear pleomorphism and necrosis . Standard treatment for ess is total abdominal hysterectomy and bilateral salpingo - oophorectomy . When there is extra - uterine spread complete debulking of all tumor is recommended . We present a patient presenting with rectal bleeding, based on a very late recurrence of a formerly misdiagnosed low grade ess metastasized to the colon . A 78 year old woman presented with rectal bleeding and recurrent lower urinary tract infections in july 2009 . Her medical history showed a total abdominal hysterectomy and bilateral salpingo - oophorectomy because of a uterus myomatosus with severe blood loss in 1983 and a laparotomy with excision of a retroperitoneal cyst in 1992 . Our patient underwent a colonoscopy which revealed a malignant circumferential obstructing tumor 28 cm from the anal verge (fig . Computed tomography showed a suspicious tumor of 8 cm, at the site of the sigmoid, closely related to the urinary bladder . Urine analysis revealed some atypical cells, but no signs for urinary tract infection or malignant cells . A hartmann's resection was performed with a partial cystectomy which was closed primarily and the patient received a urinary catheter for one week . One month postoperative she underwent intraperitoneal stomal revision, because of a retracted necrotizing colostomy . Pathologic examination showed a submucosal low grade ess of 8 cm in diameter arising from the colon with nodular invasion of the mesocolon and three lymph nodes which all contained metastases of ess (figs . 2 and 3). Resection margins were free from tumor . In the context of this finding revision of the original resections of the retroperitoneal cyst and the uterus and adnexa this revision revealed, both histomorphological as well as immunophenotypical, an equivalent aspect as the newly resected colon tumor . Final pathologic diagnosis therefore, was a very late colonic recurrence of a low grade uterine ess . Endometrial stromal sarcomas are rare tumors, contributing for 0.2% of all uterine malignancies and 715% of all uterine sarcomas . Initially, histological classification consisted of carcinosarcomas (40%), leiomyosarcomas (40%), ess (15%) and undifferentiated sarcomas (5%). Currently, the carcinosarcomas have been reclassified as a differentiated or metaplastic form of endometrial carcinoma according to the international federation of gynaecology and obstetrics (figo) classification . Ess are difficult to diagnose because of close resemblance to normal proliferative endometrial cells, and because of the rarity of the condition . Prior to surgical resection ess are often misdiagnosed as leiomyoma or other uterine benign diseases and after resection also histological examination frequently misses the diagnosis . Incidence peaks occur in premenopausal women between 40 and 55 years of age who are asymptomatic or present with vaginal bleeding or pelvic or abdominal pain . Ess can occur in patients with endometriosis and in patients with a prolonged exposure to estrogens, e.g. Patients with polycystic ovarian disease, or after estrogen use or tamoxifen therapy . It is thought that the exposure to estrogens causes a proliferative effect on the endometrial stroma . Patients with stage i ii tumors have a 5-year overall survival of 89.3% compared to 50.3% in patients with stage iii iv tumors . Tumor behaviour is apprehensive for a tendency of late recurrence . In patients with stage i disease, the median time to recurrence is 65 months compared to 9 months in patients with stage iii recurrences occur in 3656% of patients, most frequently in the pelvic cavity or the lungs . Metastatic spread to the cerebrum and bones and invasion in the great vessels have also been described . Recurrences arising in the small bowel and colon are intimately associated with endometriosis . As the rectosigmoid has the highest incidence of intestinal endometriosis, ess arising within endometriosis our patient developed a very late recurrence in the rectosigmoid, although she had no history of endometriosis . Pathologic revision did not reveal any signs of endometriosis, but the presence of a small endometriosis spot as an origin for the metastasis cannot be excluded . Standard treatment for ess is total abdominal hysterectomy with bilateral salpingo - oophorectomy, even at premenopausal ages . They are often sensitive to hormones and it is presumed that patients preserving their ovaries have a higher risk of recurrence and poorer overall outcome . Some studies advocate considering re - exploration for removal of ovaries while others state that ovarian preservation has no effect on recurrences or overall survival . In premenopausal women ovaries are frequently preserved, which does not affect the risk of recurrence in stage i patients . Lymph node metastases have been found in 7% of patients with low - grade ess . Performing a lymph node dissection, regardless of the finding of any microscopic positive nodes, five - year survival is 85.7% in lymph node positive patients compared to 95.2% in lymph node negative patients . Post - operative radiotherapy decreases the risk of local recurrence, especially in grade ii postoperative treatment with chemotherapy could might be an option in patients with hormone - unresponsive tumors and for inoperable patients . Hormonal therapy can be an important treatment because of the hormonal sensitivity of the tumor . Adjuvant progestagens should be considered in patients with ess . In patients with completely resected low - grade ess tamoxifen and estrogens are contraindicated because of the possible stimulative effect on disseminated endometrial stromal cells . Our patient underwent a hysterectomy in 1983 because of a uterus myomatosus accompanied with severe blood loss and excision of a retroperitoneal cyst in 1992 . That means our patient probably had stage i ess in 1983, which was initially not recognized as ess, but was probably regarded as a cell rich variant of leiomyoma . After nine years she developed the first retroperitoneal recurrence . At this time the tumor was detected as a malignant mesenchymal tumor, but was not recognized as ess . A recurrent ess localized in the submucosa of the colon, in the absence of endometriosis, with fixation to the urinary bladder is a very rare finding . The accompanying lymphogenic spread is uncommon . During the first surgery, at 52 years of age, our patient underwent a hysterectomy for a uterus myomatosus with bilateral oophorectomy . She never took hormonal replacement therapy except for local estrogens for a few weeks and she was not familiar with endometriosis . Prior to her third surgery in 2009, she was diagnosed with recurrent lower urinary tract infections . Urine analysis revealed some atypical cells, presumably due to tumor invasion in the urinary bladder . Final pathologic examination of the operatively removed tumor however, could not confirm spread of tumor into the bladder wall, since no urinary bladder wall was recognized in the specimen . Patient was discussed in our multidisciplinary oncology panel and because of the controversy about adjuvant therapy in the treatment of ess, in combination with the radical surgery and the long disease free period after the primary presentation and the first recurrence, no additional treatment was advised and follow up will take place on a regular base once a year . Tumor behaviour is apprehensive for a tendency of late recurrence . In patients with stage i disease, the median time to recurrence is 65 months compared to 9 months in patients with stage iii recurrences occur in 3656% of patients, most frequently in the pelvic cavity or the lungs . Metastatic spread to the cerebrum and bones and invasion in the great vessels have also been described . Recurrences arising in the small bowel and colon are intimately associated with endometriosis . As the rectosigmoid has the highest incidence of intestinal endometriosis, ess arising within endometriosis our patient developed a very late recurrence in the rectosigmoid, although she had no history of endometriosis . Pathologic revision did not reveal any signs of endometriosis, but the presence of a small endometriosis spot as an origin for the metastasis cannot be excluded . Standard treatment for ess is total abdominal hysterectomy with bilateral salpingo - oophorectomy, even at premenopausal ages . They are often sensitive to hormones and it is presumed that patients preserving their ovaries have a higher risk of recurrence and poorer overall outcome . Some studies advocate considering re - exploration for removal of ovaries while others state that ovarian preservation has no effect on recurrences or overall survival . In premenopausal women ovaries are frequently preserved, which does not affect the risk of recurrence in stage i patients . Lymph node metastases have been found in 7% of patients with low - grade ess . Performing a lymph node dissection, regardless of the finding of any microscopic positive nodes, five - year survival is 85.7% in lymph node positive patients compared to 95.2% in lymph node negative patients . Post - operative radiotherapy decreases the risk of local recurrence, especially in grade ii postoperative treatment with chemotherapy could might be an option in patients with hormone - unresponsive tumors and for inoperable patients . Hormonal therapy can be an important treatment because of the hormonal sensitivity of the tumor . Adjuvant progestagens should be considered in patients with ess . In patients with completely resected low - grade ess tamoxifen and estrogens are contraindicated because of the possible stimulative effect on disseminated endometrial stromal cells . Our patient underwent a hysterectomy in 1983 because of a uterus myomatosus accompanied with severe blood loss and excision of a retroperitoneal cyst in 1992 . That means our patient probably had stage i ess in 1983, which was initially not recognized as ess, but was probably regarded as a cell rich variant of leiomyoma . After nine years she developed the first retroperitoneal recurrence . At this time the tumor was detected as a malignant mesenchymal tumor, but was not recognized as ess . A recurrent ess localized in the submucosa of the colon, in the absence of endometriosis, with fixation to the urinary bladder is a very rare finding . The accompanying lymphogenic spread is uncommon . During the first surgery, at 52 years of age she never took hormonal replacement therapy except for local estrogens for a few weeks and she was not familiar with endometriosis . Prior to her third surgery in 2009, she was diagnosed with recurrent lower urinary tract infections . Urine analysis revealed some atypical cells, presumably due to tumor invasion in the urinary bladder . Final pathologic examination of the operatively removed tumor however, could not confirm spread of tumor into the bladder wall, since no urinary bladder wall was recognized in the specimen . Patient was discussed in our multidisciplinary oncology panel and because of the controversy about adjuvant therapy in the treatment of ess, in combination with the radical surgery and the long disease free period after the primary presentation and the first recurrence, no additional treatment was advised and follow up will take place on a regular base once a year . In conclusion, low - grade ess are uncommon tumors of the uterus with an indolent clinical course and favourable prognosis . Total abdominal hysterectomy with bilateral salpingo - oophorectomy is the treatment of choice . In patients with a history known for ess, a recurrence if recurrence occurs, radical surgery should be performed and adjuvant hormonal therapy may be considered . There was no financial support for the conduct of the study and the writing of the manuscript . Written informed consent was obtained from the patient for publication of this case report and accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal on request.
Estimation of lung volume is used to help categorize the type, severity, and progression of lung diseases, and their response to therapy.1 the overestimation of total lung capacity (tlc) by body plethysmograpy compared with high - resolution computed tomography (hrct) (or, as some see it, the underestimation by hrct compared with plethysmography) is well described in the literature.24 the degree to which tlc measured by body plethysmography differs from hrct in copd is not understood . This may have significant implications for determining patient eligibility for therapies such as lung volume reduction surgery, characterizing a prospective lung transplant recipient s lung volume dimensions, or assessing the response to treatment.5 our objective was to describe the degree to which tlc determination by body plethysmography differs from hrct . A retrospective review was performed on 71 sequential patients referred to our outpatient clinic for the evaluation of copd between september 2008 and december 2008 . Patients who met the following criteria were extracted: (1) obstructive physiology evidenced by forced expiratory volume in 1 second (fev1)/forced vital capacity (fvc) 70 and tlc 80, (2) no or minimal radiographic abnormalities, or (3) complete pulmonary function tests (pfts) and hrct within 3 months . All patients underwent pfts that were performed on a body plethysmograph (vmax spectra 22d/62j; carefusion, yorba linda, ca) according to the guidelines of the american thoracic society / european respiratory society . Tlc was obtained as per american thoracic society / european respiratory society standards.6,7 patients were excluded if they failed to meet criteria for reproducibility, which was defined as the patient demonstrating at least three functional residual capacity (frc)pleth values that agree within 5% (two patients), or if they failed to perform three to five technically satisfactory panting maneuvers at frequencies at or around 1 hertz (two patients). Hrct scans with 0.75 and 5 mm contiguous slices were performed at full inspiration using a 16 or 64 mdct scanner (somatom sensation; siemens medical systems, erlangen, germany). Patients underwent helical computed tomography (ct) of the entire lung at maximum inspiration in the supine position . Custom software (pulmonary workstation plus, vida diagnostics, inc, coralville, ia) was used to determine total lung volume, tissue volume, and air volume in milliliters, and mean lung density in hounsfield units (hu) for each patient . Percent emphysema was determined as the percentage of voxels below thresholds of 950 and 910 hu . Total volume at full inspiration represents the tlc by hrct for all analysis (figure 1). Hrct scans were excluded from analysis for radiographic abnormalities other than emphysema including parenchymal consolidation, pleural effusion, and previous lung volume reduction surgery (lvrs) or transplant surgery (two patients). Patients were also excluded from analysis when the vida software was unable to process the scan due to technical difficulties, including failure to identify the tracheobronchial tree (six patients). Student s t - test was used for comparison of tlc by plethysmography and hrct . Multiple linear regressions were used to determine which variables accounted for the ability to predict the difference between tlc measured by plethysmography and hrct . A retrospective review was performed on 71 sequential patients referred to our outpatient clinic for the evaluation of copd between september 2008 and december 2008 . Patients who met the following criteria were extracted: (1) obstructive physiology evidenced by forced expiratory volume in 1 second (fev1)/forced vital capacity (fvc) 70 and tlc 80, (2) no or minimal radiographic abnormalities, or (3) complete pulmonary function tests (pfts) and hrct within 3 months . All patients underwent pfts that were performed on a body plethysmograph (vmax spectra 22d/62j; carefusion, yorba linda, ca) according to the guidelines of the american thoracic society / european respiratory society . Tlc was obtained as per american thoracic society / european respiratory society standards.6,7 patients were excluded if they failed to meet criteria for reproducibility, which was defined as the patient demonstrating at least three functional residual capacity (frc)pleth values that agree within 5% (two patients), or if they failed to perform three to five technically satisfactory panting maneuvers at frequencies at or around 1 hertz (two patients). Hrct scans with 0.75 and 5 mm contiguous slices were performed at full inspiration using a 16 or 64 mdct scanner (somatom sensation; siemens medical systems, erlangen, germany). Patients underwent helical computed tomography (ct) of the entire lung at maximum inspiration in the supine position . Custom software (pulmonary workstation plus, vida diagnostics, inc, coralville, ia) was used to determine total lung volume, tissue volume, and air volume in milliliters, and mean lung density in hounsfield units (hu) for each patient . Percent emphysema was determined as the percentage of voxels below thresholds of 950 and 910 hu . Total volume at full inspiration represents the tlc by hrct for all analysis (figure 1). Hrct scans were excluded from analysis for radiographic abnormalities other than emphysema including parenchymal consolidation, pleural effusion, and previous lung volume reduction surgery (lvrs) or transplant surgery (two patients). Patients were also excluded from analysis when the vida software was unable to process the scan due to technical difficulties, including failure to identify the tracheobronchial tree (six patients). Student s t - test was used for comparison of tlc by plethysmography and hrct . Multiple linear regressions were used to determine which variables accounted for the ability to predict the difference between tlc measured by plethysmography and hrct . Demographics, pulmonary function data, and hrct results obtained from the 59 patients are reported in tables 13 respectively . Pulmonary function testing and hrct were performed within 3 20 days of each other . The subject population included 18 patients (31%) with global initiative for chronic obstructive lung disease (gold) ii disease, 22 patients (37%) with gold iii disease, and 19 patients (32%) with gold iv disease.8 total lung volume as measured by plethysmography correlated significantly with that measured by hrct (r = 0.92, p <0.01) (figure 2). Tlc measured by plethysmography (average 6.46 1.28 l) was larger than that determined by inspiratory hrct (5.34 1.20 l) in most patients (p <0.05). In bland altman analysis, the average of the two measurements is thought to represent the true value . As the true value increases the difference between the two modalities also increases (figure 3). The mean tlc measured by plethysmography was 1.12 l greater than tlc measured by hrct . We found fev1 to be significantly correlated with percent emphysema and mean lung density, a result which agrees with that reported by baldi et al.9 fev1 was not significantly correlated with lung volume measured by plethysmography or hrct, however, or with the difference between plethymography and hrct (table 4). As has been reported by others,1013 there was fair correlation between tlc measured by both plethysmography and hrct with indices of airflow obstruction (fev1/fvc and fvc%), static lung volumes (residual volume, percent predicted [rv%], total lung capacity, percent predicted [tlc%], functional residual capacity, percent predicted [frc%], inspiratory capacity, percent predicted), and percent emphysema . Both tlc by plethysmography and hrct showed weak but significant inverse correlations with diffusion impairment (table 5). The difference between tlc as measured by plethysmograhpy and hrct correlated significantly with static lung volumes (rv%, tlc%, and frc%) (table 5, figures 4 and 5). In patients with more severe air trapping and hyperinflation, the difference between tlc measured by plethysmography and that determined by hrct increased . Using multiple linear regressions, rv% and frc% independently predicted the differences between tlc measured by plethysmography and hrct (table 6). Thoracic gas volume (tgv) can be measured in several different ways . In patients with copd, body plethysmography is usually preferred to nitrogen washout and gas dilution techniques because it is argued that the latter methods are unable to measure poorly ventilated or unventilated areas of the lung.1416 while body plethysmography readily measures trapped air not in communication with the airways, it is not without its own inherent errors, particularly in patients with increased airway resistance . Tgv is calculated using boyle s law which states that pressure and volume are inversely proportional when temperature is constant . Within the body box, pressure at the mouth may not reflect true pressure in the alveoli if airway resistance increases.17,18 if alveolar pressure is underestimated, thoracic gas volume will be overestimated . Rodenstein et al demonstrated this error in the measurement of lung volumes by plethysmography when airflow obstruction occurs, and found thoracic gas volume measured from pressure swings at the mouth (tgvm) to be 1 or more liters greater than thoracic gas volume measured from pressure swings in the esophagus (tgves), an indirect measurement of pleural pressure.19 the authors went on to qualify this overestimation of tgv in the setting of airflow obstruction as being dependent on panting frequency.20 tgvm was 1 l greater than tgves at panting frequencies of 2 hz in asthmatics, while no difference was seen in those without asthma . At higher frequencies, the discrepancy further increased in asthmatics, again with no difference in those without asthma . At lower panting frequencies, no difference was found between the two groups.21 this relationship between panting frequency and the overestimation of lung volume by plethysmography has also been described in patients with copd.22,23 hrct is an important method for routine testing for copd . With dedicated post - processing software, rapid and reproducible estimates of tissue and air volume, mean lung density, percent emphysema, and airway anatomy are now available.24,25 standardized computer - generated hrct instructions have improved reproducibility and accuracy.26,27 lung volume measurement by hrct remains fraught with predictable and unpredictable errors.28 supine positioning during hrct results in a reduction in the size of the various subdivisions of the lung.2932 in normal subjects, vital capacity (vc) falls less than 10% when passing from the upright to the supine position,33,34 but a reduction of up to 25% has been described in patients with diaphragm dysfunction and respiratory muscle weakness, conditions commonly seen in patients with copd.35 however, postural changes in vc in patients with copd have not been well described . Unpredictable errors in the measurement of lung volumes by hrct include difficulty with maximum inspiratory maneuvers and breath - holding techniques during scanning.36 using spirometric gating, patients with severe airflow obstruction have not been shown to reproducibly achieve maximum inspiratory volumes during hrct.37 in similar patient populations, previous authors have shown strong correlations between tlc measured by plethysmography and hrct (zaporozhan et al, coxson et al, and gierada et al reported r = 0.90, 0.88, and 0.87, respectively).24 given what we know about the limitations of plethysmography and hrct in copd, we expected to find a relationship between severity of airflow obstruction and the differences in the measurement of tlc between plethsmography and hrct . If tlc by plethysmography is potentially overestimated in the setting of increased airway resistance, we postulated that as fev1 decreased there would be greater differences between tlc measured by plethysmography and hrct . We further hypothesized that increased airways resistance and subsequent air trapping and hyperinflation38 would be associated with the overestimation of lung volumes by plethysmography . A recent paper by odonnell et al compared lung volumes by plethysmography and helium dilution with hrct in copd,39 with plethysmographic tlc found to be significantly greater than hrct values, and plethysmographic overestimation of tlc reported to be greatest among subjects with fev1 <30% of predicted . This was not found to be the case in the current study as differences between tlc measured by plethysmography and hrct did not change across gold stages . This relationship between airflow obstruction and the plethysmographic overestimation of tlc may not have been seen because the current study s population included a range of fev1 from normal to very severe, and may not be powered sufficiently for subgroup analysis among patients with very severe airflow obstruction . Odonnell et al collected data from subjects at three hospitals where hrct and plethysmographic technique may not have been standardized . Patients recruited from one of the hospitals undergoing evaluation for lvrs had substantially lower average fev1 than those from the other two hospitals . Subtle differences in hrct and plethysmographic technique at this hospital may have driven the relationship between airflow obstruction and the overestimation of plethysmographic tlc . Our data were collected at a single center and our methodology was standardized for all patients . No mention was made by odonnell et al as to the effect of hyperinflation and air trapping on lung volume determination . Our data suggest that more significant air trapping (rv% and frc%) and hyperinflation (tlc%) is present in the copd patients with the greatest difference in tlc measured by plethsmography and hrct . Sub - maximal inspiratory maneuvers would have resulted in an underestimation of tlc by hrct compared to plethysmography . Another limitation of our study is that the pulmonary function tests were not necessarily performed on the same day as the hrct, weakening the correlation between these modalities . Understanding the variability with which tlc by plethysmography differs from hrct has important implications in the evaluation of patients with copd . Global and regional measurements of lung volumes can be important in identifying patients who are most likely to benefit from lvrs.40 similarly, precise volume determination is necessary to accurately identify suitable donor and recipient lungs for transplantation.5 finally, the ability to precisely measure lung volume may be central to determining the importance of hyperinflation in copd, and the impact of therapies geared to reducing end - expiratory lung volumes . Total lung capacity measured by plethysmography is larger than that determined by hrct in copd . The degree to which tlc measured by plethysmography differs from that determined by hrct is correlated with air trapping and hyperinflation . The method used to obtain tlc in severely hyperinflated patients needs to be considered when precise measurements are required for clinical decision making.
Medical schools in many parts of the world have begun to incorporate disaster - related topics into their curricula . In 2003, the association of american medical colleges (aamc) in the united states recommended that bioterrorism education be included in all medical school programs . In germany, federal laws have been enacted requiring that medical students be familiar with disaster medicine principles . Analysis of the peer - reviewed literature indicates that few curricula for medical student disaster medicine education have been published . The aamc report on bioterrorism training is more of an outline of educational objectives and activities than a specific curriculum . Its target group is us medical students in their last year of training, and builds on their growing knowledge and skills . Covering a few aspects of medical disaster management, parrish and colleagues provide a description of a medical school course for 2nd year us students by briefly listing major subject headings . First taught by military experts, this course includes simulation exercises, such as a refugee scenario, and an infectious disease outbreak tracking exercise . In this format, however, operation execution appears to be the priority with less focus on medical care delivery . Markenson et al . Suggest core competencies and subject areas for terrorism, disaster, and public health emergency preparedness for health - care students based on professional school outlines . Referencing a proficiencies list, curricular matrices are proposed, but no steps or samples for implementation are presented . In a course for senior dental students, glotzer and et al . Describe core competencies and dentists roles within disaster response efforts focusing on bioterrorism; participation in a 4-h disaster life support course of the american medical association is also recommended to provide guidance regarding incidents with weapons of mass destruction . Because a fully developed and comprehensive medical school disaster medicine curriculum was not available, the german government commissioned the development of a core medical student disaster medicine curriculum that could serve as a standardized template . Pilot versions of the curriculum were initially developed and utilized with faculty and student feedback over a 2-year period . The final curriculum resulted from these pilots, which were formally developed according to the six - step approach to curriculum development . This included general and targeted needs assessment, definition of goals and objectives, choice of educational methods, and pilot implementation and evaluation . The pilots contents were mainly based on government - funded research projects in which all curricula from german medical schools and postgraduate public health physician courses were evaluated for inclusion of disaster medicine - related themes . The physicians of all (n = 477) german county disaster management agencies and district health authorities were also surveyed for additional input into needed disaster medicine competencies . With a nearly 100% response rate from the physicians surveyed, more than 92% expressed the necessity for better education . This resulted in the development of educational concepts for improving disaster preparedness at the under- and postgraduate level [7, 8]. Key components compiled here, disaster medicine topics of contemporary relevance, and expertise gained from student emergency medicine training were then combined to generate an independent lecture series . This was finally adapted into a disaster medicine curriculum for a 4th - year german medical school program . Students are thus expected to have had 3 years of clinical training in medical school when participating in the course . The implementation experience with the pilots was straightforward: the university dean and the department chair fully supported the idea of providing an introduction to the practices of disaster medicine at the student level . Course realization could thus readily be achieved: a core curricular team of physicians was assembled and scheduled all tasks . Internal and external professionals with expertise in subject areas were identified and invited to become teaching faculty . After coordination with medical school program planners, facilities for the in - house curriculum units (rooms, equipment) were organized with the lecture support staff . For the external units, collaborations with rescue and disaster response agencies were established to permit common exercises . For exchange and drill of non - physician professionals and students, agencies provided equipment, faculty, and their trainees to serve as mock victims for a mass casualty simulation . Finally, feedback on the curriculum from all participants was utilized to make alterations and improvements as needed . When the government requested a medical student disaster education plan, we offered a clear vision for goals and objectives of a comprehensive curriculum of core issues of disaster medicine based on our experience from the pilot courses: the first aim was not only to increase medical students familiarity with the ideas and practices of disaster medicine, but to enhance their understanding of what occurs in a disaster, and where the limits and challenges faced by those delivering medical care under austere conditions lie . Second, improving knowledge of public health - relevant information on radiological / nuclear, biological, chemical, explosive, and other disaster - related health risks appeared to be important . Third, the opportunity to refresh and extend familiarity with basic and advanced life support, and to provide some limited experience with triage decision making was an additional benefit . Finally and as a long - term goal, we wished to enhance student interest in pursuing further disaster medicine expertise . It was determined that scientific evidence wherever available should be incorporated and combined with professional expertise to build the content basis of the new curriculum [9, 10]. Following analysis of international guidelines on disaster response training, peer - reviewed publications [35, 12, 13], and programs such as the us department of homeland security, federal emergency management agency (fema), and american medical association, learning objectives were fixed . After cross - referencing objectives with quality assurance criteria for medical education [1416], topics were structured into a framework represented by subject headings . To identify the content details, a systematic literature search in medline, embase, and the cochrane library with the key terms student, education, training, curriculum, course, competency, disaster, catastrophe, mass casualty, medicine, medical, preparedness, plans, and outcome was conducted . Due to the relatively small number of relevant publications, complementary internet searches were performed, and resources from websites such as from the fema program were included . Curricular committee consensus on those entities regarded to be most important was required for item inclusion . A blueprint for the core contents was then constructed in the form of modules with the goals as a matrix . Because learning and memory benefit from multifaceted, stimulating environments [17, 18], we also decided to diversify the instructive strategies . This led us to include interactive discussions, teaching of problem - solving strategies, reviews of real - life experience, role playing, and exposure to exercises in real - world activities were assigned to suitable content modules, with a minimum of one - third of the curricular time being reserved for hands - on training . These units were designed to be performed with other professionals, such as fire fighters . To evaluate the effectiveness of the curriculum, the results of a knowledge - based, pre - program written examination were compared with post - program examination results . Because the course was created to be part of the obligatory 4th - year german medical school program, the testing at course completion also served to fulfil requirements of the german educational system . Course evaluations for faculty and students were drafted to be used for further curricular developments [6, 14]. As a way to approach administrative authorities (also outside of germany), fig . 1 shows the input and adoption of the final curricular version after review by interdisciplinary professionals and governmental bodies involved in disaster health care . Our final curricular proposal was accepted by the leading board of the german civil protection committee . Small modifications were then made according to the wishes of the president and members of the german society of disaster medicine and the civil protection committee . The revision was then sent to governmental authorities, whose feedback produced another iteration in the process . The outcome was fine - tuned into formal alignment with global standards for quality improvement in medical education . These included alignment with medical school mission statements, instructional methods of preparing students for life - long, self - directed learning, or the provision of learning environments that resemble real - world scenarios . Following final civil protection committee and governmental approval, a directive was provided to all directors of universities with medical schools instructing them to implement the curriculum (february 2007). 1final curriculum development indicating stages of refinements and input from various professional and governmental bodies final curriculum development indicating stages of refinements and input from various professional and governmental bodies pilot versions of the curriculum were initially developed and utilized with faculty and student feedback over a 2-year period . The final curriculum resulted from these pilots, which were formally developed according to the six - step approach to curriculum development . This included general and targeted needs assessment, definition of goals and objectives, choice of educational methods, and pilot implementation and evaluation . The pilots contents were mainly based on government - funded research projects in which all curricula from german medical schools and postgraduate public health physician courses were evaluated for inclusion of disaster medicine - related themes . The physicians of all (n = 477) german county disaster management agencies and district health authorities were also surveyed for additional input into needed disaster medicine competencies . With a nearly 100% response rate from the physicians surveyed, more than 92% expressed the necessity for better education . This resulted in the development of educational concepts for improving disaster preparedness at the under- and postgraduate level [7, 8]. Key components compiled here, disaster medicine topics of contemporary relevance, and expertise gained from student emergency medicine training were then combined to generate an independent lecture series . This was finally adapted into a disaster medicine curriculum for a 4th - year german medical school program . Students are thus expected to have had 3 years of clinical training in medical school when participating in the course . The implementation experience with the pilots was straightforward: the university dean and the department chair fully supported the idea of providing an introduction to the practices of disaster medicine at the student level . Course realization could thus readily be achieved: a core curricular team of physicians was assembled and scheduled all tasks . Internal and external professionals with expertise in subject areas were identified and invited to become teaching faculty . After coordination with medical school program planners, facilities for the in - house curriculum units (rooms, equipment) were organized with the lecture support staff . For the external units, collaborations with rescue and disaster response agencies were established to permit common exercises . For exchange and drill of non - physician professionals and students, agencies provided equipment, faculty, and their trainees to serve as mock victims for a mass casualty simulation . Finally, feedback on the curriculum from all participants was utilized to make alterations and improvements as needed . When the government requested a medical student disaster education plan, we offered a clear vision for goals and objectives of a comprehensive curriculum of core issues of disaster medicine based on our experience from the pilot courses: the first aim was not only to increase medical students familiarity with the ideas and practices of disaster medicine, but to enhance their understanding of what occurs in a disaster, and where the limits and challenges faced by those delivering medical care under austere conditions lie . Second, improving knowledge of public health - relevant information on radiological / nuclear, biological, chemical, explosive, and other disaster - related health risks appeared to be important . Third, the opportunity to refresh and extend familiarity with basic and advanced life support, and to provide some limited experience with triage decision making was an additional benefit . Finally and as a long - term goal, we wished to enhance student interest in pursuing further disaster medicine expertise . It was determined that scientific evidence wherever available should be incorporated and combined with professional expertise to build the content basis of the new curriculum [9, 10]. Following analysis of international guidelines on disaster response training, peer - reviewed publications [35, 12, 13], and programs such as the us department of homeland security, federal emergency management agency (fema), and american medical association, learning objectives were fixed . After cross - referencing objectives with quality assurance criteria for medical education [1416], topics were structured into a framework represented by subject headings . To identify the content details, a systematic literature search in medline, embase, and the cochrane library with the key terms student, education, training, curriculum, course, competency, disaster, catastrophe, mass casualty, medicine, medical, preparedness, plans, and outcome was conducted . Due to the relatively small number of relevant publications, complementary internet searches were performed, and resources from websites such as from the fema program were included . Curricular committee consensus on those entities regarded to be most important was required for item inclusion . A blueprint for the core contents was then constructed in the form of modules with the goals as a matrix . Because learning and memory benefit from multifaceted, stimulating environments [17, 18], we also decided to diversify the instructive strategies . This led us to include interactive discussions, teaching of problem - solving strategies, reviews of real - life experience, role playing, and exposure to exercises in real - world activities were assigned to suitable content modules, with a minimum of one - third of the curricular time being reserved for hands - on training . These units were designed to be performed with other professionals, such as fire fighters . To evaluate the effectiveness of the curriculum, the results of a knowledge - based, pre - program written examination were compared with post - program examination results . Because the course was created to be part of the obligatory 4th - year german medical school program, the testing at course completion also served to fulfil requirements of the german educational system . Course evaluations for faculty and students were drafted to be used for further curricular developments [6, 14]. As a way to approach administrative authorities (also outside of germany), fig . 1 shows the input and adoption of the final curricular version after review by interdisciplinary professionals and governmental bodies involved in disaster health care . Our final curricular proposal was accepted by the leading board of the german civil protection committee . Small modifications were then made according to the wishes of the president and members of the german society of disaster medicine and the civil protection committee . The revision was then sent to governmental authorities, whose feedback produced another iteration in the process . The outcome was fine - tuned into formal alignment with global standards for quality improvement in medical education . These included alignment with medical school mission statements, instructional methods of preparing students for life - long, self - directed learning, or the provision of learning environments that resemble real - world scenarios . Following final civil protection committee and governmental approval, a directive was provided to all directors of universities with medical schools instructing them to implement the curriculum (february 2007). 1final curriculum development indicating stages of refinements and input from various professional and governmental bodies final curriculum development indicating stages of refinements and input from various professional and governmental bodies pilot versions of the curriculum were initially developed and utilized with faculty and student feedback over a 2-year period . The final curriculum resulted from these pilots, which were formally developed according to the six - step approach to curriculum development . This included general and targeted needs assessment, definition of goals and objectives, choice of educational methods, and pilot implementation and evaluation . The pilots contents were mainly based on government - funded research projects in which all curricula from german medical schools and postgraduate public health physician courses were evaluated for inclusion of disaster medicine - related themes . The physicians of all (n = 477) german county disaster management agencies and district health authorities were also surveyed for additional input into needed disaster medicine competencies . With a nearly 100% response rate from the physicians surveyed, more than 92% expressed the necessity for better education . This resulted in the development of educational concepts for improving disaster preparedness at the under- and postgraduate level [7, 8]. Key components compiled here, disaster medicine topics of contemporary relevance, and expertise gained from student emergency medicine training were then combined to generate an independent lecture series . This was finally adapted into a disaster medicine curriculum for a 4th - year german medical school program . Students are thus expected to have had 3 years of clinical training in medical school when participating in the course . The implementation experience with the pilots was straightforward: the university dean and the department chair fully supported the idea of providing an introduction to the practices of disaster medicine at the student level . Course realization could thus readily be achieved: a core curricular team of physicians was assembled and scheduled all tasks . Internal and external professionals with expertise in subject areas were identified and invited to become teaching faculty . After coordination with medical school program planners, facilities for the in - house curriculum units (rooms, equipment) were organized with the lecture support staff . For the external units, collaborations with rescue and disaster response agencies were established to permit common exercises . For exchange and drill of non - physician professionals and students, agencies provided equipment, faculty, and their trainees to serve as mock victims for a mass casualty simulation . Finally, feedback on the curriculum from all participants was utilized to make alterations and improvements as needed . When the government requested a medical student disaster education plan, we offered a clear vision for goals and objectives of a comprehensive curriculum of core issues of disaster medicine based on our experience from the pilot courses: the first aim was not only to increase medical students familiarity with the ideas and practices of disaster medicine, but to enhance their understanding of what occurs in a disaster, and where the limits and challenges faced by those delivering medical care under austere conditions lie . Second, improving knowledge of public health - relevant information on radiological / nuclear, biological, chemical, explosive, and other disaster - related health risks appeared to be important . Third, the opportunity to refresh and extend familiarity with basic and advanced life support, and to provide some limited experience with triage decision making was an additional benefit . Finally and as a long - term goal, we wished to enhance student interest in pursuing further disaster medicine expertise . It was determined that scientific evidence wherever available should be incorporated and combined with professional expertise to build the content basis of the new curriculum [9, 10]. Following analysis of international guidelines on disaster response training, peer - reviewed publications [35, 12, 13], and programs such as the us department of homeland security, federal emergency management agency (fema), and american medical association, after cross - referencing objectives with quality assurance criteria for medical education [1416], topics were structured into a framework represented by subject headings . To identify the content details, a systematic literature search in medline, embase, and the cochrane library with the key terms student, education, training, curriculum, course, competency, disaster, catastrophe, mass casualty, medicine, medical, preparedness, plans, and outcome was conducted . Due to the relatively small number of relevant publications, complementary internet searches were performed, and resources from websites such as from the fema program were included . Curricular committee consensus on those entities regarded to be most important was required for item inclusion . A blueprint for the core contents was then constructed in the form of modules with the goals as a matrix . Because learning and memory benefit from multifaceted, stimulating environments [17, 18], we also decided to diversify the instructive strategies . This led us to include interactive discussions, teaching of problem - solving strategies, reviews of real - life experience, role playing, and exposure to exercises in real - world environments . Activities were assigned to suitable content modules, with a minimum of one - third of the curricular time being reserved for hands - on training . These units were designed to be performed with other professionals, such as fire fighters . To evaluate the effectiveness of the curriculum, the results of a knowledge - based, pre - program written examination were compared with post - program examination results . Because the course was created to be part of the obligatory 4th - year german medical school program, the testing at course completion also served to fulfil requirements of the german educational system . Course evaluations for faculty and students were drafted to be used for further curricular developments [6, 14]. As a way to approach administrative authorities (also outside of germany), fig . 1 shows the input and adoption of the final curricular version after review by interdisciplinary professionals and governmental bodies involved in disaster health care . Our final curricular proposal was accepted by the leading board of the german civil protection committee . Small modifications were then made according to the wishes of the president and members of the german society of disaster medicine and the civil protection committee . The revision was then sent to governmental authorities, whose feedback produced another iteration in the process . The outcome was fine - tuned into formal alignment with global standards for quality improvement in medical education . These included alignment with medical school mission statements, instructional methods of preparing students for life - long, self - directed learning, or the provision of learning environments that resemble real - world scenarios . Following final civil protection committee and governmental approval, a directive was provided to all directors of universities with medical schools instructing them to implement the curriculum (february 2007). 1final curriculum development indicating stages of refinements and input from various professional and governmental bodies final curriculum development indicating stages of refinements and input from various professional and governmental bodies the creation of the final curriculum resulted in a course of 14 modules with each unit requiring approximately 2 h time: modules 13 (table 1a) introduce the essentials of disaster medicine terminology, disaster assistance and law, management, and multidisciplinary coordination and communication systems . As key competencies, the tasks of assistance agencies, functional response roles, scopes of responsibility, lines of authority, logistical needs for mass casualties, and triage methods are taught . Students are familiarized with operational issues of disaster response strategies including problem - based learning discussions (pbld). Table 1modules 114modules, learning activityeducational goalcore contentsa medical student disaster medicine curriculum, modules 13module 1, lecture and pbldintroduction to disaster medicine, terminologyto learn definitions of disaster medicine and to develop an understanding for general disaster managementglossary and common disaster medicine definitions, differences between disaster and emergency medicine, different phases of disaster managementdisaster assistanceprinciples of disaster assistancedisaster assistance organization, assistance agencies and structuretypologyto consider the heterogeneity of disastersnatural and technological disasters, terrorism, man - made disasters, civilian disorders, environmental and other threatslaws and regulationsto understand the legal environment and regulations for civil protection and disaster preparednessregulative and administrative issues for civil disaster protection, warfare disaster protection laws, rescue service and hospital laws, governmental resources, and authoritiesmodule 2, lecture and pblddisaster medical managementto realize the architecture and organizational management necessary for coping with mass casualty incidents and large - scale acutely ill patients in a coordinated waymass casualty disposition, treatment area, transport issuesfunctional operationstasks of rapid - intervention - units (sanitary / psychosocial care)incident command systemsdisaster contingency plans command and control structures, functional operations centercoordination structurescoordination, integration, and cooperation of multi - agency rescue and assistance responsefunctional rolesfunctional response roles, e.g., lead emergency physician, organizational leader, and technical command postinformation managementcommunication, coordinationmodule 3, lecture and pbldspecific disaster medicineto develop skills in principles of tactically managing mass casualties and large numbers of patients suffering, e.g., from combined conventional trauma and thermal injuriespatient assessment and triage: triage levels, tags, registration; primary emergency care, multi - tasking and operational management phases (e.g., patient collection, treatment area, transport)tactic disaster medical managementlogistical requirements for care of burn - injuries, mine blast, and missile - hit victims (including high - speed bullet injuries), mass trauma managementb medical student disaster medicine curriculum, modules 48module 4, pbld and interactive reviewhospital preparedness and disaster management planningto follow orders and principles of hospital alarm and evacuation planshospital disaster lawshospital alarm planshospital preparedness plans for- management of external disasters with mass casualties suffering from multiple injuries, intoxication, infections, and/or radioactive contamination- management of in - house disasters with fire incidents and hospital evacuationmodule 5, pbld and interactive reviewpresentation of past disasters and review of assistance experiencesto evaluate and understand feasibility issues of providing medical support and health care in the field and under disaster conditions based on experience from worldwide disaster assistance operationspresentation of past disasters and disaster assistance experience gained in the field, e.g., from lead emergency physicians, operations in earthquake assistance, explosions, highly contagious infectious diseases, repatriation flights, and with the german federal armed forces medical corps in world crisis regionsmodule 6, experiential trainingpreclinical and clinical triage exerciseto perform triage decisions in reality simulationtriage training exercise: real or virtual scenario simulation rescue exercise, e.g., explosion with mass casualties and blast, mechanical, and thermal injuries of all triage levelsmodule 7, experiential trainingevacuation exerciseto apply operational principles and steps of action for evacuation procedurescommand post exercise, real or virtual: e.g., evacuation organization and evacuation of a hospital, school, part of town, etc.module 8, lecture and pbldlife - saving disaster emergency medical careto learn concepts of life - saving emergency disaster medical careunder disaster conditions: provision of life - saving emergency medical care to adults and children, e.g., shock therapy, pain treatment, and sedationc medical student disaster medicine curriculum, modules 911module 9, lecture and pbldspecific disaster emergency medical care for various situations including bioterrorism incidentsto get to know principles of specific disaster medical caredisaster emergency medical first aid, specific measuressurgical and medical treatments of burn and thermal injuries and illnesses from explosive, warfare and biological agentsepidemiology and approaches to terrorist attacks, weapons, and highly contagious infectious diseases, sentinel casesmodule 10, lecture and pbldradiological and nuclear threats, accidents with radioactive material, radiation illness and syndrome, decontaminationto learn principles and basic medical care for management of incidents with radiologic / nuclear agents and contaminated victimsspecific dangers of radiological / nuclear agents, associated illnesses and radiation syndromeself protection, protection and detection equipment, special intervention unitsfirst aid medical treatment, isolation and radioactive decontamination, decontamination operations in case of mass trauma combined with contamination injuriesexperiential trainingdecontamination after radiation exposureto be exposed to practical aspects and procedures of decontaminationdecontamination exercise or decontamination demonstration, e.g., in nuclear power plant or with the fire brigadesmodule 11, lecture and pbldchemical and toxicological threats from hazardous materials and goods, transport risks, acute poisoning and toxic syndromesto get to know principles of medical countermeasures for management of incidents with dangerous chemical substances, hazardous materials and goodsidentification and risk assessment of hazardous materials, chemicals and goods, and associated toxic syndromesmanagement of acute intoxications and poisonings, threats from specific poisonsself protection, precautionspoisoning epidemiology, risk assessment for mass intoxicationsfirst aid medical treatment, e.g., enhanced elimination of toxins, use of antidotes, adjunctive services, e.g., poison emergency centers and toxicology laboratoriesexperiential trainingdecontamination after chemical poisoningto experience exposure to decontamination procedurestriage in case of mass casualties with toxic syndromesdecontamination measures and exercised medical student disaster medicine curriculum, modules 1214module 12, lecture, pbld, and interactive reviewethics and professionalismto develop familiarity with ethical codes and the duty of care relevant to disaster conditionsgeneva convention and amended protocols, ethical codes of conduct, humanitarian imperatives, social, moral, and ethical challenges of disastersquality assuranceto understand quality improvement efforts and risk management programs for disaster medical responsequality control performance indicators, incident monitoring, tools for risk and critical event assessment, structured improvement approachesappropriate documentationmodule 13, pbld and interactive reviewto comprehend concepts of psychic stress responsecase presentations for identification of critical incident stress reactions and review of therapeutic interventionspsychosocial careto learn techniques to deal with psychic reactions caused by exposure to disaster scenariostreatment approaches to acute and delayed critical incident stress reactions, acute and chronic stress syndromes, post - traumatic stress disordersto recognize need for help and to initiate psychosocial supportstructure and tasks of psychosocial emergency intervention unitsoperational strategies of psychosocial treatmentmodule 14, course completionclosing examinationto demonstrate gain of knowledge and skillsstudent final examination, oral and/or written testevaluation of educational successto assess educational value of coursecomparison of pre - program versus post - program student test resultslearner and educator assessment of courseto maintain continuous curriculum development and improvement in course qualitystudent and faculty summative and formative course evaluation modules 4 and 5 (table 1b) discuss hospital alarm and preparedness plans, with a focus on in - house and external incidents requiring high surge capacity . As core competencies, notification and mobilization orders are taught, and concepts of an institutional emergency plan are incorporated . Interactive review of past major community - threatening events and experiences from worldwide disaster and relief operations are utilized to built recognition of disaster health - care and medical assistance feasibility issues . Students learn the risks and limitations of relief operations, while focusing on the challenge of implementing care in the field . Modules 6 and 7 (table 1b) address knowledge and skills for triage and evacuation through participation in exercises . Students conduct triage and assume functional roles in real or virtual experiential training units . To provide a hands - on experience and to increase student understanding of what occurs in a mass casualty management situation, we simulate a triage exercise in a small - group format (four students / two emergency physicians) as shown in fig . 2 . In brief, our interdisciplinary executed scenario is conducted at connected hallways and the hospital grounds in the setting of poor illumination, smoke, and loud noises . Well - trained, life - like moulage volunteers simulate casualties presenting with various disorders, from superficial injuries to moribund conditions, medical illnesses, and emotional trauma . To maximize the accuracy of the scenario, simulation patients have 20% severe, 20% moderate, and 40% light injuries, as is most typically seen in real - world events . In the multi - task exposure, students are first subjected to an instructive exercise, regarding role assignment, scenario description, protection and triage directives, and radio communication orders . They are not provided with any patient information prior to interaction with the mock victims, but are made aware of a constrained timeframe for the tasks . To be authentic, students must put on full protective gear, helmets, and gloves . The hands - on exercise is then run in a step - by - step manner, starting with patient search: after they are located, patients must be checked for vital functions and examined . Following differential diagnosis, they must be triaged, and treatment goals and management priorities must be defined . The compressed timeframe exercise ends with a radio announcement . As a debriefing exercise, students receive performance assessment with respect to future roles as physician, collaborator, and communicator in a contextual feed - back . 2algorithm of student immersion in mass casualty rescue simulation exercise algorithm of student immersion in mass casualty rescue simulation exercise modules 8 and 9 (table 1b and c) teach life - saving and specific emergency medical care for various disaster situations, containing first aid and specific approaches to injuries, burns, explosions, blasts, and illnesses from warfare and biological agents . Emphasising terrorist attacks and highly contagious infectious diseases, core competencies for care of explosive, warfare, and biological incidents are included . Minimally acceptable treatment and limitations for patient - tailored therapy in case of mass casualties are discussed . Modules 10 and 11 (table 1c) teach the dangers and management of radiological / nuclear and chemical hazardous materials . Options for protection, first aid, decontamination, enhanced elimination, and antidotes are reviewed . Students learn how victims with combined trauma and toxic injury are triaged . At a nuclear power plant or fire department, the design and state of preparedness of such facilities are evaluated, and equipment is demonstrated . To provide a practical experience of a decontamination procedure and to get students personally involved in how it feels to work in protective gear, we offer a combined unit of didactic, interactive, and hands - on training at the fire department (fig . 3). After incident presentation, a lecture on radiological / nuclear and chemical hazardous substances is given . Technical equipment, mobile units, protective gear, and contamination detection devices are demonstrated . Use of protective gear is explained and a decontamination suite described . As hands - on exercises, students put on full protective gear and gas masks . Being both protected and hampered by this gear, they must attempt to manage the airway, intubate, and ventilate mannequins . This hands - on program serves as a real - world simulation experience, reproduced by (1) heat load under the multilayer over - garment, (2) enhanced personal respiratory efforts, (3) restricted visual fields because of the gas mask, and (4) interference with manual dexterity by the chemical protective gloves . After debriefing, the module ends with an interactive review and analysis of past incidents . 3flowchart of the module with the fire fighters or at a nuclear power plant focussed on education of incident management with radioactive and hazardous chemical substances flowchart of the module with the fire fighters or at a nuclear power plant focussed on education of incident management with radioactive and hazardous chemical substances modules 12 and 13 (table 1d) deal with challenges of ethical, professional, psychosocial, and quality control criteria of medical disaster management themes . Students learn critical aspects of acute and delayed stress disorders (anxiety and grief), and the psychosocial resources, such as crisis intervention units, available to provide specialised assistance . To understand quality improvement efforts, students gain familiarity with assessment tools and incident monitoring to evaluate functional components of disaster response systems . Module 14 (table 1d) is designed to assess the educational success of the course . Students demonstrate the acquired knowledge and attitudes in an oral and/or written examination . According to the german regulations, each medical school has the freedom to design the format of the examinations chosen for their student evaluations . To our knowledge, this curriculum is one of the first publications of a comprehensive medical student disaster medicine curriculum that covers major contemporary aspects of basic disaster medicine management . The developed course offers a cross - professional design in a readily adaptable structure, is focussed on actual scenarios, and has been aligned with quality criteria for improved teaching approaches [14, 16, 19]. At a time when medical schools in numerous countries are struggling to find a place for disaster medicine in their programs, this educational topic is worthy of research and discussion . Our work may be used as an educational resource, with the opportunity to adapt the basic template to various locations, threats, and systems . An introduction to care principles will be of value to preparedness in any locale, even though disaster response plans and conditions may differ among countries, or states and cities of a single country . The curriculum also provides resources for endeavors of organizations such as the section in disaster medicine of the european society for emergency medicine, which has as a current goal the development of a standard european disaster medicine curriculum for medical school undergraduates . The analysis of the implementation process and curricular success has been pre - defined for a period of 5 years, and was begun 3 years ago . However, all medical schools that have implemented the curriculum have so far reported that they now teach disaster medicine components with a scope and depth much broader than within emergency medicine, pointing out the additional value for disaster preparedness and disaster medical management . Seven of them report that they have implemented a fully independent course, and five of the medical schools base their teachings on an educational program that is in complete conformity with the curriculum developed . Because derivation and details for medical student competency definitions differ widely [3, 4, 6, 11], we used a consensus - building approach for outlining competencies and thus the framework for the educational goals of this curriculum . Requests of the education committee working group of the world association for disaster and emergency medicine, disaster medicine - related competencies as indicated in emergency medicine or radiology curricula, courses recommended by professional organizations, the literature and expert opinion were addressed as far as available [7, 2123]. Deriving subject matter from thus pre - defined goals provided the advantage of readily defining the main targets for core contents . The competency structured design also permits flexibility in addressing a multiplicity of disaster situations, including floods, hurricanes, earthquakes, and surveillance - related health problems, as well as care for vulnerable populations such as children and hospitalised patients . Educational goals were weighted in terms of time allocation to assist medical school planners operating with more limited discretionary curricular time . In our opinion and circumstances, a 14-module course composed of 2-h units provides adequate student education and mock disaster exposure in a reasonable period . Some themes important for disaster preparedness are also addressed in other medical school programs, such as in medical microbiology, toxicology, and environmental medicine . The topics we targeted for this curriculum, however, do not overlap with other student programs . Emergency medicine curricula for medical students, when present in medical school curricula at all, do not typically focus on disaster medicine or disaster response . Introduction to emergency medicine focuses on maximum possible, individual patient - tailored care [9, 21, 23] and if included this is intended to address disaster medicine conditions in which extraordinary treatment demands exceed resources, and where incidents of great magnitude requiring external assistance for management may reach epidemiological dimensions . When planning the experiential units, we attempted to ensure that costs were minimized: for cost containment, selection of a teaching cadre with background in emergency or disaster medicine who collaborates with local disaster response agencies is most effective . Additional teachers can join in, and after receiving instructor training, faculty development may be supported through ongoing participation in areas of disaster medicine interest . Exercise scenarios may not be available in all medical school environments, but can, in most cases, be established through a joint training / learning venture with existing response agencies . Because agencies are obliged to carry out simulation exercises and drill their own staff, curriculum units can be performed with mutual benefit . Exercise costs can thus be kept low, and mainly derive from transport to external locations . For an estimation of costs, table 2 provides activity - based descriptions for organizing and implementing the curriculum . Table 2task assignment for estimation of organizational burden and curricular implementation costscurricular activitymission descriptionprogram managementdevelopment of a local organizational structure responsible for curricular activities: team recruitment, appointment of managers and departmental staff in - charge, task assignmentidentification of internal and external experts and invitation as teaching cadreestablishment of collaboration with local disaster response agencies and planning of common external units, exercises, and simulationsteaching and preceptor assignment for internal and external unitsinstitution of curriculum within medical school program and coordination of units with program plannersorganization of facilities and equipment for in - house units with university s lecture support stafforganization of transport to external unitsmanagement of administrative and reporting issuesif necessary: establishment of financial recording, billing, and reimbursement systemfaculty development / continuous professional growthfuture faculty development through involvement in existing teaching activities, plans, and team meetingsfaculty instructor trainingoptional: professional growth through participation in related internal and external developmental programs for university educators and disaster medicine preparednessquality assurance / continuous curricular developmentdevelopment of data collection systemanalysis of ongoing evaluations from learners and educatorsuse of course assessments and experiences for orientation, end - of - course team meetings, and future progressstudent supportprovision of curricular schedule and learning objectivesprovision of lecture compilation (syllabus or cd) as course content workbook and base for self - directed learningdevelopment of data collection system and analysis of student examination datadevelopment of reporting system for examination gradesmaintenance of partnerships / collaborationscontinuous encouragement and support for internal and external partnerships and collaborationsrunning of administrative / overhead expendituresif necessary: management of direct and indirect expenditures, such as telecommunication costsother pursuitsdesigning of plans for course revisions or curricular enhancements, such as integration of e - learning methods task assignment for estimation of organizational burden and curricular implementation costs there are several limitations to this work . . Nevertheless, educational efforts for important medical topic areas have to be started at some point in medical school, and this can be evaluated over time . Of course, the provision of one - time disaster medicine training will only allow for a passing familiarity with terms and concepts . To retain competencies, it will be necessary to refresh and assess knowledge and skills periodically, and we are planning to implement educational refresher courses in our german national program . Moreover at present, we cannot comment on how well the curriculum performed, and what the overall success of the course will turn out to be . Moreover, exposing students to mock disaster scenarios and simulation exercises is far from real - world conditions . Certainly, students will not intubate patients in full protective gear in real disasters and will of course not be included in first rescue response teams in compromised environments . Nevertheless, based on our experience as university educators, they benefit from the knowledge and skill - building hands - on exercises that may serve as a basic training for future medical practice in all kinds of emergency situations . Our students also receive clear instruction that they are not trained search and rescue personnel . For our course, we thus try to prevent the possibility that simulation - based medical education experiences could cause student over - confidence and misjudgement of competency . In its first design, our curriculum consists of traditional, face - to - face, instructor - student interactions and hands - on experiences, and does not use electronic (e-) learning methods . E - technology holds the promise for distance education of disaster response concepts and virtual reality simulations . Because blended curricular formats integrating e - methods may enhance inter - professional exchange without compromising pedagogy, they may be included in future curricular versions . A curriculum is provided for medical student disaster medicine education at an advanced level of medical school training, along with information regarding the process involved in creating such a program at a national level . We wish to emphasize that the curriculum has been designed for medical students in a particular system and that there are various concepts for basic disaster medicine education . Due to its comprehensive, interdisciplinary nature, instructive design, and flexible structure, however, the curriculum can benefit other health - care professional systems by serving as a model curriculum, and facilitating refinement and testing of other existing, but perhaps as yet unpublished models.
Analyses were conducted for a nationally representative sample of adult ambulatory care visits, using publicly available data from the 2007 namcs . The namcs is a national survey, with the use of a multistage probability sampling design (10). All ambulatory care visits of office - based physicians were randomly sampled from physician practice settings across the country . Physicians were sampled from one of 112 geographically based probability sampling units in the u.s . (11). The study population consisted of all ambulatory care visits (n = 32,778) to physician offices in 2007 . Because adult ambulatory care was the focus of this analysis, all patients aged 18 years were included in the study sample (n = 27,169). Patients with diabetes (n = 3,403) were identified by physicians' answers of yes to the survey question does patient now have diabetes? Appropriate institutional review board approval was obtained from the cleveland state university . The categories of race were white (non - hispanic), african american (non - hispanic), hispanic, or other . Insurance type included private insurance, medicare, medicaid, self - pay, or other . Records regarding has the patient been seen in your practice before? Indicated established or new patients . Two covariates (educational attainment and median household income) were included in all models because of their significance in previous studies (12,13). The percentage of population in patient's zip code with a bachelor's degree or higher was classified as 31.7% vs. <31.7% . Median household income was dichotomized as $52,388 vs. <$52,388 . Primary care physician or a designated provider for the patient was identified by the answer of yes to are you the patient's primary care physician? Practice settings included private practice, free - standing clinic, community health center, and other . Electronic medical record (emr) use was dichotomized as yes (partial or all emr) versus no . Other covariates regarding factors accounting for the compensation included physician productivity, patient satisfaction, and quality of care and were dichotomized as yes or no . Outcome variables were defined as the use of any diagnostic testing or patient education . Cholesterol was not included, because of the lack of accurate bmi measures among this sample aged 18 years study cohort . Patient education was defined as any counseling on diet / nutrition, exercise, and stress management . Preventive care services were defined as any provision of diagnostic tests or patient education, including glucose, urinalysis, a1c, blood pressure, diet / nutrition, exercise, and stress management . On the basis of data records, three outcome variables were created and dichotomized as yes (indicating the provision of one or more diagnostic tests, patient education, and preventive services) versus no . The namcs sampling weights were obtained from the national center for health statistics (11). National estimates of the overall numbers of visits and descriptions for all variables were obtained by using the weighted measures that accounted for the multistage sampling design (10,11). The association among study variables was analyzed and tested with likelihood ratios and/or the adjusted wald test (14). Three multivariate logistic regression models were constructed for the comparison of diagnostic testing, patient education, and preventive care services among visits . Visits were stratified according to the sex (male or female) of diabetic patients . Patient - level independent variables were age, race, insurance type, residential location, visit status, number of chronic conditions, and time spent with physicians . Physician practice - level independent variables included the physician type, practice setting, ownership, emr use, and laboratory testing availability as well as whether physician productivity, patient satisfaction, or quality of care accounted for compensation . Other covariates were socioeconomic indicators of the residential location (educational attainment and median household income in patient's zip code). All statistical analyses were conducted using stata (version 10; statacorp, college station, tx). The complex survey design was accounted for in all analyses to ensure proper representation of the study population and to render nationally representative estimates . The categories of race were white (non - hispanic), african american (non - hispanic), hispanic, or other . Insurance type included private insurance, medicare, medicaid, self - pay, or other . Records regarding has the patient been seen in your practice before? Indicated established or new patients . Two covariates (educational attainment and median household income) were included in all models because of their significance in previous studies (12,13). The percentage of population in patient's zip code with a bachelor's degree or higher was classified as 31.7% vs. <31.7% . Primary care physician or a designated provider for the patient was identified by the answer of yes to are you the patient's primary care physician? Practice settings included private practice, free - standing clinic, community health center, and other . Electronic medical record (emr) use was dichotomized as yes (partial or all emr) versus no . Other covariates regarding factors accounting for the compensation included physician productivity, patient satisfaction, and quality of care and were dichotomized as yes or no . Cholesterol was not included, because of the lack of accurate bmi measures among this sample aged 18 years study cohort . Patient education was defined as any counseling on diet / nutrition, exercise, and stress management . Preventive care services were defined as any provision of diagnostic tests or patient education, including glucose, urinalysis, a1c, blood pressure, diet / nutrition, exercise, and stress management . On the basis of data records, three outcome variables were created and dichotomized as yes (indicating the provision of one or more diagnostic tests, patient education, and preventive services) versus no . The namcs sampling weights were obtained from the national center for health statistics (11). National estimates of the overall numbers of visits and descriptions for all variables were obtained by using the weighted measures that accounted for the multistage sampling design (10,11). The association among study variables was analyzed and tested with likelihood ratios and/or the adjusted wald test (14). Three multivariate logistic regression models were constructed for the comparison of diagnostic testing, patient education, and preventive care services among visits . Visits were stratified according to the sex (male or female) of diabetic patients . Patient - level independent variables were age, race, insurance type, residential location, visit status, number of chronic conditions, and time spent with physicians . Physician practice - level independent variables included the physician type, practice setting, ownership, emr use, and laboratory testing availability as well as whether physician productivity, patient satisfaction, or quality of care accounted for compensation . Other covariates were socioeconomic indicators of the residential location (educational attainment and median household income in patient's zip code). All statistical analyses were conducted using stata (version 10; statacorp, college station, tx). The complex survey design was accounted for in all analyses to ensure proper representation of the study population and to render nationally representative estimates . Patients with diabetes accounted for 12.5% of the total visits (table 1). In 2007, there were 27,169 (nonweighted) adult visits to physician's offices (799,362,170 weighted visits). Compared with individuals without diabetes, patients with diabetes were older and were more likely to be african american or hispanic american and covered by medicare insurance . The average number of chronic conditions was 2.9 for patients with diabetes and 0.6 for other individuals . Based on the zip code, 18.3% of diabetic patients and 25.6% of others resided in areas with 32% of the population having a bachelor's or higher degree . Median household income of $52,388 was 19.7% among diabetic patients and 26.9% in other individuals . Nearly one - half of visits for diabetes and one - third of visits for other conditions were to primary care physicians . Patient satisfaction or quality of care in compensation was more likely for visits for diabetes than for other visits . Patients with diabetes were more likely than others to have diagnostic tests or patient education . Characteristics of the study sample and physician practice data are means se or% . Data were adjusted for the complex survey design and for the person - level analytic weights . * p <0.01 for the comparisons between patients with and without diabetes . In multivariate analyses for men (table 2), patients aged 65 years had a lower likelihood of preventive care services . After adjustment for other covariates, race or insurance was not associated with preventive services . The number of chronic conditions predicted a slightly higher likelihood of preventive care . Living in rural areas primary care physicians were associated with a higher likelihood of diagnostic tests (odds ratio [or] 19.5 [95% ci 10.7635.59]) or patient education (2.4 [1.504.06]). Emr use predicted a higher likelihood of diagnostic tests (1.9 [1.133.44]) and patient education (1.9 [1.342.86]). On - site laboratory tests were associated with a higher likelihood of diagnostic testing (4.5 [2.996.99]). If physician compensation relied on productivity, there was a reduced likelihood of diagnostic testing (0.5 [0.310.91]) and patient education (0.4 [0.300.78]). Multivariate analysis of diabetes preventive services for men data are or (95% ci). Socioeconomic indicators of educational attainment and household income in zip code areas were included in all models . In multivariate models for women (table 3]), older women were less likely to have diagnostic tests or patient education . The race / ethnicity, payment resources, visit type, and time spent with physicians were not significant predictors of preventive services . The number of chronic conditions was associated with a slightly higher likelihood of patient education . Primary care physicians were linked with a higher likelihood of diagnostic tests (19.2 [10.2036.24]) or patient education (2.2 [1.343.73]). Emr use was associated with a higher odds of patient education (1.8 [1.222.71]). On - site laboratory tests predicted a higher likelihood of diagnostic testing (4.9 [2.958.18]). If the productivity accounted for compensation, the odds of preventive services was reduced (0.4 [0.260.81]). Multivariate analysis of diabetes preventive services for women data are or (95% ci). Socioeconomic indicators of educational attainment and household income in zip code areas were included in all models . Previous studies have reported the underuse of recommended preventive care services for patients with diabetes (5,15). This is the first study to provide a national picture of diabetes preventive services in ambulatory care visits . With regard to preventing the long - term complications of diabetes however, the 2007 namcs data suggest that variations in diabetes preventive care are part of a larger pattern of physician practices and not simply the variation of patient race or insurance . The policy implication is to increase the awareness of or training in diabetes preventive care . Patient - level predictors of diabetes preventive services, such as age or chronic condition, could be explained by physician practices, in response to patient health - related behaviors and the u.s . Preventive task force (uspstf) guidelines (16,17). In terms of practice - level predictors, the 2007 namcs data suggest that primary care physicians and practice features, i.e., the availability of emrs and an on - site laboratory, have a significant impact on diabetes preventive care . The finding associated with emr use is consistent with a recent study in primary care practices . Health services researchers at harvard medical school examined the association between the performance and structural capabilities of 412 primary care practices (18). They reported that emr use was linked with a higher performance across multiple healthcare effectiveness data and information set measures (18). In addition, this study identified a significant link between an on - site laboratory and the likelihood of diagnostic testing . The policy implication that the availability of emr systems and a laboratory facility has a positive impact on diabetes preventive services is obvious . These results indicate a great opportunity to strengthen structural capabilities of primary care practices to improve diabetes management . Strengthening structural capabilities of primary care practices existing variations in primary care practices include settings, sizes, and the availability of emrs or an on - site laboratory . For example, a rural setting and small size may limit the financial ability of physician practices to expand structural capabilities for better performance compared with large practices . It is unrealistic to expect primary care physicians on their own to realize successful implementation and use of emr systems (19). Nevertheless, small or rural practices play an irreplaceable role in serving their related populations and are an integral part of the system . Furthermore, the cost - effectiveness of preventive care for diabetic patients is largely determined by long - term health benefits rather than the cost of improving system performance (20). Thus, systematic strategies are pivotal for implementing accurate emr systems across primary care practices for achieving more efficient diabetes preventive care . Physician compensation based on the productivity or quantitative patient management is identified as a striking barrier to preventive care . Unfortunately, primary care physicians have been managing more preventive care items (21). An examination of the required time for a primary care physician to provide recommended preventive services suggests that time constraints have limited the ability of physicians to comply with the recommendations (22). In this study we found that patient education was performed eight times less than diagnostic tests . Thus, competing demands on physician's time may have compromised recommended preventive services for vulnerable diabetic patients with complex conditions . The policy implication regarding the necessity of strategic partnerships for diabetes management and preventing the long - term complications is clear . First, namcs measurements were largely based on reports by physicians and their staff for individual visits . One previous study suggested that physician self - reports overestimate the visit duration (23). However, the possibility and consequence of the busiest physicians being less likely to participate in surveys should be considered . A recent study by researchers at northwestern university reported that the majority of physician - documented medical records were accurate (24). The strength of this analysis is examining patient- and physician practice level independent predictors of diabetes preventive care, while adjusting for the visit duration . Second, the namcs data are limited to national - level information on ambulatory encounters or non hospital - based physician office visits . Current findings do not apply to other types of visits, such as visits to hospital outpatient and emergency departments . Nevertheless, namcs data provide a representative national picture regarding the nature of ambulatory care in the u.s . Therefore, the extent of comprehensiveness in diabetes preventive care for broad population groups in the u.s . Is adequately represented by namcs measures . Finally, namcs measures include services that are ordered or provided but contain no data on the uspstf rating of a or, a complete comparison of each preventive service between what is recommended and what is delivered is not possible . Previous comparisons with direct observation measures of patient visits suggest that namcs data are more accurate for diagnostic procedures than for behavioral counseling; thus, patient education may be underestimated (23). To minimize potential bias, level independent predictors of diabetes preventive services, rather than a report of each preventive care item ordered / provided . We suspect that if more accurate measures of preventive services ordered versus provided were available, the currently identified picture of less than comprehensive diabetes preventive care may be more dramatic . Given the disparities identified in diabetes preventive care services, more analyses are needed to examine the degree of preventive care for the vulnerable older population . Meanwhile, this study establishes evidence for national primary care policy to move beyond its historical focus on providers and individuals . Improving primary care infrastructure is necessary to meet projected increases in work volume, driven by the aging population, as well as medical knowledge and technology advancement . This improvement is important because system changes are more effective than patient- and physician practice recommended changes are to establish policies and provide support for implementing effective emr systems that convene multiple stakeholders, aligning with the needs of diabetes care management to prevent long - term complications (25). Achieving cost - effective quality of diabetes preventive care seems to be sufficient justification for strengthening strategic partnerships and structural capabilities of primary care practices.
Mitochondrial membranes possess quality control systems that remove non - assembled polypeptides and prevent their possibly deleterious accumulation in the membrane (arnold & langer, 2002). Several transcription factors are synthesized as membrane proteins and activated upon the specific proteolytic cleavage of solvent - exposed fragments that subsequently enter the nucleus (brown et al ., 2000). Elucidation of these membrane proteases will lead to a better understanding of crucial biological processes and diseases . Stomatin is one of the major integral membrane proteins of human erythrocytes, but its function is not fully understood . In humans, the absence of the protein is associated with a form of hemolytic anemia known as hereditary stomatocytosis (stewart et al . Stomatin - like proteins are found in almost all species of eukaryotes, eubacteria and archaea (tavernarakis et al ., 1999). Interestingly, p - stomatin genes and stopp (stomatin operon partner protein) genes probably form an operon in at least 19 species of prokaryotes and archaea (green et al ., 2004). In the genomic sequences of the hyperthermophilic archaeon pyrococcus horikoshii, ph1510 and the n - terminal region of ph1510 (residues 16236, 1510-n) is a thermostable serine protease with a catalytic ser lys dyad (ser97 and lys138), and it specifically cleaves the c - terminal hydrophobic region of the p - stomatin ph1511 (yokoyama & matsui, 2005). The cleavage is involved in a certain regulatory system, but little is known about its physiological role . In order to understand the functions of 1510-n and ph1511, the crystal structure of 1510-n was determined at 3.0 resolution in dimeric form (yokoyama et al ., 2006). Each active site around ser97 of 1510-n is in a hydrophobic environment suitable for hydrophobic substrates . The monomer of 1510-n shows a structural similarity to one monomer of escherichia coli clpp (wang et al . Clpp is a protease with a catalytic ser his asp triad and degrades misfolded proteins in order to maintain quality control of proteins . Despite the structural similarity between the monomers of 1510-n and clpp, their oligomeric forms are different . The difference in oligomeric form and catalytic residues between 1510-n and clpp would lead to a functional difference; 1510-n is likely to have a regulatory function, whereas clpp is involved in protein quality control . It is not known how 1510-n recognizes and degrades the p - stomatin ph1511 . A structural analysis of 1510-n in complex with its substrate this article reports a crystal structure of the k138a mutant of 1510-n at a resolution 2.3 higher than that of the wild - type structure reported previously (yokoyama et al . Although the structure does not contain a substrate peptide, a novel dimer structure was observed . The k138a mutant of 1510-n was prepared as described previously (yokoyama & matsui, 2005; yokoyama et al ., 2006). For the structure determination of 1510-n in complex with its substrates, crystallization attempts were carried out using 1510-n k138a mixed with a 16-aa synthetic peptide containing the sequence of ph1511 degraded by 1510-n protease (its amino acid sequence is dksnvivlmlpmemlk). The protein solution contains 5 mg ml of k138a and 5 mg ml of peptide in 50 mm tris hcl (ph 8.5) containing 0.1 m nacl, 0.05%(w / v) n, n - dimethyldodecylamine n - oxide (ldao, fluka) and 10%(v / v) dimethyl sulfoxide . Crystallization trials were carried out using the hanging - drop vapor - diffusion method by mixing equal volumes of the protein and reservoir solutions . Crystals were grown at 293 k using a reservoir solution containing 18%(w / v) peg 4000, 0.1 m mgcl2 and 0.1 m sodium acetate (ph 4.5). A crystal was flash - frozen in a 100 k stream of nitrogen gas . The cryoprotectant solution used was 20%(w / v) peg 4000, 25%(w / v) sucrose, 0.1 m mgcl2 and 0.1 m sodium acetate (ph 4.5), with tris hcl (ph 7.5) added to a final concentration of 0.1 m. the crystal diffracted synchrotron x - rays to 2.3 resolution . The asymmetric unit contains one 1510-n k138a molecule, showing a high value for the crystal volume per unit molecular mass of 3.6 da and a solvent content of 0.66 . X - ray diffraction data were collected at beamline bl6a of the photon factory in kek (tsukuba, japan) using a quantum-4r ccd detector, and were processed and scaled using hkl2000 (otwinowski & minor, 1997). The structure was determined by the molecular replacement method using the program cns (brnger et al . The crystal structure of chain b of 1510-n [protein data bank (pdb) accession code 2deo] was used as a search model (yokoyama et al . The model was subjected to rigid - body refinement with 203.0 data using the program refmac (murshudov et al ., 1997) in the ccp4 suite (collaborative computational project, number 4, 1994), and then the model gave an r factor of 0.385 . The model was subjected to several cycles of crystallographic refinement using refmac, followed by manual model fitting using the program coot (emsley & cowtan, 2004). Data collection and refinement statistics are summarized in table 1 . The buried surface area was calculated using cns (brnger et al . Figures were produced using pymol (http://www.pymol.org), molscript (kraulis, 1991) and raster3d (merritt & bacon, 1997). The atomic coordinates and structure factors have been deposited in the rcsb protein data bank with the accession code 3bpp . The crystal structure of the k138a mutant of 1510-n was solved at 2.3 resolution using the molecular replacement method . Although diffraction data were collected using the cocrystal of 1510-n k138a and the substrate peptide, no electron densities corresponding to the peptide clear and continuous electron densities are observed for all the residues except for the residues 127129 with low densities . The 2f o f c electron densities around tyr146 are shown in fig . 1 . As shown in table 1, the stereochemistry of the model was evaluated using procheck . One residue, ser97, is in a disallowed region (= 24, = 107). Ser97 is the catalytic residue and is located as a second residue of a type ii turn (hutchinson & thornton, 1994). Clpp protease shows a similar active - site structure to 1510-n, and the residue (ser97) corresponding to ser97 of 1510-n is also in a disallowed region (wang et al ., 1997), as well as that of wild - type 1510-n (yokoyama et al . The determined structure of the 1510-n k138a mutant contains one monomer in the asymmetric unit . In contrast, the wild - type 1510-n has two molecules in the asymmetric unit (yokoyama et al ., 2006). In the sds page analysis of 1510-n, a major protein band at around 45 kda (putative dimer) showed proteolytic activity (yokoyama & matsui, 2005). From the gel filtration analysis, the expected molecular mass was 4050 kda, indicating that 1510-n forms a dimer (data not shown). The crystal of 1510-n k138a belongs to space group i422, and one molecule makes contact with five symmetry - related molecules . The buried surface area (bsa) between one monomer and each symmetry - related molecule was calculated, and two large bsa values above 1000 were found (table 2). Thus two possible sets of dimer were observed (type 1 in fig . 2 and type 2 in fig . 3), and both dimers are related by a crystallographic twofold axis . As shown in table 2, the bsa for the type-1 dimer is 1180, and the dimer (fig . 2) exhibits almost the same structure as the wild - type dimer (yokoyama et al . Difference of c atoms of the k138a and wild - type dimers is 1.7 . Difference of the k138a monomer and the wild - type chain b is 1.5 . The relatively large difference is derived from large deviation of the regions around arg66 (which corresponds to the l1 loop in the wild - type structure) and arg121 (located at the base of the l2 loop). On the other hand, the bsa for the type-2 dimer of the k138a mutant is 1498 . In the structure 3 (among 20 residues forming the dimer interface, nine residues belong to the l2 loop). In the k138a mutant, the novel type-2 dimer (1498) shows a buried surface area larger than the type-1 dimer (1180). In wild - type 1510-n, the corresponding type-2 dimers are formed using symmetry - related molecules, but the r.m.s . Difference of c atoms of the wild - type and k138a dimers is large (4.1). In wild - type 1510-n, the residues 122139 corresponding to the l2 loop are disordered and are not involved in the formation of the dimer interface . Thus the bsa of the type-2 dimer (917) is narrower than that of type 1 (1217) in the wild type . In the k138a structure, the molecular surface representation indicates that the active site around s97 and a138 (mutated from lys) is in a hydrophobic environment (fig . In particular, the aromatic residues tyr101, tyr142, phe143 and tyr146 form a cluster along with met70 and met71 (fig . 5). The hydrophobic region is probably the binding site of hydrophobic substrates, and 1510-n preferentially degrades hydrophobic substrates in the region . The type-1 dimer exposes the hydrophobic active site to the solvent or the membrane (fig . Meanwhile, the type-2 dimer uses the hydrophobic active site to form the dimeric interface (fig . 3). Then the type-2 dimer is blocked off to the solvent or the membrane, and therefore it would be inactive . The 1510-n protease specifically degrades the c - terminal hydrophobic region of ph1511, 1511-c (189266), in which the residue number is presented in the parentheses (yokoyama & matsui, 2005). Although 1510-n k138a does not degrade the substrate 1511-c (189266), trace amounts of putative acyl - enzyme intermediates are detected in the sds therefore, 1510-n k138a would bind to the substrate in solution . Owing to the crystal packing constraint that all monomers forming the type-1 dimer also form the type-2 dimer, the 16-aa synthetic peptide might not enter the active site in the k138a crystal . A lon protease from methanococcus jannaschii forms a dimer in the crystal, and the active sites of the two monomers therefore, the authors suggest that the observed dimeric structure is an artifact (i m et al ., the same discussion may be possible for the type-2 dimer, but the type-2 dimer may exist as an inactive form . The type-1 dimer is probably in an active form for proteolysis because the structures in both the wild type and the k138a mutant are almost the same . In the crystal structure of the wild - type 1510-n, the structure of the k138a mutant contains the l2 loop in spite of the high average b factors of 86.2 (the average b factors of all protein atoms are 47.6). Interestingly, the l2 loop is significantly kinked at around ala138 (figs . 2 and 5). Clpp protease has a long handle region corresponding to the l2 loop, but because of the kink around ala138 the structure of the l2 loop is quite different from the handle region of clpp . Lys138 of 1510-n is located at an n - terminal end of the following -helix . Considering the -helical dipole moment, the side chain of lys138 with positive charge would destabilize the neighboring structure . Cys120 is also located at the base of the l2 loop and is located in the vicinity of ser97 (fig . The side chain of cys120 points to the protein interior . In wild - type 1510-n, however, the side chain of cys120 is relatively solvent exposed . Three proline residues (pro122, pro136 and pro137) located at the base of the l2 loop would also contribute to the kinked structure of l2 . The 1510-n protease specifically cleaves the c - terminal hydrophobic region of the p - stomatin, ph1511 (yokoyama & matsui, 2005). Although the function of stomatin is not clear, stomatin is a lipid raft protein and could function as an oligomeric scaffolding protein or as an active signalling component involved in vesicle trafficking (umlauf et al . Being degraded, 1510-n would attack the c - terminal hydrophobic region of ph1511 . If both the type-1 and the type-2 dimers are present and can change mutually at physiological conditions, the interconversion of the dimers may regulate the protease activity (active type 1 and inactive type 2). Lys138 or cys120 would be involved in the possible conformational change of l2 . In order to elucidate how 1510-n degrades p - stomatin ph1511, structure determination of 1510-n and
In at least some of immune thrombocytopenia (itp) patients, thrombocytopenia is worsened by inappropriately low levels of platelet production, unable to compensate for the increased peripheral immune - mediated destruction of platelets . Until recently, management of itp adult patients with platelet counts <3010/l and/or bleeding signs has relied upon the use of drugs or procedures (i.e. Splenectomy) aimed at decreasing b - cell and t - cell immune - response to autologous platelets and megakariocytes . The availability of thrombopoietin - receptor agonist (tpo - ra) has offered new opportunities for treating adult itp patients acting on megakariocyte survival and production of platelets . Thrombopoietin - receptor agonist increase thrombopoiesis activating the c - mpl receptor on bone marrow megakariocytes, thus providing an additional stimulus to increase their survival and platelet production . Response rates up to 90% in long - term follow - up studieshave been reported with both romiplostim and eltrombopag . However, platelet counts usually return to pretreatment levels upon discontinuation of treatment and long - term, if not life - long, administration was envisioned in order to maintain effective thrombopoiesis . In recent years sporadic patients have been reported as being able to discontinue treatment with either romiplostim or eltrombopag without relapsing off - therapy . Most studies were not designed to assess feasibility of tpora discontinuation and thus true prevalence of patients able to maintain remission off therapy cannot be inferred . A french observational study confirms that romiplostim can be successfully discontinued: of the 28/54 responding patients, 8 remained in complete remission off therapy at a median follow up of 13,5 months (range 5 - 27). Data from a recently presented interim analysis of a phase 2 study of platelet response and remission rates in itp patients receiving romiplostim show that 11 of 38 evaluable patients (29%) were able to maintain remission (i.e. 24 weeks of platelet counts 5010/l) off any treatment . We report our experience with the peptibody romiplostim in a small series of patients unresponsive to immunosuppressive therapy who were treated with this tpo - ra for variable lengths of time . Some of these patients were able to discontinue therapy after achieving response, without relapsing at long - term follow - up . Between september 2009 and may 2014 a total of 30 patients (11 m; mean age at romiplostim treatment 50.7 yrs, range 18 - 82) received romiplostim at two collaborating centers . Patients with shorter follow - up on treatment with romiplostim or on eltrombopag are not included in the present analysis . Patients were defined either as non responders or refractory according to the international working party (iwp) proposed standardization of terminology for itp . All patients are enrolled in a local italian registry (rel - itp registry), and informed consent to demographic and clinical data use is given at enrollment . The rel registry has received approval by the human subjects research review board of both participating hospitals . Of the 27 responders: 1 patient (#4) was lost to follow - up; 1 patient (#5) discontinued romiplostim due to acute myocardial infarction, at the time of sae, romiplostim dose was 10 g / kg / week and platelet counts were 8410/l; in 5/27 patients (#6 - 10) romiplostim was used as bridge to splenectomy; 5/27 patients (#11 - 15) lost response at 11, 6, 11, 12 and 11 months respectively; neutralizing anti - romiplostim antibodies could be searched for in 3/5 patients; in three (#13, 14 and 15) the test yielded a positive result; the remaining two patients could not be tested; in 2/27 patients (#16 and 17) romiplostim administration is ongoing (56 and 12 months respectively); 13/27 patients (#18 - 30) achieved stable, safe platelet counts and were able to stop romiplostim after a mean of 43.3 weeks (range 12 - 122) on therapy with sustained response maintained at a mean of 28.8 months (range 15 - 55). Strict platelet monitoring was maintained during follow up off therapy . In 7/13 patients a decreased platelet count compared to a previous control was found weeks from discontinuation of weekly romiplostim administration . These patients were re - exposed to a limited number of one shot low (1 or 2 mcg / kg) dose of romiplostim and regained a stable response, as shown for four patients in figure 1 . Patient #29 developed lower limb superficial phlebitis while on romiplostim 1 g / kg on alternate weeks and warfarin was started; platelets were 13510/l at the time of the event; screening for lupus anticoagulant, anti - phospholipids antibodies, anti - beta2glycoprotein1 was negative . Three patients (#18,20,28) had received the last rituximab course 4, 6 and 33 weeks before starting romiplostim; one patient (#19) received a first course of romiplostim as bridge to splenectomy having failed steroids and cyclosporine; a week after surgery he relapsed with severe thrombocytopenia and was re - treated with romiplostim . He received a total of 7 romiplostim doses administered over a 22 week period of time; romiplostim was discontinued at 22 weeks from splenectomy since stable, 100109/l platelet counts were achieved . Bone marrow biopsies for detection of fibrous changes during romiplostim use were not performed since most of our patients received treatment for less than 18 months . Overall, our experience with romiplostim in real life practice is in line with reported findings from controlled studies with 90% of patients (27/30) responding . However, in our experience the percentage of patients able to discontinue romiplostim without relapsing is noteworthy: 48%, if we consider the whole number of romiplostim responders (13 of 27 responders) and 65% if we consider only patients who received long - term treatment (13 of 20 patients) i.e. Not considering patients receiving romiplostim as bridge to splenectomy, lost to follow - up, or who discontinued the drug because of sae . In 5 chronic itp patients out of 13 long term responders, romiplostim seemed to be the only treatment responsible for sustained response . Indeed, it has been proposed that tpo - ra therapy may result in improved immune regulation by regulatory t cells (tregs) possibly restoring immune tolerance.14 similar immune - modulating effects have also been described for other treatment modalities (e.g. Rituximab, high - dose dexamethasone, intravenous immunoglobulin) which result in sustained responses in a fraction of itp patients.15,16 in the remaining 8 patients, response might have been either spontaneous (2 newly diagnosed and 3 persistent disease patients) or secondary to rituximab (2 patients) or splenectomy (1 patient). Nevertheless, use of romiplostim may be a treatment option for patients with persistently low platelet counts awaiting for platelet recovery, either it be spontaneous or secondary to disease - modifying treatments for which a possible late - response can be anticipated . No specific patient s features (e.g. Age, itp duration, lines of previous treatment) nor pattern of early response (e.g. Dose requirements, time to response) seemed to consistently predict for sustained response off therapy . However, it seems that patients achieving safe platelet counts at lower dosages are probably worth a try of therapy tapering and discontinuation . Manufacturer dose adjustment rules allow romiplostim to be discontinued when appropriate and clinical judgment was used to manage patients . This has been specifically tested and demonstrated for eltrombopag and it is conceivable also for romiplostim: one of the patients presented by newland received a single romiplostim dose after 11 weeks off therapy and regained high platelet counts; thereafter, hemostatic platelet counts were maintained without any therapy for 2 years . All 7 patients who were reexposed to romiplostim weeks after weekly administration was stopped (table 2 and figure 1), regained a platelet response . Knowledge that tpo - ra can be safely administered on a on - demand basis without loss of effectiveness offers the possibility of re - exposing relapsed patients without the threat of these patients not being able to regain a response . Moreover, need of on demand therapy does nt seem to predict for lost of long - term response to romiplostim . This new finding is probably worth being taken into account in planning prospective studies of feasibility of romiplostim discontinuation . The finding that at least 3 of 5 patients who abruptly lost response to romiplostim tested positive for neutralizing antibodies suggests that development of these antibodies may be a more frequent event than expected . Being a retrospective observational study, our results should be interpreted with caution, but still they suggest that in a significant fraction of patients, in real world practice, sustained remission without disease recurrence may be obtained after a relatively short - term administration of romiplostim . To date, because of their known mechanism of action, tpo - ra are thought to be effective only while the treatment is ongoing and the proper dose is being administered . Our observations support the proposal of using romiplostim as bridge to recovery, either it be spontaneous (e.g. In newly diagnosed or persistent itp), tpo - ra induced or representing a late - response to previously administered treatments, rather than long - term treatment . First of all, concern about tpo - ra long - term sides effects (marrow fibrosis, increased risk of thrombotic events), especially in younger patients, would be greatly reduced . Moreover, treatment costs, which currently also limit their use would be less of an issue . If results on persistent remissions after short - term romiplostim use were to be confirmed in larger patient population, this approach could be used to defer or avoid splenectomy and to spare patients the untoward effects of prolonged immunosuppressive treatments . This would be especially beneficial in newly diagnosed severe itp failing first line therapy with steroids . Future biological and intervention studies are needed in order to identify predictive factors of long term sustained response, allowing clinicians to tailor the best treatment for each itp patient.
Colorectal polyps are histologically classified as neoplastic or nonneoplastic . Most colorectal cancers (crcs) arise from neoplastic adenomatous polyps, and serrated polyps have recently been shown to have a different potential than traditional adenomatous polyps to develop into malignant crc 1 2 3 . Serrated polyps are pathologically classified into three groups by the world health organization: hyperplastic polyps (hps), sessile serrated adenoma / polyps (ssa / ps), and traditional serrated adenomas (tsas). Of these three types, ssa / ps are considered the main premalignant lesions in the serrated neoplastic pathway to crc, which represents 20 to 30 percent of all sporadic crc cases 4 . Foci of cytological dysplasia in ssa / ps should be noted by the pathologist as a ssa / p with cytological dysplasia . In contrast, tsas have diffuse but typically mild cytological dysplasia, and the classification systems for these polyps are still under development . Therefore, histological interpretation of these serrated polyp types differs among pathologists, which has led to a lack of knowledge regarding their true frequency . Recently, it has been reported that polyps have the potential to develop into sporadic crc via a series of molecular alterations, including braf mutation and cpg island methylation with epigenetic inactivation of the mlh1 mismatch repair gene, resulting in microsatellite instability (msi) 5 6 . Many studies have suggested that tsas and ssa / ps have significant malignant potential and are associated with subsequent development of metachronous polyps 7 8 9 10 11 . Clinically, ssa / ps are considered to be strongly associated with interval cancers after colonoscopy, which can originate from a rapidly progressing precursor lesion that evades detection . Failed attempts at endoscopic resection (er) allow for the continued growth of these lesions . However, relatively little is known about the epidemiology of ssa / ps and tsas, particularly the coexistent cancer rates . This study was performed to determine the adenocarcinoma rates associated with ssa / ps and tsas based on the epidemiology of a large cohort . The rates were compared with those for other colorectal polyps resected during the same observation period . This was a retrospective single - center cohort study of consecutive patients with colorectal polyps who underwent er from march 2003 to october 2014 at toyonaka municipal hospital . Next, we analyzed the clinicopathological findings, complete en bloc resection rate, and frequency and characteristics of coexistent carcinoma in serrated polyps resected by er based on the pathology reports . The typical indication for er of a colorectal polyp is size> 5 mm . However, the final decision to perform er is made by each physician based on the patient s condition and bowel preparation . At our institution, diminutive polyps were allowed to be followed up without resection based on the judgment of each endoscopist . However, flat and depressed lesions that were difficult to distinguish from adenoma or carcinoma were generally resected . Patients who did not achieve a clean colon on initial colonoscopy underwent repeat er to attain a clean colon, except in the case of diminutive polyps . Locations of resected colorectal polyps were classified into two groups: proximal, defined as the cecum, ascending colon, and transverse colon; and distal, defined as the descending colon, sigmoid colon and rectum . Polyp size was endoscopically measured using standard clinical practices, such as by visual estimation, the open biopsy forceps method or use of a polypectomy snare . The macroscopic appearance of the polyp was classified according to the paris endoscopic classification 12, and the data were annotated as follows: protruded or pedunculated (ip) and intermediate or broad - based forms (semi - pedunculated [isp], sessile [is], and superficially elevated [iia]). Resected polyps were evaluated by experienced pathologists who worked full time at our hospital during the observation period . Basically, the histopathologic diagnosis was based on the morphologic features observed in hematoxylin- and eosin - stained sections . Ssa / ps were diagnosed based on the following findings: crypt dilation, irregularly branching crypts, and horizontally arranged basal crypts (inverted t- and/or l - shaped crypts) 13 . Tsas were diagnosed based on the following findings: villiform growth, serrated epithelium associated with nuclear and cytological atypia, ectopic crypt formation, and eosinophilic cytoplasm . Hps were diagnosed based on findings of a serrated epithelial architecture in the upper part of the crypts, proliferative zones limited to the lower part of the crypts, and the absence of cytologic dysplasia . All continuous variables are presented as the mean standard deviation (sd), except for nonparametric variables, which are presented as the median and range . Categorical variables were compared using the chi - squared test or fisher s exact test, as appropriate . A receiver operating characteristic (roc) curve analysis was used to determine the cutoff value for polyp size for coexisting adenocarcinoma . The typical indication for er of a colorectal polyp is size> 5 mm . However, the final decision to perform er is made by each physician based on the patient s condition and bowel preparation . At our institution, diminutive polyps were allowed to be followed up without resection based on the judgment of each endoscopist . However, flat and depressed lesions that were difficult to distinguish from adenoma or carcinoma were generally resected . Patients who did not achieve a clean colon on initial colonoscopy underwent repeat er to attain a clean colon, except in the case of diminutive polyps . Locations of resected colorectal polyps were classified into two groups: proximal, defined as the cecum, ascending colon, and transverse colon; and distal, defined as the descending colon, sigmoid colon and rectum . Polyp size was endoscopically measured using standard clinical practices, such as by visual estimation, the open biopsy forceps method or use of a polypectomy snare . The macroscopic appearance of the polyp was classified according to the paris endoscopic classification 12, and the data were annotated as follows: protruded or pedunculated (ip) and intermediate or broad - based forms (semi - pedunculated [isp], sessile [is], and superficially elevated [iia]). Resected polyps were evaluated by experienced pathologists who worked full time at our hospital during the observation period . Basically, the histopathologic diagnosis was based on the morphologic features observed in hematoxylin- and eosin - stained sections . Ssa / ps were diagnosed based on the following findings: crypt dilation, irregularly branching crypts, and horizontally arranged basal crypts (inverted t- and/or l - shaped crypts) 13 . Tsas were diagnosed based on the following findings: villiform growth, serrated epithelium associated with nuclear and cytological atypia, ectopic crypt formation, and eosinophilic cytoplasm . Hps were diagnosed based on findings of a serrated epithelial architecture in the upper part of the crypts, proliferative zones limited to the lower part of the crypts, and the absence of cytologic dysplasia . All continuous variables are presented as the mean standard deviation (sd), except for nonparametric variables, which are presented as the median and range . Categorical variables were compared using the chi - squared test or fisher s exact test, as appropriate . A receiver operating characteristic (roc) curve analysis was used to determine the cutoff value for polyp size for coexisting adenocarcinoma . A total of 21,048 resected polyps from 15,326 patients (70.0% men) were identified based on pathology reports from march 2003 to october 2014 at toyonaka municipal hospital . There were 15,984 traditional adenomatous polyps (75.9%; 95% confidence interval [ci]: 75.4 76.5), 621 ssa / ps (3.0%; 95% ci: 2.7 3.2), 136 tsas (0.6%; 95% ci: 0.6 0.8), 1122 hps (5.3%; 95% ci: 5.0 5.6), and 3,186 other polyps, including nonneoplastic lesions, inflammatory polyps, carcinoid tumors, lipomas, juvenile polyps and unclassified polyps (data not shown) (table 1, fig . 1). Ssa / ps: sessile serrated adenoma / polyps; tsas: traditional serrated adenomas; hps: hyperplastic polyps; ci: confidence interval flowchart showing patients with resected colorectal polyps . Ssa / ps: sessile serrated adenoma / polyps; tsas: traditional serrated adenomas; hps: hyperplastic polyps; ca: carcinoma . The clinical and endoscopic findings of ssa / ps revealed a male sex (68.6%) and sessile type (73.1%) predominance, with 61.7% of the polyps located in the proximal colon (table 2). The clinical and endoscopic findings of tsas also revealed a male (77.2%) and sessile type (65.4%) predominance, with 77.2% of the polyps located in the distal colon (table 3). The mean sizes of the ssa / ps and tsas were 8.8 and 10.7 mm, respectively . The complete en bloc resection rates of ssa / ps and tsas were 65.2% and 62.4%, respectively (table 2 and table 3). Ssa / ps: sessile serrated adenoma / polyps; c: cecum; a: ascending colon; t: transverse colon; d: descending colon; s: sigmoid colon; r: rectum tsas: traditional serrated adenomas; c: cecum; a: ascending colon; t: transverse colon; d: descending colon; s: sigmoid colon; r: rectum among the ssa / ps, 25 (4.0%) showed cytological atypia (table 2). Of these, 8 had coexistent adenocarcinoma . Among the tsas, 1 (0.7%) showed coexistent adenocarcinoma (table 4, fig . 1, and supplementary table 1). Table 4 and supplementary table 1 provide the details of the ssa / ps and tsas with adenocarcinoma, and fig . The optimal tumor size cut - off value for coexisting adenocarcinoma was 10 mm (auc: 0.84) (fig . Ssa / ps: sessile serrated adenoma / polyps; tsas: traditional serrated adenomas; c: cecum; a: ascending colon; t: transverse colon; d: descending colon; s: sigmoid colon; r: rectum; m: mucosa; sm: submucosa . Typical pathologic images of ssa / p (a) and tsa (b) with adenocarcinoma . Receiver operating characteristic (roc) curves used to predict coexisting adenocarcinoma in patients with serrated polyps . Adenocarcinoma was found in 8.2% of the traditional adenomatous polyps (1,318/15,984) (fig . 1). Therefore, the coexisting adenocarcinoma rate was significantly lower in ssa / ps (p <0.0001) and tsas (1 polyp; p = 0.0014) than in traditional adenomatous polyps . Most of the resected serrated polyps were between 6 and 20 mm, while all the serrated polyps with adenocarcinoma were at least 10 mm . Serrated lesions of the colorectum are histologically characterized by a serrated appearance of the crypt epithelium . Therefore, until recently, serrated polyps were not differentiated from hyperplastic polyps and were not thought to require polypectomy or surveillance . In the 1980 s, autopsy studies demonstrated that the percentage of caucasian adults who had at least one serrated lesion ranged from 25% to 50% 15 16 17 18; these studies were conducted before specific serrated lesion subtypes were recognized . In 1990, the term serrated polyps was first used by longacre and fenoglio - preiser 19 . In 1996, torlakovic and snover identified undifferentiated serrated polyps characterized by their large size and proximal location and histologically classified them based on their architectural distortion 20 . Since then, serrated polyps have become a recognized and established separate entity, but an understanding of the different subtypes did not become widespread until approximately a decade ago . Recent studies have reported prevalence rates for ssa / ps ranging from 0.6% to 13.8% based on detection by colonoscopy 21 22 23, and most studies have reported rates between 1% and 5% . However, ssa / ps are more difficult to detect than traditional adenomatous polyps by endoscopy because they are flat, covered with a mucus cap, and similar in color to the surrounding mucosa . Therefore, the frequency of serrated polyps varies depending on study design and the endoscopist, and consequently, there has been a tendency to underreport the number and malignant potential of serrated polyps . The lack of standardization is another reason for the low rate; the diagnosis varies depending on the individual pathologist s definition of ssa / ps . Unfortunately, other than the report by lash et al . 21, there have been few reports of large samples of endoscopically resected colorectal polyps . Lash et al . Reported that 1.7% of a population of 179,111 patients with 290,810 mucosal polyps had ssa / ps (with and without dysplasia), which were more commonly detected in women and primarily in the right colon . They also reported that 0.9% (21/2416 lesions) of the patients with ssa / ps had adenocarcinoma . In the current study, the overall frequency of ssa / ps was 3.0% of 21,048 total resected polyps . Tsas are much less common than ssa / ps, accounting for only 0.6% of polyps . Compared with the data reported by lash et al ., the frequency of ssa / ps in our study was slightly higher, but the adenocarcinoma coexistence rate (1.3%) was similar . They speculated that the low frequency likely reflected the conservative criteria they used for the inclusion of polyps as ssa / ps . We hypothesize that the different frequencies of ssa / ps are due to different diagnostic criteria and treatment strategies used by each endoscopist or pathologist, which have impeded an accurate estimation of the prevalence of ssa / ps; this remains a major problem regarding ssa / ps that must be solved in many institutions, including high - volume centers . Kim et al . Recently reported that 21 (18.8%) of 112 tsas examined contained an admixture of serrated and traditional adenomatous dysplasia, and 9 (8.0%) of these tsas showed intramucosal adenocarcinoma 24 . The frequency of coexisting adenocarcinoma in the tsas in the present study was lower than that reported by kim et al . However, they analyzed samples from surgical pathology files, and the polyps were relatively larger (3 63 mm) than ours; both factors likely contributed to the difference in the coexisting adenocarcinoma incidence in tsas . In addition, the total number of tsas evaluated in the present study was relatively small; therefore, we believe further studies are needed to determine the risk of malignancy in tsas . A tumor size of 10 mm was found to be the optimal cut - off value for coexisting adenocarcinoma . In addition, males accounted for a large proportion of the patients with ssa / ps (68.6%), but conversely, females accounted for 80.0% of those with coexisting adenocarcinoma . In female patients with serrated polyps larger than 10 mm, thus, the coexisting adenocarcinoma rate of ssa / ps in the high - risk group was similar to that among patients with traditional adenomatous polyps . Therefore, endoscopically detected and resected polyps with a serrated phenotype are not always at high risk of becoming malignant . However, the rate of progression from a serrated lesion to crc is unknown and may depend on msi - h or braf mutation status . . There has been one report of a patient who developed crc from ssa / ps in only 8 months 25 . However, another report indicated that it took 15 years for crc to develop from ssa / ps 21 . Therefore, it is difficult to predict which serrated polyps have the potential to become malignant . Moreover, schreiner et al . Reported that large serrated polyps were associated with an increased risk for synchronous advanced neoplasia 7 . However, there is insufficient information available regarding the natural history and epidemiology of ssa / ps and tsas . Furthermore, there is no surveillance strategy for serrated lesions after polypectomy . In the current study, the complete en bloc resection rates for ssa / ps and tsas were 65.2 and 62.4%, respectively . Pohl et al . Reported that the predictors of incomplete resection included the endoscopist, increased polyp size, and ssa / p rather than traditional adenoma 26 . In this study, the overall rate of complete resection of sessile serrated lesions was 69.0%, which is similar to our data presented here . The rate of incomplete resection was higher for these polyps than for traditional adenomatous polyps (7.2%) 26 . We experienced one case of local ssa / ps recurrence due to positive findings at the cut end (data not shown). It is difficult to perform a complete en bloc resection because ssa / ps are flat, similar in color to the surrounding normal mucosa, and contain unclear edges . Rex et al . Recently reported that the rate of recurrence after er was similar for ssa / ps and traditional adenomas 27 . These findings suggest that the best surveillance strategy after er for serrated polyps might be the same as that for traditional adenomatous polyps . Generally, most colorectal polyps <5 mm were not resected in this study; resecting all of the small polyps would have been time consuming, and we previously thought that such small lesions developed slowly and required a long period of time to become invasive cancers . We believed that hyperplastic polyps were harmless, and we thus tended to ignore them, even when they were larger than 5 mm . Unfortunately, it was difficult to stratify the patients based on initial colonoscopy or initial er, but if we did not achieve a clean colon in the initial colonoscopy, the patients underwent a repeat er to attain a clean colon, except in cases of small polyps . Therefore, the current findings may not reflect the precise frequency of serrated polyps among patients with colorectal polyps, but the data are valid regarding the frequency of clinically resected colon polyps . We did not evaluate interobserver variation in assessments of macroscopic appearance, polyp size or ssa / p versus hyperplastic polyp differentiation . 2 shows the trends in the percentages of resected hyperplastic polyps and serrated polyps among all resected colorectal polyps in the current study . We first considered that ssa / ps diagnosed in the early period may have been misdiagnosed as hyperplastic polyps, but the percentage of hyperplastic polyps remained constant at approximately 5% during the observation period . In contrast, the percentage of serrated polyps rapidly increased after 2009, but since then, the percentages of hyperplastic polyps and serrated polyps have been similar . A new pathologist (dr . Adachi) replaced the previous pathologist in 2009, perhaps suggesting that the frequency of polyps diagnosed as ssa / ps greatly depends on the pathologist . In addition, our hospital introduced narrow - band imaging endoscopy in 2007; therefore, use of advanced endoscopy and the change in pathologist may have been the main reasons for the higher rate of resection of serrated polyps in the latter period . In conclusion, the overall frequency of ssa / ps was 3.0% among all resected polyps in the present study; the frequency of serrated polyps varied in previous studies depending on study design . Tsas were much less common than ssa / ps, accounting for only 0.6% of the polyps in this study . Among the resected colorectal polyps, the rate of coexisting adenocarcinoma in serrated polyps was significantly lower than that in traditional adenomatous polyps, but resection perhaps should be recommended to females with serrated polyps 10 mm because of the increased risk of coexistent carcinoma . F: female; m (sex): male; c: cecum; a: ascending colon; t: transverse colon; s: sigmoid colon; r: rectum; m (histological depth): mucosa; sm: submucosa; tsa: traditional serrated adenoma; ssa / p: sessile serrated adenoma / polyp endoscopic images of patients with serrated polyps with adenocarcinoma (cases a i, supplementary table 1, h: after injection of saline into the submucosa). Trends in percentages of resected hyperplastic polyps and serrated polyps among all resected colorectal polyps in the current study.
Depression, a commonly seen psychological health problem across the world, prevents the functionality, creativity, happiness, and satisfaction of individuals, reduces their quality of life, and leads to losses in the work force (1). Pregnancy, one of the important processes in women, is a natural event as well as a period during which many biological and psychosocial changes are experienced . The risk of the many factors that may cause depression is high because of its stress and anxiety (1 - 3). Besides, the neuroendocrinological and psychosocial changes that pregnancy causes are too many compared to other periods of life (4, 5). High levels of norepinephrine and cortisol decrease blood flow into the uterus and thus cause severe obstetric and neonatal problems for both the pregnant woman and the fetus (6 - 8). These problems may be listed as follows: spontaneous abortion, antenatal bleeding, increased uterine artery resistance, preeclampsia, eclampsia, fetal death, low apgar score, newborns with low birth weight and high levels of cortisol, neonatal growth retardation, and babies that require neonatal intensive care (6, 9, 10). International studies emphasize that many cases of depression are among women aged 18 - 44 years and that depression comprises fecundity periods such as pregnancy, birth, and puerperality (1, 8). The incidence of depression and its symptoms ranges between 8% and 38% (7, 11 - 14) in the world . This incidence varies between 12% and 36% in turkey (1, 2, 15). Among the factors that increase depression risk during pregnancy are history of depression, younger ages of mothers, low socioeconomic status, being exposed to violence before and during pregnancy, disharmony between couples, living alone, having experienced a miscarriage in the past, undesired pregnancy or ambivalent thoughts about pregnancy, having many children, and a lack or absence of social support (1, 2, 14). Social support is described as financial, emotional, and mental support given to somebody by others (3, 16). Social support positively and directly affects one s health whether there is stress or not and protects psychological well - being by decreasing or balancing the damages brought about by the stressors caused by life events (3, 17). The conducted studies indicate a close correlation between increased depression levels and insufficient levels of social support during pregnancy (10, 18, 19). Insufficient social support during pregnancy deteriorates the psychological health of the pregnant woman and affects negatively her quality of life, has a poor effect on eating habits, and leads to an increase in the use of alcohol, smoking, and substance use (3, 10). Depression, one of the health problems frequently happens among pregnant women, is a crucial problem, which should be carefully dealt with, diagnosed early and treated soon because it has an adverse effect on the wellness of the pregnant women, paves the way for postpartum depression, may become chronic, and increases the risk of attempted suicide (1, 2, 20). Depression can be prevented and treated if health care professionals can detect the factors that increase the risk of depression during pregnancy at an early period (1, 21). The aim of the current study was to determine depressive symptom levels of the pregnant women and the sociodemographic and obstetric risk factors that might lead to depression, as well as exploring the correlation between social support and pregnancy depression . This study aimed to determine the frequency of depression symptoms, and its risk factors . The population of this study consisted of the pregnant women who presented at the maternity hospital of trabzon from may 21 to june 13, 2008 . The study was conducted in a large hospital in northeastern turkey and almost all pregnant women in this region, particularly those living in the vicinity of trabzon province, received antenatal care in that hospital . Trabzon maternity hospital is a public hospital with approximately 6188 deliveries (3170 vaginal delivery and 3018 cesarean) per year . It was chosen for this study as it is the largest obstetrics hospital in the area with a 300 beds . The present study design was cross - sectional based on time and descriptive according to the purpose of the study (22, 23). The sample of this study was calculated according to the formula in which the number of individuals in the population is unknown (equation 1). Where, p = 0.22 (according to research conducted earlier, the incidence of depression in pregnancy in turkey is 22%), q = 1 - 0.22 = 0.78, d = 0.052 = 0.025, and n = 3.84 (0.78 0.22) / 0.025 = 263 (22, 23). According to the results of the above formula, 3 more women were added to the sample in case of probable data loss and as a result, a total 266 pregnant women comprised the research sample . In the literature, it is stated that a maximum of 10% data more than the determined sample size can be enrolled (22, 23). Pregnant women who accepted to participate in the study were selected by simple random method . The inclusion criteria besides being pregnant were as follows: their labor had not started and without any pregnancy complications (placenta previa, preeclampsia, fetal distress, etc .) Without any known psychiatric or neurological disorders that would interfere with the completion of the measurements . We aimed to recruit all pregnant women who met the inclusion criteria, but those who used psychiatric drugs during pregnancy (3 women), those whose labor had started (28 women) and those who did not accept participating in the study for any reason (10 women) were excluded from the study . The questionnaire form included a descriptive information form that included sociodemographic information, the beck depression inventory (bdi), and the multidimensional scale of perceived social support (mspss). It included questions related to the sociodemographic characteristics of the study participants and their medical and obstetric history . After reviewing the relevant literature (1, 2, 6, 11, 15, 24 - 26), this form was developed by the researcher to study the background and sociodemographic characteristics of the pregnant women . After the reviewing the literature, the researchers designed a three - part questionnaire form . The first part was composed of a descriptive information form, which addressed sociodemographic, obstetric characteristics, and some special situations of pregnancy . The second part was bdi to determine pregnancy depression risks and the third part comprised the mspss . (1961) (27) and includes 21 self - evaluation statements about the symptoms of depression and each item scores from 0 to 3 . The aim of the inventory is not to diagnose depression but to measure the severity of the depression symptoms objectively . A score between 0 and 9 indicates no depression; a score between 10 and 16 indicates a mild level of depression; a score between 17 and 24 indicates a moderate level of depression; and a score of 25 or higher indicates a severe level of depression (major). The turkish adaptation, reliability, and validity tests of the inventory were performed by hisli and the cut - off point in the study was accepted as 17 . A score of 17 detects depressive symptoms that require medical treatment with an exactness of 90% (28). It is rated based on a 7-point likert scale with responses ranging from absolutely no to absolutely yes (1 - 7 points). There are 3 subscales of mspss (family support, friend support, and significant other support) and each subscale is composed of four statements . The lowest score to be obtained from each subscale is 4 whereas the highest score is 28 . The lowest score to be obtained from the scale is 12 whereas the highest score is 84 . A higher score indicates a higher level of perceived social support while a lower score indicates no perceived social support or a lack of perceived social support (29). The turkish adaptation, reliability, and validity tests of the scale were confirmed by eker et al . Each pregnant woman was contacted by that observer and provided with a detailed explanation of the purpose and procedure of the study . The questionnaire form, bdi, and mspss used for the data collection were filled in by the researchers using face - to - face interviews with the pregnant women in a separate and quiet room . If the pregnant women were unable to complete the questionnaire form on their own, the researchers read out the questionnaire items to the women and recorded the answers . Smirnov test was used to determine normal distribution of the data . In the statistical evaluation, some characteristics of the pregnant women (age, education, etc .) And mean bdi score were found not to conform to the normal distribution . Data were evaluated using the mann - whitney u, kruskal - wallis tests, and the percentage, mean, standard deviation, median, mean rank, minimum and maximum values were also calculated . The relationship between pregnant women bdi total scores and social support and subscale total score was evaluated using the pearson correlation analysis . Written informed consent was obtained from pregnant women, and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki as reflected in a prior approval by the institution of human research committee . The aim of the research was explained to the pregnant women and they were informed that if they preferred not to continue, they could withdraw from the study any time they wished . The present study design was cross - sectional based on time and descriptive according to the purpose of the study (22, 23). The sample of this study was calculated according to the formula in which the number of individuals in the population is unknown (equation 1). Where, p = 0.22 (according to research conducted earlier, the incidence of depression in pregnancy in turkey is 22%), q = 1 - 0.22 = 0.78, d = 0.052 = 0.025, and n = 3.84 (0.78 0.22) / 0.025 = 263 (22, 23). According to the results of the above formula, the research sample would be 263 pregnant women . In this study, 3 more women were added to the sample in case of probable data loss and as a result, a total 266 pregnant women comprised the research sample . In the literature, it is stated that a maximum of 10% data more than the determined sample size can be enrolled (22, 23). Pregnant women who accepted to participate in the study were selected by simple random method . The inclusion criteria besides being pregnant were as follows: their labor had not started and without any pregnancy complications (placenta previa, preeclampsia, fetal distress, etc .) Without any known psychiatric or neurological disorders that would interfere with the completion of the measurements . We aimed to recruit all pregnant women who met the inclusion criteria, but those who used psychiatric drugs during pregnancy (3 women), those whose labor had started (28 women) and those who did not accept participating in the study for any reason (10 women) were excluded from the study . The questionnaire form included a descriptive information form that included sociodemographic information, the beck depression inventory (bdi), and the multidimensional scale of perceived social support (mspss). It included questions related to the sociodemographic characteristics of the study participants and their medical and obstetric history . After reviewing the relevant literature (1, 2, 6, 11, 15, 24 - 26), this form was developed by the researcher to study the background and sociodemographic characteristics of the pregnant women . After the reviewing the literature, the researchers designed a three - part questionnaire form . The first part was composed of a descriptive information form, which addressed sociodemographic, obstetric characteristics, and some special situations of pregnancy . The second part was bdi to determine pregnancy depression risks and the third part comprised the mspss . (1961) (27) and includes 21 self - evaluation statements about the symptoms of depression and each item scores from 0 to 3 . The aim of the inventory is not to diagnose depression but to measure the severity of the depression symptoms objectively . A score between 0 and 9 indicates no depression; a score between 10 and 16 indicates a mild level of depression; a score between 17 and 24 indicates a moderate level of depression; and a score of 25 or higher indicates a severe level of depression (major). The turkish adaptation, reliability, and validity tests of the inventory were performed by hisli and the cut - off point in the study was accepted as 17 . A score of 17 detects depressive symptoms that require medical treatment with an exactness of 90% (28). It is rated based on a 7-point likert scale with responses ranging from absolutely no to absolutely yes (1 - 7 points). There are 3 subscales of mspss (family support, friend support, and significant other support) and each subscale is composed of four statements . The lowest score to be obtained from each subscale is 4 whereas the highest score is 28 . The lowest score to be obtained from the scale is 12 whereas the highest score is 84 . A higher score indicates a higher level of perceived social support while a lower score indicates no perceived social support or a lack of perceived social support (29). The turkish adaptation, reliability, and validity tests of the scale were confirmed by eker et al . Each pregnant woman was contacted by that observer and provided with a detailed explanation of the purpose and procedure of the study . The questionnaire form, bdi, and mspss used for the data collection were filled in by the researchers using face - to - face interviews with the pregnant women in a separate and quiet room . If the pregnant women were unable to complete the questionnaire form on their own, the researchers read out the questionnaire items to the women and recorded the answers . Smirnov test was used to determine normal distribution of the data . In the statistical evaluation, some characteristics of the pregnant women (age, education, etc .) And mean bdi score were found not to conform to the normal distribution . Data were evaluated using the mann - whitney u, kruskal - wallis tests, and the percentage, mean, standard deviation, median, mean rank, minimum and maximum values were also calculated . The relationship between pregnant women bdi total scores and social support and subscale total score was evaluated using the pearson correlation analysis . Written informed consent was obtained from pregnant women, and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki as reflected in a prior approval by the institution of human research committee . The aim of the research was explained to the pregnant women and they were informed that if they preferred not to continue, they could withdraw from the study any time they wished . After these explanations, 266 pregnant women consented to participate in the study voluntarily . Of studied pregnant women, 84.9% were housewives, 54.5% had primary school or secondary school certificates, 51.9% were primigravida, and 68.8% were in the third trimester . Table 1 presents the distribution of depressive symptom severity and the mean bdi scores of the pregnant women . The mean bdi scores of the pregnant women was found to be 11.12 6.65 . Depression symptom severity of 18.2% of the pregnant women was at a level that required treatment and the mean bdi score in this group was 21.62 5.24 (table 1). (%) or mean sd . The effect of sociodemographic characteristics of the pregnant women on the mean bdi score is presented in table 2 . There were significant differences between the mean bdi scores and variables of educational degrees, employment status and husbands educational level, among the groups (p <0.05), whereas no difference existed between mean bdi scores and variables of age, husbands occupation, perceived economical income, length of marriage and family type among the groups (p> 0.05) (table 2) the effects of obstetric characteristics of the pregnant women upon mean bdi scores are presented in table 3 . There were statistically significant differences between mean bdi scores and variables of having an undesired pregnancy, having a chronic disease before pregnancy, experiencing pregnancy - related problems, having a child with disability or having relatives whose children were disabled (p <0.05). The mean bdi scores of those who had a miscarriage, cesarean delivery, felt unready for motherhood, and those who knew the sex of the baby but were dissatisfied with it were low though not statistically significant (p> 0.05) (table 3). Mann - whitney u test . Kruskal - wallis test . Table 4 showed the effect of some special situations of the pregnant women upon the mean bdi score . The correlation between smoking during pregnancy and the mean bdi score was statistically significant (p <0.05). However, mean bdi scores were high, though not statistically significant, among pregnant women whose type of pregnancy was different, were exposed to physical - psychological violence by their husbands, were betrayed by their husbands, and those that did not like their appearance due to weight gain during pregnancy (p> 0.05) (table 4). The highest perceived social support of the pregnant women came from significant others / husband (24.63 5.29), family (24.10 5.59) and friends (19.22 7.19). A high significant correlation existed between the mean total mspss score and the mean bdi score of the pregnant women (p <0.001) (table 5). Abbreviations: mspss, multidimensional scale of perceived social support . Found to be extremely and negatively significant . Using the pearson correlation analysis . Depression, one of the frequently seen health problems among women, is experienced by women in fecundity periods and its incidence increases with pregnancy . In the studies that investigated the incidence of depression during pregnancy in different cultures, the rate of depression was found to be 7.5% in china, 8.1% in korea, 17.9% in hungary, 30% in canada, 20% in the usa, and 19.6% in brazil (5, 11 - 14, 31). As for turkey, the incidence of depression during pregnancy ranges from 12% to 36% (1, 2, 15). Our study detected an 18.2% rate in the type of pregnancy depression that required medical treatment . The depression level detected by the current study and mean depression scores were similar to some studies while different from others; the reason for this discrepancy may be due to the different culture of the studied societies and or the use of the different measurement methods to detect depression . It is emphasized in some studies that these factors, including age, low socioeconomic status, negative life experiences, lack of a job with satisfactory income, family problems, low educational status of pregnant women and their husbands augment the severity of the depression symptoms (20, 32 - 34). In our study, the educational status of the pregnant women and their husbands as well as the employment status of the pregnant woman were detected as the factors that increased the severity of the depression symptoms . (34) noted that pregnancy depression was seen more among pregnant women who had low educational level and worked at a job with a unsatisfactory income . Current or past history of pregnancy (miscarriage or abortion), unplanned pregnancy, having a chronic disease and emotional and physical problems experienced during pregnancy are obstetric risk factors for pregnancy depression (2, 8, 20, 25). Similar to the literature, our study indicated that unplanned pregnancy, having a chronic disease and pregnancy - related problems increased the severity of depression among pregnant women (p <0.05). Many studies highlighted that pregnancy depression is found more among women who have an unplanned and undesired pregnancy, have a chronic disease and face problems in the current pregnancy (15, 18, 24, 35). Our study pointed to the fact that pregnant women with a disabled child or relatives with child disability had increased depression symptom severity . The literature states that pregnant women who themselves have a child with mental / physical disability or have first degree relatives or close friends having a child with mental / physical disability is an important risk factor that affects pregnancy depression (21). In the studies of raina et al . (36), karadavut and uneri (26), and pistav akmese et al . (37) in which mothers with children with disability were investigated, it was found that these mothers had a trait anxiety level above the average level because ambiguities related to what kind of problems the disabled children will meet in the future may cause trait anxiety and depression in the family . The type of marriage may be regarded as a risk factor for pregnancy depression . In the literature review, no study was found in which the type of marriage (disapproved marriage: running away and getting married to somebody that the family members are opposed to; or approved marriage: getting married to somebody that the family members approve of) had been examined . In our study, nearly 14% of the participating women were in a disapproved marriage and their mean bdi scores were found to be higher than in the other type of marriage . If the fact that these pregnant women in disapproved marriages were adolescent at the time of the marriage is taken into consideration, the higher mean bdi scores of these women may relate to the possibility that they had poor / insufficient social support from their families . Weight gain during pregnancy, experiencing domestic violence, history of physical, emotional and sexual violence, betrayal by the husband, smoking, and alcohol and substance use are some of the social factors that affect pregnancy depression (14, 21, 38, 39). The studies conducted show that weight gain during pregnancy causes dissatisfaction among pregnant women and consequently depressive symptoms increase during pregnancy (4, 39, 40). (18), lancaster et al . (34), karmaliani et al . (38), and leigh et al . (41) reported that depression was seen more among women who gained excessive weight, had smoked and were subjected to psychological and physical violence during pregnancy . Likewise, in our study, it was found that a statistically significant correlation existed between smoking and depression symptom levels (p <0.05). Depression symptom severity was higher among pregnant women who were subjected to psychological and physical violence, betrayed by their husbands, gained excessive weight during pregnancy and not satisfied with it; yet, the differences were not statistically significant . Social support provided by the husband, family and/or friends during pregnancy comforts pregnant women emotionally and mentally and enables them to use social sources more, helping them to cope with stressors and anxiety more easily and paving the way for their transition into motherhood roles (1, 17, 19, 25, 35). In the similar studies, it is emphasized that there is a correlation between social support during pregnancy and depression and anxiety levels and also lack of social support augments levels of depression and anxiety (1, 3, 19, 20). The total mspss score of the pregnant women in our study was 67.89 14.26 . It was found out that the pregnant women got the highest social support first from significant persons in their lives (their husbands) (24.63 5.29), second from their families (24.10 5.59), and finally from their friends (19.22 7.19). Furthermore, the social support scores obtained from these 3 groups affected mean bdi scores of the pregnant women (p <0.001). This finding was in agreement with the literature . To conclude, this means that mean bdi scores decreases as mspss scores increases . In other words as the perceived social support increases, the psychological problems caused by stressful life events decrease . In a study in canada, it was reported that both pregnancy depression risks and postpartum depression risks considerably increased among those with low social support levels during pregnancy (17). (18) carried out in germany measured the social support scores of 896 pregnant women who were in the first trimester of pregnancy . After this follow up, it was found that the pregnant women with low social support had higher levels of depression symptoms and a decreased quality of life and smoked more during the pregnancy compared to those with high social support . In the randomized study of leigh et al . (41) conducted in australia on 367 pregnant women, it was found that depressive symptom levels were higher among the women who had poor or no social support compared to those with moderate and high levels of social support . In conclusion, it was found that one - fifth of the pregnant women had a depressive symptom level (17 and 18.8%) that required medical treatment and such sociodemographic and obstetric factors as the pregnant women s educational level, employment status, husbands educational level, presence of a chronic disease, having problems during pregnancy, whether the pregnancy was planned or not, and having a child with disability or having relatives who had children with disability affect severity of depressive symptoms . In addition, a significant correlation was found between the social support given during pregnancy and decreased depressive symptom severity . The study was conducted in one city with a selected group of women who resided in the city center and therefore, its generalizability is considerably limited as the sample group was small . Also, the other limitation of the study was that detailed psychiatric examinations and diagnosis were not performed but only the women's depression and anxiety levels were measured with scales and inventories . We are of the opinion, however, that the study will shed light on relevant studies in the future as it provides information about the situation and frequency of depression in the pregnant women of one city in our country.
Pgp, the most well characterized drug transporter in the abc protein superfamily is encoded by the abcb1, previously named mdr1, gene.12 among oncologists, pgp may be most wellknown for its role in mediating chemotherapeutic multidrug resistance . Justifiably, when pgp was first discovered in a multidrug resistant cell line, the gene encoding it was designated the multidrug resistance (mdr) gene . Pgp causes multidrug resistance by using energy derived from atp hydrolysis to transport substrates across the plasma membrane often against a steep concentration gradient.13 because the transport is unidirectional, from within the cell to the extracellular space, tumor cells expressing pgp have relatively low intracellular concentrations of anticancer drugs that are transported by (substrates for) pgp compared to tumor cells that do not express pgp . Thus, tumor cells expressing pgp are resistant to a variety of structurally and functionally diverse anticancer drugs that are pgp substrates (table 1).12, 13 anticancer drugs or drug classes and their status as pgp substrates.41, 42 alkylating agents = chlorambucil, cyclophosphamide, lomustine, others . Antimetabolites = cytarabine, 5fluorouracil, gemcitabine, methotrexate, others . Despite its important role in mediating chemotherapeutic drug resistance, it is doubtful that pgp actually evolved to protect tumor cells from anticancer drugs . It was not until many years later that researchers began investigating a possible physiologic function for the transporter . Expression of pgp was identified in nonneoplastic tissues initially in humans and rodents and much later in companion animal species.5, 14 the highest levels of pgp expression by cells occurs in tissues that either serve as barriers to drug absorption (apical border of intestinal epithelial cells), enhance drug elimination from the body (biliary canalicular or renal tubular epithelial cells), or on capillary endothelial cells at socalled sanctuary sites (blood brain barrier; testes; and placenta).12 because of its strategic location and its highly efficient drug efflux function, pgp limits oral absorption, enhances excretion, and prevents entry of substrate drugs into specialized tissues.5, 12, 13 from an evolutionary perspective, it is presumed that pgp functions in a protective capacity for mammalian organisms by decreasing their exposure to potentially toxic xenobiotics.12 thus, it should not be surprising that animals with defective pgp function are highly susceptible to toxicosis when treated with drugs that are pgp substrates.15, 16, 17 substrates for pgp include not only anticancer drugs (table 1), but a wide variety of other drugs as well (table 2). Selected pgp substrates that are not anticancer drugs.6, 16, 43, 44 the asterisk indicates drugs for which evidence in the dog (specifically) exists; otherwise data was from human or rodent studies . Much of what is known about pgp's role in drug disposition in veterinary patients has been generated from studies and clinical observations of dogs affected by a wellcharacterized mutation in the abcb1 (mdr1) gene . This particular polymorphism [abcb11; nt230(del4)mdr1; the mdr1 mutation] consists of a 4 basepair deletion mutation at the 5 end of the abcb1 gene.16 the deletion results in a shift of the reading frame that generates several premature stop codons terminating protein synthesis before 10% of the protein product is synthesized . Dogs with two mutant alleles (mdr1 mutant / mutant) exhibit a pgp null phenotype, similar to abcb1 (mdr1) (/) knockout mice.18 heterozygotes, dogs with one mutant allele and one wildtype allele (mdr1 mutant / normal) have an intermediate phenotype with decreased pgp function compared to wildtype (mdr1 normal / normal) dogs.9 affected dogs include many herding and some sighthound breeds (table 3).19, 20, 21, 22, 23, 24 intrinsic pgp dysfunction in these dogs dramatically illustrates the important role pgp plays in the disposition of substrate drugs, particularly drug distribution and biliary drug excretion.6, 9, 17, 25, 26 percent of dog breeds that are heterozygous for abcb11 and are presumed to be at highest risk for drug drug interactions involving pglycoprotein because pgp is a component of the blood brain barrier, distribution of pgp substrate drugs to the brain is greatly increased in dogs homozygous for the mdr1 mutation (mdr1 mutant / mutant) dogs and moderately increased in heterozygous dogs (mdr1 mutant / normal).6, 16, 25, 26 macrocyclic lactones such as ivermectin can cause neurological toxicosis in any animal at high doses (greater than 2 mg / kg).27 mdr1 mutant / mutant dogs experience severe neurological toxicosis at much lower doses (~120 g / kg) because the defective blood brain barrier allows ivermectin to accumulate in brain tissue of these animals.16, 28 other pgp substrates (eg, loperamide, ondansetron, acepromazine, butorphanol, milbemycin, selamectin, and moxidectin) are more likely to cause neurological toxicosis in dogs with the mdr1 mutation compared to wildtype dogs.6, 11, 26 at typical doses, loperamide causes central nervous system depression in mdr1 mutant / mutant dogs, but not in wildtype dogs.6 acepromazine and butorphanol cause more profound and prolonged cns depression in dogs with the mdr1 mutation compared to wildtype dogs.1 collectively, these examples underscore pgp's critical role in drug disposition at the blood brain barrier . Pgp is expressed on both renal tubular cells and biliary canalicular cells but whether or not it plays a clinically relevant role in renal drug excretion in dogs is not known.5 conversely, ample evidence exists demonstrating the important role pgp plays in biliary drug excretion in dogs . Studies comparing biliary excretion of the radiolabeled pgp substrate tcsestamibi in mdr1 normal / normal and mdr1 mutant / mutant dogs demonstrate that mdr1 mutant / mutant dogs are unable to excrete this compound into bile.9 tcsestamibi is essentially undetectable in gallbladders of mdr1 mutant / mutant dogs but is highly concentrated in gallbladders of mdr1 normal / normal dogs (fig 1). Similarly, in studies involving mdr1 knockout mice, biliary excretion of the pgp substrate irinotecan was significantly decreased, roughly 40%, in mdr1 knockout mice compared to wildtype mice.29 extrinsic pgp deficiency, because of druginduced inhibition of pgp, can also decrease biliary excretion of pgp substrates . In rats for example, concurrent administration of a pgpinhibitor decreases the biliary clearance of doxorubicin, a pgp substrate, resulting in increased plasma concentrations of doxorubicin.8 similarly, administration of verapamil, a pgp inhibitor, to rats decreased biliary excretion of the pgp substrate irinotecan by half.30 decreased biliary excretion of chemotherapy drugs would increase the patient's overall exposure to the drug, with a corresponding increase in the likelihood of druginduced toxicity such as myelosuppression and adverse gastrointestinal effects . Ventral images of the abdomen acquired at 120 minutes after intravenous injection of tcsestamibi to an mdr1 normal / normal dog (a) and an mdr1 mutant / mutant dog (b). Intense tcsestamibi activity (arrow head) is present in the gallbladder in the mdr1 normal / normal whereas a void of activity is observed in the location of the gallbladder in the mdr1 mutant / mutant dog.9 reproduced from: coelho jc, tucker r, mattoon j, roberts g, waiting dk, mealey kl biliary excretion of technetium99msestamibi in wildtype dogs and in dogs with intrinsic (abcb11delta mutation) and extrinsic (ketoconazole treated) pglycoprotein deficiency . Diminished biliary drug excretion is considered to be the mechanism responsible for the increased sensitivity of mdr1 mutant / mutant and mdr1 mutant / normal dogs to the myelosuppressive and gastrointestinal effects of chemotherapeutic drugs that are pgp substrates . A prospective study demonstrated that these dogs are significantly more likely to develop hematologic toxicity, both neutropenia and thrombocytopenia, after treatment with the pgp substrate vincristine (0.50.7 mg / m) than mdr1 normal / normal dogs.17 the authors considered a similar study assessing doxorubicin toxicosis in dogs with the mdr1 mutation, but abandoned the study because the first two mdr1 mutant / mutant dogs that received doxorubicin (30 mg / m) died from overwhelming neutropenic sepsis . In dogs, vincristine is eliminated primarily via biliary excretion of parent drug with some urinary excretion of parent drug and metabolites.31 in other species, biliary excretion of doxorubicin is a critical component of its overall clearance.8 therefore, mdr1 mutant / mutant and mdr1 mutant / normal dogs should receive reduced doses of pgp substrate chemotherapeutic agents (eg, doxorubicin, vincristine, vinblastine) in order to avoid severe toxicosis . The fact that mdr1 mutant / mutant and mdr1 mutant / normal dogs tolerate full doses of nonpgp substrate chemotherapeutic drugs such as alkylating agents, platinum compounds and antimetabolites lends credence to the contention that deficient pgpmediated biliary excretion is the mechanism responsible for the increased sensitivity of mdr1 mutant / mutant and mdr1 mutant / normal dogs to chemotherapeutic drugs that are pgp substrates . Increased brain penetration of pgp substrates such as macrocyclic lactones, loperamide, acepromazine, and butorphanol causes greater cns depression in mdr1 mutant / mutant and mdr1 mutant / normal than in mdr1 normal / normal dogs.6, 11 for these drugs, cns depression is an adverse effect observed (and expected) when these drugs are administered at high or extremely high doses to any patient . For chemotherapeutic drugs, however, intrathecal administration of vincristine in human patients is associated with a central neuropathy consisting of ascending progressive radiculomyeloencephalopathy.32 vincristineinduced central neurotoxicity occurred after intravenous, not intrathecal, administration in a collie (mdr1 mutant / mutant) described in a recent case report.33 because extensive workup including advanced imaging, cerebrospinal fluid analysis and infectious disease (neospora, toxoplasma) titers ruled out other potential causes for the dog's neurological signs, it was concluded that vincristine was able to penetrate this dog's blood brain barrier because of pgp deficiency . Thus, it is important to keep in mind enhanced neurological toxicity as a sequelae of pgp deficiency for those chemotherapeutic agents that are both neurotoxic and substrates for pgp . Because pgp contributes to multidrug resistant tumor phenotypes in cancer patients, numerous drug candidates have been developed to inhibit pgp in an effort to improve outcome in patients treated with chemotherapy.10, 34 in human clinical trials involving pgp inhibitors unexpected and undesired pharmacokinetic interactions ensued between the inhibiting agents and the chemotherapeutic drugs . As a result, patients experienced severe adverse effects necessitating discontinuation or dose reductions of the chemotherapeutic drug(s).4, 35 for example, the potent pgp inhibitor valspodar was concurrently administered with the pgp substrate vinblastine to human patients with advanced malignancies . Because of severe toxicoses (myelosuppression and gastrointestinal) related to relative increases in vinblastine exposure, many patients required significant dose reductions (~50%) for subsequent vinblastine treatments.10 a different clinical trial in human patients with advanced cancer, this one using paclitaxel as the cytotoxic pgp substrate and valspodar as the pgp inhibitor, yielded similar results . Drug interaction.36 numerous other studies have documented these types of drug drug interactions whereby a pgp inhibiting drug increases a patient's overall exposure to a pgp substrate cytotoxic drug including vinca alkaloids, doxorubicin and paclitaxel (reviewed by varma).37 the current consensus regarding overcoming the phenomenon of pgpmediated chemotherapeutic resistance is that pharmacological pgp inhibitors cannot discriminate between pgp expressed by normal tissues, which protects the patient, and pgp expressed in cancerous tissue, which harms the patient.4, 35, 36, 37 therefore, despite in vitro success in using pgp inhibitors to enhance a chemotherapeutic drug's ability to kill tumor cells, this strategy has been abandoned in clinical practice because of enhanced toxicity in the patient . Pgp inhibitors used in the human clinical trials just described were specifically synthesized to be used in combination with pgp substrate anticancer drugs . However, many drugs commonly used in veterinary patients are also capable of inhibiting pgp function (table 3). Examples include a variety of structurally and functionally unrelated compounds including ketoconazole, spinosad, cyclosporine, omeprazole and others.4, 9, 38 it is essential for veterinarians working with animals receiving cytotoxic pgp substrates to understand that pharmacological pgp inhibition, socalled acquired or extrinsic pgp deficiency, can mimic what occurs in dogs with the mdr1 mutation, socalled endogenous or intrinsic pgp deficiency . For example ketoconazole increases brain penetration of ivermectin in mdr1 normal / normal dogs causing the same neurological toxicity one would expect to see in a collie with the mdr1 mutation (unpublished data). Ketoconazole's ability to inhibit pgpmediated biliary excretion of pgp substrates has been demonstrated using the radiolabelled pgp substrate tcsestamibi (fig 2).9 mdr1 normal / normal dogs had 1.8fold greater biliary clearance of tcsestamibi before treatment with the pgp inhibitor ketonazole than after treatment with ketoconazole . Similar effects would be expected for chemotherapeutic drugs whose clearance involves pgp mediated biliary excretion (ie, vinca alkaloids, doxorubicin, taxol). Because these drugs have a narrow therapeutic index, the delayed clearance resulting from the drug drug interaction would produce unexpectedly severe adverse effects in the patient relative to the dose administered . Time activity curves of gallbladder to liver activity ratios [using mean counts per pixel per regions of interest (roi)] for abcb1 wild / wild dogs before (: mean g / l ratio + sd, n = 6) and after (: mean g / l ratio sd, n = 6) administration of ketoconazole (5 mg / kg po q12 h 9 doses).9 reproduced from: coelho jc, tucker r, mattoon j, roberts g, waiting dk, mealey kl biliary excretion of technetium99 msestamibi in wildtype dogs and in dogs with intrinsic (abcb11delta mutation) and extrinsic (ketoconazole treated) pglycoprotein deficiency . J vet pharmacol ther this is presumed to be what happened to a dog (mdr1 normal / normal) being treated with vinblastine for a grade ii mast cell tumor . The dog experienced unexpectedly severe adverse effects (grade 4 neutropenia and grade 3 vomiting) after receiving a standard dose of vinblastine (2 mg / m) concurrently with ketoconazole . The severe vomiting and neutropenia led to sepsis, multiple metabolic disturbances and cardiac arrest . In humans, nearly 30% of an intravenous dose of vinblastine is actively excreted into the bile by pgp.39 ketoconazole also inhibits cytochrome p450 (cyp)mediated metabolism of vinblastine.2 thus, this patient experienced an overdose of vinblastine, despite administration of the recommended dose, because of ketoconazole's inhibitory effects on cyp and pgpmediated vinblastine elimination . Concurrent use of ketoconazole with other pgp substrate chemotherapeutic agents (table 1) would be expected to cause similar adverse effects . There is currently both anecdotal and experimental evidence that drug drug interactions involving pglycoprotein occur in mdr1 normal / normal dogs . However, it is likely that mdr1 mutant / normal dogs (table 3) would have an even higher risk of clinically significant pglycoproteinmediated drug drug interactions because of intrinsically diminished pglycoprotein function . Pgp inhibitors can act (i) by blocking the drug binding site either competitively, noncompetitive or allosterically; (ii) by interfering with atp hydrolysis which is required for pgp function; or (iii) by altering integrity of cell membrane lipids.4 from a clinical perspective, the most important mechanism involves competitively and noncompetitively blocking drug binding sites . It is essential to note that pgp has more than one drug (substrate) binding site, complicating our understanding of drug drug interactions involving pgp . Sitedirected mutagenesis studies have demonstrated that certain mutations alter pgp's affinity for some substrates, but not others.40 thus, it is possible that some pgp inhibitors might affect transport of vinblastine, for example, but not doxorubicin . Consequently it is not possible to predict which pgp substrate drugs in table 1 would be safe to use concurrently with pgp inhibiting drugs in table 4 . Selected pgp inhibitors.4, 45, 46, 47, 48, 49 evidence of pgpmediated drug drug interactions in dogs at clinically used doses . Many years passed between the initial discovery of pgp and the realization that pgp contributed substantially to the disposition of chemotherapeutic drugs not only within tumor cells, but in the patient . The importance of pgp mediated drug drug interactions has only recently been identified as a clinically important problem in human patients . Thus, drug package inserts may not contain information about pgpmediated drug drug interactions . Improved understanding of pgp drug binding sites and pgp dependent drug clearance and distribution mechanisms will enhance our ability to more specifically predict pgpmediated drug drug interactions . Until then, veterinarians will need to take a proactive role in considering potential drug interactions involving pgp.
Central serous chorioretinopathy (csc) is defined as a circumscribed neurosensory retinal detachment caused by focal retinal pigment epithelium (rpe) incompetence . It is generally a self - limiting condition, in which patients complain of metamorphopsia, micropsia, relative central scotoma, and a moderate decrease of visual acuity . Fluorescein angiography (fa) typically shows single or multiple inkblot early leakage points at the rpe followed by progressive localized subretinal leakage . Optical coherence tomography (oct) shows a localized dome - shaped macular detachment, with a normal neurosensory retina . Fine and owens reported the only case in the literature of csc in a child . When the clinical presentation is typical, diagnosis is forthcoming, based on ophthalmoscopic and fa findings . In atypical patients, however, a diagnosis of exclusion and further examination are warranted . A healthy 12-year - old female presented with a 3-day history of acute - onset blurred vision in her right eye without any prodromal symptoms . Initial best - corrected visual acuity was 20/40 in the right eye and 20/15 in the left eye . Fundus examination of the right eye revealed a normal coloration of the optic nerve and a retinal elevation at the fovea and inferior macula secondary to subretinal fluid without any signs of intraocular inflammation (fig . Right eye oct showed an accumulation of subretinal fluid which did not show an extension to the optic nerve head, excluding the presence of optic nerve pit (fig . Angiography demonstrated multifocal leakage at the level of the rpe at the papillomacular bundle region . Late phases of the fa showed subretinal leakage and pooling, with a very discrete hyperfluorescence of the optic nerve head (fig . Based on these findings, we decided to observe this patient without any treatment or intervention . After three months, visual acuity spontaneously improved to 20/15 and fundus evaluation was without any abnormal findings . Repeated fa and oct showed complete resolution of the multifocal leakage and subretinal fluid (fig . The patient has been periodically checked for 30 months without recurrence or any signs of inflammation in either eye . Csc is a clinical entity affecting middle - aged patients, usually males, in whom clinical appearance and fa are usually sufficient to confirm the diagnosis [1, 4,5,6]. In patients outside the usual age range of this condition (20 - 50 years), localized serous retinal detachment (srd) in childhood could be secondary to inflammatory, neoplastic, or developmental conditions . Infectious diseases or inflammatory conditions that produce multifocal choroiditis, which may simulate csc, include presumed ocular histoplasmosis syndrome (pohs), punctate inner choroidopathy (pic), and vogt - koyanagi - harada (vkh) disease . Pohs has characteristic punched - out choroidal lesions in the mid - periphery and peripapillary atrophy, which were not present in our patient . Pic, considered to be one of the white dot syndromes (wds), affects young and middle - aged myopic women (age range 15 - 55 years). Pic presents with blurred central vision and photopsias, and posterior pole examination reveals multiple (12 - 25) small (100300 mm) opaque round lesions with an srd occasionally overlying them . Other wds include multifocal choroiditis with panuveitis, multiple evanescent wds, acute multifocal posterior placoid pigment epitheliopathy, and birdshot chorioretinopathy . All of these have characteristic choroidal lesions and are mostly associated with a degree of anterior chamber inflammatory cells and vitritis, which were not associated with the lesions in our patient . Based on the multifocal leakage at the site of the srd and the discrete hyperfluorescent appearance of the optic nerve head on the late phases of the angiogram, the most important differential diagnosis is vkh disease . Vkh disease can have very different clinical manifestations depending on the stage of the disease . There are some reports occurring in children, and this group has been estimated to represent 3% of all patients with vkh disease [9, 10]. In our case, in contrast to the classic fa findings in csc, a late - phase, very discrete hyperfluorescence of the optic nerve head was found . However, the course of the disease seen in this case was incompatible with any presentation of vkh disease, since all patients with vkh disease present more than one sign of inflammation . Furthermore, vkh disease always requires treatment with no reported spontaneous resolution present in any case . Clinically observable chorioretinal pigmentary changes follow healing of the leakage spots, while in our case, no residual fundus changes were noticed after spontaneous resolution of the subretinal fluid . Optic nerve pit, which can manifest with serous macular detachment that communicates with the optic nerve, was not clinically detected in this patient . Moreover, our patient showed multifocal leakage on fa which is not associated with optic pit maculopathy . Another cause of fluid at the macular area is x - linked retinoschisis - this was ruled out since it is a bilateral condition that only affects males . Hemopoietic or intraocular neoplasia was excluded clinically and by complete blood count as the etiology of srd in this patient . In a report on 312 cases of csc, it was demonstrated that systemic steroid use, pregnancy, stress and type a personality were risk factors as shown in previous studies [4, 5]. In a study of csc in women only, it was reported that a higher than average age at onset, namely 53 years (range 40 - 60 years), and high levels of cortisol play important roles . An important cause - effect relationship between serum cortisol levels and csc presentation has been established . In the present case, cortisol was normal (22.1 g / dl, normal range 6.99 - 25 g / dl). Another contributing risk factor is high mean arterial blood pressure [4, 5]. Our patient had a mean arterial blood pressure of 100/60 mm hg (measured twice / day for 1 week). In conclusion, based on the clinical appearance and the natural course of the disease, this case is compatible with csc . To our knowledge, this is only the second case of a presumed idiopathic csc in a prepubertal female and the first one documented by fa and oct, as well as other studies to exclude secondary causes . Csc may affect females at a young age and should be included in the differential diagnosis of multifocal localized srd in that age group . None of the authors have any financial / conflicting interests or funding sources to disclose . The content of this case report has not been published or submitted for publication elsewhere nor has it been presented at any conference.
Metabolic diseases (mds) and neuromuscular disorders (nmds) comprise a large heterogeneous group of diseases, that directly (via intrinsic muscle pathology or defective metabolic pathways) or indirectly (via nerve pathology), impair muscle function and result in exercise intolerance . Although the value of exercise tests in patients with md / nmd has been acknowledged for several decades [13], the role of exercise stress tests as a diagnostic or evaluative tool in children and adults with md / nmd is relatively underresearched . Moreover, exercise stress tests are not standard for most centers to be performed in clinical care [47]. In this paper, we provide two standardized exercise tests with preliminary metabolic profiles in children with a diagnosed md / nmd for this purpose . Exercise stress tests in patients with a metabolic disorder involved in atp synthesis show a clear specific metabolic profile during exercise . These metabolic profiles can be useful as a reference for identifying patients for a possible md or nmd . Therefore, the aim of the current study was to describe the metabolic profiles during exercise in children with a diagnosed nmd or md . This information might be helpful for clinicians in the diagnosis and follow - up of patients with these disorders . In this retrospective chart review, patients with an established diagnosis involving general atp synthesis or a dystrophinopathy, who were referred for exercise stress testing to the departments of metabolic diseases and child development and exercise center, university medical center utrecht, the netherlands, were included . Thirteen patients (9, 4, age 515 years) with an established diagnoses were studied in detail . Diagnoses were glycogen storage disease (gsd) type 1a (1x), gsd type 3 (1x), gsd type 7 (1x), medium - chain acyl coa dehydrogenase deficiency (mcad (2x)), short - chain acyl coa dehydrogenase deficiency (scad (1x)), multiple acyl coa dehydrogenase deficiency (madd (2x)), ketothiolase deficiency (1x), mitochondrial myopathy (1x), hypokalemic episodic paralysis (1x), and dystrophinopathy (becker muscular dystrophy (bmd) (2x). Two exercise stress tests, respectively, a cardiopulmonary exercise test (cpet) and a prolonged exercise ergometry test (pxt) were performed following a standardized protocol . Blood samples were taken, immediately before and directly after the cpet and pxt, and analyzed for lactate, creatine kinase (ck), ammonia, acylcarnitines, and organic acid . Urine samples were collected up to three hours after the exercise test and further analyzed for creatinine, organic acid, amino acid, tetraglucose, purine, and pyrimidine . A cpet (to determine the peak oxygen uptake [vo2peak] and peak workload [wpeak]) and a submaximal pxt (90 minutes at 30% of wpeak) were performed in the morning . After a light breakfast, the patients performed a symptom - limited cpet on a bicycle ergometer . Workload was increased in constant increments of 10, 15, or 20 watts every minute, depending on the patients' length and was in some conditions adjusted for the physical condition of the patient . This protocol was continued until the patient stopped due to volitional exhaustion, despite strong verbal encouragement . During the tests, all subjects breathed through a facemask (hans rudolph inc ., kansas city, mo), connected to a calibrated respiratory gas analysis system (oxygen champion / pro, care fusion, houten, the netherlands). This system measured breath - by - breath minute ventilation (ve), oxygen uptake (vo2), and respiratory exchange ratio (rer = vco2/vo2) using conventional equations . During the maximal exercise test, heart rate (hr) peak hr (hrpeak), vo2peak, vo2peak / kg, and peak rer were taken as the average values over the last 30 seconds of the test . A pxt was performed one week after the maximal exercise test to prevent interference from the previous test . The pxt consisted of a 90-minute cycling at a constant work rate of 30% wpeak, as described previously . Blood samples were obtained from an indwelling catheter inserted into a vein of the dorsum of the hand . Five milliliters of blood from each sample was placed in lithium - heparin tube, except for determination of ffa and ammonia; respectively, normal blood (without li - heparin) and edta (another anticoagulant) blood was used for the analysis . After taking the blood, blood taken before and after the cpet was analyzed for lactate, creatine kinase (ck), ammonia, acylcarnitines, and organic acid . Urine samples taken before and after the cpet, were analyzed for creatinine, organic acid, amino acid, and tetraglucose, until three hours after the exercise test . During the pxt, blood samples were taken at regular time intervals (t = 0, 30, 60, 75, 90, 105, and 120 minutes after the start of the exercise). Concentrations of glucose, lactate, ck, free fatty acids (ffa), ammonia, 3-oh - butyric acid and 3-keto - butyric acid, acylcarnitines, and organic acid were determined . Before and up to three hours after exercise, urine samples were obtained and analyzed for creatinine, organic acid, amino acid, and tetraglucose . Glucose, lactate, ck, and ammonia were determined with a beckman coulter dxc chemical analysis machine (fullerton, usa). Enzymatic method was used for determination of ffa, 3-ketobutyric acid, and 3-oh - butyric acid . After lipoprotein lipase hydrolyzed triglyceride into fat acids and glycerol, free glycerol was measured colorimetrically . Organic acid concentration in urine and plasma was determined by gas chromatography - mass spectrometry as their trimethylsilyl derivates (hewlett packard 5890 series ii gas chromatograph linked to a hp 5989b ms - engine mass spectrometer (hewlett packard, avondale, pa)). Analysis of acylcarnitine in plasma as their butyl esters was performed by electrospray tandem mass spectrometry (esi - ms - ms; micromass quattro ultima, micromass ltd ., uk) equipped with an alliance hplc system (waters, milford, ma, usa). Analysis of amino acids in plasma and urine was done with amino acid analyzer (ion - exchange chromatography - ninhydrin). As expected, patients with a md / nmd showed abnormal results on the cpet (tables 1 and 2). Patient 2 (gsd-3) stopped the cpet because of myalgia in the lower limbs, compared to reference values for healthy children [10, 11], the patients with gsd-3, mcad, scad, and mitochondrial myopathy (patients 4, 6, and 10, resp .) Had a significantly reduced hrpeak . Rerpeak was significantly lower in the patient with gsd-3 (patient 2) and also in the patient with mcad (patient 4) and surprisingly increased to 1.0 in the patient with gsd-7 (patient 3). Vo2peak and vo2peak / kg were significantly lower in the patients 3, 4, 10, and 13 . These were patients with gsd-7, mcad, mitochondrial myopathy, and hypokalemic episodic paralysis, respectively . A remarkably high vo2peak / kg was observed in one of the patients with bmd (patient 12). The patients with gsd-1a and mitochondrial myopathy (patient 1 and 10, resp .) Had significantly increased lactate concentrations at rest . Patient 2, with gsd-3, had an increased ck values at rest and after exercise . The 2 patients with bmd (patients 11 and 12) showed persistently highly elevated ck levels . Biochemical profiles of the md / nmd patients during the pxt varied with the disorder (table 3)., patient 1 and 10), showed significantly increased concentrations of blood lactate at all time points . The patient, with gsd-7 had significantly increased ammonia concentrations with no rise in lactate during exercise . During and after exercise, the ck value of the patient with gsd-7 (patient 3) was significantly increased as well as in the 2 patients with bmd (patients 11 and 12). Acylcarnitines c6, c8, c10, c12, and c14:1 were all increased in two patients with madd (patients 7 and 8) in rest as well as during exercise . The patient with ketothiolase deficiency (patient 9) had increased c5:1 and c5-oh acylcarnitine during rest and exercise, as well as several increased organic acids in the urine . In the patient with mitochondrial myopathy (patient 10), c5 carnitine was increased in the urine during and after exercise . In the patient with scad (patient 6), there was no c4 carnitine found . In all other md / nmd patients, no altered acylcarnitines, carnitines, for organic acids concentrations the purpose of this study was to describe metabolic profiles during exercise using cpet and pxt including extensive blood and urine analyses in children with a diagnosed md / nmd . This information might be helpful for clinicians in the diagnosis and follow - up of these disorders . Because of the heterogeneity of the disorders, there was a large variation in the cpet and pxt results between patients . These differences reflect the different pathophysiology of the various disorders (e.g., defects in different metabolic pathways) and heterogeneity within disorders . For example, a low rise in lactate after cpet is suggestive for a gsd, and a very high increase in lactate, combined with a very low vo2peak, might be suggestive for a mitochondrial myopathy . Further studies should develop an algorithm for the interpretation of exercise data in md / nmd patients, comparable to the interpretative algorithms for cardiac and pulmonary limitations during exercise [12, 13]. The diagnostic yield of exercise stress testing in children with unexplained exercise intolerance seems relatively low . Among 29 patients referred for exercise intolerance of unknown origin, only 3 patients could be diagnosed with a md / nmd: 2 patients with a becker muscular dystrophinopathy and one patient with a hypokalemic episodic paralysis . However, many of these patients have undergone extensive medical screening before they were referred for exercise testing . It is our opinion that the expense of exercise testing including extensive blood and urine analyses is justified because it could be useful for guiding the diagnostic workup and can differentiate between patients with medically unexplained exercise intolerance and patients with a md / nmd . In patients with a md involved in atp synthesis, only during certain periods of metabolic stress (e.g., exercise, fasting, or illness), abnormal quantities of metabolites in blood and urine can be found, and symptoms are present . The current paper provides two standardized exercise tests with preliminary metabolic profiles for this purpose . Furthermore, several of the tested md / nmd patients (patients 3, 7, 8, and 10) were referred for exercise testing to assess their exercise capacity for physical activity recommendations . Based on their exercise results, sufficient amounts of physical activity are necessary for an optimal physical, psychosocial, and emotional development in children . In addition, for patient 12, we gave an exercise restriction based on the findings . However, during several races he developed myoglobinuria, and he had quite high resting values of ck . A muscle biopsy in the workup after the tests revealed a duplication in exon 24 - 29 of the dystrophin gene, and the diagnosis of bmd was made . Based on these results, the boy was advised to stop high - level cycling because of the increased risk of renal failure due to myoglobinuria . One of the limitations of this clinical report is the small and heterogeneous population . Therefore, multicentred studies are needed to increase the sample size for each of the disorders . Further, it is important that these profiles are established in children as not all metabolic profiles seen in adults are valid in children . For example, a recent study showed that the well - known second - wind phenomenon in patients with mcardle's disease (gsd5), which is considered as a diagnostic feature of this disease, was not observed in children with mcardle's disease . In this paper we describe the metabolic profiles during exercise in 13 children with a diagnosed md / nmd . Metabolic profiles during exercise were of assistance in diagnosing 3 patients with rare presentations of md / nmds . In addition, exercise stress testing was helpful for the prescription of appropriate levels of physical activity.
Primary non - hodgkin's lymphoma of bone (plb) is a rare disease that accounts for less than 2 percent of all lymphomas in adults (1). The histologies of the vast majority of cases present as diffuse large b - cell lymphoma, while t - cell lymphomas are extremely rare (2 - 10). Although there are a few publications regarding the characteristics, treatment options, and prognosis of plb, the optimal management remains unclear . Recently, several reports have supported the combined modality of chemotherapy and radiotherapy (11 - 13). Multiple myeloma is a b - cell neoplasia that is usually associated with osteolytic lesions and paraprotein in the plasma and/or in the urine (14 - 16). Because of the uniquely different cell lines of the two lymphoid malignancies, concurrent disease has rarely been reported . To our knowledge, the simultaneous development of primary t - cell lymphoma of bone and multiple myeloma has never been reported . In a view of the same bone lesion in the case of plb and multiple myeloma, we experienced the importance of reevaluation of disease regarding the possibility of other underlying diseases when it was deteriorated despite adequate treatment . A 48-yr - old male presented with right ankle pain, mass, and general weakness over 2 months . On physical examination, he appeared acutely ill but no other specific findings were noted . The complete blood cell counts showed leucocytes 8.710/l, hemoglobin 15.5 g / dl, and platelets 24510/l . Blood chemistry showed total protein 6.6 g / dl, albumin 4.6 g / dl, lactate dehydrogenase 236 u / l, blood urea nitrogen 14.2 mg / dl, and creatine 0.9 mg / dl . Magnetic resonance imaging (mri) of the right ankle revealed a heterogeneous osteolytic and extraosseous mass in the right tibia (fig . The bone biopsy showed diffuse infiltration of large tumor cells with vesicular prominent nuclei, abundant cytoplasm, and numerous mitotic figures . Immunohistochemical staining showed an lca, cd3, cd45ro, and cd15 phenotype (fig . Chest and abdomen computed tomography (ct) showed no abnormal findings such as lympadenopathy or hepatosplenomegaly . The tc - methylene diphosphonate (mdp) bone scan revealed hot uptake in the right distal tibia without any other bony involvement (fig . The peripheral blood smear was normal, and the bone marrow biopsy showed no lymphoma involvement . The patient was diagnosed as having primary t - cell lymphoma of bone, stage ie . Based on the international prognostic index, the patient received 6 cycles of chop chemotherapy (cyclophosphamide 750 mg / m i.v . After addition of the involved - field radiation therapy up to 5,700 cgy as once daily fraction and two more cycles of chemotherapy, the tumor mass was further decreased, but still remained on mri . F-18 fluorodeoxyglucose pet - ct showed the remaining hypermetabolic lesion on tibia, and also revealed hot uptake at the inferior ramus of the right pubis (fig . We thought that the disease was progressive and treated the patient with salvage chemotherapy including ice regimen (ifosfamide 5 g / m i.v . Day 2, and carboplatin auc of 5 i.v . Days 1 - 3) and the dhap regimen (dexamethasone 40 mg i.v . Days 1 - 4, cytarabine 2 g / m i.v . After three cycles of mine chemotherapy, restaging work - up showed a partial response . However, at the time of the fourth chemotherapy cycle, he complained of generalized bone pain . Complete blood cell counts revealed bicytopenia (leucocytes 4.510/l, hemoglobin 8.8 g / dl, platelets 8510/l), and the peripheral blood smear showed atypical lymphocytes (2%). Unexpectedly, the bone marrow biopsy showed diffuse infiltration of plasma cells with about 80% of bone marrow cells . Urine immunoelectrophoresis identified the monoclonal band as light chain lambda, and the free serum light chain lambda level in urine was 2,810 mg / l (range, 5.71 - 26.3 mg / l). Bone marrow biopsy showed diffuse uptake of cd138, which was not expressed in the lymphoma mass (fig . We concluded that the multiple myeloma was newly developed in the patient with primary t - cell lymphoma of bone . The general condition of the patient was rapidly deteriorated with thrombocytopenia and acute renal failure . The patient received a combination chemotherapy with cyclophosphamide and prednisolone, and plasmapheresis was performed to correct paraproteinemia and hypercalcemia . Despite aggressive supportive care, the patient died of renal failure . Plb generally presents as a solitary, extensive, destructive bone lesion (1). It is a very rare disease that accounts for less than 2 percent of all lymphomas in adults and accounts for approximately 3 - 5% of all malignant bone tumors and 4 - 7% of all extranodal non - hodgkin's lymphomas . Pathologic fracture and systemic' b' symptoms (fever, weight loss, and night sweats) also develop at the time of diagnosis (4, 5). A palpable mass due to soft tissue extension is seen in about one - half of cases (10). It is usually characterized by lytic bones lesions located in the metaphysis of long bones or in the axial skeleton (2 - 7). Histopathologically, the majority of plb are diffuse large b - cell type, while t - cell lymphomas are extremely rare (4 - 7). Management strategies include a combined modality of chemotherapy and radiotherapy (11 - 13). Although prognosis, survival, and complete remission rates of b - cell plb were well - described and high complete remission rates have been reported, the treatment response and prognosis of t - cell plb remain obscure and data are lacking (7 - 9). It is characterized by the neoplastic proliferation of a single clone of plasma cells producing a monoclonal immunoglobulin . This clone of plasma cells proliferates in the bone marrow and often results in skeletal osteolytic lesions . Common clinical features include recurrent bacterial infection, anemia, hypercalcemia, and renal insufficiency, and treatments include chemotherapy and radiotherapy (16). An interesting feature of this case is the development of multiple myeloma over approximately 1.5 yr following the appearance of a primary t - cell lymphoma of bone . He suffered from two lymphoid neoplasms of distinctly different cell lines: the first originating from t - cells and, 1.5 yr later, the second of b - cell origin . Bryant et al . Reported plasma cell myeloma in a patient with a cutaneous t - cell lymphoma (17). The investigators assumed that the malignant plasma cells evolved under the sustained inducer stimulus of the neoplastic t - cells . Wickenhauser et al . Also reported a newly - detected multiple myeloma in a patient with a long - standing anaplastic cutaneous t - cell lymphoma (20). In the present case, through immunohistochemical cd138 staining, we confirmed that the multiple myeloma originated from cells that differed from previously detected lymphoma cells . Neoplastic t - lymphocytes are known to retain immunoregulatory capabilities similar to those of their normal counterpart (18, 19). We also speculated that the proliferation of t - helper cells might have resulted in a sustained stimulation of b - cells, followed by proliferation of certain b - cell clones . It is also possible, although with a low order of possibility, that the two infrequent malignancies have arisen independently of each other . In this case, it is difficult to determine whether the bone lesion originated from multiple myeloma or lymphoma, based only on imaging studies . In conclusion, it is important to think about lymphoid malignancies of different pathology when an underlying lymphoid neoplasm shows rapid progression . In addition, considering the scarcity of the association between t - cell lymphoma and multiple myeloma, detection and reporting of new cases are of great value in the study of the potential relationships of immunoregulatory derangements caused by primary lymphoid tumors.
The peptide bond linking adjacent amino acids in protein structures can adopt either the cis or the trans conformation . The cis conformation occurs rarely in polypeptides because of the higher intrinsic energy compared to the trans conformation . Despite their infrequent occurrence, cis peptide bonds are very important in a variety of biological processes, such as protein folding, regulation, cell signaling and splicing of protein molecules (1). Recent studies have indicated that prolyl cis / trans isomerization can act as a molecular timer to help control the cellular process, making it a new target for therapeutic interventions (2). Furthermore, cis peptide bonds, especially the ones between non - proline residues, are located near the active sites of proteins, or have roles in the function of the protein molecules 1 . (5) trained a support vector machine (svm) using only the primary sequence as input in order to discriminate between the two conformations of proline peptide bonds . (6) predicted the isomerization of proline peptide bonds using multiple sequence alignment profiles and secondary structure as input . The cops algorithm (7) aimed to predict the peptide bond formation between any two amino acids employing an extension of the chou - fasman parameters . Most of the aforementioned studies focus only on the proline residues, ignoring the rare but highly important non - proline cis peptide bonds . Here, we make a further distinction of the peptide bonds into four classes, namely cis imide (cis - pro), cis amide (cis - nonpro), trans imide (trans - pro) and trans amide (trans - nonpro), by developing the pbond web server . Hence, pbond not only predicts the peptide bond conformation between any two amino acids, but also designates potential cis - nonpro formations . Furthermore, a reliability index is computed, which represents the confidence assigned to each prediction . A majority voting scheme is also available, which provides consensus prediction of 10 svm classifiers . Pbond has been developed using 3,050 high - quality protein sequences with resolution <2.0, r - factor <0.25 and sequence identity <25% . The pbond web server graphical interface, as shown in figure 1, consists of six fields . The numbers placed next to every field in the figure follow the same notation as below . 1 . Processing: the user may choose to process either a single sequence or upload multiple sequences for batch processing; there is no upper limit to the number of sequences submitted for batch processing . 2 . Upload sequence: amino acid sequences can be provided in fasta format either by pasting them in the text box or uploading them within a text file . More specifically, multiple sequence alignment profiles, in the form of position - specific scoring matrices (pssms), are obtained after running psi - blast (8) against one of the provided protein databases; the choice of the database highly affects the computational time, whereas only slight perturbations are expected in terms of performance . Next, the predicted secondary structure of every residue in the query sequence is computed using psipred (9); real valued predictions of solvent accessibility are obtained from rvp - net (10); six widely used physicochemical properties are also employed for every residue (volume, hydrophobicity, polarity, charge, aromatic and aliphatic character)., 11 ., centered at each residue, whose peptide bond with the preceding amino acid we are trying to predict; outside this range, the influence of the surrounding residues towards the peptide bonds formation decreases . 4 . Feature selection: next, the user may choose either to employ the whole feature vector for the prediction or an optimal reduced set of features (12) identified in our previous study (11). Voting: the user may choose to invoke either a single svm model or 10 svm models, each one trained with a different dataset . In the latter, each model independently assigns a label (cis - pro, cis - nonpro, trans - pro, trans - nonpro) to every residue in the query sequence and then a linear time majority voting algorithm calculates the consensus of the 10 predictions . Submit sequence: if a valid e - mail address is supplied, the results are submitted in a compressed file; otherwise, if the e - mail field is left blank, the prediction results can be downloaded directly from the web page, where they will be available for 10 days . The output of the pbond server consists of a compressed file containing either a single text file, or multiple text files, in case of batch processing . These files contain plain text with the predictions for every sequence uploaded, along with a reliability index for every prediction . The first and last five peptide bonds of every sequence are labeled as n / a since there are not enough residues in the sliding window to make a prediction . Due to the scarcity of cis peptide bonds (both cis - pro and cis - nonpro), a severe class imbalance problem emerges, posing a tradeoff between the identification of as many potential cis formations and certain false positive predictions . Hence, special attention was given during the training and evaluation of the pbond server so that important cis formations are not neglected . For this purpose, the predictive models of pbond server have been trained using fully balanced datasets, in which all four classes are equally represented . The evaluation of pbond has been performed on fully balanced disjoint data segments coming from the initial unbalanced dataset 13 ., 14 .. table 1 presents the performance achieved using the initial feature vector, with and without performing majority voting . Sensitivity and positive predictive value (ppv) are also provided for the two general classes (cis / trans). The performance achieved using the initial input vector is in general quite poor, even though voting slightly improves the results . In table 2, the performance achieved using the optimal reduced set of features is shown, with and without the employment of the majority voting algorithm . It is clear that the feature selection improves the classification outcome to a certain extent; a further increment in the results is achieved using the consensus prediction of the 10 models . Furthermore, the reliability index associated with every prediction can be used for post processing the prediction results . A detailed comparison of the available prediction methods in the literature and pbond is presented in table 3 . Based on the physicochemical properties of the 6 surrounding amino acids, frmmel et al . Although the reported results are promising, such refined dataset (242 proline bonds) diminishes the credibility of the proposed method . (5) as well, focused only on the proline residues and employed single sequence information coded in binary form in order to predict the conformation of the peptide bond . The prediction accuracy achieved by this method is 70% and 77% when evaluated with independent datasets and the jackknife test, respectively . (6) provided multiple sequence alignment profiles coupled with secondary structure information as input to an svm in order to predict the proline cis / trans isomerization . The overall reported accuracy is 71% after performing five - fold cross validation . Only pahlke et al . (7) aimed to predict the peptide bond conformation between any two amino acids, using the secondary structure of amino acid triplets; however, the reported results (overall accuracy 66%) are quite unsatisfactory . This could be attributed to the refined length of the sliding window, as well as to the small number of employed features . The performance of pbond compares well with previously published studies, albeit validated on different datasets and using different evaluation methods . Moreover, pbond is able to identify the scarce but highly important non - proline cis peptide bonds . Tpe and cp provided valuable comments and suggestions throughout the study and helped in the manuscript preparation . Tpe and cp provided valuable comments and suggestions throughout the study and helped in the manuscript preparation.
Orbital roof fractures secondary to blunt trauma are rarely encountered, but the exact incidence of orbital trauma remains unknown . Traumatic encephaloceles in the orbital cavity are even rarer, with only 15 cases reported till date . The commonest cause is a frontal impact or a lateral blow to the orbital rim . The two common reasons for delayed diagnosis of orbital injuries are that ecchymosis and swollen eyelids commonly follow head trauma and the patient is generally obtunded following head injury . A complete neuro - ophthalmological and a thorough palpation of the orbital rims in all patients with ecchymosis and proptosis are mandatory . Plain x - rays of the skull, orbits, and optic foramina along with computed tomography (ct) scanning in both coronal and axial sections gives adequate information as to the cause of proptosis and helps in selecting the operative technique if required . Neurological examination revealed that he was unconscious with a glasgow coma scale (gcs) score of 10 [e2v3m5]. He had bilateral periorbital hematoma more on the right side . Visual acuity and extraocular muscle motility of both eyes could not be evaluated completely due to periorbital hematoma, soft tissue swelling, and reduced level of consciousness of the patient . Cranial ct axial sections of the head revealed hematoma in the left frontal lobe causing a midline shift along with a depressed fracture of the left frontozygomatic region and a bilateral transverse fracture of the orbital roof [figure 1]. He underwent surgical evacuation of left frontal contusions, elevation, and removal of depressed segments of the left frontozygomatic bone . A dural tear was also found at the depressed fracture site and duraplasty was done using a pericranial patch . Postoperatively on day 2, he developed rapid progressive proptosis and severe congestion of conjunctiva [figure 2] contralateral to the operated site . A repeat ct scan of the head was performed in which right frontal contusion had developed with edema [figure 3]. Orbital ct coronal and axial scan was also performed which revealed the formation of an encephalocele toward the right orbit [figure 4]. The right globe was displaced laterally, inferiorly, and anteriorly . The optic canal and the optic nerve were intact . The patient underwent right frontal craniotomy wherein the right orbital roof was decompressed, and dural tear and the protruding brain tissue into right orbit were identified (housepian's approach). After excision of the protruding part of the brain microsurgically, the dura was reapproximated with a locked running stitch, and a water - tight closure was achieved . The postoperative period was encouraging, and the right eye proptosis and congestion regressed rapidly [figure 5a c]. The patient recovered well and was discharged from the hospital on the 16 post - traumatic day when he was conscious with a gcs score of 15 and had intact vision although with mild limitation of movement in the right eye . At a follow - up after three months, cosmetic and neurological recovery was excellent [figure 5d]. At the last follow - up after nine months, the patient was in good health with no neurological deficits . The final visual outcome was excellent, and the patient recovered full vision and had complete range of extraocular motions in both eyes . First preoperative computed tomography scan head showing left frontal lobe contusion (a), bilateral tranverse fracture of the orbital roof (b, arrows), and left frontozygomatic depressed fracture (c) rapid progressive proptosis and severe congestion of conjunctiva of right eye computed tomography of the head shows right frontal contusion with edema and postoperative changes in left frontozygomatic region computed tomography of the orbit shows formation of the encephalocele toward the right orbit [white arrow] and displacement of the right eye globe laterally, inferiorly, and anteriorly [black arrow] sequential photographs of patient's eye shows rapid recovery (a) preoperative condition, (b) postoperative day 10, (c) postoperative day 15 and (d) 3 month follow up excellent result nature has provided orbits as protection for the eyes . The orbital walls along with the eyeball and septum form a closed cavity . Following trauma, there is edema of the retro - ocular tissues resulting in congestion of the veins which in turn further increases the edema . Edema and venous congestion push the eyeball forward, stretching the extraocular muscles and the nerves . This further compresses the draining veins . Beyond a certain limit, the increase in intraorbital pressure gradually occludes the retinal veins, and later the retinal artery, resulting in visual failure . This sequence of events is accelerated if there is a depressed fracture of the orbital wall, roof, and rim, which has further compromised the orbital space . Isolated traumatic orbital fractures can be classified as blow - in or blow - out fractures . The former involves fractures in which the bone fragments are displaced into the orbit, causing an increase in the intraorbital pressure and/or impingement syndromes, which may lead to irreversible neurological injury especially in chronic cases . The latter involves the disruption of an orbital wall (usually medial or inferior wall is damaged), and the resultant decompression of the periorbital structures by bony fragments leading to entrapment syndromes . Both blow - in and blow - out fractures can be termed as pure if the orbital rim is spared or impure if the orbital rim is fractured . Traumatic encephaloceles related to orbital roof fractures are even rarer, with the first case reported in 1951 . The main symptoms and signs are diplopia, exophthalmos, orbital edema, subconjunctival hemorrhage, restricted movements of the eye, and loss of vision . Patients can present acutely, or the symptoms and signs may gradually appear as the fracture in the orbital wall grows over time . Growing fractures of the orbital roof have been reported several times . In a patient with persistent ocular symptoms and a history of orbital trauma, a growing fracture of the orbital wall must be suspected . Our patient was the victim of an acute traumatic encephalocele related to orbital roof fracture, which is unusual and has been rarely reported . For such cases, early diagnosis and treatment are very important as the raised intraorbital pressure may damage the optic nerve irreversibly . Visual failure, if present, would be either due to direct injury to the eyeball, or vascular injury to the optic nerve . The accepted indications of emergency orbital surgery following orbital trauma are as follows: progressive loss of vision: (immediate loss of vision is considered to be due to vascular insufficiency and is not supposed to be benefitted by surgical decompression).progressive proptosis: either due to an expanding intraorbital retro - ocular hematoma or due to progressive swelling of the retro - ocular tissues.radiologically demonstrated bony spicule pressing on the optic nerve in the canal . Progressive loss of vision: (immediate loss of vision is considered to be due to vascular insufficiency and is not supposed to be benefitted by surgical decompression). Progressive proptosis: either due to an expanding intraorbital retro - ocular hematoma or due to progressive swelling of the retro - ocular tissues . All the above indications focus on visual failure . However, visual failure is not the only problem in orbital trauma . Acute proptosis also causes stretching of the nerves supplying the extraocular muscles and results in their paresis or palsy . Therefore, relief of the raised intraorbital pressure should also be undertaken on a priority basis . A paralyzed eye with intact vision restriction of extraocular movement due to entrapment of the inferior oblique, inferior rectus, and medial rectus muscles is also possible . Such cases can be dealt with on an elective basis, the treatment of which has been discussed in great detail by mustarde reconstruction of orbital roof fracture or defect using titanium mesh, screws, and mixture of bone powder mixed with fibrin glue has been reported, claiming good cosmetic results, but they are not easily or urgently available to poor patients, like in our case . Simple substitutes such as temporalis fascia graft as was used in our case can achieve equally good results . Whenever orbital roof fractures associated with frontal contusions are identified in an acute brain - injured patient, an orbital encephalocele should be suspected . In our opinion, if the encephalocele is confirmed, a surgical approach via the subfrontal route is indicated with resection of herniated contused brain tissue, dural closure, and reconstruction of the orbital roof using temporalis fascia graft . Excellent results with regard to orbital symptoms (mainly exophthalmos) and cosmesis can be expected even without titanium mesh reconstruction if unavailable.
When imagining time and their own life events, humans do not only retrieve or predict when events have occurred or will occur, but also automatically project themselves on an imagined mental time line . Self - time travelling can thus be regarded as the ability to transpose one's habitual self - location in time to different temporal locations in the past or the future . Emphasizing the role of perspective taking, this ability to change one's own temporal egocentric perspective has been proposed to share a common mechanism with the ability to change one's own spatial egocentric perspective . In both domains, people would use existing representations as templates for processing and understanding new information, in order to plan their short- and long - term behaviors . On this account, the same processes that subserve simulation of the self at a different location in space would also subserve simulation of the self at a different point in time . An interesting question, inspired by this parallel, is whether, similarly to taking another person's spatial perspective, people can also adopt the temporal perspective of another person when travelling through time . Studies investigating spatial perspective taking indicate that people can overcome their own position in space to adopt another person's spatial perspective (e.g.,). When the scene includes another person, for instance, people may spontaneously describe spatial relations from that person's perspective despite the very real presence of their own [5, 6]. These and other findings suggest that spatial perspective taking may induce an alterocentric remapping, that is, remapping of objects and locations to an alterocentric frame of reference . To our best knowledge, no study has so far investigated whether a similar remapping may take place in the temporal domain . In other words, whether similarly to taking another person's spatial perspective, people can take another person's temporal perspective when travelling through time . To address this issue, in the present study we directly compared self- and other - perspective time travelling with the goal of testing whether / how the representation of time varies as a function of perspective taking . The psychological ability to traverse temporal distances is dependent upon a cognitive representation of time that has been suggested to be spatial in nature (e.g., [2, 812]; for an overview see). In this conceptualization, time travel is characterized by three basic components: (a) temporal location, that is, the point in (space-) time from where the travel originates [8, 14, 15]; (b) motion direction, that is, the direction along which projection takes place (either backwards or forwards with respect to the temporal location) [8, 14]; and (c) temporal duration, that is, the temporal interval to be travelled (for a description of similar concepts, see also). Within this framework, it has been demonstrated that rather than being mapped to space in a uniform manner, spatiotemporal representation presents areas of different granularity . For instance, christian and colleagues found that when asked to temporally locate events on a time line, participants used more space (-time) to represent one year in the present than in the past or the future . In a similar vein, arzy and colleagues showed that, irrespective of the temporal location, participants were faster and more accurate when asked to retrieve an event or a face located forwards rather than backwards (relative future effects in; see also). In addition, the speed of self - projection in time has been shown to depend logarithmically on the temporal distance between the imagined self - location in time and the location of the imagined event / face to retrieve . These patterns relate to the self - referenced topography of space - time mapping . To investigate whether similar patterns also apply to other - projection in time, here, we asked participants to imagine themselves or someone else at a specific point in time (i.e., past, present, or future) and to operate a notional time machine for travelling either backwards or forwards as to reach a target destination (i.e., one, three, or five years back / ahead). We used the travel duration as a proxy for how space - time representation depended on the perspective taking (self- versus other - perspective). Based on previous evidence that the amount of space used to represent time varies as a function of self - relevance, we expected that travelling in the self - perspective would take longer than travelling in the other - perspective . Moreover, we hypothesized that this effect of perspective would be greater when the travel originated in the present than in the past or future . In spite of these differences, however, we also expected similarities between self- and other - perspective time travelling . In particular, we predicted that, for both self- and other - perspective, travel duration would increase as a function of the temporal distance to be travelled . Finally, we expected that, in both perspectives and regardless of temporal location, travelling would be facilitated for the forwards motion direction (see relative future effect). Twenty - five participants (15 females, aged between 20 and 26 years, mean sd 23 1.7 years) from the university of turin took part in the study . All participants were right - handed and had normal or corrected to normal vision and no history of neurological or psychiatric disorders . All participants gave written informed consent before inclusion in the study, which was conducted in accordance with the principles of the revised helsinki declaration and approved by the ethical committee of the university of turin . Participants were seated at a desk approximately 50 cm away from a 17 computer screen (refresh rate = 60 hz). At the beginning of the experiment, they were told that their task was to operate a notional time machine to travel through time . Next, they were given instructions about the temporal perspective (self versus other) and the time locations at which to imagine themselves . On other - perspective trials, a female coexperimenter was seated at a desk placed perpendicularly with respect to participant's desk (at a distance of ~1.5 m), in front of a computer monitor connected to participant's monitor . While sitting at their desk, participants could not see what was displayed onto the monitor in front of the coexperimenter . To control for possible effects related to age difference, participants were recruited as to be similar in age to the coexperimenter . At the beginning of the experiment, each trial began with the instruction to imagine oneself (for self - perspective trials) or the other person (for other - perspective trials) at specific past (i.e., the day of your / coexperimenter's 10th birthday), present (i.e., today), or future location in time (i.e., the day of your / coexperimenter's 50th birthday). As participants were in their late adolescence / early adulthood, past and future locations corresponded to two stages of development markedly distinct from their own (i.e., a point in middle childhood and a point in middle adulthood, resp . ). From these locations, participants were instructed to move one, three, or five years, either backwards or forwards . To support the experience of travelling through time, an animated star - field display was projected onto the screen in front of the participant [17, 19, 20]. The display consisted of approximately 1000 randomly positioned white dots on a black background (see figure 1). The dots (i.e., stars) were animated (25 fps) so as to appear to move, on a linear trajectory, either toward (i.e., centripetally) or away from (i.e., centrifugally) the center of the display, corresponding to the experience of backwards and forwards self - motion (figure 1). Journeys in the past were accompanied by backwards optic flow, while those in future were accompanied by forwards flow (for a similar paradigm see). On other - perspective trials, the same star - field display was projected onto the screen in front of participant and onto the screen in front of the coexperimenter . In both self- and other - perspective trials, the participant was instructed to look at the screen in front of him / her for the entire duration of the experiment . At a self - paced interval, participants were requested to press a button (i.e., space bar) to begin the time travel (i.e., initiate the optic flow) and press it again once they felt they had reached the target (i.e., stop the optic flow). The time travel duration was calculated as the interval between these two events . Finally, to ensure participants' compliance with the task requests, in 20% of the experimental trials (a total of 72 trials per each participant, with 18 trials per each block), we asked participants to estimate the actual duration of their last time travel (in ms). Each of the four blocks comprised 90 trials for a total of 360 trials . For each perspective condition (self, other), ten trials for each temporal location (past, present, and future) by motion direction (backwards, forwards) by temporal duration (one, three, and five years) combination were administered . Within each block, temporal location presentation was fully randomized and administered in miniblock of 6 trials (i.e., one trial for each temporal duration by motion direction combination). To familiarize participants with the procedure, at the beginning of the experiment a practice session was administered (6 self - perspective and 6 other - perspective - trials, one trial for each motion direction by temporal duration combination). Pittsburgh, pa, usa) running on a pc was used to present trials and record the duration of the time travel (i.e., the time elapsed between starting and stopping the time machine). The experiment lasted about 60 minutes . To cope with the high variability within the range of travel duration (39421304 ms), we converted individual temporal intervals data into z - scores, based on means and standard deviations computed over all trials per each participant . Since each standardized coefficient scales appropriately to adjust for the disparity in the variable sizes, this procedure makes it possible to bring all of the variables into proportion with one another without losing the possibility to directly compare participants' performance across conditions . The mean z - scores were then averaged separately for each trial type (i.e., temporal location by each motion direction by temporal duration in both perspectives). The investigation of standardized data distribution using one - sample kolmogorov - smirnov goodness - of - fit tests did not show any significant difference, suggesting that data distribution within the sample was gaussian (0.196 <ps> 0.998). Travel duration z - scores were then submitted to a repeated measures anova with temporal location (past, present, and future), motion direction (backwards, forwards), temporal duration (1, 3, and 5 years), and perspective (self, other) as within subjects factors . Main effects were used to explore the means of interest (post hoc t - test), and bonferroni's corrections (level of p <0.05) were applied . In addition, to obtain an indirect measure of participants' compliance with the task requests over the experimental session, we analyzed the data from the control task to test whether participants' ability to reproduce time intervals decreased / increased over the four blocks . To this aim, we first computed a temporal accuracy estimation index, defined as the difference between the actual duration of the travel and the corresponding temporal estimation given by the participant at the control task trial (i.e., 20% of the total amount of trials: 18 trials per each block for a total of 72 trials by each participant). Then, we submitted this index to a repeated measures anova with block (i.e., first, second, third, and fourth) as within subjects factor . Participants' travel duration varied as a function of temporal location so that travels originating in the present moment were longer than travels originating in the past or in the future (main effect of temporal location: f(2,48) = 3.621; p = 0.034; partial eta square = 0.131; mean z - scores = 0.078, 0.004, and 0.082, resp . ; ps <0.05). Moreover, temporal duration of journeys was longer for travelling forwards than backwards (main effect of motion direction: f(1,24) = 7.305; p = 0.012; partial eta square = 0.233; mean z - scores = 0.038 versus 0.038, resp . ; ps <0.05). Regardless of the temporal origin of the journey and its direction, the travel duration increased as a function of the number of years to travel (main effect of temporal duration: f(2,48) = 277.747; p <0.001; partial eta square = 0.920; mean z scores = 0.86 for 1 year; 0.01 for 3 years; 0.86 for 5 years; see figure 2, ps <0.05). As predicted, neither motion direction nor temporal duration main effect were further qualified by significant interactions by perspective . In contrast, the effect of temporal location varied between self- and other - perspective (perspective by temporal location interaction: f(2,48) = 6.014; p = 0.005; partial eta square = 0.200). For self - perspective trials, indeed, travel duration was longer when the travel started either in the past or in the present rather than in the future (0.034 and 0.163 versus 0.101, resp . ; see figure 2; ps <0.05). For other - perspective trials, in contrast, no similar modulation of travel duration by temporal location was reported (0.007, 0.025, and 0.062 for present, past, and future location; figure 2; ps> 0.05). Moreover, travel duration was on average longer for the self - perspective than for the other - perspective when the travel started in the present, but not when it started in the past or in the future (self / present versus other / present = 0.163 versus 0.007, resp . ; p <0.05; self / past versus other / past = 0.034 versus 0.025 and self / future versus other / future = 0.101 versus 0.062, resp . ; ps> 0.05). Finally, the exploration of the significant temporal location by temporal duration interaction (f(4,96) = 3.780; p = 0.007; partial eta square = 0.136) revealed that there was no difference across temporal locations when travels lasted either 1 year or 3 years (1 year: past = 0.897; present = 0.793; future = 0.912 and 3 years: past = 0.016; present = 0.057; future = 0.075; ps> 0.05). In contrast, when participants were requested to cover a 5-year distance, the trip took longer when it started in the past or present rather than in the future (5 years: past = 0.926 and present = 0.968 versus future = 0.74; ps <0.5). Neither the main effect of perspective (f(1,24) = 1.588; p> 0.05; partial eta square = 0.062) nor the remaining two-, three-, or four - way interactions were found to be significant (all fs <1.484; 0.213 <ps> 0.942; all partial eta squares <0.058). Control task . Anova revealed no significant effect of block (f3,72 = 2.212; p> 0.05; partial eta square = 0.084) on temporal accuracy estimation index, suggesting that participants' compliance to the task request remained stable throughout the experiment . In this study, we assessed whether and how self- and other - projections in time map onto similar or different spatiotemporal representations . For the properties of this underlying representation to emerge, we used as a proxy the time taken to move through time (i.e., travel duration) and manipulated three basic properties of time journeys: temporal location (past, present, or future), motion direction (backwards or forwards), and temporal duration (one, three, or five years). Our results suggest that self- and other - projections hinge on different temporal representations depending on the temporal location, that is, on where in time the mental travel originates . Specifically, for self - perspective, participants took longer to cover identical distances when the travel started in the past or in present compared as to when it started in the future . For other - perspective, in contrast, travel duration was not modulated by temporal location . This effect may reflect the tendency to form higher - level construals of information about remote future event . In this respect, trope and liberman suggest that the greater the temporal distance from a future event, the more likely is the event to be represented abstractly in terms of a few general features that convey the perceived essence of events rather than in terms of more concrete and incidental details of the event . In the same vein, d'argembeau and van der linden provided evidence that projecting oneself in a specific positive or negative experience results in a richer representation when the event is expected to be experienced in the near future rather than in a more distant future . On this account, participants would travel more rapidly from a remote future location because they would simulate future events in more abstract and general terms . This is further supported by the consideration that, in contrast with remembering of past events whose features are already integrated, simulating remote future events requires the combination of disparate details gleaned from a variety of episodic sources . Forming abstract representations of remote future event may thus serve a specific adaptive role in reducing the costs required to integrate unrelated details into a coherent future representation . Over and above this, self- and other - projection also scaled differently for mental travels taking place in the proximity of the present moment . For the present location, regardless of motion direction, one year was indeed longer in the self - time rather than in the other - time . This was not the case for one - year travels originating from a past or future location . This pattern is in agreement with the finding that participants represent self - time as occupying a greater amount of space than an equivalent period related to others . Of direct relevance to the present study, christian and colleagues asked three different groups of participants to time - travel from the present moment to their own birthdays or to the birthdays of either a close friend or a hypothetical stranger of a similar age as theirs . It was found that, irrespective of motion direction (either in the past or in the future), time relevant to self was represented as occupying more space than time relevant to others (i.e., best friend or unfamiliar other). On a closer examination, however, this effect of self - relevance was only evident for temporally close events (i.e., 10 years before or after the present moment), but not more distant events (i.e., the day of 8th or 58th birthday). Taken together with our results, this suggests that a distinctive relationship bounds the self to the now . As observed by nez and cooperrider, indeed, within an internal perspective, the ego is always and inherently colocated within the now . It is perhaps therefore not surprising that self - projection is more embedded in the representation of the present time compared to other - projection . On a related note, this could explain why recent autobiographical memories tend to be recalled from the first - person perspective, while more remote memories, particularly early childhood memories, are more likely to be recalled from a third - person perspective [2427]. Despite these differences, however, self- and other - projections also shared many similarities in structure . First, for both self- and other - perspectives, travel durations increased as a function of temporal duration (one year <three years <five years). With only one exception (i.e., five years from past and present lasts longer than five years from future), this effect was not modulated by the temporal location (i.e., past, present, and future). This is consistent with the idea that computation of temporal quantities rests on a common system for magnitude processing, which is (at least in part) independent of self - relevant content specificity and episodic memory processes . The finding that 5-year travel duration lasted less when it originated in remote future might be taken to suggest that, as far as remote future is concerned, the spatial representation of time is compressed towards the anchoring point . Experiments using multiple temporal locations in remote past and future might help to address this issue . Second, in both self- and other - perspectives, independent of temporal location, travelling forwards took longer than travelling backwards . The neural system subserving mental time travel has been proposed to have evolved to anticipate and pilot our behavior rather than primarily encoding the past [8, 29, 30]. In line with this, processing of events has been shown to be future oriented across present, past, and future self - location [8, 14]. For example, when asked to judge whether an event takes place before (relative past) or after (relative future) an imagined self - location in time, participants are typically faster and more accurate for relative future than for relative past events . At first sight, the finding that travelling forwards took longer than travelling backwards may seem contradictory to response facilitation for future events . However, both phenomena may be parsimoniously interpreted as resulting from an anisometric pattern of internal spatial representation of relative past and future events, such that the representational medium is compressed towards the relative past and dilated towards the relative future . Compression towards the relative past would explain why travelling backwards lasts less, but also why judgements are more difficult (i.e., slower and less accurate) to the relative past . Along the same lines, dilation towards the relative future may account for both the increase in travel duration for travelling forwards and the relative future response facilitation . An alternative, not mutually exclusive, explanation of the motion direction effect refers to discrepancy between external time direction (i.e., roughly, the time of the calendar) and subjective time direction (i.e., the personal time which is measured by the traveler's wristwatch;) potentially experienced when travelling backwards . When participants are requested to time - travel towards relative future, the external time and their subjective time move towards the same direction (see also, for similar concepts). The subjective time may therefore be expected to add to the external time, extending the travel duration . In contrast, when participants travel towards relative past, the external time goes backwards, while their subjective time moves forwards ., the effect of motion direction would reflect the relative (forward) motion of the traveler making journeys end later in the future and earlier in the past . At this stage, both these hypotheses remain speculative and require further study for elaboration and validation . A growing body of evidence indicates that self - projection in space and in time might rest on a common neural network and share similar cognitive processes and representations . In the visuospatial domain, it has been documented that people can transpose their own actual point of view and navigate space from the perspective of someone else . The current findings suggest that a similar ability might also exist in temporal domain, supporting the notion of temporal perspective taking . By contrasting time travelling from self- and other - perspective, we found evidence that temporal representation underlying one's own projection shares many of the same characteristics of the temporal representation underlying another person's projection . Despite the fact that a greater sensitivity to temporal location for representing time in self- rather than in other - perspective emerges, when considering more abstract properties as direction and magnitude, self- and other - time exhibit a similar structure . Further research is warranted to clarify whether and to what extent these effects are sensitive to the degree of similarity between the self and the other person . For example, it will be important for future research to determine how travelling in time from the perspective of a younger or older person impacts on travel duration (depending, e.g., on whether the past / future of the participant overlaps with the present of the other person). A second issue to be addressed in future studies self - projection in time has shown to recruit a network of brain areas in distinct time periods including the occipitotemporal, temporoparietal, and anteromedial temporal cortices [4, 8]. For example, it has been reported that during mental time travel the left lateral parietal cortex is differentially activated by nonpresent subjective times compared with present (past and future> present). Capitalizing on the finding that left parietal cortex supports first - person perspective simulation, these results have been interpreted to suggest that the parietal cortex is specifically related to transformations in subjective time . Moreover it has been demonstrated that temporal self - projection into the personal past recruits greater ventral medial prefrontal cortex (mpcf) whereas self - projection into another person's perspective recruits greater dorsal mpcf . Asking participants to simulate mentally past, present, and future time from their own versus another person's perspective might help to clarify how transformations in subjective and nonsubjective time are represented in left parietal cortex and to elucidate the exact contribution of the ventral and dorsal subregions of mpcf to self- versus other - projection . Finally, the hypothesis of a partial overlap between self- and other - mechanisms for projection in time could be tested in neuropsychiatric patients with temporal orientation failures . To the extent that self- and other - projection rely on a common neural mechanism, self - referenced and other - referenced disorientation may be expected to share common fundamental characteristics.
The development of bioinformatics has been initially driven not only by the enormous quantity of data that the biologist community was able to produce during the last decades, but also by the necessity of finding approaches to organize and better analyze these huge datasets . Although the protein structures constitute small datasets with respect to many other data encountered in biology, they nevertheless represent a challenge for the data analysis, as the relative positions of atomic coordinates in a protein structure take values in the continuous three - dimensional (3d) space . The large variability of protein features is obvious from the variety of physicochemical properties among a given family of proteins . Furthermore, the full understanding of a protein function requires, in addition of the knowledge of its structure, the knowledge of the internal dynamics and thus of the conformational landscape of the protein, which correspond to large datasets . Graphs are traditionally used for modeling biological datasets, as for the analysis of protein protein and molecular interaction networks, for description of drug function, for the description of interactions within a protein, for the description of the hierarchy of local minima in the conformational space . In the description of protein conformational space, the determination of such a graph is hampered by the need to (i) simplify the protein local geometry without loss of information and (ii) find a generic approach for graph determination, while preserving the specificity of each protein . In contrast, the description of protein structure and dynamics through graphs would allow one to (i) relate structure description, conformational variability, and protein function; (ii) unify the structural and dynamical representations; and (iii) obtain, for a given protein, a model that could be interfaced with the graphs described at the cellular level, as the interactome network . In order to investigate the points quoted above, we have been using several processing tools to describe the graphs underlying the structural and dynamical features of the d - ala: d - lac (vana) ligase:(i)the self - organizing maps, to convert the conformational space in a two - dimensional (2d) map;(ii)the louvain greedy algorithm, to determine kinetic clusters in the conformational space;(iii)the girvan newmann algorithm, to determine contact communities within the protein structure, which was already used in other structural objects; and(iv)the analysis of hydrogen bonds within the protein structure, using a machine - learning approach (random forest).the conformational space has been explored using an enhanced sampling approach: the temperature - accelerated molecular dynamics (tamd). The self - organizing maps, to convert the conformational space in a two - dimensional (2d) map; the louvain greedy algorithm, to determine kinetic clusters in the conformational space; the girvan newmann algorithm, to determine contact communities within the protein structure, which was already used in other structural objects; and the analysis of hydrogen bonds within the protein structure, using a machine - learning approach (random forest). The d - ala: d - lac ligase (vana) is present in cases of resistance to the glycopeptide antibiotic vancomycin in enterococcus faetium and staphylococcus aureus . Vana synthesizes a modified precursor d - ala - d - lac instead of the usual d - ala - d - ala, synthesized by using a d - ala: d - ala ligase . This depsipeptide is then fixed at the end of the n - acetyl - muramyl - l - ala - d - glu - l - lys - d - ala - d - lac monomers involved in the building of the peptidoglycan, giving rise to a fully efficient cell wall while preventing the binding of vancomycin . The x - ray crystallographic structure of vana (figure 1a) includes the domains n - terminal (residues a2g121 shown in blue), central (residues c122s211 shown in red and yellow), and c - terminal (residues g212a342 shown in black and green). The -loop (shown in green in figure 1a, residues l236a256) is part of the c - terminal domain and closes the binding site where the ligase enzymatic reaction occurs . The two - layer -sandwich (residues a149q208) is a region opposite to the -loop in the structure and colored yellow in figure 1a . The binding site is located at the interface between n - terminal, central, and c - terminal domains . Concerted motions of the opposite domain and of the -loop allow the opening of the binding cavity to release the product of the catalytic reaction and accept new ligands . (a) three - dimensional (3d) view of the x - ray crystallographic structure of vana, colored according to its domains: the n - terminal [a2g121] shown in blue, the c - terminal [g212a342] shown in black, which includes the -loop [l236a256] shown in green, and the central domain [c122s211] shown in red, which includes the opposite domain [a149q208] shown in yellow . The disulfide bridge c52c64, located in the n - terminal domain, is shown with magenta labels (bottom right). (b) localization of the collective variables (cv) used for the different tamd calculations on a cartoon view of vana extracted at the end of a 10 ns md trajectory . The three structural cv are shown in orange and the five cv obtained from contact communities calculations are shown in cyan . The bioinformatics approaches described above have been applied to md and tamd trajectories recorded on vana . Several graph models describing the structural architecture, internal dynamics, and the opening of the -loop, have been established . These models give an extended view of the structural and dynamical features of vana and agree with the experimental knowledge available for the protein function . The starting point of the simulations was the x - ray crystallographic structure of the d - ala: d - lac ligase (vana) from enterococcus faecium bm4147 vana (pdb i d: 1e4e). The co - crystallized ligands, adp and phosphinate (1(s)-aminoethyl-(2-carboxypropyl)phosphoryl - phosphinic acid), located in the active site were removed . The c52c64 disulfide bridge, observed in the crystal was disrupted to be as close as possible to the physiological state of the d - ala: d - ala ligase . The force field charmm22 including the correction map (cmap) was used . Explicit tip3p solvent water molecules were added to the systems using a cutoff of 10 . The molecular dynamics (md) and the temperature - accelerated molecular dynamics (tamd) trajectories were recorded using namd 2.7b2 . A cutoff of 12 and a switching distance of 10 were defined for nonbonded interactions . Long - range electrostatic interactions were calculated with the particule mesh ewald (pme) protocol . Before starting the initial md trajectories, it was first minimized using 1000 steps, then thermalized by heating the system from 0 to 300 k over 30 ps, with a time step of 1 fs . The system then is equilibrated in the npt ensemble for 100 ps with a time step of 2 fs before a 40 ns md simulation . The temperature was maintained at 300 k using a langevin thermostat, and the 1 atm pressure was regulated using the langevin piston nose the shake algorithm kept all covalent bonds involving hydrogens rigid, so an integration time step of 2 fs was used for all md simulations . Atomic coordinates were saved every picosecond . At the end of the first 10 ns of the md trajectory, five independent 30-ns temperature - accelerated molecular dynamics (tamd) the tamd approach is an enhanced sampling approach, based on the parallel evolution of the protein coordinates x in a classical md simulation and of the target values z for the collective variables (x):1where x are the physical variables (atomic coordinates) of the system, (x) are the collective variables, and z the instantaneous target values of the collective variables . M is the mass matrix, v(x) is the empirical classical potential of the system, (t) denotes white noise (i.e., gaussian processes with mean 0 and covariance of p(t)p(t) = (t t), with p = x, z),> 0 is the so - called spring force constant, and> 0 are friction coefficients of the langevin thermostats, = kbt, and = kbt, where kb is the boltzmann constant and t and t represent the temperatures . Equation 1 describes the motion of x and z under the extended potential2it was shown in ref (29) that, by adjusting the parameter, so that z(t) (x(t)), and the friction coefficient so that the value of z moves slower than that of x, one can generate a trajectory z(t) in z - space that effectively moves at the artificial temperature t on the free - energy hyper - surface f(z), which is defined at the physical temperature t. hence, by construction, the limiting equation for z(t) in eq 1 samples the distribution e. then, using t> t in eq 1) accelerates the exploration of the free - energy landscape by the z(t) trajectory, as energy barriers can be crossed more easily . The value for the artificial friction on the z variables can be determined following the principle that the separation of time scales between x and z must be such that the x have time to equilibrate before the z values move substantially . In practice, we proceeded as suggested in ref (57), i.e., we ran short standard md trajectories with the collective variables restrained at (x) = z fixed, and monitored the mean force estimators gj(n) defined for each collective variable j as3where j(x(ti)) is the instantaneous value at time ti of the collective variable . The time required for gj(n) to reach a plateau (see figure s1 in the supporting information) allows one to extract the characteristic time of relaxation of the cartesian variables to a fixed value of the variables z, and hence an estimate of to ensure the time - scales separation /. As the estimator (described in eq 3) converges in 5000 simulation time steps (0.002 ps), a friction of 50 ps, corresponding to a characteristic time of 0.02 ps, is sufficient to allow system relaxation . The tamd approach was implemented in namd using a tcl script . In tamd, the evolution of the usual md equation, at 300 k, was coupled to the evolution of collective variables at a much higher temperature . Several sets of collective variables were used, which were all geometric centers located in different protein regions . The friction coefficient, = 0.5 ps, and the physical thermal energy, = 0.6 kcal / mol, are the parameters of the conventional langevin thermostat, which allow one to obtain a simulation temperature of 300 k. the restraint force constant is set to = 100 kcal/(mol). Tamd trajectories were run using a value of 20 kcal mol for the artificial thermal energy of the langevin thermostat attached to the collective variables . This thermal energy corresponds to an artificial temperature t of 10 060 k. despite the high temperature values used for the langevin thermostat attached to the collective variables, it is not expected that the folded structure of vana would be destabilized, as a large friction (= 50 ps) is used for this thermostat, along with the high force constant (= 100 kcal/(mol) to restraint the collective variable coordinates to the collective variables . In that way, we reduce the risk of system instability due to large deviation of the collective variables (x) from their target values z. the following method has been used to determine the contact communities of vana along each recorded trajectory . At each trajectory frame, a contact is set up for all -carbon pairs closer than 12, and the frequency of contacts is calculated along the trajectory . The protein structure is then considered as a graph, where the residues c constitute the vertices and the edges are weighted by the frequency of contacts between c atoms along the trajectories . Newman algorithm, as implemented in the program python, allows one to divide, in an iterative way, the graph into contact communities . First, all possible shortest paths are calculated between the c and the betweenness of each edge, which is defined as the number of shortest paths crossing this edge, is computed . The algorithm then removes the edge exhibiting the most important betweenness and includes the two edge vertices into the same community . Several runs of the algorithm are performed to remove the edge of highest betweenness until no edges remain . At the end of the process, the initial dynamic map of frequency of contacts has been split into contact communities of amino acids that are strongly connected . The self - organizing maps (som) approach was used to cluster the conformations generated along md and tamd trajectories . The som algorithm allows the mapping of the conformational space on a periodic subspace of reduced dimensions: a 50 50 map . 341 341 pairwise square euclidean distance matrices d were calculated for the 341 c atoms of vana, for each frame of the trajectory . To compress the data, a covariance matrix c was computed from each d. its four eigenvectors, corresponding to the first four significant eigenvalues ni were kept . For each trajectory frame t, the resulting compressed 4 341 matrix d vi=1,...,4, stored as a vector vt, contains the conformational descriptors and is used to cluster the protein conformations . The som was trained in two phases with the following parameters: (i) a map size of 50 50 with periodic boundaries, initialized randomly with a constant learning rate of 0.5 and a radius of 6.250 for the first phase (180 000 iterations), and (ii) an exponential decrease of learning rate (starting at 0.25) and radius (starting at 3.125) for the second phase (360 000 iterations). After the random initialization of the map, vectors of conformational descriptors vt described above, were presented to the map in random order, and the neuron closest to the presented vt was updated, as well as the neighbor neurons to preserve the coherence of the clustering . At the end of the calculation, each neuron of the som contains a average vector vt corresponding to a mixture of clustered protein conformations . The unified distance matrix (u - matrix) representation was computed to display the som topology on a bidimensional matrix . In the u - matrix, each node shows the local similarity between the corresponding neighboring som neurons, i.e., the mean distance between the node and its eight neighbors . A flooding algorithm was then used to aggregate the u - matrix basins, and to reject outside the regions corresponding to nonsimilar neurones, leading to a continuous map representation while preserving the inherent som topology . The som were additionally processed in two ways in order to determine graphs describing (i) the kinetics of the conformational space sampled and (ii) the opening path between the closed and open conformations of vana . The graph related to the kinetics of the conformational space sampled was determined in the following way . The som neurons define the microstates, and each structure along a given md or tamd trajectory is assigned to a given neuron . The element tij of the transition matrix, depicting the transition between neurons i and j, is defined as the number of i j transitions divided by the number of starts from neuron i. the transition matrix can be represented as a weighted graph, with the weight of the vertex ij being given by tij . The obtained graph is then partitioned using the greedy algorithm of louvain, in order to maximize the graph modularity . The modularity is a value between 1 and + 1, measuring the density of edges inside the partitions, compared to the density of edges outside the partitions . The greedy algorithm of louvain optimizes the modularity in two phases . In the first phase, each som neuron then, for each som neuron u, the variation of modularity is evaluated when u is removed from its cluster and placed to the cluster of each of its neighbors . If no gain of modularity is possible, u remains in its cluster . In the second phase, a new graph is built by merging the som neurons belonging to the same cluster the weights of the resulting graph are computed by summing the weights of the links between nodes in the corresponding two clusters . The opening path between the vana states displaying open and closed -loops was determined in the following way . Edges between som neurons were weighted by the value of the corresponding element of the u - matrix, which measures the local similarity between protein conformations . The starting point was the som node u corresponding to the starting point of all trajectories, with closed -loop . The final point of the path was chosen as the medoid of the som kinetic cluster 15 which will be described in section 3.3 . The medoid is the neuron whose average distance to all the neurons in the cluster is minimal . The shortest path is computed using the dijkstra algorithm, using the similarity between neurons as a distance . Finally, the path defined from som neurons was converted to a series of vana conformations by replacing each neuron by the vana conformation exhibiting the smallest euclidean distance between its vector of conformational descriptors vt and the average of the neuron vector vt. the path describing the opening has been analyzed to detect the most critical hydrogen bonds for the conformational change . For that purpose, along the opening path, a representative conformation was extracted from each kinetic cluster obtained above using the louvain greedy algorithm . This representative conformation was chosen as the medoid of the path conformations belonging to this kinetic cluster . On each of these vana conformations, hydrogen bonds have been detected using criteria based on a survey of small - molecule crystal structures . A hydrogen bond is supposed to be established if the donor acceptor and the hydrogen acceptor distances are respectively smaller than 4.0 and 3.0 . A random forest (rf) machine learning approach was used to calculate the importance of each hydrogen bond for predicting to which kinetic cluster the representative conformation belongs . The information on established and disrupted hydrogen bonds was encoded as a boolean vector for each conformation populating the path . The predicted value for each vector was the identifier of the kinetic cluster . The rf calculation was performed using the python package scikit - learn (scikit-learn.org). The number of trees in the forest was set to 10, with a gini criterion to measure the quality of a split . The number of features used when searching for the best split was set to 40, which is approximately the square root of the length of the boolean vectors (40). Once the training done, the importance of each hydrogen bond to define a kinetic cluster has been computed . The substrates, atp, d - ala, d - lac, d - alanyl - phosphate (d - ala(p)), the transition - state analogue phosphinate or phy, the product of the reaction, d - ala - d - lac, and the allosteric binder, were formatted in mol2 with chimera 1.4 and marvinsketch 5.1 (www.chemaxon.com/products/marvin/marvinsketch) for docking . Ucsf dock 6.5 was used to perform ligand docking vana conformations along the opening path obtained as described at the end of the section 2.5 . Chimera was used to add hydrogens, check atom assignment, and assign partial charges consistent with the amber - ff99sb force field . Chimera was also used to produce mol2 format files for the ligands and the selected conformations of the receptor . The dms software program generated the molecular surface of the receptor, using a radius probe of 1.4 . Spheres then were calculated around the receptor with the dock 6.5 command sphgen with radius probe values varying between 1.4 and 4 . Spheres were selected within a radius of 10 around the geometric center defined by the residues e15, k170, r289, n303, e304, n306, which are close to positions observed for the ligands (adp, phosphinate) in 1e4e . The grid encoding van der waals and electrostatic interactions was precalculated with the grid tool in a box containing the selected spheres . The dock program builds up to 500 flexible ligand docking orientations, on the precalculated grid interaction map . The ligand poses were then re - scored with the implementation of the hawkins molecular mechanics generalized born surface area (mm - gbsa) score, implemented in ucsf dock 6.5 . The use of the enhanced sampling approach tamd requires the definition of collective variables . In the present work, these variables were chosen as geometric centers of -carbons located in various vana regions . These regions were detected (table 1) from an analysis of the x - ray crystallographic structure of vana (pdb i d: 1e4e) or from the contact communities determined by the girvan starting from these regions, two sets of geometric centers were determined (see table s1 in the supporting information): structural collective variables (cvn - xr, cvo - xr, and cv-xr) and dynamical collective variables (cv-com, cve0-com, cve1-com, cvm - com, and cvo - com). Five independent 30-ns temperature - accelerated molecular dynamics (tamd) simulations were launched using various combinations of both sets of collective variables (see table s2 in the supporting information). The first five domain definitions are derived from the analysis of the x - ray (xr) crystallographic structure1e4e . The structural collective variables cvn - xr, cvo - xr, and cv-xr (table s1 and figure 1) were respectively defined on the n - terminal domain, opposite domain, and -loop, chosen from a direct observation of the pdb structure 1e4e . This choice is supported by several observations on x - ray crystallographic structures and md trajectories . First, the -loop, containing cv-xr, displays diverse orientations in x - ray crystallographic structures of d - ala: d - ala ligases . Second, the opposite region (residues 149208) was chosen to define cvo - xr, as this region moves apart from the protein core, as published in a previous work . The dynamical collectives variables were derived from the contact communities calculated using the girvan newman algorithm along a 30-ns md trajectory: these communities are described in more detail below . The corresponding geometric centers are located in the -loop (cv-com), in the n - terminal and c - terminal domains (cve0-com, cve1-com), and in the middle (cvm - com) and opposite (cvo - com) domains (see table s1 and figure 1). The contact community analysis based on the girvan newman algorithm allowed one to divide vana in five communities either in md or in tamd simulations, except in tamd_on, where four communities were observed (see figure 2). These communities are variable from one simulation to another, but involve similar protein regions for all trajectories (see table s3 in the supporting information), even though different sets of collective variables were used during each tamd trajectory . The two ends_0-com and ends_1-com communities are interlaced in the protein sequence, and contain residues from the structural definition of the n- and c - terminal regions . The opposite - com community is located in the opposite domain, while the -com community corresponds to the -loop and part of the c - terminal . The last community, middle - com (see table s3), located in the middle of the protein and partially superimposed with the central structural domain central - xr (table 1), is detected in all trajectories except tamd_on . The definition of contact communities are slightly different from the definitions of structural domains, except opposite - com, almost superimposed to the domain opposite - xr (table 1). The good fit of opposite - com to opposite - xr is expected as the opposite domain was previously detected from an analysis of md trajectories . The same color code was kept for the communities both on the 3d structures and on the graphs: the communities mainly located in the n - terminal region (numbers 0 and 1) are shown in blue and red; the middle (number 2) community is shown in magenta, if it exists; the opposite region is shown in yellow (number 3); the -loop and the main part of the c - terminal are shown in green (number 4). Projection of the communities calculated on a 30-ns trajectory of vana for (a) md, (c) tamd_on, (e) tamd_n, (g) tamd_on, (i) tamd_md, and (k) tamd_5cv . Also shown is a graph of the interconnectivity calculated between the different communities for (b) md, (d) tamd_on, (f) tamd_n, (h) tamd_on, (j) tamd_md, and (l) tamd_5cv . The collective variables (cv) used for tamd trajectories are represented by orange balls when they were derived from structural calculations and cyan balls if they were obtained from the communities calculations . The contact communities graph is connected by edges (figure 2), which depict the frequency of contact between -carbons belonging to two different communities . Thus, the edge thickness gives a qualitative indication of the relative influences that the communities have on each other . Overall, the same pattern of influences between communities is observed in all trajectories (figure 2). The community corresponding to the -loop is always strongly linked with the opposite community, as reflected by the high betweenness . The opposite domain is itself connected to the ends communities detected into the n- and c - terminal domains (shown in red and blue in figure 2). The definitions of structural, dynamical collective variables and of contact communities determined on the trajectory tamd_n are depicted (figure 3) using a color code . The definitions corresponding to the opposite domain (yellow) and to the -loop (green) are similar for the three sets of definition . Also, similar middle or central domains (magenta) are detected between dynamical collective variables and contact communities . Definition of collective variables (cv) and of contact communities displayed on the vana sequence . The first line contains the definition of structural collective variables (cvn - xr, cvo - xr, cv-xr: see table s1) determined from an analysis of the structure 1e4e . The second line contains the definition of dynamical collective variables (cve0-com, cve1-com, cvm - com, cvo - com, cv-com: table s1) determined from a community analysis using the girvan the third line contains the definition of communities (ends_oc, ends_1c, middle_c, opposite_c, _c: see table s3) determined by the girvan the following color code is used . For the structural cv: cvn - xr (blue), cvo - xr (yellow), and cv-xr (green). For the dynamical cv: cve0-com (blue), cve1-com (red), cvm - com (magenta), cvo - com (yellow), and cv-com (green). For the tamd_n communities: ends_oc (blue), ends_1c (red), middle_c (magenta), opposite_c (yellow), and _c (green). The existence of helices and strands has been monitored along the md and tamd trajectories (see table s4 in the supporting information). Most of the secondary structure elements are present more than 80% of the time, at the exception of 5 -strands, which are destabilized in the md as well as in the tamd trajectories . Thus, the folded structure of vana is not specifically altered by the use of the tamd, as has been already noticed in section section 2.2 . The 180 000 frames of vana generated either along the md or tamd trajectories were subjected to a som clustering . The analysis of som permits one to determine six clusters of conformations (see figure 4). For each cluster, the average vana conformation has been drawn in tube representation, where the tube width and color depend on the conformational local variability (root - mean - square fluctuation (rmsf),) within the cluster . The color varies from blue (rmsf close to 1) to red, corresponding to the maximal fluctuation in a given cluster (e.g., cluster 1, 13; cluster 2, 13.3; cluster 3, 15.7; cluster 4, 7.9; cluster 5, 8.0; cluster 6, 8.4). A permanent feature of the entire conformational landscape of vana is the large internal mobility of the -loop . This agrees with the apo form of vana simulated: the -loop tendency to open is expected to play an important role in the substrate processing . The root mean square deviation (rmsd) from the starting conformation of the trajectories is shown in a prune - green heat map (in). The conformation sets associated with the medoid of each cluster are depicted in putty cartoons . On the cartoons, the root - mean - square fluctuation (rmsf) of the backbone is represented by the width of the main chain and by a blue green red color scale corresponding to the rmsf values within the corresponding som cluster . The average conformation of this cluster is characterized by three regions displaying large local rmsf: the -loop, the opposite domain, and three loops [residues i43v48], [residues p71h76], [residues n83h84]. A first series of clusters, represented by clusters 1, 2, and 3, displays significant opening of the -loop, with the loop being the most open in clusters 1 and 3 . In all of these clusters, the protein internal mobility remains concentrated on the -loop (with maximal rmsf values of 13 in cluster 1 and 15.7 in cluster 3) and the other regions are much less mobile, except the opposite domain (maximal rmsf value of 8.0), the other maxima remaining 45 . Thus, after only 30 ns of simulation, the tamd trajectories have been able to reach conformations displaying a wide opening of the -loop . These conformations are similar to the x - ray crystallographic structures published on the ttddl d - ala: d - ala ligase (pdb i d: 2yzg). The second series of clusters, which is represented by clusters 5 and 6, displays conformations with semiopen or semiclosed -loop, similar to the x - ray crystallographic structure of the d - ala: d - ala ligase in ref (47) (pdb i d: 2zdg). The averaged conformations of clusters 5 and 6 display large mobility of the -loop, as well as that of a few regions of the protein: the opposite domain and the three loops previously detected in cluster 4: [residues i43v48], [residues p71h76], [residues n83h84]. The various trajectories explored the u - matrix differently (see figure 5). The larger cluster, cluster 4, was sampled by the different trajectories, but each one sampled distinct areas . The md trajectory explored mainly cluster 4, keeping the coordinate rmsd value as low as 2.5, with respect to the starting point (figure 4), and performing few incursions into cluster 6 . This result agrees with the previously recorded md trajectories in the absence of the disulfide bridge c52c64 . The starting points are shown in pink and the ending ones are shown in magenta . The blue green red color scale represents the local root - mean - square deviation (rmsd), from the starting structure for each structure (values shown are given in). Although all tamd trajectories started from the same conformation, the different choices for the collective variables, as well as the random evolution of md simulations, induced distinct explorations of the conformational space . In that respect, the trajectories tamd_on and tamd_5cv visited mainly cluster 4, containing the starting conformation . The trajectories tamd_n and tamd_on explored clusters 1, 2, and 3, corresponding to the opening of the -loop . The trajectory tamd_mn explored regions 5 and 6 . Therefore, it seems that the geometric center of the -loop is a required collective variable to obtain the loop opening . Frames extracted from tamd_n are plotted in figure s2 in the supporting information, and reveals that, before the full opening, the -loop undergoes a sideways movement . Overall, the cluster analysis of md and tamd trajectories provides an exploration of several possible models for -loop mobility . Indeed, protein conformations with fully open loop are obtained along with conformations displaying mobile closed -loop, corresponding to several conformational states explored by apo vana . The opening of the vana binding cavity was monitored by following the values of the angles and between the centers of mass of the entire protein vana (c), of the opposite domain (o), of the n - terminal (n), and of -loop () (figure 6a). The values of and angles were projected on the u - matrix (see figures 6b and 6c). An increased value for corresponds to an opening of the -loop, while an increased value for corresponds to a displacement of the opposite domain apart from the vana structure core . (a) tube representation of vana with the -loop in green and the opposite domain in yellow . Their own centers of mass is marked with a ball of the same color and respectively called and o. the center of mass of the entire protein vana is called c (shown in red) and the center of mass of the n - terminal region, called n (shown in blue). (b, c) projections of the angles on the som using a prune - green heat map: (panel (b)) and (panel (c)). Some of the structural clusters previously determined from the som analysis (figure 4) display homogeneous angle values while other clusters show much more heterogeneous values (see figures 6b and 6c). Cluster 3, which contains some of the most open conformations of vana (figure 4) is very homogeneous . It exhibits the widest opening (55) for the angle (figure 6c), while (figure 6b) is shrunk with a value of 52, showing the opposite domain moving apart, with respect to the protein core, while the -loop is still closed . Unlike cluster 3, clusters 1 and 2, containing open -loops, the and are mostly mirrored, with large values (green regions in figure 6c) corresponding to small values (violet regions in figure 6b) and vice versa . This is the sign of an anticorrelation between the -loop and opposite domain displacements . Nevertheless, some regions of figures 6b and 6c in clusters 1 and 2 display the same color, corresponding to simultaneous shrinkage or expansion of the two protein domains . For the conformations displaying the most closed -loop, sampled in clusters 4, 5, and 6, there is mainly little opening of the angles and . To analyze the kinetics of the conformational exchange in vana, the protein conformations were clustered by the louvain greedy algorithm, taking into account the time order of the dynamic simulations, as described in section 2 . In that way, the conformations populating each kinetic cluster were sampled along the same trajectory, which is a sign that the different tamd trajectories explored various aspects of the conformational kinetics . The division of som according to the kinetic clusters (figure 7) display patterns quite similar to the ones observed for the projection of the angle or on the som (see figures 6b and 6c), which proves that the overall system kinetics is mainly determined by these angle variations . However, the kinetics clustering brings additional information, with respect to the conformational clustering performed by som . Indeed, three clusters (1, 5, and 7) display nonconnected regions on the som, respectively labeled 1 and 1, 5, 5, and 5, and 7, 7, and 7 on figure 7, putting in evidence fast conformational equilibrium between distinct conformational regions . The representative conformations extracted from the nonconnected regions of each of three clusters, display conformational variability in precise regions of vana, as the l and loops and the opposite domain (o). Different types of movements for these regions are observed within the three clusters, as shown by the superimposed representative conformations (figure 7). Kinetic clustering of the vana conformation using the louvain greedy algorithm on the som neurones . For the three clusters, including nonconnected regions (1, 5, and 7), the disconnected regions are labeled, respectively, as 1 and 1, 5 to 5, and 7 to 7. the representative conformations corresponding to each disconnected region are drawn superimposed in cartoons . Starting from the kinetic clustering of som map and using a procedure described in section 2, a path relating the conformations of vana with closed and open -loop has been traced on the u - matrix (see figure 8a). The opening path starts from the kinetic cluster 5 (figure 7), passes through clusters 7, 2, and 3, and ends up in cluster 15 . The conformations sampled along this path correspond to a slight translational move of the -loop (conformational cluster 2 in figure 4) and then to a rotation of the loop on the side (conformational cluster 1 in figure 4). Note that the path through conformational clusters 2 and 1 presents the advantage of permitting a large opening, which allows the substrates to easily enter into the active site . The medoids of each clusters, labeled from a to f, are shown in red and their minimum spanning link is shown in red . (b) gbsa score (in kcal / mol) for the molecules involved in the enzymatic reaction: the substrates d - ala, d - ala-(p), d - lac; the reaction intermediate homologous, phy; the product of the enzymatic reaction d - ala - d - lac; and an allosteric inhibitor . The gbsa score is plotted along the conformations labeled from 0 to 60, extracted from the opening path . Since the opening of the vana binding site is directly related to the protein function, we analyzed the path with respect to the interaction of vana with the substrates, inhibitors, and reaction intermediate . The relative importance of hydrogen bonds within vana along the path then was statistically evaluated, and connected to experimental observations . Several ligands (d - ala, d - ala(p)), d - lac, phy, d - ala - d - lac, and an allosteric inhibitor) were docked into the vana conformations extracted from the path and the poses scored using the gbsa interaction energy (figure 8b), according to the procedure described in section 2 . The score profile displayed by the allosteric inhibitor (green curve in figure 8b) is quite negative and constant . Similarly, the score profile of d - ala (red curve in figure 8b) is also negative and does not display much variation along the path, which is in agreement with the fact that d - ala is not specific of vana, but rather binds to all proteins of the d - ala: d - lac ligase family . In contrast, the other ligands d - ala(p), d - lac, phy, and d - ala - d - lac all display profiles, becoming mostly negative in cluster e of the path, after the -loop opening (see figure 8b). Before this opening, the reaction product d - ala - d - lac (orange curve in figure 8b) displays repulsion for vana, which agrees with the release of the product after reaction . The intermediate of reaction, phy, displays a behavior similar to that of the other compounds . Six conformations, labeled a to f, were picked up in each of the kinetic clusters crossed by the path (figure 8a). On these conformations, a random forest approach, described in section 2, was used to determine the relative importance of hydrogen bonds for the kinetic cluster prediction (figure 9). The most important hydrogen bonds are mainly located in the n - terminal domain, in the opposite domain, and in the -loop, which reflects the displacements of these domains described above . In addition, some important hydrogen bonds are observed in the c - terminal region . Most important hydrogen bonds for the prediction of the kinetic cluster along the opening path . The protein structure is displayed in trace, with the opposite domain (residues [149208]) colored orange and the -loop (residues [236256]) colored green . The hydrogen bonds within the -loop and the opposite domain are colored cyan, and the hydrogen bonds between these protein domains and other protein regions are colored red . The hydrogen bonds connecting residues from different regions have been colored red in figure 9 . From this outline, the breaking of interactions between protein domains can be followed along the opening path in order to give a description of the kinetic events . The two interactions e250k22 (between -loop and n - terminal region) and e207-y137 (between the opposite domain and the n - terminal region) are broken in the protein conformation labeled c (figure 9). On the other hand, hydrogen bonds e207y137, k203d132, r174d105, and, to a lesser extent, r174e104 are formed in the two conformations e and f at the end of the path . The change from the first set of hydrogen bonds to the second set gives a description of the opening, involving only few residues, and can be compared to the patterns of experimental mutations observed for vana . The e250a mutation induces a slight decrease in experimental catalytic efficiency, which would agree with the importance of the e250k22 interaction along the opening of the -loop . The only limited decrease experimentally observed could arise from a possible reorganization of the vana structure, which would be due to the presence of residues compensating for the mutation effect . Besides, in the x - ray crystallographic structure of vana, it was observed that the residues e15, s177, and h244 are involved in a network of hydrogen bonds preventing the entrance of water molecules that could impair the catalytic reaction by hydrolyzing the ligands . The residues k22 and e250 detected in the present analysis, are located, respectively, in the vicinity of e15 and h244, and could play a similar role . The analysis of the trajectories in the frame of graph theory has permitted the determination of an opening path of vana, allowing the entrance of substrates in the binding site . The path found agrees with the interaction energy profiles observed for various vana ligands . The d - ala: d - lac ligase vana was analyzed by molecular modeling and various algorithmic tools, in order to obtain a phenomenological description of the protein internal dynamics and conformational landscape, based on graph models . The comparison of md and tamd trajectories reveals the efficiency of tamd to perform enhanced sampling of the protein conformational space . As expected, the regions of conformational space explored during tamd trajectories are closely dependent on the collective variables used . In particular, the opening of the -loop seems to be favored if a geometric center of the -loop is included into the collective variables . The exploration of the conformational landscape has permitted us to describe two different modes of -loop opening: in one mode, opens through a translation, whereas in the other, a translation of is followed by a rotation . The partial opening of the -loop has been previously observed spontaneously in md trajectories in the presence of the crystallographic disulfide bridge c52c64 . The moving of the opposite domain, closely related to the opening of the active site, was also observed in these md trajectories . Mentioned that this disulfide bridge was unexpected, because vana is a bacterial intracellular enzyme that should behave in a reducing environment incompatible with the formation of the bridge . The enhanced sampling approach taken here made it possible to observe the opening in the absence of disulfide bridge . The dynamics features observed along the opening path, as the mobility of the opposite domain, are similar to the observations previously made in the presence of the disulfide bridge . The protein internal dynamics along the opening of the active site seems to be closely related to the relative mobility of the -loop and of the opposite domain, as shown by the conformational clustering (figure 4), by the importance of the angles and (figure 6), to describe the protein kinetics (figure 7), and by the analysis of hydrogen bonds along the opening path (figures 8 and 9). Md and tamd trajectories of d - ala: d - lac ligase vana have been analyzed using various algorithms . Graph models describe the protein architecture and behavior in the conformational landscape, as well as along the conformational change related to the opening of the -loop . The contact communities detected by analysis of the contacts along the trajectories display a pattern of connections relating the -loop to the middle domain, which acts as a hub to establish connection to the opposite and the n- and c - terminal domains . This pattern is conserved in most of the trajectories, whereas contrasted internal dynamics are observed in these protein regions over the conformational space (figure 4). Indeed, the -loop is always quite mobile whereas other protein regions display large (clusters 5 and 6) to small (clusters 1, 2 and 3) internal mobility (figure 4). The various graphs obtained on the contact communities, or on the som, display characteristics similar to those observed in other bioinformatics graphs obtained in different contexts, for example, in hub, middle - com, observed in the graph of contact communities (figure 2). The graph of hydrogen bonds along the opening path reveals that all residues establishing discriminating hydrogen bonds are connected to <4 other residues (figure 9), a property of small world also encountered in chemo - informatics networks based on the ligand - set similarities . Several approaches have been proposed in the literature to describe the conformational space of proteins as graph of local minima . The analysis performed in ref (22) is based on principal component analysis (pca) of protein motion . However, the pca - based analysis detects only linear correlation, whereas som can capture nonlinear correlations . The method proposed here is related to the conformational space network (csn), which was proposed by yin et al . However, these authors used discrete structural class to cluster conformations . Similarly, in ref (20), the structures were clustered using an all - atom rmsd cutoff of 2.0 . In the present paper, we defined the so - called microstates as the elements of the som grid . In addition, from an analysis of conformational transitions between som neurons, a method to detect the kinetics cluster is proposed, and put in evidence fast conformational exchange . The graphs proposed here could be used in a systematic way in proteins for which structural information can be obtained, in order to insert these protein structural graphs into larger graphs as the ones observed in protein such model stacking would permit to relate directly phenotypic information to physicochemical interactions at the atomic level . In the case of vana, the graphs provide a model of the open / closed motion of the -loop, allowing one to perform the synthesis between various information . The influence of specific residues and/or conformations in such graphs provides candidates for directed mutagenesis studies . The md and tamd trajectories allows an exploration of the vana conformational space, which induces the observation of the -loop opening . As the closed loop blocks the entrance of the active site, understanding the way the loop is opening gives a qualitative view of the kinetics of the vana enzymatic function . In the enhanced sampling approach, the time scale of opening events observed along tamd trajectories is biased and cannot be used to give quantitative information on the opening kinetics . However, on the other hand, the conformations extracted along the opening path of the -loop, can be used for docking purposes . Indeed, during the -loop opening, the entire architecture of the vana structure, as well as the active site geometry change . Docking ligands on the active site pocket modified by the -loop opening would block this site into an inactive conformation and would orient the docking prediction toward effective inhibitors of the vana function . The protein conformations sampled during the opening path are available from the authors upon request . The d - ala: d - lac ligase vana have been exhaustively investigated by molecular dynamics and enhanced sampling simulations, in order to propose outlines of (i) protein architecture and (ii) protein conformational landscape . These two types of analyses have been conducted in parallel and give consistent results . The conformational landscape of vana is characterized by a large mobility of the -loop, which displays different translational and rotational motions, with respect to the remaining part of the protein . This conformational view of the landscape is completed by a slightly different kinetic view, which fully agrees with an angular description of the relative mobility of the opposite domain and -loop . The importance of the relative motions of the opposite domain and -loop is further enforced by the contact communities analysis of the protein structure, showing a large influence between these two regions . Overall, the numerical and statistical tools used here provide parallel descriptions of the protein structure and of the protein conformational landscape, which are in global agreement.
Our approach employs the one - bead - one - compound approach to library design, which capitalizes on split - pool combinatorial synthesis . This approach also allows for an evolutionary approach to catalyst optimization, in analogy to the strategy employed in the directed evolution of enzymes . For our initial screening for example, we elected to run reactions to low conversion initially, allowing preliminary assessment of catalyst krel values . We define krel as the relative rate of epoxidation at each olefinic site of 1 (2,3-position to give 3; 6,7-position, 4; 10,11-position, 5). It is well appreciated that reaction conversion (i.e., the extent of overoxidation) can influence product distributions and muddle the extraction of meaningful krel comparisons among catalysts . Catalysts at a later stage under synthetically meaningful conditions, with sufficient stoichiometry of terminal oxidant to achieve good yields of products (vide infra). We began with evaluation of a small library of resin - bound peptide catalysts we had synthesized in a parallel fashion, as well as a diverse split - and - pool library for the oxidation of 1 . Notably, both the parallel synthesis library and many members of the unbiased split - pool library displayed some selectivity, mostly toward the 2,3-position of 1 (table 1, selected results). These observations were auspicious, given that many catalysts exhibited significant differences from the product distribution observed with catalytic propionic acid (5:4:3 = 2.6:2.2:1.0; fig . Catalyst 6 displayed the most significant deviation from the distribution afforded by propionic acid (5:4:3 = 1.0:1.0:3.9; table 1, entry 1). To enhance selectivity for 3, a small one - bead - one - compound (oboc) combinatorial library was synthesized and biased toward peptide 6, with four variable positions adjacent to the catalytic aspartic acid (fig . Biasing of the library was accomplished as follows: at each variable position, approximately 50% of the functionalized polystyrene beads were coupled to the parent residue (corresponding to catalyst 6) at each position, while the remaining 50% of the beads were evenly distributed for coupling to either seven different residues (positions i+1 and i+2) or six different residues (i+3 and i+4 positions; fig . 2). The resulting library possesses a theoretical size of about 3,000 unique peptide sequences (n = 7788; see supplementary information). Upon completion, the theoretical distribution of peptides in this library follows the histogram shown in fig . 2b: 6% of beads contain the parent sequence (6), 25% contain a single substitution, 38% contain two substitutions, etc . Approximately 228 beads from this library were tested, including catalyst 7, which favored epoxide 3 with approximate doubling of selectivity (5:4:3 = 1.3:1.0:8.2, table 1, entry 2). This result represented a further departure of the performance of a peptide - based catalyst from the control catalyst 2 . For the new oboc library, now biased toward 7, we fixed the i+1 residue as pro and i+2 position as asn(trt), hypothesizing that each was important for selectivity . The identities of three adjacent residues (i+3, i+4 and i+5) were then varied . Biased library 2 (table 1, entries 3 and 4) produced catalysts 8a and 9a, which exhibited even greater site - selectivity, again nearly doubling the preference for 3 . In contrast, truncated analog 10 exhibits reduced selectivity of 1.3:1.0:5.0 (entry 5). We then assessed the performance of the catalysts upon resynthesis and evaluation in solution, and under synthetically meaningful conditions (e.g., 1.0 theoretical equiv oxidant, controlled by stoichiometry of dic). First, as noted below, we observe that catalysts provide better results when they are dissolved in solution, as opposed to when they are immobilized on beads . Second, and perhaps of greater significance, was our observation of significant enantioselectivity in these experiments . We had hypothesized that catalyst - dependent regioselectivity might also be coupled to the observation of enantioselectivity for a given monoepoxide isomer . The notion that the same intrinsic transition state organization associated with a specific regioselectivity might also translate into the organization required for enantioselectivity finds analogy in our previous rate - based selections for catalysts that also turned out to be enantioselective . With these considerations in mind, we resynthesized 8a and 9a as their corresponding c - terminal methyl esters, 8b and 9b . When examined under conditions designed to lead to full conversion to monoepoxide (1.0 theoretical equiv oxidant: 1.0 equiv dic, 2.0 equiv h2o2), methyl esters 8b and 9b both provide excellent overall selectivity . Catalyst 8b delivers 3 with essentially total regioselectivity, and in 82% ee (table 2, entry 1a). Catalyst 9b also exhibits high regioselectivity, with 3 showing a slightly improved 86% ee (entry 1b). Strikingly, these peptide - based catalysts exhibit site- and enantioselectivities that compare favorably to the sharpless catalyst for oxidation of 1 (equation 1, above). Given the analogy, we also evaluated catalyst 9b for asymmetric epoxidation of other terpenes to assess its generality toward allylic alcohols . Peptide 9b maintains excellent site - selectivity for geraniol (table 2, entry 2;> 100:1 site - selectivity, 87% ee). Nerol shows somewhat better enantioselectivity (entry 3, 93% ee) while retaining site - selectivity for the 2,3-cis - allylic position . Prenol also gives a favorable result, with the corresponding epoxide formed with 92% ee (entry 4). Catalyst 9b shows no selectivity for the 2,3-olefin of farnesyl methyl ether, supporting a hydroxyl directed mechanism . Initial evaluation of the libraries also revealed catalyst 11, which exhibited a surprising, albeit modest, selectivity for the 6,7-position of 1, to favor 4 (table 1, entry 6; 5:4:3 = 1.5:1.9:1.0). Catalyst 11, with d - pro in the i+1 position, was unique in that we had not observed even this modest level of alternate regioselectivity previously . We therefore prepared a new library, biased toward the structure of 11, wherein the i+1 d - pro was fixed and the adjacent three residues were varied . Therein, we found catalyst 12a, which displayed improved selectivity for 4 (table 1, entry 7; 5:4:3 = 1.5:2.9:1.0). When resynthesized as the c - terminal methyl ester 12b, and evaluated in chcl3, this catalyst yielded a product distribution of up to 1.0:4.1:1.3 (5:4:3). Intriguingly, this study did not reveal catalysts that exhibited dramatically improved selectivity for 4, although several comparable catalysts were present (see supplementary table s1). The structures of the peptides unveiled by this library prompted us to prepare analogs of catalyst 12 in a hypothesis - driven manner . We projected that a c - terminal amide might better resemble the catalysts in their on - bead screening form (wherein peptides are linked to the resin as the amide). Catalysts 12c (with c - terminal n - bu amide) and 12d (with c - terminal gly - ome) were examined . While both catalysts favored production of epoxide 4, catalyst 12d exhibited greater selectivity (5:4:3 = 1.2:8.0:1.0) under optimized conditions (fig . Furthermore, this product ratio simplifies an otherwise difficult separation of products by chromatography, allowing isolation of 4 in 43% yield as a> 94:6 mixture of 4:5 isomers . The appreciable, catalyst - dependent selectivity for the 6,7-position of farnesol is quite unusual . As in the case of the 2,3-selective catalysts, experiments with farnesyl methyl ether exhibited little to no selectivity (data not shown). Thus, the observed 6,7-selectivity may be largely the result of a hydroxyl - directed mechanism, despite the distance of the hydroxyl, six bonds away from the reacting olefin . Yet, unlike the results obtained with the best 2,3-selective catalysts, the production of 4 through the action of catalyst 12d occurs with only modest, but measurable 10% ee . The observation that both high site - selectivity and enantioselectivity can track together qualitatively with 2,3-selective catalysts, but do not appear as tightly coupled with 6,7-selective catalysts is intriguing . This may be due to the higher number of rotatable bonds between the directing hydroxyl group and the preferred site of oxidation . We have observed, in our screening studies (in correspondingly unoptimized experiments), catalysts that give somewhat lower site - selectivity (supplementary table s2, 5:4:3, ~1:34:1), but slightly higher enantioselectivity (~2030% ee). Yet, our data sets are not large, and this concept requires further study for a full assessment . Even so, the observed 6,7-site - selectivity exhibited by catalyst 12d greatly contrasts with the product distribution induced by m - cpba, and any other reported catalyst, to our knowledge (fig . 3b). That catalyst 12d shows a preference for the 6,7-position of 1 stimulated its assessment for oxidation of a more complex polyene, geranylgernaniol (13), which presents four competing olefins for functionalization . Fig . 3c) with m - cpba and with the catalysts described above . With geranylgernaniol (13; catalyst 9b, as with the oxidation of 1, exhibits high regioselectivity to give the 2,3-monooxide of geranylgeraniol (14). Catalyst 12d again exhibits striking site - selectivity for an internal alkene, which we have assigned as the 6,7-monooxide 15 . The monoepoxide 14, as well as the other monoepoxides (i.e., the 10,11- and 14,15-epoxides; see supplementary information) are also produced in the reaction, but as minor products . The catalytic, site - selective epoxidation of geranylgeraniol (or farnesol), at a position other than the 2,3-olefin, has not previously been reported to our knowledge . The challenge of the task is manifest in the nearly equivalent reactivity of the 14,15-, 10,11- and 6,7-olefins . Thus, the observations shown in figure 3 may represent an important step forward, enabled by the iterative peptide - based catalyst screening approach . In summary, we have discovered different peptide catalysts capable of furnishing different isomers of epoxyfarnesol and related compounds that are minor products of generic peracid - based olefin epoxidation . Peptide 9b appears to operate via a hydroxyl - directing mechanism, analogous to sharpless asymmetric epoxidation and with comparable selectivity . Peptide 12d, provides unprecedented selectivity for a catalyst for the internal olefin of farnesol, with substantial site - selectivity, but modest enantioselectivity . Discovery of catalysts for complex selectivity situations, wherein myriad possible products may be formed as a function of a catalytic mechanism of bond formation, is a state - of - the - art challenge for asymmetric catalysis endeavors . The application of diversity - based approaches, such as that described above, may prove fruitful, and may also offer some analogy to the directed evolution strategies employed by natural and engineered enzymatic systems . To a test tube with stir bar and teflon - lined screw cap was added peptide 9b (0.1 equiv); allylic alcohol (1.0 equiv . ); a freshly prepared / sonicated solution containing hobth2o (0.1 equiv .) And dmap (0.1 equiv .) In dcm (to a concentration of 0.2 m of substrate); and 30% aqueous h2o2 (2.0 equiv . ). The test tube was placed in ice (if running reaction below room temperature) and allowed to chill before adding dic (1.0 equiv . ). The reaction tube was then sealed with a screw - cap under ambient conditions (without exclusion of air) and brought to a cold room (4 c) where the reaction was stirred vigorously . After 7 h following addition of dic, the reaction was quenched with a saturated aqueous solution of na2so3 and stirred for a few moments, sitting in ice, before allowing to warm to room temperature . Saturated aqueous nahco3 and hexane or etoac were added, the mixture was vortexed, allowed to settle, and then the organic layer was removed, followed by two to three additional hexane or etoac extracts, peformed similarly . If site - selectivity was to be determined, an aliquot of the combined organics was removed, diluted with additional hexanes and analyzed by gc . The gc sample was returned to the organics, which were then concentrated in vacuo or under a stream of n2, and purified by flash silica gel column chromatography . To a test tube with stir bar and teflon - lined screw cap was added peptide 12d (0.1 equiv); substrate (1.0 equiv . ); about two thirds of a freshly prepared / sonicated solution containing hobth2o (0.1 equiv .) And dmap (0.1 equiv .) In chcl3 (the chcl3 was passed through basic alumina prior to mixing with dmap / hobt); and 30% aqueous h2o2 (2.0 equiv . ). The test tube was placed in ice and allowed to chill before adding dic (1.0 equiv .) Followed by the remaining chcl3 solution (to a concentration of 0.2 m of substrate), rinsing down the sides of the reaction tube . The reaction tube was then sealed with a screw - cap under ambient conditions without exclusion of air and placed in an proh bath maintained at 12 c to 18 c by cryostat . 2548 h following addition of dic, the reaction was quenched with a saturated aqueous solution of na2so3 and stirred for a few moments, still at low temperature, before allowing to warm to room temperature . Saturated aqueous nahco3 and etoac were then added and the mixture was vortexed, allowed to settle, and then the organic layer was removed, along with three additional etoac extracts, peformed similarly . An aliquot of the combined organics was removed, diluted with additional etoac, and analyzed by gc . Additional experimental procedures, descriptions of compounds, and analytical methods may be found in the supplementary information . To a test tube with stir bar and teflon - lined screw cap was added peptide 9b (0.1 equiv); allylic alcohol (1.0 equiv . ); a freshly prepared / sonicated solution containing hobth2o (0.1 equiv .) And dmap (0.1 equiv .) In dcm (to a concentration of 0.2 m of substrate); and 30% aqueous h2o2 (2.0 equiv . ). The test tube was placed in ice (if running reaction below room temperature) and allowed to chill before adding dic (1.0 equiv . ). The reaction tube was then sealed with a screw - cap under ambient conditions (without exclusion of air) and brought to a cold room (4 c) where the reaction was stirred vigorously . After 7 h following addition of dic, the reaction was quenched with a saturated aqueous solution of na2so3 and stirred for a few moments, sitting in ice, before allowing to warm to room temperature . Saturated aqueous nahco3 and hexane or etoac were added, the mixture was vortexed, allowed to settle, and then the organic layer was removed, followed by two to three additional hexane or etoac extracts, peformed similarly . If site - selectivity was to be determined, an aliquot of the combined organics was removed, diluted with additional hexanes and analyzed by gc . The gc sample was returned to the organics, which were then concentrated in vacuo or under a stream of n2, and purified by flash silica gel column chromatography . To a test tube with stir bar and teflon - lined screw cap was added peptide 12d (0.1 equiv); substrate (1.0 equiv . ); about two thirds of a freshly prepared / sonicated solution containing hobth2o (0.1 equiv .) And dmap (0.1 equiv .) In chcl3 (the chcl3 was passed through basic alumina prior to mixing with dmap / hobt); and 30% aqueous h2o2 (2.0 equiv . ). The test tube was placed in ice and allowed to chill before adding dic (1.0 equiv .) Followed by the remaining chcl3 solution (to a concentration of 0.2 m of substrate), rinsing down the sides of the reaction tube . The reaction tube was then sealed with a screw - cap under ambient conditions without exclusion of air and placed in an proh bath maintained at 12 c to 18 c by cryostat . 2548 h following addition of dic, the reaction was quenched with a saturated aqueous solution of na2so3 and stirred for a few moments, still at low temperature, before allowing to warm to room temperature . Saturated aqueous nahco3 and etoac were then added and the mixture was vortexed, allowed to settle, and then the organic layer was removed, along with three additional etoac extracts, peformed similarly . An aliquot of the combined organics was removed, diluted with additional etoac, and analyzed by gc . Additional experimental procedures, descriptions of compounds, and analytical methods may be found in the supplementary information.
Through our service we have witnessed dramatic advancements in biology and the related areas in the past 20 or more years . For example, using genome sequence data for eubacteria, archaebacteria and eukaryotes, some authors constructed a tree of life, which is the phylogenetic tree of the three super - kingdoms (1,2). Others reported a way to predict the number of genes at least in the bacterial world (3). The dramatic advancements prove our simple idea that the more data we collect and serve the more people make use of it for various purposes . On the other hand, the recent development of sequencing machines such as 454 (by 454 life sciences), solexa (by illumina, inc .) And solid (by applied biosystems) makes us worrisome as well . According to some estimate, 510 tera bases will be sequenced by solexa at one sequencing facility in a month in the near future . With the further development of the sequencing technology the whole genome of a person may repeatedly be submitted in the near future, as few examples warn (4). To cope with the expected situation of sequencing genes and genomes, we think that the new computer and tool serve our data submitters and users better and make our job more effective and efficient . In this article we will report on the data submissions to ddbj in the past year, replacement of our computer system with an upgraded one, a new data retrieval tool and a new home page . In the period from july 2006 to june 2007, ddbj collected and released the original data of 1 880 115 entries or 1 134 086 245 bases that were classified into the 19 international nucleotide sequence database collaboration (insdc) divisions (5). More than 90% of the submissions came from japanese researchers, and the rest were mainly from chinese and korean researchers . The released data includes the high - throughput cdnas (htc) of cricket, gryllus bimaculatus submitted from tokushima university (6). The data amount is 32 010 entries that can be obtained through anonymous ftp with the file name, gryllus_bimaculatus_htc_070726_1.seq.gz . Also included is 700 mb of the high - quality draft genome data of medaka, oryzias latipes, which was submitted from university of tokyo and national institute of genetics (7). The data was carefully assembled and upgraded from the wgs data that was reported in our previous paper (8). The given accession numbers are baaf03000000 (hd - rr, version 0.9), baaf04000000 (hd - rr, version 1.0) baae01000000 (hni) and acaaa0000001-acaaa0356693 (5 sage tags). Although draft genome sequences of two fugu (blowfish) species are available, the high - quality draft genome of medaka will be quite useful particularly for the study of vertebrate evolution . The submitters of the genome data discussed, for example, that the medaka genome preserved its ancestral karyotype for more than 300 million years (7). It is also noted that the current number of bacterial species / strains in the complete bacteria genome data repository, the genome information broker, (gib, http://gib.genes.nig.ac.jp/) (9), at ddbj is 569 and keeps on growing rapidly . The species added in the past year include methanococcus maripaludis (by joint genome institute), saccharopolyspora erythraea (by university of cambridge), francisella tularensis subsp . Tularensis (by ut southwestern medical center), desulfotomaculum reducens (by joint genome institute), burkholderia vietnamiensis (by joint genome institute), herminiimonas arsenicoxydans (by genoscope), geobacillus thermodenitrificans (by nankai university), corynebacterium glutamicum (by rite) and many others . We also serve a complete virus genome data repository, gib for viruses (gib - v, http://gib-v.genes.nig.ac.jp/) that now contains 31 486 virus genomes and genomic segments . In july 2007, we celebrated the 20th anniversary of the public release of the dna data . Our first release in july 1987 contained only 66 entries or 108 970 bases that were typed in from published papers . These numbers may be impressive in the comparison with the corresponding ones as of june 2007, 13 371 690 entries or 8 988 178 758 bases . This tremendous increase in the numbers perhaps reflects the remarkable advancement of research in biology and the related areas in japan in the past 20 years . The ever - increasing amount of the data also makes us worry about our hardware and software facilities . In march 2007, we completely replaced our computer system with an upgraded one . (i) the increase in the number of entries in making the flat files from 300 000 to 1 000 000 entries / day, (ii) the decrease in processing time in making a huge flat file; in case of four rice chromosomes, from 110 to 13 min, (iii) the decrease in processing time from 120 to 13 min for updating the live - list that lists the accession numbers and dates of the public release of the released entries; it is weekly updated to exchange the information about the currently released data with the embl bank and genbank, (iv) the increase in the number of ests in data processing from 40 000 to 800 000 entries / h and (v) the increase in the number of queries accepted at once by 1.5 times . Therefore, we will be able to cope with the increase in the number of data submissions for the next several years . Recently, we have installed a high - speed keyword search tool, all - round retrieval for sequence and annotation (arsa, http://arsa.ddbj.nig.ac.jp/top-e.html). The search logic behind arsa is called sigma, which was invented by arikawa and his colleagues (10,11). For a given query sigma makes it possible to retrieves all the right entries by checking the contents of a database just once, no matter how the query is complicated . Sigma does not need an index file, which means that search can be made against the currently available data . The search engine operates in parallel for divided data, which makes the search even faster . Arsa also has a large scalability with an increasing amount of data . In theory, one search can be completed within 10 s irrespective of the data size and the query formula . If the data increases more than 10 times larger than the current amount, however, we may have to increase the number units in the shunsaku accordingly to keep the present search speed . A special feature of arsa is that it can also incorporate the terms defined by the feature / qualifier of insdc . While this feature is very helpful for us to annotate the submitted data, it enables our user to perform data retrieval by using terms in the feature / qualifier . For example, you can search for cdss (protein coding sequences) located on human y chromosome, as shown in figure 1 . In the figure, the query formula is given on the top, and a part of the hit entries is given below with the accession numbers . By clicking one of the numbers you can see its contents . You can download the search result in flat file, fasta or xml, and also choose the items in the search results to be displayed on the computer screen and directly download them in tab - limited format . We also provide you with webapi (http://xml.nig.ac.jp/>http://xml.nig.ac.jp/) (12) so that you can customize arsa by writing a program in perl or java . We will soon include kegg (http://www.genome.ad.jp/kegg/) in arsa and make the 24 databases simultaneously retrievable for common keywords . Since the present hp holds many contents that have been added in an irregular sequence without much consideration for consistency, it is not really convenient now for our data submitters and users . The main point of the updating thus is to reach the almost every content with three clicks or less, which is now a common practice in making use of a hp . In the new hp when you click one of our main services, data submission, data retrieval, ftp / soap, statistics and inquiry, you can get the whole view of all contents for each service at once, and easily go to the one of them that you wish . We hope the new hp on the new computer system and tool will be more attractive to our data submitters and users worldwide.
About 20 years ago, for reasons now lost in the mists of the 20th century, i wrote a review about the ribosome for nature . Ribosomes had been discovered in the mid-1950s and, until the late 1960s, ribosome research was a major part of molecular biology . By the late 1960s it had emerged that ribosomes are the polymerases that catalyze protein synthesis under mrna control . Satisfied with that level of understanding, most who had worked on protein synthesis during the' golden age' of molecular biology sought greener pastures in the years thereafter, and interest in the ribosome waned . The thesis of my review, which was entitled' the ribosome returns', was that the ribosome field was poised for advances so dramatic that it would regain the prominence it had last enjoyed in the mid-1960s . In 1988 first, the discovery of ribozymes in the late 1970s had stimulated the interest of biochemists and molecular biologists in rna - containing objects generally, and the ribosome is the most important rna - containing object of them all . Second, the shortage of structural information that had for so long plagued the ribosome field seemed ready to end . A month or so ago, i agreed to write a successor to' the ribosome returns' for journal of biology, but shortly thereafter i started having second thoughts . As yogi berra is alleged to have said, " it is hard to make predictions, especially about the future " . By writing a successor to' the ribosome returns' i would be in the embarrassing position of calling attention to an ancient review, the very title of which was a prediction . Below i provide a personal account of what happened in the ribosome field between 1988 and 2000 and my assessment of where the field stands today . By 1988, a lot had been learned about the three - dimensional organization of the ribosome . The shapes of the two ribosomal subunits, and of the complex they form during protein synthesis, were known at low resolution (figure 1), and it was understood that protein synthesis occurs in the gap between the two subunits . The secondary structures of the ribosomal rnas (rrnas) had been worked out, and sites on rrnas where ribosomal proteins bind had been identified . In addition, the structures of several ribosomal proteins and a few rrna fragments were known at atomic resolution in isolation . However, no one was so deluded as to imagine that structural information of this sort would ever explain ribosome function . The ribosome at low resolution the images shown here are photographs of plaster models of the two ribosomal subunits made by james lake . They were derived from his em images of the two ribosomal subunits from e. coli . (a) the large subunit (left) and the small subunit (right) with some of their landmarks indicated . The only two approaches for addressing the need for structural information that seemed promising in the 1980s were x - ray crystallography and electron microscopy . The first ribosome crystals, reported by yonath, wittmann and colleagues in 1980, diffracted poorly; but, as the years went by, crystals were obtained of ribosomes and ribosomal subunits from many prokaryotic species (for example), and the resolutions of the diffraction patterns of the best of them improved . The unit cells of ribosome crystals are very large and, consequently, ribosome crystals diffract x - rays so weakly that useful data can be collected from them only at synchrotron light sources . In 1980 the technology for doing macro - molecular crystallography at synchrotrons was primitive, but major advances were made in the 1980s and thereafter (for example), and by 1988 the technology needed for ribosome crystallography was falling into place . Electron microscopy seemed promising because methods were being developed for obtaining three - dimensional electron density maps of biological objects from their two - dimensional electron microscopic (em) images . Although the theory of image reconstruction is simple, its application to objects like the ribosome, for which the images to be reconstructed are those of isolated, randomly oriented particles, was fraught with difficulties . Nevertheless, by 1988 it seemed likely that ribosome reconstructions would eventually emerge with resolutions high enough to allow trnas to be visualized bound to the ribosome . Once that threshold was crossed, it seemed to me that em would start contributing to our understanding of protein synthesis . My optimism notwithstanding, nothing published between 1988 and 1995 would have led the unbiased observer to conclude that the ribosome was likely to return any time soon . The advances made in em reconstructions did not seem dramatic, and the papers published on ribosome crystallography were records of frustration . Ribosome crystallography had run aground on the shoals of the classic problem in macromolecular crystallography, the so - called phase problem, and it was unclear if it would ever get unstuck . Crystal structures are three - dimensional models of molecules that are generated by fitting chemical structure into experimentally determined, three - dimensional maps that display the distributions of electrons in those molecules . Electron density maps can be computed from crystal diffraction data only if the phases associated with each of the tens of thousands of reflections in such datasets are known . If there is no prior knowledge about the three - dimensional structure of a macromolecule, phases must be measured experimentally . In the end, the experimental technique that contributed the most to solving ribosome crystal structures was the heavy atom multiple isomorphous replacement (mir) method that perutz devised in the 1950s to solve the structure of hemoglobin . However, in 1988, it was unclear that mir, or any other approach to phasing, such as anomalous scattering (as), would ever work for the ribosome . Everything else being equal, the larger a macromolecule, the harder it is to phase its diffraction pattern experimentally; and by crystallographic standards, ribosomes were and are huge . Ad hoc ribosome meetings have been held at different venues around the world for decades . In my estimation, none of them was more important than the ribosome conference that took place in victoria, bc, canada, in the summer of 1995 . There, frank and his colleagues presented the reconstructions they had just obtained from em images of ribosomes embedded in vitreous ice . Even though the resolutions of these reconstructions were modest by today's standards, about 25, their superiority over their predecessors, which had been derived from images of negatively stained particles, was striking . No significant progress was reported on the crystallographic front at victoria, but it was clear that this area too was heating up . A group at yale, of which i was a member, had just begun working with ribosome crystals, and gossip at the meeting revealed that we were not alone . Most notably, by the time i left victoria, i was convinced that the ribosome phase problem was soluble . Molecular replacement is a computational method for using the structure of one macromolecule to estimate the phases of the reflections in the diffraction pattern produced by a crystal of another macromolecule of related structure . Why not use a cryo - em reconstruction of the ribosome to phase ribosome diffraction patterns by molecular replacement? Although this approach would yield phases only up to the resolutions of the em reconstruction used, which was likely to be low by crystallographic standards, once the proverbial foot was in the proverbial door that far, higher resolution electron density maps would surely follow . The first successful phasing of a ribosomal diffraction pattern was reported in 1998 . That paper presents a 9 resolution electron density map of the large ribosomal subunit from haloarcula marismortui, which was obtained using crystals that had been first described 13 years earlier . The phases used to compute that map were measured by miras methods using crystals into which heavy metal cluster compounds had been soaked . The crucial step in all phasing experiments is determination of the locations in the unit cell where heavy metals and/or anomalous scatters reside . This is normally done using patterson methods, but in this instance, in order to prove that these sites had been correctly located, a second, independent approach was used . Phases were obtained by molecular replacement using an em electron density map of the h. marismortui large ribosomal subunit . Using those phases, a difference electron density map was computed that displayed only the electrons belonging to the heavy atoms in the unit cell . The patterson - derived positions for these heavy atoms corresponded exactly to the positions found by molecular replacement . Even though little molecular detail can be made out in any 9 resolution electron density map, the fundamental accuracy of this 9 resolution electron density map was beyond question . Because nothing motivates scientists more powerfully than the knowledge that their problem can be solved, ribosome crystallography advanced rapidly thereafter . By the summer of 1999, the resolution of the electron density maps available for the large ribosomal subunit from h. marismortui had improved to 5.0, and using heavy atom isomorphous replacement methods, ramakrishnan and colleagues had obtained a 5.5 resolution electron density map for the small ribosomal subunit from thermus thermophilus . A 7.5 resolution electron density map of the 70s ribosome from t. thermophilus appeared that same year . In august, a 2.4 resolution structure was published of the large ribosomal subunit from h. marismortui . (figure 2 shows what the improvements in the resolutions of the electron density maps for this object between 1998 and 2000 meant in terms of their interpretability .) At the beginning of september, an imperfect, 3.3 resolution structure of the small ribosomal subunit from t. thermophilus was presented, and 3 weeks thereafter a significantly more accurate, 3.1 resolution version of that same subunit appeared that had been independently determined . The effect of improvements in resolution on ribosome electron density maps . (a - c) views of the entire surface of the large ribosomal subunit of h. marismortui that interacts with the small ribosomal subunit . (a) an em reconstruction of that subunit that has a resolution of 20 . (b) an x - ray crystallographic image of the same particle, also at a resolution of 20 . (c) the same as (b) except for the resolution, which is 9 . (d) a view of the face of the large subunit at a resolution of 5, with the positions of l1 and l10 indicated in yellow . (e) electron density corresponding to a helix of 23s rrna at a resolution of 5 . (f) the electron density for a helix at a resolution of 2.4 . Since 2000, many ribosome crystal structures have been deposited in the protein data bank (pdb). I think of seven of them as founder structures: that is, the first atomic resolution structure obtained from a particular ribosome crystal by a particular laboratory . The three structures that appeared in 2000 are founder structures as are, first, a structure of the large ribosomal subunit from deinococcus radiodurans, second, two independently determined structures for the 70s ribosome from t. thermophilus [18 - 20], and third, a structure of the 70s ribosome from escherichia coli . The rest of the ribosome structures in the pdb are those of founder particles with substrates, substrate analogs, protein factors and inhibitors bound, and all of them were generated using effectively the same crystals that produced the corresponding founder structures . (once the structure of some crystal has been solved, experimental phase determination is not required to solve the structures of derivatives of that crystal .) Since 2000, the goal of ribosome crystallographers has been the construction of a movie of protein synthesis, the individual frames of which are atomic resolution structures of the ribosome in every state it visits as protein synthesis progresses . This movie is far from complete, mostly because the crystals needed to determine most of its frames are still not available; but it is not for lack of trying . Besides not having all the frames needed to make the complete movie of protein synthesis, what else do we not know about ribosomes crystallographically? In the first place, there are no crystal structures for eukaryotic ribosomes, and in a world controlled by sponsors fixated on homo sapiens this is not a good thing . In addition, surprisingly, there is still no complete crystal structure for a large ribosomal subunit . Neither of the two lateral arms of the large ribosomal subunit (figure 1) can be visualized at high resolution in crystal structures of isolated large subunits; they wiggle too much . The conformation of the l1 arm is stabilized when the small subunit binds to the large and, for that reason, its structure is known in at least some of the conformations it adopts during protein synthesis . Much less the l11 protuberance includes the complex that ribosomal protein l10 forms with four to six copies of ribosomal protein l7/l12 (the number depends on species). The l10 complex is very important functionally, but only its l10 portion has ever been visualized crystallographically, even partially, and its distal components are so flexible that they may never be visualized, either crystallographically or by em . However, structures are available for much of the l10 complex in isolation, and when those structures are added to the structure of the rest of the large subunit structure in silico, the effect is startling, to put it mildly (figure 3). We have no idea what this part of the ribosome does to promote protein synthesis . There are structures available for most of the complex that l10 forms with the several copies of l7/l12 that interact with it in the ribosome as isolated proteins . These structures have been added to the structure of the large ribosomal subunit from h. marismortui in silico . The small ribosomal subunit is much more dynamic conformationally than the large ribosomal subunit, and its flexibility is vital for its function (see below), but we have crystal structures for the entire object in several of its states . It is much easier to study the functional complexes of the ribosome by electron microscopy than it is to investigate them crystallographically . Electron microscopists do not need crystals, and the amounts of material they consume are orders of magnitude less than crystallographers require . Finally, image sorting techniques now exist that make it possible to obtain reconstructions of ribosomes in specific functional states, starting with images of samples in which only a fraction of the particles present are in that state; pure samples are no longer required . Consequently, as far as the making of the movie of protein synthesis is concerned, the electron microscopists are well ahead of the crystallographers and, in addition, em images of eukaryotic ribosomes exist (for example). Three - dimensional electron density maps obtained by em are qualitatively similar to crystallographic electron density maps, and they are commonly used to generate (quasi) atomic resolution models of ribosomes, even though none of them has a resolution high enough to allow an atomic resolution model of the ribosome to be constructed from it de novo . Em - derived models of the ribosome are generated by fitting crystal structures into em electron density . This procedure leads to difficulties only in those parts of a structure where you are most in need of information, namely where the structure of the em object diverges from the crystal structure(s) used to model it . The other major problem faced by consumers of em images is variation in resolution, which makes comparison of images difficult . Since 1995, substantial advances have been made in image reconstruction, and newer reconstructions generally have higher resolutions than older reconstructions . One reason is that the labor per image required to generate a reconstruction has fallen, and resolution improves as the number of images merged grows . The reconstruction process assumes that the images under analysis are different views of the same three - dimensional object . No increase in the number of images processed can overcome resolution limitations caused by particle - to - particle variations in structure, unless the variation involves discrete conformational states, in which case image sorting may save the day . Like gaul, protein synthesis divides naturally into three parts: initiation, elongation and termination (figure 4). During initiation, the two subunits of the ribosome are assembled into a complex that has an aminoacylated initiator trna and an mrna bound, ready to make the first peptide bond of a protein . During termination, a completed protein is released from its trna, and the ribosome assembly dismantled so that its components can be recycled . The steps of the elongation phase, which is the part of the process we will discuss here, constitute a cycle that must be repeated for every peptide bond formed . (a movie showing the three phases of protein synthesis has been created by the ramakrishnan group at the mrc laboratory of molecular biology and can be accessed from their website .) The purpose of the references provided here, which are entirely to recent articles and reviews, is to give the reader an entre into the relevant literature, rather than an outline of the history of the field . The intiation of translation is complete once an aminoacyl trna charged with formylated methionine has been placed in the p site bound to the initiating aug codon of an mrna . Elongation begins when a second aminoacyl trna recognizes its cognate codon and binds in the a site . This is followed by transfer of the amino acid from the trna in the p site to form a peptide bond with the amino acid attached to the trna in the a site, and translocation of the two trnas into the e and the p site, respectively . This sequence is iterated until a stop codon is encountered, the completed protein is released, and the ribosome disassembles, at termination of the translation cycle (not shown). The ribosome catalyzes two chemical reactions: the aminolysis of the ester bonds that link nascent polypeptides to trnas during protein synthesis, and the hydrolysis of those same bonds . The amino group used for the aminolysis reaction is the -amino group of an amino acid that is ester - linked through its -carboxyl group to the 3' oh of the 3'-terminal nucleotide of a trna . The products of this reaction are a peptide that is one residue longer than it was before the reaction, ester - linked to the trna that carried the amino acid into the reaction, and a trna that has nothing attached to it . Because this reaction transfers a peptide from one trna to another, it is referred to as the peptidyl transferase reaction . In the second reaction, which occurs only during the termination phase of protein synthesis, the nucleophile is water, instead of an -amino group, and peptides are transferred to water (that is, the last trna is released from the newly synthesized protein). Both the peptidyl transferase reaction and the hydrolysis reaction occur at the same site on the ribosome, its peptidyl transferase center (ptc), about which we now know quite a lot . (1) the ptc is located in the center of the subunit interface surface of the large ribosomal subunit . (2) although rna - protein interactions are essential for stabilizing the conformation of the ptc, it is composed entirely of rna: the ribosome is a ribozyme . (3) the catalytic properties of the ptc are not modulated by interactions between the two subunits . (4) the ptc includes a site that accommodates peptidyl trnas, the p site, and a site to which aminoacyl trnas bind, the a site (see figure 4). (5) both the a site and the p site of the ptc interact primarily with the 3' terminal cca sequence that is common to all trnas . Thus, (6) when the a site of the ptc is empty and its p site is occupied, the ptc adopts a conformation that protects the ester bonds of peptidyl trnas from nucleophilic attack . A conformational change accompanies the binding of aminoacyl trnas to the a site (as well as the binding of release factors to the ribosome) that exposes the ester bond in the p site to nucleophilic attack . (7) beyond positioning substrates properly, the ptc seems to do little to enhance the rate of peptide bond formation . (8) the group that makes the largest chemical contribution to the rate of the peptidyl transferase reaction is the 2' oh of the 3'-terminal a of the trna in the p site, which facilitates the removal of a proton from the attacking amino group and the addition of a proton to the leaving group, which is the 3' oh of the trna bound in the p site . (9) on the ribosome, the peptidyl transferase reaction proceeds at a rate that is about 10times faster than the rate of similar reactions in solution . (10) once substrates are bound appropriately to the ptc, the peptidyl transferase reaction occurs at a rate that is at least ten times faster than the overall rate of protein synthesis in living cells, which is about 20 s. (11) at neutral ph, the ester bond of an aminoacyl trna is a high energy bond, but the ester bond in a peptidyl trna is not . Given that the net effect of the peptidyl transferase reaction is the destruction of a high energy ester bond and the creation of a lower energy peptide bond, the forward direction of the peptide formation is strongly favored thermodynamically . This description of the peptidyl transferase reaction raises as many questions as it answers . What keeps nascent peptides from inhibiting their own synthesis by filling up the ptc? How does the peptidyl trna product of the peptidyl transferase reaction move from the a site, where it forms, to the p site, where it must reside if another amino acid is to be added to the nascent peptide chain? Product clearance is thought to be the simplest of these issues . As nascent peptides form, they insert into a cavity called the peptide exit tunnel, which extends from the back of the ptc all the way through the body of the large ribosomal subunit . It is not until the length of nascent peptides exceeds about 40 amino acids that their amino - terminal sequences emerge on the far side of the ribosome and start engaging with the apparatus that ensures protein folding and/or export . As far as we know, nascent poly - peptides diffuse down the tunnel in response to the nudge they are given as each peptide bond forms, but there are hints that it may be more interesting . Two recent crystal structures have provided insights into how ribosomes carrying completed proteins are recognized and the ester bond linking the protein to trnas is hydrolyzed, which is the ultimate step in product clearance . The trna movements that reset the ptc after each round of peptide bond formation are still only partially understood . Discussions of this process, which is called translocation, are best begun by reminding the reader that trnas are l - shaped rnas that vary considerably in sequence but are nearly identical in shape . One arm of the l, the acceptor stem, includes the 3' terminal cca sequence mentioned earlier . The other arm, the anticodon stem, terminates with a loop that includes an anticodon, which is the 3'-nucleotide sequence that pairs with mrna codons during protein synthesis . (aminoacyl trna synthetases ensure that the amino acids that get esterified to the acceptor stems of trnas are the ones encoded by mrna triplets complementary to the anticodon sequences of those trnas .) Messenger rnas bind to the small ribosomal subunit in the region where its head joins its body (figure 1), and the place on the small subunit where trna anticodons interact with mrna codons is called the decoding center . The a site and the p site of the decoding center are the locations where the anticodons of aminoacyl trnas and peptidyl trnas, respectively, are bound to the small ribosomal subunit just before peptide transfer occurs . (trnas are l - shaped because there is a prominent ridge on the large subunit separating the ptc from the decoding center of the small subunit that only an l - shaped molecule can surmount .) Translocation on the large subunit precedes translocation on the small subunit, and it seems to be a spontaneous, diffusive process . After peptide bond formation, the acceptor stems of both trnas in the ptc move towards the l1 arm of the large ribosomal subunit . The cca sequence of the discharged trna moves from the p site of the ptc to the so - called e (exit) site of the large ribosomal subunit, which can bind only deacylated trnas, and the cca - peptide moiety of the peptidyl trna in the a site moves to the p site of the ptc . The a - to - p motion of peptidyl trnas is accompanied by a 180 rotation of cca sequences relative to the bodies of trnas . Large - subunit translocation correlates with a rotation of about 10 of the small subunit relative to the large in the direction of the l1 arm, which is called ratcheting . The ratchet motion also seems to result from thermal diffusion, and it is unclear how tightly ratcheting is coupled to large - subunit translocation . Nevertheless, the data suggest that both must occur before small subunit translocation can take place . Large - subunit translocation leaves the ribosome in a hybrid state, in the sense that the acceptor stem of the peptidyl trna is in a p site of the ptc while its anticodon end occupies the a site of the decoding center; the acceptor stem of the discharged trna is in the large - subunit e site while its anticodon is in the p site of the decoding center (figure 4). First, it advances the ribosome by three nucleotides along the mrna to which it is bound in the 3' direction, which places a new codon in the a site of the decoding center . Second, it resolves the hybrid state by making the anticodon end of peptidyl trna move from the a site to the p site of the decoding center, and the anticodon end of the deacylated trna move from the p site of the decoding center to the e site of the small subunit . The anticodon of the trna that moves from the a site to the p site of the decoding center remains associated with its codon in the mrna so that the register in which the mrna is being translated is maintained . It is unclear whether the anticodons of trnas in the e site actually interact with mrna or not; there are biochemical data indicating they do, but the structural data are ambiguous . Although small - subunit translocation can occur spontaneously, the spontaneous process is painfully slow . Like all the major steps in protein synthesis, it is catalyzed in the cell by a g protein . The g protein in this case, which is ef - g in bacteria and ef-2 in eukaryotes, binds to the ribosome with a gtp bound that is hydrolyzed in the process . Em images of ef - g / ribosome complexes, which are all we have, show that this tadpole - shaped molecule binds to the ribosome with its head (which includes its gtpase site) bound to the large ribosomal subunit at the base of the l11 arm . That part of the ef - g binding site includes the sarcin - ricin loop (srl) of 23s/28s rrna (figure 3) that, for reasons still unclear, is critically important for the activity of all of the g - protein factors that interact with the ribosome during protein synthesis . The distal end of the tail of the tadpole inserts into the a site of the decoding center of the small subunit . The binding of all proteins that interact with the ef - g binding site is promoted by the l10 complex and the rest of the l11 arm, but the details remain to be worked out . In solution the gtpase activity of ef - g is very low, but it increases dramatically when the factor binds to the ribosome . Thus, shortly after ef - ggtp binds to the ribosome, its gtp hydrolyzes . This causes ef - g to undergo a major conformational change that seems to push the anticodon stems of trnas across the decoding center, dragging the mrna with them . Two additional events ensue: the ribosome unratchets; and ef - ggdp is released into solution . Biochemical data suggest, and em structures confirm, that the conformational changes that accompany ef - g - assisted translocation are more complicated than this account of translocation seems to require, but until the relevant atomic resolution structures become available, we are unlikely to understand them properly . Once translocation is complete, the ribosome is ready to bind a new aminoacyl trna, and if there is a deacylated trna in the e site of the ribosome, aminoacyl trna binding is accompanied by release of that trna into solution . Biochemical data suggest that these two processes interact with each other, and structural data show that trna release correlates with conformational changes in the l1 arm of the large subunit . Aminoacyl trnas are delivered to the ribosome by a second g protein, which is called ef - tu in bacteria and ef-1 in eukaryotes . The complex that ef - tu forms with aminoacyl trna (and gtp), the so - called ternary complex, resembles ef - g in its overall shape, with the anticodon stem of the ternary complex corresponding to the tail of the ef - g tadpole and its ef - tu / acceptor stem portion resembling the head . As far as we know, the ternary complex binds to the ribosome the same way that ef - ggtp does . Its ef - tu / acceptor stem portion associates with the srl region of the large subunit and its anticodon stem extends into the a site of the decoding center . If the anticodon of the trna in a ternary complex base - pairs properly with the mrna sequence in the a site of the decoding center, in other words if the codon and anticodon are cognate, a conformational change occurs that stimulates gtp cleavage and release of ef - tugdp from the ribosome . The aminoacyl trna left behind is oriented so that its aminoacyl end is far from the a site of the ptc; the large reorientation required to place its acceptor stem in the ptc is called accommodation . Once accommodation has occurred, the system is ready for the formation of the next peptide bond, which ensues quickly thereafter . The binding of cognate aminoacyl trnas to the ribosome is the rate - limiting step in protein synthesis, and from the point of view of information transfer, it is the most important . Given that diffusion is the process that brings ternary complexes to the ribosome, for every encounter with a cognate complex that results in accommodation there will be many encounters with non - cognate ternary complexes that must not result in accommodation if mrnas are to be translated correctly . The reason is that once the wrong aminoacyl trna enters the a site of the ptc, the wrong amino acid will be inserted into the nascent protein: the ptc does not discriminate . The only way in which trna selection can lead to accurate translation is if the ribosome binds cognate complexes much more tightly than non - cognate complexes, and it does . Ternary complex selection depends on base - pairing between the sequence of the codon in the a site of the decoding center and trna anticodon sequences . However, it has been realized for decades that the difference in free energy between the formation of a perfect watson - crick helix three base - pairs long and the formation of a three - base - pair duplex in which one of the base juxtapositions is non - canonical is not large enough to explain the accuracy of translation . The crystal structures and em structures have both provided important insights into this aspect of protein synthesis . We know from crystal structures of trnas bound to the ribosome in the accommodated state that when a cognate interaction occurs in the a site of the decoding center, the conformation of the small subunit changes so that two of its rna bases form base - triples with the first two base - pairs of the codon - anticodon helix that form in that center . These interactions, which strongly stabilize the two base - pairs in question, cannot occur unless the bases juxtaposed in that helix form watson - crick pairs . For that reason, as had long been suspected, the difference in free energy between cognate pairing and non - cognate pairing is much bigger on the ribosome than it is in solution . The orientation of the anticodon stem of a trna in the pre - accommodation state is very different from its orientation after it has accommodated . This question has been answered by em images that show that before accommodation, the anticodon stems of trnas bend in such a way that their anticodon sequences will interact with the decoding site the same way they do after accommodation . Thus, the response of the a site of the decoding center to anticodons appears to be the same no matter whether the trna carrying them is in the pre- or post - accommodation conformation . A vitally important consequence of the conformational change just alluded to is that, by some mechanism we do not understand, it activates the gtpase activity of ef - tu . Thus, if a cognate interaction has occurred in the decoding center, the ef - tu in a ternary complex will quickly hydrolyze its gtp and leave the ribosome so that the aminoacyl trna can accommodate . If the interaction between codon and anticodon is non - cognate, gtp hydrolysis is much slower, and the probability is correspondingly high that the non - cognate ternary complex will diffuse away from the ribosome intact, before gtp hydrolysis occurs . Suppose that against the odds, a non - cognate ternary complex delivers its aminoacyl trna to the ribosome in the same way that a cognate complex does . Before that aminoacyl trna can engage in peptide - bond formation, it must accommodate and, as far as we know, the only interactions that keep accommodating aminoacyl trnas from diffusing away from the ribosome are their codon - anticodon interactions . If that interaction is non - cognate, it will not be stabilized by interactions with the small subunit . It follows that non - cognate aminoacyl trnas are more likely to fall off the ribosome during accommodation than cognate aminoacyl trnas . Thus, base - pairing is used to discriminate cognate from non - cognate aminoacyl trnas twice every time an aminoacyl trna is delivered to the a site of the ptc . The understanding of decoding that has emerged from the combination of structural and biochemical investigations is a spectacular example of what everyone hoped would happen once the structure of the ribosome was understood at atomic resolution . However, as the above account shows, our understanding of even that aspect of the elongation cycle remains incomplete . Among the many other mysteries still unsolved are the mechanisms of action of lepa, which is a protein factor similar to ef - g that promotes reverse translocation, an unexpected phenomenon believed to contribute to fidelity; and the tmrna system that rescues ribosomes that have become stuck when translating a broken mrna molecule . The devotees of the ribosome will not run out of interesting problems to investigate for a while yet . My understanding of protein synthesis has been shaped by conversations with my colleagues, especially thomas steitz and rachel green.
Hypertrophic cardiomyopathy (hcm) is a genetic cardiovascular disease characterized by primary hypertrophy of a non - dilated ventricle . Hypertrophic obstructive cardiomyopathy (hocm) is generally regarded as a left ventricular (lv) outflow tract (lvot) pressure gradient (pg) of approximately 50 mm hg at rest or with provocative maneuvers.1) complications of hocm include sudden cardiac death, heart failure, and arrhythmia.2) in hocm patients, the cardiovascular system is exposed to acute hemodynamic burdens during pregnancy, which may lead to unfavorable complications.3) therefore, special monitoring and management is required for hocm patients during pregnancy . We report on a case of a 27-year - old female patient of hocm with high lvot pg over 100 mm hg, as known as severe lvot obstruction,4) who maintained pregnancy and successful delivery through close monitoring using transthoracic echocardiography (tte). A 27-year - old primigravid female was referred at 9 weeks gestation for further management of known hcm . She was diagnosed with hcm, but no further treatment was administered at that time . She had no previous medical history, and no family history of any cardiovascular disorder or sudden cardiac death . Initially she felt palpitations rarely, but after pregnancy, she complained of exertional dyspnea and palpitation upon fast walking or overeating as 9 weeks gestation . Physical examination showed blood pressure (bp) of 93/64 mm hg and regular pulse rate of 108 beats per minute . She had a grade iii / vi systolic harsh murmur at the third intercostal space on the left sternal border (erb's area). Tte showed asymmetrical hypertrophy (maximal thickness at septum 15 mm) with dynamic lvot obstruction due to systolic anterior motion of the mitral valve, and a severely enlarged left atrium (volume index; 57 ml / m). Hyperdynamic lv systolic function (ejection fraction 78%), high lvot pg (peak / mean pg; 75/47 mm hg at rest, 103/52 mm hg during valsalva maneuver), eccentric moderate mitral regurgitation (mr) grade iii / iv, and pseudonormalization of lv filling pattern (e / e'; 24) were also observed . In summary, tte showed hocm with high lvot pg and mr grade iii / iv . Initially, twenty - four - hour holter monitoring showed basically normal sinus rhythm with rare atrial premature complexes (apc) and ventricular premature complexes (vpc). After reaching 15 weeks of pregnancy, she complained of exertional dyspnea upon fast walking and dizziness on orthostatic position change, such as standing up quickly from a seated position . Repeat tte performed at 17 weeks of gestation showed an increase in pg of 119/52 mm hg at resting and 147/70 mm hg on the valsalva maneuver . She experienced dizziness and palpitations, which occasionally seemed to last for an entire day . Average heart rate was 91 beats per minute, faster than before, frequent apc's (5568/130599, 4.26%) up to non - sustained atrial tachycardia and occasional vpc's (522, 0.40%) up to triplets were checked . Due to an increase of lvot pg on tte and detection of frequent arrhythmias, we recommended lifestyle modification including avoiding dehydration or excessive effort, and referral from a private clinic to our obstetrics department for a multidisciplinary approach . As fetal ultrasound findings were normal, obstetricians recommended pharmacologic therapy . Bisoprolol 1.25 mg twice a day was prescribed at 22 weeks of pregnancy, and the chest discomfort was improved . At 34 weeks gestation, tte showed lvot pg of 125/63 mm hg at resting and 152/72 mm hg on the valsalva maneuver (fig . 2). Considering her symptoms and need for fetal growth, delivery was planned at 37 weeks of gestation . Tte performed just before delivery showed high lvot pg of 115/66 mm hg at resting and pg of 152/83 mm hg on the valsalva maneuver . She continued taking bisoprolol until she proceeded to delivery and avoiding use of sympathomimetics or inotropics was recommended . Two days later, caesarean section with general anesthesia was performed to give a birth to a healthy baby without cardiovascular complication . Just after delivery, tte showed a decrease in lvot pg of 87/33 mm hg at rest and she was unable to perform the valsalva maneuver (fig . 3). Thirteen months after delivery, a follow - up tte still showed high lvot pg of 100/31 mm hg at resting and 169/45 mm hg on the valsalva maneuver . Because of drug - refractory symptoms and severe lvot obstruction, we decided to perform an alcohol septal ablation for improving quality of life . Hcm is the most common genetic cardiovascular disorder, with prevalence of approximately 0.2% (i.e., 1:500) in the general population.5) this case showed how to monitor and manage a pregnancy with severe lvot obstruction (pg over 100 mm hg). Pregnancy causes physiologic changes in the cardiovascular system which included increases in blood volume (up to 40%) and cardiac output (3050%), and reductions in systemic vascular resistance and bp.6) in the first and second trimesters, the increase of cardiac output is influenced by a larger stroke volume . But later in pregnancy, a faster heart rate affects increase of cardiac output . Enlarged ventricular cavity which is induced by increasing volume reduce the lvot obstruction theoretically, however, cardiac output countervail against this effect, the lvot gradient will increase with advancing gestation.6)7) hcm is considered a world health organization (who) class ii iii in modified who classification of maternal cardiovascular risk, implying that depending on individual condition, there is a low to high risk of complications.8) cardiovascular complication rate in pregnancy with hcm appears to increase with history of previous cardiac events, poor functional class (new york heart association iii or iv), severe lv systolic dysfunction, and lvot obstruction.9) the higher the lvot gradient is before pregnancy or during the first trimester, the higher the likelihood that symptoms will progress . In addition, the subset of patients with severe lvot obstruction (those with a gradient of 100 mm hg) are at the highest risk of hemodynamic deterioration during pregnancy.4) pharmacologic management is recommended in symptomatic patients with hcm such as angina or dyspnea.10) beta blockers are mainly recommended as first - line agents because of their negative inotropic effects and decreasing adrenergic - induced tachycardia . Oral diuretics may be added with congestion symptoms.10) our patient showed low bp during pregnancy and a small amount of amniotic fluid was observed on fetal ultrasound at 26 weeks of gestation . Accordingly, only low dose beta blocker was administered and a modified lifestyle, avoiding aggravating factors such as excessive activity and dehydration was recommended . In our case, the patient had extremely high lvot pg more than 100 mm hg, resulting in a high risk of maternal cardiovascular complications . Because of her symptoms, close follow - up monitoring was performed using tte every two weeks during her third trimester . And we planned a caesarean section with consideration of maternal symptoms, fetal growth, and lvot pg on tte . As a result, regular tte monitoring was performed and severe cardiovascular complications and disease progression were prevented . In conclusion, we report on a case of a 27-year - old pregnant female with hocm who presented with extremely high lvot pg . She was monitored closely with regular follow - up of tte during pregnancy to determine the timing of caesarean section, which eventually resulted in a successful delivery . Complications such as sudden cardiac death, heart failure, and arrhythmia may occur in hocm patients either throughout the course of pregnancy or during labor and delivery . Tte is a relatively safe method which does not cause harm to either the mother or the fetus . Therefore, close monitoring using tte and management with medical treatment seems essential in pregnant patients with hocm before and during pregnancy.
Mandibular nerve block is commonly used for diagnosis and treatment of trigeminal neuralgia involving the mandibular division . Neurolytic blockade with absolute alcohol / phenol is usually required after diagnostic block to provide long term pain relief . Common complications of this block are hypoesthesia, dysesthesia, chemical neuritis, sensory loss in mandibular nerve distribution and very rarely facial and occulomotor palsy and masseter muscle weakness leading to difficulty in chewing . We report a rare complication in a patient with mandibular neuralgia, who developed prolonged severe vertigo and ataxia after neurolytic blockade of mandibular nerve with absolute alcohol . A 65 year old male patient presented to our pain clinic with complaint of right sided facial pain for the last 2 years . The pain was excruciating and confined to right lower jaw . He was diagnosed as a case of right sided trigeminal neuralgia involving the mandibular division (v3). He was on regular medication with oral carbamazepine 200 mg three times daily and gabapentin 300 mg twice daily . His symptoms had worsened in last few days and the pain became refractory to treatment . Clinical examination revealed pulse rate 70/minute and blood pressure of 140/90 mmhg . As the pain distribution was in the mandibular division, involving whole right lower jaw up to ear, neurolytic block of the right mandibular nerve was planned . The patient was placed in supine position with head tilted to left - side . After aseptic preparation and draping of the part, skin and subcutaneous tissue was infiltrated with 3 ml of 2% lignocaine . A 9.0 cm 22 gauge (g) needle was introduced perpendicularly in the middle of coronal process at midpoint of inferior border of right zygomatic arch . The needle was withdrawn slightly and directed posteriorly towards the ear so that it passed the posterior border of the pterygoid plate . The needle was then carefully advanced 0.5 cm each time till the patient reported paresthesia over mandibular region . After negative aspiration of blood, diagnostic block with 3 ml of 2% lignocaine was given . Five minutes after eliciting hypoesthesia in mandibular distribution, 1 ml of absolute alcohol (aa) was given for the neurolytic block . After about 5 minutes of the block, when the patient was allowed to sit up, he complained of severe giddiness (vertigo) and was not able to sit and stand up . Vertigo was associated with severe nausea and vomiting, however, patient`s hearing and speech was normal . The patient was immediately placed in supine position and vital examination revealed pulse rate of 60/minutes and blood pressure of 210/110 mmhg . The patient was given 500 ml of intravenous fluid (dextrose /saline) with 8 mg ondansetron intravenous and 10 mg nifedipine sublingually for treating vomiting and hypertension, respectively . After 30 minutes, the patient was reassessed and was found to have severe vertigo while sitting, and ataxia while walking . His blood pressure had decreased to 170/90 mmhg and his nausea and vomiting stopped . In view of persistent vertigo and ataxia, complete neurological evaluation was performed however, no significant abnormality was found except for vertigo and ataxia with tendency to fall on right side . It was decided to admit the patient in the ward for further investigation, observation and management . Computerized tomography (ct) scan brain was performed to rule out any central cause of ataxia, which was found to be normal . Patient slept well in the night and was reassessed the next morning . By this time, patient had improved significantly and was not having vertigo, ataxia or nausea and vomiting . As the patient`s vertigo and ataxia had significantly improved and he was able to walk normally, he was discharged from the hospital with advice to come for regular follow - up after one week . The patient was found to be pain free with slight hypesthesia in the right mandibular distribution . Usual complications of mandibular nerve block are hypoesthesia, dysesthesia, sensory loss in mandibular nerve distribution, facial swelling and masseter muscle weakness . Accidental injuries to the auditory tube while trying to locate or block the mandibular nerve are possible, as it is in close proximity . If this occurs during or after the test injection following local anesthetic, one should defer the injection of neurolytic agent . Diagnostic test injection with 3 ml of 2% lignocaine was uneventful and there was no resistance to injection or symptom of auditory tube irritation, i.e. Pain in ear, deafness etc . Patient developed severe vertigo and ataxia five minutes after the aa injection when he was allowed to sit - up, stand and walk . He also developed hypertension (blood pressure-210/110 mmhg) and two episodes of severe nausea and vomiting . Reported the first and only case report of vertigo during mandibular nerve block with aa . They attributed this to multiple factors: firstly their patient had received multiple neurolytic blocks before, which probably had resulted in tissue inflammation and adhesion formation; secondly, the patient had pain in the ear during the advancement of the needle and there was high resistance to aa injection, indicating injury to auditory tube; and lastly, their patient also developed prolonged horizontal nystagmus along with vertigo . The clinical manifestations in our patient were however different as he had not received any neurolytic block before, had no pain during injection and there was no resistance during injection of either lignocaine or aa . We used a 22 g, 9 cm sharp tip spinal needle for the block, due to the non availability of blunt tip needle . We may have inadvertently injured the cartilaginous part of auditory tube with the block needle and a small amount of aa might have entered the auditory canal and reached the vestibular apparatus resulting in vertigo and ataxia . In patients who receive repeated neurolytic mandibular nerve blocks, abnormal degeneration in the tissues surrounding the nerve should be considered and all agents should be injected precisely and slowly . Whenever there is a suspicion of injury to auditory canal, it is advisable to defer neurolytic blockade with aa . Patients should be closely observed for at least an hour after the block when aa is used because stimulation of the vestibular apparatus may be delayed . In the case reported, we did not observe any pain or resistance to injection to lignocaine or alcohol which may have alarmed us about the complication . Vertebro basilar ischemia or posterior circulation stroke may present as vertigo, ataxia, diplopia, dysartheria and weakness of one or both side of body . Our patient was 65 years old and developed vertigo, ataxia and hypertension after the neurolytic mandibular block . Our patient did not have nystagmus, diplopia, and dysartheria and was not a known hypertensive . Features favoring vestibular ataxia, and not the central cerebellar ataxia included: presence of severe vertigo, associated with body movement; presence of severe nausea and vomiting; severe ataxia with tendency to fall toward affected side; and increase in imbalance while walking with closed eyes . Neurology consultation was taken and ct scan brain was performed to rule out any possibility of stroke which was found to be normal . We have presented a patient of trigeminal neuralgia, who developed prolonged severe vertigo, ataxia and hypertension after the mandibular nerve block with aa, which gradually improved over 24 hours . We emphasize that in trigeminal neuralgia patients, who usually require repeated neurolytic mandibular block, the possibility of severe vertigo and ataxia should be kept in mind, as it may be associated with increased risk and complications, requiring prolonged observation and management . Use of guided techniques (nerve locator, fluoroscopy, ultrasound or ct guidance) can be considered in some selected patients.
Most ingested foreign bodies pass through the gastrointestinal tract spontaneously without necessitating any treatment; however, about 20% require endoscopic or surgical removal1). Most swallowed toothbrushes have been found in the esophagus or the stomach of the affected patient, and there has been no previously reported case of a toothbrush in the colon . Here, we report a case of a swallowed toothbrush found in the ascending colon with a complicating small hepatic abscess, which was successfully managed with laparotomy . A 31-year - old man presented with a one - week history of intermittent right upper abdominal pain . He saw his local doctor when symptoms began, and was prescribed ranitidine and antacid for dyspepsia with no improvement . He had been treated for 13 years due to a diagnosis of schizophrenia . On examination,, his blood pressure was 110/70 mmhg, and he was not jaundiced or anemic . The coagulation profile was abnormal with a prothrombin time (international normalized ratio) of 1.4 and an activated partial thromboplastin time of 40 s. an electrocardiogram was unremarkable . An abdominal radiograph showed parallel rows of short metallic radiodensities in the right mid - abdomen (figure 1). On ct images, a metallic foreign body was observed in the right colon and a thrombosis of the left portal vein was found to be associated with perfusion changes in the left hepatic lobe, suggesting a chronic intra - abdominal inflammation (figure 2). Colonoscopic examination revealed a toothbrush penetrating the wall of the hepatic flexure with the head pointing toward the ascending colon (figure 3). We attempted to remove the toothbrush after grasping it with a snare; this attempt failed due to limited space in the hepatic flexure and risk of trauma to extraluminal organs or vessels . The patient was subsequently taken to the operating room for an exploratory laparotomy . During laparotomy, the toothbrush was found to have caused a fistula between the hepatic flexure of the colon and the liver with a complicating small hepatic abscess . The toothbrush at 19 cm in length was removed, the perforation in the colon was resected in a small wedge, and anastomosis was performed . The peritoneal cavity was irrigated with normal saline, and a 10-fr drain tube was placed in the abscess cavity site . Upon further questioning, the patient reported ingesting a toothbrush approximately one year earlier . At that time the toothbrush is a rather unusual foreign body to be found in the gastrointestinal tract . In a recent report, a medline search of the years 1988 to 2000 found 11 articles with approximately 40 cases of toothbrush ingestion2). Almost all of the patients were female, ranging from 15 to 23 years of age3, 4). Most of the patients had been diagnosed with psychiatric problems such as bulimia or anorexia nervosa . The characteristic radiographic image of a swallowed toothbrush shows parallel rows of short metallic radiodensities due to the metallic plates that hold the bristles in place4). The toothbrushes were easily identified in the esophagus and stomach of the patients by upper endoscopy . In several cases, there were significant complications related to pressure necrosis, including gastritis, mucosal tears, and perforation3). Consequently, prompt removal is recommended to prevent the development of complications . In 1983, other cases have reported failure of endoscopic removal due to the size or shape of the toothbrush6, 7). In addition, cases of esophageal perforation during endoscopic toothbrush extraction8) and of laparoscopic - assisted toothbrush extraction via gastrostomy after an unsuccessful attempt at endoscopic removal7) have been reported . However, surgical removal is often required due to the geometric qualities of the toothbrush . All of the swallowed toothbrushes were found in the esophagus or the stomach of the affected patient, and there has been no previously reported case of a toothbrush in the colon . To our knowledge, this is the first documented case of a swallowed toothbrush found in the colon and persisting in the gastrointestinal tract for approximately one year . For a small foreign body penetrating the wall of the colon with no sign of peritoneal irritation, it may be possible to remove the foreign body and repair the wound endoscopically . In our case, we found the endoscopic approach unsuccessful due to the size of the toothbrush and the narrow space of the hepatic flexure . There was also a complicating abscess and a risk of trauma to extraluminal organs or vessels during endoscopic removal . The case presented demonstrates a very unusual complication of toothbrush ingestion, unlike any that have been reported previously . Thus, a typical finding of an unusual foreign body in the colon, especially in patients with psychiatric problems, should make the attending physician consider the possibility of a swallowed toothbrush . Because no cases of spontaneous passage have been reported, prompt removal
Lichen myxedematosus (lm) is a primary mucinosis that is not accompanied by thyroid disease . In the updated classification of lm given by rongioletti et al . [1, 2], the nodular type is one subset of lm that is characterized by multiple nodules on the limbs and trunk with mild or absent papular eruption . Herein, we report a case of nodular - type lm for which local injection of corticosteroids was effective . An 18-year - old japanese woman noticed a progressive appearance of nodules on both forearms and on the left cubital fossa and left thigh one year before her initial consultation at our department for further evaluation in 2008 . Physical examination revealed elastic hard, slightly elevated, shiny and yellowish to skin - colored nodules of 6 to 10 mm in size on her extremities without confluence into plaque . 1), three on the left forearm, four on the left cubital fossa, and two on the left thigh . The nodules were not itchy . Laboratory findings for liver function, free t3, free t4, thyroid - stimulating hormone, fasting blood sugar, and hba1c were all within normal limits . Xanthoma and dermatofibroma were suspected based on the clinical appearance, and one nodule on the right forearm was taken for skin biopsy . Histopathologically, collagen fibers were separated in the reticular to deep dermis without epidermal changes (fig . Basophilic mucin deposits between collagen fibers with proliferation of fibroblasts and infiltration of lymphocytes and plasmacytes around vessels were seen in the dermis . Histochemically, the mucin was positive for alcian blue and negative after hyaluronidase digestion (fig treatment with betamethasone dipropionate ointment was ineffective, but one nodule was completely eliminated by local injection of 0.02 ml triamcinolone acetonide (8 mg / ml). Since triamcinolone acetonide was subsequently recalled and was unavailable for a period, we tried local injections of 0.1 mg prednisolone sodium succinate three times for each nodule . After triamcinolone acetonide reappeared in the japanese market, local injection of 0.02 to 0.05 ml triamcinolone acetonide was performed and another three injected nodules were completely eliminated . Lm (papular mucinosis) is a disorder characterized by lichenoid papules, nodules and/or plaques due to dermal mucin deposition, and a variable degree of fibrosis without thyroid dysfunction . In an updated classification [1, 2], lm is divided into the following clinicopathologic subsets: (1) a generalized papular and sclerodermoid form (scleromyxedema) with monoclonal gammopathy and systemic manifestations that may be lethal . Histologically, the skin shows a microscopic triad of mucin deposition, fibroblast proliferation, and fibrosis; (2) localized lm, which is a papular or nodular / plaque eruption without sclerotic features, monoclonal gammopathy, or systemic involvement; here, the skin shows mucin deposition with variable fibroblast proliferation; and (3) atypical forms, which have atypical features or features intermediate between scleromyxedema and localized lm . Localized lm is further subdivided into the following four subtypes: (1) discrete lm, which has firm, smooth, waxy or flesh - colored papules of 2 to 5 mm in size, numbering a few to hundreds on the limbs and trunk in a symmetric pattern . The papules are isolated or form confluent nodules or plaque and rarely resolve spontaneously; (2) acral persistent papular mucinosis, which is characterized by a few to multiple, ivory to flesh - colored papules of 2 to 5 mm in size on the back of the hands, wrists, and occasionally the distal aspect of the forearms . Papules persist or increase without spontaneous resolution and occur predominantly in females; (3) cutaneous mucinosis of infancy, characterized by firm, opalescent papules located on the upper arms, especially the elbows, and the trunk without spontaneous resolution, and (4) nodular - type lm, characterized by multiple nodules on the limbs and trunk, with a mild or absent papular component . Our case was diagnosed as nodular - type lm based on the clinical and histological features . The patient had 8 nodules of 6 to 10 mm in size on the legs and forearms without a symmetric pattern, whereas papules on the limbs and trunk more commonly occur in a symmetric pattern in discrete lm, and occur on the back of the hands and extensor surface of the distal forearms in acral persistent papular mucinosis . Histological findings, such as mucin deposition with variable fibroblast proliferation and the absence of monoclonal gammopathy and m protein, provided further reasons for the diagnosis of nodular - type lm . Only three previous cases [4, 5] characterized by nodular - type lm have been reported, including one from japan . The mean onset age of the four reported patients with nodular - type lm, including our case, was 30.5 years (range 1848 years), which suggests that this subtype appears at a younger age compared to discrete lm and acral persistent papular mucinosis . Moreover, three of the four cases were male (table 1). A standard treatment for localized lm is not described in the literature, but in our case nodules showed complete remission following local injection of triamcinolone acetonide, which suggests that this drug may be of some benefit for this condition.
Although the conventional surgical treatment is still the most utilized and effective treatment for colorectal malignancy, the procedure may lead to prolonged hospital stay, increased medical costs, and medical resource overuse due to the aggressiveness of the approach and the high risk of developing postoperative nutrition disorders . Patients are often dissatisfied with their functional recovery and the incidence of complications is 20 to 30% [1, 2]. Although the incidence of conventional complications such as anastomotic leakage has decreased with the improvements of surgical instruments and techniques, postoperative psychiatric complications such as delirium are frequent, particularly in the elderly (70 years). As reported previously, about 1050% of advanced - age patients undergoing surgical treatment may develop delirium postoperatively [3 - 5]. Postoperative delirium is a reversible and fluctuating acute brain syndrome characterized by changes in consciousness, orientation, attention, memory, sensory perception, thinking, emotion, and volition . The occurrence of this condition may lead to prolonged hospital stay and unfavorable prognosis, and may develop into chronic brain syndrome (dementia). Although the mechanism of delirium remains unclear, it has been clearly demonstrated that multiple factors are involved [7, 8]. Besides advanced age, a recent study showed that systemic stress and inflammatory response might play an important role in the development of this condition [9, 10]. It has been reported that the serum levels of an inflammatory biomarkers, including interleukin-6 (il-6), were positively correlated to the incidence of delirium . Moreover, therefore, reduction of the perioperative stress and inflammatory response may minimize the occurrence of delirium . It has been found in several clinical trials that fast - track surgery (fts) not only facilitates the physical rehabilitation of the patients with colorectal malignancies, but also prevents upregulation of proinflammatory cytokines including il-6, with reduced stress response and inflammation [13, 14]. Moreover, krenk et al . Have shown that delirium was not observed in fast - track hip and knee arthroplasty in elderly patients . However, there is little data on whether fts can prevent or protect elderly patients with colorectal carcinoma from developing delirium after colorectal surgery . In the present randomized trial, we studied whether fts could prevent or reduce the occurrence of postoperative delirium as well as other complications in elderly patients with colorectal carcinoma and evaluated the role of il-6 in postoperative delirium . A total of 240 elderly patients with colorectal carcinoma, with ages ranging from 70 to 88 years (mean, 75.18 years; 150 men and 90 women), admitted to the fourth hospital of hebei medical university for open curative resection between 2008 and 2011, were included in the present study . These patients were randomly assigned into the traditional therapy group (n = 120) and the fts group (n = 120). Eligible patients were randomly assigned to each group in a 1:1 ratio (fig . 1flow diagram of study flow diagram of study patients with a history of dementia, parkinson s disease, alcohol intake of 250 g / day, long - term use of sleeping pills or anxiolytics, and those who received anesthesia within the past 30 days were excluded from the study during the initial screening . Those enrolled patients who were subsequently given intraoperative blood transfusion or were admitted to the intensive care unit (icu) for further treatment after operation were also excluded from analysis (fig . 1). Of the 240 study participants, 115 were diagnosed with colon cancer and 125 with rectal cancer . Patients in the two groups had comparable baseline characteristics including gender, age, site of lesion, tnm classification of malignant tumours (tnm staging), and surgical procedure (table 1). In the current study, a preoperative routine cranial magnetic resonance imaging (mri) scan was performed for all the patients in both groups, and a repeat mri scan performed for those who developed postoperative delirium to exclude cerebrovascular stroke or other central nervous system (cns) conditions as the etiology of delirium.table 1clinical data of the fast track surgery (fts) and traditional therapy groups (n = 233)characteristicsfts (n = 117)traditional (n = 116) p valueage75.66 4.1874.78 4.010.054gender male76700.467 female4146site of lesions colon57580.845 rectum6058tnm staging i13190.633 ii5247 iii3738 iv1512surgical operation colectomy52530.930 dixon3936 miles2627hypertension26330.275diabetes11140.511 clinical data of the fast track surgery (fts) and traditional therapy groups (n = 233) this study was approved by the ethics committee of the fourth hospital of hebei medical university . The length of hospital stay (los) and time to pass flatus were documented . The incidence of complications including pulmonary infection, urinary tract infection, intestinal obstruction, anastomotic leakage, heart failure, and deep venous thrombosis (dvt) were also monitored and documented . All patients were followed up and all parameters were obtained from all patients . The diagnosis of postoperative delirium as the focus of this study is described below . The perioperative managements for patients of both groups including preoperative preparation, anesthesia, pain control, and postoperative managements are compared and summarized in table 2 . The management of the fts group differed from the traditionally managed group in the several ways: (1) bowel preparation with oral purgatives instead of a mechanical enema; (2) thoracic epidural anesthesia and postoperative analgesic maintenance via the epidural catheter (ropivacaine, 2 mg / ml maintained for 48 h, controlled to 610 ml (1220 mg) per hour and opium - derived agents were excluded); (3) no nasogastric tube insertion; (4) no drainage tube placement with the exception of low rectal anastomosis; (5) water was allowed from 6 h postoperation, liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days (pod) with regular diet on pod 3; (6) early urine catheter withdrawal (at pod 12); and (7) early out - of - bed mobilization (i.e., walking).table 2comparison of fast - track and traditional perioperative care protocolstraditionalfast trackpreoperative preparationliquid diet for 3 daysoral purgativesmechanical enema(1time / day) for 3 consecutive daysno mechanical enemafasting at 8 hnormal meal until 6 h before surgerydrink deprivation 4 h before surgerynormal carbohydrate drink until 2 h before surgeryroutine nasogastric tube insertionno nasogastric tube insertionoral antibiotics administration for 3 daysno antibioticsanesthesiageneralthoracic epiduralpain controlfentanyl0.25 mg / mlropivacaine2mg / mlmidazolam0 . 5 mg / mlnefopam1.0 mg / mlvia pceavia pciafor 48 hfor 48 hopium - derived agents were excludedroutine drainage tube placementno routine drainage tube placementpostoperative managementdiet: liquid diet intake after recovery of bowel movementdiet: water was allowed from 6 h postoperation, liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days, regular diet on pod 3urinary catheter withdrawal at 3 to 5 daysurinary catheter withdrawal on pod 12out - of - bed mobilization at 3 to 5 daysout - of - bed mobilization on pod 1 comparison of fast - track and traditional perioperative care protocols the mental status and cognitive function was evaluated in accordance with the delirium rating scale - revised-98 (drs - r-98). The drs - r-98 evaluates the cognitive domain of delirium by recourse to specific evaluations for attention, orientation, short - term memory, long - term memory, and visuospatial ability . Drs - r-98 has a high sensitivity (91100%) and specificity (85100%) for detection of delirium . The drs - r-98 scoring was performed on the day of admission and then daily from pod 1 for 5 days . The presence or absence of delirium was evaluated by a psychiatrist and a nurse based on the criteria specified in drs - r-98 . Fasting peripheral venous blood samples (5 ml) were collected 1 day preoperatively and on pod 1, 2, and 3 . The blood samples were centrifuged at 3,000 rpm for 5 min, and the sera obtained were preserved at 20c for later use . The serum il-6 levels were determined using sandwich elisa with the reagents purchased from invitrogen, camarillo, ca, usa . The results are expressed as mean sd . A total of 240 elderly patients with colorectal carcinoma, with ages ranging from 70 to 88 years (mean, 75.18 years; 150 men and 90 women), admitted to the fourth hospital of hebei medical university for open curative resection between 2008 and 2011, were included in the present study . These patients were randomly assigned into the traditional therapy group (n = 120) and the fts group (n = 120). Eligible patients were randomly assigned to each group in a 1:1 ratio (fig . 1flow diagram of study flow diagram of study patients with a history of dementia, parkinson s disease, alcohol intake of 250 g / day, long - term use of sleeping pills or anxiolytics, and those who received anesthesia within the past 30 days were excluded from the study during the initial screening . Those enrolled patients who were subsequently given intraoperative blood transfusion or were admitted to the intensive care unit (icu) for further treatment after operation were also excluded from analysis (fig . 1). Of the 240 study participants, 115 were diagnosed with colon cancer and 125 with rectal cancer . Patients in the two groups had comparable baseline characteristics including gender, age, site of lesion, tnm classification of malignant tumours (tnm staging), and surgical procedure (table 1). In the current study, a preoperative routine cranial magnetic resonance imaging (mri) scan was performed for all the patients in both groups, and a repeat mri scan performed for those who developed postoperative delirium to exclude cerebrovascular stroke or other central nervous system (cns) conditions as the etiology of delirium.table 1clinical data of the fast track surgery (fts) and traditional therapy groups (n = 233)characteristicsfts (n = 117)traditional (n = 116) p valueage75.66 4.1874.78 4.010.054gender male76700.467 female4146site of lesions colon57580.845 rectum6058tnm staging i13190.633 ii5247 iii3738 iv1512surgical operation colectomy52530.930 dixon3936 miles2627hypertension26330.275diabetes11140.511 clinical data of the fast track surgery (fts) and traditional therapy groups (n = 233) this study was approved by the ethics committee of the fourth hospital of hebei medical university . The length of hospital stay (los) and time to pass flatus were documented . The incidence of complications including pulmonary infection, urinary tract infection, intestinal obstruction, anastomotic leakage, heart failure, and deep venous thrombosis (dvt) were also monitored and documented . All patients were followed up and all parameters were obtained from all patients . The diagnosis of postoperative delirium as the focus of this study is described below . The perioperative managements for patients of both groups including preoperative preparation, anesthesia, pain control, and postoperative managements are compared and summarized in table 2 . The management of the fts group differed from the traditionally managed group in the several ways: (1) bowel preparation with oral purgatives instead of a mechanical enema; (2) thoracic epidural anesthesia and postoperative analgesic maintenance via the epidural catheter (ropivacaine, 2 mg / ml maintained for 48 h, controlled to 610 ml (1220 mg) per hour and opium - derived agents were excluded); (3) no nasogastric tube insertion; (4) no drainage tube placement with the exception of low rectal anastomosis; (5) water was allowed from 6 h postoperation, liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days (pod) with regular diet on pod 3; (6) early urine catheter withdrawal (at pod 12); and (7) early out - of - bed mobilization (i.e., walking).table 2comparison of fast - track and traditional perioperative care protocolstraditionalfast trackpreoperative preparationliquid diet for 3 daysoral purgativesmechanical enema(1time / day) for 3 consecutive daysno mechanical enemafasting at 8 hnormal meal until 6 h before surgerydrink deprivation 4 h before surgerynormal carbohydrate drink until 2 h before surgeryroutine nasogastric tube insertionno nasogastric tube insertionoral antibiotics administration for 3 daysno antibioticsanesthesiageneralthoracic epiduralpain controlfentanyl0.25 mg / mlropivacaine2mg / mlmidazolam0 . 5 mg / mlnefopam1.0 mg / mlvia pceavia pciafor 48 hfor 48 hopium - derived agents were excludedroutine drainage tube placementno routine drainage tube placementpostoperative managementdiet: liquid diet intake after recovery of bowel movementdiet: water was allowed from 6 h postoperation, liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days, regular diet on pod 3urinary catheter withdrawal at 3 to 5 daysurinary catheter withdrawal on pod 12out - of - bed mobilization at 3 to 5 daysout - of - bed mobilization on pod 1 comparison of fast - track and traditional perioperative care protocols the mental status and cognitive function was evaluated in accordance with the delirium rating scale - revised-98 (drs - r-98). The drs - r-98 evaluates the cognitive domain of delirium by recourse to specific evaluations for attention, orientation, short - term memory, long - term memory, and visuospatial ability . Drs - r-98 has a high sensitivity (91100%) and specificity (85100%) for detection of delirium . The drs - r-98 scoring was performed on the day of admission and then daily from pod 1 for 5 days . The presence or absence of delirium was evaluated by a psychiatrist and a nurse based on the criteria specified in drs - r-98 . Fasting peripheral venous blood samples (5 ml) were collected 1 day preoperatively and on pod 1, 2, and 3 . The blood samples were centrifuged at 3,000 rpm for 5 min, and the sera obtained were preserved at 20c for later use . The serum il-6 levels were determined using sandwich elisa with the reagents purchased from invitrogen, camarillo, ca, usa . The results are expressed as mean sd . The measurement data are represented as mean sd . The intergroup comparison was performed using the wilcoxon rank - sum test . The age, gender, site of lesions, tnm staging, surgical procedures, and co - morbidity including hypertension and diabetes between the two groups were comparable, without statistically significant differences (table 1). Four patients were excluded from the traditional group: two patients received an intraoperative blood transfusion and the other two were admitted to icu because of pulmonary infection . Three patients were excluded from the fts group: one received an intraoperative blood transfusion, and the other two were admitted to icu because of pulmonary infection . Thus, a total of 233 patients including 116 patients in the traditional group and 117 patients in the fts group were included this study (fig . 1). The mean los of the fts group and traditional therapy group was 9.01 1.75 and 13.21 1.32 days, respectively (p <0.001). The time to pass flatus in the fts group was significantly shorter than in the traditional therapy group (48.50 9.59 vs. 77.66 7.18 h; p <0.001). On the pod 1, the level of serum albumin in the fts group was higher than that in the traditional therapy group (28.05 2.82 vs. 26.26 4.12; p <0.001). Meanwhile, the glucose in the fts group was lower than that in the traditional therapy group (8.30 2.49 vs. 10.25 2.43; p <0.001). No significant difference of liver or renal function was observed between the two groups after operation (table 3).table 3comparison of postoperative recovery and complications between the fts and traditional groupfts (117)traditional (116) p valuelos (day)9.01 1.7513.21 1.32<0.001functional recovery time to pass flatus (h)48.50 9.5977.66 7.18<0.001 serum albumin (g / l)28.05 2.8226.26 4.12<0.001 glucose (mmol / l)8.30 2.4910.25 2.43<0.001 alt (iu / l)34.65 12.2534.88 11.820.738 ast (iu / l)30.43 10.7829.47 10.400.356 cr (mol / l)77.05 23.8075.11 25.040.675 bun (mmol / l)5.63 3.605.62 3.080.831complications (cases) infection of incision680.570 pulmonary infection6190.006 urinary infection5130.047 anastomotic leakage321.000 intestinal obstruction460.736 heart failure4130.022 dvt470.340 alt alanine transaminase, ast aspartate transaminase comparison of postoperative recovery and complications between the fts and traditional group alt alanine transaminase, ast aspartate transaminase the postoperative complications were followed and documented based on clavien dindo classification system, including infection, intestinal obstruction, anastomotic leakage, heart failure, and dvt . Six pulmonary infections were observed in the fts group, significantly fewer than the 19 cases in the traditional therapy group (p = 0.006). The incidence of urinary infections was lower in fts group than that in the traditional therapy group (5 vs. 13; p = 0.047). The incidence of heart failure was much higher in the traditional therapy group than that in the fts group (13 vs. 4, p = 0.022). However, no statistical differences were found in the incidences of incision infection, bowel obstruction, anastomotic leakage, and dvt between the two groups . The results are summarized in table 3 . Using the drs - r-98 scoring system, a total 19 cases of postoperative delirium were observed, 15 in the traditional therapy group (12.9%) and 4 in the fts group (3.4%, p = 0.008) (table 4). In the traditional therapy group, nine incidents of delirium were observed at pod 1, five at pod 2, and one at pod 3, while for the fts group, three incidents of delirium were observed at pod 1 and one at pod 2 (table 4).table 4incidence of postoperative delirium in the fts and traditional grouppodftstraditional p valuepod 1390.073pod 2150.211pod 3010.498total (% of analyzed cases)4 (3.4%) 15 (12.9%) 0.008 incidence of postoperative delirium in the fts and traditional group repeat mri scanning for those with postoperative delirium did not show any cns changes (data not shown). This indicates that postoperative delirium was not due to apparent organic cns involvements, e.g., cerebrovascular stroke, after the surgery . The treatments for delirium included intensive nursing care, and for, severe cases, haloperidol (0.5 mg) was intramuscularly administered and repeated if necessary at an interval of 30 to 60 min . The delirium - related symptoms resolved in all 19 patients after the treatments . As proinflammatory cytokines particularly il-6 have been reported to be involved in the development of postoperative delirium [10, 19 - 21], we determined the serum il-6 levels in both the fts and the traditional groups . The preoperative baseline il-6 levels in both groups were very similar . For both group, the serum il-6 level peaked at pod 1, followed by a rapid decline thereafter . This il-6 peak correlated with the highest incidence of delirium on pod 1 (fig . 2). Although the il-6 level in fst group decreased to approximately the baseline level by pod 3, the il-6 level of the traditional group was still well above the baseline level (fig . 2) the serum il-6 levels in the traditional therapy group at pod 1, 2, and 3 were all significantly higher than those in the fts group (p <0.001 in all 3 pod days). Together, these results showed that enhanced il-6 level correlated with the development of postoperative delirium in the elderly patients undergoing colorectal surgery.fig . 2serum il-6 levels in the fts group and traditional group . * * p <pre preoperation, pod postoperative day serum il-6 levels in the fts group and traditional group . * the postoperative complications were followed and documented based on clavien dindo classification system, including infection, intestinal obstruction, anastomotic leakage, heart failure, and dvt . Six pulmonary infections were observed in the fts group, significantly fewer than the 19 cases in the traditional therapy group (p = 0.006). The incidence of urinary infections was lower in fts group than that in the traditional therapy group (5 vs. 13; p = 0.047). The incidence of heart failure was much higher in the traditional therapy group than that in the fts group (13 vs. 4, p = 0.022). However, no statistical differences were found in the incidences of incision infection, bowel obstruction, anastomotic leakage, and dvt between the two groups . Using the drs - r-98 scoring system, a total 19 cases of postoperative delirium were observed, 15 in the traditional therapy group (12.9%) and 4 in the fts group (3.4%, p = 0.008) (table 4). In the traditional therapy group, nine incidents of delirium were observed at pod 1, five at pod 2, and one at pod 3, while for the fts group, three incidents of delirium were observed at pod 1 and one at pod 2 (table 4).table 4incidence of postoperative delirium in the fts and traditional grouppodftstraditional p valuepod 1390.073pod 2150.211pod 3010.498total (% of analyzed cases)4 (3.4%) 15 (12.9%) 0.008 incidence of postoperative delirium in the fts and traditional group repeat mri scanning for those with postoperative delirium did not show any cns changes (data not shown). This indicates that postoperative delirium was not due to apparent organic cns involvements, e.g., cerebrovascular stroke, after the surgery . The treatments for delirium included intensive nursing care, and for, severe cases, haloperidol (0.5 mg) was intramuscularly administered and repeated if necessary at an interval of 30 to 60 min . As proinflammatory cytokines particularly il-6 have been reported to be involved in the development of postoperative delirium [10, 19 - 21], we determined the serum il-6 levels in both the fts and the traditional groups . The preoperative baseline il-6 levels in both groups were very similar . For both group, the serum il-6 level peaked at pod 1, followed by a rapid decline thereafter . This il-6 peak correlated with the highest incidence of delirium on pod 1 (fig . 2). Although the il-6 level in fst group decreased to approximately the baseline level by pod 3, the il-6 level of the traditional group was still well above the baseline level (fig . 2) the serum il-6 levels in the traditional therapy group at pod 1, 2, and 3 were all significantly higher than those in the fts group (p <0.001 in all 3 pod days). Together, these results showed that enhanced il-6 level correlated with the development of postoperative delirium in the elderly patients undergoing colorectal surgery.fig . 2serum il-6 levels in the fts group and traditional group . * * p <0.001 traditional vs. fts group . Pre preoperation, pod postoperative day serum il-6 levels in the fts group and traditional group . * in the present randomized trial, patients of advanced age (70 years) with colorectal carcinoma were treated with either fts (117 patients) or traditional approaches (116 patients). The patients managed with fts had significant shorter los and fast recovery of bowel movement (table 3). Importantly, there was a decrease in the incidence of postoperative complications including pulmonary infection, urinary infection, and heart failure (table 3), which is consistent with previous reports [22, 23]. Notably, fts treatment was also associated with a decreased frequency of postoperative delirium (table 4). Fts did not cause the same degree of increase in serum level of the proinflammatory cytokine il-6 (fig . 2). Delirium is an acute brain syndrome most commonly seen in the elderly, particularly in those with underlying organic brain pathology and/or functional impairments . The incidence of postoperative delirium varies depending on the surgical and anesthetic approaches used and ranges from 10 to 24% in gastrointestinal surgery [4, 5, 25, 26]. Predisposing factors include age; comorbid disease; and cognitive, visual, and hearing disorders [7, 8, 27, 28]. In our trial, all the patients in both groups were of advanced age (70 years) and were more prone to developing delirium after surgery . The precipitating factors include the type and extensiveness of the surgical procedure, anesthetic protocol, opioid pain killer usage, inflammatory response, infection, pain, sleep disturbance, nutritional condition, and electrolyte homeostasis [29, 30]. Since the highest frequency of delirium occurred early on pod 1, we believe that preoperative preparation, surgical approach, anesthesia, and pain control protocols were among the most important factors, although other subsequent complications (e.g., postoperative infection) might also play a role most likely later during the course . In our study, patients in both groups underwent similar open colorectal surgery (table 1), the extensiveness of the surgery and trauma incurred in both groups were similar and unlikely to be a contributing factor . Pain and sleep disturbance reported in both groups were comparable (data not shown), so they were not a likely explanation for the discrepancy of incidence of delirium between the two groups . It has been reported that early feeding in open colon resection in the elderly resulted in shorter los and reduced postoperative morbidity, so it is possible that early feeding in the fst group might be contributing to the overall lower postoperative complications including delirium . The two groups received different preoperative preparation (table 2). In the traditional group, mechanical enema and nasogastric tube insertion were applied, while in the fts group, only oral purgatives was given and no nasogastric tube was used . It is conceivable that these less - invasive procedures used in the fts group could potentially decrease the stress response, and be a factor in the lower occurrence of postoperative delirium . A major difference in the management of patients in the two groups was the different anesthesia approach, pain control, and drug selection (table 2). It is unclear whether general anesthesia per se might play a role in postoperative delirium, but it was possible that it might cause more extensive systematic stress response than epidural anesthesia applied in the fts group [32, 33]. In addition to anesthesia methods, intraoperative and postoperative medication may affect the mental status of patients [34, 35]. For patients with traditional therapy, opioid drugs including morphine, dolantin, and fentanyl that were used during general anesthesia and postoperative pain management might contribute to the occurrence of delirium . Epidural anesthesia and analgesia may block the sympathetic response that has been reported to be involved in delirium . The analgesic agent used in fts group was ropivacaine, which can alleviate moderate to severe postoperative pains effectively and safely . Together, these measures can help reduce the various stimuli to the patients during surgical injury, minimize postoperative inflammatory reaction, and facilitate functional recovery . Therefore, integrated measures should be applied to prevent elderly colorectal patients from developing postoperative delirium . Excessive release of proinflammatory cytokines such as tnf-, il-1, il-6, and il-8 during systemic inflammation can affect brain functions and promote the development of delirium . To investigate whether fts reduced the incidence of delirium by minimizing the inflammatory response, serum il-6 levels were also determined . We found that enhanced il-6 level correlated with the development of postoperative delirium and peaked on pod 1 in the elderly patients undergoing colorectal surgery (fig . 2 and table 4). Fts significantly reduced the level of il-6 increase as compared to the traditional therapy group (fig . 2). This is consistent with the previous reports that elderly patients who develop postoperative delirium may exhibit an elevated serum il-6 level [39, 40]. It is well known that il-6 is an endogenous cytokine released by monocytes, t cells, and vascular endothelial cells and is a major inducer of the inflammation . Il-6 can promote the differentiation of lymphocytes and amplify the inflammatory response leading to tissue damage . In this study, there was a correlation between elevated levels of il-6 and delirium, both of which were present at reduced levels in the fts treatment group . Less invasive preoperative preparation (no mechanic enema, no nasogastric tube insertion, etc . ), use of epidural instead of general anesthesia, and avoidance of opioids for anesthesia and pain control could all contribute to the reduced level of il-6 in the fts group . It has been reported that opioids can stimulate il-6 production in both animal and human studies [43 - 47], so anesthesia and analgesia without utilizing opioid drugs may be an important factor for reduced il-6 levels and reduced postoperative delirium in patients managed with fts . Although we only examined il-6 level in the peripheral blood, which may not directly represent the intracerebral conditions accurately, however, it has been shown that il-6 can readily cross the blood fts shortens the los, facilitates the recovery of bowel movement, and reduces occurrence of postoperative delirium and other complications in elderly patients with colorectal carcinoma . The lower incidence of delirium in the fts group is likely attributable to the reduced systemic stress and inflammatory response mediated by il-6.
Invasive species can be expensive pests, causing changes to local ecosystems that can interfere with ecosystem services and extirpate native organisms (mack et al ., 2000, sakai et al ., 2001). Parasite relationships that are important to ecosystem productivity, stability, and biodiversity (anderson and may, 1978). Host parasite dynamics within the native populations can be altered by the introduction of nonindigenous parasites because native host populations might lack the defenses necessary to combat the introduced pathogens (bar - david et al ., 2006). Likewise, increases in the abundance of native parasites can occur if populations of the introduced host are capable of being suitable hosts and/or reservoirs for parasites of native species (hudson et al . . Given that invasives can alter native species diversity via their introduced parasites (strauss et al ., 2006), it is important to investigate the parasite host interactions when areas become invaded (peeler et al . The cuban treefrog (ctf), osteopilus septentrionalis, is an invasive amphibian species that is ideally suited to address any such loss or new acquisition of parasites because they are extremely abundant and easily captured . The ctf is an invasive anuran native to cuba, the cayman islands, island de pinos, and the bahamas . It was introduced to puerto rico, various islands in the lesser antilles, alabama, georgia, maryland, minnesota, hawaii, and florida (schwartz and henderson, 1991, meshaka, 2001, johnson, 2004). The original introduction of ctfs into florida is believed to have occurred in the keys and spread throughout florida as a result of highway construction (barbour, 1931, meshaka, 2001, krysko et al ., 2011). Because o. septentrionalis is often larger than the florida - native treefrogs, has a broad dietary niche (meshaka, 1996), and can reproduce throughout the year with average clutch sizes of 20004000 eggs, although ctfs have inhabited florida for nearly 100 years, we are unaware of any published surveys of its parasites in this introduced range . Thus, we quantified macroparasites in ctfs from a natural area near tampa, florida, usa where the ctfs have been established since 1992 (campbell et al ., 2010) and compared these parasites to those previously reported in the ctf from its native range, as well as the parasites of native anurans . We hypothesized that ctfs from tampa harbor parasite species from their native range and that an exchange of parasites between native hosts and ctfs has resulted in ctfs acquiring new parasite species . During the summers of 20052008, 330 ctfs were collected from polyvinyl chloride (pvc) pipes encircling 37 wetlands spanning 4000 acres within or near the morris bridge wellfield (mbwf) within the flatwoods wilderness park in northeastern hillsborough county, florida (280701.08n 821811.15w). The mbwf is primarily second - growth pine flatwoods forest matrix with numerous borrow pits, hardwood swamps, freshwater marshes, and cypress domes (guzy et al ., 2006, host snout - vent length (svl) and wet weight were recorded . For hosts> 41 mm, sex was determined by the presence / absence of nuptial pads and evidence of mature reproductive organs . All hosts <42 mm were considered juveniles due to a lack of discernable reproductive organs . The body cavity was opened by a longitudinal incision from vent to throat and all internal body organs were examined for macroparasites . Species of parasites were counted and preserved in 70% ethanol . Identification and confirmation of parasites were provided by charles bursey at pennsylvania state university's shenango campus and by dr . The following voucher specimens were deposited in the united states national parasite collection beltsville, maryland: cylindrotaenia americana (no . 105165), oswaldocruzia lenteixeiria (no . Statistical analyses were conducted using r statistical software (r development core team, 2014). Prevalence, mean intensity, and mean abundance were calculated in accordance with definitions provided by bush et al . We tested for differences among males, females, and juveniles in their parasite mean abundance and mean intensity based on a negative binomial error distribution using the glm.nb function (mass package). Differences among males, females, and juveniles in their parasite prevalence and richness were determined using binomial and poisson error distributions, respectively, by using the glm function (mass package). Parasite evenness was calculated using simpson's diversity index, and differences in parasite evenness among males, females, and juveniles were tested using a normal distribution and the lm function . A bonferroni's alpha adjustment was used to keep the experiment - wise error rate at 0.05, which means that a p value 0.017 was considered statistically significant when we tested for differences among males, females, and juveniles . Three hundred thirty o. septentrionalis were collected and necropsied (68 males, 150 females, and 112 juveniles; mean mass [g] se: males 6.28 0.32, females 10.79 0.66, and juveniles 2.14 0.08, respectively). The overall parasite prevalence was 74.2%, and the overall mean abundance was 14.7 2.5 . At least nine species of parasites were isolated from the ctfs, which included six host records (table 1). Metacercariae, and acuariid larvae had the highest prevalences and mean abundances; the remaining helminths were relatively rare (table 2). Oswaldocruzia lenteixeirai and aplectana sp . Were the only species that have been reported in the ctf's native and introduced ranges (table 3). Host type (male, female, or juvenile) significantly affected parasite mean abundance (x = 29.44, p=<0.001) and mean intensity (x = 29.55, p=<0.001) (fig . 1) but did not affect prevalence (x = 3.55, p = 0.170), evenness (f2,201 = 0.632, p = 0.532), or richness (x = 6.96, p = 0.031) (fig . 2). Juveniles had lower mean abundance and mean intensity, but the male and female adults did not significantly differ in these two categories (p> 0.58; fig . 1). On the other hand, juveniles did not significantly differ from adults, in prevalence and evenness, but they did significantly differ from adult females for parasite richness (x = 6.71, p = 0.01; fig . 2). There was no significant difference between adult males and females in parasite evenness, richness, or prevalence (p> 0.42, fig . 2). Adults averaged (se) 19.36 (3.67) parasite individuals, 1.39 (0.07) parasite species, and 77.52% (2.83) prevalence, whereas juveniles averaged 5.71 (1.57) individuals, 1.05 (0.09) species, and 67.86% (4.41) prevalence, respectively . Our results indicate that there was no significant difference in abundance, intensity, evenness, richness, or prevalence between adult male and female ctfs despite males having greater home ranges because of extensive mate searching (vargas - salinas, 2006)., 2009, raffel et al ., 2010, raffel et al ., 2011). Our results suggest that ctfs acquire parasites through time such that older hosts have more parasites than younger hosts . Are cosmopolitan cosmocercid nematodes that usually reside in the gastrointestinal tract of amphibians and reptiles (baker, 1987, anderson, 2000). Identity to species is based on males; since only juveniles and females were found in the present study, specific identity was not possible . Gravid females can be viviparous or ovoviviparous and shed eggs and larvae in feces . At this time, the complete life cycle of aplectana spp . Is unknown (anderson, 2000), but a single study suggests that tadpoles can ingest juvenile nematodes, but that direct penetration of the skin of frogs is unsuccessful (vhora and bolek, 2013). In contrast, preliminary data from our lab show that post - metamorphic ctfs can be experimentally infected via skin penetration . Although there have been three reported aplectana spp . In north america, only one, aplectana hamatospicula, has been found in both cuba and the u.s.a . In cuba, this species has been found in 14 species of anurans, but it only occurs in one species native to florida (baker, 1987, vhora and bolek, 2013). Acuariids, nematodes belonging to the order spirurida, have a heteroxenous life cycle, with birds and arthropods as definitive and intermediate hosts, respectively (anderson, 2000). Meshaka (1996) showed that the ctfs diet consists of an array of arthropods, thus the most likely mode of infection is by consumption of an infected arthropod intermediate host . Ctfs most likely acquired metacercaria through infection by cercariae during the tadpole stage, when they are often found in sympatry with trematode - infected snails . However, we cannot rule out the adults being infected with cercariae when they returned to the water to reproduce or by postmetamorphic frogs consuming trematode - infected terrestrial snails . C. americana is a tapeworm that is found in at least 30 species of amphibians and reptiles in north america, three of which are native species in our study site . Because c. americana is also found in south america, europe, asia, and the caribbean, it is impossible to determine whether or not this species was introduced to or acquired in florida (mcallister, 1991, goldberg et al ., 2002). The life cycle of c. americana is not yet entirely understood, but transmission can occur in two ways: 1) directly, through coprophagy, or 2) indirectly, in which an infected invertebrate intermediate host (probably an insect) is ingested by the definitive host (prudhoe and bray, 1982). O. lenteixeiria is a common species of strongylid nematode reported from about forty species of amphibian and reptile final hosts, including ctfs, in the caribbean (coy otero and baru, 1979, baker, 1987, moravec and kaiser, 1995). The third stage larva penetrates the skin or is ingested by the final host where it matures into an adult in the intestines (prez vigueras, 1938). This species is also known to parasitize brown anoles, anolis sagrei, in the caribbean, but has never been reported from this common nonindigenous lizard in florida (goldberg and bursey, 2000). Rhabdias is a cosmopolitan nematode genus found as adults in lungs of amphibians and reptiles . (runey et al ., 1978, anderson, 2000, langford and janovy, 2009). At present, eight species of rhabdias are known from north america, and at least two are from the insular caribbean (bursey et al . Since the few specimens found in our study could not be identified to species, it is impossible to determine whether this nematode was acquired or introduced . Are known to occur in north america and in the caribbean (morgan, 1943, mcallister and bursey, 2007). Generally, adult nematodes are found in the stomachs of mammals, reptiles, and birds that have ingested insect intermediate hosts . Although amphibians have not been reported as definitive hosts for physaloptera, this group, as well as lizards, may harbor larvae that attach to the gastric mucosa, but do not encyst (goldberg et al ., 1993, some studies suggest that these hosts should not be considered paratenic since larvae do not encyst and often do not persist long in the host (goldberg et al ., 1993, therefore, ctfs may not serve as functional paratenic hosts for the unencysted physaloptera larvae found in the present study as well as for another larval physalopterid, abbreviata sp ., reported from ctfs in cuba (coy otero and ventosa, 1984). Are commonly found in terrestrial insects or isopods, but adults are found in the small intestines of carnivorous birds . Furthermore, amphibians and reptiles may serve as paratenic hosts by consuming arthropods infected with cystacanths (nickol, 1985). Pentastomids (worm - like arthropods bearing two pairs of retractable hooks in the mouth region) have a heteroxenous life cycle, and adults are commonly found in the respiratory tract of vertebrates (par, 2008). Although all classes of vertebrates and some invertebrates can serve as intermediate hosts, only fishes are reported to be unsuitable as final hosts (riley, 1986, barton and riley, 2004). It is likely that intermediate hosts are infected by ingesting eggs that are shed in the feces of an infected final host . After the larva penetrates the gut, it continues to migrate in the abdomen / coelem, and often ends up in the visceral tissue where it undergoes several molts until it becomes an infective nymph (par, 2008). Although a few anurans can harbor adult pentastomids, most anurans are considered to be intermediate hosts with the final host being reptiles, a group that makes up approximately 90% of pentastomid species (riley, 1986, barton and riley, 2004, par, 2008). Despite the fact that we found pentastomid nymphs in the lungs of the ctfs, we were unable to further identify this group because the pentastomid maturation was either incomplete or stunted in the ctf host . Perhaps the best - described effect of invasive species on host parasite interactions is the enemy release hypothesis (erh) (torchin et al ., 2001, erh suggests that invasive species have a competitive advantage in their introduced range because they leave behind many of their natural enemies in their native range . First, most of the parasites in a population are in a small proportion of the hosts (shaw and dobson, 1995, shaw et al ., 1998). Thus, by chance alone, it is unlikely that a heavily infected host will be introduced and successfully become established in a new environment; it is also unlikely that a heavily parasitized host would survive translocation . Second, most infected hosts are not infected with all the parasite species in their native range (poulin, 2013). Therefore, even if parasites are introduced with the host and successfully establish, the parasites typically only represent a small portion of the host's parasites from its native range . Hence, even if a parasite with a complex life cycle is introduced with the host, it is unlikely to establish because other required hosts are typically missing . In addition to the loss of natural enemies, introduced hosts are also thought to acquire few parasites in their introduced range because these parasites lack an evolutionary history with the introduced host; therefore, the parasites might not recognize or be capable of infecting a novel host (mitchell and power, 2003, torchin et al ., in the present study, we found that ctfs harbored at least one parasite species from its native range but have also likely acquired parasites from herpetofauna native to the tampa, fl area . Of the three types of parasites reported with the highest prevalences and mean abundances, the larval acuariids appear to have been acquired post invasion because they have never been reported in the ctf native range (table 3) (baker, 1987, anderson, 2000). Only one parasite species, o. lenteixeiria, appears to be a confirmed introduced species because it is found commonly in the native range of ctfs (table 3), but there are no records of this species from frogs native to florida . Because the aplectana specimens were not identifiable to species, we cannot be certain whether it was introduced or acquired by the ctf because this genus has been reported in cuba as well as in florida . However, it is worth noting that only one species, a. hamatospicula, has ever been reported from native anurans of florida, and it was found only in one species, gastrophryne carolinensis, in 1939, which means that it has most likely been introduced (walton, 1940). Ctfs at our study site were infected with fewer species of parasites than in their native range (table 2) with the exception of o. lenteixeirai and possibly aplectana sp ., ctfs at our study site were devoid of the parasite species found in their native range (table 3). Moreover, the prevalence of o. lenteixeirai is eight to fifteen times higher in its native than introduced range (table 3); however, caution must be taken when making this comparisons between the parasites being reported in the native and introduced ranges because it is likely that several populations were surveyed across extensive areas ranging from the bahamas to the cuban archipelago . As a result, we may be comparing fewer populations of ctfs or populations over a smaller spatial extent in florida to more populations or at least populations distributed over a greater spatial extent in its native range . Because several of the previous studies do not list their sites of collection, we cannot be sure as to how many populations of cuban treefrogs were surveyed in their native range . However, we know that we collected frogs from thirty - seven wetlands over a 4000 acre park . According to the enemy release hypothesis, a loss of enemies (in this case, parasite species) could, to some extent, facilitate the establishment and subsequent invasiveness of introduced species (torchin et al ., 2001, mitchell and power, 2003, torchin et al ., 2003). (2004) advocate further investigation of the erh because very few introduced species exhibit evidence of an enemy release; rather, they undergo an exchange or reduction of enemies . For example, in comparison to an overall mean parasite abundance of 4.6 calculated from coy otero and ventosa (1984) and 12.7 calculated from goldberg et al . (1994) in the ctf native range, this study indicates that the ctf has a higher mean abundance of 14.7 in the introduced range . Thus, careful consideration should be given to the vulnerability of introduced species to all of its enemies in native and invaded locations before attributing the colonization success to erh . Though erh is a plausible explanation for the colonization success of introduced species, research is still needed address whether or not shifts in host parasite dynamics of introduced species aid in their colonization or in the decline of natives . Although comparison to natives is not imperative to substantiate the erh because the hypothesis is strictly based on the abundance and richness of parasites in an introduced species in its native versus introduced range, we acknowledge that this study would have been much stronger with the inclusion of a comparison of the parasites of the native treefrogs in florida to parasites found in the invasive cuban treefrogs in florida . This might have suggested that exposure to parasites is higher in the introduced than native range of ctfs, which might help to explain their higher parasite abundance in their introduced range . Unfortunately, at the time of these collections, the southwest florida water management district prohibited collection of native frogs from this county park . Understanding how diseases shift between introduced and native populations can be imperative to prioritizing efforts in the conservation of species, and future work should focus on collecting ctfs as well as native treefrogs from various locations throughout the introduced range of ctfs to determine if their success is at least partially attributable to a loss or introduction of parasites.
Pemphigus vegetans is a rare variant of pemphigus vulgaris which commonly involves the flexures and oral mucosa . Pemphigus vegetans is a rare form of pemphigus vulgaris comprising only 1 - 2% cases . Pemphigus vegetans is an autoimmune disease characterized by flaccid bullae or pustules that erode to form hypertrophic plaques involving predominantly skin flexures and mucous membranes . The cutaneous lesions in the hallopeau type begin as pustules and heal as vegetating plaques whereas those in neumann type, with a worse prognosis, are characterized by vegetation developing during the course of pemphigus vulgaris . We herein report an unusual presentation of localized pemphigus vegetans without mucosal lesions which initially involved only the left breast, and subsequently involved the left groin during follow - up . A 62-year - old healthy woman presented to our dermatology out - patient department with single painful red colored elevated lesion covered with pustules on the left breast for 6 months . There was no history of fever, any drug intake, eye or mucosal complaints, no past or family history of similar illness, no history of any topical application . On examination, a single well - defined polycyclic erythematous plaque of size 6 7 cm with pustules concentrated mainly at the periphery of the plaque was present on left breast . Koh mount for fungus was negative, but based on clinical appearance probable diagnosis of tinea corporis was kept, and she was started on oral fluconazole 150 mg once a week and terbinafine cream local application twice daily for 2 weeks . On subsequent follow - up after 2 weeks, there was no improvement, but the lesion in the left breast had progressed and involved the entire left breast to form a single well - defined moist, verrucous vegetating plaque of size 810 cm with multiple pustules, yellow to brown colored crusts at places, and some raw oozy areas . Plaque was surrounded by a broad rim of erythema [figure 1a]. She also developed similar verrucous lesion in the left groin on subsequent 2 weeks follow - up [figure 2a]. Nikolsky's sign was negative and tzanck smear showed eosonophils, but no acantholytic cells . Gram stain for bacteria showed only mixed inflammatory infiltrate with eosinophil predominance and acid fast bacteria staining was negative . Routine laboratory studies including complete blood count, c reactive protein, erythrocyte sedimentation rate, liver and renal function tests, and chest x - ray were all within normal limits . Biopsy specimens were obtained from the right breast with the differential diagnosis of pemphigus vegetans, pyoderma gangrenosum . Well - defined moist, vegetative plaque studded with multiple pustules healed hyperpigmented plaque moist, verrucous plaque of groin patient was started on oral prednisolone 40 mg once daily with which she showed dramatic response within a week . Histopathological examination [figures 3a c] showed suprabasal cleft with acantholytic cells, numerous eosinophils, intraepidermal eosinophilic abscesses, and dense inflammatory infiltrate in the dermis . As the biopsy was taken from an early pustule, that might be an explanation for the absence of hyperkeratosis and papillomatosis in the present case . Direct immunofluorescence (dif) revealed intercellular immunoglobulin g (igg) deposits in the epidermis [figure 4]. Suprabasal cleft with acantholytic cells and intraepidermal eosinophilic abscess suprabasal cleft with acantholysis increased number of eosinophils dif shows intercellular immunoglobulin g (igg) deposits in the epidermis in a fish - net pattern the histopathology in pemphigus vegetans exhibits epidermal hyperplasia, papillomatosis, and intraepidermal eosinophilic abscesses as the lesions age . Histopathology of pemphigus vegetans seem to differ from pemphigus vulgaris by an eosinophilic response, formation of eosinophilic microabscesses and extent of vesiculation . The immunofluoresence findings in pemphigus vegetans are indistinguishable from those of pemhigus vulgaris . Patient in discussion posed diagnostic challenge as she had limited cutaneous involvement, no mucosal involvement, and atypical site of presentation . We kept the possibility of pemphigus vegetans, hallopeau type on the basis of intertriginous involvement, pustular and vegetative lesions supported, by histopathological examination with predominant eosinophilic infiltrate . All the above findings were confirmed by direct immunofluoresence showing immunoglobulin g (igg) deposits in a fish - net pattern . Various conditions that have to be kept in the differential diagnosis include the vegetating lesions of other bullous autoimmune skin diseases, such as bullous pemphigoid or immunoglobulin a (iga) pemphigus, the chronic inflammatory plaques of hailey - hailey disease, and especially vegetating pyoderma . Both diseases show similar clinical and histopathological findings, but in pyostomatitis vegetans the immunofluorescence findings are characteristically negative . Although the lesions in pemphigus vegetans can occur at any site, solitary lesion involving unusual sites like scalp, soles and at skin graft recipient site have rarely been described in literature . Very rarely solitary vegetating plaque mimicking pemphigus vegetans could be the cutaneous presentation for paraneoplastic pemphigus, in that situation associated malignancy, severe mucosal involvement, and other criterion will be helpful in reaching the final diagnosis . Till date, to the best of our knowledge, primary involvement of the breast without mucosal involvement has not been reported . After detailed and exhaustive work, she was being diagnosed as a case of pemphigus vegetans . She was administered oral steroid, which was tapered slowly over a period of 4 weeks . She showed excellent response; complete healing of plaques with hyperpigmentation occurred over a period of 2 weeks . Till date, even after 6 months of stopping steroid she had not shown any symptom or sign of relapse . To emphasize pemphigus vegetans, although a variant of pemphigus vulgaris, can present at atypical site or show limited involvement without involving mucosa giving a diagnostic challenge . So, in routine dermatological practice resistant pustular plaque not responding to antifungal or antibacterial treatment, pemphigus vegetans can be kept as a good differential . In our case, the lesions were localized in the left breast and groin, without mucosal involvement.
Eosinophilic esophagitis (ee) is defined as a primary clinicopathologic disorder of the esophagus characterized by esophageal and/or upper gastrointestinal tract symptoms, whose clinical findings in adults include food impaction and dysphagia, whereas feeding intolerance and symptoms consistent with gastroesophageal reflux disease (gerd) are more frequently observed in children . The key histological finding is an eosinophilic infiltration of the esophageal mucosa with more than 15 intraepithelial eosinophils / high - power field (hpf) in one or more biopsies, and a dense esophageal eosinophilia is often described in association with severe squamous epithelial hyperplasia, whereas the gastric and duodenal mucosae are spared . Moreover, a diagnostic requirement is the absence of gerd, which can be assessed with an esophageal ph monitoring study or after a high - dose proton pump inhibitor (ppi) treatment . The correlation between ee, atopy, aeroallergen polysensitization and food allergens has been well documented; indeed, a correlation between food allergens and ee was first demonstrated by kelly et al . Who observed that an elemental diet improved clinical and histological findings in 10 children with ee . These findings were subsequently confirmed by other authors . Although an increasing number of children and adults are diagnosed as suffering from ee, only few controlled trials have been performed to guide clinical management; thus, clinical practice is largely based on limited data and experts' opinions . Here we report the case of a 2-year - old child who was diagnosed with ee with an atypical clinical presentation and an unusual diagnostic course . A 2-year - old child was referred to the pediatric surgical department of spedali civili of brescia complaining of dry cough with nocturnal exacerbations in the last 6 months, associated with retching and failure to thrive . She had previously been diagnosed with atopic dermatitis and persistent rhinoconjunctivitis; peripheral eosinophil count was normal . Due to a suspicion of gerd, 24-hour esophageal ph monitoring was performed resulting in a pathologic reflux index (12%), and a 3-month high - dose treatment with ppi (esomeprazole 2 mg / kg / day) was prescribed, without clinical improvement . An esophagogastroduodenoscopy (egds) was carried out; esophageal linear furrows were observed and an eosinophilic infiltration of the esophageal mucosa (> 20 eosinophils / hpf) was evident at histology (fig . 1a - c), whereas gastric and duodenal findings were endoscopically and histologically normal . On these grounds the possibility of ee was raised, and the patient was referred to the allergy unit of this hospital . Perennial and seasonal aeroallergens and food - specific serum ige (cap system, phadia, uppsala, sweden) were absent and skin prick tests for common aeroallergens were negative . Since ee in children is often associated with food allergy, and although in vitro tests with commercially available food extracts were negative, prick to prick skin tests with fresh foods were performed . However, only a very weak sensitization to tomato was detected and solanaceae exclusion from the diet did not result in any clinical improvement . Nevertheless, since acute episodes appeared closely related to food intake and since milk and egg are reported among the main causative food in ee, a restricted dietary regimen was implemented, withdrawing milk and eggs . After 2 weeks of dietary restriction the patient was accidentally exposed to milk proteins as she was fed with sliced carrots that had been prepared with a cheese grater . After 1 month the patient gained weight and reported a steady and complete resolution of all symptoms, including cough . Egg intake was not followed by clinical relapse, whereas milk dietary reintroduction was no longer attempted . In order to confirm an etiopathogenetic correlation between cow's milk sensitization and ee, patch tests with cow's milk proteins the tests were performed mixing 2% skin prick test commercial extract (lofarma) to vaseline . After 48-hour occlusion, an infiltrated erythematous papula developed at the casein application site . As a result, two months later, with the patient completely asymptomatic, an egds with esophageal biopsies was repeated and showed normal - appearing mucosa; the eosinophilic esophageal infiltration was no longer detectable (fig . After 4 months, the patients ate an ice cream made of cow's milk and persistent intense coughing appeared 3 h later . On that occasion a follow - up visit was performed 2 years after the diagnosis and the patient reported being completely asymptomatic . Dry cough with nocturnal exacerbations is a symptom described in gerd, usually defined as an atypical manifestation of the condition, but it is not generally found in ee patients, even though a particular subset of patients has emerged with airway manifestations . The relationship between gerd and ee is not always still, as ee diagnosis requires exclusion of pathologic acid reflux with a ppi trial and/or a normal esophageal ph monitoring, but pathologic acid reflux in ee is described by some authors in a significant proportion of patients . On the other hand, eosinophilic infiltration of esophageal mucosa is well described in gerd; thus, the mere finding of a pathologic esophageal ph test cannot rule out ee, and the mere presence of an eosinophilic infiltration is not diagnostic of ee . The diagnostic guidelines developed by gastroenterologists are based on biopsy specimen findings of = 15 eosinophils / hpf, but the maximal number of eosinophils / hpf that is associated with gerd - related esophagitis is still under investigation . . Underlined that the distinction between ee and gerd cannot be reliably performed on histopathologic evidence alone in children with upper aerodigestive symptoms . In our patient the symptom and the result of the ph study led us to suspect gerd and to start treatment with ppi . After a 3-month trial without clinical improvement an egds was advised and ee detected . A possible explanation for the abnormal esophageal acid exposure could be due to a esophageal motility disorder and/or to lower esophageal sphincter incompetence, possibly associated to an inflammatory infiltration, but the literature is discordant about this topic . We had a definite diagnosis only after a restricted dietary regimen with resolution of symptoms and of esophageal inflammatory infiltration . In gerd chronic cough is considered the result of acid stimulation of esophageal chemoreceptors or the effect of direct acid irritation of laryngeal mucosa . In our patient acidity we feel that esophageal eosinophilic inflammation was allergen - mediated (milk protein) rather than acid - mediated . In general, eosinophils have proinflammatory effects, upregulating adhesion systems, mediating cell trafficking, and releasing cytokines, chemokines, lipid mediators and effector proteins . More in depth, eosinophil cationic proteins participate in tissue damage eventually resulting in fibrosis . Eosinophil major basic protein, the predominant toxic protein found in eosinophilic granules, has been established as a critical cytotoxin in the pathophysiology of asthma and chronic rhinosinusitis . This protein directly induces mast cell degranulation, airway reactivity and smooth muscle contraction, and increases smooth muscle responsiveness to agonist . Deposition of major basic protein in the esophageal mucosa in patients with ee has been described, although its role in the pathophysiology of this condition has not been explored . In our patient there might have been an initial esophageal sensitization to milk proteins, with ee occurring on esophageal re - challenge with the same antigen . In this scenario alternatively, mishra et al . Showed a link between allergic hypersensitivity response in the lung and the esophagus, but to our knowledge there are no previous studies showing a simultaneous esophageal and pulmonary eosinophilia due to food allergen exposure . Indeed, since most patients with ee display positive reactions to foods on skin prick test and/or atopy patch test, a mixed immunopathogenesis, both ige- and cell - mediated, has been postulated . A traditional allergologic screening only based on in vitro and percutaneous tests would not have been sufficient to identify the responsible food allergen.
Traditionally, this variability in movement was viewed as random noise - providing minimal important information [1, 2]. With the introduction of chaos and complexity theory into the life sciences in the 1990s, this negative view of variability gait (i.e., walking) is a complicated process involving coordination of multiple systems within the body (e.g., central nervous, musculoskeletal, and cardiovascular system). To walk, a person's nervous system must send signals to control a large number of muscles while simultaneously processing sensory information in order to monitor and refine movements, all while maintaining an upright stance . Given the multitude of muscles, and neural processes involved, gait variability likely arises from a combination of factors . There is increasing evidence that gait variability is a quantifiable indicator of walking function [1, 2, 9]. In the past, gait variability was viewed as experimental artifact that should be filtered out to reveal average behavior . However, more recently, evidence has demonstrated meaningful characteristics of gait variability, associated with neural control of walking [1, 2, 9, 10]. Increases in gait variability have been observed in individuals with advanced age [1114] as well as various neurologically impaired populations, including spinal cord injury and neurological conditions, such as parkinson's disease, dementia, and multiple sclerosis . Gait variability has further been associated with motor control function [9, 19], energetic cost of walking, and falls [1214] in various populations . Currently, there is no gold standard to quantify gait variability, and a number of different analysis techniques have been used [1, 2]. Most commonly, gait variability is quantified using distributional metrics, such as the standard deviation (sd) and coefficient of variation (cv) of kinematic and spatiotemporal gait parameters [1, 2]. It is maintained that sd provides a measure of absolute variability, while cv provides a measure of relative variability . Other nonlinear metrics, such as detrended fluctuation analysis and approximate entropy, have been used to examine the temporal sequential structure of gait variability (i.e., the time - evolving structure of variability). Since different metrics quantify different aspects of gait variability and operate on different timescales, selection of the appropriate metric(s) for answering a particular research question is critical in investigations of gait variability . Multiple sclerosis (ms) is a degenerative neurological disease affecting approximately 350,000 people in the usa and upwards of 2 millions worldwide . Ms is a progressive disease in which damage to the central nervous system causes widespread dysfunction . Symptoms are highly variable among persons with ms, but typically include sensory, cognitive, and motor impairment . Ms results in progressive disability, which is indexed clinically by the expanded disability status scale (edss), a 010 scale in which 0.0 represents no impairment due to ms, 4.0 indicates the onset of significant walking impairment, 7.5 indicates wheelchair dependence, and 10.0 represents death due to ms . Walking impairment is one of the most commonly reported symptoms of ms and has been reported as one of the most impactful symptoms on the quality of life . Given that walking difficulty is a major symptom of ms, documentation of gait impairment is important for indexing disease progression and rehabilitation in ms . To date, the majority of research focusing on gait dysfunction in persons with ms has focused on subjective clinical assessments and average spatiotemporal parameters . Performance walking tests such as the timed 25-foot walk, 2-minute walk, and 6-minute walk are commonly used to measure gait function in ms and routinely demonstrate that persons with ms walk slower than their peers without ms . Examinations of spatiotemporal parameters have revealed that individuals with ms walk slower, taking shorter, slower steps, and spending more of their gait cycle in double - support than healthy controls and these impairments scale with disability [2730]. Importantly, deficits in gait have been observed in persons with ms compared to controls even at fixed walking speeds [31, 32]. In addition to average gait parameters and timed walking tests, there has been increasing research to characterize variability of gait parameters and to determine the clinical significance of gait variability in ms . The purpose of this report is to review the current state of the literature concerning gait variability in individuals with multiple sclerosis . Results were collected from a literature search for gait variability + multiple sclerosis using pubmed . Fourteen publications were found, and after review, 12 of the articles were considered relevant to the topic . Four other published papers located through the authors' personal knowledge that were not listed on pubmed were included . The following discussion separates observations about gait variability in ms into 2 sections, focusing first on variability during short duration walks (10 meters) and second, on gait variability during longer duration walks . The majority of research concerning gait variability in individuals with ms characterizes variability of gait parameters over relatively short distance walks (10 meters). Understanding walking behavior, including gait variability, during shorter walks may be applicable to many activities of daily life such as maneuvering about the home or crossing the street . Another potential explanation for why investigations focus on shorter walks is the limitations of data collection equipment . Pressure sensitive electronic walkways are commonly used to measure gait parameters but are typically less than 10 meters long . Additionally, data collection using optical motion capture for overground walking is limited by the capture size . However, the development and refinement of accelerometer - based mobility monitoring has been implemented successfully in persons with ms [3336] and may potentially provide means to measure gait variability over larger distances . One of the first investigations to report on variability of walking function examined variability in the timed 25-foot walk . Importantly, variability in walking performance was viewed as random fluctuations (e.g., noise) that must be filtered to reveal significant changes in walking function . The goal of the investigation (n = 133, edss ranging from 1.0 to 3.5) was to quantify natural variability in timed 25-foot walk performance in persons with ms over a 1-year period in order to determine a threshold for meaningful change in performance . Results demonstrated that timed 25-foot walk performance may naturally vary up to 20% over a 1-year period in persons with ms . Therefore, it is suggested that changes in timed 25-foot walk time of 20% or more indicate meaningful change in walking function . It was also found that those with greater disability had the greater variability in 25-foot walk performance than those with less variability . This observation indicates that variability in performance - based test is related to disability . There have been multiple reports documenting increased variability of kinematic and spatiotemporal gait parameters in individuals with ms compared to healthy controls during walks of up to 10 meters [18, 27, 38, 39]. In one relatively small report, individuals with ms (n = 20, age = 43 10, median edss = 3.0) demonstrated significantly greater standard deviation of hip, knee, and ankle angles (sdhip = 2.0, sdknee = 2.7, sdank = 1.5) during overground walking than healthy controls (n = 8, age = 41 9, sdhip = 1.8, sdknee = 2.3, sdank = 1.4). Another investigation reported that 43 individuals with ms with minimal disability (n = 43, age = 47 9, median edss = 2.0) demonstrated greater coefficients of variation for step time (cvst = 2.6%) and single - support time (cvsst = 3.2%) than age and gender matched controls without ms (n = 43, age = 47 10, cvst, sst = 1.9%, 2.3%). Meanwhile, others reported a greater cv of step length in persons with ms with minimal impairment (n = 9, age = 42 9, median edss = 2.0, cvsl = 1.3%) compared to controls (n = 9, age = 42 10, cvsl = 1.1%). Overall, these studies highlight that persons with ms with minimal disability have elevated gait variability compared to control participants . In order to examine whether gait variability is influenced by disability in persons with ms, socie and colleagues quantified gait variability in 88 persons with ms with a wide range of disability (edss range = 2.06.5; median = 4.5). Participants walked at a comfortable pace along a 26-foot pressure sensitive walkway that recorded spatiotemporal parameters . Congruent with the field, persons with ms demonstrated greater cv of both step time (cvst = 4.7%) and step length (cvsl = 5.1%) compared to healthy controls (n = 20, age = 51 9, cvst, sl = 1.8%, 2.0%). Additionally, a positive correlation between edss and cv and sd of step time and step length and a negative correlation between edss and cv and sd of step width was observed . That is to say, variability of step length and step time is greater in individuals with ms with greater disability, while variability of step width is smaller in persons with ms with greater disability . Similarly, another study with a smaller sample (n = 10) which examined walking on a motorized treadmill demonstrated that individuals with ms with edss scores 4.0 had greater stride length cv (5.0%) compared to a group of individuals with ms with edss scores <4.0 (cvstl = 3.2%). Together the findings of these studies suggest that increases in gait variability occur early on in the disease process and apparently worsen as disability increases . The association between disability and gait variability has only been examined cross - sectionally, so no causal relation should be assumed . It is interesting to note that none of these investigations found a significant difference in persons with ms and step width variability . The differential effect of ms on gait variability of propulsion metrics (e.g., step length, step time, etc .) And gait variability of stability metrics (e.g., step width) is congruent with the notion that stability gait parameters (e.g., step width) and propulsion gait parameters (e.g., step length, step time) are controlled by separate neural circuits that could be differently influenced by disability [41, 42]. While most research in ms has focused on gait variability within walks of up to 10 meters, there are some investigations of gait variability in ms during longer walks (> 10 meters). The 6-minute walk test is a common clinical gait test in which gait variability has been characterized in individuals with ms . Preliminary evidence suggests that individuals with ms who use assistive devices (n = 9) have greater variability of step time (cvst = 11.0%) and step length (cvsl = 11.6%) than individuals with ms who walk independently (n = 9, cvst = 2.8%, cvsl = 2.3%) and healthy controls (n = 10, cvst = 2.1%, cvsl = 1.8%) throughout the 6-minute walk test . Furthermore, individuals with ms who used assistive devices experienced significantly greater increases in variability of step time, length, width, and double - support time in the last 2 minutes of the test compared to the first 2 minutes compared with the independently ambulatory ms group and controls . The authors suggest that changes in gait variability could be related to changes in stability and fatigue over the course of the 6-minute walk test . In another study, nonlinear dynamics were used to examine variability structure over the course of 3 minutes of walking in individuals with ms (n = 10, age = 35 9, median edss = 4.0) and healthy controls (n = 10, age = 35 10). Participants with and without ms walked on a motorized treadmill at a comfortable self - selected speed . The long duration of walking allowed for the examination of the time sequential structure of fluctuations in gait . Individuals with ms demonstrated lower approximate entropy of stride length (apen = 0.55) and stride width (apen = 0.51) than healthy controls (apen = 0.70, 0.68 for stride length, width, resp . ). Lower approximate entropy values are suggested to indicate more repeatable fluctuations in ms gait and theorized to result in reduced capacity to overcome perturbations . However, the same data showed no differences in detrended fluctuation analysis, another nonlinear measure of variability, of stride length, and width variability between persons with ms and controls . It is important to note that the use of a treadmill could potentially change walking behavior, including gait variability . Future work should examine the structure of gait variability in persons with ms in overground walking . Although it is clear that persons with ms have greater amounts of gait variability in short and long walking tasks, the mechanisms underlying this change in gait are not clear . Based on the notion that the control of gait involves numerous neural processes and coordination of the trunk and limbs, it most likely is not a single individual mechanism, but rather a combination of deficits that contribute to gait variability . A potential factor for the increase in gait variability among persons with ms simulations of walking have demonstrated that an increase in neuromuscular signal noise leads to increased gait variability . Indeed, elevated neuromuscular noise has been previously suggested to contribute to impairment in ms . However, specific evidence linking gait variability to neuromuscular signal activity in ms has not been reported . Additionally, empirical evidence of noise within the neuromuscular system is lacking and has been theoretically challenged . Another facet of ms that has been studied in connection to gait variability is fatigue, which is a common symptom of ms and is related to ms gait impairment . However, there is limited evidence of associations between fatigue and gait variability . Two studies report that, despite changes in self - reported fatigue, there were no significant changes in gait variability throughout a 2448 hour period in persons with ms [18, 49]. Preliminary data from our laboratory demonstrates that step time variability and double support variability increase in the persons with ms who use assistive devices while walking during the course of a 6 mw . It was proposed that this increase in gait variability results from an increase in fatigue . Overall, it appears that fatigue may indeed be associated with gait variability in ms; however, the current evidence does not support this assertion in walks of less than 10 meters . Muscle quality (ratio of muscle strength to lean muscle mass, i.e., functional muscle strength) has been associated with gait variability in healthy older adults . Additionally, decreased muscle strength as well as deficits in balance and proprioception are related to gait variability in elderly individuals . Given that persons with ms have decreased muscle strength, proprioception, and balance and that these characteristics is related to gait impairment [50, 51], these factors may contribute to gait variability in ms . An additional factor that maybe related to elevated gait variability in persons with ms is spasticity . Spasticity, the hyperexaggerated stretch reflex, is very common in persons with ms and has been found to be related to gait dysfunction in persons with ms [52, 53]. Moreover, it is logical to speculate that the presence of spasticity in the lower limbs leads to an alteration in the outcome of the descending motor command driving walking . Regardless of the logic of this speculation, there is no data in support of this proposition . It is also possible that ataxic movement disorders that are often characterized by hypermetric and dysmetric movements are associated with gait variability in persons with ms . There is evidence that persons with cerebellar ataxia have greater gait variability than healthy controls and that the amount of gait variability is related to walking speed [54, 55]. However, there is no evidence linking ataxia and gait variability in persons with ms . Lastly, there is some evidence to suggest that attentional resources are related to gait variability in persons with ms . One investigation demonstrated that individuals with ms (n = 18, age = 39 8, median edss = 2.5) exhibited less variability of swing time (cvswt = 3.0%) in normal walking than while walking and simultaneously performing a cognitive task involving serial addition (cvswt = 4.0%). The additional neurological demand of performing a cognitive task likely decreases the amount of control over gait, leading to increased gait variability . However, gait variability has been found to have clinical import in various other special populations . For instance, gait variability is associated with falls in the elderly [8, 12, 13]. One investigation demonstrated that recurrent fallers (i.e., 1 falls / year) with ms exhibit greater variability of spatial footfall placement than nonfallers with ms . Spatial footfall placement variability was quantified in that investigation using a novel method involving fitting footfall patterns with fourier series . Importantly, traditional gait variability metrics (cv of step length, time, and width) did not distinguish between recurrent and nonfallers with ms . To our knowledge, it is also possible that elevated gait variability in persons with ms is related to the amount of energy required to walk . Recently, researchers have shown that experimental increases in gait variability are related to increased energetic cost of walking in healthy young adults . The authors propose that individuals with greater gait variability need to spend energy not only on propelling the center of mass, but also on movements to correct for the erratic foot placement . Although it is well established that that persons with ms do indeed have higher energetic cost of walking [5860], there is no data that this is associated with gait variability . It is logical to speculate that since energetic cost of walking is related to fatigue, gait variability is also related to fatigue . To date evidence in support of any association between gait variability and fatigue some reports of gait variability during short walks report minimal association between gait variability and fatigue [18, 49]. However, one study on fatigue in ms (n = 14, age = 42 8, median edss = 3.5) showed significant associations between gait variability and fatigue in ms during walks to exhaustion . In this investigation, participants with ms walked until they felt complete exhaustion . During walks to exhaustion, gait variability was significantly correlated with the motor sections of the fatigue scale for motor and cognition (fsmc). Correlations between gait variability and the cognitive portion of the fsmc were not significant, suggesting that physical fatigue is more closely related to gait variability than cognitive fatigue during walks to exhaustion in ms . For instance, only one relatively small (n = 10 ms) study has analyzed time - evolving structure of gait variability in ms . Nonlinear variability measures yield different information than distributional measures (e.g., coefficient of variation) and are indicative of motor function and health and are believed to be more sensitive to dysfunction than traditional measures [6, 9, 19]. Further investigations are needed to characterize the temporal characteristics of gait variability in ms to supplement the growing body of distributional gait variability data that has been reported . As well as expanding the scope of metrics used to analyze gait variability in ms, research is warranted to explore possible associations between gait variability and falls in persons with ms . Gait variability has been associated with falls in other clinical populations such as the elderly [1214]; however, there is limited evidence linking gait variability and falls in ms . Given that 50% of individuals with ms fall in a given year [62, 63], an association between gait variability and falls in ms is clinically relevant . Specifically, gait variability could potentially be altered through therapeutic intervention, although specific observations have not been reported . Interventions have demonstrated the ability to improve physical and cognitive performance, disability level, and quality of life in ms . Future interventions in ms, particularly those targeting mobility and gait, should document gait variability as a unique indicator of gait function . Additionally, by documenting gait variability during therapeutic interventions, researchers may also be able to identify specific mechanisms that modulate gait variability in ms . Overall, the existing body of research demonstrates that gait variability is elevated in individuals with ms [18, 27, 3840] and potentially clinically significant . Additionally, a number of factors have been linked to gait variability in ms, including disability level, assistive device use [39, 63], dual - task performance, and fatigue . However, further research is needed to more fully characterize and to understand the clinical impact of gait variability in individuals with ms.
Traumatic brain injury (tbi) is an insult to the brain that is caused by an external force . . Associations between tbi and a variety of neuropsychiatric disorders have been described since the days of yore being referred to as traumatic insanities tbi is associated with a gamut of psychiatric disorders that may be divided into disorders that are also seen in patients without brain injury such as substance abuse, mood, anxiety, psychotic, personality disorders, and those that are unique to patients with brain damage, for example, involuntary emotional expression disorder, anosognosia, aprosody, and neglect . There are very few studies that have examined the interesting area of eating disorders after tbi . Eating disorders are a persistent disturbance of eating behavior or behavior intended to control weight, which significantly impairs physical health or psychosocial functioning . They comprise a spectrum of conditions, of which bulimia nervosa and anorexia nervosa are the major categories . The etiology and pathogenesis of eating disorders per se are still highly debatable . In most patients of eating disorders nonetheless, associations of anorexia and bulimia nervosa with a history of perinatal complications and head injuries suggest a role of cerebral pathology in some cases . A number of case studies describe eating disorders with intracranial tumors, injuries, or epileptogenic foci . The changes in dietary habits following tbi have generally been documented with respect to appetite, and sensory disturbances of taste and/or smell . In a review of 54 published clinical cases of eating disorders in focal brain injuries, it was found that although simple changes in appetite and eating behavior occur with hypothalamic, and brain stem lesions, more complex syndromes, including characteristic psychopathology of eating disorders, are associated with right frontal and temporal lobe damage . In a sample of 120 patients with severe tbi, ciurli et al (2011) found wide range of neuropsychiatric symptoms: apathy (42%), irritability (37%), dysphoria / depressed mood (29%), disinhibition (28%), eating disturbances (27%), and agitation (24%). However morbid hunger or persistent hyperphagia (as seen in our patient) after tbi are rare but potentially life threatening conditions . Diagnostic review of 88 admitted patients of tbi identified 2 (3%) patients presenting with this condition . In the following case report he presented to us with changes in eating pattern that were characterized by an incessant urge to eat that was difficult to control on the sight of food . It also highlights the importance of cortical structures in the genesis of abnormal eating behaviors and challenges the conventional notion that only hypothalamus is responsible for regulating eating behaviors . S, 42-year - old, hindu, married, male, premorbidly well - adjusted presented with alcohol use in dependent pattern for 20 years . The patient also suffered from left hemiplegia following the craniotomy, which recovered partially with physiotherapy over a period of 1 year . Post head injury, the patient started having low frustration tolerance, aggressive outbursts, disinhibition, difficulty in persisting with tasks, apathy, and amotivation . Patient restarted with alcohol use in dependent pattern and also started reporting craving for food which was not present before . The patient would not be able to control his craving especially on the sight of food . On occasions corroboratory information was collected from his family members who had been cohabiting with the patient . They stated that he would eat his regular three meals a day, interspersed with a lot of snacking in between meal times which mostly included oily food, chips, biscuits, sodas, and the likes . Gradually, since the last 10 years, his eating pattern has consistently been deranged . Patient subjectively reported that even though he does not feel hungry, he could not control his urge to eat . At times, he even exhorted money from strangers giving various excuses, to obtain food . There was no history of eating inedible substances, polyuria, or polydipsia . In the last 1 year lobar function tests of the patient showed a deficit in frontal lobe and he was unable to do the clock draw test . All laboratory investigations were within normal limits except high - density lipoprotein 34 mg / dl (normal value [n] 40 mg / dl), triglycerides 230 mg / dl (n <150 mg / dl), fasting plasma glucose 119 mg / dl (n <100 mg / dl), 2 h plasma glucose 201 mg / dl (n <140 mg / dl), and hba1c-6.9% (n <5.6%). Magnetic resonance imaging scan revealed large areas of damage involving both frontal lobes in the form of gliosis and encephalomalacia, along with diffuse cerebral atrophy [figure 1a and b]. (a) t1 image and (b) t2 image showing large areas of gliosis and encephalomalacia seen involving both frontal lobes . Diffuse prominence of the cerebral sulci is seen suggesting diffuse cerebral atrophy on admission, alcohol withdrawal symptoms were managed with tapering doses of benzodiazepines . The patient was asked to make a food diary as a part of the self - monitoring activity . The patient was however not very honest with the same as he did not consider his eating to be a problem and would consume food secretly . Endocrinology consultation and dietary advice were sought for his deranged blood glucose levels and he was started on metformin . Taking into consideration his weight gain and alcohol intake he was also prescribed topiramate 100 mg along with amisulpride 200 mg to control the behavioral disturbances . At discharge patient's blood glucose was within normal limits; however, there was no respite in his eating pattern . Educating the patient and engaging him in simple behavioral interventions is being planned for the follow - up visits . In our case, it is clear that the onset of eating disorder was temporally correlated with trauma to the frontal lobe . Executive functions have been proposed to play an important role in regulating a wide array of behaviors and eating behavior has been proposed to be one of them . Frontal - subcortical (fsc) circuits, in particular, are effector mechanisms that allow the organism to act in its environment . There are five major fsc circuits out of which three originate from dorsolateral prefrontal cortex (dlpfc), anterior cingulate cortex (acc), orbitofrontal cortex (ofc). The prototypic fsc circuit is a closed loop that originates in the frontal cortex, projects onto the striatal structures (caudate, putamen, and ventral striatum), from striatum connects to substantia nigra (sn) and globus pallidus (gp), from these two structures connects to specific thalamic nuclei, from where it projects back to frontal cortex . Thus, striatum, sn, gp, and thalamus are the subcortical structures which are under the influence of frontal cortex . The dlpfc allows the organization of information to facilitate a response; the acc is required for motivated behavior, and the ofc allows the integration of limbic and emotional information into behavioral responses . Impaired executive functions, apathy, and impulsivity are hallmarks of fsc circuit dysfunction which was seen in our patient . Eating disorders could be understood as forms of dysregulation of behavior that is suggestive of pfc dysfunction . Several research studies indicate that the pfc plays a pivotal role in the control of eating behavior . Various neuroimaging studies have also indicated that pfc, particularly ofc, plays an important role in the reinforcing value of food . With functional neuroimaging studies, hunger and satiety have been represented in prefrontal - subcortical systems and taste and olfactory processing in ofc . Eating behaviors are seen to be disturbed in various illnesses that involve prefrontal - subcortical systems . Tbi can damage frontotemporal structures which can induce a lack of self - restraint in eating that is resistant to behavior modification and appetite suppressants which was seen in our patient . Literature also suggests that substance use and eating disorders can both be conceptualized as maladaptive impulsive and compulsive behaviors . The case discussed above suffered tbi on the background of alcohol dependence and further progressed on to develop dysregulated eating behavior . Thus, changes in eating habits, substance use and behavior following head trauma can be explained by involvement of frontal, prefrontal areas, and subcortical circuits . This challenges the traditional view that only hypothalamic disturbance underlies eating disorder and underscores the involvement of other brain areas and circuits that could be implicated in causation of eating disorders . Cortical lesions cause eating disorders, typically when located in temporal and frontal association areas that are strongly connected to basal and diencephalic systems controlling appetite . Implication of frontotemporal circuits is consistent with functional neuroimaging research in eating disorders and also with benign changes in eating, such as the gourmand syndrome described as a preoccupation with food and a preference for fine eating; it is a benign eating disorder associated with lesions involving parts of the right anterior cerebral hemisphere . We, therefore, conclude that the current evidence favors cortical mechanisms in the genesis of eating disorders over hypothalamic ones . Fsc circuits regulate behaviors and any illness or lesion involving them can trigger maladaptive behavior . More research is needed in this area as it could have importance not only in widening our understanding of the neurobiology of eating disorders but could also have therapeutic implications, thus improving management and long - term outcome of patients with eating disorders.

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.