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Attempting to motivate our children to go to bed at a reasonable time, parents have for generations invoked these three time - honored rewards . Health is the absence of something, namely sickness, and is only fully appreciated when we no longer have it . Yet, when we are really sick, health becomes our paramount concern . Few of us would choose to be a wealthy, wise man or woman if the price was advanced alzheimer's disease, terminal cancer, or crippling arthritis . In this context, we have a major problem as a society . Despite our amazingly advanced technologies in such areas as communication, space travel, and transportation why is it that our capacity to innovate in biomedical sciences seems to lag so dramatically behind our innovation capacity in aircraft design, for example? We serve as directors of the california institute for quantitative biology (qb3), which seeks to promote innovation in the biomedical sciences . At qb3, we look closely at the impediments to improving health through technology and explore solutions to this problem . We are convinced that many answers could be found in our research universities if they were restructured . They need to be innovative in how they manage their science, as well as in how they perform it . Universities are without peer in their ability to discover the fundamental principles of science and are responsible for much innovation in our society . The creativity and nimbleness of their science is unfortunately not matched by equal creativity and nimbleness in administration and management . Although service innovation is now widely seen as contributing to economic growth and addressing societal needs as effectively as science innovation, universities in general continue to operate using time - honored, unchallenged principles . This must change . We need to evaluate how effectively we perform our public mission, and whether our exclusive focus on specialization precludes our usefulness . To know what to change, we have to go back to the way our biomedical industrial complex is constructed . We have come a long way from the days when science was done by a gentleman scientist in a room in his mansion, supported by his own funds or those of a generous friend . Nowadays, governments put a significant part of their gross domestic product into supporting research and development . Much of this investment, about $50 billion per year in the united states alone, goes to our research universities . Governments justify this use of taxpayers money by pointing out, correctly, that unfettered research activities are the most effective way of fostering the innovation needed to address current and future challenges to society . Scientists also are motivated by the sheer beauty, elegance, and excitement of fundamental discovery, but this is an acquired taste, not usually shared by the general public! What society does expect are results that meet its needs . To get societal value from university research if investors and manufacturers see a potential market for an innovation, then the discovery moves efficiently out of the lab and into the marketplace . Not only does society get something it needs, as measured by its willingness to pay for it, but the manufacturing and marketing process creates jobs and economic growth . The social contract of science, therefore, is that university scientists are given the freedom to pursue their research interests, supported by taxpayers funds, because the taxpayers have been convinced that potentially commercializable discoveries will be made, which will result in new products and economic growth . The contract specifies roles for the three stakeholders: government, universities, and the private sector . In the life sciences, the system is not working well for all three stakeholders . Government is questioning whether it is getting sufficient value when its investment in university research generates one start - up company per $100 million and one patent license for every $12 million (battelle technology partnership practice, 2010). University scientists are being told that they must stop pursuing fundamental biological principles and direct their research toward specific societal needs . Venture capital, especially seed funds for life sciences innovation, is drying up (phillippidis, 2011). Finally, the powerhouses of the biomedical industry, the large pharmaceutical companies, have few new products coming to market and are looking at a drop in income of over $100 billion in the next few years (begley and carmichael, 2010; wilson, 2011). The crisis in the biomedical innovation world is what is frequently called a wicked problem, one that is challenging to solve because it involves the interaction of disconnected entities . To solve this crisis requires stakeholders to interact with one another more productively . We believe that research universities are ideally suited to solve wicked problems but need to adjust their operating structures to do so . A few years ago, the petroleum giant bp awarded a consortium of universities led by berkeley the largest grant in university of california history, $50 million per year for 10 yr . Despite the well - publicized travails of bp, that commitment remains firmly in place . Graham fleming, now vice chancellor for research at berkeley but earlier a qb3 leader and one of the architects of the european bioinformatics institute consortium, explains it this way . Manufacture of biofuels is a classic wicked problem, requiring for its solution the expertise of many stakeholders with disparate backgrounds and nonoverlapping goals . Manufacturing biofuels requires economists to verify a market, chemical engineers to design refineries, industrial microbiologists to optimize the enzymatic breakdown of biomass, botanists to select the optimum biomass, agronomists to define the growing regions, and hydrologists to provide them with adequate water . Even a company with the resource base of bp does not have quality expertise in all these fields . In contrast, universities do have the requisite talent, but the trick is to network the faculty, creating a team . The berkeley leadership skillfully assembled a biofuel ecosystem and so deservedly won the bp competition for the $500 million . This example demonstrates that, with inspired leadership, universities can assemble teams to address wicked problems . An obvious challenge is whether creation of an analogous bio - innovation ecosystem might help address the current troubles in the life sciences industries . Insight into how a bio - innovation ecosystem might impact the difficulties faced by the life sciences industries can be garnered by examining the aviation industry . The pharmaceutical and aircraft industries both invest huge amounts of money and time in creating new products . Aeronautical engineers may not completely understand the physics of wing lift, but they can predict what will fly with remarkable accuracy . A plane is designed by engineers, built to their specifications, rolled out on a runway, and takes off perfectly . We have such trust in our aviation knowledge and our engineers that we are not surprised . In contrast, over 90% of candidate drugs fail completely to do any good when administered to patients during phase i trials (paul et al ., 2010). If manufacturing new aircraft were similar to designing new drugs, 9 out of every 10 newly designed planes would crash on takeoff! An airplane cockpit is crammed with indicators that monitor the status of almost every important function . If something begins to go wrong, it is quickly detected and the pilot can take corrective actions and determine if they are indeed working . How do we know whether a drug for alzheimer's or schizophrenia or cancer is having an effect? Lacking quantitative biomarkers that reflect the progress of a disease makes it a huge challenge to measure drug efficacy . As much as the industry needs new drugs, it yearns for biomarkers to precisely measure efficacy and sensitively warn of unwanted toxicities (editorial, 2010). Creating such biomarker sets to monitor a single disease (e.g., alzheimer's progression) we may scoff at the old theories from galen's time of balancing the humors by medical intervention, but in truth we are not much more sophisticated now . To continue the airplane analogy, it is like repairing a defect in an airplane by breaking something else! The explanation of this paradox is emerging, albeit slowly, as we move from reductionism to looking at the human body as a set of interlocking systems . Aircraft engineers have followed a systems approach for decades and biologists are beginning to follow in their footsteps . Until biologists have assembled a systems approach to replace current molecular reductionism, we may have to settle on an empirical strategy to guide drug development, using biomarkers as indicators of benefits and dangers . Successful innovation in the life sciences will require more than assembling teams of academic scientists to develop predictive biomarkers . It will also require that three entities with fiercely independent cultures and largely nonoverlapping goals have meaningful dialogues with one another . Government, academia, and the private sector need to reconcile their separate goals and work together more cohesively . A dialogue with the private sector is the obvious way to find out whether a university discovery has practical implications (silber, 2010). . Universities have poorly defined mechanisms for determining what society, or even the life sciences industry, really needs and so, by default, let research programs be driven by faculty interest, not societal need . For a successful dialogue, the private sector must also have something to learn from the university . What the life sciences industries want is the identification of novel drug targets, disease biomarkers, and disruptive technologies for a start, but few faculty do research pertinent to those wants, even if they are aware of them . The investment community wants to prowl the halls looking for a juicy academic project that will give impressive returns on investment in a small number of years a biological google! The manufacturing companies and the investors are keenly sensitive to regulatory and reimbursement hurdles imposed by government, to tax incentives, and to grants for small businesses . In return, government wants to hear about the link between scientific innovation and tax revenue, economic growth, and job creation (national economic council, 2011). Finally, the universities need to confer with government about what fraction of research funding should be targeted for applied versus basic research (usdin, 2011). Our contention is that the dialogue can be dramatically optimized by creating a university - centered bio - innovation ecosystem . The structures of the ecosystem will facilitate three - way conversations among the three stakeholders and make those conversations disciplined and productive . (companies advise government on university funding; universities give government input on small business grants, innovation zones, and regulatory science; companies help universities identify unmet needs .) We argue that this much - needed innovation in the way research universities carry out their mission will pay off in generating more user - driven innovation in the universities, more efficient use of clinical trials data, a marked increase in efficacious drugs coming to markets, more evidence - based regulatory frameworks, enhanced economic growth through job creation, and consolidation of america's place as a leader in bio - innovation . The paucity of new therapeutic drugs coming to market cannot be attributed exclusively to weaknesses in our pharmaceutical industries . Several concrete steps are suggested: acknowledge that academia must interact more collegially with the two other stakeholders, government and the private sector.recognize that the shortage of new drugs is a wicked problem and that universities are ideal places to solve wicked problems.commit to solving wicked problems by creating networks run by academic generalists who can extract value from our silos of specialization.recruit, reward, and promote academic generalists to coordinate the activities of specialists in the solution of wicked problems.set up criteria that can assess the value of academic generalists . If successful, universities should: foster partnerships between clinical and basic scientists;link academic scientists to industry partners with complementary skills, allowing them to work together to address pressing societal problems;link academics to the resources they need to start up companies;advocate effectively in a nonpartisan manner for improvements in government funding of research and in reimbursement and regulatory policies; andalert their academic colleagues, both faculty and students, to opportunities that match their research specialties to pressing needs of society . Acknowledge that academia must interact more collegially with the two other stakeholders, government and the private sector . Recognize that the shortage of new drugs is a wicked problem and that universities are ideal places to solve wicked problems . Commit to solving wicked problems by creating networks run by academic generalists who can extract value from our silos of specialization . Recruit, reward, and promote academic generalists to coordinate the activities of specialists in the solution of wicked problems . If successful, universities should: foster partnerships between clinical and basic scientists; link academic scientists to industry partners with complementary skills, allowing them to work together to address pressing societal problems; link academics to the resources they need to start up companies; advocate effectively in a nonpartisan manner for improvements in government funding of research and in reimbursement and regulatory policies; and alert their academic colleagues, both faculty and students, to opportunities that match their research specialties to pressing needs of society . Besides supporting research and education in the quantitative biosciences, qb3 has led an aggressive effort to create a small academy of generalists devoted to solving wicked problems and to creating a bio - innovation ecosystem that includes government and the private sector . Although it is still more a work in progress than a blueprint for success, our experience to date suggests there is a role in universities for scientists who are willing to go beyond the comfort zone of their own special expertise . Linking the expertise of others to find solutions to pressing problems can be equally rewarding.
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Generalized convulsive status epilepticus (gcse) and non - convulsive status epilepticus (ncse) are important neurological conditions potentially associated with significant morbidity and mortality.1 even with the best current practice, the mortality of patients with refractory gcse is up to 50% . Therefore, there is an urgent need for new treatment options that can treat these seizures safely and more effectively than the current standard drugs.2 the most common treatment protocols for status epilepticus include an intravenous benzodiazepine, either lorazepam (lzp) or diazepam, as the initial antiepileptic drug (aed) therapy, followed by phenytoin (dph) or fosphenytoin . Phenobarbital (pb) is added if the seizures continue.3 a pharmacologically induced coma, using the barbiturates, propofol or midazolam, is also a frequently used therapy for refractory status epilepticus (rse),2,4,5 which is defined as the persistence of discrete seizures without return to baseline, despite treatment with benzodiazepines and at least two adequate aeds.6,7 however, it requires artificial ventilation and hemodynamic support, and is associated with significant complications and increased mortality.2,4 topiramate (tpm) is an aed with demonstrated efficacy for a broad spectrum of seizure types8; it has multiple activities at receptors and ion channels that may be more effective than conventional anticonvulsants in treating rse.7,9 we report our experience with the use of tpm for the treatment of benzodiazepine - refractory status epilepticus (se) or recurrent seizures in 16 patients who are under severe medical complications such as systemic infection, renal dysfunction, hepatic dysfunction, and pancytopenia from bone marrow suppression . We retrospectively identified patients with gcse or ncse who were treated with tpm at our hospital between july 15, 2006 and august 10, 2008 . The medical records were reviewed for information regarding patient characteristics, associated medical conditions, type of se (gcse or ncse), duration, abnormal laboratory findings, treatment history prior to administration of tpm, and outcome . Hepatic dysfunction was defined as an ast and alt> 200 mg / dl and renal dysfunction was defined as a serum creatinine> 2.0 mg / dl and a creatinine clearance <0.6 . Anemia was defined as a hemoglobin <12.0 g / dl, thrombocytopenia as a thrombocyte count <150,000/mm and pancytopenia as a leukocyte <4,000/mm with anemia and thrombocytopenia . Administration of aeds, other than tpm, followed our hospital s protocol: lzp (4 mg) was injected intravenously and dph (15 mg / kg) or vpr (20 mg / kg) were infused intravenously for 30 minutes . The mixture was allowed to sit for several minutes to avoid clumping and then administered via syringe into a nasogastric tube . The loading dose was individualized by patient; 612 mg / kg / day, up to 1,000 mg / day . The resolution of recurrent gtc and gcse was determined by cessation of clinical seizures, and confirmed by follow - up electroencephalopgrahy (eeg). For ncse, from the database on se patients, 16 patients were identified for inclusion in this study . No patient had a history of seizures or was receiving tpm or other aeds prior to admission . The most common previous, co - morbid disease at the onset of seizures was sepsis (n=8). The laboratory studies showed that 12 patients had hematological problems (pancytopenia 8, anemia alone 2, and anemia with thrombocytopenia 2). Seven patients had renal dysfunction, three patients had hepatic dysfunction only and another four patients had both renal and hepatic dysfunction . The majority of patients had experienced gcse (n=6) or ncse (n=7). The mean tpm treatment dose was 637.5244.6 mg (3001,000 mg). Within a few days, 13 patients could experience their seizure control (the mean duration, 3.72.6 days; range 18 days), but the seizures of the other 3 subjects did not be terminated in spite of all efforts . None of the patients had a worsening of their cbc or blood chemistry profiles with the tpm treatment . For seven patients, the tpm was administrated after the failure of the loading of high doses of dph or vpr . The administration of tpm was within 2 hours after the infusion of dph or vpr . The overall outcome was described as follows: patient death (n=11), improved (n=4), transferred to another hospital (n=1). The causes of 11 deaths were sepsis (n=7), fulminant hepatitis, sah, hypoxic brain damage, and herpes encephalitis . We now represent two illustrative cases using tpm for controlling recurrent seizures from a variety of medical derangement . A 48-year - old woman with no history of seizures was admitted to the hospital in poor health . The patient was diagnosed with paroxysmal nocturnal hemoglobinuria (pnh) 20 years prior to admission and had been treated as an outpatient with oral steroids . The routine blood tests on admission revealed pancytopenia (hemoglobin 4.4 g / dl, hematocrit 13.3%, wbc 730/mm, and a platelet count of 52,000/mm) and acute renal failure (bun 126.5 mg / dl and creatinine 9.23 mg / dl). Continuous renal replacement therapy (crrt) was performed in the intensive care unit . On the third hospital day, gcse developed . A loading dose of tpm (800 mg) was administrated via a nasogastric tube . By the next day, however, intermittent brief seizures occurred several times under a maintenance dose of tpm (300 mg bid) during the subsequent two days . The diffusion - weighted mri revealed multifocal high signal intensity involving the occipital lobes, the right basal ganglia and the subcortical white matter of the prefrontal gyri on the diffusion and t2-weighted images . The identified lesions showed increased values on the adc map, suggesting vasogenic edema or a posterior reversible encephalopathy (pres). The tpm was maintained at a dose of 200 mg bid, and there was no further seizure activity . However, aspiration pneumonia and sepsis developed, and the patient died on the 15th hospital day . A 56-year - old woman was admitted to the hospital with impaired mental status . The patient had a history of multiple myeloma (stage iiia) diagnosed three years prior to the admission vad chemotherapy using vincristine, doxorubicin and dexamethasone was started . However, generalized seizures developed during the third cycle of chemotherapy, and the patient became unresponsive with her eyes deviated to the right side . The routine blood tests revealed pancytopenia (hemoglobin 5.2 g / l, hematocrit 14.4%, wbc 4,570/mm, and a platelet count of 15,000/mm), and septic shock developed . However, the blood culture revealed staphylococcus epidermidis, and she died on the 49th hospital day from septic shock . We described 16 patients with gcse, ncse or recurrent gtc in whom tpm was administered alone or together with other aeds . Most patients with overt, convulsive status epilepticus respond to the first or second aed . However, when the patient fails to respond to this standard protocol during the initial treatment, the patient is considered refractory and requires additional, more aggressive treatment.3 rse is a life - threatening condition that carries a high mortality risk . Aggressive, early intervention permitting avoidance of a pharmacological coma can reduce the overall morbidity and mortality.4 current recommendations for the treatment of refractory status epilepticus include midazolam (mdz), pentobarbital and propofol.10 however, respiratory depression and/or hypotension may result from this treatment, which may necessitate endotracheal intubation and/or vasopressor support .10,11 oral aeds such as tpm or lev, however, have much to offer if they can pre - empt these more aggressive managements.12 tpm has at least five independent actions at the cellular level13: inhibition of kainate - evoked currents, enhancement of -aminobutyric acid (gaba)-evoked currents, blockage of voltage - activated sodium channels, blockage of voltage - activated calcium channels, and inhibition of carbonic anhydrase isoenzymes . The multiple mechanisms of action confer broad - spectrum efficacy against different seizure types and make tpm an attractive choice in the treatment of both partial and generalized se.14 since tpm has pleiotropic effects on neuronal excitability, a favorable pharmacokinetic profile and appears to have few side effects in the setting of rse, it is a reasonable therapeutic option in patients with se.3 tpm is also considered appropriate for treatment of patients with of hepatic or renal disease . It does not significantly increase the risk of hepatotoxicity and renal disease is not a contraindication to the use of tpm.15 there are some reports on the use of tpm in se patients . 16 reported a patient with drug - resistant complex partial se who responded to tpm . Towne and colleagues described six adult patients with a variety of types of se who were unresponsive to conventional treatment, including two who also failed to respond to treatment with pentobarbital.7 a suspension of tpm administered by a nasogastric tube was effective in aborting the rse in all cases . Remarkably, no adverse events were observed and all six patients who survived and were discharged from the hospital . Bensalem et al . Also described the use of tpm via a nasogastric tube in three patients with rse (two with generalized seizures and one with partial seizures).14 tpm appeared to be very effective in stopping the rse; in two of the patients that were resistant to either pentobarbital or propofol, and no adverse effects were noted . Patients who had se after therapeutic doses of at least two antiepileptic medications were given tpm, 10 mg / kg / d for consecutive days, followed by maintenance doses of 5 mg / kg / d . In each case, the se was stopped within 21 hours of the initial dose of tpm . In our study, the seizures of subjects were terminated both clinically and electrographically in 13 out of 16 patients . The timing of improvement in our patients was within 18 days of starting the administration of tpm . These findings are consistent with the pharmacokinetic properties of tpm and previous reports . In six cases, given tpm after loading with dph or vpr failed, tpm successfully stopped the seizures, even in medically complicated patients . Although seizures were stopped clinically and electrographically in the majority of our patients, the overall outcome was not very favorable in our study . . The high mortality rate of the patients studied reflects the large number of leukemia and lymphoma patients treated in our hospital . The majority of the patients studied had serious and often life threatening hematological problems such as pancytopenia or thrombocytopenia . They had a variety of medical comorbidities, including systemic infection, hepatic or renal dysfunction and hematologic / oncologic disorders . However, our results convincingly support that tpm is safe and effective in a special group of seriously ill patients . We could find that tpm was not only safe but very effective for the control of recurrent epileptic seizures or se in patients with serious medical co - morbidities . Tpm may be considered as another treatment option when conventional protocols fail . A large prospective, randomized, controlled study is warranted to investigate the efficacy and safety of tpm for the treatment of se.
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Poly(vinyl alcohol) (pva) is a biocompatible polymer with a great variety of biomedical and pharmaceutical applications . Pva in aqueous solution is able to form chemical or physical gels under a variety of conditions . The physical gelation capacity of pva solutions has been well known for a long time; however, the preparation of hydrogels by repeated freezingthawing cycles has attracted renewed interest . The high degree of swelling in water, the rubbery elasticity, the chemical and mechanical stability, the porous fibrilar network structure, and the lack of toxicity makes freezethaw pva hydrogels an attractive polymer matrix for biotechnological applications . To understand the origin of these properties and therefore the bioapplicability of this material, it is necessary to study the physical gelation process produced by the freezingthawing cycles as well as the formed network structure . Komatsu el al. (13) investigated the phase diagram of the water / pva binary system . According to this diagram, gelation occurs with or without spinodal decomposition according to the polymer concentration and temperature . The process seems to start with a clustering of chains, which is primarily caused by the association of polar groups of the dissolved polymer, followed by polymer crystallization . This means that the physical cross - links, which are responsible for the network formation, could be formed by different processes, such as hydrogen bonding, polymer crystallization, and in some cases (depending on the gelation conditions), phase separation. (14) in addition, the application of freezingthawing cycles to pva solutions leads to the formation of heterogeneous networks with different morphology and properties as compared with nonfrozen gel systems . Were the pioneers in the development of this type of polymer cryogels. (7) in earlier work, they attribute the gel formation to a partial crystallization of chain segments to microcrystalline structures . However, the intensity of the diffraction maximum that corresponds to the 101 reflection is clear for only pva hydrogels with high polymer content . For hydrogels with 10 to 15% polymer, solid - state nmr was used to identify and quantify the crystalline phase, although x - ray analysis is also possible. (29) most recent works show that gelation by freezethaw processing forms heterogeneous networks of interconnected micro- and macropores . Willcox et al.,(28) in an exhaustive study using transmission electron microscopy (tem) images and nmr, showed the formation of networks with rounded pore morphology, fibrillar network morphology, or both depending on the number of cycles as well as aging . They attribute the formation of cross - links to a kinetically frustrated crystallization in the first freezing cycle . After this process, subsequent cycles (or aging processes) lead to the creation of new (secondary) crystallites and the growing of the primary crystals; however, the mesh spacing slightly changes . This fact seems to indicate that the average distance between the primary crystallites that are formed during the first thermal treatment constitutes the main controlling factor for the network structure . In addition, auriemma and coworkers in a series of works showed that the porous structure originates from freezing in the first step because of the incomplete crystallization of water . The polymer concentration in this unfrozen water phase is higher than that in the original solution, and the polymer network is formed within this microphase because of pva crystallization . The cross - link points are therefore constituted of polymer crystallites, and the formed gels could be described by a bicontinuous structure of polymer - rich and polymer - poor regions . On the basis of dsc measurements, also inferred the possibility of the existence of covalent cross - links between the polymer chains brought about by the formation of radicals due to strong local shear during water crystal growth. (33) in summary, the pva hydrogels obtained by freezingthawing cycles appear to be constituted by a very complicated network structure that is based on different phenomena (i.e., crystallization, hydrogen bonding, liquidliquid phase separation, and covalent bonds). Therefore, a complete analysis of this structure requires the use of different characterization techniques . However, most of them require the manipulation of the hydrogel sample, provoking changes in the original structure . For example, the use of chromatographic techniques or scattering techniques to measure molecular weight or cluster size requires the dilution of the gel(24) (although it was shown that it is possible). Mechanical measurements have the limitation of water evaporation at higher temperatures. (9) the cryo - tem technique has the inconvenience of handling difficulties to avoid artifacts. (28) for the study of the crystallite content, very useful techniques such as differential scanning calorimetry (dsc) as well as x - ray diffraction are challenged by a sensitivity problem in dilute samples; for this reason, measurements were sometimes made in dry samples. (34) in this vein, solid - state nmr is a powerful tool for studying polymer networks, but it has not been extensively used in this field . The focus of this work is the use of a low - field nmr spectrometer to analyze the structure of the pva gel in detail as a function of the freezingthawing cycles, checking all of the structural models suggested in the literature . In addition, a melting process that transforms these physical gels into isotropic solutions of pva in water was also studied . Importantly, we focus on detecting protons so that sensitivity, even at the low concentrations under study, is not a serious issue . The use of an inexpensive low - field nmr spectrometer on as - prepared samples to study this complex matrix has the advantage that several of the cited inconveniences in sample characterization are avoided . By using different nmr methodologies, we are able to carry out a complete and quantitative study of the complex structure exhibited by the pva gels: multiple - quantum (mq) nmr allows us to gain direct access to residual dipolar coupling constants that persist because of the existence of cross - links and other topological constrains . Therefore, this experimental procedure is a powerful tool for quantifying the gelation process(41) and gives us quantitative detail on not only the microstructure(42) but also the dynamics(43) of the gel, that is, a complete picture of the network structure evolution depending on, for example, the number of freezingthawing cycles . In addition, we used pulsed mixed magic - sandwich echo (mse) experiments that provide a near - quantitative refocusing of the rigid contribution to the initial part of the free induction decay (fid)(44) and allow for an essentially quantitative determination of the crystallinity . To our knowledge, this is the first time that advanced nmr pulse sequences performed on a low - field spectrometer were applied to the study of the network structure and phase composition of physical hydrogels . The sample was> 99% hydrolyzed, had a weight - average molecular weight of 94 000 g / mol, and had syndio-, hetero-, and isotacticities of 17.2, 54.1, and 28.7%, respectively . Pva was dissolved in deuterated water (with polymer concentrations of 10 and 20% w / w) at 80 c under continuous stirring to avoid inhomogeneities and local gelation . Solutions were stored at 80 c overnight; then, they were cooled to room temperature for 1 h. then, pva solutions were introduced to 10 mm od nmr tubes and were flame sealed to avoid variations in the water content . Two technical considerations are important at this point: (i) the quantity of sample inside the tube has to be 8 mm in height and located in the center of the radio frequency (rf) coil to ensure good rf field (b1) homogeneity . (ii) the length of the nmr tube should be as short as possible for good control of the temperature . This is important because in this way, the whole tube can be inserted in the probe head, thus avoiding possible temperature gradients due to heat conduction . Pva hydrogels were obtained by repetition of freezingthawing cycles of solutions inside the sealed tubes that consisted of 1 h of cooling to 32 c and 1 h of thawing at room temperature . After this process, samples were immediately inserted in the nmr spectrometer and measured to avoid the effect of aging . In some cases (specified below), the first freezing cycle was extended to 12 h (overnight), and subsequent cycles were 3 h long . Solid - state h nmr spectroscopy is a powerful and widely used experimental technique in the field of polymer science. (35) in recent years, the use of inexpensive and easy - to - use low - field spectrometers has gained the attraction of scientists because it certainly produces quantitative results on polymer structure and dynamics, albeit, of course, without chemical resolution . On the basis of different experiments, which will be extensively described in the next sections, applications include quantitative studies on polymer network structure(38) and polymer crystallization,(44) that is, the main topics of this study . In this work, experiments were carried out on a bruker minispec mq20 spectrometer operating at 0.5 t with 90 pulses of 1.7 s length and a dead time of 12 s . Mq spectroscopy is one of the most versatile and robust quantitative techniques for investigating not only the structure but also the dynamics of polymer networks. (38) in solution, the fast segmental motion of the polymer chains through the different accessible conformations is isotropic and completely averages the dipolar coupling interaction that is typical for solid - state spectra . However, constraints, irrespective of their nature (e.g., cross - links, entanglements, or chain packing), lead to nonisotropic segmental fluctuations and therefore to the persistence of a weak residual dipolar coupling (dres), which is our central nmr observable . In other words, the measured effect relies on the orientation dependence of the fluctuating dipolar coupling tensor with respect to the magnetic field, which can be described by an orientation autocorrelation function of the individual chain segments . Fast segmental dynamics (in the range of nanoseconds to microseconds) lead to a loss of correlation to a plateau value that is related to the existence of preferential local orientation generated by the existence of cross - links . In fact, the measurable dres is directly proportional to a local dynamic order parameter of the polymer backbone,(38) which is defined as a time average over the fluctuations of the segment - fixed dipolar tensor over the time until the plateau region is reached . It connects the experimental observable with the network parameters: here dstat is the segmental averaged dipolar coupling constant in the static limit and k is a rescaling factor that takes into account the fast dynamics (in the range of picoseconds) inside the statistical (kuhn) segments . The nmr observable is related to r, that is, the ratio of the end - to - end vector to its average unperturbed melt state (r = r/r0), and to n, the number of statistical segments between constraints . A variety of nmr experiments was used to detect residual dipolar couplings to monitor gelation processes, and recently, h mq nmr has evolved as the most powerful tool for obtaining a direct measurement of dres and even its distribution . Details of the pulse sequence as well as the data analysis are already published elsewhere . Here temperature usually plays an important role in mq measurements(43) because as previously mentioned, the order parameter should be obtained as an average of the segmental fluctuations over all of the possible conformations on the experimental time scale (less than microseconds). Therefore, the temperature is required to be far above the polymer glass - transition temperature tg to ensure that the segmental dynamics is fast enough to sweep out the whole conformational space between topological constraints and to achieve full averaging . If this is not the case, then the obtained dres (and therefore the number of junctions in the network) will be overestimated. (41) in this work, the studied samples are hydrogels; therefore, the dissolved state of the polymer chain ensures that the segmental dynamics is indeed fast enough to detect the order parameter in the plateau regime, even at room temperature . Similar to the traditional transverse relaxation experiments, the analyzed result of the mq experiment is a time - dependent but normalized double - quantum (dq) signal function, indq, that has the advantage of being independent of any temperature - dependent true relaxation (decay) effect . It is dominated by the dipolar interactions and independent of the polymer dynamics . For this purpose, the directly measured experimental dq build - up curve (idq) must be relaxation - corrected by the use of the also measured reference intensity (iref). Initially, iref contains a signal from not only the dipolar - coupled network chains but also the uncoupled, isotropically mobile components such as sol, dangling chains, and so on . The total mq magnetization (imq) needed for normalization is the sum of idq and iref but only after subtracting the non - network contributions . This fraction has a slower relaxation and can be easily identified (figure 1a). In the studied hydrogels, even though we used deuterated water to dissolve the polymer, we found a non - negligible water signal resulting from actual residual water and exchanged oh protons . Therefore, we had to identify and subtract two exponential long - time contributions to imq (i.e., the noncoupled fraction of polymer chains (b fraction in eq 2) and the solvent signal (fraction c)), which relax with longer relaxation times (t2c * t2b *) mq nmr build - up and decay data for 10% pva sample after one freezingthawing cycle . (a) as - acquired idq and imq and (b) imq (after extraction of noncoupled contributions) and normalized dq build - up curves (idq). The solid line in a represents the exponential fit for water subtraction, and the dashed line is the exponential fit representing the subtracted noncoupled polymeric defects . In b, solid and dashed lines represent the fits assuming a gamma distribution of dres and a single residual dipolar constant, respectively . (see figure 1b .) Finally, it is important to point out that in rubber networks the distribution effects of dres (related to different end - to - end separations and polydispersities of network chains) usually do not play any role. (56) therefore, the normalized dq build - up curve of a bulk rubber can be analyzed in the quasi - static limit in terms of a single dres(38) however, the complex and heterogeneous pva network structure, in addition to specific effects related to the swollen state of the sample (which is proven to be heterogeneous in polymer networks(57)), leads to large deviations from the inverted gaussian shape in the studied pva gels . The actual coupling distribution (related to a microstructurally heterogeneous distribution of constraints) the insert in figure 1b shows the broad distribution of dipolar interactions in pva networks . It resembles a gamma distribution and is consistent with observations of very heterogeneous network structures proposed in the literature . All analyzed samples showed a similar distribution shape independently of the number of freezingthawing cycles . Because regularization analysis is subject to some limitations(42) (further work about this point is in progress), we have analyzed all build - up curves under the assumption of the gamma distribution of dipolar couplings that is predicted by the gaussian distribution of end - to - end separations (but which is screened in bulk elastomers) upon fitting, the integral is numerically evaluated within the curve fitting environment of the origin software . In using this distribution, we do not want to imply that this distribution is of any significance in the studied structure; the resulting build - up curves merely describe all of our experimental build - up data very well and have the benefit of not introducing an additional fit parameter, yet they provide a well - defined average residual coupling davg . (the width of the gamma distribution is directly related to the average .) Finally, note that the mq analysis pertains to the mobile fraction of the cryogels only; this fraction always constitutes the major part of the sample (> 80%); the rest is rigid crystallites, and the signal of which rapidly relaxes and is not observable under the conditions of the mq experiment . According to previous papers, polymer crystallites act as junctions in pva networks obtained by freezingthawing cycles . Solid - state nmr is a useful method for characterizing polymer crystallinity or phase composition in this type of system . The main concept is that the crystalline signal usually decays in the first 20 s of the free - induction decay; therefore, the signal detected after the dead time (12 s in our case) partially conceals this information . Nevertheless, in some works, the fraction of protons in a glassy state in pva gels was estimated by monitoring the fid intensity during the first 20 s, which renders these studies qualitative . Perhaps the most important is the use of solid echoes with different echo delays combined with back extrapolation(64) to correct for its inability to refocus the dipolar interactions in a multiple - spin system fully . Certainly, the best approach is the use of a spectrometer with short (1 to 2 s) dead time. (65) the use of an mse was recently proposed(44) as a more effective method for refocusing the multiple dipolar interactions that lead to the fast decay of the initial part of the fid, therefore removing the dead - time problem and obtaining a near - quantitative rigid fraction determination in polymer systems . Under the same experimental conditions, differences of 40% in the quantity of rigid phase in pva compounds figure 2 shows the mse - fids for different total echo duration (realized by increasing the interpulse spacings). The signal decay data are well represented by a gaussian function (parabolic initial decay), and its extrapolation to t = 0 allows us to conclude that the signal loss is negligible . (it could be estimated to be 2% on the absolute scale, which corresponds to 10% on the relative scale .) Parabolic (gaussian) extrapolation of the maximum mse - refocused fid intensities to t = 0 . It indicates only minor losses of rigid signal at the shortest echo time (10% on a scale relative to the total crystallinity). The inserted graph shows mse - refocused fids of 10% pva gel after six cycles measured at different echo times at room temperature (304 k). However, note that even for the mse the refocusing efficiency breaks when there is molecular motion on its time scale . In particular, this may be the case if the observed rigid component is glassy rather than crystalline and close to tg . Under any condition irrespective of temperature, however, the typical decay time upon incrementing the echo was never significantly shorter than the (relatively weak) decay seen in figure 2, which is ultimately associated with the breakdown of the pulse sequence efficiency because of higher - order imperfections . We thus conclude that all observed rigid signals are associated with crystal - like domains; otherwise, a significant signal loss upon going through tg would have been observed . Therefore, mse - fid curves were used to quantify the fraction of fast - decaying rigid polymer present in pva gels . The first fast decay (caused by the rigid polymer fraction) in the mse - fids (insert in figure 2) has a quasi - gaussian decay, whereas the slower signal decay could be fitted with an exponential function . Therefore, the first 140 s of the normalized mse - fid curves (unity signal for zero time) were fit according to eq 6 in this expression, a is the fraction of detectable rigid phase in the sample and b is an adjustable parameter for a better fit of the fast decay shape . In all of the cases, b 2 (i.e., the crystalline fraction is described by a gaussian function). In addition, to reference the hard polymer phase to the total amount of polymer (instead of the sample fraction), we corrected the a parameter according to the actual fraction of polymer in the sample obtained from saturation recovery experiments (note again that the samples contain residual water) on the basis of the fact that water has a much longer t1 relaxation time . From such experiments, and assuming no significant noe transfer between the species on the same time scale, the water content can be obtained from a biexponential fit where d is the water fraction in the sample and t1w and t1p are the longitudinal relaxation time constant of water and polymer, respectively (with t1w t1p). For example, at t = 304 k, the relaxation times were t1w = 1520 ms and t1p = 60 ms for a network with 10% pva after seven freezingthawing cycles . The fast - decaying rigid crystallite signal was not detected in these experiments (the fid was evaluated at a time beyond 50 s) such that d is determined relative to the mobile polymer fraction (i.e., (1 a) in eq 6). Finally, the rigid polymer fraction discussed below was obtained according to obviously, for these corrections, it is essential to use a recycle delay that is long enough to observe the full equilibrium magnetization of the sample (5(t1w) was used as a recycle delay in all experiments, i.e., 720 s depending on the temperature). The aim of this work is to study the structure and phase composition of pva cryo - gels by using a low - field nmr spectrometer . To revise the structural models suggested in the literature, we will separately analyze the effects of three factors on the pva network structure: (i) the number of applied freezingthawing cycles, (ii) the temperature and melting process, and (iii) the aging process . To achieve this objective, in every point, dq experiments were performed to quantify the proportion of noncoupled network defects, and the molecular weight between cross - links and mse - fid pulse sequence were applied to determine the rigid fraction of the polymer . The starting point of our study is the characterization of two isotropic solutions of 10 and 20% (w / v) of pva in water . The 10% pva solution does not give any detectable dq signal, but for the decay (iref) data, two different processes can be identified (i.e., the decay of the polymer magnetization and the water relaxation). This result demonstrates that under these preparation and measurement conditions, the dilute solution can be considered to be nonentangled and above the gelation threshold . This is not a trivial result because dq experiments are, in principle, sensitive to transient links (entanglements or hydrogen bonds), and this indeed appears indicated by the 20% pva solution; under the same preparation conditions, dq experiments did yield a detectable build - up curve . However, this is in our case due to a nondissolved or partially gelled pva subphase . To avoid this problem, 20% pva solutions were prepared under more vigorous stirring as well as higher temperatures up to 90 c . In this way, a totally isotropic solution of polymer was obtained according the dq experiments . Dq experiments were performed on pva samples subject to different numbers of freezingthawing cycles to determine the evolution of noncoupled network defects as well as the network mesh size . In figure 3, we can see that after the first freezingthawing cycle, 50% of the polymer chains are coupled and are therefore part of the network, whereas the rest are not linked to it or at least belong to elastically inactive dangling chains or loops . The number of pva chains incorporated in the gel network increases with the successive cycles until it reaches a plateau after the sixth cycle, where the network has the minimum defect content . This important result shows that independent of the number of freezingthawing cycles applied to a pva solution 25% of the polymer is not coupled and therefore should not be elastically active . This is a very significant observation that is usually not taken into consideration when mechanical, dynamic - mechanical, or rheological properties of this material are analyzed . Urushizaki et al. (66) have related the viscoelastic properties of this type of gel to the nonincorporated pva chains, and they have estimated their amount from the difference in weight between the polymer gel before and after immersion in distilled water . They found that 10% of the polymer was not incorporated after the first freezingthawing cycle . Similar results were also indirectly inferred from the measure of intradiffusion coefficients of micellar aggregates of surfactant molecules dissolved in these types of gels. (67) obviously, our experimental values are much larger because we detect not only the non - cross - linked chains but also the dangling chains and other nonelastic network defects (e.g., loops). The effect of d2o in measured noncoupled network defects is another important point to take into consideration when this fraction of polymer is analyzed . Pva monomers have an exchangeable proton (oh), which, in presence of d2o, could be replaced by deuterium that is undetectable by h nmr, whereas the proton will become detectable as water (hdo). Free water signal (h2o or hdo) is subtracted from the long - time decay of the signal because it can be identified by its own longer t2 . However, the number of noncoupled network defects could be slightly overestimated because of the protondeuterium exchange . Variation of noncoupled network defects (i.e., non - cross - linked polymer chains, dangling chains, and loops) as a function of the number of freezingthawing cycles for the studied pva solutions . In addition, the decrease in the number of defects is correlated with an increase in dres that is proportional to the network chain order parameter (eq 1), see figure 4 . The formation of cross - links (independent of their nature) renders the polymer segmental motion nonisotropic, whereby the chain segments become ordered with respect to the end - to - end distance, and residual dipolar couplings arise . According to this basic principle, the increase in dres is directly related to the (inverse) length of the chains between the constraints . Similar to the observation for the noncoupled network defects, the network chain length appears to approach a plateau after cycle number six, yet the tendency is not as clear as the former result . Even after 16 cycles, the cross - link density still appears to increase . Variation of the average residual dipolar couplings (directly related to 1/n and therefore the cross - link density or the inverse mesh size) extracted from dq experiments as a function of the number of freezingthawing cycles for the studied pva solutions . Represents a sample containing 20% pva after 3 h of freezing, and is from the same sample after 16 h of freezing . With this observation, it is important to note that samples with different polymer concentration appear to exhibit the same behavior during gelation . A possible explanation could be that in the framework of a heterogeneous scenario cross - linking occurs via partial crystallization in the nonfrozen parts of the sample; it simply means that in less concentrated samples more water freezes . This fact could explain the dependence of the dimension and shape of pores on polymer concentration and the regimes of cryogenic treatment. (20) in this context, it appears to be worthwhile to study the influence of the actual freezing temperature, which should affect the polymer concentration under the conditions at which the crystallites are formed . However, the freezing time (especially in the first cycle when the network is formed) seems to have some influence in the gel network structure . As is shown in figure 4, the apparent dipolar coupling constant is very low (48 hz) after 1 h of freezing at 32 c . If the freezing period is increased to 3 h, then the cross - link density of the gel almost doubles (82 hz), whereas longer freezing (e.g., 16 h) does not lead to further variation this may indicate that 1 h of freezing is not long enough to complete the process, and for this reason, further analyses are based on samples with a first freezing cycle of 12 h and subsequent cycles of 3 h each . Returning to the origin of the pva networks produced via freezingthawing cycles from homogeneous polymer solutions in water, it appears to be well demonstrated by now that during the first freezing cycle, freezing of some water(32) increases the concentration of pva in the still unfrozen phase. (31) crystallization of pva takes place in this concentrated microphase,(68) but it is kinetically frustrated by the gelation of the polymer solution . This fact could explain the small size of these primary crystallites (5 nm), which then act as physical junctions between the amorphous and mobile polymer chains . Results extracted from the mse - fid curves of the pva gels after the first freezingthawing cycle (figure 5) indicate that 8% of the polymer behaves like a rigid solid . This rigid fraction increases with the number of cycles until it reaches a plateau close to cycle number six, just as our other reported network properties . The maximum amount of rigid phase estimated by our nmr methodology (20% of the total polymer) is substantially higher than the crystallinity of similar samples deduced by dsc as well as some other h nmr methods . Variation of the rigid (crystalline) polymer fraction extracted from mse - fid curves as a function of the number of freezingthawing cycles for the studied pva solutions . In all cases, the crystallinity of pva cryo - gels obtained in dsc studies is estimated to be 5% (not taking into account studies of dry networks(34) because the structure should be different). However, as was pointed out by willcox et al.,(28) this magnitude could very well be underestimated because the heat of fusion of the crystals in gels may be substantially lower than the assumed pva bulk value because of their small size and large surface effects (hydrogen bonds to surrounding water, etc . ). In addition, another important point is the difficulty in measuring small quantities of crystal phase in dilute gels via calorimetric methods in general; for example, a crystallinity of 8% in a gel with a pva concentration of 20% represents only a sample fraction of 0.016 . Nevertheless, this difficulty seems to be reduced by the use of microcalorimetry . In this way, similar results were obtained by performing this type of measurement on gels with low pva concentrations,(8) although the uncertainties related to the obtained results are still large . For these reasons, the use of solid - state nmr seems to be a better methodology for quantifying the crystallinity in dilute pva gels . However, there are still some discrepancies in the total amount of rigid phase measured by the use of different nmr methodologies . Ricciardi et al. (18) estimated the crystallinity of pva gels after 11 freezingthawing cycles to be 8% by analyzing the first microsecond of the h fid . Clearly, as was pointed out in the, this experimental procedure falls victim to the dead - time problem . Depending on the spectrometer, the amount of rigid signal lost during this time could be more or less significant . (ref (15) does not specify the dead time; therefore, it is not possible to estimate the related error .) For example, our low - field spectrometer has a dead time of 12 s; therefore, the fid analysis would lead to an underestimation of the rigid polymer fraction by 50% . By using different c nmr experiments (direct polarization as well as hc cross polarization), willcox et al. (28) established that after the first freezingthawing cycle, pva gel contains a rigid polymer phase of 5 2%, increasing up to 12 4% after cycle number 12 . As was pointed out by the authors,(28) the relative fraction comparing different samples could be estimated with a high accuracy, but the used methodology (in particular, the use of the intrinsically nonquantitative cross polarization) does not give a reliable absolute crystallinity . In conclusion, the key to obtaining the most accurate crystal fraction in these dilute polymer samples is to use a solid - state nmr method that allows us to detect the rigid part fully by refocusing the multispin dipolar interactions with the mse, thus solving the dead - time problem and minimizing the signal loss . Of course, detecting the more - sensitive and 100% abundant protons instead of the naturally abundant c is an additional advantage, which more than compensates for the use of an intrinsically less - sensitive low - field spectrometer . Summarizing all results discussed so far, the network structure of pva cryo - gels is very apparently based on the formation of rigid polymer areas (most probably polymer crystallites) during the cooling process . After the first cycle, almost half (8%) of the final amount (20%) of the rigid phase is already formed . In this stage, subsequent freezingthawing cycles increase the rigid polymer fraction, which has two consequences: first, the noncoupled polymer fraction is further reduced (i.e., more pva segments become elastically active), and second, the network mesh size is further reduced but only slightly during further freezingthawing cycles . Therefore, we believe that subsequent freezingthawing cycles not only increase the size of the crystals formed during the first freezing process but also lead to the creation of other more imperfect crystals . Independent of the polymer concentration of the gel, the melting of smaller and more imperfect secondary crystals takes place at lower temperature and over a large range, leading to a continuous decrease in the detected rigid polymer phase with the temperature (figure 6). At 7580 c (in good agreement with the main endotherm observed in dsc measurements), this quantity drops below the detection limit . This should be correlated with the melting of the main (primary) crystals (estimated to 5% of the polymer fraction) that support the network structure of the gel . These results are consistent with dsc measurements because they show an increase in the melting endotherm that corresponds to the initial crystals as well as to the appearance of a second endotherm at lower temperatures when the number of freezingthawing cycles was increased for a given pva solution . Variation of the rigid polymer fraction (extracted from mse - fid experiments) as a function of temperature for pva gels formed after seven freezingthawing cycles . A network with 10% pva was first heated until 324 k () and then kept at room temperature for 12 h before the second heating procedure was performed (). A solution with 20% pva was submitted to two different treatments, that is, one freezingthawing cycle and seven freezingthawing cycles, respectively . All samples were frozen for 12 h in the first cycle and 3 h in the subsequent cycles . The effect of the number of freezingthawing cycles on the crystallinity is easily understandable by comparing the evolution of the rigid polymer fraction of a pva gel obtained after seven cycles with that of a pva gel after only one freezingthawing cycle (figure 6). The onset of melting after only one freezing cycle is estimated to be 50 c, leaving the rigid polymer fraction constant at lower temperatures . Therefore, subsequent cycles seem to create more, yet imperfect, secondary crystallite structures . Above 50 c, all samples exhibit a similar behavior related to the partial melting of primary crystals or to the melting of less - perfect secondary structures . Importantly, the temperature above which it is not possible to detect any more signal of rigid polymer remains invariant, yet the final fraction seems to increase slightly with the number of cycles . Therefore, according to this result, freezingthawing cycles have a direct influence on not only the number of crystals formed during the first freezing process but also the creation of other more imperfect secondary crystallites . To ensure that the results shown in figure 6 are not artifacts produced by different magnetization relaxation at different temperatures that could possibly arise from regions that undergo a glass - to - liquid rather than a melting transition, the evolution of the signal at any given temperature was studied as a function of echo delay, see figure 7 . At all of the measured temperatures, similar behavior was found, with a loss of intensity estimated to between 0.7 and 1.8% for the two extreme cases, which corresponds to a constant 10% loss on a relative scale, as is usually found . Importantly, the drop in overall signal corresponds, within the error margins, to the expected drop due to the curie factor (temperature - dependent spin polarization). In addition, a direct check is the control of the total intensity, comparing the pva gels to the corresponding pva solutions (insert in figure 7). It is seen that no significant signal is lost / undetectable (we observe almost all protons in the sample), and from this, it is clear that the loss of intensity caused by the mse in pva gels is at a minimum, with no other (hidden) phenomena that could be related to an intermediately mobile (almost glassy) rigid fraction . Given this experimental evidence, the decay of the rigid h fraction with temperature can be related to only a melting of pva crystallites . Variation of the initial signal intensity of mse - fids with increasing echo delay at different temperatures for a pva gel containing 20% polymer after one freezingthawing cycle (12 h of freezing and 1 h thawing). The insert presents the mse - fid of the pva gel measured at 304 k with the minimum echo delay (0.0022 ms) as well as the estimated intensity at zero echo time in comparison with the mse - fid of the isotropic solution of the same sample measured under the same conditions after complete melting at 90 c . Obviously, the temperature - dependent variation of the rigid polymer phase has a direct effect on the network structure, as shown in figure 8 . The melting of secondary crystals leads to an almost exponential increase in the number of network defects (obviously, in a reversible pathway, comparing the phenomenon with the effect of successive freezingthawing cycles). However, the network mesh size (as measured by the residual dipolar coupling) remains almost constant . This means that secondary crystallization is very important in the network organization, reducing the number of network defects and therefore increasing the number of polymer segments that are elastically active . However, the molecular weight between the constraints appears to be dictated mostly by the primary crystallites formed during the first freezing cycles . For this reason, the network is completely destroyed at temperatures of 80 c when the primary crystal phase is molten, leaving no observable dq signal (i.e., an isotropic pva solution is formed). On the side, we note that the differences in the davg values given in figures 4 and 8b, which indicate a 50% decreased mesh size for the samples studied in figure 8, are related to the increase in the freezing time (12 h in the first step and 3 h in the successive cycles instead of 1 h) used in each cycle, as pointed out above . Variation of (a) the network defect fraction and (b) the inverse mesh size (given by davg measured by mq nmr) with temperature for the 20% pva gel prepared after seven freezingthawing cycles (12 h first freezing cycle, 3 h subsequent cycles). Error bars represent the fitting uncertainty, whereas lines are only guides to the eye . These nmr results on the mesh size perfectly agree with the trends observed in studies of mechanical properties reported in the literature . Clearly, the elastic behavior of pva gels depends on the number of freezingthawing cycles . Therefore, the main factor that determines the improvement of the mechanical properties upon further cycles should be associated with the number of elastically active chains . The polymer concentration in the original aqueous solution has an important effect on the elastic properties; that is, the elastic modulus increases . However, in the view of our data, this does not appear to have important consequences for the actual network structure; that is, there are no large variations in the network mesh size . Therefore, the reported variations are simply related to differences in polymer concentration . In addition, different works demonstrate a slight decrease in the elastic modulus with the temperature, until a major drop occurs at 50 c.(65) this is obviously related to the partial melting of secondary and more imperfect crystallites that leads to an increasing amount of network defects (elastically inactive chains). At temperatures close to 80 c, the complete loss of crystallinity essentially liberates all chains, and the system drops below the gel point . In view of the important role of the substantial polymer amount that is not elastically active (free and dangling chains, loops), it may be worthwhile to reconsider detailed frequency - dependent mechanical measurements, where these components should contribute to an increased loss modulus over the frequency range covering their relaxation . The capacity of our investigated systems to be transformed from an isotropic aqueous solution to a physical cryo - gel by the application of freezingthawing cycles, including thermal reversibility back to an isotropic pva solution, is in clear contradiction with the assertion that covalent bonds could form between the polymer chains during gelation . If covalent cross - links were an important contribution to the final gel properties, then residual couplings should persist even above the melting temperature of the crystallites, which is not the case . However, the presence of (rapidly tumbling) microgels above the main melting point cannot be ruled out and is in fact indicated by the relatively high viscosity (as checked by a simple tilt test). However, we point out that even the pristine 20% pva solution exhibited some detectable dq signal, indicating extended gel - like structures, unless it was heated to> 90 c . Therefore, an undetectably small fraction of crystallites (or extended hydrogen - bonded aggregates) may always be present, even at> 80 c, and hold the system close to the gel point, depending on the sample and its thermal history . From our perspective, permanent bonds can be ruled out as a major factor determining the cryo - gel structure . Finally, we address aging phenomena that are a part of the data shown in figure 6 and were not commented on until now . After heating a sample containing 10% pva to 50 c and recording the loss of crystallinity, the sample was stored for 12 h, and another measurement was performed at room temperature . The amount of rigid polymer fraction after 12 h of storage was again considerably increased, as can be seen in figure 6 . Yet, this new crystalline fraction melts at rather low temperatures, which again indicates the growth of secondary crystallites . Similar prominent aging effects have been reported in the literature . For more clear evidence, a sealed sample containing 20% pva was submitted to seven freezingthawing cycles (with 12 h of freezing in the first cycle) and was characterized after different storage times at room temperature . According to figure 9, aging clearly increases the secondary population of imperfect crystals . It also increases the amount of elastically active polymer segments but has no significant effect on the network mesh size . Therefore, aging effects on pva gel properties are again mainly related to an increasing fraction of polymer incorporated into the network structure and not to a decrease in mesh size . Effect of aging on (a) the rigid polymer fraction, (b) the noncoupled defects fraction, and (c) the average residual dipolar coupling of a gel with 10% pva obtained after seven freezingthawing cycles (12 h of freezing for the first cycle and 3 h for the following cycles). This physical gel is supported by rigid polymer areas (pva crystallites) that act as junctions between the remaining mobile and entropically elastic polymer chains and thus determine the network mesh size . In our study, the crystallites are quantitatively detected as a rigidlike fraction of quickly relaxing (and fully refocused to overcome the dead - time problem) magnetization, and the mesh size is deduced from mq experiments that measure residual dipolar couplings, the latter arising from constraints on the motion of the mobile chains (i.e., cross - links) that render the segmental motion locally anisotropic . The most important crystal fraction forms in the first freezing cycle, and subsequent freezingthawing cycles produce only a slight increase in the initial crystal size as well as induce the formation of more imperfect, secondary crystallites . This secondary crystal fraction (which constitutes around two - thirds of the total crystal phase in samples submitted to a large number of cycles, extended aging, or both) plays an important role by increasing the number of elastically active polymer chains, but it does not appreciably affect the mesh size . Pva cryo - hydrogels are shown to be totally thermoreversible; that is, pva physical networks can be transformed into an isotropic aqueous solution by increasing the temperature . During this process, the more imperfect crystals are molten first, and the number of network defects increases exponentially . In contrast, the network mesh size does not undergo significant variations until the primary crystal phase is molten at temperatures of 80 c . Our experiments did not reveal any evidence of the possible formation of covalent cross - links between pva chains during gelation; no residual couplings were detected after the melting of this primary crystal fraction . Independent of the number of cycles, the aging, or the polymer concentration in the original pva solution, a considerable fraction of polymer (at least 25%) is not elastically active, which is a result that is not yet not taken into account in the large body of literature addressing the formation and the properties of pva cryo - hydrogels . This finding is central to the interpretation and understanding of the elastic properties of this useful material and must be taken into account in a reinterpretation of the origin of the changes of the elastic moduli of pva cryogels . Finally, it is important that the in - depth study of both processes (i.e., the pva gelation by the application of freezingthawing cycles) as well as the destruction of the network structure by heating were, for the first time, entirely based on the use of an inexpensive low - field solid - state nmr spectrometer . We show that the combination of different advanced nmr strategies, that is, mq spectroscopy and component analysis of mse - refocused free - induction decays, is vital for obtaining quantitative information on network structure and phase composition (crystallinity), respectively, to arrive at conclusions that can be matched with information extracted from other diverse techniques . In addition, the use of low - field solid - state nmr spectroscopy not only is useful for extracting structural information but also could be a powerful tool for investigating the polymer dynamics of these complex systems.
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A 35-year - old woman was admitted complaining of a headache over the last two months . She had undergone a right temporal lobectomy for generalized tonic - clonic seizures 10 years earlier . Laboratory tests were normal except for an increased erythrocyte sedimentation rate of 45 mm / hr . A pre - contrast ct showed a linear calcification surrounded by a lobular - shaped mass of increasing attenuation in the right frontal lobe (fig . The mass was accompanied by extensive edema surrounding it, with a midline shifting to the left side . On mr imaging, the mass showed a heterogeneous dark signal intensity on t2-weighted images (fig . Moreover the mass had an irregular margin and was classified as an intra - axial mass with cortical involvement . Three regions of interest (roi) were determined based on the gradient - echo echo planar imaging source images and the matching t2 and gadolinium - enhanced t1-weighted images . The roi with the highest regional cerebral blood volume (rcbv) was selected and was found to be almost the same as that of the contralateral side (rcbv ratio = 1.04) (fig . Localized proton mr spectroscopy was performed using a multivoxel point - resolved spectroscopy sequence with an echo time of 144 msec . The spectroscopic data showed a slightly elevated choline (cho) and slightly decreased creatine (cr) and n - acetyl aspartate (naa) peaks with a small increase in the choline / creatine (cho / cr) ratio in the enhancing lesion (fig . This diagnosis was based on the strong enhancement of the mass accompanied with severe peritumoral edema and a mass effect . However, the perfusion data and mr spectroscopy findings suggested that the mass was benign . The mass was surgically removed and the pathology revealed a foreign body - associated granulomatous inflammation with marked lymphocytic and histiocytic infiltration, microcalcification, and fibrous scar tissue formation (fig . 1j, k). Following surgery, the chemical agents used in neurosurgery to achieve intra - operative hemostasis can cause foreign body reactions . Historically, several terms such as textiloma, gossypiboma, gauzoma, and muslinoma have been employed, reflecting the hemostatic materials involved . In our case the signal intensities of the foreign body granulomas are variable on the t1- and t2-weighted images . . Variable signal intensity on t2-weighted images may reflect a complex pathology including acute or chronic inflammation, granuloma formation, fibrosis, collagen deposition, and degeneration of foreign materials . In our patient, the low signal intensity proportion of the t2-weighted images may be explained by the fibrosis seen in the histologic specimen . In the case of ring - enhancing mass lesions, these represent degenerated foreign material and may be helpful in distinguishing a muslinoma from brain abscess (7). This was true of our patient based on the mr imaging findings showing intense mass enhancement with severe surrounding edema . However, on perfusion mri, the rcbv did not increase and, on multi - voxel mr spectroscopy, the mass cho / cr ratio increased only slightly, which was not consistent with a high - grade tumor . Previous reports on perfusion and mr spectroscopic findings in patients with non - neoplastic tumor - mimicking masses showed mildly decreased - to - increased perfusion, and small rises in cho / cr ratios, with decreased naa . Perfusion mri and mr spectroscopic findings from our patient were very similar to those of patients from previous reports with non - neoplastic mass lesions (8, 9). Retrospectively, we think we could have excluded the possibility of malignancy in our patient based on the perfusion mri and mr spectroscopy, although benign tumorous conditions may not be totally excluded by such exploratory modalities . Although the perfusion mri and mr spectroscopic findings are not specific for foreign body granulomas, these techniques may be helpful in excluding a high - grade tumor diagnosis, which may be the most important differential diagnosis . By combining clinical history of any previous operation, conventional mr imaging findings, and perfusion mri and mr spectroscopy, it may be possible to correctly diagnose a foreign body granuloma . In summary, conventional mri findings in foreign body granuloma patients may sometimes mimic those of brain tumors . To differentiate the diagnoses, the performing a perfusion mri and mr
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(the woodlands, tx) as previously described (6). Sglt2 and db / db backcrosses were performed at the jackson laboratory (bar harbor, me) and shipped to yale for studies . Mice were housed at yale university school of medicine and maintained in accordance with the institutional animal care and use committee guidelines . Mice were housed at 22 2c on a 12-h light / dark cycle with free access to food and water . Mice were fed regular chow (rc; 18% fat, 58% carbohydrate, 24% protein by calories; td2018; harlan teklad, madison, wi) or 4 weeks of high - fat diet (hfd; 55% fat, 24% carbohydrate, 21% protein by calories; td93075; harlan teklad). The comprehensive laboratory animal monitoring system (columbus instruments, columbus, oh) was used to evaluate activity, energy expenditure, feeding, drinking, and respiratory quotient over the course of 48 h. data are the 24-h average normalized to body weight . For the urine collection studies, mice were housed for 24 h with free access to food and water in wire - bottomed cages designed to separate and collect urine and feces . Mice were injected intraperitoneally with 1 mg / kg glucose, and blood was collected by tail bleed at set times for plasma insulin and glucose measurements . Hyperinsulinemic euglycemic clamps were performed as previously described (23), with minor modifications . Specifically, clamp duration was extended to 180 min and the insulin dose was increased to 20 mu kg min . Mice were given a primed / variable infusion of glucose to reach and maintain hyperglycemia during the 120-min experiment . Blood was collected at set intervals by tail bleeds for determination of plasma glucose and insulin . Because of differences in fasting plasma glucose and insulin levels between genotypes, infusion rates were chosen to achieve matched changes in plasma glucose (glucose), and the change in plasma insulin from baseline (insulin) was used to assess the pancreatic -cell response . Isolated islet studies were conducted as previously described (24). In brief, pancreata were removed from anesthetized mice and minced in cmrl media with type p collagenase to disperse cells . Islets were hand picked under a light microscope and allowed to recover overnight in media . Eighty islets were picked and loaded into a perifusion chamber with acrylamide gel column beads (bio - gel p4 g; bio - rad, hercules, ca) and perfusion buffer (krebs - ringer buffer with 3 mmol / l glucose and 0.2% fatty acid - free bsa). Perifusions were conducted with a bio - rep (miami, fl) perifusion instrument that allows for precise temperature and flow control and collection of samples into 96-well format . After a 1-h equilibration period, islets were perifused with the indicated agonists, and samples were collected at 1-min intervals for 40 min . At the end of the perifusion, islet dna was isolated and quantified for normalization of insulin and glucagon data using a picogreen dsdna quantitation kit (invitrogen) according to the manufacturer s instructions . Preparation and staining of all histologic samples were conducted at the department of pathology histology core at yale . Sections were cut at 5 m, dried, deparafinized, and rehydrated with distilled water . For kidney studies, hematoxylin and eosin (h&e) stains were analyzed in a blinded fashion by a pathologist . For pancreas studies, transferase - mediated dutp nick - end labeling (tunel) reaction was performed according to the manufacturer s instructions (millipore s7100; millipore, billerica, ma), after which slides were incubated with polyclonal anti - insulin (dako 0564) and visualized by an alkaline - phosphatase based method (dako k5355). Frequency of cell death was calculated as the number of insulin / tunel - positive cells corrected by relative islet area (islet area / pancreas area) for 10 to 15 fields per section and three sections per mouse . Islet number was determined by counting all islets within a given field, corrected to pancreas area . Approximately 30 fields were taken at random, and three sections per mouse were analyzed . Sections were staggered to reduce the possibility of analyzing the same islet twice . For proliferation studies, slides were incubated with anti - ki-67 (biocare medical crm325) and anti - insulin (dako a0564) and visualized with peroxidase and fluorescent secondary antibodies (dako k4003, invitrogen a11073). Frequency of cellular proliferation was calculated in a similar fashion as frequency of cell death . Pancreas weights were not available, so relative -cell volume was calculated as the ratio of islet area / pancreas area . Plasma and urine glucose concentrations were measured by a glucose oxidase method using a beckman glucose analyzer ii . Louis, mo), and fatty acids were measured by a spectrophotometric technique (wako nefa kit, osaka, japan). Hepatic glycogen content was measured as previously described, except that a 10:1 volume / weight dilution was used for homogenization (25). Insulin and glucagon from isolated islets were measured with the lincoplex platform (millipore). Plasma chemistries were measured with a roche cobas mira automated chemistry analyzer at the yale mouse metabolic phenotyping center analytical core . Plasma creatinine levels were determined by high - performance liquid chromatography / mass spectrometry / mass spectrometry . For time course data, two - way and one - way anova were used where appropriate . A value of p <0.05 was considered significant . (the woodlands, tx) as previously described (6). Sglt2 and db / db backcrosses were performed at the jackson laboratory (bar harbor, me) and shipped to yale for studies . Mice were housed at yale university school of medicine and maintained in accordance with the institutional animal care and use committee guidelines . Mice were housed at 22 2c on a 12-h light / dark cycle with free access to food and water . Mice were fed regular chow (rc; 18% fat, 58% carbohydrate, 24% protein by calories; td2018; harlan teklad, madison, wi) or 4 weeks of high - fat diet (hfd; 55% fat, 24% carbohydrate, 21% protein by calories; td93075; harlan teklad). The comprehensive laboratory animal monitoring system (columbus instruments, columbus, oh) was used to evaluate activity, energy expenditure, feeding, drinking, and respiratory quotient over the course of 48 h. data are the 24-h average normalized to body weight . For the urine collection studies, mice were housed for 24 h with free access to food and water in wire - bottomed cages designed to separate and collect urine and feces . Mice were injected intraperitoneally with 1 mg / kg glucose, and blood was collected by tail bleed at set times for plasma insulin and glucose measurements . Hyperinsulinemic euglycemic clamps were performed as previously described (23), with minor modifications . Specifically, clamp duration was extended to 180 min and the insulin dose was increased to 20 mu kg min . Mice were given a primed / variable infusion of glucose to reach and maintain hyperglycemia during the 120-min experiment . Blood was collected at set intervals by tail bleeds for determination of plasma glucose and insulin . Because of differences in fasting plasma glucose and insulin levels between genotypes, infusion rates were chosen to achieve matched changes in plasma glucose (glucose), and the change in plasma insulin from baseline (insulin) was used to assess the pancreatic -cell response . Isolated islet studies were conducted as previously described (24). In brief, pancreata were removed from anesthetized mice and minced in cmrl media with type p collagenase to disperse cells . Islets were hand picked under a light microscope and allowed to recover overnight in media . Eighty islets were picked and loaded into a perifusion chamber with acrylamide gel column beads (bio - gel p4 g; bio - rad, hercules, ca) and perfusion buffer (krebs - ringer buffer with 3 mmol / l glucose and 0.2% fatty acid - free bsa). Perifusions were conducted with a bio - rep (miami, fl) perifusion instrument that allows for precise temperature and flow control and collection of samples into 96-well format . After a 1-h equilibration period, islets were perifused with the indicated agonists, and samples were collected at 1-min intervals for 40 min . At the end of the perifusion, islet dna was isolated and quantified for normalization of insulin and glucagon data using a picogreen dsdna quantitation kit (invitrogen) according to the manufacturer s instructions . Preparation and staining of all histologic samples were conducted at the department of pathology histology core at yale . Sections were cut at 5 m, dried, deparafinized, and rehydrated with distilled water . For kidney studies, hematoxylin and eosin (h&e) stains were analyzed in a blinded fashion by a pathologist . For pancreas studies, transferase - mediated dutp nick - end labeling (tunel) reaction was performed according to the manufacturer s instructions (millipore s7100; millipore, billerica, ma), after which slides were incubated with polyclonal anti - insulin (dako 0564) and visualized by an alkaline - phosphatase based method (dako k5355). Frequency of cell death was calculated as the number of insulin / tunel - positive cells corrected by relative islet area (islet area / pancreas area) for 10 to 15 fields per section and three sections per mouse . Islet number was determined by counting all islets within a given field, corrected to pancreas area . Approximately 30 fields were taken at random, and three sections per mouse were analyzed . Sections were staggered to reduce the possibility of analyzing the same islet twice . For proliferation studies, slides were incubated with anti - ki-67 (biocare medical crm325) and anti - insulin (dako a0564) and visualized with peroxidase and fluorescent secondary antibodies (dako k4003, invitrogen a11073). Frequency of cellular proliferation was calculated in a similar fashion as frequency of cell death . Pancreas weights were not available, so relative -cell volume was calculated as the ratio of islet area / pancreas area . Plasma and urine glucose concentrations were measured by a glucose oxidase method using a beckman glucose analyzer ii . Louis, mo), and fatty acids were measured by a spectrophotometric technique (wako nefa kit, osaka, japan). Hepatic glycogen content was measured as previously described, except that a 10:1 volume / weight dilution was used for homogenization (25). Insulin and glucagon from isolated islets were measured with the lincoplex platform (millipore). Plasma chemistries were measured with a roche cobas mira automated chemistry analyzer at the yale mouse metabolic phenotyping center analytical core . Plasma creatinine levels were determined by high - performance liquid chromatography / mass spectrometry / mass spectrometry . Comparisons between groups were made using unpaired, two - tailed student t tests . For time course data, the rc - fed sglt2 mice weighed approximately 10% less than wild - type (wt) mice at age 16 weeks (table 1). To determine the effect of sglt2 deletion in the presence of increased body weight and adiposity, a second group of sglt2 and wt mice were fed an hfd for 4 weeks . Hfd mice had an increased body weight compared with controls for both genotypes, but unlike rc - fed mice, hfd / sglt2 mice only trended toward a lower body weight (p = 0.12; table 1). There were no differences in fat mass between rc / wt and rc / sglt2 mice, and hfd / sglt2 animals trended toward reduced adiposity (p = 0.08; table 1). Physiologic overview of sglt2 mice data were collected from 16-week - old, ad libitum fed mice . Mice were housed in custom cages for 24 h for urine output and glucosuria measurements . N = 1216 per genotype for body weight and fat data; n = 68 for remaining data . Daily urine volume was 4.5-fold greater in sglt2 than in wt mice, which translated to a 500-fold increase in glucosuria, with similar findings in the hfd mice (table 1). Despite increased urine volume and glucosuria, plasma chemistries were unchanged in sglt2 mice (supplementary table 1), in agreement with a recently published description of the sglt2 knockout model (6). There was no difference in the blood urea nitrogen (bun)/creatinine ratio between wt or sglt2 mice, suggesting that volume depletion could not account for the lower weight (supplementary table 1). Histologic analysis of kidney sections revealed normal glomerular structures and tubulointerstitial compartments, without significant histopathologic changes in the proximal or distal nephron of sglt2 mice (supplementary fig . 1), and there was no difference in kidney weight between genotypes (table 1). To determine the effect of sglt2 deletion on whole - body metabolism and behavior, sglt2 and wt mice, there was no difference in activity, energy expenditure, or food intake between genotypes (supplementary fig . C), although water intake was elevated during this time for sglt2 mice (supplementary fig . In contrast, there were marked differences in each parameter between mice during active hours (dark cycle). Activity, energy expenditure, and food and water intake were all significantly increased in sglt2 mice during the dark cycle (fig . Notably, there was a 20% increase in feeding and a 190% increase in drinking compared with controls . Despite the increase in caloric intake, sglt2 mice maintained a lower body weight through a combination of 7% higher energy expenditure during the dark cycle and 13% (1.9 kcal / day) caloric loss of carbohydrate in the urine . A: dark cycle averages are shown for metabolic cage data for activity, energy expenditure (ee), feeding, drinking, and respiratory quotient (vco2/vo2) for wt and sglt2 mice . D: changes in plasma glucose following intraperitoneal injection of 1-mg / kg glucose (wt vs. sglt2 p <0.01 for rc and p <0.001 for hfd). E: changes in plasma insulin levels during experiment shown in d (wt vs. sglt2 p <0.01 for rc and hfd). F: auc for glucose calculated from data in d. g: auc for insulin calculated from data in e. n = 812 mice per group . * p <0.05, * * p <0.01, and * * * p <0.001 . Data represent the mean sem . Under hfd conditions, mice consume significantly less carbohydrate . Interestingly, unlike rc - fed mice, there were no differences in activity, energy expenditure, or food intake in the light or dark cycle during hfd (supplementary fig . Drinking, however, was clearly increased relative to hfd controls throughout the day, reflecting continuous glucose loss . In comparison with rc / sglt2 mice, hfd / sglt2 mice had lower water consumption (36.9 2.5 vs. 45.6 2.9 l / h; p <0.05) and urine output (5.2 0.5 vs. 6.5 0.4 ml / day; p <0.05), again suggesting that carbohydrate content of the chow had a large influence on glucosuria . The lower respiratory quotient in sglt2 mice (fig . 1a) reflects a higher rate of lipid relative to carbohydrate oxidation, as might be expected for mice that are carbohydrate - depleted by profound glucosuria . Indeed, there was a 2.5-fold decrease in hepatic glycogen content in sglt2 compared with wt mice (5.1 1.1 vs. 12.7 2.0 g / mg tissue; p <0.05). This was even more dramatic on the hfd, where the respiratory quotient was 0.78 and approached that of nearly a complete dependence on lipid oxidation (supplementary fig . Compared with rc / wt mice, rc / sglt2 mice had significantly lower fasting plasma glucose concentrations and a nonsignificant reduction in plasma insulin concentrations (9.0 0.6 and 6.4 1.8 u / ml insulin; p = 0.17; fig ., fasting glucose was drastically reduced in sglt compared with wt mice and was indistinguishable from rc / wt controls (fig . Plasma insulin concentrations were 50% lower in the hfd / sglt2 compared with wt mice, as would be expected for the lower plasma glucose levels . Hfd / sglt2 mice had a twofold increase in fasting insulin compared with rc / wt mice (fig . Intraperitoneal glucose tolerance tests (ipgtts) were performed to determine the effect of sglt2 knockout on glucose homeostasis . Plasma glucose concentrations remained lower in sglt2 than in wt mice for the entire experiment (p <0.01 by two - way anova; fig . The area under the curve (auc) for glucose was 20% lower for sglt2 than for wt mice (fig . More pronounced effect was observed during hfd studies (p <0.001 by two - way anova; fig . 1d), where auc glucose was 40% less in sglt2 mice and was indistinguishable from wt / rc controls (fig . Changes in plasma insulin concentrations roughly mirrored the differences observed for plasma glucose (wt vs. sglt2 rc and hfd p <0.01, two - way anova; fig . The insulin auc was 37% less for rc / sglt2 compared with rc / wt mice, while after hfd, the insulin auc was 53% less for sglt2 mice (fig thus, loss of sglt2 significantly improved basal and ipgtt - challenged glucose homeostasis in rc- and hfd - fed mice . Increased glucosuria is associated with decreased fasting plasma glucose and insulin in db / db - sglt2 mice . A: body weights are shown for db / db - sglt, db / db - sglt2, and db / db - slgt2 mice at approximately 18 weeks of age . C: urine volume at 24 h. d: glucosuria at 24 h (n = 56 per group). * p <0.05, * * p <0.01, and * * * p <0.001 . Data represent the mean sem . To determine whether loss of sglt2 was protective in the setting of extreme obesity and insulin resistance, the db / db mice are leptin receptor deficient and develop profound hyperphagia, obesity, hypercorticosteronemia, insulin resistance, and eventually, diabetes . At approximately age 18 weeks, body weights were similar for both heterozygous (db / db - sglt2) and homozygous (db / db - sglt2) sglt2 deletion compared with controls (db / db - sglt2; fig . 2a), whereas body fat was 5% less in db / db - sglt2 mice (fig . There was a progressive effect of sglt2 deletion on urine volume, with db / db - sglt2 having a 2.6-fold and db / db - sglt having a fourfold increase (fig . 2c). The db / db mice displayed profound glucosuria, totaling approximately 500 mg / day (fig . Still, there was a three- and 4.4-fold increase in db / db - sglt2 and db / db - sglt2 mice, respectively, indicating that lowering the renal glucose threshold had a progressive effect on glucosuria (fig . Astonishingly, total daily glucose loss for db / db - sglt2 mice was 2.2 g or 5% of the animals body weight . Despite increased glucosuria in db / db - sglt2 mice, only the db / db - sglt2 mice had significantly reduced plasma glucose concentrations (fig . Strikingly, this 75-mg / dl reduction normalized glycemia to the same level as rc / wt mice (wt: 131 6 mg / dl vs. db / db - sglt2: 124 8 mg / dl). Plasma insulin levels were 50% lower in db / db - sglt2 crosses (fig . Plasma insulin levels were 10-fold higher than rc / wt mice (wt: 9.0 0.6 u / ml vs. db / db - sglt2: 112 13 u / ml), indicating the persistence of insulin resistance in the db / db model . Lastly, sglt2 deletion had no effect on plasma fatty acid levels in db / db mice (fig . 2 g). Euglycemic clamp studies were performed to evaluate the effect of sglt2 deletion on insulin sensitivity and tissue - specific glucose metabolism . Because of extreme insulin resistance in db / db mice, plasma glucose concentrations were clamped at 150 mg / dl using a 20 mu kg min insulin infusion (supplementary fig . D). The glucose infusion rate (gir) was 2.7-times greater in db / db - sglt2 than in db / db - sglt2 mice (table 2). Quantitative measurement of glucosuria during the clamp was not possible for control or db / db - sglt2 mice because of small urine volumes, but ranged from 0 to 15% of total gir for db / db - sglt2 mice (data not shown). Thus, the higher gir could only partially be accounted for by loss as glucosuria and suggested improvements in insulin sensitivity . There was no difference in whole - body or tissue - specific glucose uptake in any group (table 2), but clamped endogenous glucose production was modestly reduced in db / db - sglt2 compared with db / db - sglt2 mice (table 2). Hyperinsulinemic euglycemic clamp studies whole - body and tissue - specific glucose metabolism during hyperinsulinemic infusion . The db / db - sglt2 (n = 9), db / db - sglt2 (n = 8), and db / db - sglt2 (n = 7). P <0.05 compared with db / db - sglt2 . Because glucose toxicity can lead to -cell failure, we assessed glucose - stimulated insulin secretion by hyperglycemic clamp . 2e); therefore, all mice were clamped 200 mg / dl above their basal plasma glucose levels . Similar to euglycemic clamps, the gir required to maintain hyperglycemia for db / db - sglt2 mice was approximately twofold greater than for controls (fig . The total increase in plasma glucose concentrations above baseline during the clamp was similar for all three groups (fig . No first - phase insulin response was observed, consistent with compromised islet function in db / db mice (fig . 3d). However, in response to the same net change in plasma glucose concentration (auc glucose, fig . 3c), db / db - sglt2 mice displayed a significant increase in absolute plasma insulin levels during the clamp (p <0.001, one - way anova; fig . 3d), and auc insulin was 2.4-fold greater compared with db / db - sglt2 mice (fig . Calculation of the change in plasma insulin (insulin) from baseline also demonstrated an approximate twofold increase in glucose - stimulated insulin secretion for db / db - sglt2 compared with db / db - sglt2 mice (fig . Thus, the greater than 10-fold increase in insulin secretion for db / db - sglt2 compared with the modest fivefold increase for controls during the hyperglycemic clamp was consistent with improved -cell function in vivo . A: glucose infusion rates (gir) required to maintain hyperglycemia during 120-min clamp experiment . B: changes in plasma glucose during the clamp in response to gir in a. c: auc calculations are shown for the change in plasma glucose (glucose) from baseline during the clamp . D: changes in plasma insulin in response to changes in plasma glucose in b (db / db - sglt2 vs. db / db - sglt2; p <0.01). F: net change in plasma insulin levels from baseline (insulin) during the clamp (n = 69 per group). Significance between curves was determined by one - way anova . * p <0.05, * * p <0.01 . Isolated pancreatic islets from the db / db - sglt2 and db / db - sglt2 mice were perifused to determine if changes in individual islet function had occurred . During isolation, islet yield was much greater for db / db - sglt2 mice, so islets of similar size were selected for comparison . Surprisingly, there was no difference in glucose - stimulated insulin secretion (633 104 vs. 743 53 auc insulin; p = 0.37; fig . 4a) or glucagon suppression (0.28 0.12 vs. 0.15 0.04 auc glucagon; p = 0.35; fig . 4b) between groups, suggesting that individual islet function, per se, could not account for the observed in vivo differences . Perifusion studies conducted for wt and sglt mice on rc and hfd also demonstrated no differences in insulin or glucagon secretion (supplementary fig . Pancreatic -cell mass is increased in db / db - sglt2 mice due to reduced frequency of cell death . A: insulin secretion from isolated, perifused islets in response to low / high glucose and kcl . B: glucagon suppression during the same experiment performed in a. c: relative -cell volume (islet area / pancreas area) determined from histologic analysis of pancreata . E: frequency of -cell proliferation determined as the number of ki67/insulin - positive cells per relative islet area . F: frequency of -cell death was calculated as the number of tunel / insulin - positive cells per relative islet area . Pancreata stained as follows: panel 1, hematoxylin and eosin (h&e) stain; panel 2, insulin stain; panel 3, ki-67 stain . (a high - quality digital representation of this figure is available in the online issue .) The difference between hyperglycemic clamp and islet perifusion results suggested that changes in -cell mass might account for the increased insulin secretion observed in vivo . Compared with rc / wt mice, relative -cell volume was increased 3.3- and 5.4-fold for db / db - sglt2 and db / db - sglt2 mice, respectively, suggesting compensatory hypertrophy as a result of insulin resistance (1.06 0.12, 3.52 0.52, and 5.77 0.21 islet area / pancreas area; p <0.001 by one - way anova). Importantly, there was a 63% increase in relative -cell volume in db / db - sglt2 compared with db / db - sglt2 mice (fig . 4c and g). The difference in islet mass between db / db groups appeared to be more a function of increased islet size rather than number (p = 0.22; fig . 4d). There was no difference in the frequency of ki-67positive -cells between genotypes (fig . 4e and g), suggesting that -cell proliferation rates were not increased in the db / db - sglt2 mice . However, the frequency of tunel - positive -cells was 64% less in db / db - sglt2 mice (fig . 4f and g), indicating that rates of cell death were slower . Thus, improvements in -cell function observed in vivo appeared to result from maintained -cell mass due to reduced frequency of -cell death . The rc - fed sglt2 mice weighed approximately 10% less than wild - type (wt) mice at age 16 weeks (table 1). To determine the effect of sglt2 deletion in the presence of increased body weight and adiposity, a second group of sglt2 and wt mice were fed an hfd for 4 weeks . Hfd mice had an increased body weight compared with controls for both genotypes, but unlike rc - fed mice, hfd / sglt2 mice only trended toward a lower body weight (p = 0.12; table 1). There were no differences in fat mass between rc / wt and rc / sglt2 mice, and hfd / sglt2 animals trended toward reduced adiposity (p = 0.08; table 1). Physiologic overview of sglt2 mice data were collected from 16-week - old, ad libitum fed mice . Mice were housed in custom cages for 24 h for urine output and glucosuria measurements . N = 1216 per genotype for body weight and fat data; n = 68 for remaining data . Daily urine volume was 4.5-fold greater in sglt2 than in wt mice, which translated to a 500-fold increase in glucosuria, with similar findings in the hfd mice (table 1). Despite increased urine volume and glucosuria, plasma chemistries were unchanged in sglt2 mice (supplementary table 1), in agreement with a recently published description of the sglt2 knockout model (6). There was no difference in the blood urea nitrogen (bun)/creatinine ratio between wt or sglt2 mice, suggesting that volume depletion could not account for the lower weight (supplementary table 1). Histologic analysis of kidney sections revealed normal glomerular structures and tubulointerstitial compartments, without significant histopathologic changes in the proximal or distal nephron of sglt2 mice (supplementary fig . 1), and there was no difference in kidney weight between genotypes (table 1). To determine the effect of sglt2 deletion on whole - body metabolism and behavior, sglt2 and wt mice, there was no difference in activity, energy expenditure, or food intake between genotypes (supplementary fig . C), although water intake was elevated during this time for sglt2 mice (supplementary fig . In contrast, there were marked differences in each parameter between mice during active hours (dark cycle). Activity, energy expenditure, and food and water intake were all significantly increased in sglt2 mice during the dark cycle (fig . Notably, there was a 20% increase in feeding and a 190% increase in drinking compared with controls . Despite the increase in caloric intake, sglt2 mice maintained a lower body weight through a combination of 7% higher energy expenditure during the dark cycle and 13% (1.9 kcal / day) caloric loss of carbohydrate in the urine . A: dark cycle averages are shown for metabolic cage data for activity, energy expenditure (ee), feeding, drinking, and respiratory quotient (vco2/vo2) for wt and sglt2 mice . D: changes in plasma glucose following intraperitoneal injection of 1-mg / kg glucose (wt vs. sglt2 p <0.01 for rc and p <0.001 for hfd). E: changes in plasma insulin levels during experiment shown in d (wt vs. sglt2 p <0.01 for rc and hfd). F: auc for glucose calculated from data in d. g: auc for insulin calculated from data in e. n = 812 mice per group . * p <0.05, * * p <0.01, and * * * p <0.001 . Data represent the mean sem . Under hfd conditions, mice consume significantly less carbohydrate . Interestingly, unlike rc - fed mice, there were no differences in activity, energy expenditure, or food intake in the light or dark cycle during hfd (supplementary fig . Drinking, however, was clearly increased relative to hfd controls throughout the day, reflecting continuous glucose loss . In comparison with rc / sglt2 mice, hfd / sglt2 mice had lower water consumption (36.9 2.5 vs. 45.6 2.9 l / h; p <0.05) and urine output (5.2 0.5 vs. 6.5 0.4 ml / day; p <0.05), again suggesting that carbohydrate content of the chow had a large influence on glucosuria . The lower respiratory quotient in sglt2 mice (fig . 1a) reflects a higher rate of lipid relative to carbohydrate oxidation, as might be expected for mice that are carbohydrate - depleted by profound glucosuria . Indeed, there was a 2.5-fold decrease in hepatic glycogen content in sglt2 compared with wt mice (5.1 1.1 vs. 12.7 2.0 g / mg tissue; p <0.05). This was even more dramatic on the hfd, where the respiratory quotient was 0.78 and approached that of nearly a complete dependence on lipid oxidation (supplementary fig . Compared with rc / wt mice, rc / sglt2 mice had significantly lower fasting plasma glucose concentrations and a nonsignificant reduction in plasma insulin concentrations (9.0 0.6 and 6.4 1.8 u / ml insulin; p = 0.17; fig ., fasting glucose was drastically reduced in sglt compared with wt mice and was indistinguishable from rc / wt controls (fig . Plasma insulin concentrations were 50% lower in the hfd / sglt2 compared with wt mice, as would be expected for the lower plasma glucose levels . Hfd / sglt2 mice had a twofold increase in fasting insulin compared with rc / wt mice (fig . Intraperitoneal glucose tolerance tests (ipgtts) were performed to determine the effect of sglt2 knockout on glucose homeostasis . Plasma glucose concentrations remained lower in sglt2 than in wt mice for the entire experiment (p <0.01 by two - way anova; fig . The area under the curve (auc) for glucose was 20% lower for sglt2 than for wt mice (fig . More pronounced effect was observed during hfd studies (p <0.001 by two - way anova; fig . 1d), where auc glucose was 40% less in sglt2 mice and was indistinguishable from wt / rc controls (fig . Changes in plasma insulin concentrations roughly mirrored the differences observed for plasma glucose (wt vs. sglt2 rc and hfd p <0.01, two - way anova; fig . The insulin auc was 37% less for rc / sglt2 compared with rc / wt mice, while after hfd, the insulin auc was 53% less for sglt2 mice (fig thus, loss of sglt2 significantly improved basal and ipgtt - challenged glucose homeostasis in rc- and hfd - fed mice . Increased glucosuria is associated with decreased fasting plasma glucose and insulin in db / db - sglt2 mice . A: body weights are shown for db / db - sglt, db / db - sglt2, and db / db - slgt2 mice at approximately 18 weeks of age . C: urine volume at 24 h. d: glucosuria at 24 h (n = 56 per group). * p <0.05, * * p <0.01, and * * * p <0.001 . To determine whether loss of sglt2 was protective in the setting of extreme obesity and insulin resistance, sglt2 mice were bred onto the db / db background . The db / db mice are leptin receptor deficient and develop profound hyperphagia, obesity, hypercorticosteronemia, insulin resistance, and eventually, diabetes . At approximately age 18 weeks, body weights were similar for both heterozygous (db / db - sglt2) and homozygous (db / db - sglt2) sglt2 deletion compared with controls (db / db - sglt2; fig . 2a), whereas body fat was 5% less in db / db - sglt2 mice (fig . There was a progressive effect of sglt2 deletion on urine volume, with db / db - sglt2 having a 2.6-fold and db / db - sglt having a fourfold increase (fig . 2c). The db / db mice displayed profound glucosuria, totaling approximately 500 mg / day (fig . Still, there was a three- and 4.4-fold increase in db / db - sglt2 and db / db - sglt2 mice, respectively, indicating that lowering the renal glucose threshold had a progressive effect on glucosuria (fig . Astonishingly, total daily glucose loss for db / db - sglt2 mice was 2.2 g or 5% of the animals body weight . Despite increased glucosuria in db / db - sglt2 mice, only the db / db - sglt2 mice had significantly reduced plasma glucose concentrations (fig . Strikingly, this 75-mg / dl reduction normalized glycemia to the same level as rc / wt mice (wt: 131 6 mg / dl vs. db / db - sglt2: 124 8 mg / dl). Plasma insulin levels were 50% lower in db / db - sglt2 crosses (fig . Plasma insulin levels were 10-fold higher than rc / wt mice (wt: 9.0 0.6 u / ml vs. db / db - sglt2: 112 13 u / ml), indicating the persistence of insulin resistance in the db / db model . Lastly, sglt2 deletion had no effect on plasma fatty acid levels in db / db mice (fig euglycemic clamp studies were performed to evaluate the effect of sglt2 deletion on insulin sensitivity and tissue - specific glucose metabolism . Because of extreme insulin resistance in db / db mice, plasma glucose concentrations were clamped at 150 mg / dl using a 20 mu kg min insulin infusion (supplementary fig . D). The glucose infusion rate (gir) was 2.7-times greater in db / db - sglt2 than in db / db - sglt2 mice (table 2). Quantitative measurement of glucosuria during the clamp was not possible for control or db / db - sglt2 mice because of small urine volumes, but ranged from 0 to 15% of total gir for db / db - sglt2 mice (data not shown). Thus, the higher gir could only partially be accounted for by loss as glucosuria and suggested improvements in insulin sensitivity . There was no difference in whole - body or tissue - specific glucose uptake in any group (table 2), but clamped endogenous glucose production was modestly reduced in db / db - sglt2 compared with db / db - sglt2 mice (table 2). Hyperinsulinemic euglycemic clamp studies whole - body and tissue - specific glucose metabolism during hyperinsulinemic infusion . The db / db - sglt2 (n = 9), db / db - sglt2 (n = 8), and db / db - sglt2 (n = 7). Because glucose toxicity can lead to -cell failure, we assessed glucose - stimulated insulin secretion by hyperglycemic clamp . 2e); therefore, all mice were clamped 200 mg / dl above their basal plasma glucose levels . Similar to euglycemic clamps, the gir required to maintain hyperglycemia for db / db - sglt2 mice was approximately twofold greater than for controls (fig . The total increase in plasma glucose concentrations above baseline during the clamp was similar for all three groups (fig . No first - phase insulin response was observed, consistent with compromised islet function in db / db mice (fig . 3d). However, in response to the same net change in plasma glucose concentration (auc glucose, fig . 3c), db / db - sglt2 mice displayed a significant increase in absolute plasma insulin levels during the clamp (p <0.001, one - way anova; fig . 3d), and auc insulin was 2.4-fold greater compared with db / db - sglt2 mice (fig . 3e). Calculation of the change in plasma insulin (insulin) from baseline also demonstrated an approximate twofold increase in glucose - stimulated insulin secretion for db / db - sglt2 compared with db / db - sglt2 mice (fig . Thus, the greater than 10-fold increase in insulin secretion for db / db - sglt2 compared with the modest fivefold increase for controls during the hyperglycemic clamp was consistent with improved -cell function in vivo . A: glucose infusion rates (gir) required to maintain hyperglycemia during 120-min clamp experiment . B: changes in plasma glucose during the clamp in response to gir in a. c: auc calculations are shown for the change in plasma glucose (glucose) from baseline during the clamp . D: changes in plasma insulin in response to changes in plasma glucose in b (db / db - sglt2 vs. db / db - sglt2; p <0.01). F: net change in plasma insulin levels from baseline (insulin) during the clamp (n = 69 per group). Significance between curves was determined by one - way anova . * p <0.05, * * p <0.01 . Isolated pancreatic islets from the db / db - sglt2 and db / db - sglt2 mice were perifused to determine if changes in individual islet function had occurred . During isolation, islet yield was much greater for db / db - sglt2 mice, so islets of similar size were selected for comparison . Surprisingly, there was no difference in glucose - stimulated insulin secretion (633 104 vs. 743 53 auc insulin; p = 0.37; fig . 4a) or glucagon suppression (0.28 0.12 vs. 0.15 0.04 auc glucagon; p = 0.35; fig . 4b) between groups, suggesting that individual islet function, per se, could not account for the observed in vivo differences . Perifusion studies conducted for wt and sglt mice on rc and hfd also demonstrated no differences in insulin or glucagon secretion (supplementary fig . Pancreatic -cell mass is increased in db / db - sglt2 mice due to reduced frequency of cell death . A: insulin secretion from isolated, perifused islets in response to low / high glucose and kcl . B: glucagon suppression during the same experiment performed in a. c: relative -cell volume (islet area / pancreas area) determined from histologic analysis of pancreata . E: frequency of -cell proliferation determined as the number of ki67/insulin - positive cells per relative islet area . F: frequency of -cell death was calculated as the number of tunel / insulin - positive cells per relative islet area . Pancreata stained as follows: panel 1, hematoxylin and eosin (h&e) stain; panel 2, insulin stain; panel 3, ki-67 stain . (a high - quality digital representation of this figure is available in the online issue .) The difference between hyperglycemic clamp and islet perifusion results suggested that changes in -cell mass might account for the increased insulin secretion observed in vivo . Compared with rc / wt mice, relative -cell volume was increased 3.3- and 5.4-fold for db / db - sglt2 and db / db - sglt2 mice, respectively, suggesting compensatory hypertrophy as a result of insulin resistance (1.06 0.12, 3.52 0.52, and 5.77 0.21 islet area / pancreas area; p <0.001 by one - way anova). Importantly, there was a 63% increase in relative -cell volume in db / db - sglt2 compared with db / db - sglt2 mice (fig . 4c and g). The difference in islet mass between db / db groups appeared to be more a function of increased islet size rather than number (p = 0.22; fig . 4d). There was no difference in the frequency of ki-67positive -cells between genotypes (fig . 4e and g), suggesting that -cell proliferation rates were not increased in the db / db - sglt2 mice . However, the frequency of tunel - positive -cells was 64% less in db / db - sglt2 mice (fig . Thus, improvements in -cell function observed in vivo appeared to result from maintained -cell mass due to reduced frequency of -cell death . Type 2 diabetes is characterized by impaired glucose - stimulated insulin secretion, which is strongly associated with hyperglycemia in rodent and human studies (8,9,11,26). Chronic hyperglycemia may shift the balance of -cell proliferation and apoptosis toward cell death, resulting in -cell deficiency and impaired insulin secretion (8,10,27). Although there seem to be some differences between rodents and humans with regard to the importance of -cell proliferation in response to hyperglycemia, human autopsy studies have demonstrated a decline in -cell mass in individuals with type 2 diabetes that was associated with increased -cell death (9). A major finding of our study was that reducing glucose toxicity in a model of extreme diabetes preserved islet mass and improved insulin secretion in vivo . Increasing renal glucose excretion in db / db mice by sglt2 deletion produced beneficial effects in glucose homeostasis and, importantly, islet function . The lack of difference in islet function during isolation / perifusion studies described here suggested that the primary mechanism by which reduced glucose toxicity improved glucose - stimulated insulin secretion was through preservation of islet mass rather than a change in individual -cell function . A similar effect was observed after pharmacologic inhibition of sglt2 in db / db and kk - a mice for 12 and 9 weeks, where total pancreatic insulin content and immunohistologic staining for insulin were increased in treated mice (16,28). No human studies with sglt2 inhibitors have addressed islet function directly, and so the potential for sglt2 inhibition to preserve -cell mass by reducing glucose toxicity, as it did here in rodents, remains to be determined . Hyperglycemia in insulin - resistant or -deficient models has also been shown to contribute to peripheral insulin resistance (12,29). Despite a 64% increase in the gir required to maintain euglycemia in db / db - sglt2 mice, equivalent improvements in measured indices of insulin sensitivity were not detected during euglycemic clamps . There was no difference in whole - body or tissue - specific glucose uptake, and endogenous glucose production was modestly improved by 24% in db / db - sglt2 mice . It seems likely that unaccounted for differences in gir could result from glucosuria, but estimating acute glucosuria during the clamp was technically difficult . Furthermore, db / db mice possess a complex metabolic phenotype and insulin resistance stemming from multiple factors, including hyperglycemia, hyperlipidemia, and hypercorticosteronemia (3032). An even modest improvement in insulin resistance in this model suggests that reducing glucose toxicity by renal glucose excretion may produce even more beneficial effects on insulin resistance under less severe circumstances . In fact, inhibition of sglt2 in zdf rats for 15 days improved insulin sensitivity (15). In contrast to the subtle improvement in insulin sensitivity, sglt2 knockout markedly improved glucose intolerance under hfd - induced, insulin - resistant conditions, and reduced fasting plasma glucose and insulin levels . Rc / sglt2 mice also displayed improved glucose tolerance and reduced fasting plasma glucose, without hypoglycemia . Whether this was a response to mild, undetected hypoglycemia or for other reasons remains to be determined . Of note, sglt2 mutations in humans that result in familial renal glucosuria rarely cause hypoglycemia (3335). Likewise, the incidence of hypoglycemia during clinical trials of sglt2 inhibitors is extremely low (1722). Inhibition of sglt2 has the potential to promote negative energy balance and, in turn, weight loss . The lack of differences in plasma electrolytes, phosphate, bun, creatinine, and the bun / creatinine ratio suggested that volume depletion did not account for the differences in body weight . Lack of volume depletion in sglt2 mice was also noted in a recent study; however, no difference in body weight was detected (6). Mice in the previous report were studied at between 12 and 20 weeks of age, and group sizes for the body weight data ranged from 6 to 10 . Here, mice were studied at 16 weeks, and the group size for body weight data were 16 per group . The breadth of ages cited in the previous report, as well as smaller group sizes, may account for the discrepancy . In support of reduced body weight with loss of sglt2 function, a recently described sglt2 mutant mouse with glucosuria similar to that described here in fact, evidence from the literature supports the notion of reduced body weight with sglt2 deletion . Short - term studies of approximately 2 weeks have demonstrated no effect of sglt2 inhibition on body weight in zdf rats (14,15), whereas longer - term studies of 8 weeks or more have found reductions in body weight in rodents and humans (13,16,1921). Glucosuria that produced weight loss in humans ranged from 50 to 80 g / day, or 1016% of a 2,000 kcal / day diet (19,20). In sglt2 mice, glucosuria averaged 0.48 mg / day or 13% of the daily caloric intake of wt mice in this study . Interestingly, feeding increased about 20% in sglt2 mice, suggesting that an isocaloric diet would have a greater potential for weight loss during sglt2 deficiency . Previous studies in rodents have also noted concomitant increases in feeding behavior and weight loss during sglt2 inhibition (13,16). Although rc / sglt2 mice weighed 10% less than rc / wt mice, there was no significant difference in body weight after 4 weeks hfd, although sglt2 mice weighed about 6% less . Rc and hfd mice both had increased urine production during periods of food intake, and there was overall less urine output and glucosuria on the hfd compared with rc . Together, these data suggest that sglt2 activity is greatest in the postprandial setting and that inhibition will produce greater weight loss on a carbohydrate - rich diet . In conclusion, sglt2 deletion in mice resulted in profound glucosuria and compensatory behavioral adaptations aimed at replacing water and calories lost in the urine . Sglt2 knockout led to favorable reductions in plasma glucose and insulin during hfd and in the context of genetic obesity . Perhaps the most ominous complication of diabetes is loss of islet function, because this will accelerate the deterioration of glucose homeostasis and increase the exposure to glucose toxicity . Ablation of sglt2 in db / db mice prevented loss of -cell mass and thus preserved in vivo glucose - stimulated insulin secretion, which partly accounted for the overall improvements in glucose homeostasis . Taken together, these data support sglt2 inhibition as a viable insulin - independent treatment for type 2 diabetes.
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Lentiviral (lv) vectors, such as those derived from hiv-1, show exceptional promise as gene transfer agents and have been proven to be effective vehicles for transduction of epithelial cells of various organs, including airway epithelial cells in the lung . The epithelium lining the bronchi / bronchioli is the target cell compartment for a therapeutic approach based on gene delivery in cystic fibrosis (cf), a chronic autosomal recessive disorder due to mutation in the cf transmembrane conductance regulator (cftr) gene . Lv vectors bear some fundamental characteristics which could be useful for treating the cf lung disease, such as: (1) they integrate into the host genome and determine a long - term expression of either marker or cftr gene in animal and human xenograft models [37]; (2) they can be repeatedly administered without loss of efficiency; and (3) they do not elicit a gross inflammatory response in vitro and in vivo [4, 10]. The mechanism(s) of viral vector interaction with the apical plasma membrane and internalization has been an intensely studied question, in particular for adenoviral and adeno - associated viral vectors [1116]. The expression of receptors for these viral vectors and oncoretroviruses is more abundant on the basolateral membrane than on the apical side of the respiratory epithelium and they are hardly accessible because of the airway tight junctions [12, 17, 18]. Hiv-1-derived lv vectors expressing vesicular stomatitis virus g glycoprotein (vsv - g) on their capsid have been shown previously to transduce a polarized airway epithelium only in the presence of preconditioning agents with disruptive, although transient, effects on tight junctions [4, 5, 19]. These studies indicated entry block due to absence or low numbers of vsv - g receptors on the apical membrane of the airway epithelium, as it has been identified for other viral vectors, or postentry block, concerning the endocytosis route and nuclear import of viral genomes . Lv vectors have not been investigated in so much detail in respect to their attachment to and internalization by the cells . Building on the previoulsy published work by guibinga et al ., we observed that glycosaminoglycans (gags) are involved in lv vector - mediated transduction of polarized airway epithelial cells . In that study, a good transduction efficiency of polarized cellular monostrates with a last - generation lv vector pseudotyped with vsv - g was obtained, but only at high virus to cell ratios . Given that no studies have tested directly the effect of lv vectors on the tight junction (tj) stability and organization in airway epithelial cells, we evaluated the transduction efficiency as well as acute cytotoxicity, transepithelial resistance (ter), and occludin localization at the tj level in a bronchial - derived cell line grown in polarized fashion . We find that the vsv - g - pseudotyped lv vector infection at high viral loads is cytotoxic and determines occludin loss from the apical plasmamembrane and reduction of ter . In order to rule out the toxic effect of lv particles, we used lv virions noncovalently complexed with a cationic vector (i.e. Polyethylenimine). The vsv - g pseudotyped lv vector stock was prepared as previously described [21, 22]. Briefly, the lentivirus - based gene delivery system comprises four components: (1) the packaging plasmid pmdlg / prre, which contains elements such as structural proteins and enzymes involved in the formation of the viral particles, derived from the gag - pol genes; (2) the prsv - rev plasmid, which contains posttranscriptional regulator for gag and pol expression, as well as nuclear rna export encoded by the rev gene; (3) the transfer vector prrlsin18.cppt.cmv.egfp.wpre carrying the transgene gfp with the insertion of the ppt and the woodchuck post - transcriptional regulatory element (wpre); and (4) the pmd2.g plasmid containing the heterologous glycoprotein vsv - g . Cotransfection of the four plasmid vectors was performed on 293 t cells by calcium phosphate precipitation . The supernatant containing lv particles was concentrated by two rounds of ultracentrifugation and assayed for p24 gag antigen by enzyme - linked immunosorbent assay . The viral titer was determined by hela cell infection and subsequent flow cytometry analysis . The yield of the concentrated virus was typically 10 transducing units (tu)/ml and the specific activity ranged between 1.56 and 4.17 10 tu / ng of p24 . 16hbe41o- cells, derived from human bronchial epithelium (a kind gift of professor d. gruenert, university of california at san francisco, ca, usa), were grown in mem supplemented with earle's salt, 10% fetal bovine serum, l - glutamine and penicillin / streptomycin . They were routinely grown on plastic flasks coated with an extracellular matrix containing fibronectin / vitrogen / bovine serum albumin . The extracellular matrix coating is prepared in the laboratory as follows: 10 g / ml fibronectin (bd biosciences, ca, usa), 100 g / ml albumin from bovine serum (sigma - aldrich, milan, italy), and 30 g / ml bovine collagen type i (bd biosciences) are dissolved in mem . Cells were seeded on 6.5-mm diameter snapwell, 0.4-m pore size (corning, acton, ma, usa) at 1 10 per filter coated with the same extracellular matrix . Under these conditions, cells grow as a polarized sheet of cells and develop a transepithelial resistance of 550 ohm cm on average, as measured by a voltohmmeter (millicell - ers; millipore, vimodrone, italy). Polarized cells were incubated with the lv - gfp vector at different multiplicities of infection (moi) for 4 or 24 hours and then either immediately studied for propidium iodide staining and cell viability or incubated for further 48 hours for evaluation of gfp expression . Moi refers to the number of tu per one cell . Because we seeded 1 10 cells per well, a moi of 10 is equivalent to 10 tu; and a moi of 100 refers to 10 tu, and so forth . Branched polyethylenimine (mw 25,000 da) was obtained from sigma as 50% w / v solution . The solution was titrated with hcl solution to a ph of 7.4 and used as a 4.5 mg / ml stock solution (100 mm; stoichiometrically, this solution corresponds to 10.8 10 molecules of pei per l of solution). Ten l of saline containing different amounts of pei stock solution were added to 10 l of saline containing 50 moi (5 10 tu) of lv in order to obtain pei molecules / tu ratios ranging from 5 10 to 1 10(corresponding to a range of 0.062512.5 g/l as final concentration of pei). Spermidine (sigma - aldrich) stock solution (1 m) was diluted in order to obtain a final concentration ranging from 0.08 m to 8 mm (corresponding to spermidine molecules / tu ratios ranging from 1 10 to 1 10). The suspension containing either pei / lv or spermidine / lv was incubated for 15 minutes at room temperature and then added to cells . In another experimental setting, cells were preincubated with spermidine (final concentration ranging from 0.08 mm to 8 mm) for 1.5 hours, washed, and then infected with pei / lv at the ratio of 1 10 with 50 moi lv . The medium was changed 24 hours later and after further 48 hours, a preliminary evaluation of gfp expression was carried out by epifluorescence and confocal microscopy (see below). The cells were washed twice with phosphate - buffered saline (pbs), harvested by digesting with trypsin / ethylenediaminetetracetic acid (edta), and fixed in 2% paraformaldehyde . The cells were analyzed by fluorescence - activated cell sorting (facs) with a epics xl mcl flow cytometer (beckman coulter fullerton, ca, usa). The percentage of gfp - positive cells was determined after setting the gating on 99% of an untransfected control population of cells and by subtracting the fluorescence of the untransfected control cells . Analysis of gfp production was performed by plotting the flh-1 channel (512537 nm, with peak at 525 nm) against the flh-3 channel (608632 nm, with peak at 620 nm). Propidium iodide is an effective stain to identify nonviable cells since the dye is excluded by intact cell membranes and passes through damaged cell membranes and intercalates with dna and rna to form a bright red fluorescent complex [23, 24]. Briefly, cells on transwells were incubated with 25 g / ml propidium iodide (sigma - aldrich) for 20 minutes on ice, were washed with pbs, harvested by digesting with trypsin / edta, and resuspended in pbs . In each experiment, as a toxicity control, cells were incubated with 0.1% triton x-100 (sigma - aldrich) for 5 minutes at room temperature . The percentage of propidium iodide positive cells was determined after setting the gating on 99% of an untreated control population of cells and by subtracting the fluorescence of untreated control cells . Analysis of propidium iodide positive cells was performed by plotting the red channel (flh-2; 562588 nm, with peak at 575 nm) against the flh-1 channel . Mtt (3-(4,5-dimethyl - thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) is a water - soluble yellow dye that is readily taken up by viable cells and reduced by the action of mitochondrial dehydrogenases . The reduction product is a water - insoluble blue formazan, that must then be dissolved for colorimetric measurement . Briefly, a stock solution of mtt (sigma - aldrich) in phosphate buffered saline (pbs) (5 mg / ml) was added to the upper compartment of each well reaching a final concentration of 0.5 mg / ml (in 200 l of complete medium). After 4 hours the formazan crystals were dissolved in a 10% sds/50% dimethyl - formamide solution, and 100 l of the solution were transferred in a 96-well plate and measured spectrophotometrically by an elisa reader (powerwave ht, bio - tek, milan, italy) at a wavelenght of 570 nm with a reference wavelenght of 690 nm . The relative viability was calculated in respect to untreated cells (considered as 100%). Peripheral blood mononuclear cells (pbmcs) were isolated from the whole blood of three normal donors using lymphoprep (axis - shield, oslo, norway), according to the published protocol, and pooled . Total rna was extracted from polarized 16hbe14o- cells and pbmcs using trizol reagent (invitrogen, s. giuliano milanese, italy) following the protocol suggested by the manufacturer . The concentration of rna was estimated by nanodrop 1000 spectrophotometer (thermo scientific, waltham, ma, usa) at 260 nm wavelength and the purity was confirmed by measuring the absorbance ratio at 260/280 nm wavelengths . 1 g rna was used to prepare cdna by using a revertaid first strand cdna synthesis kit (fermentas, burlington, canada). Retrotranscription was performed under the following conditions: incubation for 60 minutes at 37c followed by 5 minutes at 70c . Rna were retrotranscripted in the presence of 200 units of revertaid m - mulv reverse transcriptase, 0.4 m of oligo (dt)18 primer, 1 mm of nucleotide mix, 20 units of ribolock rnase inhibitor, and commercial buffer (fermentas). For the pcr reaction, 100 ng of cdna were amplified in the presence of 1 unit taq polymerase (fermentas), 0.4 m of each primer (eurofins mwg operon / m medical srl, milan, italy), 0.2 mm of nucleotide mix, and commercial buffer containing 2 mm mgcl2 (fermentas). The primers for occludin amplification were forward 5'-agt gag tgc tat cct ggg cat- 3' and reverse 5'-cct ttg cag gtg ctc ttt ttg-3' which produced a dna segment of 600 bp . Pcr was performed under the following conditions: initial denaturation for 2 minutes at 94c, followed by 25 cycles of denaturation (15 seconds, 94c), annealing (30 seconds, 58c) and extension (1 minute, 72c). As control for rna integrity, we performed the actin pcr reaction using the following primers: forward 5'-caa ctg gga cga cat gga-3' and reverse 5'-acg tca cac ttc atg atg ga-3', which produced a dna segment of 610 bp . Pcr was performed under the following conditions: initial denaturation for 2 minutes at 94c, followed by 35 cycles of denaturation (15 seconds, 94c), annealing (30 seconds, 56c) and extension (1 minute, 72c). The identity of amplified products was confirmed by determination of molecular size on agarose gel electrophoresis (1.5% agarose in buffer containing 40 mm tris / acetate and 1 mm edta) and visualized by ethidium bromide staining (0.5 g / ml) under ultraviolet light . For occludin immunolocalization, polarized 16hbe14o- cells were washed three times with pbs, fixed in 3% paraformaldehyde, 2% sucrose, and permeabilized with ice cold triton hepes buffer (20 mm hepes, 300 mm sucrose, 50 mm nacl, 3 mm mgcl2, 0.5% triton x-100, ph 7.4) for 5 minutes at room temperature . Cells were incubated with blocking solution (2% bovine serum albumin [bsa], 2% fbs) for 15 minutes at 37 c, then with fluorescein isothiocyanate (fitc)-conjugated mouse anti - occludin antibody (zymed laboratories inc ., san francisco, ca, usa) (dilution 1: 100) for 30 minutes at 37 c . Filters were excised and placed side up on a glass slide, and overlayed with a drop of fluorescent mounting medium (dako, milan, italy) followed by a coverslip . Cells were analyzed using nikon te2000 microscope coupled to a radiance 2100 confocal dual - laser scanning microscopy system (bio - rad, segrate, italy). The microscope was equipped with a fitc filter (excitation 395 nm, emission 509 nm). Digital images were processed using the program laser sharp 2000 (bio - rad). For gfp detection, cells were fixed and permeabilized, incubated with propidium iodide (diluted at 1: 5,000 of 1 mg / ml stock solution) for 5 minutes at 37c, and washed . Filters were mounted and observed through the fitc and tritc (excitation 488 nm, emission 620 nm) filters . Statistical significance of differences was evaluated by a two - tailed unpaired student's t - test . We have previously demonstrated lv - mediated transgene delivery and expression in polarized airway epithelial cells at high lv: cell ratios, necessitating at least 2000 moi (multiplicity of infection). Thus, we interrogated the transduction efficiency and the cytotoxicity of lv vectors in polarized 16hbe41o- cells . Cells were incubated with different mois of lv particles for 24 hours, and then either immediately studied for propidium iodide staining (to assess membrane permeability) and viability (by means of the mtt assay), or incubated for further 48 hours for evaluation of gfp expression . As shown in figure 1(a), the higher the moi the higher the percentage of gfp - positive cells . Only cells with altered plasmamembrane permeability will intake propidium iodide which will bind nuclear dna . The higher the moi the higher the percentage of propidium iodide - stained cells (figure 1(b)). The viability was decreased only at mois 500 and 2000 (figure 1(c)). To investigate acute cytotoxicity of lv particles, membrane permeability and viability were assayed also after incubation of cells with lv particles for 4 hours . At any moi, lv particles did not exert any direct permeabilizing effect on polarized cells (figure 1(e)) and decreased the viability only at moi 2000 (figure 1(f)). Under these conditions, the percentage of transduced cells was lower than in the 24-hour protocol (figure 1(d)). These data show a dose - dependent cytotoxic effect of the lv vector, in terms of both alteration of membrane permeability and cell viability . Since the lv vector shows a disturbing effect on membrane permeability, the impact of lv particles on the tightness of the epithelial monostrates was initially investigated by measuring transepithelial resistance (ter). Ter was decreased in a moi - dependent fashion when cells were incubated with lv vectors for 24 hours (figure 2(a)), whereas was not affected at 4 hours (figure 2(b)). The effect observed with 2000 moi was similar to that achieved by ethylene glycol - bis(2-aminoethyl)-n, n, n,n-tetra - acetic acid (egta) (figure 2), a ca chelator known to transiently disrupt epithelial tight junctions . Tight junctions (tjs) are multiprotein complexes composed of integral proteins (claudins, occludin, and jam [junctional adhesion molecule]) that associate with cytoplasmic plaque proteins (zo-1, zo-2, and zo-3). The former mediate cell - cell adhesion, while the latter function as a bridge between the tj and the actin cytoskeleton [27, 28]. Since occludin has been shown to be internalized upon infection with group b coxsackievirus (cvb) and hepatitis c virus (hcv), we chose to study occludin expression and localization in cells infected with the lv vector . Reverse transcription - pcr revealed the presence of an occludin specific band (figure 3(a)). Freshly isolated and unstimulated lymphocytes and monocytes have been shown to not express occludin at the mrna and protein level [31, 32]. Pbmcs obtained from normal donors were negative for occludin mrna expression (figure 3(a)), confirming the specificity of the occludin amplification in 16hbe14o- cells . Polarized 16hbe14o- cells were incubated with the lv vector at different mois and analyzed by immunofluorescence and confocal microscopy 24 hours postinfection . Untreated cells displayed sharp circumferential organization of occludin at the lateral membrane between neighbouring cells (figure 3(b)). In egta - treated cells, high lv mois (500 and 2000) determined discontinuity in the occludin pattern at tj location (figures 3(d) and 3(e), with 2000 moi causing stronger disorganization of tjs . On the other hand, 50 moi did not cause any alteration in occludin localization at the cell periphery (figure 3(c)). Taken together, these results show that high but not low viral - to - cell ratios determine disruption of tjs when tjs are probed at 24 hours postinfection . These results prompted us to investigate if lv - mediated transduction at low moi could be enhanced by the polycation polyethylenimine (pei). Cationic lipids and polymers have been used to increase retrovirus titer and to enhance transduction of target cells [3336]. Various amounts of branched 25 kda pei molecules were mixed with 50 moi of lv particles to obtain different pei molecules / tu ratios . Cells were incubated with lv alone or pei / lv for 24 hours and gfp expression was evaluated 48 hours later by cytofluorimetry . The percentage of transduced cells did not change with low pei: lv ratios as compared with lv alone, and only the 10 ratio produced a significant 2.5-fold increase in gfp - positive cells as compared to plain lv (figure 4(a)). Epifluorescence and confocal microscopy analysis of transduced cells confirmed that pei increased by approximatively 3 fold the efficiency of lv - mediated transduction, as visualized by the number of gfp - positive cells (figure 5). To see whether the enhancing effect of pei could be universal to polyamines, we sought to determine the effect of native spermidine, a polyamine which is cationic at physiological ph . A wide range of spermidine molecules / tu ratios was tested (corresponding to a molar concentration range from 0.08 m to 8 mm), but no effect on the transduction rate given by plain lv particles (used at 50 moi) was observed (figure 4(b)). Thus, the enhancing effect seems to be unique to pei . In alternative, we investigated whether spermidine could inhibit the pei / lv - mediated transduction . In this case preincubation of polarized 16hbe14o- cells with native spermidine (0.088 mm) before addition of pei / lv formulated at the highest ratio (1 10 with 50 moi lv) did not exert any alteration in the efficiency of pei / lv (figure 4(c)). In these experiments, pei / lv particles and free pei were interrogated for their cell toxicity using the propidium iodide staining and the mtt assay . Cells were incubated with vectors for 24 hours, stained with propidium iodide for 30 minutes, and analyzed by cytofluorimetry . As shown in figure 6(a), only cells incubated with the highest pei: lv ratios with 50 moi lv, (10 pei / tu) showed a significant increase in nuclei stained with propidium iodide (from 0% up to 6.5 2.4%). Intriguingly, the same amount of pei alone (1 10 molecules) caused the uptake of propidium iodide by 9.9 4.9% of cells . Figure 6(b) shows that pei / lv and free pei exerted a small toxic effect on cells at highest doses . These results suggest that the membrane permeabilizing effect of pei / lv vectors could be attributed to free pei, as previously shown by the toxicity exerted by pei alone . Ter was measured at 4 and 24 hours postinfection and was not affected by pei / lv particles (not shown). Occludin localization was not altered by incubation of cells with pei / lv at 24 hours (figures 7(b)7(d)). Also free pei (10 molecules) did not exert any effect on tj integrity (figure 7(e)). Positively charged polycations such as polybrene are known to be required for efficient infection of cells with retroviruses and retrovirus vectors, possibly by stabilizing the interaction between negatively charged virus particles and target cellular membranes [20, 39]. Indeed, polybrene has been used also in lv - mediated transduction of polarized epithelial cells obtained from the airways . In our hands, polybrene was toxic to the cells and thus it was withdrawn from the transduction protocol . For this reason, we had to use high mois (i.e. At least 2000) to achieve a meaningful transduction of polarized airway bronchial and tracheal epithelial cells . In this study, we show that high lv mois are toxic to polarized airway epithelial cells, which are considered a good approximative model for native airway epithelium [26, 4143]. High mois determined an increase in membrane permeability at 24 hours but not at 4 hours, suggesting that the direct interaction of lv particles with cells is not harmful, rather it is so for entry, transport, and viral transcription within the cytosol . Interestingly, the damage caused by lv particles was reflected also by the decrease of ter and loss of occludin at the tjs between cells . The mtt assay shows that only high lv doses decreased the cellular viability both at 4 and 24 hours . These results strongly suggest that lv particles exert their toxic action through direct interaction with tj proteins, an effect visible only at 24 hours . Some recently acquired data indicate that proficient viral infection is dependent on the interaction of viral envelope glycoproteins with tj proteins . The primary cvb receptor, the coxsackievirus and adenovirus receptor (car), is a transmembrane component of the tj and cvb enters polarized epithelial cells from the tj, causing a transient disruption of tj integrity . Cvb does not induce major reorganization of the tj, but stimulates the specific internalization of occludin within macropinosomes . Hcv envelope glycoproteins induce a loss of claudin-1, zo-1, and occludin - delineated junctional accumulation and occludin is required for late entry step of hcv into cells . Overall, based on our previous publication, we speculate that vsv - g - pseudotyped lv virions are concentrated on the apical surface of polarized airway epithelial cells by initial attachment to gags . The binding of lv particles with gags is based on nonelectrostatic interactions . In a further step, lv virions should then bind to entry receptors (not identified yet) and eventually to occludin for internalization . Loss of occludin from the tjs and their opening could be a disadvantage in lung diseases such as cf, because of the presence of bacteria and bacterial products in the airways . In order to find a transduction protocol with less viral loads, minimal toxicity and eventually no delocalization of occludin from the tjs, we have combined lv particles with the polycation pei, based on the rationale that a cationic component would charge associate with lv particles, which carry a net negative surface charge . Several previous studies have reported the use of cationic molecules to enhance viral uptake and subsequent transgene expression in vitro and in vivo, mainly for adenoviral [4752] and retroviral vectors [3336]. In particular, pei facilitated transduction efficiency by adenoviral vectors in cultured mouse myotubes and in 9l gliosarcoma cells and enhanced retroviral transduction in nih3t3 cells . To our best knowledge, ours is the first study showing that pei enhanced lv - mediated transduction of airway epithelial cells . Pei / lv particles increased membrane permeability only at the highest pei / lv ratio, an effect likely due to excess free pei, with a little effect on the viability . Indeed, pei is known to induce the formation of transient, nanoscale holes in the membranes of living cells and these holes allow a greatly enhanced exchange of materials across the cell membrane, including propidium iodide . Although we have not investigated the membrane permeability with spermidine / lv vectors, the fact that spermidine had no enhancing effect on lv - mediated transduction supports the notion that the pei's action is due to a membrane destabilizing effect . Pei / lv particles and free pei did not cause any alteration on ter and in occludin localization, indicating that pei - induced nanoscale holes in the plasma membrane do not affect tj integrity . In conclusion, pei / lv vectors are more efficient than lv alone used at low viral load in transducing polarized epithelial cells without so pronounced cytotoxicity, and, more importantly, without disrupting tight junctions . Because the transduction efficiency mediated by pei / lv is still low it needs further refinement for obtaining higher transduction rates of polarized airway epithelia, a goal which could be achieved by testing other pei architectures . For these reasons the pei / lv vector warrants further characterization for being considered as a valid tool in gene therapy of genetic lung diseases.
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When stress is applied to a crack introduced in ductile materials, dislocations are activated and propagate from the crack tip . The stress concentration at the crack tip is accommodated by dislocation activities such as dislocation glide and multiplication, which lead to an increase in fracture toughness . Fracture toughness increases with temperature, and the fracture mode changes from brittle to ductile, which is the so - called brittle - to - ductile transition (bdt). Silicon single crystals have been used as model crystals to understand the mechanism behind the bdt because they show an abrupt increase in the fracture toughness at a certain temperature and because large dislocation - free silicon single crystals are available . John pointed out that the increase in the fracture toughness at the bdt is controlled by dislocation glide in silicon . Hirsch et al . Suggested that the abrupt increase in the fracture toughness at the bdt in silicon could be explained by the assumption that the value of the critical stress intensity factor for dislocation emission at the crack tip should be close to that of the fracture toughness of silicon . A gradual increase in fracture toughness, in contrast, could be due to the low critical stress intensity factor for dislocation emission . Figure 1(a) shows a contour map of the stress component normal to the crack plane, yy, around a crack tip, with a pair of pure edge dislocations in front of the crack tip without any stress applied . The contour map was obtained by calculating the stress field based on the elastic theory derived by thomson, with the dislocation configuration as shown in fig . Two dislocations are located 45 from the crack line and 100 m away from the crack tip . The extra half - planes of these dislocations were both at the crack side with respect to their slip planes, as shown in fig . 1(b), which represents the situation after two pure edge dislocations have been emitted from the crack tip . This indicates that some extra tensile stress is necessary for the crack to propagate, cancelling the compressive stress concentration due to the dislocations . This is the origin of the increase in the fracture toughness from the viewpoint of crack tip shielding due to dislocations [2,58]. 1. (a) contour map of yy around a crack tip with two pure edge dislocations without external stress . (b) schematic representation of the edge dislocations used for the calculation of the stress field in (a). (a) contour map of yy around a crack tip with two pure edge dislocations without external stress . (b) schematic representation of the edge dislocations used for the calculation of the stress field in (a). The first macroscopic experimental verification of the shielding effect due to dislocations was reported using photoelasticity in ionic crystals . It was shown that the residual stress field around a crack after deformation was actually a compressive stress field . In addition, higashida et al . Directly observed crack tip dislocations in mgo and silicon single crystals after deformation . The burgers vectors, including their signs, were determined in this study using high - voltage electron microscopy [912]. They pointed out that the local stress intensity factor due to dislocations calculated from the dislocation configuration is negative, which indicated that the dislocations observed were of the shielding type . A recent development in high - resolution transmission electron microscopy (hrtem) makes it possible to measure strain at the atomic scale . Htch suggested geometric phase analysis (gpa) which enables the calculation of the strain distribution from hrtem images . Gpa utilizes the crystal periodicity information that is extracted by performing fourier transformations of hrtem images . It is possible to measure the elastic displacement of atoms from a non - deformed area by calculating the change in phase of the lattice image . Once the displacement of atoms is known, the elastic strain field due to the displacement of atoms can be calculated . In this study, the local strain in front of a crack tip with dislocations in single crystal silicon was measured using gpa software (hrem research), which is a plug - in for digitalmicrograph (gatan), to clarify the existence of the shielding effect of dislocations at the atomic scale . The sample used in this study was a (110) single crystal silicon wafer with a thickness of 0.63 mm . Figure 2 shows an indent on the sample surface introduced using a micro vickers indenter (mvk - e3, mitutoyo) with an applied load of 0.5 n and a dwell time of 7 s. cracks propagated from the corners of the indent along both the <100> and <110> directions . The {100} cracks that propagated along the <110> directions were shorter than the {110} cracks that propagated along the <100> directions . That is, fracture toughness on the {100} planes was higher than that on the {110} planes . Because {110} is the second cleavage plane of silicon, it is essential to elucidate the strain around {110} crack tips with dislocations . Therefore, one of the {110} crack tips was observed in this study . The tem sample used in this study was prepared using focused ion beam (fib, quanta 3d 200i, fei). First, carbon was deposited on the top surface to protect the crack from damage by ion beams during the machining process . The area around the deposited region was milled with an ion beam at an operating voltage of 30 kv . The protective carbon layer was removed and the sample was thinned by an ion beam with an operating voltage of 30 kv, which was gradually lowered to 16, 8, 5 and 2 kv . The thinning at smaller voltages was performed to remove layers damaged during the higher voltage thinning process . The sample was thinned until the maximum thickness of the sample was <200 nm . Figure 3 shows a hrtem sample after fib machining, wherein a crack is extending from the left edge to the centre . The crack is located in a very small area that is about 480 nm away from the thinnest edge . The sample was then subjected to post - fib cleaning using a nanomill system (model 1040, fischione). This cleaning was conducted with an operating voltage of 900 v for 5 min at a tilting angle 10 to clean the top and bottom of the sample . It was followed by milling with 500 v for 10 min on each side to further reduce damage . The final sample thickness around the crack tip was 65 nm, which was measured during tem observation using the electron energy loss spectroscopy (eels) log - ratio technique . Fig . Is given by the intensity of the zero - loss electron, io, and the total electron intensity, it, in an energy loss spectrum as follows: (1)t=lnitio and (2)=106feoemln2eoem, where is the mean free path of electrons for inelastic scattering, f is the realistic correction factor, eo is the incident energy of electrons, em is the average energy loss of electrons and is the collection angle . The foil sample was observed with an aberration corrected hrtem (jem - arm200f, jeol) at an operating voltage of 200 kv at the ultramicroscopy research center, kyushu university . Figure 4(a) shows a low - magnification tem image indicating the position of a crack extending from the edge of the foil sample . To ascertain the exact position of the crack tip at the atomic scale it is also necessary to use high - angle annular dark field scanning transmission electron microscopy (haadf - stem) to determine the exact position of the crack tip . Figure 4(b) shows a z - contrast haadf - stem image in which the dark area is the crack, which is clearly distinguishable from the matrix . Figure 4(c) shows an enlarged haadf - stem image of the area indicated by an arrow in fig . 4(b) at the atomic scale, indicating that the crack is observed nearly edge - on . The crack plane is (011) and the crack is opened 2 nm wide . (b) haadf - stem images of a crack tip with (c) increased magnification . (b) haadf - stem images of a crack tip with (c) increased magnification . Figure 5(a) shows an original hrtem image of the crack tip at the atomic scale in the same area as that in fig . The distorted area in front of the crack tip indicates the existence of dislocations in that region . To determine the character of those dislocations around the crack tip, the original image was filtered to clarify lattice planes . First, the original hrtem image was transformed into the frequency domain using the fourier fast transformation (fft), as shown in fig . The background, or low frequency noise, in the centre spot of the fft image was filtered out . 5(d) was then converted back to image space by performing the inverse fft, resulting in the filtered hrtem image shown in fig . 5(b) it could be seen that there are four dislocations around the crack tip: d1, d2, d3 and d4 . There are two dislocations in the distorted region in front of the crack tip and two other dislocations on the left - hand and right - hand sides of the crack . Red and green lines in the figure represent the extra half - planes of each dislocation . The dislocations denoted as d1 and d3 lie on the slip plane of (111) while those denoted as d2 and d4 lie on the slip plane of (111). The burgers vectors of the dislocations were also determined by using the finish - to - start - right - hand (fs / rh) convention . Each burgers circuit coloured in yellow in front of the crack tip contains one dislocation . These burgers circuits were then transferred to undistorted regions at the right - hand and left - hand sides above the crack tip, forming open circuits . Because the image is the projection on the (011) plane, the exact directions of their burgers vectors cannot be determined perfectly . Dislocations d1 and d3 have the same possible pair of burgers vectors, and so do dislocations d2 and d4 . The burgers vectors of d1d4 are defined to be b1 to b4 . In each case, the edge component of the burgers vectors are the same for d1, d3; and d2, d4; which are a/6[211] and a/6[211], respectively . Here, we focus on the edge component of the burgers vector, which induces mode i shielding at the crack tip . To clarify the dislocation shielding field at the atomic scale table 1.possible burgers vectors for b1, b2, b3 and b4b1, b3b2, b4first possibilitya/2[110]=a/6[211]+a/6[121]a/2=a/6[211]+a/6second possibilitya/2[101]=a/6[211]+a/6[112]a/2[110]=a/6[211]+a/6[121] fig . (b) filtered image obtained from (d), thompson's tetrahedron is also shown . Possible burgers vectors for b1, b2, b3 and b4 (a) original hrtem image at the crack tip . (b) filtered image obtained from (d), thompson's tetrahedron is also shown . The blue areas indicate compressive strain fields while the red areas indicate tensile strain fields . There are four butterfly - like points around the crack tip where the blue and red colours discontinuously changed . It should be noted here that yy is negative around the crack tip, that is, the stress intensity at the crack tip is compressive, which is caused by those dislocations in front of the crack tip . 6.strain map of yy in front of the crack tip showing intense strain at the crack tip and at the dislocations: (a) obtained by gpa and (b) calculated based on elastic theory . The strain intensity at the crack tip is compressive, which indicates the existence of the shielding effect at the atomic scale . The spatial scale is the same between (a) and (b) while the colour scale of (b) is not exactly the same as that of (a). Strain map of yy in front of the crack tip showing intense strain at the crack tip and at the dislocations: (a) obtained by gpa and (b) calculated based on elastic theory . The strain intensity at the crack tip is compressive, which indicates the existence of the shielding effect at the atomic scale . The spatial scale is the same between (a) and (b) while the colour scale of (b) is not exactly the same as that of (a). To verify the experimental results, the crack tip strain field with the dislocations the strain components in two - dimensions at the crack tip with dislocations, but without external stress, are given by the following equations, where the crack plane lies along the x - axis and the crack tip is at the origin of the complex field: (3)xx=1e[xx(yy+zz)], (4)yy=1e[yy(xx+zz)], (5)xy=12xy, (6)xx+yy=2[(z)+(z)], (7)yyixy=++(zz) (8)=jaj1zjjz1/2 + 1 + 1zjjz1/21+jaj(jj)2(zj)2jz1/2+zj1/22, (9)=jaj1zjjz1/2 + 1 + 1zjjz1/21jaj(jj)2(zj)2jz1/2+zj1/2 + 2, (10)aj=bj2i(+1), (11)=(3)(1+),for the plane stress case where z is a complex variable for the position relative to the crack tip in the form of z = x+iy, j is the position of the jth dislocation in the form of j = cx+idy, is the shear modulus (64 gpa for silicon), bj is the burgers vector of the jth dislocation in the form of bj = fx+igy and is the poisson's ratio (0.278). The strain field around the crack tip was calculated with four dislocations and without applied stress . The positions and the burgers vectors are those of the experimentally observed dislocations shown in fig . 5 . Figure 6(b) shows the calculated strain map of yy in the same spatial scale as that in fig . 6(a). Figure 6(b) also shows compressive strain fields at the crack tip and at the dislocation cores . The calculated strain map is in good agreement with the experimental result shown in fig . It is stressed that these results show experimental proof of the dislocation shielding effect observed at the atomic scale in front of the crack tip . The measured strain field around the crack tip was compressive owing to the dislocation shielding effect . The dislocations create an extra barrier in front of the crack tip, making it more difficult for the crack to propagate, which increase the fracture toughness at the bdt . The authors thank jsps kakenhi (#24686079, #15h04147) and kazato research foundation for partly supporting this research . Funding to pay the open access publication charges for the article was provided by jsps kakenhi.
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Governance, broadly defined as the process by which the authority of a country exercises government formation, sound policy making and effective policy implementation, is regarded as a crucial determinant of population health . The effect of governance on the health of a country's population is determined by the government's response to its citizens' needs, its distribution of health - related program resources, and its management of social and environmental conditions such as economic development, food security and public education . Therefore, a country's governance is viewed as a key factor in achieving the united nations (un) millennium development goals (mdg). Historical and contemporary political analyses have shown that governments are key players in devising public policies on labor markets, welfare systems, and economic growth strategies . Core capabilities of an effective government are protecting the population from violence, preventing bureaucratic corruption, and supplying and meeting needs for public goods and services . Effective governance entails high - quality public services and the ability to contribute effectively to population health without undue political influence . Hence, effective governance, together with greater investments in infrastructure related to health, can improve people's daily living environment, quality of life, and health . In contrast, less effective governance will be unable to improve the key socioeconomic determinants needed to benefit population health . Poor governance i.e., abusing citizens, failing to provide equal protection under the law, or corrupting and mismanaging resources, infrastructure or the economy, is detrimental to population health . There has been a growing quest for better governance in the pursuit of health equity at the global level . This issue is particularly important as progress toward the mdg for child mortality has been slower than expected . Lozano et al . Estimated that only 7% (9 out of 137) of developing countries were likely to meet the target child mortality rate by 2015 . However, lack of effective governance was found to be a substantial risk factor, resulting in an inability to utilize aid resources and eventually in failure to institute a good healthcare system . Another concern has been the interplay between governance and population health in the context of the trend toward country - level urbanization and the pursuit of economic growth for better living and health conditions . People who live in urban settings or in countries with relatively better economic status are likely to have better access to food, sanitation, education, employment and healthcare - all factors that may contribute to better health . A failure of governance in the course of a country's path toward urbanization and economic development can result in ineffective health policies and services, resulting in unhealthy living conditions such as informal urban settlements and slums . Such poor living conditions are usually associated with unsafe drinking water and lack of improved sanitation facilities, and may place people at risk of contracting easily preventable diseases . Attention is also being paid to the role of governance in the practice of disease control, especially during the course of urbanization . Urbanization in the demographic sense is defined as the process of increasing the concentration of people living in urban areas . Moore notes that greater population density in urban settings is associated with greater potential for disease transmission and greater susceptibility to disease epidemics . Communicable and non - communicable chronic diseases alike, including malaria, hiv / aids, and illness related to air pollution, have been increasing dramatically in rapidly urbanizing areas of africa and asia, and in various countries in other parts of the world with relatively low per capita incomes . The crowding in high - density urban environments requires good urban governance for effective disease control . Children, as a result of physiological and behavioral differences from adults, are more susceptible to environmental, political, and socioeconomic factors . As global efforts toward reducing child mortality get under way, an understanding of how governance affects child health in the context of urbanization, economic development and disease control, especially in countries with poor governance, is essential . However, attempts to estimate the effect of governance on child mortality in the context of a country's urbanization, economic development and disease control are generally hampered by a lack of comparable indicators across countries . In addition, yearly variability in all these factors, together with the continuous effect of governance indicators, is likely to lead to complications; longitudinal analysis should therefore be used . We aimed to investigate how country - level child mortality is associated with urbanization, economic development, disease control, and governance . To address this issue, we collected publicly available country - specific data and conducted cross - national comparisons and longitudinal data modeling . The objective of our study was to investigate the ecological association between governance and child mortality and to provide global health efforts with further evidence of the need for better governance . We obtained data on child health and its predictors, from publicly available databases, for the period 1996 to 2010 for 149 countries (representing 96.5% of the world's population in 2010) that offered available data for analyses . The measure used to indicate child health status in each country was the under-5 mortality rate reported by the un, which measures the probability of a child dying between birth and his or her 5th birthday (an exact age of 5 years), expressed per 1000 live births . Yearly under-5 mortality rates were extracted from the interpolated demographic indicators in the un document world population prospects (wpp). The definitions and sources of the key determinants of under-5 mortality rates investigated in this study are shown in box 1 . Governance indicators were extracted from the worldwide governance indicators (wgi) reported by the world bank . The wgi, a database published by the world bank in 1996, consists of six dimensions of governance: voice and accountability, political stability and absence of violence, government effectiveness, regulatory quality, rule of law, and control of corruption . It was updated every 2 years between 1996 and 2002 and has been updated annually since 2002 . The six governance indicators were aggregated from 441 indicators of 35 data sources for 212 countries and territories . These data were based on surveys of individuals or domestic firms with first - hand knowledge of the governance situation in the country to reflect the common perceptions of diverse data providers, including commercial businesses (averaged share of sources=31%), surveys of firms or households (27%), non - governmental organizations (16%), and the public sector (26%). The unobserved components model was applied to construct an aggregate of the six governance dimensions from these individual measures . Data were first rescaled and then averaged with weights based on the precision of the individual data sources for each of the governance indicators: commercial businesses (averaged share of weights=0.37), surveys of firms or households (0.14), non - governmental organizations (0.22), and the public sector (0.28). To facilitate cross - country and over - time comparison, the scores were normally distributed, with a mean of zero and a standard deviation of one . With such transformation, these wgi scores were used to reflect relative levels of governance among studied countries, and do not directly quantify absolute qualities of governance . Voice and accountability perceptions of the extent to which a country's citizens are able to participate in selecting their government, as well as freedom of expression, freedom of association, and a free media (world bank's world governance indicators [wgi]) political stability and absence of violence perceptions of the likelihood that the government will be destabilized or overthrown by unconstitutional or violent means, including politically motivated violence and terrorism (world bank's wgi) government effectiveness perceptions of the quality of public services, the quality of the civil service and the degree of its independence from political pressures, the quality of policy formulation and implementation, and the credibility of the government's commitment to such policies (world bank's wgi) perceptions of the ability of the government to formulate and implement sound policies and regulations that permit and promote private sector development (world bank's wgi) perceptions of the extent to which agents have confidence in and abide by the rules of society, and in particular the quality of contract enforcement, property rights, the police, and the courts, as well as the likelihood of crime and violence (world bank's wgi) control of corruption perceptions of the extent to which public power is exercised for private gain, including both petty and grand forms of corruption, as well as capture of the state by elites and private interests (world bank's wgi) proportion of a country's population living in areas classified as urban, according to the criteria used by each country (united nations' world population prospects, ministry of the interior of taiwan) the state of each country's economy was calculated from income based on the yearly expenditure - side gross domestic product (gdp) at purchasing power parity (penn world table version 8.0) proportion of children aged 1223 months in each country who had received immunization against measles and against diphtheria, pertussis, and tetanus (dpt) (world bank's world development indicators [wdi], department of health of taiwan) proportion of the population in each country with access to an improved water source (world bank's wdi, ministry of economic affairs of taiwan) proportion of a country's population with access to improved sanitation facilities (world bank's wdi, ministry of the interior of taiwan) sum of public and private health expenditures as a percentage of total gdp, which covers the provision of health services (preventive and curative), family planning activities, nutrition activities, and emergency aid designated for health but does not include provision of water and sanitation (world bank's wdi, social indicators of taiwan) annual average concentrations of particulate matter with diameter <10 (pm10) (world bank's wdi, environmental protection administration of taiwan) the world bank collected the pm10 data from the who and then estimated the country - level pm10 data using urban - population - weighted pm10 levels in residential areas of cities larger than 100 000 . For taiwan, we used the average pm10 data collected from ambient air quality monitoring stations located in areas of high population density . Urbanization was measured as the proportion of a country's population living in areas classified as urban, according to the criteria used by that country . We chose national economies for our analysis because poverty perpetuates inadequate living conditions and drives up child mortality . Population immunization rate (e.g. Against measles, and diphtheria, pertussis, and tetanus [dpt]), clean water supply, and sanitation were also considered in our analysis because they are vital elements in preventing disease outbreaks . These data were obtained from the world bank's world development indicators and country reports . High population density with inadequate medical and hygienic services contributes to a rising incidence of communicable diseases in urban environments . The evidence on the effect of health expenditures on reducing child mortality is inconclusive: some studies have found a country's health expenditures to have positive effects on population health outcomes, while others have shown minor or non - significant effects on child mortality . For this study, we included health expenditures as a variable in our factor selection process . We also included air pollution, because it can reflect a country's environmental quality during urbanization and industrialization and is associated with various adverse health effects for children, including increased hospital admissions for acute respiratory conditions, increased medication use in children with asthma, and mortality . The major sources of urban air pollution are burning of fossil fuels or biomass, power generation, industrial processes, and motor vehicle activities . Particulate matter, one of the typical urban pollutants, is associated with level of economic activity, population size, energy consumption, and pollution - related regulations and controls in urban areas . We first applied a factor analysis with a varimax rotation to reduce the number of correlated variables and identify and categorize interrelated variables . Next, the effects of selected factors on the under-5 mortality rate were analyzed using the semiparametric generalized additive mixed model, with the logarithm of the under-5 mortality rate (y) to base 10 of a country as the dependent variable, selected factors (fi) as fixed - effect variables, and the intercept as the random - effect variable: log10yj, t=0+b0j+ii, jfi, j, t+f(t)+j, t where i(1, 2, 3,) denotes the number of selected factors, j(1, 2,, 149) denotes the country code, and t(1, 2,, 15) denotes the calendar year from 1996 to 2010 . The decision on whether to keep or drop factors (fi) was based on the akaike information criterion . The time smoother f(t) was estimated by a cubic b - spline to control temporally autoregressive correlations . J, t was the error term following a normal distribution with mean zero and variance j, t . The random intercept b0j was an unstructured spatial term following a normal distribution with mean zero and estimated variance 0.1980 to control for spatial heterogeneity among countries . All statistical analyses were performed by the proc corr, factor, and glimmix procedures in sas v9.2 (sas institute, cary, nc, usa). Graphs were drawn using sigmaplot v10.0 (systat software, richmond, ca, usa). We obtained data on child health and its predictors, from publicly available databases, for the period 1996 to 2010 for 149 countries (representing 96.5% of the world's population in 2010) that offered available data for analyses . The measure used to indicate child health status in each country was the under-5 mortality rate reported by the un, which measures the probability of a child dying between birth and his or her 5th birthday (an exact age of 5 years), expressed per 1000 live births . Yearly under-5 mortality rates were extracted from the interpolated demographic indicators in the un document world population prospects (wpp). The definitions and sources of the key determinants of under-5 mortality rates investigated in this study are shown in box 1 . Governance indicators were extracted from the worldwide governance indicators (wgi) reported by the world bank . The wgi, a database published by the world bank in 1996, consists of six dimensions of governance: voice and accountability, political stability and absence of violence, government effectiveness, regulatory quality, rule of law, and control of corruption . It was updated every 2 years between 1996 and 2002 and has been updated annually since 2002 . The six governance indicators were aggregated from 441 indicators of 35 data sources for 212 countries and territories . These data were based on surveys of individuals or domestic firms with first - hand knowledge of the governance situation in the country to reflect the common perceptions of diverse data providers, including commercial businesses (averaged share of sources=31%), surveys of firms or households (27%), non - governmental organizations (16%), and the public sector (26%). The unobserved components model was applied to construct an aggregate of the six governance dimensions from these individual measures . Data were first rescaled and then averaged with weights based on the precision of the individual data sources for each of the governance indicators: commercial businesses (averaged share of weights=0.37), surveys of firms or households (0.14), non - governmental organizations (0.22), and the public sector (0.28). To facilitate cross - country and over - time comparison, the scores were normally distributed, with a mean of zero and a standard deviation of one . With such transformation, these wgi scores were used to reflect relative levels of governance among studied countries, and do not directly quantify absolute qualities of governance . Voice and accountability perceptions of the extent to which a country's citizens are able to participate in selecting their government, as well as freedom of expression, freedom of association, and a free media (world bank's world governance indicators [wgi]) political stability and absence of violence perceptions of the likelihood that the government will be destabilized or overthrown by unconstitutional or violent means, including politically motivated violence and terrorism (world bank's wgi) government effectiveness perceptions of the quality of public services, the quality of the civil service and the degree of its independence from political pressures, the quality of policy formulation and implementation, and the credibility of the government's commitment to such policies (world bank's wgi) perceptions of the ability of the government to formulate and implement sound policies and regulations that permit and promote private sector development (world bank's wgi) perceptions of the extent to which agents have confidence in and abide by the rules of society, and in particular the quality of contract enforcement, property rights, the police, and the courts, as well as the likelihood of crime and violence (world bank's wgi) control of corruption perceptions of the extent to which public power is exercised for private gain, including both petty and grand forms of corruption, as well as capture of the state by elites and private interests (world bank's wgi) proportion of a country's population living in areas classified as urban, according to the criteria used by each country (united nations' world population prospects, ministry of the interior of taiwan) the state of each country's economy was calculated from income based on the yearly expenditure - side gross domestic product (gdp) at purchasing power parity (penn world table version 8.0) proportion of children aged 1223 months in each country who had received immunization against measles and against diphtheria, pertussis, and tetanus (dpt) (world bank's world development indicators [wdi], department of health of taiwan) proportion of the population in each country with access to an improved water source (world bank's wdi, ministry of economic affairs of taiwan) proportion of a country's population with access to improved sanitation facilities (world bank's wdi, ministry of the interior of taiwan) sum of public and private health expenditures as a percentage of total gdp, which covers the provision of health services (preventive and curative), family planning activities, nutrition activities, and emergency aid designated for health but does not include provision of water and sanitation (world bank's wdi, social indicators of taiwan) annual average concentrations of particulate matter with diameter <10 (pm10) (world bank's wdi, environmental protection administration of taiwan) the world bank collected the pm10 data from the who and then estimated the country - level pm10 data using urban - population - weighted pm10 levels in residential areas of cities larger than 100 000 . For taiwan, we used the average pm10 data collected from ambient air quality monitoring stations located in areas of high population density . Urbanization was measured as the proportion of a country's population living in areas classified as urban, according to the criteria used by that country . We chose national economies for our analysis because poverty perpetuates inadequate living conditions and drives up child mortality . Population immunization rate (e.g. Against measles, and diphtheria, pertussis, and tetanus [dpt]), clean water supply, and sanitation were also considered in our analysis because they are vital elements in preventing disease outbreaks . These data were obtained from the world bank's world development indicators and country reports . High population density with inadequate medical and hygienic services contributes to a rising incidence of communicable diseases in urban environments . The evidence on the effect of health expenditures on reducing child mortality is inconclusive: some studies have found a country's health expenditures to have positive effects on population health outcomes, while others have shown minor or non - significant effects on child mortality . For this study, we included health expenditures as a variable in our factor selection process . We also included air pollution, because it can reflect a country's environmental quality during urbanization and industrialization and is associated with various adverse health effects for children, including increased hospital admissions for acute respiratory conditions, increased medication use in children with asthma, and mortality . The major sources of urban air pollution are burning of fossil fuels or biomass, power generation, industrial processes, and motor vehicle activities . Particulate matter, one of the typical urban pollutants, is associated with level of economic activity, population size, energy consumption, and pollution - related regulations and controls in urban areas . We first applied a factor analysis with a varimax rotation to reduce the number of correlated variables and identify and categorize interrelated variables . Next, the effects of selected factors on the under-5 mortality rate were analyzed using the semiparametric generalized additive mixed model, with the logarithm of the under-5 mortality rate (y) to base 10 of a country as the dependent variable, selected factors (fi) as fixed - effect variables, and the intercept as the random - effect variable: log10yj, t=0+b0j+ii, jfi, j, t+f(t)+j, t where i(1, 2, 3,) denotes the number of selected factors, j(1, 2,, 149) denotes the country code, and t(1, 2,, 15) denotes the calendar year from 1996 to 2010 . The decision on whether to keep or drop factors (fi) was based on the akaike information criterion . The time smoother f(t) j, t was the error term following a normal distribution with mean zero and variance j, t . The random intercept b0j was an unstructured spatial term following a normal distribution with mean zero and estimated variance 0.1980 to control for spatial heterogeneity among countries . All statistical analyses were performed by the proc corr, factor, and glimmix procedures in sas v9.2 (sas institute, cary, nc, usa). Graphs were drawn using sigmaplot v10.0 (systat software, richmond, ca, usa). Table 1 shows the averaged means, standard deviations, minimums, and maximums for the under-5 mortality rate, the six governance indicators, and the other eight selected determinants of child mortality across 149 countries in 2010 . On average, the difference in mortality rate between countries with the lowest and the highest rates was 89-fold, with the lowest rate being observed in singapore (2.28 per 1000 live births) and the highest rate in chad (202.89 per 1000 live births). The lowest governance scores were: voice and accountability (2.02) and regulatory quality (2.08) in turkmenistan, political stability and absence of violence score (2.73) in pakistan, government effectiveness (1.73) in comoros, rule of law (1.79) in zimbabwe, and control of corruption (1.50) in equatorial guinea . Most of the highest governance indicator scores were observed in european countries, including voice and accountability (1.61) in switzerland, political stability and absence of violence (1.44) in luxembourg, rule of law (1.97) in finland, and regulatory quality (1.90) and control of corruption (2.38) in denmark; the highest government effectiveness score was observed in singapore (2.24). Table 1.averaged means, standard deviations, minimums, and maximums for 149 countries' under-5 mortality rate (deaths at age <5 years per 1000 live births) and indicators of governance, development, and disease controlindicatorsmeansdminimummedianmaximummodeunder-5 mortality rate49.8851.652.2827.07202.89governance indicators voice and accountability score (points)0.100.972.020.141.61 political stability and absence of violence score (points)0.190.922.730.061.44 government effectiveness score (points)0.020.991.730.212.24 regulatory quality score (points)0.010.942.080.151.90 rule of law score (points)0.110.991.790.361.97 control of corruption score (points)0.091.011.500.392.38development - related indicators urbanization level (% of a country's population living in urban areas)55.9922.1511.0058.15100.0040.10 log transformed expenditure - based gdp at purchasing power parity (millions of international dollars)10.992.055.6610.9016.39disease control - related indicators clean water supply (% of population with access to an improved water source)86.8215.6844.0093.00100.00100.00 sanitation (% of population with access to improved sanitation facilities)70.9430.449.0083.00100.00100.00 immunization: measles (% of children ages 1223 months immunized against measles)87.6813.1533.0093.0099.0099.00 immunization: dpt (% of children aged 1223 months immunized against dpt)88.3813.3733.0094.0099.0099.00health expense - related indicators health expenditures, total (% of gdp)6.912.890.686.4820.804.39pollution - related indicator pm10 concentration at country level (g / m)36.5025.176.0029.00137.0027.00sd: standard deviation; gdp: gross domestic product; dpt: diphtheria, pertussis, and tetanus; pm10: particulate matter with diameter <10 . Values were estimated using the latest available data for each country . All data were from 2010 with the following exceptions: clean water supply data for argentina (2007), equatorial guinea (2006), grenada (2004), lithuania (2009), panama (2009), romania (2008), tunisia (2009), turkmenistan (2006), and venezuela (bolivarian republic of) (2007); sanitation data for argentina (2007), equatorial guinea (2006), lebanon (2005), latvia (2009), lithuania (2009), panama (2009), romania (2008), tunisia (2009), and venezuela (bolivarian republic of) (2007); and health expenditure data for zimbabwe (2001). Averaged means, standard deviations, minimums, and maximums for 149 countries' under-5 mortality rate (deaths at age <5 years per 1000 live births) and indicators of governance, development, and disease control sd: standard deviation; gdp: gross domestic product; dpt: diphtheria, pertussis, and tetanus; pm10: particulate matter with diameter <10 . Values were estimated using the latest available data for each country . All data were from 2010 with the following exceptions: clean water supply data for argentina (2007), equatorial guinea (2006), grenada (2004), lithuania (2009), panama (2009), romania (2008), tunisia (2009), turkmenistan (2006), and venezuela (bolivarian republic of) (2007); sanitation data for argentina (2007), equatorial guinea (2006), lebanon (2005), latvia (2009), lithuania (2009), panama (2009), romania (2008), tunisia (2009), and venezuela (bolivarian republic of) (2007); and health expenditure data for zimbabwe (2001). Scatter plots of 2010 national data for the under-5 mortality rate, associated with the six governance indicators, are shown in figure 1 . All six dimensions of governance show inversely linear relations with the under-5 mortality rate . Figure 1.ecological relations between national under-5 mortality rate (deaths at age <5 years per 1000 live births) and the world bank's six worldwide governance indicators across 149 countries in 2010 . (a) voice and accountability; (b) political stability and absence of violence; (c) government effectiveness; (d) regulatory quality; (e) rule of law; (f) control of corruption . Ecological relations between national under-5 mortality rate (deaths at age <5 years per 1000 live births) and the world bank's six worldwide governance indicators across 149 countries in 2010 . (a) voice and accountability; (b) political stability and absence of violence; (c) government effectiveness; (d) regulatory quality; (e) rule of law; (f) control of corruption . All of these predictors are significantly correlated, with p - values of <0.001 . High correlations are observed between the governance indicators (pearson's correlation coefficient, r=0.680.95, p<0.001); between clean water supply and sanitation (r=0.80, p<0.001); and between immunization against measles and dpt (r=0 . Table 2.pearson correlation matrix for the selected indicators of governance, development, and disease control across 149 countriesvariable(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(1) voice and accountability10.680.830.860.840.810.480.310.560.470.390.430.500.36(2) political stability and absence of violence0.6810.720.690.780.740.430.090.490.460.430.460.290.35(3) government effectiveness0.830.7210.930.950.940.580.490.640.600.460.500.410.36(4) regulatory quality0.860.690.9310.920.880.550.440.600.570.450.490.410.37(5) rule of law0.840.780.950.9210.950.540.400.610.570.450.490.420.34(6) control of corruption0.810.740.940.880.9510.550.370.570.540.400.450.450.34(7) urbanization0.480.430.580.550.540.5510.460.650.630.420.390.290.28(8) economy0.310.090.490.440.400.370.4610.420.420.320.290.170.10(9) clean water supply0.560.490.640.600.610.570.650.4210.800.690.700.280.38(10) sanitation0.470.460.600.570.570.540.630.420.8010.690.670.270.35(11) immunization: measles0.390.430.460.450.450.400.420.320.690.6910.920.220.32(12) immunization: dpt0.430.460.500.490.490.450.390.290.700.670.9210.250.33(13) health expenditures0.500.290.410.410.420.450.290.170.280.270.220.2510.31(14) pm100.360.350.360.370.340.340.280.100.380.350.320.330.311dpt: diphtheria, pertussis, and tetanus; pm10: particulate matter with diameter <10 . P - values for all correlations <0.001 . Pearson correlation matrix for the selected indicators of governance, development, and disease control across 149 countries dpt: diphtheria, pertussis, and tetanus; pm10: particulate matter with diameter <10 . P - values for all correlations <0.001 . Table 3 shows three factors with eigenvalues> 1 and thus qualified for further modeling . Governance. The relative contributions of the six indicators towards governance, ranked from high to low, were rule of law, control of corruption, government effectiveness, regulatory quality, political stability and absence of violence, and voice and accountability . Hygienic condition, including clean water supply and sanitation, and immunization rate against measles and dpt had the largest loadings for factor 2, named disease control. The relative contributions of the four disease - control factors, ranked from high to low, were immunization against measles, immunization against dpt, clean water supply, and sanitation . Urbanization and economy showed the largest loadings for factor 3, named table 3.factor loading matrix for the major factors affecting population health across 149 countriesfactor 1 (governance)factor 2 (disease control)factor 3 (development)(1) voice and accountability0.82240.18230.1557(2) political stability and absence of violence0.82580.30400.1253(3) government effectiveness0.86300.24290.3400(4) regulatory quality0.84910.23440.2976(5) rule of law0.90350.24350.2378(6) control of corruption0.89420.18550.2363(7) urbanization0.39990.27000.6174(8) economy0.15580.14690.8708(9) clean water supply0.38360.65760.4081(10) sanitation0.34510.64340.4630(11) immunization: measles0.19040.93050.1225(12) immunization: dpt0.25440.91880.0670(13) health expenditures0.29250.10070.0810(14) pm100.21160.17130.0263dpt: diphtheria, pertussis, and tetanus; pm10: particulate matter with diameter <10 . Factor loading> 0.5 . Factor loading matrix for the major factors affecting population health across 149 countries dpt: diphtheria, pertussis, and tetanus; pm10: particulate matter with diameter <10 . Factor loading> 0.5 . Table 4 lists the influence of the three qualifying factors on under-5 mortality rates . All three factors were inversely associated with the log - transformed mortality rates (p<0.001). The coefficients 1, 2, and 3 suggest that, per unit increases in governance, disease control, and development, the child mortality rate had a 0.901-fold (= 10) (95% ci 0.8770.926), 0.922-fold (= 10) (95% ci 0.9030.942), and 0.823-fold (= 10) (95% ci 0.7910.857) decrease, respectively, at fixed levels of other two factors . Table 4.estimated fixed effects of determinants on under-5 mortality rates at the country leveldeterminantscoefficients (95% ci)intercept1.4067 (1.2985 to 1.5150)governance (factor 1)0.0453 (0.0569 to 0.0336)disease control (factor 2)0.0351 (0.0443 to 0.0258)development (factor 3)0.0846 (0.1020 to 0.0672) estimated using a semi - parametric generalized additive mixed model for data from 149 countries from 1996 to 2010 . Governance, was related to the six worldwide governance indicators (i.e. Voice and accountability, political stability and absence of violence, government effectiveness, regulatory quality, rule of law, and control of corruption). Disease control, was related to hygienic condition (i.e. Clean water supply and sanitation) and immunization rate against measles and dpt . Estimated fixed effects of determinants on under-5 mortality rates at the country level estimated using a semi - parametric generalized additive mixed model for data from 149 countries from 1996 to 2010 . Governance, was related to the six worldwide governance indicators (i.e. Voice and accountability, political stability and absence of violence, government effectiveness, regulatory quality, rule of law, and control of corruption). Disease control, was related to hygienic condition (i.e. Clean water supply and sanitation) and immunization rate against measles and dpt . Factor 3, named this study contributes to our understanding of global child health issues by showing the significant association between governance and under-5 mortality rates at country level, taking into account factors of development (mainly urbanization and economy) and disease control (mainly hygienic conditions and immunization rates). As the difference in child mortality rates between countries remained as high as 89-fold in 2010, our results indicate that the effect of governance on child mortality is just as important as the effect of development and disease control . Per unit increases in governance, disease control and development could contribute to a 0.901-, 0.922-, and 0.823-fold decrease, respectively, in under-5 mortality rates at fixed levels of the other two factors . Our model predicted best for taiwan, uganda, philippines, slovakia, and norway, and worst for equatorial guinea, nepal, luxembourg, oman, and the maldives . Improvements in a country's level of development (mainly urbanization and economy) have always been the primary factors for reductions in the under-5 mortality rate . However, the recent economic downturn has threatened positive health outcomes in many countries by affecting food prices and increasing nutrition deficiencies . Our results suggest that even if a country has not benefited from urbanization or economic growth, the plausible positive role of better access to improved social and environmental conditions, enabled under effective governance, might be an alternative for a country pursuing reductions in its child mortality rate . Immunization rate against measles increased from 79.5% to 87.7% and that against dpt increased from 79.7% to 88.4%, for the studied 149 countries from 1996 to 2010 . The increased delivery of immunization services by domestic and international efforts, such as the global alliance for vaccines and immunization and the global fund to fight aids, tb and malaria, lowered child mortality rates in most countries . There is a growing quest for better governance at both the country and global level in the pursuit of health equity . Our findings indicate that the association of governance with child mortality should not be underestimated . Governance represents the characteristics of the management of a social organization . From the viewpoint of a country, its governance consists of the traditions and institutions by which authority is exercised and the country is governed . According to the concepts developed by the world bank, governance of a country represents: 1 . The process by which the government is selected, monitored, and replaced; 2 . The capacity of the government to effectively formulate and implement sound policies; 3 . The respect of citizens and the state for the institutions that govern economic and social interactions among them . Experts have suggested that countries with better governance are likely to have health policies that benefit the social determinants of health . In contrast, poor governance might negatively impact health expenditures and health services for the general population . Corruption, or the misuse of public power for private gain, has been the most studied factor of governance in relation to its detrimental impact on the healthcare system, resource distribution, and health outcomes . A study on the progress of mdg 1, a program with the goal of eradicating extreme hunger and poverty, indicated that political decisions on government expenditures played a key part in meeting the mdg 1 goal in 88 countries . For example, poor - quality public administration and inefficient healthcare spending have been shown to have a negative impact on child health (including child mortality rates) in some middle eastern and african countries . Previous evidence has also indicated that around 510% of health - related budgets disappeared before they were spent on real practices . Anti - corruption efforts can be demonstrated to be a key component of good governance and even a reason to wean countries off foreign aid . Although corruption is recognized conceptually as an important driver of population health via its interaction with various socioeconomic determinants, there has been limited research on the effects of corruption on health that also considers the demographic factors of population growth and aggregation . These factors put direct pressure on health resource use, in addition to the interplay of governance (as defined by wgi) in this changing world . Our findings presented here consider the effect of a demographic factor (i.e. Urbanization), and also incorporate governance, encompassing the concept of voice and accountability, political stability and absence of violence, government effectiveness, regulatory quality, rule of law, and control of corruption . The use of a global and longitudinal dataset of under-5 mortality rates was necessary to understand the health impacts and potential applications of health policies and resolutions . To our knowledge, this is the first study to address the effects of governance on child mortality that uses longitudinal data and adjusts for the effect of time (i.e. Calendar year), development factors (i.e. Urbanization and economy), and disease control factors (i.e. Clean water supply, sanitation, and measles and dpt vaccination rate) on a global scale our further analyses of removing the top five countries with lowest and highest governance scores indicated that association between governance and child mortality remained robust . However, our ecological dataset was limited to inter - country and not intra - country comparisons . Important contributors to the total burden of child mortality, such as nutrition and intra - country inequality, warrant a framework that explores the causal pathways between child mortality and its determinants . For example, the distribution of the economy (e.g. Gini coefficient) and other determinants of health among different social groups within a country (e.g. Urban rural and intra - urban differences) also influence inequalities in health outcomes at the intra - country level . Because intra - country data on urban rural health conditions and health risks were limited and incomplete, for this study we could not differentiate health impacts by urban rural areas or by demographic characteristics, such as age and sex, within a country . Our model may therefore have overestimated the health of subpopulations with lower socioeconomic status, and this limitation should be taken into account in future studies . Estimates vary depending on how many potential confounding factors are controlled, how many countries are included, and over what span of time data are collected . For these reasons, we used the largest possible number of confounding factors to examine whether adding predictors as fixed effects would provide better model fitness . We may have overestimated the effects of significant determinants in our models by not fully considering these unmeasured factors, because of the scarcity of available data . However, when we added confounding factors as fixed effects, our models showed the number of countries to drastically decrease . For example, the number of countries with data available decreased from 149 to 55 when we added literacy rate, nutritional status and gini coefficient into our model . Therefore, we preferred to report the findings based on our current model, which had the largest number of studied countries and increased representativeness . In addition, we treated the country itself as the random effect to control for the effects of country heterogeneity, which would act as a proxy variable for these unmeasured factors . If more comparable and comprehensible data were available, we would expect a better estimation . Governance should be taken into account in global health efforts to reduce the under-5 mortality rate in the context of global urbanization, economic growth, and disease control . Countries should target their efforts toward providing healthier physical and social living environments through better governance . Global health communities should start collecting useful information related to indicators of governance and initiate studies into the causal pathways between governance and important socioeconomic determinants of population health . Important contributors to the total burden of child mortality, such as nutrition and intra - country inequality, warrant a framework that explores the causal pathways between child mortality and its determinants . For example, the distribution of the economy (e.g. Gini coefficient) and other determinants of health among different social groups within a country (e.g. Urban rural and intra - urban differences) also influence inequalities in health outcomes at the intra - country level . Because intra - country data on urban rural health conditions and health risks were limited and incomplete, for this study we could not differentiate health impacts by urban rural areas or by demographic characteristics, such as age and sex, within a country . Our model may therefore have overestimated the health of subpopulations with lower socioeconomic status, and this limitation should be taken into account in future studies . Estimates vary depending on how many potential confounding factors are controlled, how many countries are included, and over what span of time data are collected . For these reasons, we used the largest possible number of confounding factors to examine whether adding predictors as fixed effects would provide better model fitness . We may have overestimated the effects of significant determinants in our models by not fully considering these unmeasured factors, because of the scarcity of available data . However, when we added confounding factors as fixed effects, our models showed the number of countries to drastically decrease . For example, the number of countries with data available decreased from 149 to 55 when we added literacy rate, nutritional status and gini coefficient into our model . Therefore, we preferred to report the findings based on our current model, which had the largest number of studied countries and increased representativeness . In addition, we treated the country itself as the random effect to control for the effects of country heterogeneity, which would act as a proxy variable for these unmeasured factors . If more comparable and comprehensible data were available, we would expect a better estimation . Governance should be taken into account in global health efforts to reduce the under-5 mortality rate in the context of global urbanization, economic growth, and disease control . Countries should target their efforts toward providing healthier physical and social living environments through better governance . Global health communities should start collecting useful information related to indicators of governance and initiate studies into the causal pathways between governance and important socioeconomic determinants of population health.
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To assess the correspondence between distinct basal ganglia pathways and hypothesized cognitive processes, we applied the high spatio - temporal resolution of single - unit electrophysiology to a stop - signal task based around our prior studies of rodent decision - making (fig . 1, table s1). Each rat was trained to place its nose in a central port until the onset of a go cue (1khz or 4khz tone) that directed a brief lateral head movement (to left or right; see supplemental movie). On 30% of trials the go tone was followed by a stop cue (white noise), instructing that the rat should stay in the central port (fig . Both go trials and stop trials correct performance was rewarded by delivery of a sugar pellet . As typically observed for stop - signal tasks, reaction times for failed stop trials corresponded to the faster portion of the go trial reaction time distribution (fig . This is consistent with race models: when the go process happens more quickly, a stop process is less likely to successfully suppress behavior . For our first set of recordings (experiment 1), well - trained subjects (n=4) received tetrode implants that simultaneously targeted striatum, stn, globus pallidus (gp), and snr (for further details see ref . We isolated spikes from individual neurons during task performance, from each brain region (for anatomical locations see fig . S1). A challenge when studying behavioral inhibition is to disentangle neural activity specifically linked to stopping, rather than going . To do this, we followed a latency matching procedure (see methods), which exploits the similarity in reaction times -and thus presumably the go process - between failed stop trials and fast go trials . We compared the firing rate of each neuron between these trial types, and between correct stop trials and slow go trials . We then assessed the fraction of each neuronal population that showed significant differences at each moment in time (fig . In contrast, both stn and snr contained a significant proportion of neurons with rapid responses to the stop signal (fig . 2a, red and purple filled bars). For stn, this population response was the same for correct and failed stop trials (for the two stn bins just after stop cue onset, p = 0.17 and 0.21, shuffle test) and thus resembled a fast sensory-like response to the stop cue (see fig . Strikingly however, for snr this fast change in activity was only observed for correct, rather than failed stop trials (for the two filled red snr bins, p = 0.008 and 0.005, shuffle test; see also fig . Thus, while the activity in stn was consistent with a sensory response, activity in snr instead reflected the behavioral outcome on each trial . Our gp recordings did not yield such unambiguous results . Although the initial screen indicated that some neurons may selectively respond for correct stops (fig . 2a), the direct comparison did not confirm a selective gp response in correct, rather than failed stop trials (fig . We next examined the time course of activity in these stop - related stn and snr neurons . 3a), that in some cases took the form of just a single, precisely timed extra spike (fig . These stn increases had consistently very low latencies (peak response ~15 ms; fig . 24 for similarly low stn latencies) that were not different between correct and failed stop trials (p = 0.41, paired t - test; fig . The magnitude of the peak stn response also showed no systematic population preference for correct versus failed stop trials (fig . 3b; p = 0.004 comparing stn to snr latencies, one - sided kolmogorov - smirnov test) and preferentially on correct stop trials (fig . This latency difference was seen even when the analysis was restricted to units recorded in the same session (n = 15 pairs; stn cells preceded snr cells by an average of 13.6 ms, p = 0.041, shuffling statistic). All snr neurons that responded to the stop cue on correct trials did so before the stop - signal reaction time (ssrt; grey lines in fig . 3a), a standard, inferred behavioral measure for how quick a process must be to influence stopping performance . Thus, snr activity not only distinguishes between correct and failed stop trials, it does so quickly enough to affect the trial outcome . Most of the snr units with fast stop cue responses (10/18) also markedly decreased their activity beginning just before movement initiation (fig . This suggests that the stop cue may not alter snr activity globally, but rather have a selective influence over cells and subregions involved in controlling the specific movement that needs to be inhibited . We therefore performed a second set of recordings (experiment 2) using both high - density silicon probes (n = 3 rats, fig . 4a) and more tetrodes (n = 2 rats) to target a wide range of snr locations . Combining all snr results together revealed a clear hotspot of snr cells that fired significantly more on correct than on failed stop trials, briefly after the stop cue (figs . 4, s4a d). Subregion described in anatomical studies, located dorsolaterally and extended along the rostral - caudal axis . This subregion projects to specific parts of the superior colliculus involved in orienting movements, so the stop signal influences activity in an snr subregion that is likely critical for exerting fast behavioral control . The distinct latencies of stn and snr cue responses are consistent with stop information being conveyed along the stn - snr pathway . Yet, the selectivity of this transmission to correct stop trials suggests some form of gating mechanism . In other words: given that the glutamatergic stn cells spike on failed stop trials, why are snr neurons not responsive to this input? The answer may lie in the movement - related firing rate decreases of snr neurons (figs . S3 and s4e, f). Such snr firing pauses are well - known from studies of eye and limb movements, and are thought to facilitate action through disinhibition of superior colliculus and other structures more directly linked to motor output . Snr pauses are driven by increased firing of the gabaergic striatal direct pathway neurons, plausible participants in a go process . To assess how striatal neurons may contribute to movement preparation and initiation, we looked for units that distinguish movement direction before movement onset . We found an abrupt increase in contralateral coding starting ~140ms before movements (arrow in fig . When we compared the activity of these direction - selective striatal cells (n = 74) between different trial types, we observed a rapid acceleration of firing rate just before movement onset (fig . 5b, right), that followed the same trajectory for fast go, slow go and failed stop trials . Aligned on the earlier go cue, this striatal activity remained very similar between fast go and failed stop trials, but distinct to slow go and correct stop trials (fig . These results fit well with a simple race model, in which variability in the timing of a striatal - based go process determines the outcome on stop trials . On failed stop trials, movement - related striatal activity has already begun to increase by the time of stop cue onset (figs ., the lack of snr responses to the stop cue on failed stop trials may be due to the early arrival of striatal gabaergic input, shunting away the effects of glutamatergic inputs from stn . To confirm the viability of this idea we studied gating of the stop cue responses in a simple integrate - and - fire model of an snr neuron . This neuron received excitatory pulses, mimicking stn sensory responses to stop cues, and (as in prior basal ganglia models) this excitatory input was influenced by gabaergic inhibition (see methods). For gabaergic input we adjusted synaptic strengths of inhibitory and excitatory inputs to provide a good qualitative match with the cue - evoked increases and movement - related decreases in snr firing . We define as the interval between stop cue onset and the point in the striatal output at which movements begin on go trials . If the stop cue begins long before movement onset (fig . 6a; = 200ms), striatal inhibition is low at that time and the stop cue evokes a full response in the snr cell . By contrast, if the stop cue occurs only briefly before movement onset (fig . 6a; = 50ms), a high level of striatal inhibition suppresses the snr cue response . A systematic variation of in the behaviorally relevant range yielded a gating curve that quantified the model response to the stop cue (fig . The gating phenomenon required strong divisive inhibition, e.g. Through shunting inhibition, rather than simple summation of inhibitory and excitatory conductances (fig . We then used the behavioral data of each rat to estimate the actual distribution of for both correct and failed stop trials (fig . These distributions could then be used to calculate model firing rates for these trial types (fig . Importantly, just as in real rat snr cells, the model snr cell selectively responded to the stop cue in correct but not failed stop trials . We conclude that the integration of distinct excitatory and inhibitory synaptic inputs by individual snr neurons provides a straightforward, mechanistic account of how go and stop processes can race within the brain . Race models have been central to theories of action suppression for decades, yet clear evidence that they actually describe neural processes has been elusive . Here we have demonstrated that activity in two key basal ganglia pathways for action control closely resembles a race between go and stop processes . Individual snr neurons show both movement - related pauses in firing (driven by striatum) and rapid firing rate increases following stop cues (driven by stn), and the relative timing of these influences corresponds to whether stopping was successful . These snr cells are located in a specific dorsolateral subregion, that in turn projects to collicular intermediate layers important for the control of orienting movements . Furthermore, the evidence we found for shunting inhibition of stn inputs by striatal inputs begins to reveal how mechanisms operating within single cells can contribute to sensorimotor gating . It is important to note that neurons in stn and snr with fast stop cue responses also increased spiking with the go cue that instructed contralateral movement (figs . Thus the stn - snr pathway does not solely convey signals that instruct stopping, but also other task - relevant cues . The effect of both go and stop cues was to transiently increase firing of a population of snr neurons that decrease firing with movement onset (fig . S3). We found that trials in which stn and snr responded more strongly to the go cue had longer reaction times (fig . S9), consistent with a role for stn - snr transmission in delaying behavioral output, rather than causing outright stopping (see below). A rapid, automatic inhibition of behavioral responses by task - relevant cues may help prevent responses that are impulsive or premature (i.e. When movement preparation is incomplete), and also explain why even cues that instruct subjects not to stop, but instead continue as planned, result in longer reaction times . Further, our results contribute to the ongoing debate about whether certain brain areas contribute to action inhibition, versus cue - evoked reorienting of attention, by indicating that these functions are not necessarily distinct . The very low, fixed latency of cue - evoked activity in stn is informative in several ways . First, race models often incorporate variable timing of both go and stop processes, yet we found that the stn stop cue response occurred at the same time in correct and failed stop trials . Thus, if these responses are part of a stop process, performance variability arises directly from the variable timing of the go process (i.e. Variable reaction times), at least in this version of the stop - signal task . This result is consistent with recent simulations of basal ganglia networks during inhibitory control: stn provides the same fast signal to pause action, whether stopping is actually successful or not . Second, increased stn spiking within just 15 ms of cue onset constrains the sophistication of prior information processing, and which afferents can be driving this response . Recent human studies have emphasized the role of frontal cortical inputs to stn in action suppression, but it is not clear that cue information can be passed quickly enough through cortex to cause this fast stn spiking . Our implementation of the stop - signal task encouraged very quick responses, and may have increased the importance of subcortical pathways that support sensory processing and fast orienting - like movements . In particular, many neurons in the thalamic intralaminar complex (centromedian / parafascicular nuclei, cm / pf) and pedunculopontine nucleus (ppn) have short - latency responses to salient auditory stimuli and project to a range of basal ganglia targets, including stn . Cm / pf projections to striatum are important for behavioral switching and learning following unexpected cues . We hypothesize that the stn responses we observed are one component of a broader interrupt system, mediated by cm / pf and/or ppn, that coordinates a response to salient cues across multiple timescales using multiple pathways (fig . Very fast yet transient excitation of stn and snr serves to delay actions that are close to execution, similar to previous descriptions of stn buying time during decision - making . However, the stn - driven increase in snr firing is highly transient; in our simulation it would delay, but not fully cancel, the striatum - driven decrease in firing that releases movements (fig . We also found evidence that a second, slower mechanism may act within striatum to help shut down the go process . Movementrelated striatal activity abruptly decreases in correct stop trials, compared to slow go trials (fig . 5b, c and s6), and a similar suppression of contralateral - coding striatal activity was observed in an antisaccade task . Such suppression of a go process is a key feature of interactive race striatum - based processing by itself is unlikely to account for stop - signal performance, as the reduction in striatal output was not consistently before the ssrt (fig . It thus appears that complementary mechanisms allow action suppression to be both fast (via stn) and selective (via striatum). Future studies will investigate how the striatal go process is suppressed in correct stop trials . Direct pathway neurons can be inhibited in many ways, and some (non - exclusive) possibilities include the influences of indirect pathway cells and cholinergic interneurons . We used a basic stop - signal task, designed to investigate reactive aspects of behavioral inhibition (responding to a stop cue). This task does not assess all the complexities of behavioral inhibition, such as proactive components (i.e. Preparedness to stop). It has been proposed that proactive inhibition involves yet another basal ganglia circuit, the indirect pathway from striatum through gp . In follow - up studies we plan to investigate whether systematically varying preparedness to stop reveals a clear role for gp that was not apparent here . Finally, we previously reported (using the experiment 1 data) that salient task cues cause a rapid reset of beta oscillatory phase throughout the basal ganglia, whether or not the cues actually direct behavior on a given trial . By contrast, cue - induced increases in beta power only occur for cues that are used - for example, after the stop cue on correct but not failed stop trials . This distinction corresponds closely to the difference between stn and snr described here: events that caused abrupt increases in stn firing also produced beta phase reset, while those events that additionally increased snr firing subsequently produced beta power increases . Furthermore, there is evidence that other oscillatory frequencies such as delta / theta can influence the parameters of behavioral control, such as decision thresholds . An important direction for future investigation will be to determine the mechanistic relationships between rapid firing rate changes and altered dynamic states within basal ganglia circuitry . All animal experiments were approved by the university of michigan committee for the use and care of animals . Subjects were adult male long - evans rats, housed on a 12:12 reverse light: dark cycle and tested during the dark phase . Rats were housed in groups of 34 with moderate environmental enrichment (toys, variety of bedding, 593920 cm cages) during pre - surgical training, then singly - housed after surgery . The operant chamber had 5 nose - poke holes on one wall and a food dispenser on the opposite wall . At the start of each trial, one of the three more - central holes (chosen randomly) was illuminated, indicating that the rat should poke and hold its nose in that port . After a variable hold delay (5001,200 ms), a tone (go cue; 65 db, 50 ms) instructed the rat to move promptly into the adjacent hole either to the left (1 khz tone) or right (4 khz tone); correct choices triggered immediate delivery of a sugar pellet reward (signaled by an audible click of the food dispenser). To encourage rats to respond quickly, on go trials rats had to leave the initial port within a limited hold period, and then poke the adjacent hole within a movement hold (see table s1). On stop trials (30%), the go cue was followed after a short delay (the stop - signal delay, ssd) by a stop cue (white noise burst, 65 db, 125 ms). If the rat moved before the ssd, the stop cue was not played and the trial was treated as a go trial . To successfully complete a stop trial, the rat had to maintain its nose in the initial port until the limited hold period would have expired on a go trial . At that point, the audible click of the food dispenser signaled reward delivery . Errors of any type produced a time - out (houselight on, 8 s). The computer - controlled sequence of trials was randomized and experimenters were blind to the trial sequence . However, to discourage the animal from adopting a holding - strategy without go responses, the animal had to perform a correct go trial before a stop trial could occur . Other randomization or blinding procedures were not performed in this study . After achieving stable task performance (typically ~23 months of training,> 70% correct choices on go trials) rats in experiment 1 received implants containing 21 individually drivable tetrodes targeting basal ganglia structures (str, gp, stn, and snr), and recording sessions began approximately one week later . Ssd was held constant within each recording session to facilitate electrophysiological analyses, but was adjusted between stop - signal sessions so that correct and failed stop trials were approximately equal in number . On alternate days rats performed the stop - signal task and a go / nogo task, which was identical in most respects to the stop - signal task but with an ssd of zero (on nogo trials the white noise was played instead of a go cue). During task performance wide - band (19,000 hz) tetrodes were usually moved by at least 80 m between two stop - signal sessions . In some cases, (e.g. If the number of trials was low in one recording session), tetrodes were not moved between sessions, and we only included the better session in the analysis . Individual neurons were isolated offline using wavelet - based filtering followed by standard manual spike - sorting, and classification into different presumed cell types . Experiment 2 examined whether stop responses are localized to a specific subregion of snr . To facilitate a systematic functional mapping, three rats received 8-shank, 64-channel silicon probes (neuronexus inc .) Into snr . Silicon probe shanks were coated in the lipophilic dye dio before implantation and not moved after initial surgery . For these fixed - location silicon probes we only included data from a single session per animal, to avoid including duplicate cells . Data distribution was not formally tested for normality, but we instead mostly used statistical methods that are robust for non - normal - distributed data (kolmogoroy - smirnov and shuffling tests). First, we determined the fraction of failed stop trials f. then we calculated the stopping time as the f - th percentile of the distribution of go trial reaction times . The ssrt is then the stopping time minus the ssd (for further details see ref . ). This ssrt value was also used to separate go trials in the same session into fast go and slow go . To examine the activity of each neuron we used 40 ms time bins (sliding in steps of 20 ms) to obtain spike count distributions for different trial types, near key task events . A neuron had to exceed a 3 hz firing rate in at least one time bin to be included in subsequent analyses . To best isolate activity associated with stop cues, we compared trial types for which the activity associated with movement preparation is most similar . That is, we compared failed stop trials with fast go trials, and correct stop trials with slow go trials (latency - matching). For stop trials neural activity was aligned to the onset of the stop cue, and for go trials we used the time at which the stop signal would have occurred, i.e. Go cue onset + ssd . We interpreted these as trials for the initial stopping was actually successful, but subsequent holding on for reward was not (both spike and lfp measures were consistent with this interpretation; data not shown). We therefore excluded failed stop trials with reaction times> 500 ms from all analyses . To compare whether spike rates were different between two trial types, we used a shuffle test for each time bin . We shuffled the trial type labels 10,000 times, and for each shuffle we compared the means of the two resulting spike count distributions . To yield a p - value we determined the fraction of shuffles in which the difference between the shuffled means was larger (or smaller) than the difference between the two actually observed means . We used a p - value of 0.05 to determine significant coding of trial types . It follows that 5% of a population of randomly active units should, on average, be classified as coding a binomial test was then used to determine whether the empirically measured fraction of coding units was significantly higher than that expected by chance . We corrected for multiple testing with respect to the overall time - window around task events (e.g. For fig . 2, each single test was done for a 40 ms time - window, yielding 500/40 independent tests around the task events). This correction is overly conservative for some key time points of interest, since we hypothesized a priori that firing - rates would change shortly after cue onset . 2a) indicate times when the p - value was below 0.05 without adjusting for multiple comparisons . For stn we included units that contributed to the significant responses in either correct or failed stop trials (i.e. The filled red and magenta bars for stn in fig . Did not reach significance in failed stop trials, we included those individual snr units that only contributed to the significant stop cue response in correct stop trials (i.e. The filled red bars for snr in fig . The firing rate time course of each unit was then estimated by averaging spike counts over trials of the same type (e.g. Failed stop - right trials) with a sliding 20 ms window in steps of 5 ms around important task events (e.g. The stop cue; fig . Firing rate time courses between trial types, activity of each unit was first transformed to z - scores using the mean and standard deviation of session - wide firing rate estimates (obtained from 1 s wide windows). To compare the magnitude of the stop response between correct and failed stop trials (fig . 3c), we used peak firing rates in the range 1070 ms after stop cue onset . To more precisely identify the times at which stn and snr neurons responded to the stop cue (fig . 3b), we used non - overlapping 3 ms time windows smoothed with a 3-point average for the firing rate estimation . The latency was taken as the time of peak firing within the range 1070 ms after stop cue onset . 4 and s4a d) if the corresponding trial types were significantly different in at least two out three bins (each bin 40 ms wide, centered at 40, 60 and 80 ms after the stop cue). For fig . 4 we compared trial types separately for ipsi- and contralateral conditions and marked units red if in one or both cases the activity differences were significant . Similarly, in fig . S2b, a unit is counted in the histogram if it exhibits a significant difference in activity between failed and correct stop trials for ipsi- or contralateral trials . To describe the gating of the stop cue response, we implemented an integrate - and - fire model of a single snr cell . Changes in the membrane potential v at time t were given by: mdvdt=v(t)+emrmi(t), where m is the membrane time constant, em the membrane resting potential, and rm the membrane input resistance . I is a sum of currents: i(t) = ib(t) + istr(t) + istn(t). The current ib was used to mimic the high spontaneous activity observed in snr cells: ib(t) = gb (v(t) eb) (see below for parameter settings). Synaptic input from the striatum was summarized by istr(t) = gstr(t)(v(t) estr). The time - dependent synaptic conductance gstr(t) was modeled using the activity a(t) of go - related striatal cells during movement initiation shown in fig . 5b (right panel), averaged over slow and fast go trials, as gstr(t)=a(t)i=0ta(i). The scaling factor controls the movement - related decrease in our model snr cell and was fitted to reproduce the average time course in fig . Although further increases in striatal input strength could block stn inputs without the need for shunting inhibition, this change also produced too early a pause in snr firing (i.e. A mismatch with the timing of striatal and snr changes observed in vivo). The sum i=0ta(i) is over previous activity in the current trial (starting 500ms before movement onset) and is used as a simplified description of facilitation found at striatonigral synapses . In our model this facilitation effectively lead to a broader peak of the striatal activity ramp and thereby to a broader pause in snr activity, as we observed in the experimental data . Although real snr neurons pause at a range of times relative to movement onset (fig . S3 and ref . ), for our simple model we used a fixed striatal input time course . We further assumed that stn input to snr consisted of a single spike at time ts evoked by the go or stop cue (see stn example cell in fig . Each spike gave rise to a modulated alpha function yielding a synaptic current istn (t) = h(t) (t, ts)(v(t) estn) with a scaling factor and a shunting factor h(t). The scaling factor controls the amplitude of the snr response to the stn input . We choose to fit the amplitude of snr responses to the go cue . Due to the high baseline activity in snr mediated by ib, in some trials stn inputs can shift the spike timing rather than increase the snr firing rate . When the snr baseline activity is diminished by inhibitory input, this effect is reduced so that stn input becomes more likely to evoke an snr spike (see grey line in fig . The alpha function was zero for t <0 and otherwise defined as (t, ts) = exp((t ts) /q) exp((t ts) /0.25q) with a time constant q. the shunting factor h(t) takes values between zero and one depending on the amount of inhibitory current: h(t)=(1istr(t)j)(1istr(t)j), where denotes the heaviside step function . The reference current j controls the efficacy of the shunting inhibition (fig . A spike was generated by the model snr cell if v(t) reached the threshold voltage vthr = 50mv . After one millisecond, v(t) was then reset to the equilibrium membrane potential em = 80 mv . Other parameter values were m=20 ms; rm = 10m; gb = 0.095s; eb = 0 mv; estr = 100mv; estn = 0 mv; = 1/1200; = 0.1; q = 10ms; and j = 1, 1.5, and 2 na for strong, medium and weak shunting inhibition, respectively . Further, complex effects on synaptic integration of gabaa were neglected in our model as for simplicity we set estr <em . However, we performed additional simulations with more realistic values for shunting inhibition where em vetoing effect on excitatory stn input . For the simulation without shunting inhibition h(t) was always set to one . In the model we kept the ssd constant at 300 ms, and assumed that stn input reached snr starting 30 ms after stop cue onset . The snr response to the stop cue was measured as the firing rate within the subsequent 50 ms time window . The interval between movement onset and stop cue presentation was varied in 1 ms time steps, over the range 0 500 ms . For each, 500 trials with and 500 trials without stop cue were simulated and the average firing rates were then used for the results . To obtain snr output for failed stop trials, we used the behavioral data of the rats . From the measured reaction times of s7) was used as the model parameter for failed stop trials . For correct stop trials, however, employing the reaction time distribution on go trials, it is possible to estimate what the reaction time would have been if the stop cue had not been presented . More formally, for each rat we have an empirically measured reaction time distribution for go trials fgo(t) = p(rt = t), with p denoting probability and rt being stop - aligned reaction times in 50 ms wide bins . With the corresponding probability distribution for failed stop trials ffs, the hypothetical distribution of correct stop reaction times was estimated as: fcs(t)=fgo(t)pffs(t)1p, where p denotes the overall probability of failing to stop (number of failed stop trials divided by the number of stop trials). Due to noise in the behavioral data in a few cases the estimates of fcs were negative, so we applied a lower bound for fcs(t) of zero and rescaled the whole probability distribution to maintain a total probability of one . The resulting estimates of fcs were then used as the distribution of for correct stop trials . All animal experiments were approved by the university of michigan committee for the use and care of animals . Subjects were adult male long - evans rats, housed on a 12:12 reverse light: dark cycle and tested during the dark phase . Rats were housed in groups of 34 with moderate environmental enrichment (toys, variety of bedding, 593920 cm cages) during pre - surgical training, then singly - housed after surgery . The operant chamber had 5 nose - poke holes on one wall and a food dispenser on the opposite wall . At the start of each trial, one of the three more - central holes (chosen randomly) was illuminated, indicating that the rat should poke and hold its nose in that port . After a variable hold delay (5001,200 ms), a tone (go cue; 65 db, 50 ms) instructed the rat to move promptly into the adjacent hole either to the left (1 khz tone) or right (4 khz tone); correct choices triggered immediate delivery of a sugar pellet reward (signaled by an audible click of the food dispenser). To encourage rats to respond quickly, on go trials rats had to leave the initial port within a limited hold period, and then poke the adjacent hole within a movement hold (see table s1). On stop trials (30%), the go cue was followed after a short delay (the stop - signal delay, ssd) by a stop cue (white noise burst, 65 db, 125 ms). If the rat moved before the ssd, the stop cue was not played and the trial was treated as a go trial . To successfully complete a stop trial, the rat had to maintain its nose in the initial port until the limited hold period would have expired on a go trial . At that point, the audible click of the food dispenser signaled reward delivery . Errors of any type produced a time - out (houselight on, 8 s). The computer - controlled sequence of trials was randomized and experimenters were blind to the trial sequence . However, to discourage the animal from adopting a holding - strategy without go responses, the animal had to perform a correct go trial before a stop trial could occur . Other randomization or blinding procedures were not performed in this study . After achieving stable task performance (typically ~23 months of training,> 70% correct choices on go trials) rats in experiment 1 received implants containing 21 individually drivable tetrodes targeting basal ganglia structures (str, gp, stn, and snr), and recording sessions began approximately one week later . Ssd was held constant within each recording session to facilitate electrophysiological analyses, but was adjusted between stop - signal sessions so that correct and failed stop trials were approximately equal in number . On alternate days rats performed the stop - signal task and a go / nogo task, which was identical in most respects to the stop - signal task but with an ssd of zero (on nogo trials the white noise was played instead of a go cue). During task performance wide - band (19,000 hz) tetrodes were usually moved by at least 80 m between two stop - signal sessions . In some cases, (e.g. If the number of trials was low in one recording session), tetrodes were not moved between sessions, and we only included the better session in the analysis . Individual neurons were isolated offline using wavelet - based filtering followed by standard manual spike - sorting, and classification into different presumed cell types . Experiment 2 examined whether stop responses are localized to a specific subregion of snr . To facilitate a systematic functional mapping, three rats received 8-shank, 64-channel silicon probes (neuronexus inc .) Into snr . Silicon probe shanks were coated in the lipophilic dye dio before implantation and not moved after initial surgery . For these fixed - location silicon probes we only included data from a single session per animal, to avoid including duplicate cells . Data distribution was not formally tested for normality, but we instead mostly used statistical methods that are robust for non - normal - distributed data (kolmogoroy - smirnov and shuffling tests). First, we determined the fraction of failed stop trials f. then we calculated the stopping time as the f - th percentile of the distribution of go trial reaction times . The ssrt is then the stopping time minus the ssd (for further details see ref . ). This ssrt value was also used to separate go trials in the same session into fast go and slow go . To examine the activity of each neuron we used 40 ms time bins (sliding in steps of 20 ms) to obtain spike count distributions for different trial types, near key task events . A neuron had to exceed a 3 hz firing rate in at least one time bin to be included in subsequent analyses . To best isolate activity associated with stop cues, we compared trial types for which the activity associated with movement preparation is most similar . That is, we compared failed stop trials with fast go trials, and correct stop trials with slow go trials (latency - matching). For stop trials neural activity was aligned to the onset of the stop cue, and for go trials we used the time at which the stop signal would have occurred, i.e. Go cue onset + ssd . For we interpreted these as trials for the initial stopping was actually successful, but subsequent holding on for reward was not (both spike and lfp measures were consistent with this interpretation; data not shown). We therefore excluded failed stop trials with reaction times> 500 ms from all analyses . To compare whether spike rates were different between two trial types, we used a shuffle test for each time bin . We shuffled the trial type labels 10,000 times, and for each shuffle we compared the means of the two resulting spike count distributions . To yield a p - value we determined the fraction of shuffles in which the difference between the shuffled means was larger (or smaller) than the difference between the two actually observed means . We used a p - value of 0.05 to determine significant coding of trial types . It follows that 5% of a population of randomly active units should, on average, be classified as coding a binomial test was then used to determine whether the empirically measured fraction of coding units was significantly higher than that expected by chance . We corrected for multiple testing with respect to the overall time - window around task events (e.g. For fig . 2, each single test was done for a 40 ms time - window, yielding 500/40 independent tests around the task events). This correction is overly conservative for some key time points of interest, since we hypothesized a priori that firing - rates would change shortly after cue onset . 2a) indicate times when the p - value was below 0.05 without adjusting for multiple comparisons . For stn we included units that contributed to the significant responses in either correct or failed stop trials (i.e. The filled red and magenta bars for stn in fig . As the overall population of snr units did not reach significance in failed stop trials, we included those individual snr units that only contributed to the significant stop cue response in correct stop trials (i.e. The filled red bars for snr in fig . The firing rate time course of each unit was then estimated by averaging spike counts over trials of the same type (e.g. Failed stop - right trials) with a sliding 20 ms window in steps of 5 ms around important task events (e.g. The stop cue; fig . Firing rate time courses between trial types, activity of each unit was first transformed to z - scores using the mean and standard deviation of session - wide firing rate estimates (obtained from 1 s wide windows). To compare the magnitude of the stop response between correct and failed stop trials (fig . 3c), we used peak firing rates in the range 1070 ms after stop cue onset . To more precisely identify the times at which stn and snr neurons responded to the stop cue (fig . 3b), we used non - overlapping 3 ms time windows smoothed with a 3-point average for the firing rate estimation . The latency was taken as the time of peak firing within the range 1070 ms after stop cue onset . 4 and s4a d) if the corresponding trial types were significantly different in at least two out three bins (each bin 40 ms wide, centered at 40, 60 and 80 ms after the stop cue). For fig . 4 we compared trial types separately for ipsi- and contralateral conditions and marked units red if in one or both cases the activity differences were significant . S2b, a unit is counted in the histogram if it exhibits a significant difference in activity between failed and correct stop trials for ipsi- or contralateral trials . To describe the gating of the stop cue response, we implemented an integrate - and - fire model of a single snr cell . Changes in the membrane potential v at time t were given by: mdvdt=v(t)+emrmi(t), where m is the membrane time constant, em the membrane resting potential, and rm the membrane input resistance . I is a sum of currents: i(t) = ib(t) + istr(t) + istn(t). The current ib was used to mimic the high spontaneous activity observed in snr cells: ib(t) = gb (v(t) eb) (see below for parameter settings). Synaptic input from the striatum was summarized by istr(t) = gstr(t)(v(t) estr). The time - dependent synaptic conductance gstr(t) was modeled using the activity a(t) of go - related striatal cells during movement initiation shown in fig . 5b (right panel), averaged over slow and fast go trials, as gstr(t)=a(t)i=0ta(i). The scaling factor controls the movement - related decrease in our model snr cell and was fitted to reproduce the average time course in fig . Although further increases in striatal input strength could block stn inputs without the need for shunting inhibition, this change also produced too early a pause in snr firing (i.e. A mismatch with the timing of striatal and snr changes observed in vivo). The sum i=0ta(i) is over previous activity in the current trial (starting 500ms before movement onset) and is used as a simplified description of facilitation found at striatonigral synapses . In our model this facilitation effectively lead to a broader peak of the striatal activity ramp and thereby to a broader pause in snr activity, as we observed in the experimental data . Although real snr neurons pause at a range of times relative to movement onset (fig . S3 and ref . ), for our simple model we used a fixed striatal input time course . We further assumed that stn input to snr consisted of a single spike at time ts evoked by the go or stop cue (see stn example cell in fig . Each spike gave rise to a modulated alpha function yielding a synaptic current istn (t) = h(t) (t, ts)(v(t) estn) with a scaling factor and a shunting factor h(t). The scaling factor controls the amplitude of the snr response to the stn input . Due to the high baseline activity in snr mediated by ib, in some trials stn inputs can shift the spike timing rather than increase the snr firing rate . When the snr baseline activity is diminished by inhibitory input, this effect is reduced so that stn input becomes more likely to evoke an snr spike (see grey line in fig . The alpha function was zero for t <0 and otherwise defined as (t, ts) = exp((t ts) /q) exp((t ts) /0.25q) with a time constant q. the shunting factor h(t) takes values between zero and one depending on the amount of inhibitory current: h(t)=(1istr(t)j)(1istr(t)j), where denotes the heaviside step function . The reference current j controls the efficacy of the shunting inhibition (fig . A spike was generated by the model snr cell if v(t) reached the threshold voltage vthr = 50mv . After one millisecond, v(t) was then reset to the equilibrium membrane potential em = 80 mv . Other parameter values were m=20 ms; rm = 10m; gb = 0.095s; eb = 0 mv; estr = 100mv; estn = 0 mv; = 1/1200; = 0.1; q = 10ms; and j = 1, 1.5, and 2 na for strong, medium and weak shunting inhibition, respectively . Further, complex effects on synaptic integration of gabaa were neglected in our model as for simplicity we set estr <em . However, we performed additional simulations with more realistic values for shunting inhibition where em <estr <vthr . With appropriately rescaled values for and j therefore, the key feature of inhibition was the divisive vetoing effect on excitatory stn input . For the simulation without shunting inhibition h(t) was always set to one . In the model we kept the ssd constant at 300 ms, and assumed that stn input reached snr starting 30 ms after stop cue onset . The snr response to the stop cue was measured as the firing rate within the subsequent 50 ms time window . The interval between movement onset and stop cue presentation was varied in 1 ms time steps, over the range 0 500 ms . For each, 500 trials with and 500 trials without stop cue were simulated and the average firing rates were then used for the results . To obtain snr output for failed stop trials, we used the behavioral data of the rats . From the measured reaction times of s7) was used as the model parameter for failed stop trials . For correct stop trials, however, employing the reaction time distribution on go trials, it is possible to estimate what the reaction time would have been if the stop cue had not been presented . More formally, for each rat we have an empirically measured reaction time distribution for go trials fgo(t) = p(rt = t), with p denoting probability and rt being stop - aligned reaction times in 50 ms wide bins . With the corresponding probability distribution for failed stop trials ffs, the hypothetical distribution of correct stop reaction times was estimated as: fcs(t)=fgo(t)pffs(t)1p, where p denotes the overall probability of failing to stop (number of failed stop trials divided by the number of stop trials). Due to noise in the behavioral data in a few cases the estimates of fcs were negative, so we applied a lower bound for fcs(t) of zero and rescaled the whole probability distribution to maintain a total probability of one . The resulting estimates of fcs were then used as the distribution of for correct stop trials.
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It is present, in fact, in compounds with different activities such as inhibitors of p38 map kinase, inhibitors of hiv - ep1 cellular transcription factor, inhibitors of coagulation factor xa, and -chemokine receptor ccr5 antagonists in anti - hiv therapy; in particular we have focused our work on the development of new cyp51 inhibitors, active both on fungal strains and trypanosoma cruzi . Many literature data evidenced that the pyridine group can efficaciously replace the heme - iron chelating azole moiety present in classical azole cyp51 inhibitors and, therefore, the alkylation of 4-aminopyridine (4ap) represents an important goal in organic synthesis to develop novel classes of antifungal and antiparasitic drugs [7, 8]. Due to the wide presence of these products, the alkylation of 4-aminopyridine (4ap) is therefore an important goal in organic synthesis . Some examples are the efficient condensation of 4ap with alcohols catalyzed by benzaldehyde or copper [10, 11] or magnetite, the reaction of 4ap with an acyl chloride, and the following reduction of the amide with lialh4 . The most straightforward method, however, is the direct alkylation of 4ap with alkyl halides, although it suffers from some drawbacks . The two different nitrogen atoms compete in the alkylation reaction and usually the more nucleophilic pyridine nitrogen atom reacts faster, leading to the corresponding pyridinium salt (scheme 1) [14, 15]. In these case, the use of a very strong base is therefore necessary: n - buli was successfully used by singh and coworkers, obtaining n - methyl- and n - ethyl-4-aminopyridines in 7480% yields . A viable alternative is the enhancement of the nucleophilicity of the amine nitrogen atom (versus the pyridine one), allowing the use of weaker bases . An example is the activation of 2-aminopyridine as formyl or boc derivative at the amine nitrogen atom, with subsequent deprotonation using sodium hydride, alkylation, and deprotection with trifluoroacetic acid . The deprotonation of n - boc-2-aminopyridine with nah needs a careful control of the temperature (05c) and is carried out in anhydrous dmf, with a vigorous stirring required to keep the suspension fluid . In this context, we envisaged the possibility to alkylate n - boc-4-aminopyridine (n - boc-4ap) using milder reaction conditions, that is, using electrogenerated tetraethylammonium cyanomethanide (et4nch2cn). This base, the acetonitrile anion, can be easily obtained by cathodic galvanostatic reduction of a solution of acetonitrile containing tetraethylammonium hexafluorophosphate as supporting electrolyte (scheme 2), without by - products (the reagent is the electron), and it was successfully used by us in a good variety of reactions, such as the selective n - alkylation of bifunctional compounds, the gewald reaction, the synthesis of -lactams, and the synthesis of carbamates . The actual mechanism for the formation of acetonitrile anion is not known, but a hypothesis based on the direct reduction of the tetraethylammonium cation has been reported (scheme 2). The high reactivity of this base is ascribable to the large tetraethylammonium counterion, which renders the acetonitrile anion extremely reactive . Moreover, its reaction as a base gives no by - products, as the protonation restores the molecule of solvent . The reaction of electrogenerated acetonitrile anion with 4ap, followed by an alkyl halide, leads to poor yields in desired compound with the pyridinium salt being the major product . This prevents the direct use of ch2cn with 4ap . On the other hand, if the amine nitrogen is activated as boc derivative (n - boc-4ap), the deprotonation / alkylation reaction using acetonitrile anion leads to products1 in very high yields (scheme 3 and table 1, entries 16). The classic deprotection with trifluoroacetic acid allows obtaining the desired products2 (scheme 3 and table 1, entries 16). The data in table 1 highlight that the reaction of deprotonation of n - boc-4ap using electrogenerated acetonitrile anion, alkylation with both alkyl and benzyl halides, and deprotection with trifluoroacetic acid is very efficient, with overall yields of 7886% . However, when the alkylating agent is a bromoacetophenone, the yields in alkylated product are lower and in most cases the deprotection reaction leads to the dealkylation of the starting material (table 1, entries 710). As many biologically active compounds contain the dialkylated 4-aminopyridine moiety, we tried to carry out a second alkylation on products 2a j using acetonitrile anion but, as expected, the high nucleophilicity of the pyridine nitrogen led to the synthesis of the corresponding pyridinium salt . We thus carried out this second alkylation using strong bases, the most efficient being t - buok in dmso (scheme 4), although the yields in dialkylated 4ap were not very high . Symmetrically dialkylated 4ap, 4ap was subjected to deprotonation with t - buok in dmso, adding an excess of alkylating agent . The reaction led to a mixture of mono- and dialkylated 4-aminopyridines, in moderate to acceptable yields . A selection of synthesized compounds was in vitro tested to evaluate antifungal activity against different strains of c. albicans, c. parapsilosis, and cryptococcus neoformans; data are reported in table 4 . As can be evidenced the nonsymmetrical dialkylated 4aps 3ac and 3ae showed a moderate antifungal activity towards c. albicans and c. parapsilosis with mic50 values of 32 g / ml and showed an interesting activity against cryptococcus neoformans, with mic50 values of 0.4 and 4 g / ml, respectively . Otherwise, the symmetrical dialkylated 4aps 3cc, 3ee and the boc - protected monoalkylated 4aps 1b, 1e, 1f showed poor antifungal activity with mic50 and mic100 64 g / ml . Furthermore, the symmetrical dialkylated 4aps 3cc, 3ee, and 3ff were in vitro tested to evaluate the activity against trypanosoma cruzi, trypanosoma brucei, leishmania infantum, and plasmodium falciparum; the results are summarized in table 5 . As can be evidenced, all tested compounds showed a moderate activity versus p. falciparum and an interesting activity towards l. infantum with ic50 values lower than the reference drug miltefosine; otherwise, they resulted scarcely active against t. cruzi and t. brucei . In conclusion, we demonstrated the usefulness of electrogenerated acetonitrile anion in the alkylation of n - boc 4-aminopyridines, both from the point of view of the high yields and of the cleanliness of the reaction (no by - products). The deprotection of n - boc 4-aminopyridines allowed obtaining monoalkylated 4-aminopyridine in very high yields . The following alkylation, by means of t - buok and alkyl halides, led to nonsymmetrically dialkylated 4-aminopyridine, while symmetrically dialkylated products were obtained directly from 4-aminopyridine by reaction with an excess of t - buok and alkyl halide . Furthermore, it can also be concluded that the monoalkylation of the 4ap leads to inactive products and otherwise interesting activity against fungi and some protozoa can be obtained by dual, symmetrical, or nonsymmetrical dialkylation of the amino group of 4ap; these active molecules can be considered as lead compound to develop new antifungal and antiprotozoal compounds . To a solution of di - tert - butyl dicarbonate (3 mmol) in acetonitrile (3 cm) at room temperature 4-aminopyridine (3 mmol) was slowly added . The solvent was evaporated and the crude 4-[n-(tert - butoxycarbonyl)amino]pyridine (> 95%) was used in the electrolyses without further purification . Rf (30% ethyl acetate in light petroleum ether) 0.20; h nmr (200 mhz, cdcl3) 1.53 (s,9h), 6.9 (bs, 1h), 7.32 (dd, j = 4.8, 1.6 hz, 2h), 8.45 (dd, j = 4.8, 1.6 hz, 2h); c nmr (50 mhz, cdcl3) 28.2, 81.7, 112.3, 145.6, 150.3, 151.9; eims, m / z: 194 (m, 1%), 137 (2%), 121 (5%), 120 (8%), 94 (50%), 78 (4%), 57 (100%). Constant current electrolyses (i = 25 ma cm) were performed under a nitrogen atmosphere, at 20c, using an amel model 552 potentiostat equipped with an amel model 731 integrator . All the experiments were carried out in a divided glass cell separated through a porous glass plug filled up with a layer of gel (i.e., methyl cellulose 0.5% volume dissolved in dmf - et4npf6 1.0 mol dm); pt spirals (apparent areas 0.8 cm) were used both as cathode and anode . Mecn - et4npf6 0.1 mol dm was used as solvent - supporting electrolyte system (catholyte: 20 cm; anolyte: 5 cm) after 145 c were passed, the current was switched off and 1 mmol of alkylating agent was added to the catholyte . The solution was kept under stirring at room temperature for 2 hours; then the solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography, using a mixture of ethyl acetate / light petroleum ether 2/8 in volume, obtaining the pure products . Flash column chromatography was carried out using merck 60 kieselgel (230400 mesh) under pressure . Gc - ms measurements were carried out on se 54 capillary column using a fisons 8000 gas chromatograph coupled with a fisons md 800 quadrupole mass selective detector . H and c nmr spectra were recorded at room temperature using a bruker ac 200 spectrometer using cdcl3 as internal standard . Rf (80% ethyl acetate in dichloromethane) 0.60; h nmr (200 mhz, cdcl3) 0.88 (t, j = 6.5 hz, 3h), 1.201.30 (m, 10h), 1.491.86 (m, 3h), 1.50 (s, 9h), 3.69 (app t, j = 7.6 hz, 2h), 7.24 (dd, j = 6.2, 1.6 hz, 2h), 8.51 (dd, j = 6.2, 1.6 hz, 2h); c nmr (50 mhz, cdcl3) 14.0, 22.6, 26.7, 28.2, 28.4, 29.1, 31.7, 48.7, 81.4, 118.8, 150.0, 150.1, 153.4 . Rf (50% ethyl acetate in light petroleum ether) 0.46; h nmr (200 mhz, cdcl3) 1.48 (s, 9h), 1.862.02 (m, 2h), 2.64 (t, j = 7.6 hz, 2h), 3.74 (app t, j = 7.6 hz, 2h), 7.127.33 (m, 7h), 8.49 (dd, j = 4.8, 1.6 hz, 2h); c nmr (50 mhz, cdcl3) 28.2, 30.0, 33.0, 48.3, 81.6, 118.9, 126.1, 128.3, 128.5, 141.0, 149.7, 150.3, 153.4; eims, m / z: m absent, 212 (5%), 107 (100%), 105 (5%), 91 (25%), 78 (27%), 77 (12%). Rf (60% ethyl acetate in light petroleum ether) 0.58; h nmr (200 mhz, cdcl3) 1.45 (s, 9h), 4.94 (s, 2h), 7.207.37 (m, 7h), 8.46 (dd, j = 4.8, 1.6 hz, 2h); c nmr (50 mhz, cdcl3) 28.1, 52.5, 82.1, 118.2, 126.3, 127.3, 128.7, 137.7, 150.2, 150.1, 153.5; eims, m / z: m absent, 227 (4%), 183 (14%), 91 (100%), 78 (7%), 57 (51%). Tert - butyl (2,6-dichlorobenzyl)pyridin-4-ylcarbamate 1d . Rf (50% ethyl acetate in light petroleum ether) 0.60; h nmr (200 mhz, cdcl3) 1.48 (s, 9h), 5.29 (s, 2h), 7.027.21 (m, 5h), 8.41 (dd, j = 4.8, 1.6 hz, 2h); c nmr (50 mhz, cdcl3) 28.2, 46.7, 81.7, 121.5, 128.6, 129.5, 131.7, 136.1, 148.1, 149.9, 153.3; eims, m / z: 352 (m, 1%), 252 (3%), 163 (6%), 161 (30%), 159 (42%), 78 (76%), 51 (100%). Tert - butyl (4-fluorobenzyl)pyridin-4-ylcarbamate 1e . Rf (50% ethyl acetate in light petroleum ether) 0.49; h nmr (200 mhz, cdcl3) 1.45 (s, 9h), 4.89 (s, 2h), 6.977.22 (m, 6h), 8.47 (app d, j = 6.0 hz, 2h); c nmr (50 mhz, cdcl3) 28.1, 51.8, 82.2, 116.6 (d, j = 21.5 hz), 118.4, 128.1 (d, j = 8.0 hz), 133.4 (d, j = 3.2 hz), 149.9, 150.3, 153.4, 162.2 (d, j = 245.4 hz); eims, m / z: 302 (m, 1%), 245 (4%), 201 (53%), 108 (100%), 78 (42%), 57 (100%). Tert - butyl (4-trifluoromethylbenzyl)pyridin-4-ylcarbamate 1f . Rf (50% ethyl acetate in light petroleum ether) 0.41; h nmr (200 mhz, cdcl3) 1.46 (s, 9h), 4.99 (s, 2h), 7.207.36 (m, 4h), 7.61 (app d, j = 8.4 hz, 2h), 8.49 (app d, j = 6.2 hz, 2h); c nmr (50 mhz, cdcl3) 28.1, 52.2, 82.5, 118.1, 124.0 (q, j = 271.9 hz), 125.7 (q, j = 3.7 hz), 126.6, 129.7 (q, j = 32.3 hz), 141.9, 149.8, 150.4, 153.3; eims, m / z: m absent, 251 (9%), 158 (34%), 145 (2%), 78 (25%), 69 (9%), 57 (100%). Tert - butyl (2-oxo-2-phenylethyl)pyridin-4-ylcarbamate 1 g. rf (60% ethyl acetate in light petroleum ether) 0.50; h nmr (200 mhz, cdcl3) 1.46 (s, 9h), 5.09 (s, 2h), 7.26 (d, j = 6.4 hz, 2h), 7.487.68 (m, 3h), 7.99 (d, j = 8.2 hz, 2h), 8.478.55 (m, 2h); c nmr (50 mhz, cdcl3) 28.1, 55.5, 82.5, 118.7, 127.9, 128.9, 133.9, 134.7, 150.2, 153.2, 193.7 . Tert - butyl (2-(4-fluoropheny)-2oxoethyl)pyridin-4-ylcarbamate 1 h. rf (50% ethyl acetate in light petroleum ether) 0.50; h nmr (200 mhz, cdcl3) 1.46 (s, 9h), 5.05 (s, 2h), 7.157.27 (m, 4h), 7.998.06 (m, 2h), 8.52 (dd, j = 5.0, 1.4 hz, 2h); c nmr (50 mhz, cdcl3) 28.1, 55.4, 82.6, 116.2 (d, j = 22.0 hz), 118.8, 130.6 (d, j = 9.4 hz), 131.1 (d, j = 3.2 hz), 150.1, 150.2, 153.2, 166.2 (d, j = 256.1 hz), 192.2 . Tert - butyl (2-(4-chloropheny)-2oxoethyl)pyridin-4-ylcarbamate 1i . Rf (20% ethyl acetate in dichloromethane) 0.30; h nmr (200 mhz, cdcl3) 1.45 (s, 9h), 5.04 (s, 2h), 7.21 (dd, j = 4.6, 1.6 hz, 2h), 7.50 (d, j = 8.8 hz, 2h), 7.93 (d, j = 8.0 hz, 2h), 8.51 (dd, j = 4.6, 1.6 hz, 2h); c nmr (50 mhz, cdcl3) 28.1, 55.4, 82.6, 118.8, 129.3, 133.0, 140.5, 149.9, 150.3, 153.1, 192.7 . Tert - butyl (2-(4-methoxypheny)-2oxoethyl)pyridin-4-ylcarbamate 1j . Rf (40% ethyl acetate in dichloromethane) 0.50; h nmr (200 mhz, cdcl3) 1.46 (s, 9h), 3.90 (s, 3h), 5.04 (s, 2h), 6.99 (d, j = 9.0 hz, 2h), 7.24 (dd, j = 4.8, 1.6 hz, 2h), 7.97 (d, j = 9.0 hz, 2h), 8.50 (dd, j = 4.8, 1.6 hz, 2h); c nmr (50 mhz, cdcl3) 28.1, 55.1, 55.5, 82.3, 114.1, 118.7, 127.8, 130.2, 150.2, 153.3, 164.1, 192.0 . To a solution of 1 (1 mmol) in ch2cl2 (5 cm), kept at 0c, 1 cm of cf3cooh was added . The solution was then mixed with aqueous sodium carbonate till ph 8 and extracted with ethyl acetate . The solvent was removed under reduced pressure and the mixture was purified by flash chromatography, yielding pure compound 2 . Rf (20% dichloromethane in ethyl acetate) 0.16; h nmr (200 mhz, cdcl3) 0.89 (t, j = 7.2 hz, 3h), 1.251.34 (6h), 1.611.70 (m, 2h), 2.893.12 (m, 2h), 3.153.24 (m, 2h), 5.435.48 (m, 2h), 6.57 (d, j = 5.2 hz, 2h), 8.11 (d, j = 5.2 hz, 2h); c nmr (50 mhz, cdcl3) 14.1, 22.6, 27.0, 28.8, 29.2, 29.2, 31.8, 42.9, 107.4, 155.2 . Rf (ethyl acetate) 0.46; h nmr (200 mhz, cd3cn) 1.912.06 (m, 2h), 2.71 (t, j = 7.4 hz, 2h), 3.173.27 (m, 2h), 4.9 (bs, 1h), 6.626.66 (m, 2h), 7.147.46 (m, 5h), 7.457.97 (m, 2h); c nmr (50 mhz, cd3cn) 29.8, 32.5, 42.0, 107.3, 125.9, 128.3, 128.4, 141.0, 141.5, 157.8; eims, m / z: 212 (m, 1%), 107 (100%), 91 (43%), 78 (13%). Rf (ethyl acetate) 0.57; h nmr (200 mhz, cdcl3) 4.46 (d, j = 6.0 hz, 2h), 6.7 (bs, 2h), 7.1 (bs, 1h), 7.217.41 (m, 5h), 8.0 (bs, 2h); c nmr (50 mhz, cdcl3) 46.8, 107.4, 127.2, 127.6, 128.8, 137.7, 148.6, 154.0; eims, m / z: 184 (m, 15%), 183 (15%), 107 (5%), 91 (100%), 78 (16%). Rf (ethyl acetate) 0.38; h nmr (200 mhz, cd3cn) 4.59 (s, 2h), 6.0 (bs, 1h), 6.68 (dd, j = 5.2, 1.4 hz, 2h), 7.277.47 (m, 3h), 8.41 (app d, j = 5.4 hz, 2h); c nmr (50 mhz, cd3cn) 42.2, 107.4, 128.7, 130.4, 132.8, 136.0, 147.3, 154.6; eims, m / z: 256 (m + 4, 1%), 254 (m + 2, 7%), 252 (m, 14%), 162 (10%), 160 (67%), 158 (100%), 78 (37%). Rf (ethyl acetate) 0.27; h nmr (200 mhz, cdcl3) 4.38 (d, j = 5.4 hz, 2h), 5.1 (bs, 1h), 6.5 (bs, 2h), 7.027.10 (m, 2h), 7.307.34 (m, 2h), 8.2 (bs, 2h); c nmr (50 mhz, cdcl3) 46.3, 107.8, 115.8 (d, j = 21.5 hz), 129.0 (d, j = 8.1 hz), 133.1, 148.5, 153.8, 166.8 (d, j = 205.9 hz),; eims, m / z: 202 (m, 5%), 107 (16%), 109 (100%), 78 (16%). Rf (ethyl acetate) 0.25; h nmr (200 mhz, cdcl3) 4.51 (d, j = 6.0 hz, 2h), 6.2 (bs, 1h), 6.6 (bs, 2h), 7.54 (d, j = 8.0 hz, 2h), 7.67 (d, j = 8.0 hz, 2h), 8.1 (bs, 2h); c nmr (50 mhz, cdcl3) 45.4, 107.8, 124.4 (q, j = 271.0 hz), 125.4 (q, j = 3.9 hz), 128.4 (q, j = 31.9 hz), 127.7, 143.2, 146.7, 155.0; eims, m / z: 252 (m, 80%), 183 (11%), 159 (100%), 107 (52%), 78 (31%). 1-(4-methoxyphenyl)-2-(pyridin-4-ylamine)ethan-1-one 2 g. rf (50% ethyl acetate in ethanol) 0.15; h nmr (200 mhz, cdcl3) 3.90 (s, 3h), 4.57 (d, j = 3.8 hz, 2h), 4.6 (bs, 1h), 6.58 (d, j = 6.2 hz, 2h), 6.98 (d, j = 9.0 hz, 2h), 7.98 (d, j = 9.0 hz, 2h), 8.19 (d, j = 6.2 hz, 2h); c nmr (50 mhz, cdcl3) 48.2, 55.6, 108.1, 114.1, 127.2, 130.2, 150.8, 153.6, 164.5, 191.6 . To a solution of 2 (1 mmol) in anhydrous dmso (2 cm), kept at rt under n2, 1.5 mmol of t - buok was added . This mixture was allowed to stir for 20 min at rt; then 1 mmol of alkyl halide was added and the solution was kept under stirring at rt for 4 h. the solution was then mixed with water and extracted with dichloromethane . The solvent was removed under reduced pressure and the mixture was purified by flash chromatography, yielding pure compound 3 . Rf (80% ethyl acetate in ethanol) 0.38; h nmr (200 mhz, cdcl3) 0.860.92 (m, 3h), 1.201.40 (m, 10h), 1.631.70 (m, 2h), 3.42 (app t, j = 7.6 hz, 2h), 4.59 (s, 2h), 6.51 (d, j = 5.2 hz, 2h), 7.147.38 (m, 5h), 8.18 (d, j = 5.2 hz, 2h); c nmr (50 mhz, cdcl3) 14.1, 22.6, 26.9, 27.0, 29.2, 29.4, 29.7, 31.8, 50.7, 53.4, 106.9, 126.2, 127.3, 128.8, 136.8, 148.9, 153.6 . Rf (ethyl acetate) 0.40; h nmr (200 mhz, cdcl3) 0.860.92 (m, 3h), 1.231.35 (m, 10h), 1.621.72 (m, 2h), 3.42 (app t, j = 7.8 hz, 2h), 4.58 (s, 2h), 6.56 (app d, j = 6.4 hz, 2h), 6.997.16 (m, 4h), 8.17 (app d, j = 6.4 hz, 2h); c nmr (50 mhz, cdcl3) 14.1, 19.2, 22.6, 26.9, 29.2, 29.3, 31.7, 51.3, 53.3, 107.4, 116.1 (d, j = 21.7 hz), 127.9 (d, j = 8.1 hz), 130.9 (d, j = 3.5 hz), 144.9, 155.3, 162.3 (d, j = 246.5 hz). Rf (ethyl acetate: n - hexane: methanol 50: 33: 17) 0.48; h nmr (200 mhz, cdcl3) 1.912.03 (m, 2h), 2.68 (t, j = 7.4 hz, 2h), 3.43 (app t, j = 7.8 hz, 2h), 4.54 (s, 2h), 6.44 (app d, j = 5.4 hz, 2h), 6.967.36 (m, 9h), 8.15 (bs, 2h); c nmr (50 mhz, cdcl3) 28.1, 33.0, 49.9, 52.9, 106.8, 115.8 (d, j = 21.6 hz), 126.3, 127.9 (d, j = 8.0 hz), 128.3, 128.6, 132.1 (d, j = 3.2 hz), 140.7, 147.9, 153.7, 162.1 (d, j = 245.8 hz). To a solution of 4ap (1 mmol) in anhydrous dmso (2 cm), kept at rt under n2, 2 mmol of t - buok was added . This mixture was allowed to stir for 20 min at rt; then 2 mmol of alkyl halide was added and the solution was kept under stirring at rt for 4 h. the solution was then mixed with water and extracted with dichloromethane . The solvent was removed under reduced pressure and the mixture was purified by flash chromatography, yielding pure compound 3 . Rf (80% ethyl acetate in dichloromethane) 0.20; h nmr (200 mhz, cd3cn) 1.841.99 (m, 4h), 2.65 (t, j = 7.5 hz, 4h), 3.29 (t, j = 7.5 hz, 4h), 6.30 (d, j = 5.6 hz, 2h), 7.177.31 (m, 10h), 8.13 (d, j = 5.6 hz, 2h); c nmr (50 mhz, cd3cn) 28.3, 33.1, 49.5, 106.4, 126.2, 128.3, 128.5, 141.1, 140.7, 152.4 . Rf (80% ethyl acetate in ethanol) 0.32; h nmr (200 mhz, cdcl3) 4.67 (s, 4h), 6.58 (dd, j = 4.8, 1.6 hz, 2h), 7.197.40 (m, 10h), 8.20 (dd, j = 4.8, 1.6 hz, 2h); c nmr (50 mhz, cdcl3) 53.2, 107.1, 126.4, 127.4, 128.9, 136.8, 150.2, 153.9 . Di(4-fluorobenzyl)pyridin-4-amine 3ee . Rf (80% ethyl acetate in dichloromethane) 0.40; h nmr (200 mhz, cdcl3) 4.61 (s, 4h), 6.56 (dd, j = 5.0, 1.6 hz, 2h), 6.997.19 (m, 8h), 8.22 (dd, j = 5.0, 1.6 hz, 2h); c nmr (50 mhz, cdcl3) 52.5, 107.1, 115.8 (d, j = 21.6 hz), 128.1 (d, j = 8.0 hz), 132.3 (d, j = 3.2 hz), 150.3, 153.6, 162.2 (d, j = 245.8 hz). Rf (80% ethyl acetate in dichloromethane) 0.25; h nmr (200 mhz, cdcl3) 4.73 (s, 4h), 6.58 (dd, j = 5.0, 1.6 hz, 2h), 7.367.59 (m, 8h), 8.26 (dd, j = 5.0, 1.6 hz, 2h); c nmr (50 mhz, cdcl3) 53.1, 107.1, 123.3 (q, j = 3.7 hz), 123.8 (q, j = 272.3 hz), 124.5 (q, j = 3.8 hz), 129.7, 131.4 (q, j = 32.4 hz), 137.7, 150.4, 153.4 . Organisms . For the antifungal evaluation, strains obtained from the american type culture collection (atcc, rockville, md, usa), the german collection of microorganisms (dsmz, braunschweig, germany), and the pharmaceutical microbiology culture collection (pmc, department of public health and infectious diseases, sapienza university, rome, italy) were tested . The strains were candida albicans (atcc 10231, atcc 10261, atcc 24433, atcc 90028, 3153, pmc 1002, pmc 1011, and pmc 1030), c. parapsilosis atcc22019, c. parapsilosis dsm 11224, c. tropicalis dsm 11953, c. tropicalis pmc 0908, c. tropicalis pmc 0910, c. glabrata pmc 0805, c. krusei dsm 6128, and c. krusei pmc 0613, cryptococcus neoformans (dsm 11959, pmc 2102, pmc 2107, pmc 2111, and pmc 2136), dermatophytes (trichophyton mentagrophytes dsm 4870, t. mentagrophytes pmc6509, microsporum gypseum dsm 7303, and m. gypseum pmc 7331). All of the strains were stored and grown in accordance with the procedures of the clinical and laboratory standards institute (clsi) [26, 27]. The final concentration ranged from 0.125 to 64 g / ml . The compounds were dissolved previously in dmso at concentrations 100 times higher than the highest desired test concentration and successively diluted in test medium in accordance with the procedures of the clsi . Microdilution trays containing 100 l of serial twofold dilutions of compounds in rpmi 1640 medium (sigma - aldrich, st . Louis, mo, usa) were inoculated with an organism suspension adjusted to attain a final inoculum concentration of 1.0 101.5 10 cells / ml for yeasts and 0.4 105 10 cfu / ml for dermatophytes . The panels were incubated at 35c and observed for the presence of growth at 48 h (candida spp .) And 72 h (c. neoformans and dermatophytes). The minimal inhibitory concentration (mic) was, for yeasts, the lowest concentration that showed 50% growth inhibition compared with the growth control and, for dermatophytes, the lowest concentration that showed 80% growth inhibition compared with the growth control . The mic100 was the lowest drug concentration that prevented 100% of growth with respect to the untreated control . According to csi protocols, the fluconazole mic50 and the amphotericin b mic100 were calculated (22,23). The results were expressed as the geometric mean (g m) of the mic values . For the evaluation of antiprotozoal and cytotoxic activity an integrated panel of microbial screens and standard screening methodologies were adopted as previously described on the following organisms: chloroquine - resistant p. falciparum k 1-strain; l. infantum mhom / ma (be)/67 amastigote stage; suramin - sensitive trypanosoma brucei squib-427 strain; trypanosoma cruzi tulahuen cl2 (benznidazole - sensitive) strain; human fetal lung fibroblast cells (mrc-5 sv2). Compounds were tested at 5 concentrations (64, 16, 4, 1, and 0.25 g / ml) to establish a full dose titration and determine the ic50 (inhibitory concentration 50%). The final in - test concentration of dmso did not exceed 0.5%, which is known not to interfere with the different assays.
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To develop a compact system for basic experiments, we chose a sealed x - ray tube with a molybdenum target (oxford instruments inc ., ca). The voltage we could apply ranged from 10 to 50 kvp (voltage - peak), and the maximum current was 1 ma . We clearly observed strong characteristic radiations of 17.479 kev and 19.608 kev, which corresponded to the k and k, respectively, of molybdenum . The bremsstrahlung broadband spectrum was also generated by bombarding of the target material with electrons . The x - rays generated from the target were reflected on the inner wall of each capillary and they passed through the parabolic polycapillary optics . As the shape of the each capillary was a parabola, the x - rays radiated from a focus of the parabola passed through each capillary and they were parallel to the optical axis . In other words, the polycapillary optics transferred the divergent x - rays to a parallel beam along the optical axis . The outer diameter and length of the parabolic polycapillary optics were 10 mm and 133 mm, respectively . For mo k radiation, the critical angle of each capillary was 1.7 mrad and the reflectivity of a single reflection on the capillary was greater than 0.8 . The incidence x - rays with the critical angle were reflected approximately 17 times on the inside wall of each capillary, and in this case, the reflectivity was approximately 0.02 . However, when the incidence x - rays with less than the critical angle were included, the final transmission efficiency of the generated x - rays could be as high as 0.3 . Taking into consideration the transmission efficiency, the applied tube voltage was 40 kvp which was higher than the applied tube voltage for conventional mammography . In this study, the operating condition of the x - ray tube for imaging test objects was fixed at a tube voltage of 40 kvp and a current of 0.5 ma, and the exposure time to let the image develop was three seconds . The parabolic polycapillary cut the high energy of more than 20 kev by its x - ray reflection properties, but the low energy x - ray of less than 20 kev was passed by the parabolic polycapillary . Thus, a tube voltage more than 40 kvp could be applied and the number of photons generated on the x - ray tube could be increased . In a tube voltage of 40 kvp with a current of 0.5 ma, the number of photons generated by the molybdenum target x - ray tube in front of the polycapillary optics was ~ 610 photons / sec . It meant that the number of photons on the detector was enough to make a digital image . In our experiment,, nj) with a 24 m 24 m pixel size was used for imaging . The nyquist frequency of the captured image using the ccd detector was 21 lp / mm for which the spatial resolution was limited by the pixel size of the ccd detector . In order to obtain a higher spatial resolution of 21 lp / mm in our system configuration, the pixel size of the ccd detector would have to be less than 24 m 24 m . The ccd chip was cooled down to 223 k to reduce the dark noise of the ccd detector . Figure 1 shows the experimental setup used to evaluate the performance of the small - field digital mammography . Parallel x - ray beams by the parabolic polycapillary optics entered a filter and then passed through a test sample . All images used in these experiments were corrected by post - image processing, and the contrast (imax - imin) / imin, was calculated by maximum and minimum pixel values . The objects on an image are distinguished by their contrast difference and achievable spatial resolution of the system . The mtf of an imaging system shows a performance of the spatial resolution connected with the contrast (12). We directly examined the mtf to verify the spatial resolution of the small - field digital mammographic imaging system by taking images of the line - pairs instead of indirectly obtaining the images from an edge spread function . The exposure time was three seconds when we took the linear pattern images of a phantom of a gold - line . X - ray exposure gives rise to energy accumulation in an object, and the energy can destroy biological tissue and its chemical state . When x - ray photons n with a wavelength are irradiated on the area d of a sample, then the dose of the specimen with the thickness and density can be written as: which is the radiation dose for a single x - ray wavelength or energy of hc/, and where is the linear absorption coefficient of the sample . All the x - rays that arrive on the sample contribute to the radiation dose of the sample . Therefore, the radiation dose d for a general x - ray tube source can be expressed as: we measured the radiation exposure to enter the test sample using an ionization chamber (fluke corp ., oh) with an exposure time of three seconds and measured the spectrum of incident x - rays to the test sample together . The absolute number of x - ray photons with energy hc/ was known by the radiation exposure and the x - ray spectrum, and the radiation dose of the test sample was obtained from equations and and the material information of the test sample . This method is similar to the dose measurement in which x - ray tube voltage and half - value layer (or equivalent other parameters) should be known . We obtained an image more than 10 mm in diameter by scanning a test standard phantom of breast and combining each scanned image . Breast tissue which was fixed by paraffin and proven on biopsy to be a ductal carcinoma in situ was imaged with the small - field digital mammographic imaging system . A conventional projection - type digital mammography of the breast tissue sample was taken to compare with the findings of the small - field digital mammographic imaging system . The breast tissue study was carried out in accordance with the regulations of the institutional review board of university . There were various choices of filters for our mammographic imaging system, e.g., molybdenum (mo) and rhodium (rh). The spatial resolution of 12 lp / mm for the molybdenum and rhodium filters with a 25 m thickness was achieved at a mtf of 10% as shown in figure 2 . The spatial resolution of 12 lp / mm corresponded to 41.7 m in spatial resolution . From the standpoint of the ccd detector, this spatial resolution was matched to almost two pixels . When we used molybdenum filters with 25 m and 50 m thickness, the mtf values for each filter weren't very different . However, the contrasts for the molybdenum filter with a 25 m and 50 m thick was 1.7 and 1.5, respectively . The contrast was measured with a step - like acrylic object . In the case of a 25-m molybdenum filter, the contrast for was 12% higher than that for a 50-m molybdenum filter . The image contrast was proportional to the square of the photon number detected on the ccd camera . Thus, the thick filter led to a lower contrast . When a 100-m - thick molybdenum filter was used in the mammographic imaging system, the contrast rapidly decreased to 0.48 and the spatial resolution was 11 lp / mm . Figure 4 shows the radiation dose using rhodium filters of 25-m thickness for an acrylic phantom with a 44 mm thickness (mammographic accreditation phantom, cirs, va), equivalent to a 42 mm compressed breast with 50% glandular tissue and 50% adipose tissue, as a function of the tube current and fixed voltage for molybdenum . The radiation dose, as a function of the voltage at a fixed current, increased quadratically . The radiation dose shown in the table 1 is when the parabolic polycapillary optics was not combined with the x - ray tube in our system configuration . When no parabolic polycapillary optics was used, the system became a conventional projection - type digital mammography . The radiation dose of the system without the parabolic polycapillary optics was three times higher than with the parabolic polycapillary optics . The field that was exposed to the radiation dose for the small - field digital mammographic imaging system was 3% of the field for the projection - type system . The small - field digital mammographic imaging system compared to the projection - type digital mammography had advantages in terms of the radiation dose, dose field and absorbed x - ray energy as shown in table 1 . Radiation dose was calculated from the measured x - ray energy absorbed and the measured dose field which led to mass information . In the mammography system without the parabolic polycapillary optics, the field illuminated x - ray could be controlled by lead block or collimator . In this case, the radiation field could be small . However, the radiation dose was still three times higher because the radiation dose was not proportional to the field size but to the accumulated x - ray energy and mass of the object . In our small - field mammographic imaging system, the radiation doses for the molybdenum and the rhodium filters were 0.18 mgy and 0.13 mgy, respectively . Theoretical estimation of the radiation dose (13, 14) for the phantom was 0.32 mgy at a radiation exposure of 0.15 r, a half - value layer of 0.41, and a tube voltage of 35 kvp for a mo / rh tube - filter combination . The value of 0.32 mgy was very close to the 0.38 mgy in table 1 . In addition, the american college of radiology advocates that the maximum value of the mean glandular dose must not exceed 3 mgy (15). We compared spectra for x - ray beams passing through parabolic polycapillary optics combined with the x - ray tube and direct x - ray beam as shown in figure 5a . The high energy x - rays larger than 20 kev were almost removed by propagating the parabolic polycapillary optics although low energy x - rays were also reduced simultaneously . The cut - off of the high energy could also be verified by the calculation of the x - ray reflectivity on the inner surface of the parabolic polycapillary optics (16). Figure 5b shows the x - ray reflectivity as a function of x - ray energy at the fixed incidence angle of 1.7 mrad . After two reflections, the high energy x - rays of more than 20 kev were almost removed . The x - ray generated from the x - ray tube could be reflected up to 17 times on the inner surface of the parabolic polycapillary optics . Low energy x - rays have a larger critical angle than that of high energy x - rays, but the reflectivity of the low energy x - rays decrease at a larger incidence angle than its critical angle . Thus, the intensity of low energy x - rays passing through the parabolic polycapillary optics with various incident angles was also reduced as shown in figure 5a . Additionally, the molybdenum and rhodium filters remarkably reduced the low energy of the x - ray beam which passed through the parabolic polycapillary optics removing the high energy larger than 20 kev of the generated x - rays because the critical angles for the high energy x - rays were smaller than that for molybdenum radiation . Because of the parabolic polycapillary optics and the filter, the radiation doses were remarkably reduced, and the fact that the rhodium filter allowed a lower radiation dose could be verified by measuring the energy spectra shown in figure 6 . Within the overall energy region, the number of photons for the rhodium filter was lower than that for the molybdenum filter . We were able to obtain an enlarged image more than 10 mm in diameter by scanning a test object and combining each scanned image . Each scanned image was overlapped by approximately 30% of the field in order to guarantee that no scan field was missed on the test sample . X - ray exposure to make an image was three seconds and the object was moved for next imaging during one second . Figure 7a presents the scanned and recombined phantom image of 0.32-mm - al2o3 specks which simulate the microcalcifications of breast cancer . The 44-mm - thick acrylic phantom contained fibers, specks and masses that were composed of various materials . Figure 7b shows an image of the same specks taken on projection - type digital mammography . The system produced a magnified image of 1.07 x, and the spatial resolution of the system was 11 lp / mm which was tested by a line - pair phantom . The spatial resolution was limited by the spot size of 100 m rather than the pixel size of the detector because of the cone beam . Comparing both sets of images in figure 7a, b, the ratio of background noise, which was defined as small - field mammographic imaging system / projection - type digital mammography, was 0.8 . It meant that the image contrast of the small - field mammographic imaging system might be 1.2 times higher than that of the projection - type, digital mammography . Figure 8a clearly shows microcalcifications within breast tissue which was fixed by paraffin and proven by biopsy to be ductal carcinoma in situ . A small figure located on the left upper position in figure 8b shows a full image of the breast tissue sample taken with a projection - type digital mammography, and the outer black circle in figure 8b was the same field taken with the small - field scanning mammographic imaging system . Calcifications located within two small circles in figure 8a were clearly observed . In figure 8b, it was difficult to distinguish calcifications located at the same positions because of lower image contrast . However, the same objects in figure 8b seemed to one unseparated object . Because of the high spatial resolution of 12 lp / mm for the small - field mammographic imaging system, two microcalcifications could be distinguished . However, the projection - type digital mammography could not distinguish two objects because of the separation distance was at the resolution limit of 11 lp / mm . There were various choices of filters for our mammographic imaging system, e.g., molybdenum (mo) and rhodium (rh). The spatial resolution of 12 lp / mm for the molybdenum and rhodium filters with a 25 m thickness was achieved at a mtf of 10% as shown in figure 2 . The spatial resolution of 12 lp / mm corresponded to 41.7 m in spatial resolution . From the standpoint of the ccd detector, this spatial resolution was matched to almost two pixels . When we used molybdenum filters with 25 m and 50 m thickness, the mtf values for each filter weren't very different . However, the contrasts for the molybdenum filter with a 25 m and 50 m thick was 1.7 and 1.5, respectively . The contrast was measured with a step - like acrylic object . In the case of a 25-m molybdenum filter, the contrast for was 12% higher than that for a 50-m molybdenum filter . The image contrast was proportional to the square of the photon number detected on the ccd camera . Thus, the thick filter led to a lower contrast . When a 100-m - thick molybdenum filter was used in the mammographic imaging system, the contrast rapidly decreased to 0.48 and the spatial resolution was 11 lp / mm . Figure 4 shows the radiation dose using rhodium filters of 25-m thickness for an acrylic phantom with a 44 mm thickness (mammographic accreditation phantom, cirs, va), equivalent to a 42 mm compressed breast with 50% glandular tissue and 50% adipose tissue, as a function of the tube current and fixed voltage for molybdenum . The radiation dose, as a function of the voltage at a fixed current, increased quadratically . The radiation dose shown in the table 1 is when the parabolic polycapillary optics was not combined with the x - ray tube in our system configuration . When no parabolic polycapillary optics was used, the system became a conventional projection - type digital mammography . The radiation dose of the system without the parabolic polycapillary optics was three times higher than with the parabolic polycapillary optics . The field that was exposed to the radiation dose for the small - field digital mammographic imaging system was 3% of the field for the projection - type system . The small - field digital mammographic imaging system compared to the projection - type digital mammography had advantages in terms of the radiation dose, dose field and absorbed x - ray energy as shown in table 1 . Radiation dose was calculated from the measured x - ray energy absorbed and the measured dose field which led to mass information . In the mammography system without the parabolic polycapillary optics, the field illuminated x - ray could be controlled by lead block or collimator . In this case, the radiation field could be small . However, the radiation dose was still three times higher because the radiation dose was not proportional to the field size but to the accumulated x - ray energy and mass of the object . In our small - field mammographic imaging system, the radiation doses for the molybdenum and theoretical estimation of the radiation dose (13, 14) for the phantom was 0.32 mgy at a radiation exposure of 0.15 r, a half - value layer of 0.41, and a tube voltage of 35 kvp for a mo / rh tube - filter combination . The value of 0.32 mgy was very close to the 0.38 mgy in table 1 . In addition, the american college of radiology advocates that the maximum value of the mean glandular dose must not exceed 3 mgy (15). We compared spectra for x - ray beams passing through parabolic polycapillary optics combined with the x - ray tube and direct x - ray beam as shown in figure 5a . The high energy x - rays larger than 20 kev were almost removed by propagating the parabolic polycapillary optics although low energy x - rays were also reduced simultaneously . The cut - off of the high energy could also be verified by the calculation of the x - ray reflectivity on the inner surface of the parabolic polycapillary optics (16). Figure 5b shows the x - ray reflectivity as a function of x - ray energy at the fixed incidence angle of 1.7 mrad . After two reflections, the high energy x - rays of more than 20 kev were almost removed . The x - ray generated from the x - ray tube could be reflected up to 17 times on the inner surface of the parabolic polycapillary optics . Low energy x - rays have a larger critical angle than that of high energy x - rays, but the reflectivity of the low energy x - rays decrease at a larger incidence angle than its critical angle . Thus, the intensity of low energy x - rays passing through the parabolic polycapillary optics with various incident angles was also reduced as shown in figure 5a . Additionally, the molybdenum and rhodium filters remarkably reduced the low energy of the x - ray beam which passed through the parabolic polycapillary optics removing the high energy larger than 20 kev of the generated x - rays because the critical angles for the high energy x - rays were smaller than that for molybdenum radiation . Because of the parabolic polycapillary optics and the filter, the radiation doses were remarkably reduced, and the fact that the rhodium filter allowed a lower radiation dose could be verified by measuring the energy spectra shown in figure 6 . Within the overall energy region, the number of photons for the rhodium filter was lower than that for the molybdenum filter . We were able to obtain an enlarged image more than 10 mm in diameter by scanning a test object and combining each scanned image . Each scanned image was overlapped by approximately 30% of the field in order to guarantee that no scan field was missed on the test sample . X - ray exposure to make an image was three seconds and the object was moved for next imaging during one second . Figure 7a presents the scanned and recombined phantom image of 0.32-mm - al2o3 specks which simulate the microcalcifications of breast cancer . The 44-mm - thick acrylic phantom contained fibers, specks and masses that were composed of various materials . Figure 7b shows an image of the same specks taken on projection - type digital mammography . The system produced a magnified image of 1.07 x, and the spatial resolution of the system was 11 lp / mm which was tested by a line - pair phantom . The spatial resolution was limited by the spot size of 100 m rather than the pixel size of the detector because of the cone beam . Comparing both sets of images in figure 7a, b, the ratio of background noise, which was defined as small - field mammographic imaging system / projection - type digital mammography, was 0.8 . It meant that the image contrast of the small - field mammographic imaging system might be 1.2 times higher than that of the projection - type, digital mammography . Figure 8a clearly shows microcalcifications within breast tissue which was fixed by paraffin and proven by biopsy to be ductal carcinoma in situ . A small figure located on the left upper position in figure 8b shows a full image of the breast tissue sample taken with a projection - type digital mammography, and the outer black circle in figure 8b was the same field taken with the small - field scanning mammographic imaging system . Calcifications located within two small circles in figure 8a were clearly observed . In figure 8b, it was difficult to distinguish calcifications located at the same positions because of lower image contrast . Because of the high spatial resolution of 12 lp / mm for the small - field mammographic imaging system, two microcalcifications could be distinguished . However, the projection - type digital mammography could not distinguish two objects because of the separation distance was at the resolution limit of 11 lp / mm . Previous studies (7 - 9) have demonstrated the potential for x - ray capillary optics, non - parabolic polycapillary optics, aligned between the object and computed radiography phosphor imaging plate, to be incorporated into digital mammographic image . Consequently, the post - object capillary optics had the potential to increase image contrast by eliminating scattered x - rays . A scanning process is also required to obtain an image because the size of the capillary optics is small . In the system configuration, the capillary optics played a role as a small' grid', and there was no improvement on the spatial resolution which was approximately 5 lp / mm because the divergent x - ray source of 100 m spot size illuminated the object and the pixel size of detector was large . In addition, the system configuration could not avoid a high radiation dose because of long exposure time, i.e., six seconds per one scanned image during scanning . In the scanning process, the capillary optics and the detector were combined with each other and moved together . We modified the system configuration to improve the spatial resolution and to reduce radiation dose instead of sacrificing the contrast slightly (fig . The spot size of the x - ray tube used in this system was not an important factor in effecting the spatial resolution due to the use of a parallel beam . For the parallel beam, there was no image blurring by finite spot size of the x - ray tube, and the pixel size of the detector was a critical factor to determine the spatial resolution . The illumination area of the parallel beam was small because the diameter of the parabolic polycapillary optics was small . However, there was enough space to increase the field of view by scanning the object . When the x - ray tube of 100-m - spot size without the parabolic polycapillary optics and the same ccd detector are used to make a projection - type x - ray image, we may obtain a similar spatial resolution of 12 lp / mm at a magnification of 1.48 . In the imaging system using the divergent x - ray beam, the edge gradient blurring for the spot size and the magnification may correspond with the two pixels of the ccd detector which is almost equal to the spatial resolution . If the magnification is larger than 1.48, the spatial resolution will be worse, and if the magnification is smaller than 1.48, the spatial resolution will not improve because one line - pair needs two pixels to separate the pattern at least . In this sense, only similar spatial resolution of 12 lp / mm will be available at a magnification of less than 1.48 for the divergent x - ray beam . In addition, a high radiation dose compared to the low radiation from small - field digital mammographic imaging system cannot be avoided for the divergent beam . In general, a thick filter considerably reduces low - energy x - rays including also characteristic radiations and requires a longer exposure time to obtain the same number of photons on the ccd detector compared to a thin filter . With using a thick filter, the detector noise could be increased and the image quality might be reduced if the x - ray source was not strong enough . Spatial resolutions of 12 lp / mm for molybdenum and rhodium filters with a 25-m thickness were obtained using the small - field digital mammographic imaging system . The resolution in the digital mammographic imaging system strongly depended on the pixel size of the detector . The resolution should be improved in future with advances in the manufacturing technology for ccd or cmos detectors thereby creating a smaller pixel size . The small - field digital mammographic imaging system was able to diminish the radiation dose compared to that of the conventional projection - type mammography, for which an entire object receives the radiation dose . In addition, the parabolic polycapillary optics itself plays a role of a low - pass filter to eliminate x - rays which have higher energy than molybdenum radiation . Image blur and radiation dose by x - ray beams of high energy which are not removed by conventional molybdenum and rhodium filters the x - ray beams of high energy larger than 20 kev removed contributes to improve image quality and to reduce the radiation dose . In other applications, e.g., conventional radiography, high energy x - ray, for instance, 75 kev, is required . To propagate the high energy x - ray, each diameter of glass capillary in the polycapillary optics should be very small, i.e., sub - micrometer size, because the critical angle for the high energy x - ray is extremely small . In current manufacturing technology thus, general x - ray imaging using parallel beam through the parabolic polycapillary optics can be restricted . Therefore, to obtain a parallel beam of high photon flux, the x - ray tube should have a high capacity . In conclusion, due to its high spatial resolution and low radiation dose, a small - field digital mammographic imaging system has the potential to be used for examining local volumes of breast tissue . A relatively large field image this system can be a useful diagnostic tool for examining a small field of breast cancer with a low radiation dose to the patient after conducting conventional mammography studies . To improve the small - field digital mammographic imaging system using parabolic polycapillary optics, large
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Cervical spondylosis is a common cause of chronic neck pain, radiculopathy and/or myelopathy resulting in significant disability . When patients do not respond to conservative therapy, anterior cervical discectomy and fusion (acdf) usually is used to achieve neural decompression, maintain cervical lordosis, and provide segmental stabilization . In our practice, it was often found that patients with cervical spondylosis tended to have concomitant cervical vertigo symptoms such as tinnitus, headache, blur vision, and palpitation . Recent studies have indicated that cervical spondylosis patients with cervical vertigo symptoms could be managed successfully with acdf . In this study, we report 2 cases of cervical spondylosis with concomitant cervical vertigo and hypertension that were treated successfully with acdf . This study obtained the approval of the medical ethics committee of our hospital (general hospital of armed police force, beijing, china). Case 1, a 49-year - old female, was admitted with 2 years of history of chronic neck pain, headache, and episodic vertigo and tinnitus . Episode occurred 1 to 2 times every day, and lasted 1 to 2 h each time . She felt numbness of both upper extremities and weakness of all 4 extremities for 1 year . Physical examination showed that limited neck motion, slightly diminished sensation in both arms, and brisk deep tendon reflexes . Radiographic examination of the cervical spine showed mild diminution in the height of the c5/6 disc space (figure 1). Magnetic resonance imaging (mri) scan showed cord compression by a large central herniation of c5/6 disc with signs of myelomalacia (figure 2). Lateral radiograph of cervical spine before operation showed slight narrowing of the c5/6 disc space . T2-weighted sagittal magnetic resonance imaging (mri) revealed a large herniation of c5/6 disc before operation, with marked compression of cervical cord, resulting in myelomalacia . The patient had a 2-year history of hypertension, blood pressure was 160/100 mmhg at onset, and then controlled in the normal range (110130/7080 mmhg) by oral medications (betaloc, 100 mg, once daily; enalapril, 10 mg, once daily). Case 2, a 60-year - old male, was admitted with 10 years of history of chronic neck pain and stiffness . He also experienced vertigo, palpitation, nausea and vomiting with noted numbness of upper limbs, and heaviness of 4 limbs for over 6 months before admission . Radiographic examination of the cervical spine revealed large anterior and posterior spur formation at the c5/6 and c6/7 disc levels, severe diminution in the height of the c5/6 disc space, and moderate diminution in the height of the c6/7 disc space . Mri scan showed cervical canal stenosis with marked cord compression secondary to spondylotic changes in c5/6 and c6/7 discs . The patient had a 1-year history of hypertension (maximum at 180/120 mmhg), which was controlled within the normal range by oral medications (irbesartan, 150 mg, once daily; nifedipine sustained release tablets, 20 mg, once daily; amlodiping besylate, 5 mg, once daily). Cervical spondylotic myelopathy was diagnosed and subsequently cervical anterior surgery was recommended for the 2 patients preoperatively based on clinical findings consistent with imaging study . Case 1 underwent acdf at the c5/6 disc level (figures 3 and 4). After surgery, strength in all 4 limbs was significantly recovered, and symptoms of neck pain, headache, vertigo, and tinnitus disappeared . Interestingly, her blood pressure also dropped to within the normal range after surgery even without oral medications . Lateral radiograph of cervical spine 3 months after operation showed anterior cervical plate fixation with cage fusion at c5/6 disc level . T2-weighted sagittal magnetic resonance imaging (mri) 7 days after operation showed disappearance of cord compression at c5/6 disc level . His symptoms of cervical vertigo completely disappeared, numbness of both upper limbs significantly released, and muscle strength of 4 limbs obviously increased . Similarly to case 1, her cervical vertigo never recurred, and her blood pressure remained normal without the use of antihypertensive medications . Case 2 has undergone a follow - up of 14 months, the patient never had cervical vertigo anymore, and his blood pressure remained normal throughout (120130/7585 mmhg). Clinical results indicated that acdf can eliminate the concomitant vertigo symptoms in the 2 patients . However, the exact mechanisms of resolution of symptoms cannot be elucidated precisely . Because cervical spondylosis is a common disease, vertigo occurred in patients with cervical spondylosis is of particular importance . One previous study showed that vertigo was present in 50% of patients with cervical spondylosis, whereas another study identified cervical spondylosis as the cause of dizziness in 65% of elderly patients . It has been shown that the patients with cervical spondylosis complaining of vertigo have significant lower blood flow parameters than non - vertigo patients with cervical spondylosis . Insufficient blood supply to posterior circulation is called vertebrobasilar insufficiency . The most common complaint in patients with vertebrobasilar insufficiency insufficient blood supply does not necessarily cause symptoms if there is sufficient collateral circulation, whereas a full range of symptoms commonly occur as a result of an insufficient terminal vessel . The vascular supply to the vestibulocochlear organ, being an end artery, makes this organ more susceptible to vertebrobasilar insufficiency . Neurons, axons, and hair cells in the vestibulocochlear system are known to respond to ischemia by depolarizing, causing transient hyperexcitability with ectopic discharges, manifesting as tinnitus, vertigo, and dizziness . It was hypothesized that vertebral artery insufficiency secondary to cervical spondylosis could result in vertigo . However, the mechanisms of vertigo caused by cervical spondylosis are not clear so far . The mechanical compression on vertebral artery from spurs of the luschka joint was considered as a main mechanism . The reduction of vertebral artery flow can be more obvious with the rotation and hyperextension of the head . In our current study in addition, mechanical compression cannot explain some other symptoms such as palpitation, nausea, vomiting, etc . Recent clinical studies by hong et al and li et al demonstrated that the stimulation of the sympathetic nerve fibers other than the compression of the vertebral artery induces these symptoms such as vertigo, tinnitus, nausea, and vomiting . Current study clearly indicates that hypertension can be associated with cervical spondylosis, as a secondary condition . We speculate that the mechanisms of hypertension induced by cervical spondylosis could be the same as that of cervical vertigo . It is well known that cervical disc, dura mater, and posterior longitudinal ligament are rich in sympathetic fibers . Sympathetic nerve fibers distributed around the vertebral artery have been implicated in the autoregulation of vertebrobasilar artery blood flow and cerebral blood flow . It has been evidenced that sympathetic nerve activity withdraws the blood flow, whereas sympathectomy can increase it . A previous study investigated sympathetic and parasympathetic changes incited by the stimulation of nerves around the proximal vertebral artery in cat model and found subsequent papillary changes, pulse and blood pressure changes . Elevation in levels of inflammatory cytokines has been detected in painful discs of humans, and thought to be related to degeneration and pain . The pathogenic change in the painful disc is featured with the formation of zones of vascularized granulation tissue and extensive innervation extending from the outer layer of the annulus fibrosus into the nucleus pulposus . Degenerative changes in the disc, such as loss of the normal structure and a mechanical load, can lead to abnormal motion, which can provoke mechanical stimulation . Mechanical stimuli, which are normally innocuous to disc nociceptors can, in certain circumstances such as inflammation, generate an amplified response termed peripheral sensitization . The vertebral arteries are mainly innervated by nerve fibers from the cervical sympathetic ganglia (13). Innervation in cervical disc is analogous to that in the lumbar spine, receiving innervation posteriorly from the sinuvetebral nerves, laterally from the vertebral nerve, and anteriorly from the sympathetic trunks . Recently, an animal study revealed a reciprocal neural connection between cervical spinal and sympathetic ganglia . It seems likely that stimulation of sympathetic nerve fibers in the pathologically degenerative disc and surrounding tissues produce sympathetic excitation, and induce a sympathetic reflex to cause vertebrobasilar insufficiency and hypertension . Improvement of neurological function, resolution of vertigo, and recovery of blood pressure following surgery could be attributed to the excision of degenerative and herniated cervical disc, the decompression of the dura mater and posterior longitudinal ligament, and the stabilization of degenerated segment . Chronic neck pain is one of the major symptoms in patients with cervical spondylosis, whereas some studies have indicated that resting blood pressure levels may be elevated in patients with persistent pain . In healthy individuals, these interactions are believed to reflect a homeostatic feedback loop that helps restore arousal levels in the presence of acutely painful stimuli . A role for baroreceptors in this feedback loop appears likely, with pain triggering sympathetically driven blood pressure increases, resulting in increased stimulation of baroreceptors, which in turn activates descending pain inhibitory pathways . Previous studies suggested that blood pressure / pain regulatory relationship may be substantially altered in chronic pain conditions . A retrospective study by bruehl et al suggested that chronic pain could be associated with increased risk of hypertension . Their study found that over 39% of patients with chronic pain were diagnosed with clinical hypertension, compared with only 21% of pain - free internal medicine patients . They thought that chronic pain - related impairments in overlapping systems modulating both pain and blood pressure would result in a higher prevalence of clinically diagnosed hypertension in patients with chronic pain than in comparable pain - free patients . Our study indicates a possible relation between chronic neck pain and hypertension . If chronic neck pain could lead to hypertension through sympathetic arousal and failure of normal homeostatic pain regulatory mechanisms, early treatment for resolution of chronic neck pain may have a beneficial impact on cardiovascular disease risk in patients with cervical spondylosis we report here 2 patients of cervical spondylosis with concomitant cervical vertigo and hypertension that were treated successfully with anterior cervical discectomy and fusion . Stimulation of sympathetic nerve fibers in pathologically degenerative disc could produce sympathetic excitation, and induce a sympathetic reflex to cause cervical vertigo and hypertension . In addition, chronic neck pain could contribute to hypertension development through sympathetic arousal and failure of normal homeostatic pain regulatory mechanisms . Early treatment for resolution of symptoms of cervical spondylosis may have a beneficial impact on cardiovascular disease risk in patients with cervical spondylosis.
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Although herpes simplex virus (hsv) infections are very common worldwide, herpes simplex encephalitis (hse) is a rare disease with an incidence of one case per 250,000500,000 individuals per year . Approximately 50% of patients with hse are older than 50 years.1 cytomegalovirus (cmv), another member of the herpesvirus family, is the most common cause of congenital infection, with a prevalence rate of 0.2%2.5% in all live newborns.2,3 in adults, most reported cmv infections are seen in immunocompromised patients such as those with hiv (human immunodeficiency virus)4,5 and after chemoradiotherapy.6 in these cases, the patients were also co - infected with hsv . Here, we present a patient with coinfection of cmv and hsv type ii (hsv - ii), who was admitted to the hospital for acute psychotic symptoms, but never treated with immunosuppressants before this admission . For acute psychotic symptoms of delirium, delusion of persecution, fidget, over - alertness, and aggressive behaviors, a 67-year - old chinese man was sent to the outpatient department of the shantou central hospital, guangdong province, people s republic of china, on october 15, 2013 . On october 14, he became suspicious, had delusion of persecution, and ran around . On the same day, he was sent to the local hospital where a magnetic resonance imaging was performed showing multiple hyperintensities in the left occipital lobe and temporal lobes in the t2-weighted images (figure 1a). In addition, multiple hyperintensity lesions were seen in the temporal and parietal lobes and demyelination lesions in the white matter surrounding the lateral ventricles in the fluid - attenuated inversion recovery images (figure 1b). Then, the patient was referred to the shantou central hospital, affiliated shantou hospital of sun yat - sen university on october 15 . On examination, but, physical examinations showed no abnormalities (normal blood pressure and there was no fever). The positive and negative syndrome scale score was 109 (positive syndrome score of 36, negative of 14, and general of 59). The patient had no history of head trauma and hospitalization for mental disorders or any tumors, according to the family members . Emergency treatment was started with the administration of the tranquilizer sodium phenobarbital (100 mg) to reduce dysphoria and aggressive behaviors, the mood stabilizer sodium valproate (100 mg, administered in two fractions) to stabilize the mental state of the patient, and the antipsychotic olanzapine (10 mg, administered in four fractions) for the delusion of persecution . In addition, along with supportive treatments (piracetam 20 grams in 100 ml 0.9% sodium chloride), empirical administration of antiviral treatment (ganciclovir 250 mg in 0.9% sodium chloride) was instituted under suspicion of viral encephalitis . On october 16, a blood examination was performed which showed no anemia and leukocytosis, but presented a higher percentage of neutrophil number (80.60%; reference range 40%75%), a lower percentage of lymphocytes (11.70%; reference range 20%50%), very high levels of creatine kinase (ck; 619 u / l, reference range 0174 u / l) and its isoenzyme (ckmb; 39 u / l, reference range 024 u / l). In addition, a higher level of c - reactive protein (crp, 113 mg / l; 08 chest radiographs (october 17) showed stale tuberculous lesions in the upper lobe of right lung . On october 18, while the psychotic symptoms were improved, rough breath sounds were heard in the lungs, suggesting the presence of an infection there . Therefore, the antibiotic treatment with piperacillin - tazobactam (4,500 mg in 0.9% sodium chloride) was initiated while the other treatments continued . On the same day, an electroencephalogram was performed showing slow waves characterized by strong peaks of delta and theta frequencies in the frontal lobe of the right hemisphere (figure 2). On october 19, a susceptibility - weighted imaging was performed, which more clearly showed the stale hemorrhages in the frontal lobe of the right hemisphere (figure 1c), in addition to those seen in the left occipital lobe and temporal lobes of both sides . The outcomes of cerebrospinal fluid (csf) examination came out on october 20, 21, and 22, respectively, showing blood cells in normal ranges, mild lower levels of chloride (117 mmol / l; reference range 120132 mmol / l), and glucose (2.41 mmol / l; reference range 2.84.5 mmol / l). No bacteria were found . But both anti - cmv igg and anti - hsv - ii igm were positive, supporting a diagnosis of coinfection with hsv - ii and cmv in the brain . On october 28, as required by the family members, the patient was discharged . His psychotic symptoms were significantly improved as evidenced by the reduction of positive and negative syndrome scale from 109 to 71 (positive syndrome score of 14, negative of 13, and general of 44). The patient continued the antiviral and antipsychotic treatments at home after the discharge from the hospital . He died of progressive cachexia 4 months after the discharge (february 26, 2014), although his psychotic symptoms did not relapse . The ethics committee of the shantou central hospital did not require approval for this case study . Except for one early case report,7 all other double infections of the central nervous system (cns) with cmv and hsv were reported in immunocompromised patients.46,8 the patient reported here had no history of being treated with immunosuppressants . His blood examination showed normal cell count despite of the presence of anti - cmv igg and anti - hsv - ii igm in csf, which is in accordance with the reported evidence for increased production of antibodies against several neurotropic infectious pathogens including hsv - i, hsv - ii, and cmv, in the csf of individuals with bipolar disorder9 or autism spectrum disorders.10,11 the mild signs of cns inflammation in this patient may account for the absence of seizures and focal neurological signs although he presented primarily acute psychotic symptoms and mental status changes . In line with this patient with hsv - ii infection, neurological sequelae were more frequent among patients with hsv - i compared with those infected with hsv - ii.12 in addition, cmv encephalitis in immunocompetent individuals has been associated with mild signs of cns inflammation, including absent to moderate pleocytosis.1315 in contrast, cmv in immunocompromised patients was featured with more severe clinical manifestations.5,6,16 the pathological hallmark of hse is hemorrhagic necrosis in the medial part of the temporal lobes . In addition, adjacent areas such as the orbital surface of the frontal lobe and cingulate gyrus may be involved . In line with these, magnetic resonance imaging of this patient showed hemorrhages in multiple regions of the brain, including the right frontal lobe, left occipital lobe, and the temporal lobes of both sides . The hemorrhage in the right frontal lobe may be related to the peaks of delta and theta frequencies seen in electroencephalogram of this brain region . In addition, this patient showed multiple small infarctions and demyelination in the white matter surrounding the lateral ventricles . The multiple small infarctions may be the consequence of cmv infection . In line with this speculation, animal, and human studies1720 have shown the cmv - induced damage of microvascular endothelium, vasculitis, thrombotic occlusions, and hemorrhages . Another feature of this patient is a very high level of crp in his blood . The co - existence of high crp level and the antibodies to cmv and hsv - ii reminded us of a recent human study, in which the crp level was strongly associated with viral detection rate and mixed viral / bacterial detection rate; whereas the rate of bacterial detection was not associated with the crp level.21 more significantly, plasma crp was an independent predictor of mortality in patients with chronic obstructive pulmonary disease caused by virus infections.21,22 in summary, we present a case of a 67-year - old male with coinfection of cmv and hsv - ii and without history of being treated with immunosuppressants . The presence of his psychosis symptoms and absence of neurological manifestations may represent the clinical features of coinfection of cmv and hsv - ii.
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The estimated incidence of bnc after open retropubic radical prostatectomy (rrp) ranges from 3% to 26% [1 - 8]. Robot - assisted laparoscopic radical prostatectomy (ralp) is becoming a more frequently performed procedure because it clearly has a lower rate of postoperative complications than rrp . In particular, the incidence of bnc after ralp ranges from 0.6% to 3% [9 - 12]. Several case reports have suggested that rrp, pure laparoscopic radical prostatectomy (lrp), and ralp are associated with complications related to the use of surgical clips, including bnc and the formation of bladder stones [13 - 19]. The purpose of this study was to evaluate the surgical clip - related complications that occurred after rrp, lrp, and ralp in our institution . In our institution, rrp was the method of choice for radical prostatectomy until august 2005, when the first case of lrp was performed . Thereafter, lrp was frequently performed between august 2005 and april 2008 . From may 2008 we retrospectively reviewed a database that has been maintained in our institution of the clinical, surgical, and pathological parameters of these procedures . We reviewed a total of 641 cases who underwent radical prostatectomy at our institution between january 2006 and april 2009 . Of the 641 patients, 439, 49, and 153 underwent rrp, lrp, and ralp, respectively . In all cases, the median follow - up time for the entire cohort was 19.0 months (range, 1 - 42 months). Of the rrp, lrp, and ralp patients, 25 (5.7%), 1 (2.0%), and 2 (1.3%) had bnc, respectively, and 2 of the rrp patients developed a bladder stone . In total, 6 patients with complications related to the use of surgical clips were identified in the cohort . Cystoscopic examination of patient 2 indicated bnc and the presence of a metal clip protruding into the urethra through the urethrovesical anastomosis at 4 o'clock (fig . Patients 3 and 4 were both found to have a hem - o - lok clip in the bladder neck (fig . Their bncs were resolved after a single urethral dilatation, after which the symptoms disappeared . Patients 5 and 6 were found to have a bladder stone that had formed around the metal clip in the bladder (fig . Retrospective re - evaluation of the kidney, ureter and bladder x - ray (kub) revealed the metal clip in the bladder region (patient 5: fig . 4). During the follow - up, neither patient exhibited signs of bladder stone recurrence . In all patients, one study based on a patient self - reported questionnaire, which revealed that 25.9% of rrp patients reported at least one episode of bnc that required treatment, was unable to identify factors that could predict the occurrence of bnc . In contrast, surya et al reported that many factors promote the occurrence of bnc after rrp, including urinary extravasation, excessive blood loss, and previous bladder neck surgery . Similarly, thiel et al found that 17.5% of rrp patients required stricture dilatation by a mean time of 6 months after surgery and revealed that increased age and increased blood loss were statistically associated with stricture formation . Recently, erickson et al suggested that improved surgical technique and increased surgeon experience appear to be the most important factors that reduce the incidence of bnc . Huang and lepor added a history of previous transurethral resection of the prostate, pelvic external beam radiotherapy, and a hypertrophic healing response to the risk factors for bnc after rrp . Another study found that of the 11.1% of patients who developed bnc after rrp, the biggest risk factor was current smoking (26%), with comorbidities such as hypertension, coronary artery disease, and diabetes also being significantly associated with the formation of bnc . The investigators hypothesized that local ischemia of the urethra and bladder neck caused by microvascular disease may lead to poor anastomotic healing and scar formation . Similarly, the incidence of bnc in our cohort of 153 consecutive ralp patients was 1.3% . It is not clear why the rate of bnc after ralp is significantly lower than that quoted in the rrp literature, but msezane et al suggested that it may be due to the running anastomosis, better visualization, improved instrument maneuverability, and decreased blood loss . Moreover, a comprehensive review of the literature comparing rrp, lrp, and ralp revealed that higher estimated blood loss was a significant factor in the development of bnc . Recently, a study comparing rrp and ralp suggested that the predisposing causes of bnc are likely to be the combination of a fixed circular bladder stomatization with the subsequent healing of the stoma anastomosis to the urethra . The present study shows that bnc may also arise as a result of clip migration . This has also been observed in the literature (table 2), although it should be noted that most reported complications with surgical clips relate to hemorrhage . Long et al reported the first case of metal clip migration - induced bnc after rrp in a patient who had suffered multiple episodes of urinary retention and had undergone several failed urethrotomies . Later, blumenthal et al reported the first case report of hem - o - lok migration into the vesicourethral anastomosis after ralp . Those authors believe that the relationship between surgical clip migration into the anastomosis and bnc formation is not coincidental; they postulated that the physical disruption of the anastomosis by the surgical clip contributed to poor healing and may have elicited an inflammatory reaction . Tunnard et al then described a case of a hem - o - lok clip - related complication following lrp: during bladder neck dilatation for a tight bladder neck, a hem - o - lok was found in the bladder, having migrated from the urethrovesical anastomosis . It was removed successfully but a repeat cystoscopy 3 months later revealed another hem - o - lok device that had eroded through the vesicourethral anastomosis . This was removed successfully with the aid of a holmium laser . In the present study, stones developed around the surgical clip in 2 patients after rrp . Other studies have also reported surgical clip - related complications other than bnc (table 2). These include the case described by palou et al of severe perineal pain after rrp due to the protrusion of a metal clip into the urethra at the urethrovesical anastomosis, and the case of banks et al, in which the migration of a hem - o - lok clip into the bladder led to stone formation after lrp . There was also a recent report by kadekawa et al of a case in which a metal clip had migrated into the urinary bladder after rrp . It was hypothesized that inflammation had arisen around the urinary bladder or vesicourethral anastomosis and that this also involved the metal clip, which then eroded the bladder wall and eventually migrated into the bladder . In addition, mora et al reported a case of intravesical migration and subsequent calculus formation with the spontaneous expulsion of a hem - o - lok clip after lrp . These findings suggest that foreign bodies in the bladder can act as a nidus for bladder stone formation owing to the presence of persistent chronic inflammation . The mechanism underlying the migration of a surgical clip into the urinary tract is unclear . In our series, the rate of bnc after ralp was significantly lower than that after rrp . Because improvements in outcomes are related to temporal improvements in particular procedures, this trend might account for some improvement in outcome in the ralp group . It appears that surgical clips are prone to migration and may cause, or significantly contribute to, bnc or the formation of bladder stones after radical prostatectomy . These findings raise questions regarding the use of foreign bodies in close proximity to the vesicourethral anastomosis during radical prostatectomy . At the very least, they indicate that care must be taken with the surgical clips used for inducing hemostasis near the apex of the prostate in radical prostatectomy . In addition, because the incidence of bnc after ralp is low, when unexplained voiding difficulty occurs after ralp, one should suspect that a hem - o - lok clip has migrated, especially because hem - o - lok clips cannot be detected on x - rays.
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Organizing pneumonia (op) is characterized by an indolent clinical course and a favorable prognosis although a fulminant variant has been documented . Pulmonary air leak comprising pneumothorax, pneumomediastinum, and subcutaneous emphysema is rarely a presenting feature of an op . The exact pathogenesis and an appropriate management guideline have not yet been established for this dreaded complication . Review of medical literature reveals only a few case reports of air leak in op . Here, we report a case of op secondary to bleomycin chemotherapy, where pulmonary air leak was the major presenting symptom, which has never been reported previously . A 43-year - old female presented to our emergency department with severe shortness of breath, diffuse neck swelling, and neck pain, of 1 day duration . She had a stage iii ovarian immature teratoma diagnosed 6 months back and was treated with bilateral salpingo oophorectomy followed by 6 cycles of chemotherapy with bleomycin, etoposide, and cisplatin . One week prior to the admission, she had fever and severe cough which was treated from a local hospital . On examination, she had respiratory distress with a respiratory rate of 32/min . Her spo2 was 82% in room air, blood pressure 110/70 mmhg, and heart rate 110/min . Examination of the respiratory system showed equal breath sounds on both sides with bilateral fine basal crepitations and muffled heart sounds . The values for the blood gas analysis were a ph of 7.42, pco2 of 34 mmhg, and po2 of 65 mmhg . Chest x - ray showed [figure 1] features suggestive of subcutaneous emphysema and mediastinal emphysema with bilateral haziness and alveolar opacities in lower zones . A computed tomography (ct) thorax [figure 2] with findings of ground - glass opacities and peripheral consolidation, confirmed the diagnosis of pneumonia and mediastinal emphysema . Mediastinal emphysema and diffuse haziness in chest x - ray (a - c) serial computed tomography images showing bilateral diffuse ground - glass opacities, mediastinal emphysema, and peripheral consolidation with this clinical presentation of a pulmonary air leak syndrome and radiologic picture of bilateral predominant ground glass opacities with patchy consolidations, we considered the possibility of pneumocystis jiroveci pneumonia or a drug - induced lung disease like diffuse alveolar damage / op . Since the patient had severe respiratory distress and hypoxemia, she was immediately taken up for surgical management . A video - assisted thoracoscopy was done [figure 3], and the mediastinum dissected revealing multiple paracardiac blebs . These blebs were ablated; the pneumomediastinum drained into left hemithorax, and an intercostal drainage tube was inserted in left hemithorax . Video - assisted thoracoscopy showing paracardiac blebs patient's condition improved dramatically with rising oxygen saturation . She was shifted to the intensive care unit and was treated with antibiotics, steroids, and supportive treatment . Biopsy of the lung specimen [figure 4] revealed bronchiolitis obliterans organizing pneumonia (boop). Soon the air - leak disappeared, and she was discharged from the hospital with a long - term course of steroids and macrolide . (a) histology showed plugs of fibrous tissue in terminal bronchioles, (b) trichrome stain showing fibrous proliferation op is a clinicopathologic syndrome first described by davison and epler et al . In the 1980s . Causes of secondary op include infections, drugs, connective tissue diseases, organ transplantation, and inflammatory bowel diseases . The manifestations of op are nonspecific; hence, the diagnosis is delayed in many . High index of suspicion should be kept to diagnose secondary op, especially in patients with chemotherapy, organ transplantation, etc . A unique manifestation of bleomycin toxicity is multiple pulmonary nodules in ct mimicking metastasis and having histologic characteristic of op / boop . A study conducted by mokhtari et al . Found that of the 43 patients with isolated boop in cancer patients, 3 were found to be due to bleomycin . Kofteridis et al ., iwanaga et al ., and yang et al . Have published case reports of air leak syndrome in boop . The hallmark of op is the presence of intraalveolar buds of granulation tissue consisting of fibroblasts - myofibroblasts embedded in connective tissue . These buds may extend from one alveolus to the next through the interalveolar pores and into the bronchioles obstructing the lumen (bronchiolitis obliterans). The pathogenesis proposed for air leak is that, localized plugs of fibrous tissue in the bronchiole lumen in op, produce a ball valve mechanism and alveolar over - distension, eventually leading to rupture . The air dissects through the bronchovascular sheath and ascends toward the hilum to produce a pneumomediastinum and also along the subcutaneous connective tissue to cause subcutaneous emphysema . Air leakage in the mediastinum may also occur due to the rupture of paracardiac bleb . In severe interstitial lung disease (ild), we also considered the possibility of pneumocystis jiroveci pneumonia and metastasis as a differential in this immunocompromised patient with fever, cough, radiologically bilateral ground glassing, and air leak, but investigation profile proved against this . In our patient, the treatment of op is long - term steroid, and the treatment for massive pneumomediastinum is limited mediastinotomy and drainage . Op should be included in our differential diagnosis list, whenever a patient presents with air leak . Lung biopsy should be done to confirm the diagnosis and would prove to be lifesaving as this disease responds well to steroids . Limited mediastinotomy and drainage of pneumomediastinum in massive air leak is lifesaving and gives rapid improvement.
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Over the past few decades continuous progress has been made in the development of insulin therapy . The need for basal insulin was felt around 60 years ago, and so longer - acting insulins like lectin and neutral protamine hagedorn (nph) were developed . Studies have also shown that missing two basal insulin injections per week can lead to a 0.20.3% increase in glycated hemoglobin (hba1c). At the same time, existing basal insulins were found to have limitations . In order to counter these limitations, insulin analogues were developed . There are three kinds of insulin analogues: rapid - acting insulin analogues, biphasic insulin analogues, and basal insulin analogs . The primary structure of insulin is based on a specific sequence of amino acids in the protein . The same structure slight changes were made in this insulin structure to produce different analogs with desired pharmacokinetic properties . The need of insulin analogues arises from the fact that the human insulin injections have a lag period of around 1/2 h between administration and onset of action . As a result, endogenous insulin, after secretion from the pancreas, enters the portal circulation, after which it reaches the systemic circulation . On the other hand, exogenous human insulin, after injection into sub - cutaneous tissue, enters the systemic circulation, and then about 10% of the originally administered insulin reaches the portal circulation . Thus, the portal and systemic gradient of insulin is inverted in the case of exogenous insulin . It is because of this reason that exogenous human insulin is not the best physiological way of using insulin therapy . Rapid - acting insulin analogs overcome this by being more in tune with the physiological rise and fall of glucose values after each meal . The basal human insulin that is available is nph, which lacks the desired 24 h effect . This limitation highlights the need for the development of basal insulin analogues including insulin detemir, insulin glargine and insulin degludec with duration of action of at least a day . In addition, a research study has established that insulin detemir has better glycaemic control with fewer hypoglycaemic epsiodes in children when compared to nph insulin . Longer duration of action, a flat time - action profile, low day - to - day glycemic variability, and the potential for flexible dosing . Hypoglycemia in children and adults is still a concern, but with the advent of better devices, it is easier to detect and manage hypoglycemia . The physicians thus aim to achieve tighter glucose control, as a result of which the hba1c targets are improved . Insulindetemir, for instance, produce better hba1c control with less hypoglycemia than nph insulin . Research studies have shown that in children / adolescents, an intramuscular instead of subcutaneous injection of insulin therapy leads to faster absorption and action, and also shorter duration of action . It has also been found that the rigidity of the insulin regimen interferes with the patient's lifestyle (diet / exercise), which is an integral part of any child or adolescent's life . All the basal insulin analogues have a similar onset of action (between 1 and 2 h) but the duration of action varies (glargine: 20 - 30 h, detemir: 24 h and degludec: 42 h). Both detemir and degludec have a 50 amino acid chain while glargine has a 53 amino acid chain . In glargine, there is the addition of two, and substitution of one amino acid . In detemir, there is a lack of b30 amino acid and an addition of acylated fatty acid side chain at b29 position . In degludec, there is a lack of b30; addition of glutamic acid spacer and diacylated fatty acid side chain at b29 . The mechanism of prolongation of action of glargine involves precipitation at acidic ph, while the mechanism of prolongation of action of detemir and degludec involves binding to albumin and multi - hexamer formation respectively . Both glargine and degludec have been classified as pregnancy category c molecules while detemir is classified as pregnancy category b. glargine is a pro - drug that requires conversion from its monomeric soluble form to a microcrystalline precipitate in the subcutaneous tissue to obtain its long - acting pharmacokinetics . This process may be disturbed by its rapid access to the circulation after intramuscular injection, leading to hypoglycemia . A study has shown that accidental intramuscular injection of glargine can happen in 30% of children with type 1 diabetes mellitus (t1 dm). The definition of hypoglycemia is debatable . According to the 2009 and 2014 ispad guidelines, <3.6 mmol / l (65 mg / dl) has been used most often for clinical definition, and (70 mg / dl) is the recommended lower target for blood glucose, whereas the ada working group suggests 70 mg / dl as hypoglycemia . The who guidelines for neonatal hypoglycemia suggest that the newborn / infant / child with signs of illness would be diagnosed with hypoglycemia if blood glucose is <2.5 mmol / l or 45 mg / dl; for healthy term / pre - term newborn feeding well, the hypoglycemia cut - off value is <1.1 mmol / l or 19.8 mg / dl, and for an infant / child with severe malnutrition the value is <3.0 mmol / l or 54 mg / dl . Insulin degludec is neutral, soluble ultra - long - acting insulin that forms large multi - hexamer assemblies at physiological ph following subcutaneous injection . Its molecular structure is similar to the human insulin amino acid sequence, apart from deletion of threonine at position b30 and the addition of a 16-carbon fatty acid chain attached to lysine at position b29 via a glutamic acid spacer . These subsequently release monomers that are absorbed at a slow and steady rate into the circulation . Pharmacokinetic / pharmacodynamic studies show that insulin degludec has a very long duration of action of 42 h, with a half - life exceeding 25 h, and variability of 25% with respect to glargine . A study was recently conducted which investigated once - daily insulin degludec versus insulin detemir, both in combination with bolus insulin aspart in a 26 week trial, followed by another 26 week extension, in children and adolescents with t1 dm . This trial (n = 350) was the first to look into the long - term safety of insulin degludec in children and adolescents (age: 118 years). The results showed that at 26 weeks, insulin degludec in combination with insulin aspart was noninferior to insulin detemir in combination with insulin aspart . The mean basal insulin doses at the end of treatment were reduced by 31% in the insulin degludec group . In the 26-week extension, a lower insulin dose and a significantly greater reduction in fasting plasma glucose versus insulin detemir both regimens had similar rates of overall and nocturnal hypoglycemia, the rate of severe hypoglycemia was numerically higher with insulin degludec plus insulin aspart . Of note, patients on insulin degludec had significantly lower rates (59% less) of hyperglycemia with ketosis . Insulin degludec, however, is currently not approved in patients below the age of 18 years . New formulations of basal insulin that are under development are u300 glargine, which has completed phase iii and pegylated insulin lispro, which is in phase iii, insulin degludec / insulin aspart (idegasp), which is a soluble co - formulation of insulin degludec and insulin aspart, and ideglira, which is a fixed - ratio combination of insulin degludec and liraglutide for t2 dm.
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Induction of anesthesia of the donor mouse is initiated with 5% isoflurane . The mouse is orotracheally intubated with a 20-gauge intravenous catheter and then placed on a rodent ventilator, using 100% oxygen at a rate of 125 breaths / minute and approximately 0.5 ml tidal volume (2% of its body weight). Heparin at 100 u / kg is injected into the inferior vena cava (ivc), from just below the liver . The diaphragm is cut along the ventral costal attachment toward the spine, and the thoracic cavity is exposed by cutting both sides of the chest wall to the neck . After incising the ivc at the level of diaphragm, the right atrial appendage is cut and the lungs flushed with 2 ml of cooled (4 c) lactated ringer's injection and 0.1 ml of heparin via a transverse incision at the root of the pulmonary artery (pa) trunk . Arresting ventilation at two thirds of end - tidal inflation, the heart - lung block is excised and stored on ice (4 c). Identify the pa, located at the most cranial aspect of the hilum, as well as the attached main bronchus (br). Carefully dissect the pa from the bronchus . The cuff for the bronchus is derived from a 20-gauge iv catheter, and cut in length of 1.0 mm with an extension of 0.7 mm . Using the same materials, the pv cuff varies with the weight of the donor mouse . Specifically, for mice 24 - 27 grams the cuff size is 22-gauge, 0.7 mm in length with a 0.7 mm extension . For mice weighing 27 - 32 grams, the studies utilized a 20-gauge catheter that is 0.7 mm in length with a 0.7 mm extension . The cuffs are inserted into the distal ends of the pa, pv and br and secured with a 9 - 0 suture . The donor lung is flushed and washed with sterile saline with sterile heparin before storage . The donor lung is then wrapped with sterile gauze soaked in sterile saline on ice (4 c), which keeps the lung very clean and sterile . A microvessel clamp is placed on the bronchus to prevent lactated ringer entry into the airway . Induction of anesthesia and mechanical ventilation are the same as described for the donor above . The left chest wall is shaved and prepped with 70% alcohol and the surgical field is draped . A thoracotomy incision is made in the left third intercostal space, extending the incision dorsally close to the spine, and a microvessel clamp placed on the left pulmonary vessels and bronchus adjacent to the heart . You can see pa at the cranial aspect, pv at the caudal end of hilum and br between them . Using gentle traction on the hemostat to cause mild tension on the pa, br and pv, the left lung is pulled out from the thoracic cavity while leaving the central hilar structures clamped . The pa, pv and br are isolated by blunt dissection followed by placing a 9 - 0 suture positioned loosely around the pa, pv and br . After dissecting the pa completely from its adventitial sheath, a small transverse incision of approximately one fourth of the vessel's circumference is made into the anterior wall, leaving the continuation of the dorsal part of the artery intact . The donor lung, wrapped in cold, lactated ringer - soaked cotton gauze and prepared as described above, is then positioned into the thoracic cavity, and cuffs inserted into the recipient pa, pv and br and secured with 9 - 0 suture . After positioning the transplanted lung back into the recipient thorax, the thoracotomy incision is closed using a 5 - 0 suture . Buprenorphine (0.05 - 0.15 mg / kg) is administered immediately after surgery, and for every 8 hours for 2 - 3 days post - surgery . Our experience has taught us that it requires several months of repeated practice to become proficient in the mouse lung transplant model . After proficiency is attained, we achieved a 96% (96/100 consecutive surgeries) perioperative survival rate with deaths occurring within seven days post operatively . Two deaths were due to bleeding that began intraoperatively, and pneumothraces were the cause of death in the other two mice . For all procedures, the warm ischemia time was 14.32 3.14 minutes, and cold ischemia time was 58.51 18.06 minutes . Three orthotopic lung transplant groups were studied: isograft: c57bl/6c57bl/6, allograft: c57bl/10c57bl/6 and c57bl/6c57bl/10 . We used only male mice but our technique can be also applied to female mice, because there is no significant anatomical difference between the sexes . Grading of rejection pathology was conducted in a blinded fashion utilizing standard criteria for clinical lung transplantation(table 1). Whereas we observed mild or no rejection in isograft (c57bl/6c57bl/6), both allograft combinations developed comparable acute or chronic rejection (figure 1). In contrast, ob was significantly more frequent in the c57bl/10c57bl/6 than c57bl/6c57bl/10 group by day28 (table 1). Scoring of acute rejection (" a " scores) by standard criteria as described in representative results . Data represents the mean sd of " a " scores at days 28 post transplantation . Data represents the quantity and percentage of mice in each group that developed ob at days 21 and 28 post transplantation . Panel 1b and 1c represents h&e and masson's trichrome stained bl/10 lung allograft transplanted into bl/6 mouse recipient, which developed ob and non ob, respectively . Main trouble shooting during the procedure were as follows . Blood flow disorder: flush donor lung until the color becomes white in color . Difficulty of cuff insertion into the donor: ascertain that vasculature is of sufficient length and free of attached fat and connective tissues . Bleeding at anastomotic site: use q tips to apply pressure at affected site for approximately 5 minutes . Recipient pa blood flow obstruction: mainly caused by torsion of the during cuff insertion . If air is observed within the pa then re - do of the anastomosis is necessary . Recipient pv blood flow obstruction: make sure the position and direction of the cuff . Release the compression from the bronchial cuff . Pneumothorax: this may be caused by either bronchial perforation due to manipulation of the airway, or perforation of lung surface due to trauma associated with the transplant procedure . The former is treated with surgical closure of the hole within the airway wall; and the latter is treated by oversewing the leak on the lung surface using 10 - 0 nylon suture . Induction of anesthesia of the donor mouse is initiated with 5% isoflurane . The mouse is orotracheally intubated with a 20-gauge intravenous catheter and then placed on a rodent ventilator, using 100% oxygen at a rate of 125 breaths / minute and approximately 0.5 ml tidal volume (2% of its body weight). Heparin at 100 u / kg is injected into the inferior vena cava (ivc), from just below the liver . The diaphragm is cut along the ventral costal attachment toward the spine, and the thoracic cavity is exposed by cutting both sides of the chest wall to the neck . After incising the ivc at the level of diaphragm, the right atrial appendage is cut and the lungs flushed with 2 ml of cooled (4 c) lactated ringer's injection and 0.1 ml of heparin via a transverse incision at the root of the pulmonary artery (pa) trunk . Arresting ventilation at two thirds of end - tidal inflation, the heart - lung block is excised and stored on ice (4 c). Identify the pa, located at the most cranial aspect of the hilum, as well as the attached main bronchus (br). Carefully dissect the pa from the bronchus . The cuff for the bronchus is derived from a 20-gauge iv catheter, and cut in length of 1.0 mm with an extension of 0.7 mm . Using the same materials, the pv cuff varies with the weight of the donor mouse . Specifically, for mice 24 - 27 grams the cuff size is 22-gauge, 0.7 mm in length with a 0.7 mm extension . For mice weighing 27 - 32 grams, the studies utilized a 20-gauge catheter that is 0.7 mm in length with a 0.7 mm extension . The cuffs are inserted into the distal ends of the pa, pv and br and secured with a 9 - 0 suture . The donor lung is flushed and washed with sterile saline with sterile heparin before storage . The donor lung is then wrapped with sterile gauze soaked in sterile saline on ice (4 c), which keeps the lung very clean and sterile . A microvessel clamp is placed on the bronchus to prevent lactated ringer entry into the airway . Induction of anesthesia and mechanical ventilation are the same as described for the donor above . The left chest wall is shaved and prepped with 70% alcohol and the surgical field is draped . A thoracotomy incision is made in the left third intercostal space, extending the incision dorsally close to the spine, and a microvessel clamp placed on the left pulmonary vessels and bronchus adjacent to the heart . You can see pa at the cranial aspect, pv at the caudal end of hilum and br between them . Using gentle traction on the hemostat to cause mild tension on the pa, br and pv, the left lung is pulled out from the thoracic cavity while leaving the central hilar structures clamped . The pa, pv and br are isolated by blunt dissection followed by placing a 9 - 0 suture positioned loosely around the pa, pv and br . After dissecting the pa completely from its adventitial sheath, a small transverse incision of approximately one fourth of the vessel's circumference is made into the anterior wall, leaving the continuation of the dorsal part of the artery intact . The donor lung, wrapped in cold, lactated ringer - soaked cotton gauze and prepared as described above, is then positioned into the thoracic cavity, and cuffs inserted into the recipient pa, pv and br and secured with 9 - 0 suture . After positioning the transplanted lung back into the recipient thorax, the thoracotomy incision is closed using a 5 - 0 suture . Buprenorphine (0.05 - 0.15 mg / kg) is administered immediately after surgery, and for every 8 hours for 2 - 3 days post - surgery . Our experience has taught us that it requires several months of repeated practice to become proficient in the mouse lung transplant model . After proficiency is attained, we achieved a 96% (96/100 consecutive surgeries) perioperative survival rate with deaths occurring within seven days post operatively . Two deaths were due to bleeding that began intraoperatively, and pneumothraces were the cause of death in the other two mice . For all procedures, the warm ischemia time was 14.32 3.14 minutes, and cold ischemia time was 58.51 18.06 minutes . Three orthotopic lung transplant groups were studied: isograft: c57bl/6c57bl/6, allograft: c57bl/10c57bl/6 and c57bl/6c57bl/10 . We used only male mice but our technique can be also applied to female mice, because there is no significant anatomical difference between the sexes . Grading of rejection pathology was conducted in a blinded fashion utilizing standard criteria for clinical lung transplantation(table 1). Whereas we observed mild or no rejection in isograft (c57bl/6c57bl/6), both allograft combinations developed comparable acute or chronic rejection (figure 1). In contrast, ob was significantly more frequent in the c57bl/10c57bl/6 than c57bl/6c57bl/10 group by day28 (table 1). Scoring of acute rejection (" a " scores) by standard criteria as described in representative results . Data represents the mean sd of " a " scores at days 28 post transplantation . Data represents the quantity and percentage of mice in each group that developed ob at days 21 and 28 post transplantation . Panel 1b and 1c represents h&e and masson's trichrome stained bl/10 lung allograft transplanted into bl/6 mouse recipient, which developed ob and non ob, respectively . Main trouble shooting during the procedure were as follows . Blood flow disorder: flush donor lung until the color becomes white in color . Difficulty of cuff insertion into the donor: ascertain that vasculature is of sufficient length and free of attached fat and connective tissues . Bleeding at anastomotic site: use q tips to apply pressure at affected site for approximately 5 minutes . Recipient pa blood flow obstruction: mainly caused by torsion of the during cuff insertion . If air is observed within the pa then re - do of the anastomosis is necessary . Recipient pv blood flow obstruction: make sure the position and direction of the cuff . Pneumothorax: this may be caused by either bronchial perforation due to manipulation of the airway, or perforation of lung surface due to trauma associated with the transplant procedure . The former is treated with surgical closure of the hole within the airway wall; and the latter is treated by oversewing the leak on the lung surface using 10 - 0 nylon suture . Orthotopic lung transplantation in mice is challenging due to microsurgical demands and extreme fragility of tissues . Introduction of the cuff technique has allowed for the widespread use of the orthotopic lung transplantation in rats . This became the basis for the development of the orthotopic lung transplant model in mice in our lab . In mice and rats, unlike humans, the left lung contains only one lobe and makes up only 25% of the total lung mass . This makes left - single lung transplantation feasible in the murine model without the need for a circulatory support system . Our preliminary surgeries revealed that ventilation and perfusion of the transplanted lung was highly dependent on the size of pv anastomosis . Inappropriate cuff sizing resulted in either atelectasis of the transplanted lung or dehiscence of the pv anastomosis . Okazaki et al reported the use of combined vessel ligation and clipping the bronchus . In the current study we use an aneurysm clip for all hilar structures, which we suggest may shorten warm ischemic time . One limitation of the orthotopic left lung transplant in the murine model is that recipient animals can survive after alloimmune - mediated necrosis of their allografts . Therefore, survival studies are not feasible and graft assessment depends on histological examination of the transplanted lung . Using this technique this suggests the role of minor, and not major, histocompatibility antigens in obliterative bronchiolitis pathogenesis . This model represents a novel research tool for the examination of lung transplantation and the advancement of clinical lung transplants.
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Nurses are among multidisciplinary team members who play a significant role in health care and are involved in pharmacological management of the patients conditions . They should have enough knowledge and skills regarding pharmacological and medication management, in order to establish a safe and high quality care (1). Nursing education in iran is a four year graduate model . In the first three years, students are taught in the nursing schools and also pass some clinical apprenticeship courses in hospitals under direct supervision of clinical instructors . Due to the load of teaching duties of educators, a large part of clinical courses are supervised by trainers or mentors other than those who taught students in nursing schools . The fourth year of nursing education is held in the form of internship in which nursing students take direct role in implementing the patients caring plans with little or no supervision by an instructor or a mentor . It is the nurses and nursing students responsibility to evaluate every patient before prescribing medications, administer the medication safely, monitor the patients for side effects, decide if a dose reduction is required for a patient (i. e, with renal failure) and to evaluate the efficiency of the prescribed medication . It is also essential to explain to the patients about their medicines and help the physician and pharmacist solve potential problems (2). In a study in the united kingdom approximately 25% of the nurses reported that the time spent on teaching medication management skills is inadequate . They believed that more time should be spent on this crucial area (3).some of the studies also reported that nursing students were not effectively prepared in the area of pharmacological and medication management (1, 4, 5) and emphasized that the current nursing curricula do not provide the students with sufficient opportunities to promote their performance in medication management . Consequently, many medication - related errors are made by nursing students (6 - 8) and nurses (9). A number of studies reported that nursing students have problems in calculation of medication dosage (10), frequently encounter uncertainty in the process of medication management (11), experience difficulties in application of safe injecting methods (12) and in understanding and demonstrating pharmacological concepts in clinical practice (13). Reviewing medication management education systems, some of the studies addressed the reasons behind nursing students medication errors and gaps in pharmacology (6 - 8, 14, 15). Several studies on factors affecting students learning of pharmaceutical care, reported that clinical instructors professional competence is the most determining factor in this area (5, 16, 17). It is reported that many nursing instructors are inexperienced and not competent in clinical training and clinical supervision (18). Some of the studies investigated the nursing educators challenges regarding clinical education and reported that despite their crucial role in preparing nursing students, they receive little support in developing their teaching skills, do not have opportunities for formal education and professional development related to their role (19, 20), and have no formal authority in clinical settings (21). Most of the studies in the area of nursing students pharmacological management investigated their knowledge and skills (2, 3, 11), the problems they experienced in their work (16), the type of medication errors they made (8), their confidence in medication calculations (10), or their perceptions on effective medication administration education and pharmacological education needs (22, 23). In two studies from iran, ghamari studied the nursing students perspectives on factors affecting learning of pharmaceutical care in clinical settings (5) and their medication management skills (11). These studies are also conducted overseas and no studies on nursing students views in this regard are available from iran . The current study aimed to investigate the nursing students perceptions on the status of clinical pharmaceutical and medication care education during internship program . This cross - sectional study was conducted in two semesters in the 2013 - 2014 academic year at the qom and naragh branches of islamic azad university, and kashan university of medical sciences . The study population comprised senior nursing students and sampling included all nursing students who were in their fourth year of studying in nursing in these semesters (i.e., from october 2013 to late june 2014). All of the students (those who were visitors at the institutions) were included in the population participating in the study (n = 152) and completed the questionnaire . Out of the 152 respondents, a total of 44, 29 and 79 were the students registered at the qom and naragh branches of islamic azad university, and kashan university of medical sciences, respectively . The second section including 31 items associated with clinical educators performance in teaching the process of pharmacological management . The students were asked to evaluate their educators performance during four years at university using the percentage they enhanced the students knowledge and skills in pharmacology and medication management . The range of responses included less than 50% (= 1); 50% to 75% (= 2); and 75% to 100% (= 3). They were prepared through literature review and in accordance with the principles of teaching and skills of medicine preparation and administration activities, clinical skill of administering an injection and post medication care (24). Finally, there were two questions about students satisfaction with their level of knowledge and skills in pharmacology and medication management . Currently, how much are you satisfied with your own pharmaceutical care skills? And currently, how much are you satisfied with your own pharmacology knowledge? The answers were very low (= 1), low (= 2), moderate (= 3), high (= 4) and very high (= 5). The questionnaire was reviewed by a number of nursing faculty members at the school of medical sciences of islamic azad university, qom branch, and kashan university of medical sciences and they confirmed its content validity after some modifications based on their comments . The reliability of the questionnaire was assessed through internal consistency method (25) after administration among ten senior students in islamic azad university (qom branch); and the cronbach s alpha was 0.87 . The respondents were invited to complete the questionnaires in a quiet and private environment and the researcher or a research assistant collected the questionnaires after the completion which took about 15 minutes . The study was approved by institutional review board and the research ethics committee of islamic azad university, qom branch . Afterwards all nursing students in the seventh and eighth semesters of nursing bachelor program at the qom and naragh branches of islamic azad university, and kashan university of medical sciences were invited to participate in the study . The students were assured that all the information would remain confidential and the questionnaires were anonymous . Descriptive statistics (frequency, rate of frequency, mean, and standard deviation (sd) were used in this study . Anova was performed to compare the mean of pharmaceutical care education scores in the three universities . The study was approved by institutional review board and the research ethics committee of islamic azad university, qom branch . Afterwards all nursing students in the seventh and eighth semesters of nursing bachelor program at the qom and naragh branches of islamic azad university, and kashan university of medical sciences were invited to participate in the study . The students were assured that all the information would remain confidential and the questionnaires were anonymous . Descriptive statistics (frequency, rate of frequency, mean, and standard deviation (sd) were used in this study . Anova was performed to compare the mean of pharmaceutical care education scores in the three universities . The study was conducted on 152 nursing students among whom 82.9% were female and their mean age was 22.57 1.55 years, ranging from 21 to 29 years . The mean and standard deviation of 10 weakest items on clinical educators performance in teaching pharmacological management in the three universities are presented in table 1 . The item teaching pharmacological management based on students learning needs earned the lowest rank . All data are presented as mean sd . According to the majority of students, 75% - 100% of educators are always or often eager to promote students knowledge and medication management skills . The students indicated that they were taught the skills related to medication management (i. e., hand washing, cleaning the tray before putting the drug, wearing gloves before injections, etc .) And safety practices related to medication management (i. e., asking patients names, avoiding re - using needles, informing patients about medication side - effects and management of saline solution, etc . ). Students responses to two of the questions related to their level of satisfaction with the knowledge of pharmacology and skills of medication management produced the mean and standard deviation of 2.56 1.03 and 2.87 0.91, respectively . The current study aimed to assess the status of clinical pharmaceutical and medication care education from the nursing students perceptive . Authors did not focus on specific educators; but focused on the performance of all clinical nurse educators regarding pharmacology and medication management education . The results indicated a poor quality of education in several aspects of pharmacology and medication management education . Due to the crucial importance of pharmacology and medication management in nursing and in patients health and safety, the findings signified the urgent need to take measures to overcome the problems and reduce the potential risks . According to the students, instructors had the weakest performance in the three items of teaching pharmacology and medication management based on students learning needs, teaching medication management through a patient - centered method and teaching pharmacology and medication management based on the course plan . Teaching pharmacological management in clinical setting is a very important activity which not only needs paying attention to the patients health and safety needs but also to the students learning needs . A clinical educator who teaches clinical pharmacology and medication management should be proficient in pharmacology and diagnosing and prioritizing both the patients and the students needs . The current study findings showed that clinical instructors were weak in clinical education and had weak knowledge on pharmacology and medication management . It also seems that clinical educators have no specific course plans or specific objectives especially in the field of pharmacology and medication management . For instance they might present some materials about classification of anti - tumor drugs at the first clinical apprenticeship . Previous studies in iran have also shown that the majority of nurse educators mostly focus on issues such as dosage calculation and take the prescribed dose in specific times of a day (5, 26, 27), instead of how a nurse can decide a dose reduction required for a patient with renal failure for example . This is why nurses and nursing students need a high level of knowledge on pharmacology and medication management in order to care for their patients appropriately . Although not on nursing students however, a study on nurses information needs and information seeking behaviors, have reported that nurses require pharmacological information more often than other types of information . Therefore, they sought pharmacological information nine times a week while they sought epidemiological data only twice a week (28). A study in iran showed that nursing students are often anxious about their capability to implement their knowledge and skills on physiology, anatomy and pharmacology . The results of the present study implied that the students wished to receive constructive feedbacks about their medication management practices . However, the item giving constructive feedback and advice about their own medication errors, technical errors, or inaccurate medication recordings was among the ten weakest items . This finding not only shows the weak performance of clinical instructors in this regard, but also signifies the importance of timely and appropriate feedback on the students learning . Consistently, a qualitative study on student nurses perceptions of effective medication administration education, has reported that nursing students wished not only to encounter frequent learning opportunities, but also to receive timely feedback on their performances in medication administration (22). In the present study, the item teaching pharmacology and medications management through a patient - centered approach scored as the second weak item . This finding confirms that clinical instructors usually teach through a functional or work - based approach in their clinical educations . A previous study showed that nursing students prefer to learn pharmacology and pharmaceutical care through a patient - centered approach (5). Investigated the nursing students perspective on effective clinical teaching and reported that nursing students believed that patient - centered approaches cause patients be considered as a whole, while work - based training cannot produce a meaningful learning (29). The results of the two questions about students level of satisfaction with their own knowledge of pharmacology and medication management skills indicated that they evaluated themselves as having a moderate level of knowledge and skills . In this regard, the results of previous studies highlighted that nursing students acquire insufficient level of pharmacological knowledge during nursing bachelor programs (13, 23, 30). The insufficient pharmacological knowledge of nursing students predisposes them to medication errors (8, 31). For instance, the lack of pharmaceutical knowledge and medication management skills might lead the students to administer penicillin to the patients who re penicillin - allergic due to the lack of check (32). The present study showed that although nursing schools of all the three universities were relatively similar in their educational performance in teaching pharmacology and medication management, however the scores of kashan university of medical sciences were approximately higher in most instances . This finding shows the urgent need to pay special attention to the quality of education in all nursing schools and with special focus on the islamic azad university . In conclusion, the study showed that nursing students give a relatively low score in several aspects of their instructors performance regarding teaching pharmacology and medication management . It seems that many clinical nurse educators in the studied settings are incompetent especially in teaching pharmacology and medication management while this is a critical area and needs special attention . It is necessary to plan properly for each element of clinical education (objectives, content, educators, and evaluation) to achieve the goals of nursing education . According to the above - mentioned findings and nursing instructors incompetency in some areas, it is necessary to enable nurse instructors in teaching principles and practices of pharmaceutical care and to revise nursing programs . Therefore, it is recommended that clinical nurse educators go through in - service courses to upgrade their competency not only in clinical teaching, but also in pharmacology and pharmacological management . The current study examined only the students perspective; however, the instructors perspective on the existing barriers to appropriate pharmacological and medication management education should also be assessed . In addition, more objective assessments on the students knowledge and skills and the clinical educators performance are recommended . The study also assessed the students perspective of all educators and had no focus on the performance of individual instructors . Then, the retrospective nature of the assessment might induce some biases on the results . It is recommended that the students perspective on the same issue be immediately assessed after each clinical rotation, not only the performance of all instructors, but also their specific strengths and weaknesses can be assessed . Then more appropriate in - service education programs might be designed to overcome the problems.
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A 66-year - old female was presented to the emergency room with sudden weakness on her left side and dysarthria while taking aspirin . Diffusion weighted magnetic resonance imaging (mri) showed several small acute infarctions scattered in her right frontal cortex and basal ganglia . Mr angiography revealed a severe stenosis of the right middle cerebral artery (mca). Digital subtraction angiography (dsa) confirmed the severe long segment stenosis of the mca and a significant decrease of flow velocity to the distal segment (fig . 1a). We suspected that her current medical treatment would be ineffective to prevent another stroke, and thus, we planned an endovascular treatment . An endovascular procedure was performed 1 week later when her left - sided weakness was almost recovered . She received a daily oral administration of 325 mg of aspirin and 75 mg of clopidogrel before the procedure . Endovascular procedure was performed under local anesthesia and with systemic heparinization . With a 6 fr guiding catheter located in the right internal carotid artery (ica), after the microcatheter was advanced across the lesion, the microwire was exchanged with a 300 cm exchange wire that could facilitate balloon and stent navigation . The microcatheter was removed, and a 1.520 mm gateway balloon catheter was advanced over the exchange wire . The balloon was located within the stenotic segment and inflated to approximately 6 atm . On control angiogram after balloon angioplasty next, the wingspan delivery system was prepared and advanced over the exchange wire across the target lesion . During wingspan (3.520 mm) passed cavernous ica, patient complained headache and nausea, and systolic blood pressure was elevated from normal range to approximately 160 mmhg . Immediately, a stent was deployed at the stenotic segment and a control angiogram was achieved . Although the stenotic segment was recanalized and the distal flow restored to normal velocity (fig . 1b), a tiny irregularity of posterior parietal branch of right mca and extravasation of contrast media suggested that dissections were revealed (fig . Rotational flat panel ct was performed immediately and showed a small amount of extravasation of contrast media within the subarachnoid space along the right lateral sulcus (fig . We dropped the blood pressure under normal range and reversed heparin with protamine sulfate . On the angiogram performed 10 minutes later, a small filling defect appeared within the stenosis, which was suspected as ist . Because of a subarachnoid hemorrhage caused by an iatrogenic vessel injury, iib / iiia receptor antagonist or fibrinolytic agent should not be administrated for thrombolysis . To manage the ist rebar 18 " microcatheter (ev3, irvine, ca, u.s.a .) Was passed the wingspan stent, and a solitaire stent (420 mm, ev3, irvine, ca, u.s.a .) Was deployed to cover the wingspan stent (fig . Ist was completely resolved on dsa obtained at 30 and 60 minutes after procedure (fig . Brain ct at immediate and 24 hours after procedure did not reveal any new low attenuation or progression of subarachnoid hemorrhage . In - stent restenosis occurs in approximately 30% of patients after delivery of the wingspan stent, although many of these incidents of re - stenosis remain asymptomatic . Although the actual rate of periprocedural ist of intracranial stent is not obvious, the most recent report demonstrated that the rate of acute or subacute ist after intracranial stent ranged from 10% to 14.6% [4, 6]. Contrary to chronic in - stent stenosis, acute ist can result in permanent disability of the patient . . Aspirin and clopidogrel resistance may be the actual causes of ist during the periprocedural period, but it is likely there are other factors that have not been elucidated yet . It should be noted that the incidence of ist in the wingspan and gateway system is more frequent than that in the stent for aneurysm neck remodeling . The reason can be explained that the underlying disease itself is different from cerebral aneurysms and may be more prone to acute thrombus formation . A balloon angioplasty before the wingspan placement may result in an endothelial injury or the exposure of atheromatous plaque material, promoting an in - stent thrombus formation . The present case showed that the secondary self - expandable stent had resolved acute ist without a new thrombus formation within the stent in spite of the non - administration of fibrinolytic agents . The second stent would open circumferentially to displace the thrombus, immediately reestablishing a channel of flow within the occluded vessel . Once antegrade flow was restored, thrombus dissolution via endogenous thrombolysis would follow stent deployment . Therefore, we suspected that the etiology of acute ist in our case might be not associated with aspirin or clopidogel resistance but with endothelial injury . Stent in - stent would cover the exposure plaque material from the coagulation system, protecting re - growth of the thrombus within stent . Unfortunately, we were unable to confirm drug resistance using blood assay, such as verifynow p2y12 test . The management described for the ist includes a systemic or local application of iib / iiia receptor antagonist or a recombinant tissue - type plasminogen activator with / without balloon angioplasty [4, 6, 8]. Although these thrombolytic agents are effective solutions for acute thrombolysis, they cannot be used and the mechanical technique should be considered for the recanalization of thrombotic occlusion of artery when there is a recent hemorrhage such as the present case . We have found several reports regarding the use of stent - in stent technique for recanalization of the late in - stent restenosis following the wingspan placement [5, 8, 9]. However, to the best of our knowledge, this present case is the first case that has recanalized acute ist using a stent in - stent technique and without using the thrombolytics . Iatrogenic vascular perforation is a complication of major concern that can result in fatal intracranial hemorrhages during endovascular neurointervention . When vascular perforation is detected during a procedure, the treatment should immediately begin with a reversal of anticoagulant, lowering the blood pressure and/or temporary or permanent closure of the ruptured vessel . As in this case, when an ist following a wingspan stent placement for intracranial stenosis occurs with a coincidentally hemorrhagic complication, the deployment of another stent in - stent should be considered as a treatment option.
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Six cases were reported from 2007 to 2012 to the oral and maxillofacial unit of annaswamy mudaliar general hospital, bangalore, out of which four patients were males with the lesion occurring between the second decade of life and sixth decade of life [figures 1a3c]. In all cases, the mandible was involved with the greatest size of tumor seen being 11 cm 9.6 cm [figure 1a]. All cases exhibited enlargement of cortical plates with perforation, pain and tenderness with regional lymph node enlargement, with three cases showing fixed submandibular groups of lymph nodes (level ib) and in one case parasthesia of inferior alveolar nerve . Five cases showed unilocular radiolucency and one case showed multilocular radiolucency with erosion of bone, including inferior border of mandible in one case [figure 1c]. Maximum follow - up was for 39 months with one patient being lost to the follow - up [table 1]. All patients had a chest x - ray and an abdomen ultrasound done to avoid missing distant metastasis as we were aware of the potential of this lesion to metastasize . Incisional biopsy was done for diagnosis for all cases and was diagnosed as features suggestive of ameloblastoma with areas showing dysplastic changes in the epithelium suggestive of a neoplastic change . (a) patient exhibiting large extra oral tumor with draining sinus, (b) intra oral view of tumor: (c) orthopantomogram showing bicortical involvement of mandible in one patient, wide excision was done as the lesion was small and the patient being young did not wish aggressive measures that may have had postsurgical cosmetic deformity [figure 2a c]. He chose the wait and watch policy and there is no evidence of disease after 32 months . (a) preoperative view showing tumor involving left mandible and sulcus, (b) preoperative orthopantomogram, (c) intra oral view of tumor being excised two patients underwent hemimandibulectomy and neck dissection because of increased involvement of mandible with palpable level ib nodes where fine - needle aspiration cytology showed positive for carcinoma, as also one more patient who had underwent a wide excision with hemimandibulectomy and neck dissection due to skin involvement and palpable fixed nodes at ib level [figure 1a c]. One patient had a segmental resection of the mandibleone patient had a hemimandibulectomy [figure 3a c]all the resected specimens were sent for histological examination . One patient had a segmental resection of the mandible one patient had a hemimandibulectomy [figure 3a c] all the resected specimens were sent for histological examination . (a) preoperative view of tumor involving left mandible, (b) intra oral view of tumor, (c) orthopantomogram showing involvement of left mandible the largest diameter of the tumors was 10 cm 9 cm [figure 1a] showing increased vascularity with numerous cystic spaces and areas of necrosis . Typical features found were multiple pieces of tissue with predominant tumor islands within connective tissue seen under scanner . Under higher magnification tumor islands appearing epithelial odontogenic in origin showed infiltrative pattern with each island showing peripheral columnar cells with central stellate reticulum exhibiting feature of ameloblastoma, but with atypical features such as bizarre mitosis, altered nuclear chromatin ratio, hyperchromatisim, and mild pleomorphism . Individual cell keratinization with keratin pearl formation with stroma showed predominant inflammatory component and endothelial lined blood vessels [figure 4]. Immunohistochemical findings showed that ameloblastic carcinomas reacted strongly with antibodies directed against cytokeratin chm and aei and ae3 . Histopathological picture showing bizarre mitosis, altered nuclear chromatin ratio, hyperchromatisim, mild pleomorphism, and central stellate reticulum confirming ameloblastic carcinoma no evidence of disease or metastasis was seen postsurgery in any of the patients . It is interesting to note that up to date of completion of this study 71 cases of ameloblastoma had reported to our department between february 2007 and may 2012 . In this series, ameloblastic carcinoma is a lesion with histologic behavior that dictates a more aggressive surgical approach than that of a simple ameloblastoma . Mean age for occurrence is 30.5 years, male: female ratio is 1.5:1, and 80% are located in the mandible posterior region . Involvement of the maxilla is less frequent than that of the mandible . The clinical symptoms of ameloblastic carcinoma are more aggressive than conventional ameloblastoma, with swelling, pain, rapid growth, trismus, dysphonia, expansion of the jaws, and frequently perforation of the cortex . These were mirrored in our series also . Radiographic appearance of ameloblastic carcinoma is consistent with that of ameloblastoma except for occasional presence of some focal radiopacities, reflecting dystrophic calcification . Histologically, ameloblastic carcinoma does not show uniform proliferation, and has pleomorphic and hyperchromatic cells arranged in the form of sheets and chords with reversed polarity . An additional consideration in the differential diagnosis is the squamous cell carcinoma arising in the lining of an odontogenic cyst . Histologically, this lesion tends to more closely resemble oral squamous cell carcinoma ameloblastic carcinoma . The squamous odontogenic tumor may also be mistaken for ameloblastic carcinoma, being composed of islands of squamous epithelium that lack stellate reticulum like zones, peripheral palisading, microcystic changes and dystrophic calcifications . However, the epithelium of the squamous odontogenic tumor lacks any cytologic evidence of malignant disease . Thus, the term ameloblastic carcinoma can be applied to our series, where all showed evidence of malignant disease including cytologic atypia and mitoses with indisputable features of classic ameloblastoma . Whether ameloblastoma may transform biologically and histologically from a classic ameloblastoma to a malignant lesion is controversial . Many authors have shown that metastasizing ameloblastomas are histologically indistinguishable from classic ameloblastomas and others have identified malignant features in the tumor, usually after repeated surgical excisions . The high rate of recurrence maybe due to the mode of growth and surgical mismanagement rather that any inherent malignant properties and metastases are exceedingly rare . Wide local excision is the treatment of choice as most investigators have recommended, which we had followed in all our cases . Regional lymph node dissection should be performed selectively as we did in three of our cases where in the regional lymph node was palpable . The efficacy of adjuvant radiation or chemotherapy as a postsurgical treatment is not clear and seems to have a limited value . Lanham treated a patient with doxorubicin, cisplatin, cyclophosphamide, dacarbazine, and 5 fu; but, the tumor failed to respond . Yoon et al . Have reported distant metastasis as early as 4 months and as late as 47 months after surgery . None of our patients showed recurrence, but one patient was lost to follow - up . None of our patients were sent for radiotherapy or chemotherapy . In our case series, least follow - up until date is 22 months and maximum follow - up being 39 months . This small series of cases illustrates the malignant portion in the spectrum of ameloblastomas and it is possible that ameloblastoma may show a wide variety of histologic and biologic behavior ranging from benignity to frank malignancy . Cases of ameloblastoma should be studied carefully, correlating their histologic pattern with biologic behavior to detect changes in histology that may predict aggressive behavior . When a case is diagnosed as ameloblastic carcinoma, assessment of nodal metastasis and evidence of distant metastasis treatment of ameloblastic carcinoma is wide surgical resection and if evidence of nodal metastasis is present, neck dissection should also be considered.
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Frequent episodic and chronic childhood tension - type headache (tth) is a prevalent and debilitating situation for the child and family . After clarifying the diagnosis in an interdisciplinary setting, because many parents are reluctant to choose preventive medication for their child, patient education is an important aspect of pediatric headache team services . The focus is on empowering the family to incorporate a healthy lifestyle at home and at school to actively prevent the development or triggering of headache . The most basic lifestyle factors that a discussion (between nurse / physical therapist and parent) can contribute to are sufficiency of sleep, liquids, food, activity level, and how to handle psychosocial stressors.1 for nonpharmacological behavioral treatments, patient education on relaxation therapy with or without electromyographic or thermal biofeedback has shown, over the last 20 years, substantial evidence in reducing headache frequency in both children and adults.25 passive acupunctural laser therapy has also shown positive effects in a controlled trial on headache frequency.6 andrasik et al7 compared preventive medication with amitriptyline and relaxation therapy for juvenile tth and found clinical improvements from both interventions at 1- and 2-year follow - up . Even though these effects are supported by evidence, use in practice is more limited than expected.8 a major limitation is that interventions such as relaxation therapy require approximately up to ten 1-hour sessions in which a trained expert teaches the child with the use of specialized equipment.8 as families are busy and society resources sparse, there is a need for educational approaches that are not only less costly but also more accessible and flexible, which is why internet - based self - help interventions are on the rise;9 other alternatives are warranted . Physical training compared to relaxation therapy and acupuncture has been examined in one study for adults with chronic tth.10 within - group differences showed significant positive long - lasting effects for all three treatments . Brief daily strength training using elastic bands has also been shown to reduce nonchronic headache frequency in adult office workers with neck shoulder muscle pain.11 physical exercise for children has not yet been examined, although the world health organization published global recommendations on physical activity for health in 2011 specifically for children aged 517.12 in one of our previous studies, we found reduced neck shoulder muscle strength and aerobic power together with increased pericranial tenderness, especially in the trapezius descendens, to be associated with tth in girls.13 we hypothesized that strengthening the shoulder muscles would result in reduced headache frequency and improvements in the girl s physical capabilities . This study aimed at comparing the outcomes of specific supervised strength training of shoulder muscles with the outcomes of counseling (defined patient education programs) for girls with tth . A randomized controlled trial was performed in accordance with the consort 2010 statement14 and guidelines for trials for recurrent headache.15,16 the trial was approved by the national committee on health research ethics for hospitals in the capital region of denmark, and the danish data protection agency . Registered by clinicaltrials.gov h-3 - 2009 - 081, identifier ncto115557 . From may 1, 2010 to september 1, 2013, 49 consecutive girls aged 918 years agreed to participate in the study with a 24-months follow - up period until september 2015 . They were diagnosed exclusively with either frequent episodic tth (at least ten episodes, $1 day but <15 days per month for at least 3 months) or chronic tth ($15 days per month on average for> 3 months), thus fulfilling the international classification of headache disorders 2nd edition and 3rd edition, beta version.17,18 we were unable to recruit a sufficient number of boys to meet the criteria for statistical analyses . All participants were diagnosed and recruited from the children s headache clinic, glostrup and herlev hospitals, university of copenhagen . Before inclusion, the girls with tth underwent a clinical diagnostic and neurological examination that included examination of the spine by the department s neuropediatricians and one specialist physical therapist . The diagnosis was clarified using 1- to 6-month headache reports from a diary similar to that recommended by jensen et al.19 exclusion criteria were set by the neuropediatrician from anamnestic, observational and clinical assessments . Exclusion criteria were: migraine headache with more than one episode per month for a period of 6 months; a history of trauma, arterial hypertension, or intracranial hypertension; headache secondary to a cervical or other morbidity; and headache associated with a psychiatric comorbidity . Another exclusion criterion was headache associated with a significant learning disability or complicated social situation that required special education and/or community involvement . Girls who had been or were enrolled in other treatment programs at the children s headache clinic were also excluded . No preventive headache medication and/or analgesic overuse were allowed before or during the study period . None of the participants were allowed the intake of analgesics, taken for any reason, within 12 hours before testing . At the first diagnostic visit, all participants were informed by the contact physician and nurse about the basic needs a child has for adequate food, liquids, sleep, and activity . This study followed the ethical principles of the world medical association s declaration of helsinki 2008 and 201320 and the united nations convention on the rights of the child 1989.21 the parents gave written informed consent, and the children gave oral informed assent . According to the ethical guidelines participants were informed about confidentiality and the right to withdraw from the study at any time without affecting their treatment . Self - reports of headache were monitored daily using a visual analog score (vas)/numeric scale22 in a monthly calendar . Mean headache frequency, intensity, and duration over 28 days prior to the test date were calculated . Health - related quality of life (hrqol) and disability were recorded at baseline and were administered by a nurse during the first visit at the clinic for counseling and diagnosis . A follow - up was administered by means of a mailed questionnaire 24 months after baseline . Self - reports of the pediatric quality of life inventory (pedsql 4.0; mapi research institute, lyon, france) generic core scales . Validated and validated in translation to danish,23 the questionnaire consists of 23 items comprising four dimensions: physical functioning (eight items), emotional functioning (five items), social functioning (five items), and school functioning (five items), which were scored on a five - point likert scale (04/never almost always). Parental and child reports were collected for children aged 812 and 1318 years.24 for data analysis, raw scores 04 were reciprocally transformed to 0100 scale scores following the scoring instructions.25self - reports of the pediatric migraine disability assessment (pedmidas) questionnaire translated into danish . The first three pedmidas questions relate to the impact of headache on school attendance and performance . Originally developed to assess migraine disability in pediatric and adolescent patients, pedmidas has been tested and validated for 418-year - olds with migraine by the cincinnati children s headache centre26,27 and it also addresses children with tth.28 the score is interpreted as follows: 010 little to none disability grade; 1130 grade ii, mild; 3150 grade iii, moderate; and above 50 grade iv, severe disability . Self - reports of the pediatric quality of life inventory (pedsql 4.0; mapi research institute, lyon, france) generic core scales . Validated and validated in translation to danish,23 the questionnaire consists of 23 items comprising four dimensions: physical functioning (eight items), emotional functioning (five items), social functioning (five items), and school functioning (five items), which were scored on a five - point likert scale (04/never almost always). Parental and child reports were collected for children aged 812 and 1318 years.24 for data analysis, raw scores 04 were reciprocally transformed to 0100 scale scores following the scoring instructions.25 self - reports of the pediatric migraine disability assessment (pedmidas) questionnaire translated into danish . The first three pedmidas questions relate to the impact of headache on school attendance and performance . Originally developed to assess migraine disability in pediatric and adolescent patients, pedmidas has been tested and validated for 418-year - olds with migraine by the cincinnati children s headache centre26,27 and it also addresses children with tth.28 the score is interpreted as follows: 010 little to none disability grade; 1130 grade ii, mild; 3150 grade iii, moderate; and above 50 grade iv, severe disability . Measurements were conducted at baseline, after 10 weeks of intervention and after 12 weeks of home practice / follow - up; in all 22 weeks . A bioanalytic research assistant, blinded to the participants histories, trained and experienced in the specific test methods, conducted the testing . The test procedures followed a standardized protocol and were conducted in a warm comfortable room . Body mass index (bmi) was calculated.general joint mobility was screened using the validated beighton score with nine points as a maximum . The cut - off for positive findings of hypermobility was 5/9.29tenderness of pericranial muscles was examined in a sitting position in a chair with adjustable neck and leg support using the total tenderness score (tts) system validated by bendtsen et al.30,31 a total score was calculated based on the standardized manual palpation procedures of seven bilateral sites (masseter, frontalis, temporalis, processus mastoideus, occipital insertion, trapezius, and sternocleidomastoideus) using a four - point scale . The maximum possible score was 42 points.isometric muscular testing was conducted using a computerized force transducer, model vishay nobel, type kis-2, max . 2 kn (vishay precision group, malvern, pa, usa) wall mounted on a custom - built adjustable stand . The signal from the force transducer was amplified and sampled in the computer using a national instruments, type pwr02 (including a strain gauge amplifier, type scc - sg24), and a data acquisition card daqcard-6036e (national instruments, austin, tx, usa). All signals were sampled with a frequency of 100 hz and low - pass filtered with a cut - off frequency of 10 hz . The child was positioned on a chair with the upper and lower trunk fixed with belts.for neck flexion measurements, the lower edge of the padded force transducer was positioned to correspond with the line between the eyebrows . For neck extension measurements, the testing included isometric maximal voluntary contraction (mvc) in neck flexion and extension from a neutral upright position . The child was instructed to slowly build up the force to maximum within 2 seconds and then exert maximal pressure for about 3 seconds and thereafter slowly relax again . Participants were verbally encouraged to perform maximally.for shoulder mvc and rate of force development (rfd), the child was asked to lie on a mattress in a supine position . The dominant shoulder was then positioned in external rotation and abduction, while the elbow was stretched to position the back of the wrist on the pad . The participant was instructed to press as fast and as forcefully as possible in the direction of abduction . For both the neck and shoulder test, a minimum of three attempts was given . If the third attempt was> 5% of the second or first, an extra attempt was given, though no more than five.aerobic power was determined by a monark ergomedic 939e pc bike (monark exercise ab, stockholm, sweden) using a submaximal strand test procedure and nomogram / accompanying tables for predicting maximal oxygen uptake lmin, vo2max.32 heart rate was monitored using an adjustable polar pulse belt (proterapi a / s, brndby, denmark) and recorded continuously on a computer.the number of children receiving a supply of vitamin d and/or psychologist counseling was noted . General joint mobility was screened using the validated beighton score with nine points as a maximum . The cut - off for positive findings of hypermobility was 5/9.29 tenderness of pericranial muscles was examined in a sitting position in a chair with adjustable neck and leg support using the total tenderness score (tts) system validated by bendtsen et al.30,31 a total score was calculated based on the standardized manual palpation procedures of seven bilateral sites (masseter, frontalis, temporalis, processus mastoideus, occipital insertion, trapezius, and sternocleidomastoideus) using a four - point scale . Isometric muscular testing was conducted using a computerized force transducer, model vishay nobel, type kis-2, max . 2 kn (vishay precision group, malvern, pa, usa) wall mounted on a custom - built adjustable stand . The signal from the force transducer was amplified and sampled in the computer using a national instruments, type pwr02 (including a strain gauge amplifier, type scc - sg24), and a data acquisition card daqcard-6036e (national instruments, austin, tx, usa). All signals were sampled with a frequency of 100 hz and low - pass filtered with a cut - off frequency of 10 hz . The child was positioned on a chair with the upper and lower trunk fixed with belts . For neck flexion measurements, the lower edge of the padded force transducer was positioned to correspond with the line between the eyebrows . For neck extension measurements, the testing included isometric maximal voluntary contraction (mvc) in neck flexion and extension from a neutral upright position . The child was instructed to slowly build up the force to maximum within 2 seconds and then exert maximal pressure for about 3 seconds and thereafter slowly relax again . Participants were verbally encouraged to perform maximally . For shoulder mvc and rate of force development (rfd), the dominant shoulder was then positioned in external rotation and abduction, while the elbow was stretched to position the back of the wrist on the pad . The participant was instructed to press as fast and as forcefully as possible in the direction of abduction . For both the neck and shoulder test, a minimum of three attempts was given . If the third attempt was> 5% of the second or first, an extra attempt was given, though no more than five . Aerobic power was determined by a monark ergomedic 939e pc bike (monark exercise ab, stockholm, sweden) using a submaximal strand test procedure and nomogram / accompanying tables for predicting maximal oxygen uptake lmin, vo2max.32 heart rate was monitored using an adjustable polar pulse belt (proterapi a / s, brndby, denmark) and recorded continuously on a computer . The number of children receiving a supply of vitamin d and/or psychologist counseling was noted . To avoid withholding any information relevant for the child s health, the child and parents were informed about the results of the tts and the aerobic power test by the researcher responsible . A test retest study by tornoe et al33 contains a detailed description of test procedures with analyses of repeatability and variability . This study was a blinded block - randomized controlled trial with computer - generated allocation in blocks of ten, administered by a secretary, to place participants into one of two intervention groups, a and b. both interventions were based on short, defined education courses of 10 weeks home training with 34 sessions of supervised training or counseling, approximately 140 minutes in total for each group . The contact nurse and an experienced pediatric physical therapist administered the interventions, which were followed by a 12-week follow - up period in which home practice was advised to continue without supervision . Families were welcome to request psychological counseling after the 10 weeks intervention period, which was to be supported by an interdisciplinary team conference . The physical therapist taught and supervised group a in 34 sessions of progressive specific strength training with a focus on the trapezius muscles with resistive tubing elastics (thera - band; hygenic corporation, akron, ohio, usa) and recording in a prospective diary . Training was to be conducted three times a week at home and comprised alternating four exercises two by two . The progression involved doing one set with ten repetitions and moving to three sets with ten repetitions . The tubing elastics were used in three strengths, red / niveau i, green / niveau ii, and blue / niveau iii, to provide progression of resistance . To determine the level of resistance, a 15 repetition maximum (rm) was used to estimate an exercise resistance equivalent 75%80% of one rm.34 focus was on warm - up exercises for the arms and practicing the exercises . The progressive strength training was based on research and recommendations in the american academy of pediatrics, committee on sports medicine and fitness, policy statement for pediatric training,35 faigenbaum et al36,37 and andersen et al.38 figure 3 provides an illustration of the exercises . During the 10 weeks of intervention, compliance and continuity with the training schedule was secured by carrying out intermediate checks of the training diary and discussion with the child and parents . After the 10 weeks of training, the child and family had a single counseling session with the nurse to avoid withholding help for the family . Group b was provided with need - based counseling based on recommendations for multidisciplinary headache management.39 group b, as an established and evidenced intervention, was the control group, in accordance with international guidelines.15 the interdisciplinary intervention has been proven to be effective in the treatment of children in the clinic.40 the intervention comprised one session with the nurse and two sessions with the physical therapist . The focus was on a motivational dialogue following a checklist to encourage the child and parents to practice self - care through lifestyle changes and physical awareness . The aim was to enhance the child s physical health and well - being at home and at school . The child, with parental support, was encouraged to practice and exercise at home . Table 1 provides a checklist of the topics the nurse and physical therapist used during counseling . The power analysis for this study was based on preliminary data from a case control study and was calculated as follows: two groups of 23 participants were calculated from a mean headache frequency for girls at 20 days (sd 9.6), a significance level of 5%, a power (1) at 80% and an expected difference at 40% . In order not to lose power, subgroup analyses of episodic and chronic tth descriptive statistics were used to describe the data s central tendencies, dispersal, and trends . Mean, median, standard deviation, range, and frequency were used as appropriate . The distribution of data was examined by means of normal probability plots and kolmogorov smirnov test . For all primary and secondary variables, headache frequency and headache duration were analyzed as binomial counts (count parameter equaled 28 days and 24 hours, respectively) using generalized linear models . Headache intensity, peak data, tts, and oxygen uptake were analyzed using two - sample t - test . Headache at test (yes / no) was analyzed by pearson s chi - square test . To investigate whether there was an effect of the intervention and if the effect was different after 10 weeks of intervention and after a further 12 weeks of home practice, the models included time and the interaction between time and intervention . Headache frequency, headache duration, and headache at test were analyzed by use of a repeated measurement analysis for binominal counts . The rest of the variables were analyzed by linear mixed models for repeated measurements by use of the mixed procedure by sas 9.2 . For the primary variables, an analysis based on intention - to - treat, with the last observation carried forward, was also performed . Vitamin d supplementation and counseling by psychologist were analyzed as additional explanatory variables, although the material was too small to fully adjust for four variables . Effect size was described and presented as the odds ratio (or) (95% confidence interval [ci]) of having headache on a random day . For all tests, a randomized controlled trial was performed in accordance with the consort 2010 statement14 and guidelines for trials for recurrent headache.15,16 the trial was approved by the national committee on health research ethics for hospitals in the capital region of denmark, and the danish data protection agency . From may 1, 2010 to september 1, 2013, 49 consecutive girls aged 918 years agreed to participate in the study with a 24-months follow - up period until september 2015 . They were diagnosed exclusively with either frequent episodic tth (at least ten episodes, $1 day but <15 days per month for at least 3 months) or chronic tth ($15 days per month on average for> 3 months), thus fulfilling the international classification of headache disorders 2nd edition and 3rd edition, beta version.17,18 we were unable to recruit a sufficient number of boys to meet the criteria for statistical analyses . All participants were diagnosed and recruited from the children s headache clinic, glostrup and herlev hospitals, university of copenhagen . Before inclusion, the girls with tth underwent a clinical diagnostic and neurological examination that included examination of the spine by the department s neuropediatricians and one specialist physical therapist . The diagnosis was clarified using 1- to 6-month headache reports from a diary similar to that recommended by jensen et al.19 exclusion criteria were set by the neuropediatrician from anamnestic, observational and clinical assessments . Exclusion criteria were: migraine headache with more than one episode per month for a period of 6 months; a history of trauma, arterial hypertension, or intracranial hypertension; headache secondary to a cervical or other morbidity; and headache associated with a psychiatric comorbidity . Another exclusion criterion was headache associated with a significant learning disability or complicated social situation that required special education and/or community involvement . Girls who had been or were enrolled in other treatment programs at the children s headache clinic were also excluded . No preventive headache medication and/or analgesic overuse were allowed before or during the study period . None of the participants were allowed the intake of analgesics, taken for any reason, within 12 hours before testing . At the first diagnostic visit, all participants were informed by the contact physician and nurse about the basic needs a child has for adequate food, liquids, sleep, and activity . This study followed the ethical principles of the world medical association s declaration of helsinki 2008 and 201320 and the united nations convention on the rights of the child 1989.21 the parents gave written informed consent, and the children gave oral informed assent . According to the ethical guidelines, children above the age of 15 also received special age - appropriate information . Participants were informed about confidentiality and the right to withdraw from the study at any time without affecting their treatment . Self - reports of headache were monitored daily using a visual analog score (vas)/numeric scale22 in a monthly calendar . Mean headache frequency, intensity, and duration over 28 days prior to the test date were calculated . Health - related quality of life (hrqol) and disability were recorded at baseline and were administered by a nurse during the first visit at the clinic for counseling and diagnosis . A follow - up was administered by means of a mailed questionnaire 24 months after baseline . Self - reports of the pediatric quality of life inventory (pedsql 4.0; mapi research institute, lyon, france) generic core scales . Validated and validated in translation to danish,23 the questionnaire consists of 23 items comprising four dimensions: physical functioning (eight items), emotional functioning (five items), social functioning (five items), and school functioning (five items), which were scored on a five - point likert scale (04/never almost always). Parental and child reports were collected for children aged 812 and 1318 years.24 for data analysis, raw scores 04 were reciprocally transformed to 0100 scale scores following the scoring instructions.25self - reports of the pediatric migraine disability assessment (pedmidas) questionnaire translated into danish . The first three pedmidas questions relate to the impact of headache on school attendance and performance . Originally developed to assess migraine disability in pediatric and adolescent patients, pedmidas has been tested and validated for 418-year - olds with migraine by the cincinnati children s headache centre26,27 and it also addresses children with tth.28 the score is interpreted as follows: 010 little to none disability grade; 1130 grade ii, mild; 3150 grade iii, moderate; and above 50 grade iv, severe disability . Self - reports of the pediatric quality of life inventory (pedsql 4.0; mapi research institute, lyon, france) generic core scales . Validated and validated in translation to danish,23 the questionnaire consists of 23 items comprising four dimensions: physical functioning (eight items), emotional functioning (five items), social functioning (five items), and school functioning (five items), which were scored on a five - point likert scale (04/never almost always). Parental and child reports were collected for children aged 812 and 1318 years.24 for data analysis, raw scores 04 were reciprocally transformed to 0100 scale scores following the scoring instructions.25 self - reports of the pediatric migraine disability assessment (pedmidas) questionnaire translated into danish . The first three pedmidas questions relate to the impact of headache on school attendance and performance . Originally developed to assess migraine disability in pediatric and adolescent patients, pedmidas has been tested and validated for 418-year - olds with migraine by the cincinnati children s headache centre26,27 and it also addresses children with tth.28 the score is interpreted as follows: 010 little to none disability grade; 1130 grade ii, mild; 3150 grade iii, moderate; and above 50 grade iv, severe disability . Measurements were conducted at baseline, after 10 weeks of intervention and after 12 weeks of home practice / follow - up; in all 22 weeks . A bioanalytic research assistant, blinded to the participants histories, trained and experienced in the specific test methods, conducted the testing . The test procedures followed a standardized protocol and were conducted in a warm comfortable room . . Age and anthropometric measures such as height and weight were collected . Body mass index (bmi) was calculated.general joint mobility was screened using the validated beighton score with nine points as a maximum . The cut - off for positive findings of hypermobility was 5/9.29tenderness of pericranial muscles was examined in a sitting position in a chair with adjustable neck and leg support using the total tenderness score (tts) system validated by bendtsen et al.30,31 a total score was calculated based on the standardized manual palpation procedures of seven bilateral sites (masseter, frontalis, temporalis, processus mastoideus, occipital insertion, trapezius, and sternocleidomastoideus) using a four - point scale . The maximum possible score was 42 points.isometric muscular testing was conducted using a computerized force transducer, model vishay nobel, type kis-2, max . 2 kn (vishay precision group, malvern, pa, usa) wall mounted on a custom - built adjustable stand . The signal from the force transducer was amplified and sampled in the computer using a national instruments, type pwr02 (including a strain gauge amplifier, type scc - sg24), and a data acquisition card daqcard-6036e (national instruments, austin, tx, usa). All signals were sampled with a frequency of 100 hz and low - pass filtered with a cut - off frequency of 10 hz . The child was positioned on a chair with the upper and lower trunk fixed with belts.for neck flexion measurements, the lower edge of the padded force transducer was positioned to correspond with the line between the eyebrows . For neck extension measurements, the testing included isometric maximal voluntary contraction (mvc) in neck flexion and extension from a neutral upright position . The child was instructed to slowly build up the force to maximum within 2 seconds and then exert maximal pressure for about 3 seconds and thereafter slowly relax again . Participants were verbally encouraged to perform maximally.for shoulder mvc and rate of force development (rfd), the child was asked to lie on a mattress in a supine position . The dominant shoulder was then positioned in external rotation and abduction, while the elbow was stretched to position the back of the wrist on the pad . The participant was instructed to press as fast and as forcefully as possible in the direction of abduction . For both the neck and shoulder test, a minimum of three attempts was given . If the third attempt was> 5% of the second or first, an extra attempt was given, though no more than five.aerobic power was determined by a monark ergomedic 939e pc bike (monark exercise ab, stockholm, sweden) using a submaximal strand test procedure and nomogram / accompanying tables for predicting maximal oxygen uptake lmin, vo2max.32 heart rate was monitored using an adjustable polar pulse belt (proterapi a / s, brndby, denmark) and recorded continuously on a computer.the number of children receiving a supply of vitamin d and/or psychologist counseling was noted . General joint mobility was screened using the validated beighton score with nine points as a maximum . The cut - off for positive findings of hypermobility was 5/9.29 tenderness of pericranial muscles was examined in a sitting position in a chair with adjustable neck and leg support using the total tenderness score (tts) system validated by bendtsen et al.30,31 a total score was calculated based on the standardized manual palpation procedures of seven bilateral sites (masseter, frontalis, temporalis, processus mastoideus, occipital insertion, trapezius, and sternocleidomastoideus) using a four - point scale . Isometric muscular testing was conducted using a computerized force transducer, model vishay nobel, type kis-2, max . 2 kn (vishay precision group, malvern, pa, usa) wall mounted on a custom - built adjustable stand . The signal from the force transducer was amplified and sampled in the computer using a national instruments, type pwr02 (including a strain gauge amplifier, type scc - sg24), and a data acquisition card daqcard-6036e (national instruments, austin, tx, usa). All signals were sampled with a frequency of 100 hz and low - pass filtered with a cut - off frequency of 10 hz . The child was positioned on a chair with the upper and lower trunk fixed with belts . For neck flexion measurements, the lower edge of the padded force transducer was positioned to correspond with the line between the eyebrows . For neck extension measurements, the testing included isometric maximal voluntary contraction (mvc) in neck flexion and extension from a neutral upright position . The child was instructed to slowly build up the force to maximum within 2 seconds and then exert maximal pressure for about 3 seconds and thereafter slowly relax again . Participants were verbally encouraged to perform maximally . For shoulder mvc and rate of force development (rfd), the child was asked to lie on a mattress in a supine position . The dominant shoulder was then positioned in external rotation and abduction, while the elbow was stretched to position the back of the wrist on the pad . The participant was instructed to press as fast and as forcefully as possible in the direction of abduction . For both the neck and shoulder test, a minimum of three attempts was given . If the third attempt was> 5% of the second or first, an extra attempt was given, though no more than five . Aerobic power was determined by a monark ergomedic 939e pc bike (monark exercise ab, stockholm, sweden) using a submaximal strand test procedure and nomogram / accompanying tables for predicting maximal oxygen uptake lmin, vo2max.32 heart rate was monitored using an adjustable polar pulse belt (proterapi a / s, brndby, denmark) and recorded continuously on a computer . The number of children receiving a supply of vitamin d and/or psychologist counseling was noted . To avoid withholding any information relevant for the child s health, the child and parents were informed about the results of the tts and the aerobic power test by the researcher responsible . A test retest study by tornoe et al33 contains a detailed description of test procedures with analyses of repeatability and variability . This study was a blinded block - randomized controlled trial with computer - generated allocation in blocks of ten, administered by a secretary, to place participants into one of two intervention groups, a and b. both interventions were based on short, defined education courses of 10 weeks home training with 34 sessions of supervised training or counseling, approximately 140 minutes in total for each group . The contact nurse and an experienced pediatric physical therapist administered the interventions, which were followed by a 12-week follow - up period in which home practice was advised to continue without supervision . Families were welcome to request psychological counseling after the 10 weeks intervention period, which was to be supported by an interdisciplinary team conference . The physical therapist taught and supervised group a in 34 sessions of progressive specific strength training with a focus on the trapezius muscles with resistive tubing elastics (thera - band; hygenic corporation, akron, ohio, usa) and recording in a prospective diary . Training was to be conducted three times a week at home and comprised alternating four exercises two by two . The progression involved doing one set with ten repetitions and moving to three sets with ten repetitions . The tubing elastics were used in three strengths, red / niveau i, green / niveau ii, and blue / niveau iii, to provide progression of resistance . To determine the level of resistance, a 15 repetition maximum (rm) was used to estimate an exercise resistance equivalent 75%80% of one rm.34 focus was on warm - up exercises for the arms and practicing the exercises . The progressive strength training was based on research and recommendations in the american academy of pediatrics, committee on sports medicine and fitness, policy statement for pediatric training,35 faigenbaum et al36,37 and andersen et al.38 figure 3 provides an illustration of the exercises . During the 10 weeks of intervention, compliance and continuity with the training schedule was secured by carrying out intermediate checks of the training diary and discussion with the child and parents . After the 10 weeks of training, the child and family had a single counseling session with the nurse to avoid withholding help for the family . Group b was provided with need - based counseling based on recommendations for multidisciplinary headache management.39 group b, as an established and evidenced intervention, was the control group, in accordance with international guidelines.15 the interdisciplinary intervention has been proven to be effective in the treatment of children in the clinic.40 the intervention comprised one session with the nurse and two sessions with the physical therapist . The focus was on a motivational dialogue following a checklist to encourage the child and parents to practice self - care through lifestyle changes and physical awareness . The aim was to enhance the child s physical health and well - being at home and at school . The child, with parental support, was encouraged to practice and exercise at home . Table 1 provides a checklist of the topics the nurse and physical therapist used during counseling . The power analysis for this study was based on preliminary data from a case control study and was calculated as follows: two groups of 23 participants were calculated from a mean headache frequency for girls at 20 days (sd 9.6), a significance level of 5%, a power (1) at 80% and an expected difference at 40% . In order not to lose power, subgroup analyses of episodic and chronic tth were avoided . A blinded statistician conducted the statistical analysis . Descriptive statistics were used to describe the data s central tendencies, dispersal, and trends . Mean, median, standard deviation, range, and frequency were used as appropriate . The distribution of data was examined by means of normal probability plots and kolmogorov smirnov test . For all primary and secondary variables, headache frequency and headache duration were analyzed as binomial counts (count parameter equaled 28 days and 24 hours, respectively) using generalized linear models . Headache intensity, peak data, tts, and oxygen uptake were analyzed using two - sample t - test . Headache at test (yes / no) was analyzed by pearson s chi - square test . To investigate whether there was an effect of the intervention and if the effect was different after 10 weeks of intervention and after a further 12 weeks of home practice, the models included time and the interaction between time and intervention . Headache frequency, headache duration, and headache at test were analyzed by use of a repeated measurement analysis for binominal counts . The rest of the variables were analyzed by linear mixed models for repeated measurements by use of the mixed procedure by sas 9.2 . For the primary variables, an analysis based on intention - to - treat, with the last observation carried forward, was also performed . Vitamin d supplementation and counseling by psychologist were analyzed as additional explanatory variables, although the material was too small to fully adjust for four variables . Effect size was described and presented as the odds ratio (or) (95% confidence interval [ci]) of having headache on a random day . Table 2 presents anthropometric and headache characteristics at baseline in the two intervention groups with data of headache frequency, headache intensity, headache duration, age, weight, height, and bmi . Baseline characteristics and hrqol identified deficits in the areas of physical, emotional, and school functioning . In comparison with healthy child reports from varni et al.24 at a 2-year follow - up seven families were, for various reasons, no longer available . Twenty out of 30 potential responders (equivalent to 67%) returned the questionnaires and were equally distributed between group a and b. one girl had moved from home, so one parental report was missing . Fifty - five percent now report little to none disability, and 45% report mild to severe disability . Marked improvements are predominant in the area of physical functioning, school functioning, and total score . Participants and dropouts at baseline, after 10 weeks of intervention, and after 12 weeks of home practice are presented in the results in table 5 . The reasons for dropouts were related to either perceived lack of time or lack of motivation . Six participants dropped out of the interdisciplinary intervention group and four out of the specific strength training group . Five girls in group a and six in group b received a vitamin d supplementation due to low levels of vitamin d on the baseline blood tests . One girl from group a visited the psychologist during the last 12-week home practice period as did five girls from group b. in the two groups a and b, 42% and 44%, respectively, had no headache at baseline test, and there were no significant differences over time and between groups . The mean vas score at baseline test was 1.6 (sd 2.0) for group a and 2.2 (sd 2.6) for group b. no significant differences over time and between interventions were identified . There was a statistically significant effect on headache frequency and duration for both interventions without any significant interactions between time and interventions . For both interventions, the effect on headache frequency was statistically significant after 10 weeks of intervention (p=0.001) and after 12 weeks of home practice (p=0.001). The effect on duration was likewise significant (p=0.022), while the effect on intensity was not significant for either groups . The results from intention - to - treat analyses were identical to the per protocol analyses . The odds of having headache on a random day decreased for group a during 10 weeks of intervention by 0.68 (0.470.99) (or [95% ci]) and by 0.64 (0.440.93) after 12 weeks of home practice . For group b, the odds decreased by 0.62 (0.440.89) (or [95% ci]) during 10 weeks of intervention and by 0.66 (0.450.95) after 12 weeks of home practice . As the interaction between interventions and time was not statistically significant, the pooled or after 10 weeks of intervention was 0.65 (0.500.84) (or [95% ci]) and at the same level after 12 weeks of home practice . Over the 22 weeks, 33% of the girls had an improvement percent above 30%; group a 6/20 girls, group b 7/19 girls . Twenty six percent improved above 50%, specifically, 4/20 girls in the a group and 6/19 girls in the b group . The strength training compliance was very good and all participants managed to train the entire 10 weeks as scheduled . Eighteen girls managed to progress to resistance level ii by the beginning of week 5 . Two of these girls, furthermore, managed to progress to resistance level iii by the beginning of week 8 . Over the 22 weeks, peak shoulder mvc increased 10% in 5/20 girls in the a group and in 2/19 girls in the b group . Four out of twenty in the a group had increased shoulder mvc 20%, while none in the b group had increased shoulder mvc 20% . No significant changes were found for groups . There was a highly significant reduction in neck extension mvc from both interventions after 10 weeks of intervention (p<0.001) and after 12 weeks of home practice (p<0.001). There was also a significant reduction in peak neck flexion mvc for both interventions (p=0.016). The cervicothoracic ratio between neck extension / neck flexion decreased over the 22 weeks from 1.9 to 1.7 for both groups . Predicted vo2max increased 5% in 8/20 girls and 10% in 5/20 girls in the a group . In the b group, 6/19 girls had increased vo2max 5% and 3/19 girls had increased vo2max 10% . In the a group, 4/20 had increased oxygen uptake 15% as did 1/19 in the b group . In the a group, 50% of those girls with a headache reduction of 30% had an increase in vo2max> 5% . The change was within the coefficient of variation between test and retest reported by tornoe et al.33 table 2 presents anthropometric and headache characteristics at baseline in the two intervention groups with data of headache frequency, headache intensity, headache duration, age, weight, height, and bmi . Baseline characteristics and hrqol identified deficits in the areas of physical, emotional, and school functioning . In comparison with healthy child reports from varni et al.24 at a 2-year follow - up seven families were, for various reasons, no longer available . Twenty out of 30 potential responders (equivalent to 67%) returned the questionnaires and were equally distributed between group a and b. one girl had moved from home, so one parental report was missing . Fifty - five percent now report little to none disability, and 45% report mild to severe disability . Marked improvements are predominant in the area of physical functioning, school functioning, and total score . Participants and dropouts at baseline, after 10 weeks of intervention, and after 12 weeks of home practice are presented in the results in table 5 . The reasons for dropouts were related to either perceived lack of time or lack of motivation . Six participants dropped out of the interdisciplinary intervention group and four out of the specific strength training group . Five girls in group a and six in group b received a vitamin d supplementation due to low levels of vitamin d on the baseline blood tests . One girl from group a visited the psychologist during the last 12-week home practice period as did five girls from group b. in the two groups a and b, 42% and 44%, respectively, had no headache at baseline test, and there were no significant differences over time and between groups . The mean vas score at baseline test was 1.6 (sd 2.0) for group a and 2.2 (sd 2.6) for group b. no significant differences over time and between interventions were identified . There was a statistically significant effect on headache frequency and duration for both interventions without any significant interactions between time and interventions . For both interventions, the effect on headache frequency was statistically significant after 10 weeks of intervention (p=0.001) and after 12 weeks of home practice (p=0.001). The effect on duration was likewise significant (p=0.022), while the effect on intensity was not significant for either groups . The results from intention - to - treat analyses were identical to the per protocol analyses . The odds of having headache on a random day decreased for group a during 10 weeks of intervention by 0.68 (0.470.99) (or [95% ci]) and by 0.64 (0.440.93) after 12 weeks of home practice . For group b, the odds decreased by 0.62 (0.440.89) (or [95% ci]) during 10 weeks of intervention and by 0.66 (0.450.95) after 12 weeks of home practice . As the interaction between interventions and time was not statistically significant, the pooled or after 10 weeks of intervention was 0.65 (0.500.84) (or [95% ci]) and at the same level after 12 weeks of home practice . Over the 22 weeks, 33% of the girls had an improvement percent above 30%; group a 6/20 girls, group b 7/19 girls . Twenty six percent improved above 50%, specifically, 4/20 girls in the a group and 6/19 girls in the b group . The strength training compliance was very good and all participants managed to train the entire 10 weeks as scheduled . Eighteen girls managed to progress to resistance level ii by the beginning of week 5 . Two of these girls, furthermore, managed to progress to resistance level iii by the beginning of week 8 . Over the 22 weeks, peak shoulder mvc increased 10% in 5/20 girls in the a group and in 2/19 girls in the b group . Four out of twenty in the a group had increased shoulder mvc 20%, while none in the b group had increased shoulder mvc 20% . No significant changes were found for groups . There was a highly significant reduction in neck extension mvc from both interventions after 10 weeks of intervention (p<0.001) and after 12 weeks of home practice (p<0.001). There was also a significant reduction in peak neck flexion mvc for both interventions (p=0.016). The cervicothoracic ratio between neck extension / neck flexion decreased over the 22 weeks from 1.9 to 1.7 for both groups . Predicted vo2max increased 5% in 8/20 girls and 10% in 5/20 girls in the a group . In the b group, 6/19 girls had increased vo2max 5% and 3/19 girls had increased vo2max 10% . In the a group, 4/20 had increased oxygen uptake 15% as did 1/19 in the b group . In the a group, 50% of those girls with a headache reduction of 30% had an increase in vo2max> 5% . For the b group the change was within the coefficient of variation between test and retest reported by tornoe et al.33 the participants in this study reported deficits at baseline in hrqol in physical, emotional, and school functioning areas, with school functioning obtaining the lowest score . This corresponds with a study by kaczynski et al41 at an american tertiary headache clinic, reporting that headache associated with impaired school functioning for children with tth.41 kaczynski et al41 also report that children with tth were more likely to choose passive coping strategies, such as withdrawal and nonparticipation . Another aspect they reported was that school difficulties were associated with a protective parental strategy . These results highlight the importance of examining coping strategies, family dynamics, and promoting self - active strategies . Our study documents that for the pooled sample, hrqol improvements are maintained at the 2-year follow - up, in particular in physical functioning, school functioning, and total score . One of our previous studies indicated a statistically significant association between headache and reduced strength of neck shoulder muscles and aerobic power, combined with increased pericranial tenderness, which supports the perceived deficits in physical health.13 we hypothesized that strength training of the shoulder muscles, especially the trapezius descendens, would enhance the girl s physical capability as an active coping strategy and thus provide a reduction in headache frequency . We measured health - related outcomes such as muscle strength and predicted vo2max, as well as headache . The results from this study showed a statistically significant and clinically important reduction in headache frequency and duration due to strength training and interdisciplinary counseling . Both groups also seemed to benefit from exercising or body awareness, with a significant and not anticipated reduction in neck extension mvc and neck extension / flexion ratio . The reduction indicates a positive change in muscle balance over the neck . Over the 22 weeks, the ratio decreased from 1.9 to 1.7, pointing toward a possible trend that adjustment of muscle balance precedes strength gains . Cervicothoracic neck extension / flexion ratio is reported to be stable over years and between sexes and is reported at a level of 1.71.842,43 in adults . In our case control study, 41 healthy girls had a neck extension / flexion ratio at 1.8 . Faigenbaum et al44 underscore that children, due to sedentary lifestyles, may, from a young age, not develop adequate muscle strength and motor performance skills and that a deficit in muscular fitness might affect outcomes of strength training . We have not examined asymptomatic prerequisite motor skills, but based on the results we might hypothesize that the first learning outcome these girls accomplished from training was adjusting muscle balance . The compliance with the moderate strength training program we piloted was good, and we can now conclude that it has a potential to provide important changes in physical capability both in terms of adjusting muscle balance and enhancing shoulder strength and vo2max, leading to headache reduction and health improvements . The exercise volume and intensity versus exercise variation should be considered in order to keep a stable weekly volume . Further research is still needed to examine the outcomes that children, boys and girls with tth, gain from different intensities and energy expenditures of exercising . Also, the relationship between predicted vo2max and tth in girls needs further research . Results from a study with adults with tth by sderberg et al10 showed significant effects on headache frequency from physical training . Training comprised ten supervised sessions with strength exercises comprising 335 repetitions of five exercises, with each session commencing with 10 minutes of warm - up on an ergometer bicycle.10 the exercise volume was higher than that used in the present study, and the amount of repetitions would probably not be suitable for children . Although the results achieved in muscle strength and aerobic power were minor, counseling also included mutual agreements of how to enhance the girl s level of physical activity . None of the groups showed reductions in pericranial tenderness, suggesting that the girl s myofascial pain sensibility did not alter over a period of 6 months . The significant worsening of pericranial tenderness was not anticipated and is interpreted as being due to a rather large coefficient of variation,33 which needs to be further studied . As enhanced pericranial tenderness is seen as a consequence of frequent or chronic tth,45 the lack of reduction in tenderness might indicate that expectations of recovery from a chronic headache situation have a prolonged perspective . Dropouts were explained by a perceived lack of either time and/or motivation . To limit the number of sessions and at the same time reach the child s motivation to choose active coping strategies, innovative learning strategies should be considered . Digital learning and support combined with social events for the child and family might support an interactive learning situation . Physical therapists can work to find new ways of encouraging the child to do physical exercise with, and not despite, electronic devices to counteract a sedentary lifestyle . This study did not examine knowledge improvements or motivation for change / barriers for change at an individual level . Baker et al46 recommended identifying barriers for change in interventions at both an organizational and individual level, although evidence of the effect of identifying barriers is not yet clear . Consequently, more research is needed that examines the needs of contemporary families and that explores the barriers parents and children face in order to reinvent social and interactive physical behaviors that benefit the child s health and well - being . The 1989 un convention on the rights of the child states that the best interests of children must be the primary concern in making decisions that may affect them . This was our main concern when setting up the study and meant that we did not establish a control group without treatment or a placebo treatment . We dealt with interventions as defined by the complexity of involving a variety of health - care professionals at various organizational levels with different behaviors . The study targeted families with an array of behavioral cultures, expectations, and different degrees of compliance . In complex interventions, we are challenged by the difficulty of isolating the active ingredients of an intervention and have therefore focused on who accomplished what . Some of the strengths of this study include the effort put into controlling for influencing factors, the well - examined test methodology, and the blinded statistical analyses . The fact that we only managed to include girls turned out to be a statistical strength avoiding subgroup analyses . We did not examine the situation of the contemporary family to explore the knowledge gained or the process of change, but these aspects are needed in future research involving these children and their parents . Both educating girls with tth in a moderate strength training program for a period of 10 weeks and doing need - based motivational counseling for lifestyle changes have a significant effect on headache frequency and duration with no significant between - group difference . The majority of the girls reported little to none disability, with a total score approaching normative data . The results indicate that both physical health and hrqol can be influenced significantly by physical exercise and nurse counseling . However, we need to know more about how the dosing and energy expenditure in exercising affects both girls and boys with tth . Face - to - face supervision of the exercise is recommended to ensure its quality and to prevent adverse effects . Imparting knowledge about healthy lifestyles and active coping strategies, such as physical exercise, supported by digital learning and support, social activities, and events, is a possible option for limiting the amount of sessions necessary while simultaneously supporting interactive learning . The 1989 un convention on the rights of the child states that the best interests of children must be the primary concern in making decisions that may affect them . This was our main concern when setting up the study and meant that we did not establish a control group without treatment or a placebo treatment . We dealt with interventions as defined by the complexity of involving a variety of health - care professionals at various organizational levels with different behaviors . The study targeted families with an array of behavioral cultures, expectations, and different degrees of compliance . In complex interventions, we are challenged by the difficulty of isolating the active ingredients of an intervention and have therefore focused on who accomplished what . Some of the strengths of this study include the effort put into controlling for influencing factors, the well - examined test methodology, and the blinded statistical analyses . The fact that we only managed to include girls turned out to be a statistical strength avoiding subgroup analyses . We did not examine the situation of the contemporary family to explore the knowledge gained or the process of change, but these aspects are needed in future research involving these children and their parents . Both educating girls with tth in a moderate strength training program for a period of 10 weeks and doing need - based motivational counseling for lifestyle changes have a significant effect on headache frequency and duration with no significant between - group difference . The majority of the girls reported little to none disability, with a total score approaching normative data . The results indicate that both physical health and hrqol can be influenced significantly by physical exercise and nurse counseling . However, we need to know more about how the dosing and energy expenditure in exercising affects both girls and boys with tth . Face - to - face supervision of the exercise is recommended to ensure its quality and to prevent adverse effects . Imparting knowledge about healthy lifestyles and active coping strategies, such as physical exercise, supported by digital learning and support, social activities, and events, is a possible option for limiting the amount of sessions necessary while simultaneously supporting interactive learning.
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Fat embolism syndrome (fes) is a rare and potentially lethal complication of long bone fractures . The incidence of clinically significant fes occurs only in 0.9 - 2.2% of long - bone fractures . Hypoxia or respiratory distress, deteriorating mental status, and petechial hemorrhage are the main diagnostic criteria; secondary diagnostic signs include tachycardia, fever, anemia, and thrombocytopenia typically seen in the context of long - bone fracture . Neurologic symptoms can be transient and widely varies from a diffuse encephalopathy to focal deficits . Fat emboli can pass through the pulmonary vasculature, resulting in systemic embolization, most commonly in the brain and kidneys . In this article, we aimed to highlight the diagnosis and treatment of cerebral fat embolism through an illustrative a case developing change of consciousness, respiratory failure and epileptic seizures after long - bone fracture caused by gunshot injury . A 20-year - old male patient was admitted to the emergency department with sudden change in consciousness and muscle contractions after 1 day follow - up in a hospital for long bone fractures of femur and tibia due to gun - shot injury . On admission, he had generalised tonic clonic seizures and his glascow coma scale was seven with localization of painful stimuli, incomprehensible muttering, and no eye opening . In the physical examination, blood pressure was 130/80 mmhg, pulse was 128/min, breath rate was 22/min and body temperature was 38c . No lateralising motor deficit was found . There was a long bone splint on the left lower extremity . The results of hematologic and biochemical parameters of the patient with the range of normal values of our laboratory in the paranthesis were as follows at first admission: hemoglobine: 11.1 gr / dl (13,6 - 17,2), platelet count: 75000/l (156000 - 373000), glucose: 189 mg / dl (70 - 110), aspartate aminotransferase (ast): 143 iu / l (0 - 46), alanine aminotransferase (alt): 53 iu / l (0 - 46), and creatine phosphokinase (cpk): 7866 iu / l (35 - 195). Other biochemical findings and blood gas analysis were normal . There were no abnormalities on a postero - anterior chest x - ray cervical and thoracic computed tomography (ct). An initial ct of the head was unremarkable for intracranial abnormalities . Magnetic resonance imaging (mri) was performed diffusion weighted mri trace images with a 2 mm slice gap, 5 mm slice thickness, echoplanar imaging (epi) sequence type, b value of 1000 mm / s, signal intensity dots on a dark background . Diffusion coefficient map slice of the patient revealed the hypointense punctuate to lesions in centrum semiovale and periventricular regions [figures 1b and 2b]. Diffusion weighted cranial magnetic resonance image reveals starfield pattern; bilaterally localised multiple hyperintense punctate lesions in centrum semiovale regions (arrows). The slice thickness of the images was 5 mm, echoplanar imaging was used and the b value of the slices was 500 diffusion weighted magnetic resonance image reveals starfield pattern; multiple milimetric sized hyperintense lesions (arrows) due to restricted diffusion areas in periventricular regions . Echoplanar imagings were taken with slice thickness of 5 mm and b value of 1000 diffusion coefficient map slice of the patient reveals the hypointense punctuate to lesions (arrows) in centrum semiovale region . The slice thickness of the images was 5 mm, echoplanar imaging was used and the b value of the slices was 500 diffusion coefficient map slices of the patient reveals the hypointense punctate lesions (arrows) in periventricular region . The slice thickness was 5 mm, echoplanar imaging with b value of 500 was used an electroencephalogram was performed and diffuse bioelectrical delay on the right hemisphere was found . With the above findings, the diagnosis of the case was cerebral diffuse fes . Antiepileptic therapy with phenytoin (2 150 mg) and low molecular weighted heparin therapy with enoxaparin sodium (2 4000 u) were administered . The patient had hypoxemia and the arterial blood gas analysis revealed pao2 as 65 mmhg, and saturation of arterial oxygen as 82% requiring mechanical ventilation, disturbance of conscioussness and sudden drop in hemoglobin and platelet count . Hemoglobin was 7.1 gm / dl and platelet count had decreased to 53000/l at that time . The patient was rehydrated and administered with sulbactam / ampicillin (4 1 g i.v .) For fever and a possible infection due to open injuries . Three units of erhthrocyte suspension and 3 units of fresh frozen plasma were given to patient to replace blood loss . On the 5 day of hospitalization, the level of consciousness of the patient improved and mechanical ventilatory support could be discontinued . Fes was first described in 1862 after an autopsy identified fat in the pulmonary vasculature following a crush injury . Pinney et al ., reported a fes rate of only 4% in a study of 274 consecutive patients with isolated femoral shaft fractures . Fat globules generated within the systemic circulation may cause pulmonary dysfunction, neurological changes, dermal symptoms, and dysfunction of several other organs . The incidence of symptomatic fat embolism was found much lower (0.9%) than the incidence of fat globules within the circulation (22.0%). Long bone fractures are known as recognized to be important risk - factors for fes, especially femur fracture, followed by tibia fracture . Most of the fes were reported 24 - 72 h after long bone fractures . In the present case neurological symptoms can be transient and widely varied from a diffuse encephalopathy to focal deficits . The most common clinical symptoms were reported as hypoxemia (pao2 <70 mmhg), change of consciousness, infiltrates infiltration findings on chest plain film, and sudden drop in hemoglobin and platelets . The present patient also had hypoxemia requiring mechanical ventilation, disturbance of conscioussness and a sudden drop in platelet count . Gregorakos et al ., described two teenage males suffering from prolonged coma secondary to fes due to lower extremity fractures from a motor vehicle collision . Mri showed multiple areas of increased signal intensity in the cerebral white matter in the first patient and diffuse high signal intensity in periventricular and subcortical white matter in the second one . In a patient with fes, chang et al ., reported multifocal high signal intensity changes in bilateral cerebral hemispheres and cerebellum in diffusion weighted magnetic resonance images . The majority of case reports on fes found that cerebral dysfunction associated with fes to be reversible . Parizel et al ., described a teenage female who fractured her left tibia secondary to motor vehicle injury . Diffusion weighted mri showed punctate foci of high signal intensity in subcortical white and gray matter and the patient had full neurological recovery by 1 week and repeated magnetic resonance images 4 weeks after the accident revealed disappearance of signal abnormalities . The outcome of patients with fes who receive supportive care is generally complete resolution of pulmonary, neurological, and dermatological lesions with a mortality of less than 10% . Cerebral fat embolism is extremely rare but it should be suspected in trauma patients with long - bone fractures accompanied by unexplained neurological or respiratory deterioration.
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As a result of a massive scale - up in malaria control programs by the who as part of the millennium development goals, the number of malaria deaths decreased worldwide by about 20% between 2000 and 2009 1 . Although this represents some progress in reducing the disease burden, malaria nevertheless remains a major global health threat and continues to cause high morbidity and mortality, especially in sub - saharan africa where almost 600 million people are at risk . Malaria persists due to the lack of an efficacious vaccine, the spread of drug - resistant parasites, and the emergence of insecticide - resistant anopheles mosquito vectors as well as changing agricultural practices, global warming, and human migration 2,3 . Refugees exposed to malaria and other infectious diseases in their country of origin or in camps who immigrate to developed countries may be important reservoirs and further hasten the spread of malaria to other regions 4 - 6 . Of the plasmodium parasites (p. falciparum, p. vivax, p. malariae, p. ovale curtisi, p. ovale wallikeri, and p. knowlesi) that cause human malaria, p. falciparum is the most virulent and deadly parasite 7, 8 . Most deaths due to p. falciparum infection occur in sub - saharan africa, with 92% of the annual malaria deaths occurring among infants and young children <5 years of age 1,9 - 11 . In this region, these individuals and pregnant women, especially primigravidae, are the groups with the highest risk for the development of severe and fatal complications of p. falciparum infection especially cerebral malaria (cm) and severe malarial anemia (sma), 2,9 - 11 . Unprotected travelers and other non - immune individuals are also extremely vulnerable to these complications . Immunity to infection with blood - stage plasmodium parasites is critically dependent on the type 1 cytokine ifn- and requires coordinate and timely innate and adaptive immune responses involving dendritic cells (dc), nk cells, cd4 t helper cells, and b cells 12,13 . Importantly, a balance between pro - inflammatory and anti - inflammatory responses is essential to limit the development of life - threatening immune - mediated pathology such as cm and sma 14 . Although a clearer understanding of the mechanisms involved in protective immunity and immunopathology is emerging, our understanding of the regulatory mechanisms required to maintain the balance between beneficial and deleterious responses during blood - stage malaria infection remains limited . Here, we review recent findings from our laboratory and others in experimental models of malaria in mice and in plasmodium - infected humans on the activation and expansion of cd11c dc and foxp3 regulatory t cells and their possible roles in regulating adaptive immunity to blood - stage malaria . The life cycle of the malaria parasite is complex with a sexual phase in the anopheles mosquito vector and an asexual phase in the host 12 . Sporozoites are transmitted to the host by the bite of an infected female mosquito and travel via the blood to the liver where they invade hepatocytes and initiate the exo - erythrocytic or liver stage, which is clinically silent and without any pathologic sequelae . Parasite multiplication in the liver results in merozoites that infect red blood cells (rbc), where they undergo mitosis and form erythrocytic schizonts containing many merozoites that are released and infect new rbc . As yet unknown factors trigger some merozoites to differentiate into male and female gametocytes that are taken - up during a blood meal by the mosquito for fertilization and sporozoite production 15 . The major manifestations of malaria in infected individuals include fever, malaise, splenomegaly, and anemia and are due to the cyclical multiplication of merozoites in rbc 12,14 . During p. falciparum infection, the spectrum of severe pathology is broad and includes metabolic acidosis, cm, and sma, and is typically accompanied by hypoxia, hypoglycemia, and lactic acidosis due in part to the increased metabolic demands of the parasite 12,14 . P. falciparum, the most virulent and deadly human parasite, is highly prevalent in sub - saharan africa 1 . P. vivax is the most common species causing human infection and predominates outside africa in south america and asia while infections with p. malariae and p. ovale subspecies are usually mild . Recently, human infections with p. knowlesi, a parasite naturally occurring among monkeys, have been identified in malaria endemic areas 8 . Numerous challenges such as the lack of access to appropriate tissue samples and the inability to manipulate the immune response to elucidate underlying mechanisms impede the study of malaria in humans 16 . As an alternative, experimental models in non - human primates and rodent models have been used . However, the lack of availability and high cost as well as ethical issues limit the use of non - human primates to study malaria . Infection of inbred mice with various rodent plasmodium species (p. chabaudi, p. berghei, p. vinckei and p. yoelii) that differ in virulence and pathology provide the most convenient experimental models (table 1) 12,17 . It is important to note that while no single rodent model replicates all of the features of human malaria in terms of pathophysiology or immune responses, these models have been essential in defining the basis of interactions between the host and the plasmodium parasite . Studies in mouse malaria models have revealed important and novel information leading to a clearer understanding of the immune response in protection and pathogenesis, to identify previously unrecognized genes that regulate susceptibility to malaria, and to develop malaria vaccines and novel chemotherapeutic agents 12,17 - 21 . Because p. chabaudi as is considered to share many biological and immunological features in common with p. falciparum, infection of mice with this parasite is widely used as a tool to study immune responses to blood - stage malaria 12,17,19 . Infection of resistant c57bl/6 (b6) mice with 10 - 10 p. chabaudi as infected rbc (irbc) results in a course of parasitemia characterized by parasite replication during the first week post infection (p.i .) Culminating in a moderate peak parasitemia of 30 - 35% between days 8 - 10 p.i . 12,17,19 . In a seminal study, grun and weidanz demonstrated in mice infected with p. chabaudi that: 1) the control of peak parasitemia is dependent on a cell - mediated immune response that we now know requires cd4 t cells, and 2) the requirement for b cells in the clearance and elimination of the parasite 12 . Later studies from our laboratory showed that control of acute p. chabaudi as infection is critically dependent on il-12-dependent ifn- production by nk cells and cd4 th1 cells indicating the importance of both innate and adaptive immune responses 12 . The importance of cognate interactions between cd4 th cells and b cells for malaria - specific antibody production for efficient parasite elimination later during infection was demonstrated by langhorne and her colleagues 12,17 . Identification of the importance of nk cells, cd4 t cells, and b cells in mediating control and resolution of p. chabaudi as infection has provided a framework to delineate the accessory cells and cytokines involved in activating innate immunity and triggering adaptive immunity to malaria . Studies in mice infected with avirulent and virulent strains of p. yoelii as well as in mice infected with p. berghei anka have also provided important information about the role of the immune response in protection and immunopathogenesis 14,17,21 . Dc are a heterogeneous and highly specialized population of antigen presenting cells (apc) that provide a central link between innate and adaptive immune responses and play an important role at the host - pathogen interface including in response to plasmodium parasites 22 - 25 . Dc reside in almost all tissues where they play important roles in surveillance and sensing foreign antigens . Pattern recognition receptors, such as toll - like receptors (tlrs), nod - like receptors, and c - type lectins, expressed by these cells are important in distinguishing a diverse but overlapping repertoire of conserved microbial molecules 26 . Dc precursors are generated from pluripotent stem cells in the bone marrow under the influence of lineage specific growth factors and are released into the blood for transit to lymphoid organs, including secondary lymphoid tissues such as the spleen as well as non - lymphoid tissues such as the skin, where they reside as immature cells 22 . Following encounter with antigen, immature dc undergo maturation in response to stimulation by pathogens or cytokines and other soluble mediators produced by various cell types . Dc maturation results in a decreased ability to capture and process antigen and increased surface expression of mhc - ii and co - stimulatory (cd40, cd80, cd86) and adhesion molecules 22 - 27 . Dc maturation is critical in immunity to pathogenic microorganisms because of the potent ability of these cells to polarize the differentiation of nave cd4 t cells to th cell subsets including th1, th2, th17, follicular th cells, and induced regulatory t cells 28,29 . Dc also function as accessory cells and are important in activating nk cells for cytotoxicity and cytokine secretion in response to viruses and tumors 30 . As a result of interactions with t cells, nk cells and other effector cells, dc, thus, are not only important in antigen presentation and activating cd4 t cells but also function as accessory cells to regulate and amplify innate and adaptive immune responses . The relationship between dc maturation and malaria has been investigated in studies using peripheral blood monocytes from humans and murine dc 25,31 . Initially, it was shown that dc function is compromised during malaria based on the in vitro observation that binding of p. falciparum irbc to human monocyte - derived dc inhibits maturation and reduces the capacity of the dc to function as an apc 32 . Analysis of dc recovered from african children with severe p. falciparum malaria revealed that the frequency of myeloid - derived cd11cbdca3dc is significantly increased during acute infection 33 . A recent study comparing dc subpopulations in individuals infected with p. falciparum or p. vivax and residing in areas with similar endemicity found a significant increase in plasmacytoid cd123dc and a decreased ratio of myeloid to plasmacytoid dc during infection, regardless of the infecting species 34 . In this study, the maturation of peripheral blood dc was observed to be impaired ex vivo as shown by low cd86 expression in a proportion of the p. vivax - infected patients, but there was no evidence that dc maturation was affected in p. falciparum - infected individuals . These data suggest that dc subpopulations are differentially affected in humans during blood - stage malaria and that the variability observed in the effects of malaria infection on dc function may be due to the plasmodium species, the severity of the infection, and the patient population studied . Similar observations of diverse effects of malaria infection on dc have been made in studies using various combinations of parasites and inbred mouse strains . Distinct subpopulations of dc including myeloid - derived dc (cd11ccd4, cd11ccd8, cd11ccd8), plasmacytoid dc (cd11cb220), and a regulatory subset of cd11ccd45rb dc have been observed to expand in the spleens of mice infected with various plasmodium parasites 27,31,35 - 38 . The development of transgenic mice expressing a t cell receptor (tcr) specific for a peptide of the p. chabaudi merozoite surface protein-1 (msp1) has provided a novel tool to elucidate the role of dc and other cells in the immune response to malaria 39 . Using transgenic msp1-specific tcr mice infected with p. chabaudi as, sponaas et al provided direct evidence that cd11c dc in the spleen activate plasmodium - specific cd4 t cells 38 . This study also provided evidence that splenic dc subpopulations have distinct antigen presenting functions during malaria . Although both cd8 and cd8 dc present malaria peptides and induce ifn- production by cd4 t cells, only cd8 dc stimulate antigen - specific proliferation of cd4 t cells and secretion of il-4 and il-10 . Recently, lundie et al reported another strategy to study the immune response to malaria that involves the generation of genetically - modified strains of p. berghei anka expressing mhc - i- and mhc - ii - restricted t cell epitopes specific for cognate antigens, such as ova, linked to reporter gfp 40 . Using the genetically modified parasites to infect ot - i or ot - ii transgenic mice, these investigators observed that both cd8cd4 and cd8 but not cd8cd4 dc present p. berghei - expressed antigens to cd4 t cells during malaria infection, while the cd8 subpopulation induces pathogenic cd8 t cells via cross - presentation 40 . In a separate study, blood - stage p. berghei anka infection was found to cause suppression of mhc - i - restricted immunity to non - malarial antigens as a possible consequence of systemic dc activation 41 . This observation suggests that dc are not directly suppressed by malaria parasites, but that they follow their normal maturation pathway after activation by irbc or parasite products . Subsequent findings by lundie et al showed that dc isolated from p. berghei - infected mice efficiently present p. berghei - expressed antigens to cd4 and cd8 t cells on days 2 and 3 p.i ., but their ability to present parasite antigens as well as mhc - i- and mhc - ii - restricted, non - malarial antigens is drastically impaired by day 4 p.i . These findings are consistent with earlier studies by perry et al in p. yoelii - infected mice which showed that dc become refractory to tlr - ligand - stimulated il-12 and tnf- production with increased il-10 secretion as the infection progresses 43 . Dc from infected mice were found to retain the capacity to stimulate ova - specific proliferation of nave ot - ii cd4 t cells throughout p. yoelii infection, but the responding t cell phenotype changed later during infection from cd4ifn- to cd4il-10 t cells . However, functional impairment of dc at early times p.i . During lethal infections due to p. yoelii ym, p. vinckei or p. berghei anka has been observed based on the inability of dc from infected mice to secrete high levels of il-12 and prime nave cd4 t cells 31 . It was reported that the decreased function of dc in mice infected with p. vinckei or p. berghei is mediated in part by tnf- 31 . In contrast, lundie and colleagues failed to demonstrate a role for tnf- in impaired dc function during p. berghei anka infection 42 . As will be described below, spleen dc recovered from p. chabaudi as - infected b6 mice during the first week p.i . Altogether, studies in mouse models essentially confirm the findings in human studies and provide further explanation that the conflicting data obtained in human studies may be due to several factors including the parasite species and strain, the time after infection, and the size of the inoculum . Studies performed using human peripheral blood mononuclear cells and in plasmodium - infected mice indicate that nk cells are an early and crucial source of ifn- during blood - stage malaria 12 . Findings in experimental models of infection, especially viral infections, tumors, and inflammatory diseases, provide important evidence that optimal ifn- production by nk cells requires contact - dependent and -independent interactions with myeloid - derived accessory cells such as monocytes, macrophages, and dc 30 . As will be described below, we investigated if an accessory cell is necessary to promote ifn- secretion by nk cells during p. chabaudi as infection and questioned if cd11c dc are involved . Previous studies in b6 mice showed that during early p. chabaudi as infection dc, macrophages, and nk cells rapidly accumulate and expand in the spleen where they undergo maturation and functional activation in response to irbc captured in the spleen by phagocytosis (reviewed in 44). During early infection, cd11c dc migrate from the marginal zone to the cd4 t cell - rich periarteriolar lymphoid sheaths while macrophages expand and remain in the red pulp 45 . Importantly, within the first week p.i ., spleen dc from p. chabaudi as - infected b6 mice display up - regulation of mhc - ii and co - stimulatory molecule expression, secrete pro - inflammatory cytokines including high levels of il-12, and stimulate nave cd4 t cell proliferation and ifn- production 25,27,31,45 . In contrast, splenic macrophages from p. chabaudi as - infected mice suppress t cell proliferation and il-2 secretion 46,47 . Together, these findings provide compelling evidence that cd11c dc play an important role in innate immunity during blood - stage malaria and trigger an adaptive type 1 immune response necessary for control and rapid resolution of infection . To determine if dc have a role in activating nk cells during blood - stage malaria, we compared the maturation and function of cd11c dc purified by magnetic beads from the spleens of infected wild - type (wt) b6 mice and il-12p40 and il-15 mice that lack cytokines involved in nk cell proliferation, survival and activation 44 . In previous studies, we demonstrated that the course of p. chabaudi as infection is more severe in il-12p40 mice compared to wt and il-15 mice (12, reviewed in 44). We observed that the uptake of irbc by cd11c dc early after infection was similar in wt, il-12p40, and il-15 mice . Dc from infected il-15 and wt mice expressed similar levels of cd40, cd80, and cd86 while dc from infected il-12p40 mice expressed significantly lower levels of co - stimulatory molecules . Analysis of cytokine production in response to in vitro stimulation with irbc revealed that dc recovered from il-12p40mice on day 4 p.i . Produced significantly lower levels of pro - inflammatory cytokines compared to wt dc but secreted significantly more il-2 while the levels of il-10 secreted by wt, il-12p40, and il-15 dc were similar . These data indicate that cd11c dc from infected il-12p40mice are as efficient as dc from infected wt and il-15 mice in phagocytizing irbc but that dc maturation is impaired in infected il-12p40mice . To investigate if dc from p. chabaudi as - infected mice promote ifn- production by nk cells, we established co - cultures of purified cd11c dc from infected wt mice with cd3dx5 nk cells purified from the spleens of nave wt mice and analyzed nk cell expression of intracellular ifn- expression before and after co - culture with dc by flow cytometry 44 . Ifn- levels in the supernatants of the co - cultures were also determined . As shown in fig . 1a, few cd3dx5 nk cells from nave wt mice expressed intracellular ifn- prior to co - culture with dc from infected wt mice . After co - culture with dc from infected mice, there was a dramatic increase in the frequency of dx5ifn- nk cells . Although the frequency of cd11cifn- dc also increased in the co - cultures, the major source of ifn- was observed to be nk cells, a finding corroborated when dc from infected wt mice were co - cultured with nk cells from nave ifn- mice 44 . Determination of the levels of ifn- in the supernatants confirmed that wt dc from infected mice stimulated significantly higher ifn- secretion than wt dc from nave mice (fig . We also determined the relative contribution of il-12 and il-15 to dc - nk cell interactions during malaria by co - culturing purified cd11c dc from infected il-12p40 or il-15 mice with nk cells from nave wt mice . Dc from infected il-15 mice stimulated a similar percentage of wt nk cells to express intracellular ifn- as wt dc, and ifn- levels were similar in supernatants of wt nk cells co - cultured with wt dc and il-15 dc . In contrast, dc from infected il-12p40 mice are unable to promote ifn- expression by nk cells from nave wt mice (fig . These data are consistent with the observations described above linking lethal malaria infections in mice with functional impairment of dc and indicate that soluble mediators, especially il-12, are required to induce optimal ifn- secretion by nk cells during blood - stage malaria . We also observed that dc - nk cell interaction is dependent on cell - cell contact since significantly lower ifn- production was observed when nk cells and dc were separated by a transwell insert . Reciprocal crosstalk between dc and nk cells has been demonstrated in a number of studies, with evidence of complex interactions between the two cell types 30,48 . Nk cells activated by tumors, bacterial products, or il-2 have been shown to induce dc maturation and secretion of the th1-promoting cytokine il-12 30,48 . To determine if malaria - activated nk cells induce dc maturation, we co - cultured purified dx5 nk cells from day 5 infected wt mice with cd11c dc from nave wt mice which were exposed to irbc for 1 hr prior to co - culture with nk cells to prevent lysis of immature dc by the activated nk cells 44 . Dc from nave mice co - cultured with nk cells from infected wt mice displayed significantly increased cd40 and cd86 expression compared to control dc cultured alone or with nk cells from nave wt mice (fig . Up - regulation of co - stimulatory molecule expression on dc co - cultured with malaria - activated nk cells was found to be dependent on cell contact . Co - culture with malaria - activated nk cells but not nk cells from nave mice also stimulated dc to secrete high levels of cytokines especially il-12 (fig . Dc separated from nk cells by a transwell insert showed similar levels of cytokine production as control co - cultures indicating that soluble factors produced by nk cells and not cell - cell contact are responsible for enhanced il-12 production by dc . The importance of nk cell - derived ifn- in inducing enhanced production of th1-promoting il-12 by dc was demonstrated by co - culture of wt dc with nk cells from p. chabaudi as - infected ifn- mice and by the addition of anti - ifn- monoclonal antibody to co - cultures of wt dc and wt nk cells . In the absence of nk cell - derived ifn-, importantly, dc stimulated by malaria - activated nk cells were found to be fully licensed apc and primed nave cd4 t cells to proliferate and produce the type 1 cytokine ifn- indicating th1 polarization 44 . Our observations in the mouse model of p. chabaudi as infection confirmed in vitro studies by newman et al in human peripheral blood mononuclear cells stimulated with p. falciparum irbc showing that ifn- production by nk cells only occurs following multiple contact - dependent and cytokine - mediated signals derived from myeloid - derived dc and monocytes 49 . Importantly, reciprocal activation of accessory cell maturation by malaria - activated nk cells was observed in the studies with pbmc and in our studies in mice . These findings indicate that interactions between accessory cells and nk cells during malaria are bi - directional similar to virus and tumor models 30,44,48,49 . To address if nk cells are required to induce dc maturation and function in vivo, we treated p. chabaudi as - infected wt mice with anti - asialo - gm1 antibody and examined dc maturation and th cell polarization . As we observed previously, infected b6 mice treated with anti - asialo - gm1 antibody mice display a course of infection characterized by a significantly higher peak parasitemia and significantly higher recrudescent parasitemia during the chronic phase of infection 12,44 . It should be noted that a similar course of p. chabaudi as infection is also evident in il-12p40 mice which are deficient in nk cell secretion of ifn- during early infection 50 . During infection, cd11c dc purified from the spleens of nk cell - depleted wt mice expressed lower levels of cd40 and cd86 and produced significantly less il-12 and more il-2 in response to irbc in vitro than dc from isotype control - treated wt mice . Impairment of dc maturation and il-12 production in infected nk cell - depleted wt mice resulted in inefficient priming of nave cd4 t cells for malaria antigen - specific proliferation and ifn- secretion compared to dc from isotype control - treated wt mice . Together, these data provide in vivo evidence that dc - nk cell crosstalk is required for dc maturation and function and optimal ifn- production by nk cells and cd4 t cells during p. chabaudi as infection . Recent studies have focused on the role of cd4 regulatory t cells in regulating adaptive immune responses during plasmodium infection . In particular, natural cd4foxp3 regulatory t cells have been shown to be important in determining the outcome of infection with various intracellular pathogens, including protozoan parasites 51,52 . Cd4foxp3 t cells have been shown to inhibit effector t cell responses during infection, resulting in inadequate immune control of the pathogen and persistence of low - level infection . It has also been observed that the potent ability of natural regulatory t cells to modulate effector t cell responses is important in limiting the development of tissue damage during infection with protozoan parasites 51,52 . Studies in individuals with malaria and in mice infected with various rodent plasmodium species have revealed an important expansion of cd4cd25foxp3 t cells 53 - 55 . Experimental p. falciparum infection in human volunteers showed that the magnitude of expansion of this t cell population correlates with high parasitemia levels and low pro - inflammatory responses, findings that suggest a possible link between natural regulatory t cells and the clinical outcome of malaria 53 . Experiments to determine the impact of cd4cd25foxp3 t cells in plasmodium - infected mice by depleting cd25 t cells have led to inconsistent findings 53 - 55 . Although there are several possible explanations for the inconsistencies, including the concern that antibody depletion of cd25 t cells may result in the loss of nave and effector t cells, the results of these studies are consistent with the notion that natural regulatory t cells suppress pro - inflammatory responses involved in protective immunity as well as immunopathology during malaria 53 - 55 . We investigated the role of cd4foxp3 regulatory t cells during p. chabaudi as infection using several approaches as alternatives to in vivo depletion of cd25 t cells 56 . First, we compared the course and outcome of p. chabaudi as infection and malaria - specific immune responses in wt mice and transgenic mice generated by khattri et al 57 that overexpress the foxp3 transcription factor . Compared to infected wt mice, infected foxp3 transgenic mice develop fulminant peak parasitemia levels and 100% of these mice succumb to the infection in association with deficient malaria - specific immune responses . Adoptive transfer of regulatory t cell populations purified from foxp3 transgenic or wt mice to recipient wt mice confirmed that high numbers of these cells compromised immune control of malaria . It is well established that an intact spleen is required for the development of protective immune responses to malaria infection in mice 58 . To determine if regulatory t cells localize to this tissue during p. chabaudi as infection, we adoptively transferred cd4cd25 or cd4cd25 t cells purified from the spleens of ubi - gfp / b6 reporter mice and analyzed the frequency of cd4gfp t cells in various tissues in recipient wt mice infected with p. chabaudi as one day later . This experiment demonstrated that gfpcd4 t cells preferentially accumulate in the spleen as opposed to the blood or draining lymph nodes 56 . We also observed that significantly higher numbers of gfpcd4cd25 compared to gfpcd4cd25 t cells accumulate in the spleens of infected recipient mice . Immunohistochemical staining of spleen sections from infected wt mice confirmed that foxp3 regulatory t cells accumulate in the spleen during malaria and demonstrated that this t cell population localizes almost exclusively within the t cell areas of the white pulp . Additionally, we investigated if cd4cd25foxp3 t cells expand in the spleen of wt mice during p. chabaudi as infection by analyzing the frequency of regulatory t cells at various times p.i . By flow cytometry . Consistent with previous observations, we observed a significant but transient increase in the numbers of cd4cd25foxp3 t cells in the spleen of infected wt mice, which was followed by a significant and sustained decrease due to reduced proliferation and apoptosis of cd4foxp3 t cells 56 . The t cell growth factor il-2, a cytokine produced by activated cd4 t cells, is required for the expansion, survival, and function of natural regulatory t cells and especially to maintain foxp3 expression by these cells 59 . Decreases in il-2 secretion by effector cd4 t cells has been shown to be important for contraction of foxp3regulatory t cells in mice infected with toxoplasma gondii 52 . During p. chabaudi as infection in wt mice, we observed that the kinetics of il-2 secretion by effector cd4foxp3 t cells coincided with changes in cd4foxp3 cells and the differentiation of cd4t - betifn- cells required for immune control of blood - stage malaria . 4b, the administration of an immune complex consisting of recombinant murine il-2 and anti - il-2 monoclonal antibody (clones jes6 - 1) to infected wt mice increased the severity of p. chabaudi as infection and promoted the expansion of foxp3 regulatory t cells 56,60 . Together, our findings demonstrate that a tight balance between cd4foxp3 regulatory t cells and effector cd4 th1 cells is necessary to effectively control and eliminate p. chabaudi as infection and that this balance is dependent in part on il-2 . Although our findings provide convincing evidence that shifting the balance between natural regulatory t cells and effector cd4 t cells in favor of cd4foxp3 t cells severely compromises immune control of blood - stage malaria, it has not been established if this balance is perturbed in individuals infected with p. falciparum in endemic areas 61 . Nor has the contribution of il-2 to expansion of cd4foxp3 t cells in humans infected with malaria been investigated . The life cycle of the malaria parasite is complex with a sexual phase in the anopheles mosquito vector and an asexual phase in the host 12 . Sporozoites are transmitted to the host by the bite of an infected female mosquito and travel via the blood to the liver where they invade hepatocytes and initiate the exo - erythrocytic or liver stage, which is clinically silent and without any pathologic sequelae . Parasite multiplication in the liver results in merozoites that infect red blood cells (rbc), where they undergo mitosis and form erythrocytic schizonts containing many merozoites that are released and infect new rbc . As yet unknown factors trigger some merozoites to differentiate into male and female gametocytes that are taken - up during a blood meal by the mosquito for fertilization and sporozoite production 15 . The major manifestations of malaria in infected individuals include fever, malaise, splenomegaly, and anemia and are due to the cyclical multiplication of merozoites in rbc 12,14 . During p. falciparum infection, the spectrum of severe pathology is broad and includes metabolic acidosis, cm, and sma, and is typically accompanied by hypoxia, hypoglycemia, and lactic acidosis due in part to the increased metabolic demands of the parasite 12,14 . P. falciparum, the most virulent and deadly human parasite, is highly prevalent in sub - saharan africa 1 . P. vivax is the most common species causing human infection and predominates outside africa in south america and asia while infections with p. malariae and p. ovale subspecies are usually mild . Recently, human infections with p. knowlesi, a parasite naturally occurring among monkeys, have been identified in malaria endemic areas 8 . Numerous challenges such as the lack of access to appropriate tissue samples and the inability to manipulate the immune response to elucidate underlying mechanisms impede the study of malaria in humans 16 . As an alternative, experimental models in non - human primates and rodent models have been used . However, the lack of availability and high cost as well as ethical issues limit the use of non - human primates to study malaria . Infection of inbred mice with various rodent plasmodium species (p. chabaudi, p. berghei, p. vinckei and p. yoelii) that differ in virulence and pathology provide the most convenient experimental models (table 1) 12,17 . It is important to note that while no single rodent model replicates all of the features of human malaria in terms of pathophysiology or immune responses, these models have been essential in defining the basis of interactions between the host and the plasmodium parasite . Studies in mouse malaria models have revealed important and novel information leading to a clearer understanding of the immune response in protection and pathogenesis, to identify previously unrecognized genes that regulate susceptibility to malaria, and to develop malaria vaccines and novel chemotherapeutic agents 12,17 - 21 . Because p. chabaudi as is considered to share many biological and immunological features in common with p. falciparum, infection of mice with this parasite is widely used as a tool to study immune responses to blood - stage malaria 12,17,19 . Infection of resistant c57bl/6 (b6) mice with 10 - 10 p. chabaudi as infected rbc (irbc) results in a course of parasitemia characterized by parasite replication during the first week post infection (p.i .) Culminating in a moderate peak parasitemia of 30 - 35% between days 8 - 10 p.i . Weidanz demonstrated in mice infected with p. chabaudi that: 1) the control of peak parasitemia is dependent on a cell - mediated immune response that we now know requires cd4 t cells, and 2) the requirement for b cells in the clearance and elimination of the parasite 12 . Later studies from our laboratory showed that control of acute p. chabaudi as infection is critically dependent on il-12-dependent ifn- production by nk cells and cd4 th1 cells indicating the importance of both innate and adaptive immune responses 12 . The importance of cognate interactions between cd4 th cells and b cells for malaria - specific antibody production for efficient parasite elimination later during infection was demonstrated by langhorne and her colleagues 12,17 . Identification of the importance of nk cells, cd4 t cells, and b cells in mediating control and resolution of p. chabaudi as infection has provided a framework to delineate the accessory cells and cytokines involved in activating innate immunity and triggering adaptive immunity to malaria . Studies in mice infected with avirulent and virulent strains of p. yoelii as well as in mice infected with p. berghei anka have also provided important information about the role of the immune response in protection and immunopathogenesis 14,17,21 . Dc are a heterogeneous and highly specialized population of antigen presenting cells (apc) that provide a central link between innate and adaptive immune responses and play an important role at the host - pathogen interface including in response to plasmodium parasites 22 - 25 . Dc reside in almost all tissues where they play important roles in surveillance and sensing foreign antigens . Pattern recognition receptors, such as toll - like receptors (tlrs), nod - like receptors, and c - type lectins, expressed by these cells are important in distinguishing a diverse but overlapping repertoire of conserved microbial molecules 26 . Dc precursors are generated from pluripotent stem cells in the bone marrow under the influence of lineage specific growth factors and are released into the blood for transit to lymphoid organs, including secondary lymphoid tissues such as the spleen as well as non - lymphoid tissues such as the skin, where they reside as immature cells 22 . Following encounter with antigen, immature dc undergo maturation in response to stimulation by pathogens or cytokines and other soluble mediators produced by various cell types . Dc maturation results in a decreased ability to capture and process antigen and increased surface expression of mhc - ii and co - stimulatory (cd40, cd80, cd86) and adhesion molecules 22 - 27 . Dc maturation is critical in immunity to pathogenic microorganisms because of the potent ability of these cells to polarize the differentiation of nave cd4 t cells to th cell subsets including th1, th2, th17, follicular th cells, and induced regulatory t cells 28,29 . Dc also function as accessory cells and are important in activating nk cells for cytotoxicity and cytokine secretion in response to viruses and tumors 30 . As a result of interactions with t cells, nk cells and other effector cells, dc, thus, are not only important in antigen presentation and activating cd4 t cells but also function as accessory cells to regulate and amplify innate and adaptive immune responses . The relationship between dc maturation and malaria has been investigated in studies using peripheral blood monocytes from humans and murine dc 25,31 . Initially, it was shown that dc function is compromised during malaria based on the in vitro observation that binding of p. falciparum irbc to human monocyte - derived dc inhibits maturation and reduces the capacity of the dc to function as an apc 32 . Analysis of dc recovered from african children with severe p. falciparum malaria revealed that the frequency of myeloid - derived cd11cbdca3dc is significantly increased during acute infection 33 . A recent study comparing dc subpopulations in individuals infected with p. falciparum or p. vivax and residing in areas with similar endemicity found a significant increase in plasmacytoid cd123dc and a decreased ratio of myeloid to plasmacytoid dc during infection, regardless of the infecting species 34 . In this study, the maturation of peripheral blood dc was observed to be impaired ex vivo as shown by low cd86 expression in a proportion of the p. vivax - infected patients, but there was no evidence that dc maturation was affected in p. falciparum - infected individuals . These data suggest that dc subpopulations are differentially affected in humans during blood - stage malaria and that the variability observed in the effects of malaria infection on dc function may be due to the plasmodium species, the severity of the infection, and the patient population studied . Similar observations of diverse effects of malaria infection on dc have been made in studies using various combinations of parasites and inbred mouse strains . Distinct subpopulations of dc including myeloid - derived dc (cd11ccd4, cd11ccd8, cd11ccd8), plasmacytoid dc (cd11cb220), and a regulatory subset of cd11ccd45rb dc have been observed to expand in the spleens of mice infected with various plasmodium parasites 27,31,35 - 38 . The development of transgenic mice expressing a t cell receptor (tcr) specific for a peptide of the p. chabaudi merozoite surface protein-1 (msp1) has provided a novel tool to elucidate the role of dc and other cells in the immune response to malaria 39 . Using transgenic msp1-specific tcr mice infected with p. chabaudi as, sponaas et al provided direct evidence that cd11c dc in the spleen activate plasmodium - specific cd4 t cells 38 . This study also provided evidence that splenic dc subpopulations have distinct antigen presenting functions during malaria . Although both cd8 and cd8 dc present malaria peptides and induce ifn- production by cd4 t cells, only cd8 dc stimulate antigen - specific proliferation of cd4 t cells and secretion of il-4 and il-10 . Recently, lundie et al reported another strategy to study the immune response to malaria that involves the generation of genetically - modified strains of p. berghei anka expressing mhc - i- and mhc - ii - restricted t cell epitopes specific for cognate antigens, such as ova, linked to reporter gfp 40 . Using the genetically modified parasites to infect ot - i or ot - ii transgenic mice, these investigators observed that both cd8cd4 and cd8 but not cd8cd4 dc present p. berghei - expressed antigens to cd4 t cells during malaria infection, while the cd8 subpopulation induces pathogenic cd8 t cells via cross - presentation 40 . In a separate study, blood - stage p. berghei anka infection was found to cause suppression of mhc - i - restricted immunity to non - malarial antigens as a possible consequence of systemic dc activation 41 . This observation suggests that dc are not directly suppressed by malaria parasites, but that they follow their normal maturation pathway after activation by irbc or parasite products . Subsequent findings by lundie et al showed that dc isolated from p. berghei - infected mice efficiently present p. berghei - expressed antigens to cd4 and cd8 t cells on days 2 and 3 p.i ., but their ability to present parasite antigens as well as mhc - i- and mhc - ii - restricted, non - malarial antigens is drastically impaired by day 4 p.i . These findings are consistent with earlier studies by perry et al in p. yoelii - infected mice which showed that dc become refractory to tlr - ligand - stimulated il-12 and tnf- production with increased il-10 secretion as the infection progresses 43 . Dc from infected mice were found to retain the capacity to stimulate ova - specific proliferation of nave ot - ii cd4 t cells throughout p. yoelii infection, but the responding t cell phenotype changed later during infection from cd4ifn- to cd4il-10 t cells . However, functional impairment of dc at early times p.i . During lethal infections due to p. yoelii ym, p. vinckei or p. berghei anka has been observed based on the inability of dc from infected mice to secrete high levels of il-12 and prime nave cd4 t cells 31 . It was reported that the decreased function of dc in mice infected with p. vinckei or p. berghei is mediated in part by tnf- 31 . In contrast, lundie and colleagues failed to demonstrate a role for tnf- in impaired dc function during p. berghei anka infection 42 . As will be described below, spleen dc recovered from p. chabaudi as - infected b6 mice during the first week p.i . Altogether, studies in mouse models essentially confirm the findings in human studies and provide further explanation that the conflicting data obtained in human studies may be due to several factors including the parasite species and strain, the time after infection, and the size of the inoculum . Studies performed using human peripheral blood mononuclear cells and in plasmodium - infected mice indicate that nk cells are an early and crucial source of ifn- during blood - stage malaria 12 . Findings in experimental models of infection, especially viral infections, tumors, and inflammatory diseases, provide important evidence that optimal ifn- production by nk cells requires contact - dependent and -independent interactions with myeloid - derived accessory cells such as monocytes, macrophages, and dc 30 . As will be described below, we investigated if an accessory cell is necessary to promote ifn- secretion by nk cells during p. chabaudi as infection and questioned if cd11c dc are involved . Previous studies in b6 mice showed that during early p. chabaudi as infection dc, macrophages, and nk cells rapidly accumulate and expand in the spleen where they undergo maturation and functional activation in response to irbc captured in the spleen by phagocytosis (reviewed in 44). During early infection, cd11c dc migrate from the marginal zone to the cd4 t cell - rich periarteriolar lymphoid sheaths while macrophages expand and remain in the red pulp 45 . Importantly, within the first week p.i ., spleen dc from p. chabaudi as - infected b6 mice display up - regulation of mhc - ii and co - stimulatory molecule expression, secrete pro - inflammatory cytokines including high levels of il-12, and stimulate nave cd4 t cell proliferation and ifn- production 25,27,31,45 . In contrast, splenic macrophages from p. chabaudi as - infected mice suppress t cell proliferation and il-2 secretion 46,47 . Together, these findings provide compelling evidence that cd11c dc play an important role in innate immunity during blood - stage malaria and trigger an adaptive type 1 immune response necessary for control and rapid resolution of infection . To determine if dc have a role in activating nk cells during blood - stage malaria, we compared the maturation and function of cd11c dc purified by magnetic beads from the spleens of infected wild - type (wt) b6 mice and il-12p40 and il-15 mice that lack cytokines involved in nk cell proliferation, survival and activation 44 . In previous studies, we demonstrated that the course of p. chabaudi as infection is more severe in il-12p40 mice compared to wt and il-15 mice (12, reviewed in 44). We observed that the uptake of irbc by cd11c dc early after infection was similar in wt, il-12p40, and il-15 mice . Dc from infected il-15 and wt mice expressed similar levels of cd40, cd80, and cd86 while dc from infected il-12p40 mice expressed significantly lower levels of co - stimulatory molecules . Analysis of cytokine production in response to in vitro stimulation with irbc revealed that dc recovered from il-12p40mice on day 4 p.i . Produced significantly lower levels of pro - inflammatory cytokines compared to wt dc but secreted significantly more il-2 while the levels of il-10 secreted by wt, il-12p40, and il-15 dc were similar . These data indicate that cd11c dc from infected il-12p40mice are as efficient as dc from infected wt and il-15 mice in phagocytizing irbc but that dc maturation is impaired in infected il-12p40mice . To investigate if dc from p. chabaudi as - infected mice promote ifn- production by nk cells, we established co - cultures of purified cd11c dc from infected wt mice with cd3dx5 nk cells purified from the spleens of nave wt mice and analyzed nk cell expression of intracellular ifn- expression before and after co - culture with dc by flow cytometry 44 . Ifn- levels in the supernatants of the co - cultures were also determined . As shown in fig . 1a, few cd3dx5 nk cells from nave wt mice expressed intracellular ifn- prior to co - culture with dc from infected wt mice . After co - culture with dc from infected mice, there was a dramatic increase in the frequency of dx5ifn- nk cells . Although the frequency of cd11cifn- dc also increased in the co - cultures, the major source of ifn- was observed to be nk cells, a finding corroborated when dc from infected wt mice were co - cultured with nk cells from nave ifn- mice 44 . Determination of the levels of ifn- in the supernatants confirmed that wt dc from infected mice stimulated significantly higher ifn- secretion than wt dc from nave mice (fig . We also determined the relative contribution of il-12 and il-15 to dc - nk cell interactions during malaria by co - culturing purified cd11c dc from infected il-12p40 or il-15 mice with nk cells from nave wt mice . Dc from infected il-15 mice stimulated a similar percentage of wt nk cells to express intracellular ifn- as wt dc, and ifn- levels were similar in supernatants of wt nk cells co - cultured with wt dc and il-15 dc . In contrast, dc from infected il-12p40 mice are unable to promote ifn- expression by nk cells from nave wt mice (fig . These data are consistent with the observations described above linking lethal malaria infections in mice with functional impairment of dc and indicate that soluble mediators, especially il-12, are required to induce optimal ifn- secretion by nk cells during blood - stage malaria . We also observed that dc - nk cell interaction is dependent on cell - cell contact since significantly lower ifn- production was observed when nk cells and dc were separated by a transwell insert . Reciprocal crosstalk between dc and nk cells has been demonstrated in a number of studies, with evidence of complex interactions between the two cell types 30,48 . Nk cells activated by tumors, bacterial products, or il-2 have been shown to induce dc maturation and secretion of the th1-promoting cytokine il-12 30,48 . To determine if malaria - activated nk cells induce dc maturation, we co - cultured purified dx5 nk cells from day 5 infected wt mice with cd11c dc from nave wt mice which were exposed to irbc for 1 hr prior to co - culture with nk cells to prevent lysis of immature dc by the activated nk cells 44 . Dc from nave mice co - cultured with nk cells from infected wt mice displayed significantly increased cd40 and cd86 expression compared to control dc cultured alone or with nk cells from nave wt mice (fig . Up - regulation of co - stimulatory molecule expression on dc co - cultured with malaria - activated nk cells was found to be dependent on cell contact . Co - culture with malaria - activated nk cells but not nk cells from nave mice also stimulated dc to secrete high levels of cytokines especially il-12 (fig . Dc separated from nk cells by a transwell insert showed similar levels of cytokine production as control co - cultures indicating that soluble factors produced by nk cells and not cell - cell contact are responsible for enhanced il-12 production by dc . The importance of nk cell - derived ifn- in inducing enhanced production of th1-promoting il-12 by dc was demonstrated by co - culture of wt dc with nk cells from p. chabaudi as - infected ifn- mice and by the addition of anti - ifn- monoclonal antibody to co - cultures of wt dc and wt nk cells . In the absence of nk cell - derived ifn-, importantly, dc stimulated by malaria - activated nk cells were found to be fully licensed apc and primed nave cd4 t cells to proliferate and produce the type 1 cytokine ifn- indicating th1 polarization 44 . Our observations in the mouse model of p. chabaudi as infection confirmed in vitro studies by newman et al in human peripheral blood mononuclear cells stimulated with p. falciparum irbc showing that ifn- production by nk cells only occurs following multiple contact - dependent and cytokine - mediated signals derived from myeloid - derived dc and monocytes 49 . Importantly, reciprocal activation of accessory cell maturation by malaria - activated nk cells was observed in the studies with pbmc and in our studies in mice . These findings indicate that interactions between accessory cells and nk cells during malaria are bi - directional similar to virus and tumor models 30,44,48,49 . To address if nk cells are required to induce dc maturation and function in vivo, we treated p. chabaudi as - infected wt mice with anti - asialo - gm1 antibody and examined dc maturation and th cell polarization . As we observed previously, infected b6 mice treated with anti - asialo - gm1 antibody mice display a course of infection characterized by a significantly higher peak parasitemia and significantly higher recrudescent parasitemia during the chronic phase of infection 12,44 . It should be noted that a similar course of p. chabaudi as infection is also evident in il-12p40 mice which are deficient in nk cell secretion of ifn- during early infection 50 . During infection, cd11c dc purified from the spleens of nk cell - depleted wt mice expressed lower levels of cd40 and cd86 and produced significantly less il-12 and more il-2 in response to irbc in vitro than dc from isotype control - treated wt mice . Impairment of dc maturation and il-12 production in infected nk cell - depleted wt mice resulted in inefficient priming of nave cd4 t cells for malaria antigen - specific proliferation and ifn- secretion compared to dc from isotype control - treated wt mice . Together, these data provide in vivo evidence that dc - nk cell crosstalk is required for dc maturation and function and optimal ifn- production by nk cells and cd4 t cells during p. chabaudi as infection . Recent studies have focused on the role of cd4 regulatory t cells in regulating adaptive immune responses during plasmodium infection . In particular, natural cd4foxp3 regulatory t cells have been shown to be important in determining the outcome of infection with various intracellular pathogens, including protozoan parasites 51,52 . Cd4foxp3 t cells have been shown to inhibit effector t cell responses during infection, resulting in inadequate immune control of the pathogen and persistence of low - level infection . It has also been observed that the potent ability of natural regulatory t cells to modulate effector t cell responses is important in limiting the development of tissue damage during infection with protozoan parasites 51,52 . Studies in individuals with malaria and in mice infected with various rodent plasmodium species have revealed an important expansion of cd4cd25foxp3 t cells 53 - 55 . Experimental p. falciparum infection in human volunteers showed that the magnitude of expansion of this t cell population correlates with high parasitemia levels and low pro - inflammatory responses, findings that suggest a possible link between natural regulatory t cells and the clinical outcome of malaria 53 . Experiments to determine the impact of cd4cd25foxp3 t cells in plasmodium - infected mice by depleting cd25 t cells have led to inconsistent findings 53 - 55 . Although there are several possible explanations for the inconsistencies, including the concern that antibody depletion of cd25 t cells may result in the loss of nave and effector t cells, the results of these studies are consistent with the notion that natural regulatory t cells suppress pro - inflammatory responses involved in protective immunity as well as immunopathology during malaria 53 - 55 . We investigated the role of cd4foxp3 regulatory t cells during p. chabaudi as infection using several approaches as alternatives to in vivo depletion of cd25 t cells 56 . First, we compared the course and outcome of p. chabaudi as infection and malaria - specific immune responses in wt mice and transgenic mice generated by khattri et al 57 that overexpress the foxp3 transcription factor . Compared to infected wt mice, infected foxp3 transgenic mice develop fulminant peak parasitemia levels and 100% of these mice succumb to the infection in association with deficient malaria - specific immune responses . Adoptive transfer of regulatory t cell populations purified from foxp3 transgenic or wt mice to recipient wt mice confirmed that high numbers of these cells compromised immune control of malaria . It is well established that an intact spleen is required for the development of protective immune responses to malaria infection in mice 58 . To determine if regulatory t cells localize to this tissue during p. chabaudi as infection, we adoptively transferred cd4cd25 or cd4cd25 t cells purified from the spleens of ubi - gfp / b6 reporter mice and analyzed the frequency of cd4gfp t cells in various tissues in recipient wt mice infected with p. chabaudi as one day later . This experiment demonstrated that gfpcd4 t cells preferentially accumulate in the spleen as opposed to the blood or draining lymph nodes 56 . We also observed that significantly higher numbers of gfpcd4cd25 compared to gfpcd4cd25 t cells accumulate in the spleens of infected recipient mice . Immunohistochemical staining of spleen sections from infected wt mice confirmed that foxp3 regulatory t cells accumulate in the spleen during malaria and demonstrated that this t cell population localizes almost exclusively within the t cell areas of the white pulp . Additionally, we investigated if cd4cd25foxp3 t cells expand in the spleen of wt mice during p. chabaudi as infection by analyzing the frequency of regulatory t cells at various times p.i . By flow cytometry . Consistent with previous observations, we observed a significant but transient increase in the numbers of cd4cd25foxp3 t cells in the spleen of infected wt mice, which was followed by a significant and sustained decrease due to reduced proliferation and apoptosis of cd4foxp3 t cells 56 . The t cell growth factor il-2, a cytokine produced by activated cd4 t cells, is required for the expansion, survival, and function of natural regulatory t cells and especially to maintain foxp3 expression by these cells 59 . Decreases in il-2 secretion by effector cd4 t cells has been shown to be important for contraction of foxp3regulatory t cells in mice infected with toxoplasma gondii 52 . During p. chabaudi as infection in wt mice, we observed that the kinetics of il-2 secretion by effector cd4foxp3 t cells coincided with changes in cd4foxp3 cells and the differentiation of cd4t - betifn- cells required for immune control of blood - stage malaria . 4b, the administration of an immune complex consisting of recombinant murine il-2 and anti - il-2 monoclonal antibody (clones jes6 - 1) to infected wt mice increased the severity of p. chabaudi as infection and promoted the expansion of foxp3 regulatory t cells 56,60 . Together, our findings demonstrate that a tight balance between cd4foxp3 regulatory t cells and effector cd4 th1 cells is necessary to effectively control and eliminate p. chabaudi as infection and that this balance is dependent in part on il-2 . Although our findings provide convincing evidence that shifting the balance between natural regulatory t cells and effector cd4 t cells in favor of cd4foxp3 t cells severely compromises immune control of blood - stage malaria, it has not been established if this balance is perturbed in individuals infected with p. falciparum in endemic areas 61 . Nor has the contribution of il-2 to expansion of cd4foxp3 t cells in humans infected with malaria been investigated . Studies in malaria - infected humans and in mouse models provide strong evidence that plasmodium parasites modulate dc maturation and function and induce the expansion of cd4foxp3 regulatory t cells . In turn, these regulatory t cells may modulate effector cd4 t cell responses to malaria infection suppressing protective immune responses but preventing immune - mediated pathology . Accumulating data indicate that immune control of parasite growth is a likely down - stream effect of induction of functionally mature dc that produce il-12 and induce ifn- production by nk cells and cd4 th1 cells (fig . 5). Recent studies also indicate that myeloid - derived cd11c dc induce cd4 t cells to secrete ifn- and lead to the development of experimental cm in mice infected with p. berghei anka suggesting that malaria - activated dc are also involved in mediating pathology during malaria 62 . More extensive studies are required to provide a clearer understanding of the role of dc that are deficient in producing the th1-promoting cytokine il-12 and result in a lethal infection . The possibility that such dc have a tolerogenic phenotype and secrete cytokines such as il-2, il-10, and tgf- which may induce regulatory t cells requires further investigation . Indeed, it has been observed that populations of tolerogenic human and murine dc induce or promote cd4foxp3 regulatory t cells via tgf-- and il-2-dependent mechanisms 63 - 66 . Increased knowledge concerning dc and cd4foxp3 regulatory t cells may provide strategies for devising novel immunotherapeutic approaches for manipulating the immune response to malaria and tilting the balance between protective and immunopathogenic responses in favor of the host and prevention of severe disease.
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Currently, nanoparticles (nps) are used in several scientific applications, but among these silver nanoparticles (ag - nps) are predominant . This is because of several characteristic features of this type of metal np.13 antimicrobials and nanocomposite fabrication are some of the highly useful applications, among others, of applications that were attributed to ag - np.4,5 in the fabrication of nps, it is very important to control particle size, shape, and morphology . Laser ablation in liquid media is a simple and clean method for synthesis of nps.69 in this technique, there is no need to use any chemical reagents (such as nabh4) in the fabrication process or any purification techniques to characterize the produced nps . For example, nps using fatty acids (oleic and lauric acid) and vegetable oils as stabilizers have been synthesized.1012 it was found that these materials consist of amphiphilic molecules with polar carboxylic groups which are able to adsorb nps, and their nonpolar long carbon chain prevents nps agglomeration through steric repulsion . Castor oil is a vegetable oil obtained by cold pressing the seeds and subsequent clarification of the oil by heat . It has high viscosity, high polarity, and very low vapor pressure and optical activity in comparison with other oils . Castor oil is classified as a safe and effective stimulant laxative by the us food and drug administration (fda). It is a triglyceride containing fatty acid chains, 90% of which are ricinoleic acid . Oleic and linoleic acids are the other significant components.12 with the above characteristics, it is expected that castor oil can be used as a good stabilizer in nps fabrication . In this study, we report fabrication of ag - nps in castor oil . To the best of our knowledge, fabrication of ag - nps in castor oil using laser ablation has not been reported previously . For the fabrication of ag - nps, a silver plate was ablated by laser in castor oil for 10 min . As shown schematically in figure 1, the plate with high purity (> 99.99%) was located 5 mm behind a glass cell wall filled with 10 ml of castor oil . A nanosecond - pulsed q - switched nd: yag laser (brilliant; lambda photometrics ltd, hertfordshire, uk) with 10-hz repetition rate, 5-ns pulse duration, and 360 mj / pulse energy at its original wavelength (1064 nm) was applied . A lens with focal length of 250 mm was used to focus the laser pulses onto the silver plate . During plate ablation, an atomic absorption spectrometer (aas - s series; thermo scientific, san jose, ca) was used to measure concentration of ag - nps in castor oil . The obtained concentration of ag - nps in castor oil was 0.13 mg / l . Due to the high viscosity of castor oil the prepared sample was characterized using a uv - vis double beam photospectrometer (shimadzu, columbia, sc), a transmission electron microscope (tem, hitachi h-7100; hitachi, tokyo, japan), and a fourier transform - infrared (ft - ir) spectrometer (1650; perkin elmer, waltham, ma). Figure 2a indicates the absorption spectrum of the castor oil immediately after laser ablation of the silver plate inside it . The appearance of the maximum absorption peak around 400 nm confirms that the solution contained ag - nps, and that this peak originated from the oscillation of collection of free conduction electrons of ag - nps, which is called surface plasmon resonance . The ultraviolet - visible spectrum of the ag - nps in castor oil was measured after a period of 2 months in order to check the ability of castor oil as a stabilizer . As shown in figure 2b, the spectrum does not show any significant change in comparison with the spectrum of the freshly prepared sample; however, there is a small reduction in absorption . This showed that the ag - nps produced in castor oil using the laser ablation technique were stable and did not agglomerate during storage of the sample at room temperature for quite a long period of time . The slight decrease in absorption intensity was possibly due to the sedimentation of larger particles . The spherical shape nps can be very appropriately used for drug loading and for most biological applications such as antibacterial properties.13 figure 3 shows an approximate 5.06 nm average diameter for freshly prepared ag - nps obtained from two areas . Nps are formed via nucleation, transition, and crystal growth of materials such as silver atoms, clusters, and droplets that were emitted from the silver plate upon laser ablation.14 castor oil can adsorb the produced ag - nps and long - chain hydrophobic moieties protect them through a steric stabilization mechanism . Ft - ir spectroscopy of castor oil and castor oil containing ag - nps (figure 5) shows absorption bands at 3448, 2923, 2857, 1739, and 1160 cm related to the vibrational - stretching mode of o h (intermolecular hydrogen bond), c h (sp), c h (sp), co, and c o, respectively . In addition, the appearance of the absorption band at 3780 cm can be attributed to nonhydrogen - bonded o we believe that the existence of ag - nps prevents intermolecular hydrogen bonding, and therefore, the vibrational stretching modes of free o h appear at a higher wave number.15 ag - nps have been successfully prepared in castor oil by laser ablation from a silver plate . The results obtained showed that the produced ag - nps were well dispersed and also stable for quite a long period of time . This ability of castor oil to prevent agglomeration of ag - nps is due to the presence of long - chain hydrophobic moieties.
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The rapid spread of resistance among common pathogenic microorganisms is a serious challenge around the word . This phenomenon affects antibiotics' effectiveness and limits available options for the treatment of common infections in human . Serious infections due to beta - lactamase producing microorganisms, especially in hospitalized patients are increasing now . Both enzymatic and nonenzymatic pathways cause resistance to third - generation cephalosporins, aminoglycosides, fluoroquinolones, and carbapenems . Antibiotic resistance occurs following mutation in chromosomal genes or by horizontal transfer of genes between different microorganisms . The main mechanism of antimicrobial resistance in enterobacteriaceae family is transferring of plasmid encoding extended - spectrum beta - lactamase (esbl). Esbl producers (esbl - p) are gram - negative microorganisms which almost always belong to the enterobacteriaceae species . These gram - negative bacteria secret esbl enzyme in periplasmic space and hydrolyze the beta - lactam ring in penicillins, cephalosporins, and aztreonam . In general, esbl - p pathogens can cause severe and life - threatening infections such as bacteremia, sepsis, pneumonia, and meningitis . In the united states, 26,000 infections and 17,000 deaths per 2012 were due to esbl - p species . According to the centers for disease control and prevention (cdc) report, more than 19% of healthcare - associated infections are resistant to extended - spectrum cephalosporins . In the united states, the mortality rate was 57% more common in patients with bloodstream infection caused by esbl - p than nonproducers . Prolong hospitalization, presence of invasive medical devices, receiving total parenteral nutrition, age <12 weeks, prior treatment with cephalosporins and aminoglycosides, recent surgery, and hemodialysis are defined as risk factors for colonization with esbl - p species . The incidence of infections due to resistant microorganisms is increasing in the recent years in iran . In this study, frequency and antimicrobial sensitivity pattern of multidrug resistant (mdr) gram - negative microorganisms were evaluated in a referral teaching hospital in tehran, iran . This cross - sectional study was performed between december 2014 and january 2016 in imam khomeini hospital, a referral teaching hospital affiliated to the tehran university of medical sciences, tehran, iran . Biologic clinical samples including urine, cerebrospinal fluid (csf), blood, and tracheal secretions that were referred to the central laboratory department from different wards of the hospital were analyzed according to the clinical and laboratory standard institute instructions . Antimicrobial sensitivity patterns of all isolates were recognized by using standard antibiotic disks on mueller - hinton agar . Following antibiotic disks from himedia, bioscience company, india, was used for the primary antibiogram and esbl screening; ciprofloxacin (5 g), ceftriaxone (30 g), cefotaxime (30 g), ceftazidime (30 g), amikacin (30 g), ampicillin - sulbactam (10/10 g), imipenem (10 g), and meropenem (10 g). After 24 h of incubation, if an inhibitory concentration zone was <25 mm for ceftriaxone, 27 mm for cefotaxime or 22 mm for ceftazidime, phenotypic confirmatory test was performed with double disk synergy test . For this test, cefotaxime / clavulanic acid (30/10 g) and ceftazidime / clavulanic acid (30/10 g) discs were used . Increasing of 5 mm in the inhibition zone diameter in double synergy test versus the antibiotic tested alone was considered in favor of esbl - p isolates . Non - esbl isolates that were resistant to imipenem or meropenem was categorized as probable carbapenem - resistant gram - negative microorganisms . Patients, in whom mdr gram - negative pathogens were confirmed phenotypically, were detected and followed by the clinical pharmacists during the course of hospitalization . Mdr was defined as resistance to at least three classes of antibiotics (aminoglycosides, anti - mrsa cephalosporins, antipseudomonal penicillins + beta - lactamase inhibitors, carbapenems, and nonextended spectrum cephalosporins; first and second generation cephalosporins, extended - spectrum cephalosporins; and third and fourth generation cephalosporins, cephamycins, fluoroquinolones, folate pathway inhibitors, glycylcyclines, monobactams, penicillins, penicillins + beta - lactamase inhibitors, polymyxins, phosphonic acids, phenicols, and tetracyclines). Demographic data, baseline diseases, type of biological sample, isolated microorganism, type of infection, antibiotic regimen before availability of the culture result, and change in the antibiotic regimen following receiving the antibiogram results, response to the treatment regimen, duration of hospitalization, and patient's outcome were considered variables for each recruited patient . Cultures compatible with patient clinical status were measured as true infection according to the cdc definitions for health - care associated infections . Chi - square or fisher exact test was used for comparing the categorical variables between the groups . During the study period, fifty patients with mdr gram - negative infections including confirmed esbl or probably carbapenem - resistant enterobacteriaceae (cre) were detected . The mean sd of patients' age was 59.02 17.96 years old and thirty (60%) of them were males . Tracheal secretions (17 [34%]), urine (15 [30%]), blood (8 [16%]), soft tissue (3 [6%]), peritoneal fluid (2 [4%]), csf (1 [2%]), and pleural fluid (1 [2%]) were positive in the patients . Most patients were hospitalized in intensive care unit (35 [70%]) and general ward (8 [16%]), followed by emergency, neurosurgery, and coronary care unit wards . Klebsiella species (78%), escherichia coli (20%), and enterobacter cloacae (2%) were isolated microorganisms from the patients' biological samples . All of isolated e. coli and e. cloacae but only 56% of isolated klebsiellas species were sensitive to carbapenems . The result of antimicrobial susceptibility tests revealed that 30% of the isolated microorganisms were resistant to carbapenems that may be cre . However, most of these species (86.7%) were esbl negative . According to the double disk synergy test, 17 (34%) of all isolates were esbl - p and others were esbl - negative . Esbl was positive in 58.8% and 41.2% of isolated klebsiella species and e. coli, respectively . Based on the cdc definition, the clinical condition was compatible with the isolates in 62% of all patients and 25.8% of patients with esbl - p infections . In 71% of the cases, a carbapenem alone or plus amikacin or ciprofloxacin were selected regimens for patients with esbl infections . For patients with probable cre infections, clinical response was detected in 54.5% of the patients who were treated based on the antibiogram results . Clinical response was higher in the esbl - p than the non - esbl - p infections (75% vs. 52%). However, this difference was not significant (p = 0.09). Most nonresponders (80%) had sepsis due to klebsiella species . Finally, 41.9% of the patients were discharged from the hospital and 58.2% died . Characteristics of patients with esbl - p and probably cre infections were summarized in tables 2 and 3, respectively . Resistance pattern of isolated gram - negative pathogens characteristics of patients with extended - spectrum -lactamase - producing infections characteristics of patients with probable carbapenem resistant enterobacteriaceae infections inappropriate antibiotic administration and consequent increasing number of mdr pathogens including esbl - p and cre are a serious worldwide concern in recent years . Rapid growing of esbl - p and cre among community and hospitalized patients is a global threat, especially in critically ill patients . Considering that only limited new antimicrobial agents have been introduced in recent years; in some situations, we do not have an effective weapon against these pathogens . Following extensive use of cephalosporins in last years, resistance rate of enterobacteriaceae family to these agents is increasing around the world . Cephalsporins - resistant rate of these microorganisms was 30% among 11 countries of asia in 2010 . This rate received to 87% in 2014 at latin america . Only in limited studies, the prevalence of esbl - p pathogens was evaluated in iran and ranged from 43.6% in ilam to 74% in milad hospital . However, the average rate of esbl - p microorganisms was 42.2% in iran . In a recent study, more than 50% of isolated microorganisms from bile specimens were esbl - p . In european hospitals, more than 80% of isolated e. coli and klebsiella pneumonia were belonged to the esbl - p category . In the present study, the frequency of esbl - p pathogens was lower than the previous reports from our country . In a report from three hospitals of iran, all isolated esbl - p microorganisms were sensitive to carbapenems . However, in our study, some of esbl - p species and most of esbl negative strains were cre . All cre were klebsiella species . To interpret the result of clinical responses, limitations of the study esbl - p pathogens were identified phenotypically but were not confirmed by the genotypic assay method . Genotypic assay is not easily available method in our hospitals and only is applied for research purpose . Although double disk synergy test is a common and practical method for esbl confirmation but some isolates may be missed by this test . The sensitivity of this method could be reduced by microorganisms that show low - esbl activity . It has been shown that 13.63% of esbl positive strains were not recognized by double disk method . Therefore, some of non - esbl strains in our study may be false negatives of the test . In this study, cre isolates were detected based on the results of the disk diffusion method and were not confirmed based on the phenotypic and genotypic assays . Specific methods such as bromic acid in combination with clavulanate are recommended to unmask the underlying esbls among enterobacteriaceae family with carbapenemase enzyme . Pharmacokinetic parameters such as inadequate tissue penetration of antimicrobial agents can influence the clinical responses in in vivo settings . Most of the recruited patients had at least one of the following severe comorbidities including malignancies, respiratory disorders, ischemic heart disease, heart failure, diabetes mellitus, renal failure, cerebrovascular accident, hepatitis, immunodeficiency, and sepsis . Unfortunately like other countries, cre prevalence in our country is increasing in the recent years . Empiric administration of carbapenems should be restricted to patients with risk factors of infections with esbl - p bacteria and in specific clinical situations . To limit the use of last - line antibiotics such as carbapenems, availability of accurate phenotypic, and genotypic methods for detection of esbl - p and carbapenemase strains is essential in clinical practice.
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Approximately 1 - 5% of peripheral blood t cells express the t - cell receptor instead of the conventional t - cell receptor . The versus t - cell lineage commitment during intrathymic t - cell development seems to be controlled by the signal strength provided to the t - cell receptor . In healthy donors, most blood t cells carry a specific t - cell receptor composed of v9 and v2 elements . In addition to effector functions shared with t cells, the v9v2 t cells can acquire professional antigen - presenting capacity characteristic of dendritic cells . In contrast to t cells, v9v2 t cells do not see processed antigenic peptides presented by major histocompatibility complex molecules, but rather recognize small phosphorylated non - peptide molecules (phosphoantigens) produced by many microorganisms but also by transformed eukaryotic cells . While microbial phosphoantigens are active at pico- to nanomolar concentrations, micromolar concentrations of the eukaryotic phosphoantigen isopentenyl pyrophosphate (ipp) are required for t - cell activation . Such high concentrations are not achieved in the mevalonate pathway of isoprenoid synthesis used in non - transformed cells . Interestingly, human v9v2 t cells can kill a broad variety of epithelial tumor and leukemia / lymphoma cells . The sensitivity of tumor cells to t - cell - mediated killing is increased upon treatment of tumor cells with aminobisphosphonates (n - bps), drugs that are used in clinical practice for the treatment of osteoporosis and bone metastasis in cancer patients . N - bps inhibit the ipp - processing enzyme farnesyl diphosphate synthase (fpps), thereby leading to an accumulation of ipp, which is then sensed by the t cells . T cells are poor producers of interleukin-2 (il-2), which is required for expansion of t cells . Therefore, attempts to activate tumor - reactive t cells endogenously by treating patients with n - bps must take into consideration an appropriate supply of il-2 . Alternative strategies consider the adoptive transfer of in vitro expanded tumor - reactive t cells [10 - 14]. The critical role of fpps in the control of intracellular ipp levels, and thus of the sensitivity of tumor cells toward t - cell killing, has been recently demonstrated using short hairpin rna - mediated knock - down of fpps . Knock - down of fpps caused tumor cells, which otherwise were not recognized by t cells, to be susceptible to t - cell killing . Therefore, v9v2 t cells recognize and kill tumor cells on the basis of the unbalanced isoprenoid metabolic pathway in transformed cells, a pathway that is stable in non - malignant cells . The discovery that n - bps activate t cells by inhibiting fpps, thereby leading to accumulation of ipp, has paved the way for proof - of - principle studies to activate t cells in patients with advanced cancer . In a phase i clinical trial, dieli and colleagues treated patients with hormone - refractory prostate cancer with a standard application of the n - bp zoledronate (4 mg intravenous infusion every 21 days) either with or without additional low - dose (6 10 iu) subcutaneous application of il-2 . Various parameters, including subset analysis of t cells, and serum levels of prostate - specific antigen and cytokines, were monitored over time . Although the two cohorts comprised only a few patients, statistically significant effects of zoledronate plus il-2 on the mobilization and effector cell maturation of t cells were recorded . Very importantly, the two cohorts showed distinct clinical outcomes, with clinical responses seen in six of nine patients treated with zoledronate plus il-2 but only in one of nine patients treated with zoledronate alone . Interestingly, a correlation between favorable outcome at 12 months and t - cell numbers or functional status (or both) was observed . Similarly, wilhelm and colleagues had previously shown that the combined application of n - bp plus low - dose il-2 can induce objective tumor responses in patients with lymphoid malignancies . Together, these studies support the view that application of n - bps plus il-2 is safe, induces in vivo activation / maturation of t cells, and may have beneficial effects in advanced cancer (figure 1a). (a) n - bps inhibit farnesyl diphosphate synthase (fpps), thus preventing processing of isopentenyl pyrophosphate (ipp) to farnesyl diphosphate (fpp). The combined application of n - bp plus il-2 leads to in vivo activation of t cells . (b) alternatively, t cells can be activated in vitro with n - bp or synthetic phosphoantigens in the presence of antigen - presenting cells (apc) and can be subsequently expanded to large cell numbers by an exogenous supply of il-2 for subsequent adoptive transfer into cancer patients . The cell preparation can be performed under gmp (good manufacturing practice) conditions . In a case study reported by laggner et al ., regression of lung and bone metastases was observed in a patient with advanced stage melanoma upon systemic treatment with zoledronate and localized radiotherapy . Although t - cell subsets were analyzed, it is difficult to ascertain a substantial role of t - cell activation in the resolution of metastases in this single case, particularly since il-2 was omitted in the treatment of this patient . In addition to their t - cell activating properties, n - bps also exhibit direct anti - tumor activities by both inhibiting proliferation and inducing apoptosis in tumor cells . While zoledronate seems to be the most potent t - cell - activating substance among the n - bps licensed for clinical application, derivatives of zoledronate with further improved t - cell - stimulating capacity and enhanced direct anti - tumor activity are under development . Such modified n - bps might also exert improved in vivo activation of t cells when given to patients together with il-2 . T - cell - based immunotherapeutic strategy is the adoptive transfer of in vitro expanded v9v2 t cells from tumor patients (figure 1b). Recently, efficient protocols for the large - scale in vitro expansion of v9v2 t cells based on stimulation with synthetic phosphoantigens or zoledronate have been established . First results indicate that the repetitive adoptive transfer of in vitro expanded t cells is well tolerated and may induce anti - tumor responses in patients with solid tumors, including renal cell carcinoma and myeloma . The protocol developed by dieli et al . For the in vivo activation of t cells based on zoledronate plus low - dose il-2 application is ready to be explored in larger clinical trials and in other tumor entities with poor prognosis, for example, pancreatic ductal adenocarcinoma where it might be combined with standard regimens such as gemcitabine . It is conceivable that this protocol can be further improved, for instance, by combination with tumor - targeting monoclonal antibodies . Along this line, it has been shown that b - cell lymphoma or breast tumor cell killing by fc receptor - expressing t cells is enhanced in the presence of targeting antibodies rituximab (cd20) or trastuzumab (her2/neu), respectively . Moreover, a t - cell - stimulating synthetic phosphoantigen was found to enhance the depletion of cd20 b cells by rituximab in a non - human primate model in vivo, pointing to the possible use of phosphoantigen plus anti - cd20 antibodies in the treatment of cd20 leukemias and lymphomas . Furthermore, cytokines promoting homeostatic proliferation and survival of t cells, such as il-15, or cytokines potentiating the cytolytic activity and pro - inflammatory response, such as il-21, might be combined with il-2 or used instead of il-2 . This could be considered both for in vivo application together with n - bps and for optimization of in vitro expansion of t cells . In addition, future study protocols might include the combination of in vivo activation of t cells (by n - bp or phosphoantigen plus il-2) followed by the adoptive transfer of in vitro expanded t cells, finally, it should be stressed that t - cell - based immunotherapy is not expected to replace established therapeutic protocols . Rather, it might offer additional benefit to the patient, for instance, in combination with conventional chemotherapy.
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Castleman's disease (cd) is a rare lymphoproliferative disorder caused by faulty immune regulation resulting in excessive b - lymphocyte and plasma - cell proliferation in lymphatic tissue . Marked interfollicular vascular proliferation leads to mass formation, which can encase bronchi and sometimes cause mural erosion leading to life - threatening hemoptysis . Vascular supply to these lesions have been reported to arise from the bronchial, internal mammary, and intercostal arteries . We report a case of hilar intrathoracic cd with atypical radiological features where the left inferior phrenic artery and bronchial circulation provided codominant blood supply . A 24-year - old female with a 5-month history of wheeze and nonproductive cough underwent chest x - ray, which showed a well - defined mass in the left hilar region [figure 1a d]. A contrast - enhanced computed tomography (ct) scan of the chest revealed a heterogeneously enhancing 7 5 cm mass containing flecks of calcification at the left hilum . 18 fluorodeoxyglucose positron emission tomography (18-fdg pet) scan demonstrated an inhomogeneous hypermetabolic lesion with no evidence of metastatic uptake . A ct - guided percutaneous core biopsy revealed chronic inflammatory tissue with no evidence of malignancy . Due to worsening cough and wheeze, the patient underwent a thoracotomy and surgical resection . During the operation there was massive blood loss of 2000 ml . Histopathology confirmed the mass to have marked lymphoid infiltrate with irregular b cell follicles and hyaline vascular - like change consistent with cd . Chest x - ray of a 24-year - old female (a) showing well - defined left hilar mass . Contrast - enhanced ct scan (b and c) shows heterogeneously enhancing mass with coarse calcifications within this hilar mass . 18-fdg pet / ct scan (d) shows hypermetabolic uptake within the lesion two months after the thoracotomy, the patient presented to the emergency department with a 1-day history of massive hemoptysis (> 200 ml). A ct pulmonary angiogram for clinically suspected pulmonary embolism showed no pulmonary embolism, however, multiplanar reformats showed low attenuation soft tissue in the surgical resection bed at the left hilum receiving blood supply through hypertrophied left bronchial and left inferior phrenic arteries [figure 2a f]. The patient had an embolization through a right common femoral artery approach and the common (conjoined) origin of the right broncho - superior intercostal trunk and left bronchial arteries were catheterized with a 5 fr (cordis, usa) left amplatz coronary i catheter (cordis, usa). The sharply angulated left bronchial artery could not be superselectively catheterized with a range of microcatheters . The main trunk was embolized with 300500 micron polyvinyl alcohol (pva) particles (cook, usa). The medial division of the inferior phrenic artery was seen to supply the mass via its pericardial branch . This was super - selectively catheterized with the same 5 fr catheter and a renegade microcatheter (boston scientific, usa) and embolized with the same pva particles . No spinal, esophageal, or coronary supply was demonstrated from the bronchial and left inferior phrenic arteries . There were no procedure - related complications and the patient had an uneventful recovery with no further hemoptysis during the 3 years follow - up period . Contrast - enhanced ct with mip images (a and c) showing blood supply to the lesion with bronchial artery and left inferior phrenic artery (arrows). Catheter angiogram demonstrates bronchial artery (arrow in b) and left inferior phrenic artery (arrow in d) and subsequent embolization with pva (e and f) cd may be localized or multicentric and primarily involves the mediastinum, neck, and mesentery . There are three known histological variates namely (a) hyaline - vascular, (b) plasma - cell, and (c) mixed variant . Classic features on a ct scan is a solitary, intensely, and homogeneously enhancing mass with no local invasion . The standard treatment of the localized form is surgical resection, however, where there is encasement or invasion of the adjacent structures, preoperative embolization has been advocated . Recently, embolization alone without surgical resection has been shown to terminate hemoptysis and alleviate shortness of breath due to airway compression in patients with unresectable disease . In our patient, preoperative embolization, which may have reduced the large volume intraoperative blood loss, was not considered . It was only when the patient presented with hemoptysis after surgical resection that the vascular supply was identified and an emergency referral to interventional radiology was made . Previous reports have identified bronchial, internal mammary, and intercostal arteries as feeding vessels to cd . Inferior phrenic supply to cd has not been reported before to the best of our knowledge . When basal lung segments are suspected as the source of hemoptysis, inferior phrenic supply should always be considered . Inferior phrenic supply to hilar masses is much rarer, but when it occurs, the supply is usually via the pericardial branch of its medial division . The left inferior phrenic medial branch may supply the esophagus, and it is important to exclude any esophageal supply before particulate embolization . Careful review of multiplanar reformats of the ct scan aided procedure planning and the consent process by identifying the hypertrophied arterial feeders . Cd should be considered in the differential diagnosis for all benign intrathoracic masses . Where a mass shows avid enhancement, whether homogeneous or heterogeneous, a ct angiogram,
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Nasolacrimal duct obstruction (nldo) is a common congenital abnormality that occurs in 30% of neonates (range 6 to 84%).1 only 2 to 4% of these children become symptomatic, and most cases of nldo resolve spontaneously in the first year of life.1 2 3 4 dacryocystocele or congenital mucocele of nasolacrimal duct is a relatively rare variant of nldo, representing 0.1% of children with congenital obstruction, and results from the coexistence of a distal and a proximal obstruction.1 5 nasolacrimal duct mucocele typically presents as a bulging in the lower medial canthus of the eye, associated with epiphora ., we report a case of congenital nasolacrimal duct mucocele, which recurred after surgery and was treated with endoscopic approach, followed by a literature review . The patient was a 30-year - old man with a history of bilateral congenital cyst of nasolacrimal duct diagnosed by the presence of a bulge in both lacrimal sac topographies since birth, without associated symptoms . He underwent surgery on the left side at 9 years of age and on the right side at 21 years of age, but the tumor recurred on the right side, without tearing, pain, discharge, or other symptoms . In subsequent evaluation with an ophthalmologist, a lack of upper and lower right lacrimal ducts was identified and indicated reconstruction surgery lacrimal spot, which was done in december 2007 . However, he developed ipsilateral epiphora later, requiring another two procedures dacryocystectomy in july 2011 and august 2011but without success, leading to recurrent local infections . After the last procedure, computed tomography (ct) showed a cystic expansion in the right lacrimal sac topography and dilatation of the bony canal of the nasolacrimal duct (figs . 1 and 2). Computed tomography of the paranasal sinuses, axial section, in bone window, showing hypodense cystic lesion in the right nasolacrimal duct topography . Computed tomography of the paranasal sinuses, coronal section, in bone window, showing hypodense cystic lesion in the right nasolacrimal duct topography . The patient was then referred for evaluation by the rhinology group of hospital universitrio professor edgard santos (salvador, brazil); nasal endoscopy showed no alterations . He had endoscopic dacryocystorhinostomy in december 2011, with confection of mucosal flap and osteotomy of nasolacrimal bone, noting that the lacrimal sac was already opened with drainage of thick purulent secretion . The patient remains asymptomatic and without clinical signs 1 year and 8 months after surgery . The nasolacrimal duct is formed by canalization of the caudal extremity of an epithelial cord derived from the ectoderm in the naso - optic fissure, which is often not completed at birth . Nldo at birth is common and usually asymptomatic or presents with epiphora in neonates and infants, which resolves spontaneously in most cases.2 generally, it results in blockage in the distal end of the nasolacrimal system, at the hasner valve level, although the blockage can also occur at the lacrimal spot.2 3 5 nasolacrimal mucocele, on the other hand, occurs in a small proportion of children with nldo, when there is a distal obstruction (distal membrane perforation failure) associated with a proximal obstruction, which can be functional or mechanical.2 3 5 it is characterized by a cystic mass at the medial canthus with dilation of the nasolacrimal duct that can, rarely, extend into nasal cavity.2 3 6 patients with dacryocystoceles may present with local infection or difficult breathing or breast - feeding in the breast ipsilateral to the mucocele.5 although dacryocystoceles are rare in adults, recurrent chronic keratitis of bacterial etiology (chlamydia trachomatis) has been reported in patients with trachoma, which, due to repeated infections, can lead scarring of the conjunctiva and even to lacrimal obstruction.7 it is believed that a mixture of mesodermal cells, mucus, amniotic fluid, tears, and colonizing bacteria compose the contents of the lacrimal sac, causing distention of the lacrimal system seen in dacryocystocele.7 encephalocele, hemangioma, dermoid cysts, and nasal gliomas may present similarly and must be considered in the differential diagnosis.5 dacryoceles appear as rounded, well - circumscribed lesions centered in the region of lacrimal sac in ct and magnetic resonance imaging (mri). In ct, if infected, it may show a peripheral contrast uptake.8 conservative treatment of dacryocystocele is based on a short course of topical antibiotics, warm compresses, and local massage three times a day, with a reported resolution rate of 76% . Dacryocystitis may occur within a few days or weeks and requires intravenous antibiotics to prevent sepsis . Most ophthalmologists recommend early surgical intervention in cases of respiratory compromise, dacryocystitis, cellulitis, large dacryocystoceles inducing astigmatism, or recurrent dacryocystoceles and in cases of failed conservative strategies.1 9 10 in cases of infection or respiratory compromise, drainage is required 24 to 48 hours after the start of antibioticoterapia.6 dacryocystorhinostomy is a surgery commonly performed, in which a fistula is created between the lacrimal sac and the nasal cavity to relieve the epiphora caused by nldo . The success rates vary from 75 to 95% in external access and 60 to 90% in endoscopic approach . The most common cause of failure in endoscopic surgery is obstruction of the new ostium by granulation or scar tissue.11 12 13 however, recent studies have shown better results with endoscopic dacryocystorhinostomy with confection mucosal flap in front of the middle turbinate and subsequent lateral wall osteotomy, with similar rates compared with the external access . The endoscopic technique has advantages such as no scars, less surgical trauma, less bleeding, a quicker return to work, and preservation of the medial canthus structure, providing sustainability of lacrimal pump mechanism.11 14 the success of surgery depends on creating a large bony ostium and preventing closure of this stoma . Use of mucosal flaps after wide resection of bone surrounding the sac is one technique used to prevent granulation tissue and narrowing of the canal, with good results according to the literature.11 14 15 the main advantage of external dacryocystorhinostomy (dcr) is visualization of the anatomy that facilitates the precise removal of bone in the lacrimal fossa and enables the exact anastomosis of the nasal mucosa and lacrimal sac . Nevertheless, an intranasal component that is not recognized prior to the external access increases the chance of treatment failure if the catheter does not pass over the wall of the cyst.5 11 nasolacrimal duct mucocele is a rare occurrence; however, it carries a risk of major complications (local infection, cellulitis, respiratory distress, etc . ). Surgery (dacryocystorhinostomy) is considered as the definitive treatment, and external access is still the most widespread and commonly used approach by ophthalmologists . However, the interest in an endoscopic nasal approach has increasingly grown as recent studies have shown good results with this technique, with similar success rates between the two types of procedures.
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A 61-year - old man with a history of impaired glucose tolerance, hypertension, and chemical pneumonitis without chronic lung disease sought care at a community hospital in northwestern minnesota after experiencing 2 days of fever, productive cough, and exertional dyspnea . During recent travel through parks in the western united states, he had been exposed to animal antlers and hides, wild bison, and donkeys . On hospital admission, the patient s temperature was 37.3c, blood pressure 148/72, pulse 100, respiratory rate 20, and room air oxygen saturation 89% (median 95%, range 65%100%). Laboratory tests (table 1) and chest imaging (figure 1) were performed . Blood samples for culture were obtained, and ceftriaxone (2 g) and azithromycin (500 mg) (table 2) were administered intravenously (iv) for presumed community - acquired pneumonia (cap). Initial admission was to a community hospital; on day 4, the patient was transferred to a tertiary referral center . Chest x - ray and computed tomographic scan images for a patient with inhalation anthrax, minnesota, usa . A) on hospital day 1, the x - ray image revealed a right upper lobe infiltrate and widening of the mediastinum . B) on hospital day 2, computed tomographic scan of the chest with intravenous contrast showed dense consolidation of the right upper lobe, mediastinal adenopathy (small arrow), and bilateral pleural effusions (large arrows). C) by hospital day 4, progressive infiltrates in the right lung were present . * iv, intravenous; hospital day, number of days in hospital including day of admission; ph, post - hospitalization days . Iv medication was discontinued and oral medication was started on day of discharge (hospital day 26) and continued for 35 additional days to complete 60 days of therapy . On hospital day 2, the patient had increasing tachycardia and higher oxygen requirements . A bacillus species was isolated from blood cultures and sent to the minnesota department of health public health laboratory (mdhphl) for identification, and iv ciprofloxacin was initiated . On hospital day 3, the patient s condition continued to decline (table 1). Mdhphl identified b. anthracis in the blood cultures, and meropenem and vancomycin were added to the treatment regimen . The isolate was sent to the centers for disease control and prevention (cdc, atlanta, ga, usa). On hospital day 4, the patient was transferred to a referral center with progressive respiratory failure requiring endotracheal intubation . Iv ciprofloxacin was continued, and iv rifampin and clindamycin were administered (table 2). A chest tube was placed in the right pleural space, and 550 ml of serosanguineous fluid was drained during the initial 24 hours . Pleural fluid analysis showed a leukocyte count of 3,389 cells / ml (neutrophils 38%, lymphocytes 56%, monocytes 6%), a lactate dehydrogenase level of 352, and negative gram stain results . On day 5, anthrax immune globulin (aig) was requested from cdc on day 4 and administered on day 5 without adverse reaction . The patient s disease course was complicated by nonoliguric renal failure; serum creatinine peaked at 1.5 mg / dl . On day 8, rifampin was discontinued; meropenem, which had been discontinued on day 5, was resumed for prophylaxis against nosocomial infection and improved central nervous system coverage of b. anthracis infection . After stabilization, the patient was maintained on volume control ventilation: tidal volume 500 ml, positive end the patient was extubated on day 11, and the chest tube was removed on day 13; left - sided pleural effusion did not recur . He completed a 10-day course of clindamycin and a 14-day course of meropenem . Upon discharge on day 26, cdc performed susceptibility testing using broth microdilution (technical appendix). In compliance with the investigational new drug protocol for aig administration, we obtained serial serum samples to assess levels of lethal factor (lf) and anti protective antigen (pa) igg . We used an elisa to determine serum anti - pa igg levels before and after aig administration (4). The lower limit of quantification (lloq) for this assay is 3.7 g / ml . Antimicrobial susceptibility testing (technical appendix) performed on the b. anthracis isolate showed a mic of penicillin of <0.015 g / ml and mic of ciprofloxacin of 0.12 g / ml . The patient s initial plasma lf level was 58.0 ng / ml, which declined to 1.5 ng / ml before aig administration: pleural fluid lf was 16.2 ng / ml at initial drainage and declined steadily (figure 2). Before aig administration, no anti - pa igg was discernable because these quantifications were below the lloq (figure 2). Immediately after aig administration, anti - pa igg reached maximal value of 160.5 g / ml and maintained a plateau thereafter . Plasma and pleural fluid lethal factor levels and anti - protective antigen igg (aig) levels for a patient from the time of examination in the community hospital emergency department to discharge from the tertiary referral center . Asterisks indicate that anti - protective aig levels obtained before anthrax immune globulin administration were below the lower limit of quantification . The patient s initial plasma lethal factor level was 58.0 ng / ml and declined to 1.5 ng / ml before aig administration . We describe the second us case of naturally acquired inhalation anthrax since the bioterrorism - related infections of 2001 and the third known case worldwide in which the patient received aig (2,6). Before this case, the most recent case in the united states had occurred in pennsylvania during 2006 (2). That patient had a 3-day prodromal illness, and initial plasma lf (294.3 ng / ml) and pleural fluid lf (543.2 ng / ml) levels substantially higher than those reported here . Seroconversion to anti - pa igg occurred before aig administration in the 2006 case, possibly because of a longer interval between symptom onset and aig infusion (10 vs. 6 days). The patient, who appears to have been in the fulminant phase of illness when tailored antimicrobial drug therapy was initiated on hospital day 4, died on day 7 despite aig treatment (6). (7) proposed a scoring system derived from multivariate analysis to distinguish inhalation anthrax from cap on the basis of clinical features at disease onset . In the case - patient described here, 3 of 5 identifying variables were present: elevated alanine aminotransferase / aspartate aminotransferase, normal leukocyte count, and tachycardia . The 2 remaining possible variables, low serum sodium level and nausea / vomiting were not present . This correlated with a sensitivity of 82% and specificity of 81% for diagnosing anthrax rather than cap . Mediastinal widening, exhibited in the patient in this report, has also been proposed as a characteristic that can distinguish anthrax from cap . When anthrax patients were compared with an age-, sex-, and race - matched control population, mediastinal widening occurred in 82% of anthrax patients and 8% of cap patients (8). A systematic review of inhalation anthrax cases showed improved survival if antimicrobial drugs were initiated during the prodromal rather than fulminant phase of illness: 75% of patients who survived and 10% of those who died were administered antimicrobial drugs in the prodromal phase (9). A new staging system for inhalation anthrax was proposed that divides the prodromal period into early and intermediate progressive stages (10). Early diagnosis facilitated implementation of multi antimicrobial drug therapy during the intermediate progressive stage, which is associated with increased the survival rate (67% vs. 21%) (9). For the case discussed here, systems to facilitate cooperation between a community hospital and state health agency enabled definitive identification of b. anthracis within 24 hours of culture becoming positive, leading to specific interventions including combination antimicrobial therapy, pleural drainage, and aig administration . Drainage of pleural fluid has also been associated with increased survival (83% vs. 9%) (9); it was performed shortly after the current patient s illness reached the late fulminant stage . Besides elevated lf levels in pleural fluid of recent us case - patients, patients with bioterrorism - related cases had large quantities of b. anthracis cell wall antigens, which further supports use of drainage procedures (11). Although no clinical studies have reported on efficacy of passive immunization of humans against anthrax as treatment, mortality rates have been reduced in studies of inhalation anthrax in which animals were given polyclonal antibodies against pa (12). Aig administration in the current case was associated with a reduction in toxemia, although the role of passive immunization in anthrax treatment needs further evaluation.
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Research took place in the first half of 2007, and the basic criteria in selection were male veterans who had experienced war trauma . To form group a, all veterans who were diagnosed with ptsd in mostar clinical hospital and came for a doctor's appointment were asked to participate in the study . To form group b, initial contact with veterans who do not suffer from ptsd was through veterans associations, and further recruitment the principal researcher paid a visit to the two war veteran association facilities, where the veterans assembled on a daily basis . Research notification forms were handed out, and the purpose of the study was explained . For those veterans interested in participating in the study, a visit to the researcher was arranged either at the psychiatric ward in mostar clinical hospital or at the association facility . Inclusion criteria for all male veterans were active participation in the war, being married, or cohabiting . An additional inclusion criterion was the presence of ptsd for the experimental group and absence of ptsd for the control group . Exclusion criteria were the presence of alcohol addiction or mental disorders prior to the war . However, negative correlation between marital satisfaction and alcohol abuse is well documented (doumas, blasey & mitchell, 2007) and, therefore, such participants were excluded to investigate only the influence of ptsd . Each participant was asked to inform their married or cohabiting partner of the research and ask if they were also willing to participate . All participants were informed of the study goals, told that they could refuse to participate at any point of the research, and signed the written consent . Also, all participants were given the opportunity of therapy or medical intervention, if needed . Veterans and their partner first completed the socio - demographic questionnaire and then followed this up with the harvard trauma manual bosnia - herzegovina version (allden et al ., 1998) to establish the presence of ptsd . Cut - down, annoyed, guilt, eye - opener (cage) (ewing, 1984) was used for identifying alcohol addiction . Finally, dyadic adjustment scale (das) (spanier, 1979) and brief symptom inventory (bsi) (derogatis, 1993) were applied . From 409 veterans contacted through mostar clinical hospital, 317 sixty - nine veterans (21.8%) refused to participate in the research and an additional 37 fulfilled alcohol - addiction exclusion criteria . The research continued with 211 veterans who were asked to inform their wives of the research . From a prospective 211 wives / partners, 57 veteran wives (27.0%) refused to participate . Finally, 154 couples in which husband had ptsd entered the study . From 106 veterans contacted through veterans associations, seven (6.6%) of them met the alcohol - addiction criteria and were excluded from the research . From 99 contacted wives, 22 (22.2%) refused to participate in the research . Finally, 77 couples where husband did not meet criteria for ptsd participated in the study . General demographic data, social, and economic status were determined using a general demographic questionnaire designed particularly for the aim of this research . To determine the presence of posttraumatic symptoms, harvard trauma questionnaire bosnia - herzegovina version (htq; allden et al ., the htq is a structured interview used in many studies and is particularly suitable for use in multicultural settings (norris & hamblen, 2004). For the purposes of this study, first and fourth module were used: a list of possible traumatic events contains questions relating to experiences and traumatic events that the inhabitants of bosnia and herzegovina were exposed to during various stages of the war (during the war, during the refugee period, and postwar period). It contains 46 possible traumatic events presented in the form of yes and no questions . A list of questions relating to psychosocial problems caused by trauma contains a list of 40 statements on psychosocial difficulties caused by trauma . The first 16 statements are derived from dsm - iv criteria for ptsd and represent three clusters of symptoms: re - experiencing of a traumatic event (b), avoidance (c), and arousal symptoms (d). The rest of the statements refer to participants perception of the degree in which the trauma affected their everyday abilities . Responses to each question are scored as follows: 1=not at all, 2=very little, 3=quite, 4=very much . The total score is the mean value of all 40 statements, whereas the ptsd score is the mean value of the first 16 statements . The instrument has been validated for use in a non - psychiatric medical setting in bosnia and herzegovina, with maximized sensitivity and specificity at a cut - off point of 2.06 (auc 0.981 sensitivity 100%, specificity 93.9%, predictive positive value (ppv) of 61.5, and negative predictive value (npv) of 100, cronbach's alpha=0.88) (oruc et al ., 2008). However, for the purposes of this study, a 2.5 cut - off score for ptsd was used, which is comparable with the results of patients who were clinically diagnosed with ptsd in other studies (htq; allden et al ., 1998, mollica et al ., 1992). A list of possible traumatic events contains questions relating to experiences and traumatic events that the inhabitants of bosnia and herzegovina were exposed to during various stages of the war (during the war, during the refugee period, and postwar period). It contains 46 possible traumatic events presented in the form of yes and no questions . A list of questions relating to psychosocial problems caused by trauma contains a list of 40 statements on psychosocial difficulties caused by trauma . The first 16 statements are derived from dsm - iv criteria for ptsd and represent three clusters of symptoms: re - experiencing of a traumatic event (b), avoidance (c), and arousal symptoms (d). The rest of the statements refer to participants perception of the degree in which the trauma affected their everyday abilities . Responses to each question are scored as follows: 1=not at all, 2=very little, 3=quite, 4=very much . The total score is the mean value of all 40 statements, whereas the ptsd score is the mean value of the first 16 statements . The instrument has been validated for use in a non - psychiatric medical setting in bosnia and herzegovina, with maximized sensitivity and specificity at a cut - off point of 2.06 (auc 0.981 sensitivity 100%, specificity 93.9%, predictive positive value (ppv) of 61.5, and negative predictive value (npv) of 100, cronbach's alpha=0.88) (oruc et al ., 2008). However, for the purposes of this study, a 2.5 cut - off score for ptsd was used, which is comparable with the results of patients who were clinically diagnosed with ptsd in other studies (htq; allden et al ., 1998, mollica et al ., 1992). For the purposes of screening for alcoholism, the cage consists of four yes or no questions, and two or more affirmative answers denote alcohol problem . The quality of partner / spouse relationship was determined by the das (spanier, 1979). Responses are mostly given on a 5-point scale, and some of the questions are dichotomous . The authors obtained four factors in a factor analysis, and the scale is, therefore, divided into four subscales . Brief symptom inventory (derogatis, 1993) is a 53-item self - report inventory designed to assess the current level of psychological symptoms and distress . Symptoms are assigned to nine subscales that represent domains of psychopathology and for the purposes of this study, depression and anger items are answered on a 5-point likert - type scale (04) that ranges from not at all to extremely . Reliability of the whole bsi is very high, and internal consistency on separate dimensions ranges from cronbach's alpha= 0.83 to cronbach's alpha=0.92 in the traumatized persons, and from cronbach's alpha=0.69 to cronbach's alpha=0.84 in general croatian population (stribic, 2005). Anova was used for interval variables, whereas fischer's least significant difference (lsd) post - hoc test and one - way analysis of variance were used for a more detailed analysis . Statistical analyses were performed using statistical package for social science for windows v.11 (spss inc . From 409 veterans contacted through mostar clinical hospital, 317 were married and thus were asked to participate . Sixty - nine veterans (21.8%) refused to participate in the research and an additional 37 fulfilled alcohol - addiction exclusion criteria . The research continued with 211 veterans who were asked to inform their wives of the research . From a prospective 211 wives / partners, 57 veteran wives (27.0%) refused to participate . Finally, 154 couples in which husband had ptsd entered the study . From 106 veterans contacted through veterans associations, seven (6.6%) of them met the alcohol - addiction criteria and were excluded from the research . From 99 contacted wives, 22 (22.2%) refused to participate in the research . Finally, 77 couples where husband did not meet criteria for ptsd participated in the study . General demographic data, social, and economic status were determined using a general demographic questionnaire designed particularly for the aim of this research . To determine the presence of posttraumatic symptoms, harvard trauma questionnaire bosnia - herzegovina version (htq; allden et al ., 1998) the htq is a structured interview used in many studies and is particularly suitable for use in multicultural settings (norris & hamblen, 2004). For the purposes of this study, first and fourth module were used: a list of possible traumatic events contains questions relating to experiences and traumatic events that the inhabitants of bosnia and herzegovina were exposed to during various stages of the war (during the war, during the refugee period, and postwar period). It contains 46 possible traumatic events presented in the form of yes and no questions . A list of questions relating to psychosocial problems caused by trauma contains a list of 40 statements on psychosocial difficulties caused by trauma . The first 16 statements are derived from dsm - iv criteria for ptsd and represent three clusters of symptoms: re - experiencing of a traumatic event (b), avoidance (c), and arousal symptoms (d). The rest of the statements refer to participants perception of the degree in which the trauma affected their everyday abilities . Responses to each question are scored as follows: 1=not at all, 2=very little, 3=quite, 4=very much . The total score is the mean value of all 40 statements, whereas the ptsd score is the mean value of the first 16 statements . The instrument has been validated for use in a non - psychiatric medical setting in bosnia and herzegovina, with maximized sensitivity and specificity at a cut - off point of 2.06 (auc 0.981 sensitivity 100%, specificity 93.9%, predictive positive value (ppv) of 61.5, and negative predictive value (npv) of 100, cronbach's alpha=0.88) (oruc et al ., 2008). However, for the purposes of this study, a 2.5 cut - off score for ptsd was used, which is comparable with the results of patients who were clinically diagnosed with ptsd in other studies (htq; allden et al ., 1998, mollica et al ., 1992). A list of possible traumatic events contains questions relating to experiences and traumatic events that the inhabitants of bosnia and herzegovina were exposed to during various stages of the war (during the war, during the refugee period, and postwar period). It contains 46 possible traumatic events presented in the form of yes and no questions . A list of questions relating to psychosocial problems caused by trauma contains a list of 40 statements on psychosocial difficulties caused by trauma . The first 16 statements are derived from dsm - iv criteria for ptsd and represent three clusters of symptoms: re - experiencing of a traumatic event (b), avoidance (c), and arousal symptoms (d). The rest of the statements refer to participants perception of the degree in which the trauma affected their everyday abilities . Responses to each question are scored as follows: 1=not at all, 2=very little, 3=quite, 4=very much . The total score is the mean value of all 40 statements, whereas the ptsd score is the mean value of the first 16 statements . The instrument has been validated for use in a non - psychiatric medical setting in bosnia and herzegovina, with maximized sensitivity and specificity at a cut - off point of 2.06 (auc 0.981 sensitivity 100%, specificity 93.9%, predictive positive value (ppv) of 61.5, and negative predictive value (npv) of 100, cronbach's alpha=0.88) (oruc et al ., 2008). However, for the purposes of this study, a 2.5 cut - off score for ptsd was used, which is comparable with the results of patients who were clinically diagnosed with ptsd in other studies (htq; allden et al ., 1998, mollica et al ., 1992). For the purposes of screening for alcoholism, the cage questionnaire was applied (ewing, 1984). The cage consists of four yes or no questions, and two or more affirmative answers denote alcohol problem . The quality of partner / spouse relationship was determined by the das (spanier, 1979). Responses are mostly given on a 5-point scale, and some of the questions are dichotomous . The authors obtained four factors in a factor analysis, and the scale is, therefore, divided into four subscales . Brief symptom inventory (derogatis, 1993) is a 53-item self - report inventory designed to assess the current level of psychological symptoms and distress . Symptoms are assigned to nine subscales that represent domains of psychopathology and for the purposes of this study, depression and anger items are answered on a 5-point likert - type scale (04) that ranges from not at all to extremely . Reliability of the whole bsi is very high, and internal consistency on separate dimensions ranges from cronbach's alpha= 0.83 to cronbach's alpha=0.92 in the traumatized persons, and from cronbach's alpha=0.69 to cronbach's alpha=0.84 in general croatian population (stribic, 2005). Anova was used for interval variables, whereas fischer's least significant difference (lsd) post - hoc test and one - way analysis of variance were used for a more detailed analysis . Statistical analyses were performed using statistical package for social science for windows v.11 (spss inc ., chicago, il, usa). To investigate differences in marital satisfaction, couples were further divided into four groups: group a1 where only male partner had ptsd, group a2 where both partners had ptsd, group b1 where neither partners had ptsd, and group b2 where only female partner had ptsd . Because in group b2, there were only five couples, the group was excluded from further analysis . Women and men from three groups did not differ significantly regarding their age (f= 0.523, p= 0.594; f=1,587, p=0.207, respectively) with the average age for female participants being 44and 92 (9,922) and for male participants 48 and75 (9,876). Female partners differed in the educational level (2=14,565; df = 4; p=0.010) and economic status (2=27,508; df=4; p=0.000) between the groups . Veterans also differed in education level (2=6,816; df=4; p=0.029) and economic status (24,954; df=4; p=0.000). Veterans from a1 and a2 had significantly lower education level and lower economic status than the b1 group . The differences on das score among the three groups were tested using one - way anova . The analyses showed significant differences among the three groups of participants in the total das score (table 1) both for female and male partners . Lsd post hoc test shows that there is a significant difference between all three groups of veteran's wives in marital adjustment scores . Wives whose partners do not have ptsd have significantly higher scores on das (105.813.39) than the other two groups (respectively). Also, wives in group where both partners are ptsd affected score significantly lower on das (90.3416.01) compared with wives in group where only male partner has ptsd (81.59 24.65). Lsd post hoc test for veterans marital adjustment shows that veterans who do not have ptsd (105.8415.20) have significantly higher score on das than the two groups of veterans with ptsd (94.0817.31; 89.9414.18, respectively). Veterans who are diagnosed with ptsd do not differ significantly on das score regarding their wives ptsd status . Differences in the das total score between groups notes: das, dyadic adjustment scale; ptsd, posttraumatic stress disorder . As we have found significant differences between groups in marital adjustment, further analysis was performed to examine which variables best explain dyadic adjustment . Table 2 provides the results of linear regression analysis, with total das score for women as the outcome variable, that is, total das score for men, and with b cluster, c cluster, and d cluster symptoms, depressiveness and hostility for both partners as contributing variables . Linear regression analysis for the overall das scores for wives and husbands notes: bsi, brief symptom inventory; das, dyadic adjustment scale; ptsd, posttraumatic stress disorder . Results (table 2) show that clusters b, c, and d of ptsd symptoms, hostility, and depression scores of wives and veterans explain up to 33.8% of female partner's marital satisfaction and up to 17.6% of veteran's marital satisfaction . When it comes to marital satisfaction of wives, his avoidance symptoms (6.3%), her level of depression, and her re - experiencing symptoms are significant predictors . None of the predictor variables have significant individual contribution to his marital satisfaction . Also, women from the a groups report significantly lower marital satisfaction than their husbands . In couples, where neither partner has ptsd marital satisfaction is almost the same (table 1) women and men from three groups did not differ significantly regarding their age (f= 0.523, p= 0.594; f=1,587, p=0.207, respectively) with the average age for female participants being 44and 92 (9,922) and for male participants 48 and75 (9,876). Female partners differed in the educational level (2=14,565; df = 4; p=0.010) and economic status (2=27,508; df=4; p=0.000) between the groups . Veterans also differed in education level (2=6,816; df=4; p=0.029) and economic status (24,954; df=4; p=0.000). Veterans from a1 and a2 had significantly lower education level and lower economic status than the b1 group . The differences on das score among the three groups were tested using one - way anova . The analyses showed significant differences among the three groups of participants in the total das score (table 1) both for female and male partners . Lsd post hoc test shows that there is a significant difference between all three groups of veteran's wives in marital adjustment scores . Wives whose partners do not have ptsd have significantly higher scores on das (105.813.39) than the other two groups (respectively). Also, wives in group where both partners are ptsd affected score significantly lower on das (90.3416.01) compared with wives in group where only male partner has ptsd (81.59 24.65). Lsd post hoc test for veterans marital adjustment shows that veterans who do not have ptsd (105.8415.20) have significantly higher score on das than the two groups of veterans with ptsd (94.0817.31; 89.9414.18, respectively). Veterans who are diagnosed with ptsd do not differ significantly on das score regarding their wives ptsd status . Differences in the das total score between groups notes: das, dyadic adjustment scale; ptsd, posttraumatic stress disorder . As we have found significant differences between groups in marital adjustment, further analysis was performed to examine which variables best explain dyadic adjustment . Table 2 provides the results of linear regression analysis, with total das score for women as the outcome variable, that is, total das score for men, and with b cluster, c cluster, and d cluster symptoms, depressiveness and hostility for both partners as contributing variables . Linear regression analysis for the overall das scores for wives and husbands notes: bsi, brief symptom inventory; das, dyadic adjustment scale; ptsd, posttraumatic stress disorder . Results (table 2) show that clusters b, c, and d of ptsd symptoms, hostility, and depression scores of wives and veterans explain up to 33.8% of female partner's marital satisfaction and up to 17.6% of veteran's marital satisfaction . When it comes to marital satisfaction of wives, his avoidance symptoms (6.3%), her level of depression, and her re - experiencing symptoms are significant predictors . Also, women from the a groups report significantly lower marital satisfaction than their husbands . In couples, where neither partner has ptsd marital satisfaction is almost the same (table 1) our results convincingly show that veterans ptsd is related to lower levels of marriage quality of their wives, who often encounter problems in their intimate / partner relationships . The results of our research are in accordance with a series of other studies into the effect of ptsd on partner relationships of vietnam and israeli veterans (chrysos et al ., 2005; dekel et al ., 2005; dekel & solomon, 2006; galovski & lyons, 2004; nelson goff et al ., 2006), which also determined that ptsd symptoms directly affect veterans abilities to establish and maintain intimate relationships and are related to lower marital satisfaction . Dyadic relationships become even more complex when both partners suffer from ptsd . In our sample, marital quality was significantly lower in couples where both partners had ptsd, compared with couples where only the veteran had ptsd or neither partner had ptsd . Ptsd - affected wives as well as their partners suffer from emotional numbness and withdrawal . It is questionable whether they are able to perform family roles that have been imposed on them and satisfy their partners increased needs for support although being themselves affected by ptsd (solomon, 1988). It could be assumed that these wives do not receive adequate and needed support from their husbands, which only adds to their psychological distress and dysfunctionality . In such case, family cannot provide a supportive context for the recovery of primary traumatized member (figley & blaine everson, 2010). Our study showed that husband's avoidance symptoms significantly contribute to wife's marital adjustment scores, which again is in concordance with previous results . Most researches on the functioning of marital relationships demonstrated that communication skills play a constituent role in general marital satisfaction and that emotional expression plays a key role in establishing and maintaining close and intimate relationships (harkness & zador, 2001; riggs, byrne, weathers & litz, 1998). Interaction of the symptoms of emotional numbness and emotional alienation in veterans family life creates an emotional gap and a serious functional loss to their wives (nelson goff et al ., 2006; rosenheck & thomson, 1986). Veterans inhibited intimacy and expressiveness, limited emotional expression, lack of interest in social activities, and problems with family cohesion and sexual intimacy contribute to marital discord (dekel & solomon, 2006; mason, 1990; riggs et al ., 1998). Wives depressiveness significantly contributes to her marital adjustment that is expected because it is well recognized that marital dissatisfaction and depressive symptoms are associated (balog et al ., 2003; st john & montgomery, 2009). An interesting finding is that a wife's symptoms of re - experiencing traumatic events rather than avoidance symptoms significantly explain her marital adjustment . Similar results were obtained by hamilton, nelson goff, crow, and reisberg (2009) who found that greater intrusion symptoms of female partners of operation iraqi freedom (oif) and operation enduring freedom (oef) veterans significantly predict relationship satisfaction scores . There are several possible explanations for our finding, and it is important to underline that the women in our study were exposed to civil war traumatic events and their possible sufferings are rarely recognized as such and usually go untreated . Different studies have noted that women have the tendency to report more re - experiencing than other symptoms . Also, it is our speculation that living with a war veteran is a daily reminder of traumatic event and a possible point of resentment because his trauma is more valuable . Unlike the study of evans et al . (2003), where veterans avoidance symptoms and comorbid symptoms of anger and depression play a significant role in family functioning, in our sample, examined factors did not have an individual impact, rather the total clinical manifestation of ptsd together with hostility and depressiveness impacts veterans marital adjustment . Drawing conclusions was also limited by a non - homogeneous sample in socio - demographic variables relating to educational level and economic status that can serve as mediators and mitigate the effect of ptsd on partner relationships . Even though ptsd is most frequently accompanied by comorbid mental and physical diseases (bleich & solomon, 2004; kessler, sonnega, bromet, hughes, & nelson, 1995), this research did not examine the effect of such diseases on partner relationship . A group of participants in which only wives have ptsd would reveal more on ptsd effects on partner relationships and family functioning . Information on premorbid functioning of a couple, including potential adjustment risks and protection factors, would also complete the picture . Without any doubt, couples where both partners have ptsd report the lowest marital satisfaction . This result demonstrates that in couples where both partners have ptsd, each of the partners creates his / her own system of functioning that may help them in carrying the burden of the chronic disease, but at the same time, it produces a negative impact on the spiral of mutual adjustment and marital quality (nelson goff & smith, 2005). Finally, the result of our research confirms the negative effect of war veterans ptsd on the quality of their marital relations, especially when both partners suffer from ptsd . The result also highlights the importance of recognizing ptsd in wives of traumatized veterans as well as the importance of family approach in the treatment of ptsd . There is no conflict of interest in the present study for any of the authors.
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Owing to the development of modern technology, the use of smartphones has been increasing rapidly . According to the pew research center1, smartphone users represent 56% of the entire us population, and the average amount of time spent on a smartphone per day is 5.1 hours . It was reported that the average time spent on a smartphone per day has increased rapidly as the smartphone penetration rate has soared . However, poor posture while using a smartphone for continuous periods causes musculoskeletal problems2 . Forward neck posture (forward head posture), a postural distortion, is regarded as a typical musculoskeletal disorder, and it is commonly seen in patients with cervical diseases3 . If the horizontal distance between the ear s tragus and the posterior angle of the acromion is more than 5 cm while a person is standing, the occiput and upper cervical spine lean forward excessively and the lower cervical spine and the upper vertebrae lean backward excessively . All these symptoms correspond to the features of forward head posture (forward neck posture)4 . When abnormal alignment of the vertebrae is continuously maintained due to inappropriate posture, that is, forward head posture, the vertebrae and surrounding soft tissues might be easily exposed to a sudden impact or chronic stress, and this could lead to possible changes in the blood vessels, spinal disease, organ dysfunction, degenerative diseases, and autonomic dysfunctions, such as headaches and chronic fatigue5 . The sling stabilization exercise can be used as a therapy for straight neck treatment and kim and kim6 have noted that stretching and sling exercises are effective at ameliorating cervical spine instability . The curative power of a sling stabilization exercise is found in its ability to enhance mobility, improve sensorimotor function, and increase relaxation7 . The sling stabilization exercise is a type of treatment in which patients with neck pain can use their own body weight to resist gravity; thus, this type of exercise can be performed very safely . As the sling stabilization exercise enables patients to perform unloading exercise in a gravity - reduced environment, it has a similar effect to what would be expected from an aquatic exercise . A study was concluded to determine the effect of the co - contraction of the masticatory muscles during neck stabilization exercises on changes in the thickness of the neck flexors . That study concluded that co - contraction of the masticatory muscles during neck stabilization exercise is helpful, increasing the thickness of the longus colli muscle8 . A number of different types of stretching exercises can be used to treat patients with chronic neck pain, including dynamic stretching, which a patient can practice alone for prophylaxis9, proprioceptive neuromuscular facilitation, where the release and contraction of a muscle are repeatedly practiced, and evjenth - hamberg stretching, where isometric contraction and static stretching are applied to agonist and antagonist muscles using the hold - relax technique and the contract - relax technique10 . Lee11 investigated the effects of stretching exercises on forward head posture . In that study, the subjects were divided into three groups, a balance exercise group, a stretching exercise group, and a control group, and the respective groups performed the assigned exercises for four weeks . That study found that maximum muscle contraction was increased in the balance exercise group and the stretching exercise group . It also found that the forward head posture improved in those two groups . In addition, park12 reported that the symptoms of forward head posture improved after applying static stretching and evjenth - hamberg stretching . Most of the existing studies have mainly focused on comparing stretching with other types of exercises and they have concentrated only on demonstrating that stretching is effective for the treatment of forward head posture . However, very few studies have investigated the effect of training patients to use stretching and sling stabilization exercises in order to treat straight neck symptoms . Only a few studies have focused on how to treat straight neck symptoms and correct spinal alignment . Hence, this present study was conducted to compare the pre- and post - test results of the craniovertebral angle (cva), the cranial rotation angle (cra), and cervical range of motion (rom), aiming to demonstrate whether or not training by stretching and sling stabilization exercises has any remedial value . From among people in their 20s who were suffering from straight neck syndrome, 20 patients, diagnosed with straight neck, were recruited as the study s subjects . The subjects were divided into two groups: one sling stabilization exercise group, and one stretching exercise group . All the subjects voluntarily participated in the experiment and were thoroughly briefed on the purpose of the study and the tasks to be performed . The experiment began only after the subjects fully understood the study s intention and had consented to participation . Exclusion criteria included: heart disease, a mental health condition, dementia, sensory disturbance, spinal tumor, spondylolisthesis, pelvic osteoporosis, spinal osteoporosis, lumbar herniated intervertebral disc, or any other disease that might have influenced the spine or musculoskeletal system during experiment . For rom, the angle of flexion, extension, rotation, and lateral bending were measured within the active range of motion, using a standard goniometer (table 2). When the rom was measured, the intraclass correlation coefficient (icc) was used as an index of reliability . As the icc (2, k) was 0.934, the reliability of the measurements was considered to be high . The measurements were taken twice, and the mean value of the two measurements was used for the analysis . The measurements were taken before the experiment began and then again after the 6-week intervention had ended . For the cva and cra measurements, the subjects were positioned so as to retain their natural head posture while standing at ease with both of their arms relaxed and placed alongside their torso . To measure the cervical angle, the 7th cervical (c7) vertebra, ear tragus, and the lateral canthus of each subject were linked by straight lines on the x - ray, and then the cva and cra were measured . If the x - ray showed a small cva and a large cra, the subject was noted as having greater forward head posture . For the sling stabilization exercise, the cervical flexion - extension exercise and cervical lateral bending exercise were performed with the aid of sling stabilization exercise equipment . To perform the cervical flexion - extension exercise, the subjects were instructed to maintain the head in a neutral position while in a supine position and to slowly extend the neck, using the strap of the sling . The cervical flexion exercise was performed with the subjects in the prone position . In this position, the strap of sling was adjusted so that it would be perpendicular to the surface of the ground in order to prevent the chin and shoulders from being lifted during the exercise . The subjects performed three sets of exercise, and the exercise was repeated 15 times within each of the sets . Between each set, the subjects rested for 30 seconds . To perform the cervical lateral bending exercise, the subjects slowly performed right and left lateral bending of the head while releasing their neck . The subjects performed three sets of exercise, and the exercise was repeated 15 times within each of the sets . Between each set, the subjects rested for 30 seconds . To perform the stretching exercise, this study adopted a method in which active contractions and passive relaxations of the agonist muscles were repeated, and active contraction of antagonist muscles was added, finally . Modifying the stretching exercise used by park12, this stretching exercise was performed for six weeks, and it was practiced three times each week . To stretch the sternocleidomastoid muscle, the subjects were instructed to lie in the supine position, to let their head and neck hang over the end of a hospital bed, and to place their shoulders at the edge of a bed . The therapist was told to stand at the head of the hospital bed after fixing the subject s shoulder and chest to the table using a belt . The head and neck posture were arranged so that the subjects would easily feel the tension created by the muscle contraction . When the therapist held the subject s head, both of the subject s ears were covered by both of the therapist s palms, and the subject s sternocleidomastoid muscles were held by the therapist s fingers . While holding this posture, the therapist fully rotated the subject s head to the right and pulled the head, making it perform left lateral bending . The subject pressed the therapist s right hand in order to signal that the therapist should apply a similar amount of force in the opposite direction . Using these balanced forces, the therapist guided the subject in performing isometric contractions . The subject performed the isometric contraction for six seconds, counting from one to six, and gradually increased the intensity of the contraction, in order to prevent the valsalva maneuver from occurring . While the subject relaxed for two to three seconds after the contraction, the therapist moved the subject s head in the direction in which greater stretching was possible . The therapist had to move the subject s head beyond the point which the muscle did not allow further movement, and held the stretch for 1516 seconds . Finally, the therapist asked the subject to look to the right side and to move the head in the direction where the subject could extend the head further . By resisting this type of movement, this movement was continued for six seconds and the subject was allowed to rest for 10 seconds . These movements were repeated four times, and a total of 160 seconds were assigned to the stretching exercises . For the upper trapezius, the subject was instructed to hang his head and neck over the end of a hospital bed in the supine position and the therapist used a belt to fix the subject s shoulder and chest to the bed . The therapist was required to stand at the head of the bed, to hold the back of subject s head in his right hand, and to hold up the subject s head using his wrist and arm . The subject s jaw was held by the therapist s left hand and the therapist pulled up the subject s head . In this condition, the therapist arranged the subject s cervical vertebrae to first slowly rotate fully to the right and then to engage in left lateral bending . This time, the therapist s body moved along with the subject s head . Afterwards, isometric contraction was implemented and maintained for six seconds . In order to strengthen the antagonist muscle, the therapist kept his hands in the same place and pulled the subject s jaw to the right . The therapist then asked the subject to look towards the direction in which the head was to be extended to and move the head further in that direction . By resisting these movements, the therapist strengthened the subject s antagonist muscles . The time assigned for the isometric contraction, the method used to relax the muscle, and the entire time assigned for the stretching exercise were identical to the conditions used for the sternocleidomastoid stretches . The kolmogorov - smirnov (k - s) test was used to verify the normality of the data . In order to analyze the way in which the straight neck symptoms varied depending on the type of treatment, the independent t - test the paired t - test was used to investigate variations in the pre- and post - test measusres of straight neck and spinal posture, depending on the type of treatment . A statistical significance level of = 0.05 was used . This study was approved by the institutional review board of namseoul university (cheonan, korea, nsu-140609 - 2). This study investigated whether sling stabilization and stretching exercises could cause structural changes in the cervical spine of straight neck patients . In terms of the change in the cervical angle, there was no significant difference between the sling stabilization exercise group and the stretching exercise group . When a comparison of the pre- and post - test results was conducted, focusing on changes in cra and cva, the stretching exercise group was found to show a statistically significant changes while the sling stabilization exercise group did not (table 1table 1.changes in cva and cra of the two groups (degrees)variablegrouppre - testpost - testcvasling73.16.969.15.4stretching71.36.265.35.2crasling153.011.2148.715.9stretching150.611.2142.710.5values are expressed as mean standard deviation; cva: craniovertebral angle; cra: craniovertebral angle; * <0 .05; p <0.05: significance difference between pre- and post- exercise). In terms of rom when the pre- and post - test results of the sling stabilization exercise group were compared, a significant difference was found in flexion, right rotation, left lateral bending, and right lateral bending . A significant difference was found in left rotation, left lateral bending, and right lateral bending, when the pre- and post - test results of the stretching exercise group were compared (table 2table 2.changes in rom of the two groups (degrees)variablegrouppre - testpost - testflexionsling46.59.849.68.4stretching 50.24.852.46.0extensionsling37.011.741.78.0stretching 38.46.541.05.7left rotationsling49.16.954.48.4stretching 48.46.254.54.9right rotationsling48.58.456.39.2stretching 51.53.554.56.5left lateral bendingsling34.27.242.05.4stretching 34.34.438.34.5right lateral bendingsling ex30.13.336.64.7stretching 33.84.837.35.1values are expressed as mean standard deviation; * <0.05; p <0.05: significance difference between pre- and post - exercise). Values are expressed as mean standard deviation; cva: craniovertebral angle; cra: craniovertebral angle; * <0 .05; p <0.05: significance difference between pre- and post- exercise values are expressed as mean standard deviation; * <0.05; p <0.05: significance difference between pre- and post - exercise people today commonly experience neck diseases because of repetitious jobs and inappropriate posture resulting from industrialization13 . Sling exercise has the major therapeutic advantage of underwater exercise, a reduction of the influence of gravity, on hard ground . The present study found that only the stretching exercise group displayed a significant difference in terms of cva and cra changes when the pre- and post - test results were compared . In the stretching exercise group, cva decreased from 71.336.28 to 65.335.22 degrees and cra decreased from 150.6611.21 to 142.7010.53 degrees . Yang et al.15 conducted a study of subjects with forward head posture, who performed neck flexion and extension exercises, stretching exercises, and neck stabilization exercises using sling exercise equipment . That study compared the pre- and post - test results of threshold measuring pressure pain, cva, and cra . No significant difference was found in threshold pressure pain, while significant differences were found in the cva and cra values of the rapid upper limb assessment (rula). In another study, 25 subjects were divided into two groups, a sling stabilization exercise group and a control group, and they were instructed to conduct the assigned exercise for four weeks . Cva, cra, muscle activity, and cervical alignments were measured after the completion of the exercise . When the pre- and post - test results of cva and cra were compared, the sling stabilization exercise group displayed significant differences were both cva and cra, while a significant difference was found in the cva value of the control group, which only performed the stretching exercise16 . As shown in this study, the stretching exercise returned the shortened muscle to normality, and the abnormal cervical range of motion caused by the shortened muscle also returned to normality . Thus, the stretching exercise was found to be effective at normalizing abnormal forward head posture . The sling stabilization exercise was also found to have exerted a positive influence . In the sling stabilization exercise group, the subjects were able to exercise with no influence of gravity, within their own cervical rom, under the therapist s control, using the sling equipment . Thus, the normalization of abnormal forward head posture is possible using the sling stabilization exercise . This can be attributed to the fact that the subjects living habits could not be thoroughly controlled and the therapist s contribution to the sling stabilization exercise group was less than the interference in the stretching exercise group . Therefore, the patient s weakened muscle could not be fully used, which could resulted in the lack of a significant difference in the findings . In terms of rom changes, the measurement of cervical rom, is regarded as the standard test by which a patient with a disorder such as straight neck syndrome is assessed, because it enables investigators to focus on the factors of pain and functional movement . Rom is also frequently used as a yardstick to judge whether actual remedial value has occurred . In many studies chun et al.18 reported that the older patients who had performed stretching exercises were found to have increased their cervical range of motion . The neck pain group that had been treated by both the rom technique (joint mobilization) and the physical therapy technique, displayed better results, thereby demonstrating that flexion, extension, left lateral bending, right lateral bending, left rotation, and right rotation were all improved in comparison to the other neck pain group, which had only been treated using the physical therapy technique19 . A study, in which the head - neck flexion exercise was performed using sling equipment, reported that the rom for flexion and extension was significantly improved20 . In the present study the sling stabilization exercise group showed a significant difference in the flexion, right rotation, left lateral bending, and right lateral bending results, and the stretching exercise group showed a significant difference in the left rotation, left lateral bending, and right lateral bending results . It seems that the muscular asymmetry, caused by the straight neck symptom, was returned to the symmetric state by the sling stabilization exercise . Hence, the normal alignment of the cervical spine was realized by the exercise conducted by the sling stabilization exercise group and this led to the improvement in rom . Moreover, the stretching exercise used in the stretching exercise group contributed to the normalization of muscle length by extending the shortened muscle, and this normalization of muscle length led to the improvement in rom . The results of this study coincide with the findings presented in many previous studies; thus, it is possible to infer that the stretching exercise and the sling stabilization exercise exert a positive influence on rom . The reason why not all of the rom variables in this present study showed improvements that were as significant as the improvements found in other studies, is attributed to the fact that the exercise methods used in this present study were different from those used in other studies; moreover, in this present study, the individual subjects daily living habits could not be thoroughly controlled . This study found that the sling stabilization exercise group showed significant improvement in the rom, while the stretching exercise group showed a significant improvement in cervical alignment and rom . All parts of the human body are intimately connected to one another, and imbalance causes abnormal body phenomena to appear when a problem breaks out even in a single part of human body . Because muscles in the human body control musculoskeletal posture, they play a fundamental role in movement . Thus, the work load on human muscles is always heavy . Due to this peculiar nature of muscles, they are vulnerable to irregular posture, unsafe movements, over - use, and unusual usage . Therefore, structural damage and functional disorders are apt to occur frequently in the muscles of the human body21 . One study revealed that pains around the neck might develop into a more serious disease because of the psychological stress they cause22 . In relation to cervical spine alignment and rom, this study s findings suggest that future studies should try to identify the way in which different types of therapies could be used to treat straight neck patients, in addition to current treatment approaches . Future studies should also examine how psychological treatment could be given along with various types of existing treatment, to help ensure better patient outcomes.
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We report a 32-year - old iranian male diagnosed with a case of miliary tuberculosis affecting the prostate . Computerized tomography increased the clinical suspicion of miliary tuberculosis extending to the prostate where a trans - rectal urethral biopsy was obtained . A strong clinical suspicion and availability of sophisticated tests with confirmation by biopsy, polymerase chain reaction, and culture are needed in order to avoid misdiagnosis of complicated miliary tuberculosis cases . Miliary tuberculosis is a rare form of a tuberculosis infection that results from massive lymphohematogenous dissemination of the mycobacterium tuberculosis bacilli.1 this form of infection, which accounts for 2.8% of all tuberculosis infections, usually involves the extrapulmonary organs.2 from extrapulmonary tuberculosis only 22% affects the genitourinary system, while tuberculosis of the prostate gland is seen in only 2.7% of genitourinary tuberculosis.3,4 due to the rare involvement of the prostate, very scarce literature of the subject, and non - specific symptoms, the clinical suspicion of prostate tuberculosis is difficult . Here we report a case of a young, previously healthy, immunocompetent patient with miliary tuberculosis involving the prostate . We present a case of a 32-year - old immunocompetent iranian male who presented to the emergency room with a generalized tonic - clonic seizure lasting for 3 minutes . Furthermore, he did not have any genitourinary symptoms, including dysuria, hematuria, or penile discharge . A computerized tomography (ct) scan of the head failed to show any acute central nervous system insult . Prior to this admission, he gave history of an admission 12 months ago to a hospital outside the country, where he complained of fatigue, weight loss, and fever . Reports from the hospital showed that he also had chronic moderate ascites and a left pleural effusion . However, several laboratory and imaging tests were done and it was concluded that there was no evidence of infectious disease, chronic liver disease, or malignancy . It was recommended that he may need more investigations or a laparoscopy with peritoneal tissue biopsy, which was not done . Upon the latest admission, the respiratory rate was 12 breaths per minute, the body temperature was 36.9c, and the blood pressure was 112/70 mmhg . Digital rectal examination was not performed since the patient did not have any genitourinary or gastrointestinal symptoms . During this hospitalization, the laboratory investigations, urine routine and microscopy, cultures, virology screen, including human immunodeficiency virus, as well as the autoimmune markers did not show any significant issues . Also, three sets of sputum acid - fast bacilli smears were done, which were all negative . A lumber puncture was also performed with a normal initial pressure, 0 mmcell count, glucose of 4.1 mm per liters, and protein of 764 mg per liter . At the same time a magnetic resonance image of the brain was done concluding multifocal enhancing foci suggestive of an infective process (figure 1). Due to his previous history, ct - chest, the abdominal ct showed mild peritoneal ascites with evidence of mild splenomegaly . The ascetic fluid taken was translucent and yellow in color and the serum - ascites albumin gradient 5 gram per liter (0.5 gram per deciliter) furthermore, the analysis was negative for ziehl neelsen, gram stain, and culture . The pelvis ct also showed an enlargement of the prostate and a right prostate lesion extending to the seminal vesicles (faint ring enhancing lesion measuring 32.73 cm) (figure 2). The case was discussed with the patient and he agreed to start anti - tuberculosis treatment and to have a trans - rectal biopsy of the prostate . The biopsy showed multiple slides of necrotizing granulomata suggestive of tuberculosis (figure 3). Neelsen stain was also performed, and the diagnosis of miliary tuberculosis was confirmed (figure 4). He was started on anti - tuberculosis therapy with isoniazid, rifampicin, ethambutol, pyrazinamide, and pyridoxine for 12 months . At follow - up the patient was asymptomatic with a normal physical examination and laboratory tests with no drug side effects . This complication is seen in 1%3% of all tuberculosis cases.5 although most cases of miliary tuberculosis are treatable, the mortality rate among young adults with miliary tuberculosis remains 15% 20% and for adults 25%30%.2 one of the main causes for these high mortality rates includes late detection of disease caused by non - specific symptoms . Tuberculosis of the prostate is rare and the literature is scarce . Most often, the cases are under - reported, and easily missed . Patients with prostatic tuberculosis usually present with non - specific symptoms except in rare cases . These rare cases present with irritative voiding symptoms, terminal dysuria or hemospermia.6 however this was not the case in our patient . The patient was asymptomatic and like most cases in the literature, tuberculosis of the prostate was diagnosed incidentally . As in our case, tuberculosis of the prostate is mainly diagnosed by pathologists where a trans - rectal ultrasound guided biopsy of the prostate is the mainstay of the diagnosis.7 as wang and chang have demonstrated, it was the clinical usefulness of the contrast enhanced ct for the diagnosis of tuberculosis of the prostate in which low density multiple and bilateral lesions with irregular borders are seen that prompted us to further investigate the prostate.8 once the disease is diagnosed, the patient should receive a full course of anti - tubercular therapy . Often, the presentation can be non - specific and misleading, as in our case . A strong clinical suspicion and availability of sophisticated tests with confirmation by biopsy, polymerase chain reaction, and culture are needed in order to avoid misdiagnosis of complicated miliary tuberculosis cases.
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The acridinones represented by imidazo- and triazoloacridinones are a new group of potent antitumor compounds (cholody et al ., 1990, 1992, 1996) from which one of the most active derivatives called as c-1305 has been selected for extended preclinical trials and the other one called c-1311 (for review see mazerska et al ., 1998) is currently undergoing phase ii clinical trials as drug symadextm (burger et al ., 1996; den brok et al ., 2005a, b, c). Imidazoacridinones exhibit in vivo activity against various tumor cell lines as leukemia, melanoma, colon adenocarcinoma, and colon carcinoma . Triazoloacridinones exhibit in vivo activity against leukemia, murine carcinoma, lung carcinoma, breast carcinoma, and colon carcinoma (cholody et al ., 1990, 1992, 1996; kusnierczyk et al ., 1994; burger et al ., as was previously shown (skadanowski et al ., 1999; lemke et al ., 2004; augustin et al ., 2004, 2006; wesierska - gadek et al ., 2004; koba and konopa, 2007; koba et al ., 2009), cellular dna is important target for the triazoloacridinone drugs, and hence interactions with dna are naturally the crucial point in view of the biological activity of these compounds . In previous article (skadanowski et al ., 1999; lemke et al ., 2004), it was indicated that triazoloacridinones inhibit cleavable complexes of topoisomerase ii with dna . They inhibit also nucleic acid or protein synthesis induced by g2 block of cell cycle followed by apoptosis (augustin et al ., 2004, 2006; 2004), intercalating to dna and binding in minor groove (koba and konopa, 2007; koba et al ., 2009) and/or forming of interstrand dna crosslinks (koba and konopa, 2007). In addition, it was shown that intercalation to dna takes place preferentially in guanine triplet regions inducing changes in dna structures (lemke et al ., 2005). For imidazoacridinones, it was demonstrated that intercalation to dna undergoes at physiological condition with parallel stabilization of double - stranded dna and unwinding of supercoiled dna (burger et al ., 1999; the intercalative binding mode of acridinone derivatives was also confirmed with the use of molecular - modeling studies (mazerski and muchniewicz, 2000). Similar to other dna - binding agents, treatment of tumor cells with imidazoacridinones induces topoisomerase ii - associated dna strand breaks (skadanowski et al ., 1996), arrests cells in g2 phase, and stimulates apoptosis (zaffaroni et al ., 2001; skwarska et al ., 2007) or mitotic catastrophe (hyzy et al ., 2005; skwarska et al ., 2007 however, after testing imidazoacridinone and triazoloacridinone derivatives, it has been concluded that although the intercalative binding to dna seems to be necessary for their biological activity (the most active compounds have usually the highest binding affinity), it is not sufficient (some inactive analogs also bind strongly with dna) (dziegielewski et al . Moreover, acridinones undergo enzymatic oxidation, and this reaction is important for their biological activity as intercalation to dna and covalent adducts formation (dziegielewski and konopa, 1996; mazerska et al ., 1999, 2003). In this context, noncovalent interaction of acridinones may help position drug molecules on dna for the covalent reaction . In this article, physicochemical interactions of acridinones with dna were evaluated in view of quantitative structure activity relationships (qsar). Multiple regression analysis was used to model relationships between molecular structural descriptors and biological (antitumor) activity of molecules, or between molecular structural descriptors and physicochemical interaction of acridinone derivatives with dna . The biological data expressed as anti - leukemia p388 activity and parameter describing ability to physicochemical (noncovalent) interaction with dna as value of dna - duplexes stabilization were applied in this study . The acridinone derivatives examined in this study have been selected to collect analogue compounds differing in chemical structures as well as anticancer activities (table 1). The data of acridinones antitumor activity against p388 leukemia in mice in vivo and expressed as the percentage of increase in survival time of the treated to that of the control mice with p388 leukemia at optimal dose (ils) were taken from the literature (table 1) (cholody et al ., 1990, 1992; koba and konopa, 2007; mazerska et al ., 1996). The data of physicochemical binding of acridinones to dna (as values of dna - duplexes stabilization), which were expressed as an increase in dna melting temperature in centigrade degrees of ctdna at drug to dna base pairs 0.25 m ratio were taken from the literature (table 1) (koba and konopa, 2007; dziegielewski et al ., 2002).table 1chemical structures of acridinones studiedcompoundxnr1r2r3r4r5r6ilstmc-1310c2ch2ch3ch2ch3ohhch3h18515.3c-1311c2ch2ch3ch2ch3ohhhh9313.7c-1330c2ch2ch3ch2ch3och3hhh9611.5c-1415c2ch2ch3ch2ch3hhhh557.2c-1419c2ch2ch3ch2ch3hhhoh278.3c-1558c2ch2ch3ch2ch3c(ch3)3hhh02.4c-1176c2ch3ch3hhhh909.5c-1263c2ch3ch3ohhhh11012.3c-1212c3ch3ch3hhhh2511.5c-1371c3ch3ch3ohhhh1203.5c-1554c5ch2ch3ch2ch3ch3hhh2010.5c-1266c5ch3ch3hhhh109.9c-1492c5ch3ch3ohhhh8513.1c-1233n2ch3ch3hh h779.1c-1303n2ch3ch3ohh h10213.1c-1533n2ch3ch3ohch3h108.1c-1567n2ch3ch3c(ch3)3h h06.8c-1410n2hch2ch3ohh h787.1c-1296n3ch3ch3ch3h h1811.5c-1305n3ch3ch3ohh h16515.1the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dosethe increase in dna melting temperature (expressed in centigrade degrees) at drug to dna base pairs 0.25 m ratio chemical structures of acridinones studied the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dose the increase in dna melting temperature (expressed in centigrade degrees) at drug to dna base pairs 0.25 m ratio the structure of the tested compounds was studied by molecular modeling using hyperchem 7.5 release software (kaliszan et al ., 1995; ivanciuc, 1996) and dragon software (todeschini et al ., 2000 the structures of the compounds were first pre - optimized with the molecular mechanics force field (mm+) procedure included in the hyperchem 6.03 (hypercube) http://www.hyper.com, and the resulting geometries were further refined by means of the semi - empirical molecular orbitals method am1 using the polak - ribiere algorithm and a gradient norm limit of 0.01 kcal . The following molecular descriptors taken from hyperchem software were considered among quantum and chemical indices: total energy (te), binding energy (be), isolated atomic energy (iae), electron energy (ee), core core energy (cce), heat flow (hf), energy of the highest occupied molecular orbitals (e_homo), energy of the lowest unoccupied molecular orbitals (e_lumo), and difference between homo and lumo energies referred to as eg = energy gap; ionization energy (potential) (ie) and electron affinity (ea) were calculated as a difference between the hf of positive molecular ion and electrically neutral molecule, and electronegativity (en) calculated as average arithmetic potential of ionization and ea . In addition, other parameters were used as the value of electron density of atom orbitals from the lowest to the highest (ed_min and ed_max, respectively), the highest positive electron charge on the atoms (max_pos), and the highest negative electron charge on the atoms (max_neg), the difference between the highest positive and negative charge (delta_q), distribution of dipolar moment along x, y, and z axes (x_dm, y_dm, and z_dm, respectively), total dipolar moment (tdm), mean polarizability of molecules (in atom units) mp (mean polarizability), energy equal to the length of the wave with the greatest long - wave transfer of electrons, for which the value of oscillator force was different from zero (el)the value of wave figures were converted to ev and the value of the most intensive electron transfer (emax the maximum value of oscillator force calculated with the use of am1 method as well as oscillator maximum force used for the transfer (os_emax). Moreover, they include the following descriptors: surface area of the molecule available for solvent (sa), molecule volume (v), hydration energy (he), the calculated distribution coefficient logarithm (logp), refraction (r), and polarizability (p). On the other hand, using dragon software, over 1,300 molecular descriptors were calculated and considered for qsar analysis . They include molecular parameters from different group and class of descriptors as constitutional, topological, walk and path counts, connectivity indices, information indices, 2d autocorrelations, edge adjacency indices, topological charge indices, eigenvalue - based indices, geometrical, 3d - morse, whim, getaway, functional group counts, atom - centred fragments, charge, molecular properties and other group of descriptors, and describing some properties of compound as geometry, symmetry, topology, electronic, steric or thermodynamic and other properties . The definitions of these descriptors are reviewed by todeschini (todeschini et al ., 2000). The statistical qsar analysis was performed with the use of stepwise regression analysis that used statistica 8.0 software (statsoft, tulsa, ok, usa). The objective of stepwise regression is to construct a multivariate regression model (qsar equation) for a certain property, y, based on several selected explanatory variables . In stepwise regression, the first selected explanatory variable has the highest correlation with dependent variable, y. then, explanatory (independent) variables are consecutively added to the model in a forward selection procedure . A new variable is added to the model if a significant change in residuals of the model can be observed . The significance is evaluated using a statistical test, usually f - test (the value of the f - test of significance, f). In addition, the multiple correlation coefficients (r), the standard error of estimate (s), and the significance levels of each term and of whole equation (p) are calculated for the derived qsar equations . Whenever a new variable is included into a model, a backward elimination step follows in which an f - test detects the earlier selected variables, which can be removed from the model without any significant change on the level of the residuals . Stepwise regression is very much popular in qsar studies, since the stepwise procedure is simple and based on the classical multiple linear regression (mlr) approach . One of the drawbacks of the method is the fact that no optimal variable selection is guaranteed, since the new variables are found based on the previously included variables into the model (put et al ., 2006). During model building, the model fit can be improved proportional to the model complexity . Therefore, the more the factors are included into the model, the better the model fits the training data . Usually, the model fit is evaluated by the root mean - squared error (rmse), computed for the training data . The determination of the optimal complexity of the model requires an estimation of its predictive ability, to prevent overfitting to the calibration data . After all, the main goal of qsar models is to obtain a reasonable prediction of the retention for future samples . To evaluate the prediction by means of an internal validation procedures, cross validation can be used . The predictive ability of a model is characterized by the cross - validated root mean - squared error (rmsecv); test values were calculated with the matlab software (mathworks, natick, ma, usa). The rmsecv as values, which quantify the predictive power of the qsar model, were calculated by the leave - one - out method and leave - ten - out method . The acridinone derivatives examined in this study have been selected to collect analogue compounds differing in chemical structures as well as anticancer activities (table 1). The data of acridinones antitumor activity against p388 leukemia in mice in vivo and expressed as the percentage of increase in survival time of the treated to that of the control mice with p388 leukemia at optimal dose (ils) were taken from the literature (table 1) (cholody et al ., 1990, 1992; koba and konopa, 2007; mazerska et al ., 1996). The data of physicochemical binding of acridinones to dna (as values of dna - duplexes stabilization), which were expressed as an increase in dna melting temperature in centigrade degrees of ctdna at drug to dna base pairs 0.25 m ratio were taken from the literature (table 1) (koba and konopa, 2007; dziegielewski et al ., 2002).table 1chemical structures of acridinones studiedcompoundxnr1r2r3r4r5r6ilstmc-1310c2ch2ch3ch2ch3ohhch3h18515.3c-1311c2ch2ch3ch2ch3ohhhh9313.7c-1330c2ch2ch3ch2ch3och3hhh9611.5c-1415c2ch2ch3ch2ch3hhhh557.2c-1419c2ch2ch3ch2ch3hhhoh278.3c-1558c2ch2ch3ch2ch3c(ch3)3hhh02.4c-1176c2ch3ch3hhhh909.5c-1263c2ch3ch3ohhhh11012.3c-1212c3ch3ch3hhhh2511.5c-1371c3ch3ch3ohhhh1203.5c-1554c5ch2ch3ch2ch3ch3hhh2010.5c-1266c5ch3ch3hhhh109.9c-1492c5ch3ch3ohhhh8513.1c-1233n2ch3ch3hh h779.1c-1303n2ch3ch3ohh h10213.1c-1533n2ch3ch3ohch3h108.1c-1567n2ch3ch3c(ch3)3h h06.8c-1410n2hch2ch3ohh h787.1c-1296n3ch3ch3ch3h h1811.5c-1305n3ch3ch3ohh h16515.1the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dosethe increase in dna melting temperature (expressed in centigrade degrees) at drug to dna base pairs 0.25 m ratio chemical structures of acridinones studied the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dose the increase in dna melting temperature (expressed in centigrade degrees) at drug to dna base pairs 0.25 m ratio the structure of the tested compounds was studied by molecular modeling using hyperchem 7.5 release software (kaliszan et al ., 1995; ivanciuc, 1996) and dragon software (todeschini et al ., 2000 the structures of the compounds were first pre - optimized with the molecular mechanics force field (mm+) procedure included in the hyperchem 6.03 (hypercube) http://www.hyper.com, and the resulting geometries were further refined by means of the semi - empirical molecular orbitals method am1 using the polak - ribiere algorithm and a gradient norm limit of 0.01 kcal . The following molecular descriptors taken from hyperchem software were considered among quantum and chemical indices: total energy (te), binding energy (be), isolated atomic energy (iae), electron energy (ee), core core energy (cce), heat flow (hf), energy of the highest occupied molecular orbitals (e_homo), energy of the lowest unoccupied molecular orbitals (e_lumo), and difference between homo and lumo energies referred to as eg = energy gap; ionization energy (potential) (ie) and electron affinity (ea) were calculated as a difference between the hf of positive molecular ion and electrically neutral molecule, and electronegativity (en) calculated as average arithmetic potential of ionization and ea . In addition, other parameters were used as the value of electron density of atom orbitals from the lowest to the highest (ed_min and ed_max, respectively), the highest positive electron charge on the atoms (max_pos), and the highest negative electron charge on the atoms (max_neg), the difference between the highest positive and negative charge (delta_q), distribution of dipolar moment along x, y, and z axes (x_dm, y_dm, and z_dm, respectively), total dipolar moment (tdm), mean polarizability of molecules (in atom units) mp (mean polarizability), energy equal to the length of the wave with the greatest long - wave transfer of electrons, for which the value of oscillator force was different from zero (el)the value of wave figures were converted to ev and the value of the most intensive electron transfer (emax the maximum value of oscillator force calculated with the use of am1 method as well as oscillator maximum force used for the transfer (os_emax). Moreover, they include the following descriptors: surface area of the molecule available for solvent (sa), molecule volume (v), hydration energy (he), the calculated distribution coefficient logarithm (logp), refraction (r), and polarizability (p). On the other hand, using dragon software, over 1,300 molecular descriptors were calculated and considered for qsar analysis . They include molecular parameters from different group and class of descriptors as constitutional, topological, walk and path counts, connectivity indices, information indices, 2d autocorrelations, edge adjacency indices, topological charge indices, eigenvalue - based indices, geometrical, 3d - morse, whim, getaway, functional group counts, atom - centred fragments, charge, molecular properties and other group of descriptors, and describing some properties of compound as geometry, symmetry, topology, electronic, steric or thermodynamic and other properties . The statistical qsar analysis was performed with the use of stepwise regression analysis that used statistica 8.0 software (statsoft, tulsa, ok, usa). The objective of stepwise regression is to construct a multivariate regression model (qsar equation) for a certain property, y, based on several selected explanatory variables . In stepwise regression, the first selected explanatory variable has the highest correlation with dependent variable, y. then, explanatory (independent) variables are consecutively added to the model in a forward selection procedure . A new variable is added to the model if a significant change in residuals of the model can be observed . The significance is evaluated using a statistical test, usually f - test (the value of the f - test of significance, f). In addition, the multiple correlation coefficients (r), the standard error of estimate (s), and the significance levels of each term and of whole equation (p) are calculated for the derived qsar equations . Whenever a new variable is included into a model, a backward elimination step follows in which an f - test detects the earlier selected variables, which can be removed from the model without any significant change on the level of the residuals . Stepwise regression is very much popular in qsar studies, since the stepwise procedure is simple and based on the classical multiple linear regression (mlr) approach . One of the drawbacks of the method is the fact that no optimal variable selection is guaranteed, since the new variables are found based on the previously included variables into the model (put et al ., 2006). During model building, the model fit can be improved proportional to the model complexity . Therefore, the more the factors are included into the model, the better the model fits the training data . Usually, the model fit is evaluated by the root mean - squared error (rmse), computed for the training data . The determination of the optimal complexity of the model requires an estimation of its predictive ability, to prevent overfitting to the calibration data . After all, the main goal of qsar models is to obtain a reasonable prediction of the retention for future samples . To evaluate the prediction by means of an internal validation procedures, cross validation can be used . The predictive ability of a model is characterized by the cross - validated root mean - squared error (rmsecv); test values were calculated with the matlab software (mathworks, natick, ma, usa). The rmsecv as values, which quantify the predictive power of the qsar model, were calculated by the leave - one - out method and leave - ten - out method . The chemical structures of the 20 compounds considered for this study and their antitumor and noncovalent dna - binding activities are presented in table 1 . In this study, qsar analysis using multiple regression method was performed for biological activity data (ils) or physicochemical interaction with dna (tm), and non - empirical parameters (molecular descriptors). Owing to a large number of non - empirical parameters (over 1,300), treated as independent variables and according to qsar strategy and multi - parameter regression rule in derived multi - parameter regression equation, the number of independent variables must be 56 times less than the number of cases considered in this study . In practice, for obtaining statistically significant equation, one independent variable (in our case structural descriptor) falls, generally out of five to maximum six cases considered, in dependent - variable activity (in our case, activity of acridinones). In the research done, the data set of 20 acridinone derivatives (dependent variables) was taken to qsar analysis, and for this reason the derived qsar equations were maximally limited to four statistically significant independent variables (structural descriptors). Moreover, correlations were limited to the value of regression coefficient r 0.8 and an additional criterion, considered as relevant to particular independent variables, was established at the significance level p 0.05 . The calculated equations are presented in table 2 and characterized by four statistically significant independent variables with a good value of regression coefficient r 0.8 (r = 0.9384 and r = 0.8388 for quantitative structure antitumor activity relationships and quantitative structure ability to dna - duplexes stabilization relationships, respectively). Moreover, all the regression coefficients are highly statistically significant (p <0.05) as is the whole equation (p <7 10 for quantitative structure antitumor activity relationships and p <9 10 for quantitative structure ability to dna - duplexes stabilization relationships, respectively). The values of the multiple correlation coefficient, r; the standard error of the estimate, s; and the value of the f - test of significance, f, are also statistically significant.table 2multiple regression qsar equation (dependent variable = k0 + k1a + k2b + k3c + k4d)dependent variablecoefficients and statistically significant molecular descriptorsstatistical parametersk0k1ak2bk3ck4dr (r)sfptm97.44 55.096.59 1.50gats7e3.03 0.88i0.64 0.30h-047147.44 83.58mp0.8388 (0.7036)2.158.907 10p = 1 10p = 5 10p = 4 10p = 5 10p = 1 10ils88.80 153.448914.33 1225.69g3m36.31 6.02logp4691.69 1227.99g2p4744.01 1451.51g3p0.9384 (0.8806)21.0327.659 10p = 1 10p = 3 10p = 2 10p = 2 10p = 5 10multiple correlation coefficient (determination coefficient)standard error of estimatevalue of the f - test of significancesignificance level multiple regression qsar equation (dependent variable = k0 + k1a + k2b + k3c + k4d) multiple correlation coefficient (determination coefficient) standard error of estimate value of the f - test of significance statistically significant parameters used in qsar analysis (values of molecular descriptors are presented in the table 3) as logarithm of theoretically calculated n - octanol water partition coefficient (logp) from the class of hydrophobic descriptors, 3st component symmetry directional whim index weighted by atomic masses (g3 m), 2st component symmetry directional whim index by atomic polarizabilities (g2p), and 3st component symmetry directional whim directional index / weighted by atomic polarizabilities (g3p) from class of whim (weighted holistic invariant molecular) descriptors had the influence upon antitumor activity of acridinones . The whim descriptors are molecular descriptors based on statistical indices calculated on the projections of the atoms along principal axes . They are built in such a way as to capture relevant molecular 3d information regarding molecular size shape, symmetry, and atom distribution with respect to invariant reference frames (todeschini et al ., 2000). In general, the obtained data indicate that hydrophobic and total molecular symmetry properties are important for antitumor activity of acridinones . These observations are in partial agreement with the data obtained by mazerska (mazerska et al ., however, impact of lipophilicity on the biological activity of these compounds was observed only in the case of derivatives with 8-hydroxyl group, which undergo metabolic activation (mazerska et al . Moreover, non - hydroxyl or 9-hydroxyl derivatives also exhibited lipophilic properties, but its effect was not crucial when metabolic activation did not occur . Relocation of hydroxyl group from position 8 to 9 drastically decreases antitumor activity (c-1311 8-hydroxyl, c-1419 9-hydroxyl). In addition, hydrophobic properties of acridinones can play important role in transport and accumulation of these compounds in cells in view of fastening of metabolic activation (skadanowski et al ., on the other hand, diaminoalkyl side chain has also crucial influence on antitumor activity of acridinones . For compounds without 8-hydroxyl group, the increase in number of carbon atoms between nitrogen atoms from two to three or five (c-1415, c-1176, and c-1233 two; c-1212 and c-1296 three; and c-1266 five) generally decreases antitumor activity of imidazo- and triazoloacridinones . In case of derivatives bearing 8-hydroxyl group, the increase in number of carbon atoms (c-1311, c-1263 two, c-1371 three, and c-1492 five) rather do not augment antitumor activity for imidazoacridinones, while good increase in antitumor activity is observed in case of triazoloacridinones (c-1303, c-1410 two, and c-1305 three carbon atoms). These observations indicate that length and conformation of diaminoalkyl side chain attached to flat aromatic acridinone ring have some influence on proper conformation of acridinone derivatives molecule and is in accordance with earlier modeling studies (mazerski and muchniewicz, 2000) the observations also proved our suggestion that total molecular symmetry properties in context of interaction with dna as intercalation and dna groove binding are crucial for antitumor activity of acridinones.table 3values of molecular descriptors used in qsar analysiscompoundmolecular descriptorsgats7eih-047mpg3mlogpg2pg3pc-13101.073.70130.660.161.980.150.15c-13110.923.06160.660.152.190.150.15c-13301.193.16160.660.152.150.150.15c-14150.902.32140.670.151.160.150.15c-14190.892.01130.660.152.190.150.16c-15582.132.28130.650.150.150.150.15c-11760.942.50160.680.161.120.160.16c-12630.903.34150.670.162.870.160.16c-12121.012.61160.670.161.790.160.16c-13710.942.11150.670.152.820.150.15c-15540.832.66130.660.151.010.150.15c-12660.862.60130.660.150.950.150.16c-14920.863.10150.660.151.970.150.15c-12330.992.99160.680.171.120.170.16c-13030.872.48150.670.162.140.160.16c-15330.911.11150.670.161.780.170.16c-15672.153.53150.660.150.20.150.15c-14100.862.39110.670.162.160.160.16c-12960.943.08190.670.161.060.170.16c-13050.812.44180.670.172.090.160.16 values of molecular descriptors used in qsar analysis on the other hand, statistically significant parameters values of molecular descriptors are presented in the table 3such as dipole moment (i) from class of electronic descriptors, mean atomic polarizability scaled on carbon atom (mp) from class of constitutional descriptors, geary autocorrelation - lag 7 weighted by atomic sanderson electronegativities (gats7e) from class of 2d autocorrelations descriptors, and h attached to c1(sp3)/co(sp2) (h-047) from class of atom - centered fragments descriptors had the influence upon physicochemical (noncovalent) dna - duplexes stabilization of acridinone derivatives . The obtained data indicate that electronic and topological properties are important for noncovalent dna - duplexes stabilization of these compounds . It is known that drug dna binding induces changes in dna structure and topology and is closely connected with conformation of drug molecule and its electronic and topological properties . The presence of a hydroxyl group in position 8 of acridinone ring slightly increases the affinity for dna compared to unsubstituted or alkyl - substituted derivatives, possibly because of additional hydrogen bonds with the dna phosphate backbone . As it was mentioned earlier (mazerski and muchniewicz, 2000), the charged diaminoalkyl side chain of acridinone compounds can interact with dna in the minor groove, in addition to intercalation . In addition, some other data (koba and konopa, 2007) indicated that intercalation is not involved in stabilization of secondary structure of dna . However, for the biologically non - active compounds, c-1558 and c-1567, bearing a t - butyl group in position 8, the tm values were 2.4 and 6.8c, respectively, indicating that stabilization of the dna duplex by these compounds especially by c-1567 probably derived from electrostatic interactions of the side chain with dna and not from its intercalation to dna . This means that intercalation to dna is necessary for the biological activity of acridinones via positioning the drug molecules within dna before the covalent reaction and formation of interstrand dna crosslink (koba and konopa, 2007). This also indicated that topological and electronic properties of acridinone derivatives are important for their physicochemical interactions with dna . Moreover, the molecular modeling studies (mazerski and muchniewicz, 2000) evidenced that when acridinone c-1311 is intercalated between gc, the highly reactive position 8 on acridinone core is in close proximity to nucleophilic n7 position on guanine . It is plausible to postulate that drug molecule first intercalates into dna and then, after in situ activation, binds covalently to the neighboring base . These observations are compatible with recent findings demonstrating that electrochemically activated c-1311 forms covalent adducts with deoxyguanine (mazerska et al ., 2003). On the other hand, the structure of acridinones suggests that there are at least two possible sites for enzymatic oxidation / activation, which potentially could be involved in the covalent binding to dna . One is the diaminoalkyl side chain at position 5 which is necessary for covalent binding of mitoxantrone to dna (skadanowski and konopa, 2000). The other one is the potential quinone imine group formed by hydroxyl group in position 8 (8-oh) and heterocyclic nitrogen atom in acridinone nucleus (mazerska et al ., 2003). Recently proposed mechanism of oxidation involves highly unstable carbocations generated in these two positions (mazerska et al ., 2003). It is suggested that c-1311 carbocations react rapidly with nucleophiles present in the environment, including dna bases forming covalent adducts . These observations indicate that topological and electronic properties of acridinone derivatives are also important for their covalent interactions with dna . Moreover, the calculated values of ils and tm obtained for other compounds (table 4) and the plots of the experimental data versus the calculated data (fig . 1a b) for dna - duplexes stabilization of acridinones expressed as tm (the increase in dna melting temperature at drug to dna base pairs 0.25 m ratio) and antitumor activity of acridinones expressed as ils (survival time of treated to control mice with p388 leukemia at optimal dose) proved good correlation and predictive potency of proposed qsar models . In addition, the rmsecv as value, which quantifies the predictive power of the proposed qsar model, are calculated by the leave - one - out and the leave - ten - out methods and presented in the table 5 . The obtained values of rmsecv test (22.79 and 22.27 for quantitative structure antitumor activity relationships as well as 2.39 and 2.41 for quantitative structure ability to dna - duplexes stabilization relationships) performed for all the four statistically significant independent variables proved the predictive power of the derived qsar models.table 4values of experimental and calculated data for dna - duplexes stabilization and antitumor activity of acridinonescompoundtm exp.tm calc.ils exp.ils calc.c-131015.312.62.718517213c-131113.713.60.193903c-133011.512.10.696897c-14157.28.81.655532c-14198.38.70.4274316c-15582.42.80.4055c-11769.58.90.6904644c-126312.312.50.21101100c-121211.510.21.3257045c-13713.58.55.01201137c-155410.511.10.6204727c-12669.910.70.81028c-149213.113.50.485823c-12339.110.00.9778811c-130313.110.13.01028319c-15338.15.72.4102313c-15676.86.30.5033c-14107.17.30.278846c-129611.514.02.518315c-130515.112.32.81651705mean value of 1.413the increase in dna melting temperature (expressed in centigrade degrees) at drug to dna base pairs 0.25 m ratiodifference between experimental and calculated valuesthe percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dosefig . 1correlation between the experimental data and the calculated data from the derived multiple regression qsar equation for a dna - duplexes stabilization of acridinones expressed as tm (the increase in dna melting temperature at drug to dna base pairs 0.25 m ratio) and b antitumor activity of acridinones expressed as ils (survival time of treated to control mice with p388 leukemia at optimal dose)table 5values of the cross - validated root - mean - square error rmsecv testqsar model for dependent variablermsecv testleave - one - out methodleave - ten - out method12341234tm3.362.532.562.393.442.632.642.41ils53.3942.1028.4822.7954.2342.3528.7422.2714 represents rmsecv test performed only for one, combined two and three, and for all the four significance descriptors in qsar models, respectively . In the case of qsar model, for tm as dependent - variable values, 14 were obtained for only gats7e, gats7e combined with i, gats7e combined with i and h-047, gats7e combined with i, h-047, and mp descriptors . In the case of qsar model for ils as dependent - variable values, 14 were obtained for only g3 m, g3 m combined with logp, g3 m combined with logp and g2p, and g3 m combined with logp, g2p and g3p descriptors values of experimental and calculated data for dna - duplexes stabilization and antitumor activity of acridinones the increase in dna melting temperature (expressed in centigrade degrees) at drug to dna base pairs 0.25 m ratio difference between experimental and calculated values the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dose correlation between the experimental data and the calculated data from the derived multiple regression qsar equation for a dna - duplexes stabilization of acridinones expressed as tm (the increase in dna melting temperature at drug to dna base pairs 0.25 m ratio) and b antitumor activity of acridinones expressed as ils (survival time of treated to control mice with p388 leukemia at optimal dose) values of the cross - validated root - mean - square error rmsecv test 14 represents rmsecv test performed only for one, combined two and three, and for all the four significance descriptors in qsar models, respectively . In the case of qsar model, for tm as dependent - variable values, 14 were obtained for only gats7e, gats7e combined with i, gats7e combined with i and h-047, gats7e combined with i, h-047, and mp descriptors . In the case of qsar model for ils as dependent - variable values, 14 were obtained for only g3 m, g3 m combined with logp, g3 m combined with logp and g2p, and g3 m combined with logp, g2p and g3p descriptors statistically significant equations describing structure antitumor activity relationships and structure ability to physicochemical (noncovalent) interaction with dna relationships in acridinone derivatives group were derived . It has been found that hydrophobic and total molecular symmetry properties are important for antitumor activity of acridinone derivatives, and electronic and topological properties are important for physicochemical (noncovalent) dna - duplexes stabilization of these compounds.
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Field work for this study was conducted in late july, august, and early september of 20112013 at study sites located in ellsworth, mcpherson, and reno counties in central kansas (fig . The habitat at all study sites was qualitatively similar and consisted of native midgrass prairie vegetation intermixed with both naturally occurring and planted trees . The inset map indicates the location of the main map in the united states . Phonotaxis by e. erro was studied in the field by broadcasting appropriate acoustic stimuli from a loudspeaker, a technique that has been widely used by researchers working on other species of acoustic parasitoids (e.g., cade 1975; soper et al . The acoustic stimuli were designed to represent the calling song of the host cicada neotibicen dorsatus . Details of the methods used to construct these signals are provided in stucky (2015). Briefly, the calling songs of 20 different male n. dorsatus were recorded and analyzed to determine the values of three acoustic parameters: peak frequency, chirp length, and chirp rate (here, chirp is equivalent to the term pulse group defined in stucky (2015); see cole (2008) for a spectrogram and oscillograms of the n. dorsatus calling song). The mean values of these parameters were used to construct representative model calls that represented the average mating call of male n. dorsatus from the general study area . Model calls were constructed either directly from the recordings of n. dorsatus or by generating synthetic signals from amplitude - modulated sine waves . The synthetic signals had a frequency of 5.64 khz, chirp length of 20.3 ms, and a chirp rate of 37 chirps per second . 5.64 khz is higher than the observed mean peak frequency of n. dorsatus (4.31 khz), but e. erro are much more responsive to signals of 5.64 khz than 4.31 khz (b. stucky, unpublished data), so the higher frequency was used . Neotibicen dorsatus has a broadband call with peak acoustic energy from 3 khz to 7 khz (cole 2008), so the synthetic signals were still broadly representative of the natural calling song . For direct behavioral observations, sounds were broadcast from a tweeter speaker (pyle ph44, pyle audio inc ., brooklyn, ny) mounted in the top of a wooden box, and flies were observed as they arrived at the speaker and while they remained on or near the broadcast apparatus . I did not attempt to estimate the numbers of female and male flies that were attracted during these behavioral observations because flies sometimes depart and return to a sound source multiple times during a broadcast (b. stucky, personal observation), which makes manually counting flies as they arrive unreliable . Instead, acoustic live traps (stucky 2016) were used to objectively quantify the comparative phonotactic responsiveness of male and female e. erro . For each trapping session, three traps were deployed at a field site and the synthetic n. dorsatus call was simultaneously broadcast from all three traps for 30 min . The traps were arranged linearly on the ground with 10 m between adjacent traps . To provide additional information about e. erro s phonotactic behavior, the relative signal amplitudes among the three traps were generally not the same and differed by as much as 18 db between the loudest and quietest traps . Because of this, the signals were rotated among the traps every 10 min so that, after 30 min, all three traps had broadcast at all three amplitudes for the same amount of time . After every 10 min of broadcasting, the flies captured by each trap were counted and sexed (i.e., captured flies were counted and sexed before the signals were rotated among the traps and again at the end of the last broadcast). Absolute broadcast amplitudes ranged from 85 to 117 db spl (reference 20 pa) at 1 m from the loudspeaker, as measured with a radioshack model 33 - 2055 spl meter (radioshack corporation, fort worth, tx) set to c - weighting . For comparison, the natural call of male n. dorsatus, measured using the same spl meter, is typically 100 db spl at 0.5 m (b. stucky, unpublished data). To avoid pseudoreplication, no captured flies were released until after trapping was completed at a location . Traps were only operated in the afternoons when the host cicadas were naturally active . Trapping in 2011 was part of a larger study of e. erro s phonotactic behavior, the full results of which will be reported in a separate paper . To test whether nongravid female flies (defined here as females with no live first - instar larvae in their incubatory pouches) perform positive phonotaxis to host calls, i dissected the abdomens of 20 female flies that were captured at broadcasts of the n. dorsatus calling song and noted the presence or absence of larvae . In addition, in 2012 i tested the phonotactic responses of two female flies that had been reared from infected n. dorsatus collected in the field . Each fly was tested separately . For each test, the flies were released into a large, outdoor, rectangular screen cage (1.8 m wide on each side and 2.1 m high in the center) and given several minutes to acclimate to the enclosure . A portable digital audio player (samsung galaxy player 4.0) was held on the side of the cage opposite to the side on which the fly was perched, and the model call of n. dorsatus was broadcast from the audio player s onboard speaker . Depending on the location of the fly, the distance between the fly and speaker at the start of the broadcast varied between 1.8 and 3.1 m. playback was terminated once a fly performed complete phonotaxis to the speaker or after 2 min if the fly did not travel to the sound source . The overall proportion of male flies captured by the acoustic traps across all years and study sites was analyzed using an exact binomial test . To further investigate the comparative phonotactic behaviors of female and male flies, the numbers of flies arriving at the call broadcasts were analyzed using poisson regression (i.e., generalized linear models with log link function and poisson - distributed response) (dobson and barnett 2008). The predictor variable of primary interest was fly sex, but field site, year, and relative amplitude were also considered as predictors . To account for possible variations in male and female responses across the study conditions, the pairwise interactions of fly sex with each of field site, year, and amplitude were also evaluated . The effect of each explanatory variable was tested by comparing a reduced model against the full model using the difference of their deviance statistics (i.e., likelihood ratio tests) (dobson and barnett 2008). Confidence intervals (ci) for the overall estimated proportions of males and nongravid females arriving at call broadcasts were calculated using the wilson method (also known as the score confidence interval) (wilson 1927; agresti and coull 1998). All statistical analyses were done using r version 3.1.3 (r core team 2015). Traps broadcasting the model n. dorsatus call were operated at three field sites for a total of 24 trap - hours and captured a total of 110 emblemasoma erro (table 1). Of these, 76 (69.1%) were female and 34 (30.9%) were male . Considering all trapping data together, significantly more females than males were captured by the traps (exact binomial test: p <0.001; 95% ci for the proportion of male flies: 0.2300.401). Total numbers of female and male e. erro captured in acoustic traps for each study yearyearfemalemaletotal2011229312012367432013181836total7634110 total numbers of female and male e. erro captured in acoustic traps for each study year the results of the poisson regression analysis of the numbers of flies captured in the acoustic traps are given in table 2 . As expected from the binomial test of the aggregated data, there were significant differences in the numbers of male and female flies that were captured (p <0.001), but the proportions of males and females also varied among the study years (interaction of fly sex and year: p = 0.0177). The proportion of male flies captured in 2013 was much higher than in 2011 and 2012 (table 1); there was no obvious reason for this pattern . There were no differences in the proportions of males and females trapped among the field sites (interaction of fly sex and field site: p = 0.369). Signal amplitude had a strong effect on the number of flies captured by a trap (p <0.001), but amplitude had no effect on the proportions of male and female flies that were trapped (interaction of amplitude and fly sex: p = 0.386). Because broadcast amplitude had no effect on the relative phonotactic responses of male and female e. erro, the effect of amplitude on fly phonotaxis is not considered further here and will instead be analyzed as part of a separate paper addressing acoustic signal perception by e. erro . Analysis of deviance table for the poisson regression model of the number of flies captured in the acoustic trapsvariabledeviance ()d.f.pamplitude124.3251<0.001sex16.4511<0.001year3.29720.192field site0.21520.898sexyear8.07120.0177sexfield site1.99320.369sexamplitude0.75110.386significance values for individual predictor variables were calculated using type ii tests as implemented in the car package for r (fox and weisberg 2011). Analysis of deviance table for the poisson regression model of the number of flies captured in the acoustic traps significance values for individual predictor variables were calculated using type ii tests as implemented in the car package for r (fox and weisberg 2011). On three occasions, male flies were also occasionally captured in the field on the outside of mesh cages housing calling male n. dorsatus cicadas, which confirmed that they perform phonotaxis to the natural mating calls of male cicadas as well as to the loudspeakers . Twenty female flies were collected at model n. dorsatus call broadcasts and dissected to determine whether they were gravid (i.e., whether they contained first - instar larvae). Of these 20 flies, 6 (30%) had no larvae in their incubatory pouches and 14 (70%) contained first - instar larvae (95% ci for the proportion of nongravid females: 0.1450.519). The six nongravid flies all contained eggs only . In some cases, the eggs were quite small and clearly at an early point in their development . Collection dates ranged from late july to early september; however, because e. erro is multivoltine in the central great plains (stucky 2015), collection date is not a reliable predictor of fly age . Regardless, it was clear that none of the nongravid females had exhausted their egg supply . In the tests of the phonotactic responses of the two reared female e. erro that had not been exposed to either male flies or potential hosts, both flies performed complete flight phonotaxis to the broadcast speaker . The first fly was tested 3 times, and it traversed the width of the cage to arrive at the speaker in the first two tests . For the third test, the fly did not travel to the speaker, but the broadcast still appeared to trigger walking about the wall of the cage and several short flights . The second fly was tested 4 times, and performed complete flight phonotaxis to the speaker all 4 times . Neither fly showed any obvious response to the speaker when it was not broadcasting the model n. dorsatus call . Male e. erro that were attracted by the broadcast loudspeaker typically either perched on the top of the loudspeaker box or immediately pursued one or more flies that had previously arrived at the loudspeaker . When another fly was visually detected by a male, the male would often rapidly approach the second fly either on foot or in the air and make physical contact with it . Such contact generally appeared to be an attempt at copulation, because the pursuing male would grasp the second fly and mount it in the coupling position typical of many diptera (mcalpine 1981). Male flies attempted to mount both males and females and seemed unable to discriminate between the sexes prior to making physical contact . When a male attempted to mount another fly, it usually resulted in both flies departing from the loudspeaker box, so it was rarely possible to determine whether attempted couplings were ultimately successful . During these and other field experiments with e. erro, i observed three apparently successful matings between flies that had been attracted to model cicada call broadcasts . Two matings involved male and female files that were observed copulating inside one of the live traps after both had been captured . The durations of these matings were not precisely timed, but in the second case, the pair remained together for at least 25 min . In the third observation of mating in the field, two flies that landed near the loudspeaker began copulating, then flew off joined together . Attempts were also made to observe mating behavior in captivity in the lab, but these efforts were mostly unsuccessful . However, putative male mating behavior in captivity was similar to that observed at the sound broadcasts in the field, with males pursuing potential mates either by walking or by flight . Only one successful mating was observed in captivity, between a pair of flies that had been reared from a parasitized n. dorsatus (fig . 2). These flies remained in copula for at least 90 min . Traps broadcasting the model n. dorsatus call were operated at three field sites for a total of 24 trap - hours and captured a total of 110 emblemasoma erro (table 1). Of these, 76 (69.1%) were female and 34 (30.9%) were male . Considering all trapping data together, significantly more females than males were captured by the traps (exact binomial test: p <0.001; 95% ci for the proportion of male flies: 0.2300.401). Total numbers of female and male e. erro captured in acoustic traps for each study yearyearfemalemaletotal2011229312012367432013181836total7634110 total numbers of female and male e. erro captured in acoustic traps for each study year the results of the poisson regression analysis of the numbers of flies captured in the acoustic traps are given in table 2 . As expected from the binomial test of the aggregated data, there were significant differences in the numbers of male and female flies that were captured (p <0.001), but the proportions of males and females also varied among the study years (interaction of fly sex and year: p = 0.0177). The proportion of male flies captured in 2013 was much higher than in 2011 and 2012 (table 1); there was no obvious reason for this pattern . There were no differences in the proportions of males and females trapped among the field sites (interaction of fly sex and field site: p = 0.369). Signal amplitude had a strong effect on the number of flies captured by a trap (p <0.001), but amplitude had no effect on the proportions of male and female flies that were trapped (interaction of amplitude and fly sex: p = 0.386). Because broadcast amplitude had no effect on the relative phonotactic responses of male and female e. erro, the effect of amplitude on fly phonotaxis is not considered further here and will instead be analyzed as part of a separate paper addressing acoustic signal perception by e. erro . Analysis of deviance table for the poisson regression model of the number of flies captured in the acoustic trapsvariabledeviance ()d.f.pamplitude124.3251<0.001sex16.4511<0.001year3.29720.192field site0.21520.898sexyear8.07120.0177sexfield site1.99320.369sexamplitude0.75110.386significance values for individual predictor variables were calculated using type ii tests as implemented in the car package for r (fox and weisberg 2011). Analysis of deviance table for the poisson regression model of the number of flies captured in the acoustic traps significance values for individual predictor variables were calculated using type ii tests as implemented in the car package for r (fox and weisberg 2011). On three occasions, male flies were also occasionally captured in the field on the outside of mesh cages housing calling male n. dorsatus cicadas, which confirmed that they perform phonotaxis to the natural mating calls of male cicadas as well as to the loudspeakers . Twenty female flies were collected at model n. dorsatus call broadcasts and dissected to determine whether they were gravid (i.e., whether they contained first - instar larvae). Of these 20 flies, 6 (30%) had no larvae in their incubatory pouches and 14 (70%) contained first - instar larvae (95% ci for the proportion of nongravid females: 0.1450.519). The six nongravid flies all contained eggs only . In some cases, the eggs were quite small and clearly at an early point in their development . Collection dates ranged from late july to early september; however, because e. erro is multivoltine in the central great plains (stucky 2015), collection date is not a reliable predictor of fly age . Regardless, it was clear that none of the nongravid females had exhausted their egg supply . In the tests of the phonotactic responses of the two reared female e. erro that had not been exposed to either male flies or potential hosts, both flies performed complete flight phonotaxis to the broadcast speaker . The first fly was tested 3 times, and it traversed the width of the cage to arrive at the speaker in the first two tests . For the third test, the fly did not travel to the speaker, but the broadcast still appeared to trigger walking about the wall of the cage and several short flights . The second fly was tested 4 times, and performed complete flight phonotaxis to the speaker all 4 times . Neither fly showed any obvious response to the speaker when it was not broadcasting the model n. dorsatus call . Male e. erro that were attracted by the broadcast loudspeaker typically either perched on the top of the loudspeaker box or immediately pursued one or more flies that had previously arrived at the loudspeaker . When another fly was visually detected by a male, the male would often rapidly approach the second fly either on foot or in the air and make physical contact with it . Such contact generally appeared to be an attempt at copulation, because the pursuing male would grasp the second fly and mount it in the coupling position typical of many diptera (mcalpine 1981). Male flies attempted to mount both males and females and seemed unable to discriminate between the sexes prior to making physical contact . When a male attempted to mount another fly, it usually resulted in both flies departing from the loudspeaker box, so it was rarely possible to determine whether attempted couplings were ultimately successful . During these and other field experiments with e. erro, i observed three apparently successful matings between flies that had been attracted to model cicada call broadcasts . Two matings involved male and female files that were observed copulating inside one of the live traps after both had been captured . The durations of these matings were not precisely timed, but in the second case, the pair remained together for at least 25 min . In the third observation of mating in the field, two flies that landed near the loudspeaker began copulating, then flew off joined together . Attempts were also made to observe mating behavior in captivity in the lab, but these efforts were mostly unsuccessful . However, putative male mating behavior in captivity was similar to that observed at the sound broadcasts in the field, with males pursuing potential mates either by walking or by flight . Only one successful mating was observed in captivity, between a pair of flies that had been reared from a parasitized n. dorsatus (fig . 2). These flies remained in copula for at least 90 min . All results of this study were consistent with the hypothesis that emblemasoma erro uses the acoustic sexual signals of its hosts as a means for locating potential mates . In the field, both male flies and nongravid female flies performed positive phonotaxis to acoustic stimuli mimicking the calls of their host cicadas . Unmated female flies with no previous exposure to male flies or host signals were also phonotactically responsive . Once male flies arrived at a sound source, they pursued and tried to mate with other flies that were also attracted to the acoustic stimulus . Although it was not known with certainty whether the nongravid female flies captured in this study were seeking mating opportunities, these flies were obviously incapable of parasitizing hosts, and the observation of three mating pairs in the field indicates that at least some sexually receptive females perform phonotaxis to cicada calls . Traveling to calling cicadas is an expenditure of both time and energy, and it is difficult to imagine any other compensatory benefit to either males or nongravid females besides finding mates . This study cannot rule out the possibility that female e. erro also play a role in attracting males for mating (e.g., with a pheromone signal), but it seems unlikely . Observations of male e. erro suggest that even at very close range, they cannot easily discriminate between receptive females and other females and males . Indeed, their approach to finding a suitable female appears to be mostly trial and error, as has been observed for some other sarcophagid species (thomas 1950; sharma 1975; adham et al . 1980). In any case, the acoustic stimulus by itself is clearly sufficient to attract male flies . On three occasions, only male e. erro were captured by an acoustic trap, and at the beginning of a call broadcast, it was not uncommon for one or more male flies to arrive before any females . There is remarkably little information available about the mate - finding behaviors of other sarcophagids . The literature does suggest, though, that there are two main strategies used by these flies for locating mates . In the first strategy, male flies aggregate at visual markers that are unrelated to adult or larval food resources, such as the tops of hills, and females visit these locations to mate (chapman 1954; dodge and seago 1954; povoln and verves 1997). This behavior, often referred to as hilltopping, is common among many species of calyptrate flies (chapman 1954; dodge and seago 1954; alcock and schaefer 1983). In the second strategy, both males and females travel to adult feeding sites or larviposition sites to seek mates . This has been observed, for example, in sarcophagid species that feed on dung and carrion (thomas 1950; martn - vega and baz 2013; rivers and dahlem 2013). Males of at least one species, sarcophaga bullata parker, also produce a pheromone that is attractive to females (girard and budris 1975; girard et al . The mate - finding behavior of e. erro appears to be a highly specialized version of the second sarcophagid mate finding strategy described in the previous paragraph . Both males and females are attracted to larviposition sites (i.e., male cicadas) to find mates, and both sexes use cicadas mating calls as the cue for locating these sites . The two mate - finding strategies used by sarcophagids are not mutually exclusive, of course, so it is entirely possible that e. erro males also aggregate at visual markers . The ability to facultatively switch strategies could be especially advantageous at times when hosts are not abundant, such as early in the season . Once they arrive at a calling cicada, the mating behaviors of male e. erro are similar to other members of their family . Although sarcophagid species vary in their strategies for initially bringing the sexes together, at close range, most sarcophagid mating systems seem to depend on males that actively search for females visually and, once a potential mate is spotted, attempt to intercept the female and initiate mating with little or no courtship behavior (thomas 1950; moradeshaghi and bohart 1968; sharma 1975; adham et al . 1980; spofford and kurczewski 1985; alcock 2000; gilbert and kim 2007; see spofford and kurczewski 1985; for an exception). Several authors have commented on the apparent inability of male sarcophagids to discriminate among receptive females, unreceptive females, other males, or even other species (thomas 1950; sharma 1975; adham et al . Only 30% of the flies captured in the live traps were male, but this might underestimate the true proportion of males that were attracted to the sound broadcasts . During trap operation, males could often be seen perching on the outside of the trap, from where they would watch for and attempt to intercept other flies that arrived . These males usually did not enter the trap and they sometimes persisted on the outside of the trap until the broadcast terminated . Carrion - feeding sarcophagid species in which both males and females are attracted to carcasses to seek mates can be caught with carrion - baited live traps, and, in at least one study, the percentage of males captured in this way was very similar to that observed here for e. erro (martn - vega and baz 2013). In that study, male carrion - feeding flies that were attracted to the carrion - baited traps stationed themselves near the food resource to intercept mates and did not always enter the traps (martn - vega and baz 2013), much like the behavior of e. erro males at the acoustic traps . Among the species of acoustic parasitoids that have been studied so far, e. erro is the only one for which there is strong evidence that hearing plays a significant role in the lives of the adult males . Yet males of all species appear to have physiologically functional ears (lakes - harlan and lehmann 2015), so why have other acoustic parasitoids not evolved a similar strategy to locate mates? For tachinid acoustic parasitoids of the tribe ormiini, the answer might lie with the biology of their hosts . As far as is known, these flies all parasitize crickets or katydids (orthoptera: ensifera) that produce their calling songs at night (lehmann 2003). Like sarcophagids, males of many tachinid species depend on vision for short - range mate finding (wood 1987), and this requirement might preclude them from using nocturnal host calls as long - range mate finding cues . Indeed, there is evidence that some ormiines use hilltopping to locate mates (lederhouse et al . Also, several authors have posited that the ormiine ear might function to evade bat predation (robert et al . 1992, 1996), and while there is experimental evidence supporting this hypothesis for females of one species, ormia ochracea (bigot) (rosen et al . 2009), the behavioral response of males to bat ultrasound has not been reported . There are no obvious explanations for the absence of acoustic mate finding in emblemasoma auditrix (shewell), the only other species of emblemasoma for which any detailed life history information is available . As with e. erro, female e. auditrix hunt for host cicadas acoustically, and the host cicadas for both species are active during the day (soper et al . 1976; lakes - harlan et al . 2000). Despite thorough investigation of multiple hypotheses, including the use of host signals to find mates, lakes - harlan et al . (2014) were unable to identify any adaptive function for hearing in male e. auditrix . The mating system of e. auditrix is not well understood, but it appears to be a variant of hilltopping behavior in which males aggregate in patches of vegetation and use visual cues to pursue potential mates (lakes - harlan et al . Although they both host on cicadas, e. auditrix and e. erro are in several respects quite different from one another . Emblemasoma auditrix is highly specialized and only attacks a single cicada species, okanagana rimosa (say) (lakes - harlan et al . Emblemasoma erro, on the other hand, has a broader host range, and its hosts usually have a considerably longer seasonal presence than o. rimosa (b. stucky, in preparation). Emblemasoma auditrix is univoltine while e. erro in the central great plains is multivoltine (stucky 2015). It seems plausible that these contrasting natural history traits could favor one mate finding strategy over another, but the extent to which they, or other factors, might have influenced the evolution of these flies mating systems is not known . Use of its hosts communication signals as a mate - finding cue appears to be unique among the few well - studied species of acoustic parasitoids, but there is convincing evidence that some species of nonacoustic eavesdropping parasitoids also use this strategy . At least four species of tachinid flies [euclytia flava (townsend), gymnosoma rotundatum (linnaeus), hemyda aurata robineau - desvoidy, and trichopoda pennipes (fabricius)] and one species of phorid fly (apocephalus paraponerae borgmeier) eavesdrop on the pheromone signals of other insects to locate both potential hosts and potential mates (mitchell and mau 1971; harris and todd 1980; aldrich 1985; feener et al . (it is not entirely clear, however, whether host finding by a. paraponerae is a case of true eavesdropping (hermann et al . 1984; feener et al . 1996)). Some parasitoid wasps that eavesdrop on pheromones have also adopted this strategy, such as the pteromalid tomicobia tibialis ashmead (bedard 1965; rice 1969). A few other cases in which male eavesdropping parasitoids have been attracted to host pheromones have also been reported, but without direct observation of mating or sexual behaviors following chemotaxis (kennedy 1979, 1984; dixon and payne 1980; zaki 1985; micha and wyss 1996; gabry et al . In some studies, male behavior is difficult to interpret because it is not always clear if the host signal by itself is attractive to males or if females that arrive at the signal source are subsequently responsible for attracting males . Regardless, e. erro is clearly not alone in its use of host communication signals as a cue for locating potential mates . Every summer, the grasslands of the central great plains ring with the mating calls of male neotibicen dorsatus trying to attract female cicadas . By producing their calls, these male cicadas unwittingly betray their locations to female emblemasoma erro searching for hosts for their offspring . This study reveals that the acoustic signals of n. dorsatus serve yet another purpose in e. erro s life cycle . For these flies, the call of n. dorsatus is also a love song that both male and female e. erro exploit as a means for finding mating opportunities . Emblemasoma erro and olfactory eavesdroppers with analogous mate - finding behaviors demonstrate that for at least some eavesdropping parasitoids, insect communication signals do not merely provide an efficient way to find potential hosts
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Persistent left superior vena cava (plsvc) is a congenital but benign vascular anomaly with a prevalence of 0.3 - 0.5% in the general population . Plsvc is one of the most common cardiovascular anomalies but is generally asymptomatic, and it is thus often identified accidentally during cardiovascular ultrasound / ct scan screening or coronary interventional angiography . Anatomically, plsvc causes the aberrant backflow vein to drain into the retrograde positioned coronary sinus . Some researchers have called attention to the risk of atresia of the coronary sinus among the anomalies associated with plsvc, but there are no recommendations regarding anticoagulation therapy for patients with plsvc . Nevertheless, coronary sinus atresia (csa) due to plsvc is a rare complication . In patients with hematological malignancies, the insertion of a central venous catheter (cvc) is required for the patient to undergo intensive chemotherapy . Hematologists must make a decision whether to conduct a cvc insertion for patients with a given complication . There are a few reports of csa in plsvc induced by central venous catheterization, pacemaker wire placement, or cardiac surgery . The placement of a cvc might evoke arrhythmia due to an additive enhancement of the patient's arrhythmogenic condition . Considering such a situation, it may not be advisable to insert a cvc in a plsvc patient with a hematological malignancy . We treated a 42-year - old male with stage iv hodgkin's lymphoma with bone marrow involvement . The initial chemotherapy (abvd, doxorubicin 25 mg / m, bleomycin 9 mg / m, vinblastine 6 mg / m, and dacarbazine 250 mg / m, day 1 and 15) was effective, resulting in complete remission after 6 cycles of abvd . Although the abvd chemotherapy had been administered via a peripheral vein, salvage chemotherapy was initiated via an implantable venous access port catheter because his peripheral blood accesses were ultimately unusable . He received an indwelling port catheter from the left subclavian vein, since the right subclavian approach was not possible . 1a), which was found at the time for the first time in his life . The patient successfully completed a total of 3 courses of salvage chemotherapy constructed with arac, carboplatin, etoposide, and methylpredonisolone (aces). Although obstruction of the coronary venous drainage caused by plsvc has been suggested, it is not always thrombogenic . A small plsvc flow can easily connect to hemostasis of the venous circulation return . In our patient, the plsvc cavity had enough space for a port catheter to be cannulated and positioned (fig . . Ideally the back flow and diameter of the plsvc to the coronary sinus should be evaluated by cardiovascular ultrasound or cardiography . Although plsvc is a rare congenital anomaly, some risks including thrombosis and occlusion should be annotated when the patient receives a cvc insertion . The relationship between csa and plsvc remains unclear; however, the hematologist should pay attention to the malpositioning of the cvc tip . Informed consent to participate in the study was obtained from the participant . Written informed consent was obtained from the patient for publication of this case series and any accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal.
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Gait is the most natural motion performed by humans in their ordinary life and has the highest frequency in human activities . Numerous musculoskeletal muscles and nerves of the lower extremities respond together during gait1, 2 . During gait, movement occurs through a series of interactions among the heel bones, the soles, and the ends of the feet . Damages to the feet is associated with impact force, control force, and the distribution of plantar pressure during grounding of the heel bones3 . Flat - arched feet, in particular, have been associated with altered foot function, including prolonged calcaneal eversion, increased tibial internal rotation, increased forefoot abduction, reduced efficiency of gait, and reduced shock absorption4 . The medial longitudinal arch (mla) plays an important role in shock absorbance and energy transfer during walking5 . Although the etiology of this deformity can be arthritic or traumatic in nature6, it is most commonly associated with posterior tibial tendon dysfunction7 . Load during gait increases the number of steps and the double support time, decreases step length, raises energy consumption, and makes the repulsive force against the earth or the lower extremity joints greater8, 9 . During gait, the kinds or directions of loading change the distribution of pressure delivered to the center of the body and the feet, which may trigger abnormal gait by causing fatigue fracture or affecting muscle activity or postural alignment9, 10 . Based on the fact that individuals with the flatfeet more easily feel muscle fatigue of the lower extremities and have a higher risk of damages to the musculoskeletal system than individuals with normal feet, the aim of this study was to examine differences between flatfeet and normal feet while subjects walked on an ascending slope like when climbing a mountain . The subjects in the present study, people with normal feet (n=15) or flatfeet (n=15), were between the ages of 21 and 30 . Sufficient explanations of this study s intent and the overall purpose were given, and voluntary consent to participate in this study was obtained from all of the subjects . All procedures were reviewed and approved by the institutional ethics committee of eulji university hospital . Flatfoot was confirmed by posture analysis (gps400, redbalance, italy). As described by clarke11 strake s line is the line that passes between the medial border of the forefoot and the medial border of the hindfoot, and marie s line is the line that passes between the center of the 3rd toe and the center of the hindfoot . There is also a bisector line between strake s line and marie s line . We categorized subjects into the normal foot group if their medial soles passed to the lateral side of marie s line, and we categorized subjects into the flat foot group if their medial soles passed between the bisector line and strake s line . All subjects received a sufficient explanation about the research and provided consent to participation . A treadmill (ac5000 m, scifit, uk) was used to examine kinematic features during gait . The average gait velocities of the men at slow, normal, and fast rates are 3, 4, and 5 km / h using a slope of 10%, respectively, and those of women are 2.7, 3.7, 4.7 km / h using a slope of 10%, respectively12 . Subjects walked for one minute to determine their natural gait velocity before the experiment . Muscle activity data were collected and analyzed using a wireless surface electromyograph (telemyo 2400 t, noraxon co., usa). The electrode diameter was 11.4 mm, and the distance between the electrodes was 20 mm . The sampling rate for the emg signal was set at 1000 hz, the bandwidth was set between 20450 hz, and the notch filter was set at 60 hz . Emg was conducted after removing the horny layer with sand paper, and cleansing the areas with an alcohol swab . To measure muscle activations in the lower extremities during gait, electrodes were attached to the abductor hallucis, tibialis anterior, medial gastrocnemius, lateral gastrocnemius, peroneus longus, vastus medialis, vastus lateralis, and biceps femoris muscles . The frequency range of the emg signal was band - pass filtered between 20 and 500 hz, and the sampling frequency was 1024 hz . We normalized the signals of muscles to the maximal voluntary isometric contraction (mvic). The general subject characteristics (age, height, and weight) were tested for homogeneity using the independent t - test . Data were analyzed by repeated measures anova in spss for windows (version 17.0), and the differences between groups at the different gait velocities were examined with the independent t - test . Statistical significance was accepted for p values less than 0.05 . The general characteristics of the subjects are shown in table 1table 1.general characteristics of each group (meanse)eg (n=15)cc (n=15)number of individuals (male / female)5/106/9age (years)21.41.322.10.6height (cm)164.21.6167.42.1body weight (kg)61.2.357.22.4foot length (mm)254.24.2257.22.7ankle width (cm)5.60.36.21.2 . Muscle activities of the flat - footed subjects were significantly different from those of the normal - footed subjects at all of the different gait velocities (p <0.05) (table 2table 2 . Comparison of variable with different velocity on a 10% slope (% mvc)groupslownormalfastrectus femoriseg*20.83.224.13.230.63.7cg*21.42.523.73.929.94.7vastus medialiseg*19.92.325.15.838.06.2cg*21.71.624.21.431.84.1vastus lateraliseg*20.51.823.81.629.53.6cg*20.72.424.31.929.13.7tibialis anterioreg*23.40.724.81.430.24.5cg*22.51.825.22.130.03.4peroneus longuseg*20.83.127.02.031.00.7cg*22.11.327.63.432.94.3medial gastrocnemiuseg*32.24.534.24.635.83.2cg*33.31.034.61.337.11.5lateral gastrocnemiuseg*33.41.734.41.636.22.3cg*32.71.234.11.338.71.7abductor halluciseg*14.64.217.34.020.23.6cg*15.23.421.45.026.43.2*p<0.05, eg: experimental group; cg: control group), especially those of the vastus medialis and abductor hallucis muscles (p <0.05) (table 3table 3 . Results of between - subject comparisons of the effects of muscle activation during treadmill gait on a 10% slopetype iii ssdfmsrectus femorisgroup0.210.2error225.2288.0vastus medialis*group23.9123.9error155.7285.5vastus lateralisgroup0.010.0error68.4282.4tibialis anteriorgroup0.410.4error70.7282.5peroneus longusgroup11.6111.6error111.7283.9medial gastrocnemiusgroup6.316.3error311.12811.1lateral gastrocnemiusgroup1.711.7error63.5282.2abductor hallucis*group98.2198.2error381.12813.6*p<0.05). In the subjects with the flatfeet, the function of their feet may did not activate properly as a result of overuse when conducting activities that put repetitive loads on the feet13 . When the subtalar joints are excessively pronated, the medial tibia areas of the knees slip backward at a rapid speed, rotate and enter under the medial femoral condyles, which may cause damage to the medial side of the knees6 . The results of analysis of the muscle activities of the subjects during gait on an ascending slope showed that overall, their muscle activities increased according to the rise in gait speed, and there were significant differences between the two groups in the vastus medialis and abductor hallucis muscles . In the subjects with flatfeet, extensor muscles, in particular, the vastus medialis, play a role in lowering speed and absorbing impact through eccentric contraction . The subjects with flatfeet received more loads on the vastus medialis than those with normal feet due to weakened plantar flexion muscles . Drawing in a slant line of the patellas by the vastus medialis muscles has an important meaning in stabilization and direction of the patellas when they pass or slip through the intercondylar areas of the femurs14 . The abductor hallucis muscles, which are situated below the medial longitudinal arches of the feet, stop at the sesamoid bones of the distal phalanxes from the heel bones and provide dynamic stability to the medial longitudinal arches15 . In this study, the changes in activity of the abductor hallucis muscles according to the changes in speed and gradient were smaller in the subjects with flatfeet than those with normal feet . This indicates that the abductor halluces muscles of individuals with flatfeet do not properly function as muscles for dynamic stability of the medial longitudinal arches . The present study verified that in individuals with flatfeet, the abductor hallucis muscles affected descent of the navicular bones by inducing blockage16 and fatigue of the tibial nerves17.
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To fabricate the vertical wse2/mos2 heterojunction devices, wse2 was grown using a physical vapor deposition process on a si / sio2 (300 nm sio2) substrate . A total of 0.2 g wse2 powder (alfa aesar, 13084) was added into an alumina boat as precursor . The blank si / sio2 substrates (1 cm 5 cm) were loaded into a home - built vapor deposition system in a horizontal tube furnace (lindberg / blue m) with 1 in . The system was pumped down to a vacuum of 10 mtorr in 10 min, and refilled with 150 sccm of ultrahigh purity argon gas (airgas, 99.9999%) then heated to desired growth temperature within 30 min . After that, the growth kept at the desired temperature for 30 min, and then terminated by shutting off the power of the furnace . The mos2 flakes were then exfoliated onto the wse2 flakes through a micromechanical cleavage approach . The metal electrodes (for probe contact or wire bonding purposes) were patterned on the si / sio2 substrates by using electron - beam lithography and electron - beam deposition of ti / au (50/50 nm) thin film . Ni / au (5/50 nm) contact electrodes were then deposited to form the ohmic contact to mos2 and au (50 nm) was deposited to form the ohmic contact to wse2 . The microstructures and morphologies of the nanostructures are characterized by a jeol 6700 scanning electron microscope (sem). The cross - sectional image of the heterostructure device is obtained by an fei titan transmission electron microscope (tem). The dc electrical transport measurements were conducted with a lakeshore probe station (model ttp4) and a computer - controlled analogue - to - digital converter (national instruments model 6030e). The confocal micro - pl and raman measurements were conducted on a horiba labhr confocal raman system with 600 g / mm grating, 50 diffraction - limited objective (n.a . = 0.75), with an ar laser (514 nm) or a he ne laser (633 nm) excitation . The scanning photocurrent measurements were conducted with the same horiba labhr confocal raman system combined with the same electrical measurement system . The el measurements were performed on a home - built confocal pl measurement system combining with the same electrical measurement system with a temperature control in ar environment . Unless mentioned in the main text, all measurements were conducted at room temperature . = 0.5) and captured by a liquid - nitrogen - cooled ccd camera (princeton instruments pylon 400f). The spectra were taken by using an acton 2300i spectrometer with 150 g / mm grating and liquid - nitrogen - cooled ccd.
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Breast cancer (bc) is the most common malignancy (1, 2) and the second leading cause of cancer - related mortality in women (3). Global statistics indicate increasing rate of bc incidence, especially in developing countries (4). Although bc is one of the most common cancers in women, its prevalence widely varies in different parts of the world, which could be due to differences in lifestyles, such as nutrition and unhealthy behaviors, reproductive history, or genetic history (4 - 7). According to estimates, there are 180,000 new cases of bc in the united states every year (8). Also, of every three women with cancer, one has bc (1). In iran, although the incidence of breast cancer is still low comparing with western countries (iran, 23.65 per 100 000 and united states, 140.8 per 100 000 caucasian women) (10), new statistics indicate increasing rates of new bc cases (11). Results of several studies indicate that genes are not the main source of chronic diseases such as bc; for instance, a study conducted on identical twins shows that the effect of genes on bc is only 20% (12), while the effect of lifestyle and environmental factors in causing chronic diseases including bc is 90 - 95% (13). Risk factors associated with bc can be divided into two groups: risk factors that can be modified, and risk factors that cannot . Nutrition, smoking and alcohol use are among risk factors that can be modified . Because of vast differences in breast cancer in different areas, diet is considered one of the causes of breast cancer (7, 14, 15). Changing growth factors, such as insulin, may increase the risk of bc (16). There is evidence that cancer is a preventable disease, and requires quitting smoking, restricting alcohol use, increasing intake of fruits and vegetables, limiting calorie intake, exercise, avoiding exposure to direct sunlight, minimizing red meat consumption, use of whole grains, vaccination, and frequent follow - ups (17, 18). Moreover, according to studies, compliance with the above - mentioned can lead to longer survival of breast cancer patients (12). According to researches, there is an inverse relationship between use of liquid oils with high levels of poly - unsaturated fatty acids (pufa) n-3 such as olive oil and bc . However, use of animal fat increases the risk of bc (19, 20). The alcohol increases serum estrogen level and disrupts metabolism and absorption of folate, which increases risk of bc (16, 21, 22). One of the objectives of the world health organization by 2020 is promotion of healthy lifestyle, reduction of factors harmful to people s health, including wrong nutrition and use of alcohol (23). The prevalence of cancers, particularly breast cancer is increasing, and bc can be prevented through identifying nutritional factors and modifying the diet . Thus, this study is designed to compare nutritional and unhealthy behaviors in women with and without bc referred to mahdieh imaging center of hamadan in 2013 . This cross - sectional study was conducted on 116 women with bc and 116 healthy women presenting to mahdieh imaging center of hamadan (hamadan, iran) from october to december 2013 . After approval of the study protocol (date 20/7/2013), the permission obtained from the research deputy of shahid beheshti university of medical sciences was presented to the director of mahdieh imaging center of hamadan . Random sampling method was used, and subjects were selected among breast cancer patients aged 20 - 60 years, regular menstrual periods, no known medical conditions such as diabetes, thyroid disorders and polycystic ovary syndrome, no hormone replacement therapy and no changes in diet in the past three years . The two groups were matched for variables related to breast cancer including age, weight, body mass index (bmi) and age of menarche . Sample size was estimated of 116 individuals in each group with 95% confidence interval . Every ethical consideration was observed . Participants were informed about the purpose and methods of the study and the interested women were invited to participate in this study . Pathologically, cancer was confirmed in one point in the breast in women with bc . Individuals without breast cancer - including those referred to mahdieh imaging center for other reasons and there was no known cancer according to self - report were included in our control group . The subjects at first studied the directions to answer the questions, which contained some explanations about different food groups and then answered the researcher s questions . Questions included consumption of fruits, vegetables, meat, cereals, nuts and oilseeds, low - fat dairy, whole - grain bread, fish, the kind of oil including liquid oil, solid or animal fat, fast foods, white sauce, sausages, chicken wings, neck and skin, salty foods, potato chips and snacks, soda drinks, and fried foods . Answers to questions included a daily, weekly, monthly and annual food consumption as well as never option . The items about cereals, fruits and vegetables, meat and dairy was considered as positive and taking a higher score is associated with the higher consumption of such foods and items including fat and sugar are considered as negative and their higher score represents their lower consumption . Total score was measured by considering from 4 points for daily to zero for never regarding the positive items and vice versa for the negative items . The score of zero to 33 indicated poor nutritional status, the score of 34 to 66 represented a moderately appropriate nutritional status and the score of 67 to 100 represented an appropriate nutritional status . Unhealthy behavior questionnaire was a researcher - made questionnaire with 8 items and 22 points . The scores were converted to 0 - 100, where 0 - 33, 34 - 66 and 67 - 100 respectively showed the low, moderate and the high risk behaviors . Content validity was used to validate this questionnaire, and retest was used to determine its reliability . Pearson s correlation coefficient using test - retest method within a 10 day interval was equal to 0.82 . Content validity was used for validity, and test - retest was used for reliability of diet, and unhealthy behaviors questionnaires . Pearson s correlation coefficient was 0.82 for diet questionnaire, which its validity and reliability have been confirmed in various studies (24 - 26). Data were analyzed with spss version 17 using t - test, chi - square test and mann - whitney - u test . The t - test was used to compare age, weight, body mass index, menarche age, mean scores of type of feeding, mean scores of different groups of food on the food pyramid and mean scores of type of food between the 2 groups . Mann - whitney - u test was used to compare husband s education, education, occupation, husband s occupation, monthly income and the chi - square test was used to compare marital status between the two groups and an = 0.05 was considered as the level of statistical significance . There was no difference between the two groups in terms of age, weight, bmi, menarche age . The results of the current study indicated a statistically significant difference in terms of nutritional status between patients with and without bc (p <0.001) (table 2). The nutrition score was not less than 33 in either of the groups; it was 34 - 66 in 62% of people with cancer and in 34.5% of non - cancer individuals and 67 - 100 in 38% of patients with cancer and in 65.5% of non - cancer individuals . The mean nutritional score was 56.14 in patients with cancer and 66.25 in healthy individuals . The findings indicate more appropriate nutrition in healthy people than in those with bc . The frequency of consumption of food groups based on the food pyramid is shown in table 3 . In this regard, there is a statistically significant difference between the two groups . Consumption of fruits, vegetables, meat and dairy was significantly higher in healthy people than in patients with cancer, but consumption of cereals, fat and pastry was higher in people with cancer than in healthy people . There was a statistically significant difference between the two groups in terms of the type of food . In terms of food, consumption of whole wheat bread, fish, cheese, yogurt and nuts was higher in healthy people than in patients with bc, but consumption of fast food, white sauce, rice and liquid and solid frying oil was higher in patients with cancer than in healthy people . Data are presented as mean sd . Data are presented as mean sd . The analysis of the data showed a significant relationship between bc and inappropriate diet (p <0.001) and the findings indicate more inappropriate nutrition in bc than in those with healthy people . Also the results of this study showed insignificant differences in terms of diet, food groups and the type of food used among bc patients and non- bc individuals (p <0.001), which represents the effect of diet on bc, but no relationship was observed between bc and unhealthy behaviors . The results of previous studies also show the relationship between diet and bc (24), which is consistent with our findings . In our study, consumption of fruits and vegetables was higher in healthy people than in patients with cancer . The results of previous studies indicate that elevated fruit and vegetable consumption reduces bc risk (25, 26). Also, survival of people who use fruits and vegetables five or more times during a week is higher than those who eat fruits less than five times (21, 25, 27 - 29). Vegetable consumption decreased 6% of the breast cancer risk and fruit consumption was associated with 12% decrease (30). This reduction was higher in women with a high intake of vitamin b6 and vitamin b12 (31). Also receiving high levels of folate has a protective role against bc (32) and may be associated with lower risk of bc among moderate to high alcohol - drinkers (31). Folate is one of the b vitamins that exists in many food sources especially in dark green vegetables, fruits, liver, potato, dairy, bread and cereals (32, 33) and among food sources, fruits and vegetables are rich sources of folate (30). The relationship of folate intake, and fruit and vegetable consumption with reduction in bc risk has been raised . This may be attributed to the effect of folate in vegetables (21, 34, 35). In the current research, the rate of meat and dairy consumption was higher in healthy people than in patients with cancer . Fung et al . Found that meat and dairy consumption in premenopausal ages is associated with increased risk of bc (30), which is contrary to our findings . Also the direct relationship between receiving carbohydrate and bc has been suggested (36), which is consistent with the current study . However, in some studies, an inverse relationship was expressed between them (37, 38), which is contrary to the present study and in some other studies no relationship was found between them (39, 40). In some studies on protein intake, a direct significant relationship is mentioned between protein intake and increased risk of bc (36, 40), which is in contrast to our findings and some other studies have found no relationship between protein intake and increased risk of bc (41). In our study, diet including chicken, fish and turkey is associated with a lower risk of breast cancer (30), which was consistent with our findings ., there is a very close relationship between fat intake and increased risk of breast cancer (42, 43), which is consistent with our study . But in some studies, there were no relationship between them (38), which is contrary to our findings . The rate of cereal consumption was higher in healthy individuals than in patients with cancer . However, some studies have raised that whole - grain intake is associated with a lower risk of bc (30), which is contrary to our findings . Various studies conducted showed that the incidence of bc in smokers is 4.6 times higher and this rate is 3.2 times higher in women who are exposed to cigarette smoke for two hours a day for 25 years (14). The published results from these studies have generally suggested that women who regularly consume alcohol may be at a slightly increased risk of the disease, but the findings reported for tobacco are inconsistent . Alcohol and tobacco consumption are known to be associated with another, and published results have not always allowed adequately for possible confounding between these exposures (9). There is potential for confounding between the possible effects of alcohol and of tobacco on bc, as drinking and smoking are closely associated, one with another (45 - 48). Given the high prevalence of breast cancer and its increasing trend, prevention and reduction of mortality due to breast cancer is very important . According to the results of this study, women with bc have more improper diet than healthy individuals; thus, diet is likely to be one of the important factors affecting bc and modifying diet can prevent bc or reduce its mortality . Therefore, emphasis on modified diet is recommended as a non - medical option for prevention of bc . Considering high prevalence of bc and the direct relationship between nutritional factors and bc, modifying diet is recommended . Training and giving awareness is necessary for women and girls, especially about appropriate diet and unhealthy behavior . This study has been done on women referred to mri center that maybe - cannot represent all of the women and the results cannot be generalized to all the women of the child - bearing age and dietary assessment is mostly based on self - reports . Considering high prevalence of bc and the direct relationship between nutritional factors and bc, modifying diet is recommended . Training and giving awareness is necessary for women and girls, especially about appropriate diet and unhealthy behavior . This study has been done on women referred to mri center that maybe - cannot represent all of the women and the results cannot be generalized to all the women of the child - bearing age and dietary assessment is mostly based on self - reports.
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The study was conducted in neurophysiology division of defence institute of physiology & allied sciences (dipas), delhi, from april to july 2010 . Ten healthy young male volunteers (21 - 30 yr) with average education level of viii - x standard of the indian education system (10 yr of total education) drawn randomly from the indian army participated in the study . They were free from any neurological disorder especially hearing loss, sleep - related and cardiac problems and diabetes . They were not taking any sort of psychotropic, psychoactive or other drugs during the course of the study and were abstained from alcohol and smoking during the experiment . The purpose of the study was explained to them and written informed consent was obtained . Study design: the study was divided into two phases: pre- and post - intervention . A complete set of parameters comprising erp p300, cnv, mlr and raven's advanced progressive matrices (rapm) were recorded at a fixed time in the morning (0700 - 0800 h) except 36 h sd (1900 - 2000 h). The first night was adaptation to make the subject comfortable to sleep with the necessary attachments . The first recording (baseline, bl) was performed on the morning before the same night sd, the second recording on the morning after one night's sd (24 h sd), third recording after 36 h sd (36 h sd). A 36 h total sd was obtained by keeping the subject awake for 12 h after 24 h of deprivation . Fourth recording was taken after recovery sleep (rec sd); fifth recording after 60 days of meditation practice, on the morning of 24 h sd (med+24 h sd). Sixth recording was performed after 36 h sd (med+36 h sd) and seventh recording on the morning after recovery sleep (med+rec sd). Each volunteer slept for about 7.5 to 8 h during adaptation and baseline nights and 8.0 to 9.0 h during recovery night . The participants were allowed to play video games, watch movies, and read books according to their choice as a tool for maintenance of continuous wakefulness24 . During 36 h sd (daytime), subjects were on regular duty schedule without any extra laborious workload . This was done to assure the homogenous work pattern for them through the period of experiment with no irregular activity . Actigraphy was performed using an actiwatch (aw 64, mini mitter, usa) to monitor the activity profile throughout the period of experiment . The threshold activity was set such that even a nap could be detected (actiware v. 5.0, mini mitter, usa). Evoked potentials: scalp was gently cleaned with nprep skin prepping gel (weaver and company, usa) and ten20 conductive eeg paste was applied to different locations on the scalp according to the internationally accepted 1020 electrode placement system . Ag - agcl disc electrodes (nicolet biomedical, usa) were used for recording . All the recordings were done with subjects in supine position and eyes open with restricted eye movements . Auditory (sound) stimulus was given to the left ear by a headphone (telephonics, usa). P300: electrodes were placed at a1 and a2 (reference), fpz (common ground), cz and pz (active). Two different click sounds, one non - target and the other target, were presented to the subject using standard auditory odd - ball paradigm . The subject was asked to count the rare stimuli with attention and concentration, so that he could prompt the number of clicks he had counted at the end of the session . The stimulus characteristics were - intensity: 80 db spl, sensitivity: 100 v, frequency: 2 khz, sweep pattern: gated, sweep rate: 0.9/sec, lff: 1.0 hz and hff: 30.0 hz for non - target and intensity: 80 db spl, sensitivity: 100 v, frequency: 750 hz, sweep pattern: gated, sweep rate: 0.9/sec, lff: 1.0 hz and hff: 30.0 hz target stimuli . After recording, latencies (msec) of peaks n1, p2, n2 and p300 and amplitudes (v) of p200 and p300 were marked . Cnv: electrodes were placed at e1 linked with e2 (eye movement), a1 (reference), fpz (common ground), c3, c4, cz and fz (active). The subject worn a pair of led goggles (nicolet biomedical, usa) and a headphone (for visual and auditory stimuli respectively). The subject was given two stimuli: the first the subject was asked to be alert and to expect a second stimulus to come forth . After 1 sec of s1, the second imperative stimulus (s2) was given in the form of trains of visual led flashes and the subject was asked to press a button to stop the flash . One single sweep was 4 sec long, and eight such sweeps were averaged for 1 set of waveforms . The stimulus characteristics were - intensity: 70 db spl, sensitivity: 2 mv, frequency: 1 khz, sweep pattern: gated, sweep rate: 0.1/sec, lff: 0.01 hz and hff: 30.0 hz . After recording, latencies (msec) mlr: electrodes were placed at a1 and a2 (reference), fpz (common ground) and cz (active). The volunteer was instructed to concentrate towards the stimuli coming in a synchronized fashion to his left ear . The stimulus characteristics were: intensity: 75 db spl, sensitivity: 100 v, frequency: 500 hz, sweep pattern: gated, sweep rate: 39.1/sec, lff: 10.0 hz and hff: 100.0 hz . Latencies (msec) of peaks na and pa and amplitude (v) of na - pa were marked . Subjects were explained the modality of the test in detail and asked to practice with set - i accordingly . Each problem page (in both sets) contained nine design cells displayed in a square manner of 3 by 3 (a matrix), with the bottom - right cell vacant . The subjects had to predict the possible design pattern for the vacant cell and choose the same from a list of eight patterns provided below the matrix . After completing set - i, they were asked to solve the matrices in the set - ii (actual test set) within a time frame of 30 min . The reason for time - locking was to make the subject more attentive towards the test . This was a 25 min session of spiritual exercise performed by moderate workers on a twice daily basis for 60 days . Each session consisted of four stages with volunteer sitting in upright position and back erect - first quietly with eyes closed for 6 min . This was followed by the pranayama udgeet - the respiratory exercise for the next 6 min, in which the volunteer sat with hands in upright position and mouth shut . The index finger was placed on the forehead; with next three fingers covering his eyes and the thumb pressing the ear lid . This is to ensure blockage of energy exchange with the universe except through respiration . In this state the person chanted next 6 min the om was chanted while sitting erect on back and exhaling slowly with the eyes closed . Last 6 min were spent in absolute stillness, wherein the volunteer sat with eyes closed and breathing slowly, again in first posture . At the end, participants raised their hand above their head, rasped the palms first slowly, and then briskly, placed the palms over the eyes and the session was completed . Statistical analysis: statistical analyses were done using spss 13.0 (ibm, usa). Analysis of statistical significance for changes during various conditions was performed using one way anova for repetitive measurements for cnv, p300-erp and mlr . Each value at a given condition was compared to that in another condition using multiple comparison (post hoc) t - tests . Rapm was analyzed using friedman's rank test for analysis of significant difference within groups, followed by mann whitney's u test to analyze between group significance . Pearson's product - moment correlation coefficient (r) was computed to see for significant correlation between rapm with mlr - na, p300-p3, p300-p3 amplitude (amp), cnv - m1, cnv - m1 amp, cnv - m2 and cnv - m2 amp . The determined r was then converted to student's t - scores for testing of ho (significance). Subjects were explained the modality of the test in detail and asked to practice with set - i accordingly . Each problem page (in both sets) contained nine design cells displayed in a square manner of 3 by 3 (a matrix), with the bottom - right cell vacant . The subjects had to predict the possible design pattern for the vacant cell and choose the same from a list of eight patterns provided below the matrix . There was no time restriction for solving the practice set . After completing set - i, they were asked to solve the matrices in the set - ii (actual test set) within a time frame of 30 min . The reason for time - locking was to make the subject more attentive towards the test . This was a 25 min session of spiritual exercise performed by moderate workers on a twice daily basis for 60 days . Each session consisted of four stages with volunteer sitting in upright position and back erect - first quietly with eyes closed for 6 min . This was followed by the pranayama udgeet - the respiratory exercise for the next 6 min, in which the volunteer sat with hands in upright position and mouth shut . The index finger was placed on the forehead; with next three fingers covering his eyes and the thumb pressing the ear lid . This is to ensure blockage of energy exchange with the universe except through respiration . In this state the person chanted next 6 min the om was chanted while sitting erect on back and exhaling slowly with the eyes closed . Last 6 min were spent in absolute stillness, wherein the volunteer sat with eyes closed and breathing slowly, again in first posture . At the end, participants raised their hand above their head, rasped the palms first slowly, and then briskly, placed the palms over the eyes and the session was completed . Statistical analysis: statistical analyses were done using spss 13.0 (ibm, usa). Analysis of statistical significance for changes during various conditions was performed using one way anova for repetitive measurements for cnv, p300-erp and mlr . Each value at a given condition was compared to that in another condition using multiple comparison (post hoc) t - tests . Rapm was analyzed using friedman's rank test for analysis of significant difference within groups, followed by mann whitney's u test to analyze between group significance . Pearson's product - moment correlation coefficient (r) was computed to see for significant correlation between rapm with mlr - na, p300-p3, p300-p3 amplitude (amp), cnv - m1, cnv - m1 amp, cnv - m2 and cnv - m2 amp . The determined r was then converted to student's t - scores for testing of ho (significance). P300: n1, p2 and n2 components of p300 erp did not register any significant change, though there was increase in latencies after 24 and 36 h of sd . Latency of p300 registered significant increase after 36 h sd (table i) compared to baseline (p<0.01), and significant reduction was noted after recovery sleep (p<0.05). Amplitude of p300 component increased following 24 h sd as well as 36 h sd (p<0.01), and returned to its baseline range after recovery sleep (p<0.01) (table i). Effect of meditation practice on p300 after sleep deprivation (sd) cnv: latencies of n1 and m1 did not alter significantly . However, m2 was delayed significantly after 24 h sd (p<0.05) and the delay was greater after 36 h sd (p<0.01) compared to baseline (table ii). The latency returned to baseline after recovery sleep (p<0.05, recovery compared to 36 h sd). Amplitude of m1 increased after 24 h sd significantly (p<0.05), which was even higher following 36 h of prolonged deprivation (p<0.001), compared to baseline . After recovery sleep, there was significant reduction in the amplitude of m1 compared to 36 h sd (p<0.05). Amplitude of m2 showed significant elevation after 36 h sd (p<0.001) compared to baseline . After recovery sleep, significant reduction in amplitude of m2 was observed compared to both 24 h (p<0.05) and 36 h (p<0.001) of sd (table ii). In comparison to baseline, cnv reaction time was prolonged following 24 and 36 h of sd, the latter being significant (p<0.01). After recovery sleep, reaction time again registered significant decrease compared to 36 h sd (p<0.01). Effect of meditation practice on contingent negative variation after sleep deprivation (sd) mlr: latency of the peak na registered no significant change after 24 h sd but significant change following 36 h sd compared to baseline (p<0.01) and recovery (p<0.05). Latency of pa and amplitude of na - pa did not register any significant change at any stage (table iii). Effect of meditation on middle latency response after sleep deprivation (sd) psychological assessment: total sd decreased number of correct responses in rapm set - ii session after 24 h and 36 h (p<0.01) compared to baseline . This decline was recovered (p<0.01, compared to 36 h sd) after recovery (fig . ). Number of correct responses in rapm set - ii session showed significant negative correlation with amplitude of m1 component of cnv (r = -0.66; p<0.05) at baseline . Mlr - na, p300-p3, p300-p3 amp, cnv - m1, cnv - m2 and cnv - m2 amp . 1), p300 erp registered no significant changes in terms of latencies of n1, p2, n2 and p300 components . Cnv: there was no significant change in latencies of n1, m1 and m2 components of cnv (table ii). Amplitude of m1 also did not alter significantly, though there was a significant increase in m2 amplitude after 36 h sd (p<0.05). Mlr: after two months of meditation practice, there was no significant change in any components of mlr (table iii). Neuropsychological assessment: total sd slightly decreased number of correct responses in rapm set - ii session after sd compared to baseline (fig . ). P300: n1, p2 and n2 components of p300 erp did not register any significant change, though there was increase in latencies after 24 and 36 h of sd . Latency of p300 registered significant increase after 36 h sd (table i) compared to baseline (p<0.01), and significant reduction was noted after recovery sleep (p<0.05). Amplitude of p300 component increased following 24 h sd as well as 36 h sd (p<0.01), and returned to its baseline range after recovery sleep (p<0.01) (table i). Effect of meditation practice on p300 after sleep deprivation (sd) cnv: latencies of n1 and m1 did not alter significantly . However, m2 was delayed significantly after 24 h sd (p<0.05) and the delay was greater after 36 h sd (p<0.01) compared to baseline (table ii). The latency returned to baseline after recovery sleep (p<0.05, recovery compared to 36 h sd). Amplitude of m1 increased after 24 h sd significantly (p<0.05), which was even higher following 36 h of prolonged deprivation (p<0.001), compared to baseline . After recovery sleep, there was significant reduction in the amplitude of m1 compared to 36 h sd (p<0.05). Amplitude of m2 showed significant elevation after 36 h sd (p<0.001) compared to baseline . After recovery sleep, significant reduction in amplitude of m2 was observed compared to both 24 h (p<0.05) and 36 h (p<0.001) of sd (table ii). In comparison to baseline, cnv reaction time was prolonged following 24 and 36 h of sd, the latter being significant (p<0.01). After recovery sleep, reaction time again registered significant decrease compared to 36 h sd (p<0.01). Effect of meditation practice on contingent negative variation after sleep deprivation (sd) mlr: latency of the peak na registered no significant change after 24 h sd but significant change following 36 h sd compared to baseline (p<0.01) and recovery (p<0.05). Latency of pa and amplitude of na - pa did not register any significant change at any stage (table iii). Effect of meditation on middle latency response after sleep deprivation (sd) psychological assessment: total sd decreased number of correct responses in rapm set - ii session after 24 h and 36 h (p<0.01) compared to baseline . This decline was recovered (p<0.01, compared to 36 h sd) after recovery (fig . ). Number of correct responses in rapm set - ii session showed significant negative correlation with amplitude of m1 component of cnv (r = -0.66; p<0.05) at baseline . Mlr - na, p300-p3, p300-p3 amp, cnv - m1, cnv - m2 and cnv - m2 amp . 1), p300 erp registered no significant changes in terms of latencies of n1, p2, n2 and p300 components . Cnv: there was no significant change in latencies of n1, m1 and m2 components of cnv (table ii). Amplitude of m1 also did not alter significantly, though there was a significant increase in m2 amplitude after 36 h sd (p<0.05). Mlr: after two months of meditation practice, there was no significant change in any components of mlr (table iii). Neuropsychological assessment: total sd slightly decreased number of correct responses in rapm set - ii session after sd compared to baseline (fig . ). P300: n1, p2 and n2 components of p300 erp did not register any significant change, though there was increase in latencies after 24 and 36 h of sd . Latency of p300 registered significant increase after 36 h sd (table i) compared to baseline (p<0.01), and significant reduction was noted after recovery sleep (p<0.05). Amplitude of p300 component increased following 24 h sd as well as 36 h sd (p<0.01), and returned to its baseline range after recovery sleep (p<0.01) (table i). Effect of meditation practice on p300 after sleep deprivation (sd) cnv: latencies of n1 and m1 did not alter significantly . However, m2 was delayed significantly after 24 h sd (p<0.05) and the delay was greater after 36 h sd (p<0.01) compared to baseline (table ii). The latency returned to baseline after recovery sleep (p<0.05, recovery compared to 36 h sd). Amplitude of m1 increased after 24 h sd significantly (p<0.05), which was even higher following 36 h of prolonged deprivation (p<0.001), compared to baseline . After recovery sleep, there was significant reduction in the amplitude of m1 compared to 36 h sd (p<0.05). Amplitude of m2 showed significant elevation after 36 h sd (p<0.001) compared to baseline . After recovery sleep, significant reduction in amplitude of m2 was observed compared to both 24 h (p<0.05) and 36 h (p<0.001) of sd (table ii). In comparison to baseline, cnv reaction time was prolonged following 24 and 36 h of sd, the latter being significant (p<0.01). After recovery sleep, reaction time again registered significant decrease compared to 36 h sd (p<0.01). Effect of meditation practice on contingent negative variation after sleep deprivation (sd) mlr: latency of the peak na registered no significant change after 24 h sd but significant change following 36 h sd compared to baseline (p<0.01) and recovery (p<0.05). Latency of pa and amplitude of na - pa did not register any significant change at any stage (table iii). Effect of meditation on middle latency response after sleep deprivation (sd) psychological assessment: total sd decreased number of correct responses in rapm set - ii session after 24 h and 36 h (p<0.01) compared to baseline . This decline was recovered (p<0.01, compared to 36 h sd) after recovery (fig . ). Number of correct responses in rapm set - ii session showed significant negative correlation with amplitude of m1 component of cnv (r = -0.66; p<0.05) at baseline . Mlr - na, p300-p3, p300-p3 amp, cnv - m1, cnv - m2 and cnv - m2 amp . 1), p300 erp registered no significant changes in terms of latencies of n1, p2, n2 and p300 components . Cnv: there was no significant change in latencies of n1, m1 and m2 components of cnv (table ii). Amplitude of m1 also did not alter significantly, though there was a significant increase in m2 amplitude after 36 h sd (p<0.05). Mlr: after two months of meditation practice, there was no significant change in any components of mlr (table iii). Neuropsychological assessment: total sd slightly decreased number of correct responses in rapm set - ii session after sd compared to baseline (fig . ). The present study used a novel approach to evaluate the psychophysiological and functional measures of cognition together . The study also attempted to establish the relationship between the psychophysiological components i.e. Rapm scores with the latencies and amplitudes of p300, cnv and mlr . In our study p300 component of erp showed significant increase after 36 h of sd in terms of latency . This is indicative of reduced ability of concentration, discrimination of a target stimulus in a train of non - target stimuli; matching the stimulus against the memory representation of the relevant (target) stimuli and decision making (whether the presented stimulus is a target one). P300 is elicited when a form of selective attention is required by the subject in order to identify infrequent occurrences of a target (relevant) stimulus, which is presented unpredictably24 . The delayed p300 latency also indicates that there is improper classification and recognition of stimulus as relevant to the assigned task . Lee et al10 have reported significantly prolonged latency of n2 and decreased amplitudes of n1, p200 and p300 . In our study delay in latencies of n1, p2 and n2 components of this erp n2 preceding p300 may directly affect the absolute timing of decision processes in sensory discrimination; and it is also sensitive to the cognitive attributes to the stimulus27 . Amplitude of p300 increased after 24 and 36 h sd, the latter being significant compared to baseline . P300 amplitude depends upon the selective attention of a subject, which is compromised after sd . After sd a person gets inattentive to a considerable extent which results in unpredictable presentation of a predictable stimulus . Another possible explanation may be that, stimuli which signal some internal response (like memory task in p300) or provide useful information (like target and non - target discrimination criterion in p300), result in an erp component (like p300) with definitely higher amplitude24 . Cnv appears to represent the summation of at least two, probably more, component waveforms . One previous study has reported increase in amplitude of cnv components after 24 h sd in epileptics28 as found in our study . In our study, the early component of cnv (m1) was delayed non - significantly following sd . M1 is a stimulus related frontally predominant component, perhaps reflecting the orienting response . On the other side, m2, the late cnv component is a centrally predominant component thought to be related to the premotor potential associated with the motor response29 . A delayed m1 indicates subject's deficient ability to get warned after the warning stimulus (s1) and to get oriented about s1 as well; and delayed m2 indicates improper preparation for the subsequent motor response . Latency of m2 was significantly increased following 24 h sd and 36 h sd compared to baseline . A significantly prolonged reaction time implies a state of deficient motor response, subject's incapability of perceptual processing and preparatory judgment . Na is believed to be generated subcortically and pa, bilaterally within the primary auditory cortex30 . After continuous wakefulness, the conduction at the subcortical level may be delayed; or there may be some changes, which appear to resemble changes in na latency after subcortical lesion . The reversal of the situation after recovery sleep i.e., reduction of na latency after recovery sleep proposes that, the effect of prolonged wakefulness is ameliorated by 8 - 9 h of recovery sleep . In humans mid latency responses may actually consist of overlapping potentials originating from primary auditory cortex, ascending activating system and/or subcortical structures31 . It is possible that sd may lead to changes in neural processing at some or all of these levels . As mentioned earlier23, mlr has got some attention related cortical components, which may deteriorate after sd . Rapm measures two main components of intelligence; the ability to think clearly and make sense of complexity known as eductive ability, and the ability to store and reproduce information called reproductive ability32 . It appears to measure a type of reasoning ability with very high predictive validities in most occupational groups . It may be posible that 36 h sd interferes with educative and reproductive ability, which leads to deficient problem solving and analytical ability . The educative and reproductive abilities involve important components of cognitive function like attention, vigilance, concentration and working memory . It was therefore, not surprising that a significant negative correlation was obtained between the amplitude of m1 (early cnv) and number of correct responses in rapm set - ii . A plausible explanation for this effect is that, following sd, a greater effort in terms of trying to concentrate harder is needed for the same extent of task complexity . The greater amount of subjective effort is reflected in increased m1 amplitude and the poor performance is depicted by reduced rapm scores . In conclusion, the present study validates and extends the value of p300, cnv and mlr as the markers for quantification of the degree of cognitive impairments after total sd . It also shows that total sd involves deficit in sensory perception as well as inefficiency in motor responses, which leads to cognitive impairment in an integrative manner, as is evident from significant changes in p300, cnv, mlr and rapm scores . Thus, cognitive impairment after total sd is an assembly of dysfunctions in different regions of brain rather than being a single component; and the event related (p300 and cnv) and evoked potentials (mlr) can analyze the situation real - time, which is further confirmed by rapm scores, the psychophysiological assessment tool.
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Cardiac resynchronisation therapy (crt) is most effective in heart failure patients with left bundle branch block as it specifically aims to resynchronise the delayed electrical activation of the left ventricle [12]. However, crt is indicated in a much broader spectrum of patients with heart failure . A large european survey among 141 hospitals demonstrated that approximately 25% of crt implantations are upgrade procedures . Patients with an existing cardiac implantable electronic device (cied) have a class i indication for upgrading to crt when they experience a high percentage of ventricular pacing and remain in new york heart association (nyha) class iii to ambulatory iv with a left ventricular ejection fraction (lvef) 35% . Other reasons for upgrading to crt include (i) worsening of heart failure (class) in patients with pre - existing wide qrs complexes, (ii) electrical remodelling due to heart failure or antiarrhythmic drug therapy with widening of the qrs complex, and (iii) evolving crt guidelines . Previous literature has shown that cardiac device replacement or upgrade procedures are associated with more complications and are more complex than de novo implantations due to the possibility of venous thrombosis and the risk of damaging or extracting old leads [5, 6]. The fear of a complicated procedure might prevent upgrading to crt in patients with a clear indication . Moreover, reluctance about the need for an upgrade procedure in the future might make clinicians more willing to implant a de novo crt device in heart failure patients with only a class iib indication for crt but a clear indication for a conventional (single or dual chamber) device . We aimed to evaluate procedural data and periprocedural and long - term complication rates of upgrade procedures compared with de novo crt implantations in our university medical centre . This retrospective study analysed data from all patients who had undergone an upgrade procedure from a single or dual chamber pacemaker or implantable cardioverter defibrillator (icd) to a crt device at the university medical centre of utrecht (umcu) in the period 20062012 . An equally sized reference group of de novo implantations was randomly constructed out of patients implanted in the same period (n = 358). Procedures were performed by experienced operators, defined as performing at least 75 icd / pacemaker implantations annually with at least one - third of these being crt device implantations . Periprocedural complications (30 days) and long - term device - related complications (1 year) were divided into complications with and without consequences . Complications with consequences were defined as those in need of invasive procedures (e.g. Lead revision / replacement, extraction of the system, pericardiocentesis) or any other action deviating from standard clinical care (prolonged hospitalisation, postponement of implant after start, transfer to the coronary or intensive care unit or the need to start anticoagulants). Categorical variables are presented as percentages and the p - value was calculated using pearson s chi - square test . If distribution was skewed, variables are presented as median with 25th to 75th percentile interquartile range (iqr) and the p - value was calculated by the mann - whitney u test . Normally distributed variables are presented as means with a standard deviation and a p - value calculated by the student s t test . The occurrence of specific periprocedural complications was compared between de novo and upgrade procedures using a chi - square test . The hazard ratio for the risk of any periprocedural complication with consequences for the type of procedure (upgrade versus de novo implantation) was calculated by a crude logistic regression analysis . To identify any confounding effect of baseline characteristics on the risk of periprocedural complications after an upgrade procedure and de novo crt implantation, multiple bivariate logistic regression analyses were performed, including procedure type and one baseline characteristic . The crude hazard ratio for procedure type and risk of periprocedural complications was adjusted for all baseline characteristic differences in a multivariable logistic regression analysis . The comparison of the occurrence of long - term complications by procedure type was performed by the chi - square test . Patients with censored data due to loss to follow - up or death within 1 year of follow - up were excluded from this comparison . This retrospective study analysed data from all patients who had undergone an upgrade procedure from a single or dual chamber pacemaker or implantable cardioverter defibrillator (icd) to a crt device at the university medical centre of utrecht (umcu) in the period 20062012 . An equally sized reference group of de novo implantations was randomly constructed out of patients implanted in the same period (n = 358). Procedures were performed by experienced operators, defined as performing at least 75 icd / pacemaker implantations annually with at least one - third of these being crt device implantations . Periprocedural complications (30 days) and long - term device - related complications (1 year) were divided into complications with and without consequences . Complications with consequences were defined as those in need of invasive procedures (e.g. Lead revision / replacement, extraction of the system, pericardiocentesis) or any other action deviating from standard clinical care (prolonged hospitalisation, postponement of implant after start, transfer to the coronary or intensive care unit or the need to start anticoagulants). Categorical variables are presented as percentages and the p - value was calculated using pearson s chi - square test . If distribution was skewed, variables are presented as median with 25th to 75th percentile interquartile range (iqr) and the p - value was calculated by the mann - whitney u test . Normally distributed variables are presented as means with a standard deviation and a p - value calculated by the student s t test . The occurrence of specific periprocedural complications was compared between de novo and upgrade procedures using a chi - square test . The hazard ratio for the risk of any periprocedural complication with consequences for the type of procedure (upgrade versus de novo implantation) was calculated by a crude logistic regression analysis . To identify any confounding effect of baseline characteristics on the risk of periprocedural complications after an upgrade procedure and de novo crt implantation, multiple bivariate logistic regression analyses were performed, including procedure type and one baseline characteristic . The crude hazard ratio for procedure type and risk of periprocedural complications was adjusted for all baseline characteristic differences in a multivariable logistic regression analysis . The comparison of the occurrence of long - term complications by procedure type was performed by the chi - square test . Patients with censored data due to loss to follow - up or death within 1 year of follow - up were excluded from this comparison . This analysis included 134 patients who underwent an upgrade procedure to a crt device and 134 randomly selected patients receiving a de novo crt device . Of these 268 patients, 264 (98.5%) received a crt defibrillator (crt - d), three (2.2%) patients were upgraded to a crt pacemaker (crt - p) and one (0.7%) patient received a de novo crt - p . Fifty - four (40%) patients where upgraded from a double chamber device and 81 (60%) from a single chamber device . Seventy - six (57%) upgrade patients were paced from the right ventricle at baseline . Upgrade patients were more often males (p = 0.011) and of older age (p = 0.001) than the random sample of patients receiving a de novo crt implantation . They more often had ischaemic cardiomyopathy (p = 0.049) and a history of atrial fibrillation (p = 0.008) and were more often on amiodarone (p <0.001) and a vitamin k antagonist (p = 0.022). No baseline characteristic had a significant confounding effect on the hazard ratio for procedure type and the risk of periprocedural complications.table 1baseline comparison between patients receiving an upgrade to a crt device and those receiving a de novo crt devicebaseline characteristicstotal population (n = 268)upgrade(n = 134)de novo implants (n = 134) p - value age (years) 69(6174)71(6375)67(6072) 0.001 male (n(%))202(75)110(82)92(69) 0.011 bmi26(2429)26(2429)26(2430)0.450 nyha class (n,%) 0.080 i2(1)1(1)1(1) ii36(13)13(10)23(17) iii213(80)110(82)103(77) iv17(6)10(8)7(5)ischaemic cmp (n,%) 148(55)82(61)66(49) 0.049 lvef (%) 23 (7)24 (7)23 (7)0.239bnp141(68262)146(73273)130(50226)0.112atrium fibrillation (n,%) 60(22)39(29)21(16) 0.008 cardiac medication (n,%) diuretic231(88)117(88)114(87)0.816beta - blocking201(76)100(76)101(76)0.940ace inhibiting227(86)110(83)117(89)0.158 other medication amiodarone54(21)39(29)15(11) <0.001 use of anticoagulation (n,%) vitamin - k antagonist191(72)104(78)87(65) 0.022 other57(21)23(17)34(25)0.101inr (n = 191) 1.7(1.32.1)1.7(1.42.1)1.5(1.31.9)0.053 comorbidity diabetes mellitus (n,%) 62(23)29(22)33(25)0.540copd (n,%) 34(13)17(13)17(13)0.981renal function (egfr)57(19)55(20)59(19)0.090 subclavian / anonyma vein thrombosis (n,%) 11(4)11(8)0(0) 0.001 time since initial cied implantation (months) 57(31115) standard deviation deviation, range; 25th to 75th percentile inter quartile range . Ace angiotensin i converting enzyme, bmi body mass index, bnp brain natriuretic peptide, cied cardiac implantable electronic device, cmp cardiomyopathy, copd chronic obstructive pulmonary disease, inr international normalised ratio, lvef left ventricular ejection fraction, nyha new york heart association, sd standard deviation . Nyha iii and iv versus i, ii baseline comparison between patients receiving an upgrade to a crt device and those receiving a de novo crt device standard deviation deviation, range; 25th to 75th percentile inter quartile range . Ace angiotensin i converting enzyme, bmi body mass index, bnp brain natriuretic peptide, cied cardiac implantable electronic device, cmp cardiomyopathy, copd chronic obstructive pulmonary disease, inr international normalised ratio, lvef left ventricular ejection fraction, nyha new york heart association, sd standard deviation . Nyha iii and iv versus i, ii procedural data are presented in table 2 . Seventy - three (27%) procedures were extended by study - related lv pressure (lv dp / dt max) measurements to optimise lead positions and device settings . Median radiation time was 25 (20) minutes and median hospitalisation after implantation, including rehospitalisation for another attempt to complete crt implantation after initial lv lead placement failure, was 2 (2) days . There were no significant differences in these procedural data between upgrade and de novo crt implantation procedures . At baseline, ten (8%) patients undergoing an upgrade procedure had a subclavian (or anonyma) vein thrombosis . In six of these patients the vein was recanalised, in two a contralateral implant was chosen and in two patients the occlusion could be passed . In 124 (92%) ten (8%) patients did not receive a right atrial lead because of permanent atrial fibrillation . Of the upgrade patients 91 (68%) received two new leads, three leads were placed in 16 (12%) patients and 27 (20%) patients only received an additional lv lead.table 2procedural dataprocedural data (median, iqr)totalupgradede novo p - valuelength procedure (min)156(78)145(71)163(81)0.062standard procedure (n = 194)139(69)135(75)142(61)0.515procedure with lv dp / dt max measurements 187(66)171(72)195(42)0.176radiation time (min)25(20)25(19)25(21)0.595total hospitalisation time (days)2(2)1(2)2(2)0.418 leads added or implanted (intention to) (n,%) <0.001127(20%) 0(0)291(68%) 10(8%) 316(12%) 124(92%) n of operating specialists 1.000 n = 1 (experienced cardiologist)224(85)112(85)112(85) n = 2 (fellow with experienced cardiologist or two experienced cardiologists)40(15)20(15)20(15) of which 39.7% upgrade procedures . Starting after implantation including additional hospitalisation for later lv lead placement after initial failure or re - hospitalisation due to implantation or device related complications . Of which 39.7% upgrade procedures . Starting after implantation including additional hospitalisation for later lv lead placement after initial failure or re - hospitalisation due to implantation or device related complications . 4 patients had missing data on implanting physician . The periprocedural complication rate is presented in table 3 . In total 21 (7.8%) patients experienced a complication with consequences . The crude hazard ratio for periprocedural complications (with consequences) for upgrade procedures compared with de novo crt implantations was 1.692 (p = 0.260) and when adjusted for baseline differences between the two groups it was 1.779 (p = 0.249) (table 4). One patient developed acute decreased saturation during an upgrade procedure probably based on cardiac asthma . Saturation did not improve with pharmacological treatment or intubation, and resuscitation was required, which the patient did not survive . In seven (2.6%) patients transvenous lv lead placement was unsuccessful due to difficulty in visualising or cannulating the coronary sinus . Phrenic nerve stimulation was seen in 14 (5.3%) patients and usually managed by adjustments in the pacing configuration, but in one case lead revision was needed shortly after implantation . Pocket haematoma was seen in 12 (4.5%) of the total 268 patients; of these four upgrade patients needed to be re - hospitalised or needed blood transfusion . Table 3periprocedural complicationscomplication (n,%) total (n = 268)upgrade (n = 134)de novo (n = 134) perforation / dissection entrance vein 2(0.7)1(0.8)1(0.8) cs dissection conservative8(3.0)4(3.0)4(3.0)with consequences cardiac tamponade conservative1(0.4)1(0.8)pericardiocentesis1(0.4)1(0.8) cardiac arrest during implant 1(0.4)1(0.8) haemorrhage during implant 2(0.7)2(1.5)conservative1(0.4)1(0.8)transfer to ccu lv lead placement failure (transvenous) 7(2.6)2(1.5)5(3.7) pneumothorax conservative2(0.7)2(1.5)drainage3(1.1)1(0.8)2(1.5) pocket haematoma conservative8(3.0)4(3.0)4(3.0)re - hospitalisation / transfusion 4(1.5)4(3.0)0(0) pns conservative13(4.9)4(3.0)9(6.7) lead revision1(0.4)1(0.8) thrombosis v. subclavian post implant 1(0.4)1(0.4) death (implant related) 1(0.4)1(0.8) total 56(20.9)24(17.9)32(23.9) with consequences 21(7.8)9(6.7)12(9.0) ccu coronary care unit, cs coronary sinus, lv left ventricular, pns phrenic nerve stimulation . Leading to postponement of implant or conversion to another entrance vein . Due to temporary av block without an escape rhythm for which a short period of resuscitation followed by temporary pacing by the icd lead after which recovery of conduction periprocedural complications ccu coronary care unit, cs coronary sinus, lv left ventricular, pns phrenic nerve stimulation . Due to temporary av block without an escape rhythm for which a short period of resuscitation followed by temporary pacing by the icd lead after which recovery of conduction p <0.05 for comparison between de novo and upgrade procedures . Table 4hazard ratio for upgrade versus de novo crt implantation procedures and the risk of periprocedural complications, adjusted for differences in baseline characteristics between upgrade and de novo patients . Hazard ratio for upgrade versus de novo crt implantation procedures and the risk of periprocedural complications, adjusted for differences in baseline characteristics between upgrade and de novo patients . Ten (4.2%) patients had a long - term device - related complication with consequences; there was no difference between upgrade (3.5%) and de novo (4.9%) patients . Lead dysfunction in need of lead replacement occurred once in a de novo and an upgrade patient . Lead dislodgment in need of lead revision was seen in three de novo patients and in one upgrade patient . Device - related endocarditis requiring extraction of the system was seen once in both a de novo and in an upgrade patient.table 5long - term device - related complications during 1 year of follow - upcomplication (n,%) total n = 236 upgrade n = 113de novo n = 123 pns conservative11(4.7)6(5.3)5(4.1)lead revision1(0.4)1(0.9) lead dysfunction conservative6(2.5)3(2.7)3(2.4)lead replacement2(0.8)1(0.9)1(0.8) lead dislodgement conservative4(1.7)1(0.9)3(2.4)lead revision4(1.7)1(0.9)3(2.4) infection conservative2(0.8)-2(1.6)ab / hospitalisation1(0.4)-1(0.8)extraction2(0.8)1(0.9)1(0.8) total 33(14.0)14(12.4)19(15.4) with consequences 10(4.2)4(3.5)6(4.9)loss to fu514htx33death 27198n before death or htx long - term complication 3 2 1 ab antibiotics, htx heart transplantation, fu follow - up, pns phrenic nerve stimulation . Excluded were those patients who were loss to follow up (n=5) or died / received an htx during follow up before any long term complication could occur (n = 32). One upgrade patient died during procedure. P <0.05 for comparison between de novo and upgrade procedures . Long - term device - related complications during 1 year of follow - up ab antibiotics, htx heart transplantation, fu follow - up, pns phrenic nerve stimulation . Excluded were those patients who were loss to follow up (n=5) or died / received an htx during follow up before any long term complication could occur (n = 32). This analysis included 134 patients who underwent an upgrade procedure to a crt device and 134 randomly selected patients receiving a de novo crt device . Of these 268 patients, 264 (98.5%) received a crt defibrillator (crt - d), three (2.2%) patients were upgraded to a crt pacemaker (crt - p) and one (0.7%) patient received a de novo crt - p . Fifty - four (40%) patients where upgraded from a double chamber device and 81 (60%) from a single chamber device . Seventy - six (57%) upgrade patients were paced from the right ventricle at baseline . Upgrade patients were more often males (p = 0.011) and of older age (p = 0.001) than the random sample of patients receiving a de novo crt implantation . They more often had ischaemic cardiomyopathy (p = 0.049) and a history of atrial fibrillation (p = 0.008) and were more often on amiodarone (p <0.001) and a vitamin k antagonist (p = 0.022). No baseline characteristic had a significant confounding effect on the hazard ratio for procedure type and the risk of periprocedural complications.table 1baseline comparison between patients receiving an upgrade to a crt device and those receiving a de novo crt devicebaseline characteristicstotal population (n = 268)upgrade(n = 134)de novo implants (n = 134) p - value age (years) 69(6174)71(6375)67(6072) 0.001 male (n(%))202(75)110(82)92(69) 0.011 bmi26(2429)26(2429)26(2430)0.450 nyha class (n,%) 0.080 i2(1)1(1)1(1) ii36(13)13(10)23(17) iii213(80)110(82)103(77) iv17(6)10(8)7(5)ischaemic cmp (n,%) 148(55)82(61)66(49) 0.049 lvef (%) 23 (7)24 (7)23 (7)0.239bnp141(68262)146(73273)130(50226)0.112atrium fibrillation (n,%) 60(22)39(29)21(16) 0.008 cardiac medication (n,%) diuretic231(88)117(88)114(87)0.816beta - blocking201(76)100(76)101(76)0.940ace inhibiting227(86)110(83)117(89)0.158 other medication amiodarone54(21)39(29)15(11) <0.001 use of anticoagulation (n,%) vitamin - k antagonist191(72)104(78)87(65) 0.022 other57(21)23(17)34(25)0.101inr (n = 191) 1.7(1.32.1)1.7(1.42.1)1.5(1.31.9)0.053 comorbidity diabetes mellitus (n,%) 62(23)29(22)33(25)0.540copd (n,%) 34(13)17(13)17(13)0.981renal function (egfr)57(19)55(20)59(19)0.090 subclavian / anonyma vein thrombosis (n,%) 11(4)11(8)0(0) 0.001 time since initial cied implantation (months) 57(31115) standard deviation deviation, range; 25th to 75th percentile inter quartile range . Ace angiotensin i converting enzyme, bmi body mass index, bnp brain natriuretic peptide, cied cardiac implantable electronic device, cmp cardiomyopathy, copd chronic obstructive pulmonary disease, inr international normalised ratio, lvef left ventricular ejection fraction, nyha new york heart association, sd standard deviation . Nyha iii and iv versus i, ii baseline comparison between patients receiving an upgrade to a crt device and those receiving a de novo crt device standard deviation deviation, range; 25th to 75th percentile inter quartile range . Ace angiotensin i converting enzyme, bmi body mass index, bnp brain natriuretic peptide, cied cardiac implantable electronic device, cmp cardiomyopathy, copd chronic obstructive pulmonary disease, inr international normalised ratio, lvef left ventricular ejection fraction, nyha new york heart association, sd standard deviation . Seventy - three (27%) procedures were extended by study - related lv pressure (lv dp / dt max) measurements to optimise lead positions and device settings . Median radiation time was 25 (20) minutes and median hospitalisation after implantation, including rehospitalisation for another attempt to complete crt implantation after initial lv lead placement failure, was 2 (2) days . There were no significant differences in these procedural data between upgrade and de novo crt implantation procedures . At baseline, ten (8%) patients undergoing an upgrade procedure had a subclavian (or anonyma) vein thrombosis . In six of these patients the vein was recanalised, in two a contralateral implant was chosen and in two patients the occlusion could be passed . In 124 (92%) ten (8%) patients did not receive a right atrial lead because of permanent atrial fibrillation . Of the upgrade patients 91 (68%) received two new leads, three leads were placed in 16 (12%) patients and 27 (20%) patients only received an additional lv lead.table 2procedural dataprocedural data (median, iqr)totalupgradede novo p - valuelength procedure (min)156(78)145(71)163(81)0.062standard procedure (n = 194)139(69)135(75)142(61)0.515procedure with lv dp / dt max measurements 187(66)171(72)195(42)0.176radiation time (min)25(20)25(19)25(21)0.595total hospitalisation time (days)2(2)1(2)2(2)0.418 leads added or implanted (intention to) (n,%) <0.001127(20%) 0(0)291(68%) 10(8%) 316(12%) 124(92%) n of operating specialists 1.000 n = 1 (experienced cardiologist)224(85)112(85)112(85) n = 2 (fellow with experienced cardiologist or two experienced cardiologists)40(15)20(15)20(15) of which 39.7% upgrade procedures . Starting after implantation including additional hospitalisation for later lv lead placement after initial failure or re - hospitalisation due to implantation or device related complications . Of which 39.7% upgrade procedures . Starting after implantation including additional hospitalisation for later lv lead placement after initial failure or re - hospitalisation due to implantation or device related complications . The periprocedural complication rate is presented in table 3 . In total 21 (7.8%) patients experienced a complication with consequences . The crude hazard ratio for periprocedural complications (with consequences) for upgrade procedures compared with de novo crt implantations was 1.692 (p = 0.260) and when adjusted for baseline differences between the two groups it was 1.779 (p = 0.249) (table 4). One patient developed acute decreased saturation during an upgrade procedure probably based on cardiac asthma . Saturation did not improve with pharmacological treatment or intubation, and resuscitation was required, which the patient did not survive . In seven (2.6%) patients transvenous lv lead placement was unsuccessful due to difficulty in visualising or cannulating the coronary sinus . Phrenic nerve stimulation was seen in 14 (5.3%) patients and usually managed by adjustments in the pacing configuration, but in one case lead revision was needed shortly after implantation . Coronary sinus dissection (3%) pocket haematoma was seen in 12 (4.5%) of the total 268 patients; of these four upgrade patients needed to be re - hospitalised or needed blood transfusion . Table 3periprocedural complicationscomplication (n,%) total (n = 268)upgrade (n = 134)de novo (n = 134) perforation / dissection entrance vein 2(0.7)1(0.8)1(0.8) cs dissection conservative8(3.0)4(3.0)4(3.0)with consequences cardiac tamponade conservative1(0.4)1(0.8)pericardiocentesis1(0.4)1(0.8) cardiac arrest during implant 1(0.4)1(0.8) haemorrhage during implant 2(0.7)2(1.5)conservative1(0.4)1(0.8)transfer to ccu lv lead placement failure (transvenous) 7(2.6)2(1.5)5(3.7) pneumothorax conservative2(0.7)2(1.5)drainage3(1.1)1(0.8)2(1.5) pocket haematoma conservative8(3.0)4(3.0)4(3.0)re - hospitalisation / transfusion 4(1.5)4(3.0)0(0) pns conservative13(4.9)4(3.0)9(6.7) lead revision1(0.4)1(0.8) thrombosis v. subclavian post implant 1(0.4)1(0.4) death (implant related) 1(0.4)1(0.8) total 56(20.9)24(17.9)32(23.9) with consequences 21(7.8)9(6.7)12(9.0) ccu coronary care unit, cs coronary sinus, lv left ventricular, pns phrenic nerve stimulation . Leading to postponement of implant or conversion to another entrance vein . Due to temporary av block without an escape rhythm for which a short period of resuscitation followed by temporary pacing by the icd lead after which recovery of conduction . Periprocedural complications ccu coronary care unit, cs coronary sinus, lv left ventricular, pns phrenic nerve stimulation . Leading to postponement of implant or conversion to another entrance vein . Due to temporary av block without an escape rhythm for which a short period of resuscitation followed by temporary pacing by the icd lead after which recovery of conduction . . Table 4hazard ratio for upgrade versus de novo crt implantation procedures and the risk of periprocedural complications, adjusted for differences in baseline characteristics between upgrade and de novo patients . Hazard ratio for upgrade versus de novo crt implantation procedures and the risk of periprocedural complications, adjusted for differences in baseline characteristics between upgrade and de novo patients . Ten (4.2%) patients had a long - term device - related complication with consequences; there was no difference between upgrade (3.5%) and de novo (4.9%) patients . Lead dysfunction in need of lead replacement occurred once in a de novo and an upgrade patient . Lead dislodgment in need of lead revision was seen in three de novo patients and in one upgrade patient . Device - related endocarditis requiring extraction of the system was seen once in both a de novo and in an upgrade patient.table 5long - term device - related complications during 1 year of follow - upcomplication (n,%) total n = 236 upgrade n = 113de novo n = 123 pns conservative11(4.7)6(5.3)5(4.1)lead revision1(0.4)1(0.9) lead dysfunction conservative6(2.5)3(2.7)3(2.4)lead replacement2(0.8)1(0.9)1(0.8) lead dislodgement conservative4(1.7)1(0.9)3(2.4)lead revision4(1.7)1(0.9)3(2.4) infection conservative2(0.8)-2(1.6)ab / hospitalisation1(0.4)-1(0.8)extraction2(0.8)1(0.9)1(0.8) total 33(14.0)14(12.4)19(15.4) with consequences 10(4.2)4(3.5)6(4.9)loss to fu514htx33death 27198n before death or htx long - term complication 3 2 1 ab antibiotics, htx heart transplantation, fu follow - up, pns phrenic nerve stimulation . Excluded were those patients who were loss to follow up (n=5) or died / received an htx during follow up before any long term complication could occur (n = 32). One upgrade patient died during procedure. P <0.05 for comparison between de novo and upgrade procedures . Long - term device - related complications during 1 year of follow - up ab antibiotics, htx heart transplantation, fu follow - up, pns phrenic nerve stimulation . Excluded were those patients who were loss to follow up (n=5) or died / received an htx during follow up before any long term complication could occur (n = 32). Our single - centre experience did not find any significant differences in either procedure length, radiation duration, total hospitalisation, or in periprocedural and long - term device - related complication rates when comparing upgrade procedures with de novo crt implantations . Previously, the raft study showed that crt - d reduced rates of death and hospitalisation for heart failure when compared with icd therapy only . However, device - related hospitalisation was significantly higher for the crt - d group compared with the icd - only group . The increased risk of complications of a triple chamber device compared with conventional single and dual chamber pacemakers or icds was also highlighted by a large nationwide cohort of 5918 danish patients who underwent a cied implantation, in which patients receiving a crt device showed the highest complication rate . Therefore, the decision to implant a triple chamber device needs to be carefully considered . For patients with a class iib indication for crt but a clear indication for a conventional icd the alternative to a de novo crt implantation is to await the potential need for an upgrade to crt . However, as was shown by poole et al ., crt upgrade is a high - risk procedure as well, with an 18.7% risk of any complication during a follow - up of six months . The fear of complications associated with an upgrade procedure might prevent the upgrade to crt in patients with a clear indication . This was suggested by the results of scott et al . Who performed a single - centre retrospective observational study evaluating the implantation of icds for a period of 5 years . In this period 549 new icds were implanted of which 73% single or dual chamber icds and 27% crt - d . When they applied alternative indication criteria (lvef 30%, qrs 130 ms, nyha i iv), 42.6% of the icd recipients met the criteria for crt at initial implant . However, the upgrade rate to crt at 5 years was only 5.1% . This is in line with the results of the european survey which showed that there were no significant differences in complication rates when comparing 692 upgrades with 1675 de novo crt procedures at 141 centres in europe . However, this was a randomised trial and such trials commonly report less complications than in a real - life setting due to more strict patient selection and more experienced operators . On the contrary, the previously mentioned danish population - based cohort of cied implantations, including 445 crt - d implantations, showed that crt upgrade procedures or lead revisions were associated with more complications than crt de novo implantations . As both our study and the danish cohort study reflect the results of a real - life setting the differences are not likely to be based on patient selection . In our retrospective study, patient selection was equal for both de novo and upgrade procedures in that it followed standard clinical care in which the benefit of crt is balanced against the risk of complications . It might be that the operators in our tertiary centre were more experienced in implanting crt devices . Although most procedures in the danish cohort were also performed by operators with an annual volume of at least 75 icd or pacemaker implantations, it is not clear whether the experience in implanting a crt device was as high as in our centre . Another point of concern for many clinicians is the fear of a more complex procedure when a single or dual chamber pacemaker or icd needs to be upgraded, compared with a de novo implantation of a crt device, due to the possibility of damaging or extracting old leads . Four upgrade patients experienced lead dysfunction after the procedure, which is comparable with numbers in previous literature [5, 10]. These were three old leads (two right atrial leads and one right ventricular lead) and one newly placed right atrial lead . Moreover, this was done due to damage to the old lead during the procedure in only one of 16 upgrade patients who received a completely new system . Therefore, our data seem to indicate that when an upgrade procedure is performed by an experienced operator the risk of damaging old leads is small . Furthermore, we found no significant difference in procedure duration comparing upgrade procedures with de novo implantations, but there was a slight trend towards an even shorter procedure length for upgrade procedures . This suggests comparable complexity as one would expect the upgrade procedure to require a smaller amount of time, as in most upgrades less leads are added than implanted during a de novo implantation . Complicating factors such as subclavian vein thrombosis only slightly increased the median procedure duration compared with standard procedures (159 (95) minutes versus 139 (69) minutes). Moreover, in our upgrade group the procedure success was 98.5% and did not significantly differ for the randomly selected de novo crt implantation procedures . A meta - analysis of 9082 patients in 25 crt trials showed an implantation success rate of 94.4% indicating that the success rate of upgrade procedures was most definitely not lower compared with de novo procedures . Decisions on device type (conventional or triple chamber pacemaker or icd) can be difficult in patients with an indication for a conventional pacemaker or icd without a clear class i or iia indication for crt . Based on fulfilment of indication criteria for crt the chance of benefit from crt in these patients is low and the complication rate increases with the number of leads implanted . However, when a conventional pacemaker or icd is opted for the need for upgrading can eventually arise . This means the patient is exposed to two procedures, where it is generally believed that an upgrade procedure is more complex and associated with a higher complication rate . This single - centre experience of a dutch university hospital shows neither a higher complexity nor a higher complication rate of upgrade procedures to crt compared with de novo crt implantations . Therefore, crt upgrade procedures should be performed when indicated without fear for a higher complication rate . However, as upgrading means an additional procedure is needed, future research should focus on identifying patient characteristics that predict the necessity for upgrading to crt, especially for those patients in need of upgrading within the life expectancy of the initial device . Due to the retrospective design of the study only complications properly documented in the electronic patient file were identified and not all baseline data were available for all patients . However, the advantage of the retrospective design is that it reflects a real - life setting of these device implantations . Furthermore, although none of the baseline characteristics showed a confounding effect on the risk of periprocedural complications, it is possible that some data that were not collected confounded the results, for instance steroid use . Finally, as this study shows the results of a single - centre experience, the results can only be applicable for a comparable centre . In a device implantation centre with experienced operators, crt upgrade procedures are not associated with more complications nor do they seem more complex compared with de novo crt implantations.
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Fried seeds subjected to grinding by adding sufficient water to it for 30 - 45 min . The mixture was filtered through muslin cloth and pea nut milk is separated . Solvent {ethanol: acetone (1:1)} was added to the milk and then subjected to refrigeration for 30 min . Indomethacin was dissolved in chloroform and bio - polymer solution was prepared by adding water to it . The drug solution was added to bio - material solution and mixed by magnetic stirrer until emulsion is formed . Release of drug through egg shell membrane: egg shell membrane was separated using con . The egg shell membrane attached to the tube . Measured quantity of micro particles 5 ml solution was withdrawal and 5 ml buffer solution was added to make the volume . Same procedure was repeated for different formulated films of aceclofenac with phosphate buffer (ph-7.4). The dissolution studies were perform and subjecting it for dissolution for 3 hrs and analyze the samples by uv spectrophotometer at 320 nm .% cummulative release vs time in ph 1.2% cummulative release vs time in ph 7.4 fried seeds subjected to grinding by adding sufficient water to it for 30 - 45 min . The mixture was filtered through muslin cloth and pea nut milk is separated . Solvent {ethanol: acetone (1:1)} was added to the milk and then subjected to refrigeration for 30 min . Indomethacin was dissolved in chloroform and bio - polymer solution was prepared by adding water to it . The drug solution was added to bio - material solution and mixed by magnetic stirrer until emulsion is formed . Release of drug through egg shell membrane: egg shell membrane was separated using con . The egg shell membrane attached to the tube . Measured quantity of micro particles 5 ml solution was withdrawal and 5 ml buffer solution was added to make the volume . Same procedure was repeated for different formulated films of aceclofenac with phosphate buffer (ph-7.4). The dissolution studies were perform and subjecting it for dissolution for 3 hrs and analyze the samples by uv spectrophotometer at 320 nm .% cummulative release vs time in ph 1.2% cummulative release vs time in ph 7.4 five different formulations were formulated using various proportion of bio loaded sustained release bio - micro particles was performed . It had t50% and t 80% of 1.56 hrs and 2.45 hr formulation fg-5 shows percentage yield t50% and t80% of 1.62 hrs and 2.61 hrs at hence these two formulations are the best among the five prepared formulations.
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Although modern treatment approaches lead to marked improvements in outcome of patients with chronic heart failure (chf) during the last decades, this group of patients is still affected by high mortality rates.1 consequently, strategies to identify patients at very high risk for cardiovascular events may help to intensify treatment strategies tailored to the need of the individual patient . In addition, exploration of novel markers predictive of mortality and cardiovascular events helps to improve the knowledge of underlying pathophysiologic mechanisms . Oxidative stress, resulting from an imbalance between reactive oxygen species (ros) production and antioxidative defenses, is known to be a key mediator in the pathogenesis of chf.2, 3 potential sources of increased ros generation in the failing heart are the mitochondrial electron transport chain (etc), nadph oxidases, nitric oxide synthases, and xanthine oxidase.4, 5, 6 excessive release of ros leads to left ventricular remodeling and dysfunction by cellular damage through a number of pathways including direct damage to proteins, membranes, dna, and rna or indirect damage through the activation of proinflammatory and proapoptotic pathways.7 besides promoting cellular cholesterol efflux and reverse cholesterol transport, highdensity lipoprotein (hdl) exerts versatile protective functions on the cardiovascular system including antioxidative activity by binding and removing oxidative molecules.8 thus, hdl is believed to play an important role in diminishing elevated oxidative stress levels . However, it has become evident that protective properties of hdl become functionally impaired under certain conditions such as inflammation and tissue injury, and oxidatively modified hdl may even promote prooxidative processes.9, 10, 11, 12, 13, 14, 15 a previously conducted casecontrol study published by patel et al showed an inverse correlation between the prevalence of chf and the ability of hdl to promote reverse cholesterol transport or to prevent lipid oxidation.16 in addition, paraoxonase1 (pon1) activity, which represents an hdlbound key mediator to prevent oxidative modifications, has previously been linked to outcome in chf.17 in the light of these findings, and considering the crucial role of oxidative processes implicated in the pathophysiology of chf, it is tempting to speculate that hdl functionality not only is significantly impaired but may also be associated with outcome in this highly vulnerable population . Based on the aforementioned background, we aimed to determine the prognostic value of antioxidant capacity of hdl in patients with chf . Patients enrolled in this prospective observational study were recruited at the outpatient department for heart failure at the medical university of vienna between january 2008 and july 2013 . Inclusion criteria were defined as new york heart association functional classification (nyha) 2 and the presence of either an nterminal probtype natriuretic peptide level 500 pg / ml or an echocardiographic left ventricular ejection fraction (lvef) <40% at time of enrollment . Exclusion criteria were defined as age <18 years, presence of a severe lifethreatening condition other than chf (eg, malignancies), chronic inflammatory diseases, and refusal of informed consent . Treatment and diagnosis were in accordance with current heart failure guidelines.18 baseline demographics and clinical history were assessed by trained researchers using a standardized questionnaire . The study complies with the declaration of helsinki and was approved by the ethics committee of the medical university of vienna . A combination of death due to cardiovascular events and heart transplantation (htx) was chosen as primary study endpoint . Mortality was assessed browsing local community registries (statistik austria), and death certificates were reviewed and categorized into cardiovascular and noncardiovascular causes of death . Hdl antioxidative capacity, the ability of hdl to inactivate previously oxidized ldl, was measured using a wellvalidated cellfree assay comparable to the cellbased assays as previously described.13, 14, 16, 19, 20, 21, 22 the assay is based on the conversion of 2,7dichlorodihydrofluorescein diacetate (dcf) into its fluorescent form in the presence of oxidized ldl . In accordance with prior study we used apolipoprotein (apo)bdepleted patient serum, which includes hdl, apoa1, apoa2, and hdlassociated particles and determined the ability to attenuate or aggravate previously oxidized lowdensity lipoprotein cholesterol (ldl).20 briefly, purified ldl (merck, millipore, darmstadt, germany) was diluted in pbs to a final cholesterol concentration of 100 g / ml prior to a 6hour oxidation in 100 mol / l cuso4 (merck) at 37c . Oxidized ldl (final concentration 1.4 g / ml) and hdlcontaining supernatant (15 l) and dcfda (final concentration 2.9 g / ml) were added to separate wells of 96well black microplates with clear bottoms (corning, amsterdam, the netherlands) and incubated at 37c with pbs to a final volume of 175 l for 1 hour . The fluorescence signal was measured at an excitation of 485 nm and emission wavelength of 530 nm using a synergy h1 hybrid microplate reader (biotek, winooski, vt). The highdensity oxidative index (hoi) was determined by calculating the ratio of fluorescence in the presence of apobdepleted patient samples divided by the fluorescence of oxidized ldl alone and logtransformed before analysis . A hoi <1.0 stands for antioxidative hdl function, whereas a hoi 1.0 indicates prooxidative hdl.12, 13, 21, 22 this definition is based on the functional properties of hdl and describes whether the respective apobdepleted patient sample attenuates or aggravates experimental oxidative stress . To correct for interassay variability across different plates, fluorescence value was normalized by the fluorescence of a pooled serum control from 3 healthy volunteers added on each plate . Interassay coefficient of variation (cv) was 7.8%, whereas intraassay cv was 5.9% . Pon1 activity was determined using a highly sensitive fluorometric assay (enzchek paraoxonase assay kit, invitrogen, carlsbad, ca). Ntprobnp was analyzed on an elecsys 2010 ntprobnp elisa (roche diagnostics, mannheim, germany). Plasma concentrations of interleukin6 (il6) were measured using a human quantikine highsensitivity elisa (r&d systems, minneapolis, mn). Categorical data are presented as counts and percentages, continuous data as median and interquartile range (iqr). Receiver operating characteristic (roc) curves and the fisher exact test were applied to estimate the optimal risk cutoff of the hoi . Cox proportional hazard regression models were used to estimate the effect of variables on outcome . Results were reported as the hazard ratio (hr) for categorical variables and as hr per 1 increase of a logtransformed standard deviation (hr per 1 sd) for continuous variables . In the multivariable model hoi was adjusted for established chf risk predictors and potential confounders associated with the hoi, including age, sex, nyha class, ntprobnp, lvef (continuous), estimated glomerular filtration rate, body mass index, hdlcholesterol, crp, il6, pon1 activity, diabetes mellitus, and atrial fibrillation . To account for competing risk of endpoints, data were analyzed for causespecific and subdistribution hazard models as recommended by austin et al.23 in addition, cumulative incidence functions for the cardiovascular endpoint and noncardiac death are shown in figure s1 . Kaplanmeier curves were constructed to estimate event rates according to an hoi above or below 1 . The harrell cstatistic was applied to evaluate the discriminatory power to predict cardiovascular events, and models were compared with the likelihoodratio test . An improvement in individual risk stratification was analyzed using net reclassification improvement (nri) and integrated discrimination increment (idi).24 risk categories for the categorical nri were set as 0% to 30% (low), 30% to 60% (middle), and> 60% (high).25 a pvalue of <0.05 (2tailed) was considered statistically significant . All statistical analyses were performed using spss 20.0 (ibm spss, armonk, ny), stata 12 (statacorp, college station, tx), and r (r development core team, vienna, austria) including the predictable package.26 patients enrolled in this prospective observational study were recruited at the outpatient department for heart failure at the medical university of vienna between january 2008 and july 2013 . Inclusion criteria were defined as new york heart association functional classification (nyha) 2 and the presence of either an nterminal probtype natriuretic peptide level 500 pg / ml or an echocardiographic left ventricular ejection fraction (lvef) <40% at time of enrollment . Exclusion criteria were defined as age <18 years, presence of a severe lifethreatening condition other than chf (eg, malignancies), chronic inflammatory diseases, and refusal of informed consent . Treatment and diagnosis were in accordance with current heart failure guidelines.18 baseline demographics and clinical history were assessed by trained researchers using a standardized questionnaire . The study complies with the declaration of helsinki and was approved by the ethics committee of the medical university of vienna . A combination of death due to cardiovascular events and heart transplantation (htx) was chosen as primary study endpoint . Mortality was assessed browsing local community registries (statistik austria), and death certificates were reviewed and categorized into cardiovascular and noncardiovascular causes of death . Hdl antioxidative capacity, the ability of hdl to inactivate previously oxidized ldl, was measured using a wellvalidated cellfree assay comparable to the cellbased assays as previously described.13, 14, 16, 19, 20, 21, 22 the assay is based on the conversion of 2,7dichlorodihydrofluorescein diacetate (dcf) into its fluorescent form in the presence of oxidized ldl . In accordance with prior study we used apolipoprotein (apo)bdepleted patient serum, which includes hdl, apoa1, apoa2, and hdlassociated particles and determined the ability to attenuate or aggravate previously oxidized lowdensity lipoprotein cholesterol (ldl).20 briefly, purified ldl (merck, millipore, darmstadt, germany) was diluted in pbs to a final cholesterol concentration of 100 g / ml prior to a 6hour oxidation in 100 mol / l cuso4 (merck) at 37c . Oxidized ldl (final concentration 1.4 g / ml) and hdlcontaining supernatant (15 l) and dcfda (final concentration 2.9 g / ml) were added to separate wells of 96well black microplates with clear bottoms (corning, amsterdam, the netherlands) and incubated at 37c with pbs to a final volume of 175 l for 1 hour . The fluorescence signal was measured at an excitation of 485 nm and emission wavelength of 530 nm using a synergy h1 hybrid microplate reader (biotek, winooski, vt). All patient samples were run in duplicate, and mean fluorescence was recorded . The highdensity oxidative index (hoi) was determined by calculating the ratio of fluorescence in the presence of apobdepleted patient samples divided by the fluorescence of oxidized ldl alone and logtransformed before analysis . A hoi <1.0 stands for antioxidative hdl function, whereas a hoi 1.0 indicates prooxidative hdl.12, 13, 21, 22 this definition is based on the functional properties of hdl and describes whether the respective apobdepleted patient sample attenuates or aggravates experimental oxidative stress . To correct for interassay variability across different plates, fluorescence value was normalized by the fluorescence of a pooled serum control from 3 healthy volunteers added on each plate . Interassay coefficient of variation (cv) was 7.8%, whereas intraassay cv was 5.9% . Pon1 activity was determined using a highly sensitive fluorometric assay (enzchek paraoxonase assay kit, invitrogen, carlsbad, ca). Ntprobnp was analyzed on an elecsys 2010 ntprobnp elisa (roche diagnostics, mannheim, germany). Plasma concentrations of interleukin6 (il6) were measured using a human quantikine highsensitivity elisa (r&d systems, minneapolis, mn). Categorical data are presented as counts and percentages, continuous data as median and interquartile range (iqr). Receiver operating characteristic (roc) curves and the fisher exact test were applied to estimate the optimal risk cutoff of the hoi . Cox proportional hazard regression models were used to estimate the effect of variables on outcome . Results were reported as the hazard ratio (hr) for categorical variables and as hr per 1 increase of a logtransformed standard deviation (hr per 1 sd) for continuous variables . In the multivariable model hoi was adjusted for established chf risk predictors and potential confounders associated with the hoi, including age, sex, nyha class, ntprobnp, lvef (continuous), estimated glomerular filtration rate, body mass index, hdlcholesterol, crp, il6, pon1 activity, diabetes mellitus, and atrial fibrillation . To account for competing risk of endpoints, data were analyzed for causespecific and subdistribution hazard models as recommended by austin et al.23 in addition, cumulative incidence functions for the cardiovascular endpoint and noncardiac death are shown in figure s1 . Kaplanmeier curves were constructed to estimate event rates according to an hoi above or below 1 . The harrell cstatistic was applied to evaluate the discriminatory power to predict cardiovascular events, and models were compared with the likelihoodratio test . An improvement in individual risk stratification was analyzed using net reclassification improvement (nri) and integrated discrimination increment (idi).24 risk categories for the categorical nri were set as 0% to 30% (low), 30% to 60% (middle), and> 60% (high).25 a pvalue of <0.05 (2tailed) was considered statistically significant . All statistical analyses were performed using spss 20.0 (ibm spss, armonk, ny), stata 12 (statacorp, college station, tx), and r (r development core team, vienna, austria) including the predictable package.26 the final study cohort comprised 320 patients with a diagnosis of chf consisting of 147 (46%) patients with ischemic etiology and 173 (54%) patients with nonischemic etiology of chf . Detailed baseline characteristics according to patients with an hoi above or below 1 are shown in table 1 . In brief, patients had a median age of 65 years (iqr 5771 years), 260 (81%) were male, 145 (45.3%) were in nyha class iii / iv, and patients had a median ntprobnp level of 1214 (iqr 4772654) pg / ml . Baseline characteristics baseline characteristics according to patients stratified by a highdensity lipoprotein oxidative index below or above 1 . Comparisons between groups were performed using chisquared test and mannwhitney u test as appropriate . Continuous data are presented as median and interquartile range, categorical data as counts and percentages . Ace indicates angiotensinconverting enzyme; adp, adenosine diphosphate receptor; arb, angiotensin receptor blockers; bpm, beats per minute; chf, chronic heart failure; copd, chronic obstructive pulmonary disease; crp, creactive protein; egfr, estimated glomerular filtration rate; hdl, highdensity lipoprotein; hoi, highdensity lipoprotein oxidative index; ldl, lowdensity lipoprotein; lvef, left ventricular ejection fraction; mi, myocardial infarction; ntprobnp, nterminal probtype natriuretic peptide; nyha, new york heart association functional classification; sbp, systolic blood pressure . With respect to antioxidative capacity of hdl, we found that 223 (69.7%) patients had a preserved antioxidative function with an hoi <1, and 97 (30.3%) patients presented prooxidative hdl serum measurements with an hoi 1 . Patients with an hoi 1 had significantly higher levels of creactive protein (crp; 0.47 [iqr 0.130.62 mg / l], p=0.003), hba1c (6.2% [iqr 5.7% to 7.1%] vs 6.0% [5.6% to 6.7%], p=0.035), ntprobnp (1393 pg / ml [iqr 6633392 pg / ml] vs 1095 pg / ml [iqr 4032229 pg / ml], p=0.025) and were more often in nyha class iii / iv (55% vs 41%, p=0.025). Furthermore, patients with an hoi 1 showed a significantly lower pon1 activity (0.029 u/l [0.0250.036 u/l] vs 0.033 u/l [0.0280.04 u/l], p<0.001). With respect to inflammatory markers, hoi showed weak significant correlations with il6 (r=0.16, p=0.01) and crp (r=0.248, p<0.001). No differences in hoi (p=0.73) or pon1 activity (p=0.409) were found between patients with ischemic and nonischemic etiology of chf . There was no difference in serum hdlcholesterol levels between patients with an hoi 1 or an hoi <1 . However, pon1 activity showed a weak but significant correlation with hdlcholesterol levels (r=0.177, p=0.001). The median followup period was 2.8 years (95% ci 1.84.9 years), corresponding to a total of 1096 patient years . During this observation time, 88 (27.5%) patients reached the combined cardiovascular endpoint, and 116 (36.3%) patients died from all causes . The optimal risk cutoff of the hoi using roc analysis and fisher exact test was 0.995 with a sensitivity of 42.5% and a specificity of 73.9% . An hoi 1 had a significant association with survival free of cardiovascular mortality and htx of patients in unadjusted cox regression analysis with a hr of 2.28 (95% ci 1.483.51, p<0.001; table 2). Corresponding kaplanmeier curves stratified by an hoi 1 or <1 are shown in figure 1 . The association of hoi 1 with cardiovascular events remained significant after multivariable adjustment for age, sex, nyha class, ntprobnp, lvef, estimated glomerular filtration rate, body mass index, hdlcholesterol, crp, il6, pon1 activity, diabetes mellitus, and atrial fibrillation with an adjusted hr of 1.83 (95% ci 1.142.92, p=0.012; table 2). Analysis for subdistribution and causespecific hazard models showed a significant association of an hoi 1 with the cardiovascular endpoint but not with noncardiac death in multivariable analysis (table s1). No difference in the predictive value of an hoi 1 was found between ischemic and nonischemic etiology of chf (p for interaction 0.60). However, we observed a significant interaction between il6 and an hoi 1 versus <1 (p=0.044). Stratification into tertiles of il6 revealed that the hoi 1 versus <1 had increasing hrs with higher levels of il6 and a significant association with the cardiovascular endpoint in the highest il6 tertile (2.09 pg / ml) with a crude hr of 2.91 (95% ci 1.625.24, p<0.001; table s2). Pon1 activity showed a significant inverse association with the cardiovascular endpoint with a hr per 1 sd of 0.72 (95% ci 0.570.9, p=0.004; table 2) in an unadjusted cox regression, but this association turned nonsignificant after multivariable adjustment with an adjusted hr per 1 sd of 0.95 (95% ci 0.371.21, p=0.68; table 2). Unadjusted and multivariable cox proportional hazard models were used to estimate the effect of highdensity lipoprotein oxidative index on survival of patients . Ci indicates confidence interval; hdl, highdensity lipoprotein; hr, hazard ratio; htx, heart transplantation . Adjusted for age, sex, new york heart association functional classification, nterminal probtype natriuretic peptide, left ventricular ejection fraction, estimated glomerular filtration rate, body mass index, highdensity lipoprotein, creactive protein (crp), interleukin6 (il6), paraoxonase activity, type 2 diabetes mellitus, and atrial fibrillation . Results for paraoxonase activity are represented as hazard ratio per increase of 1 standard deviation . Kaplanmeier plots showing the crude cumulative survival free from allcause mortality (a and b) and cardiovascular mortality (c and d) according to the highdensity lipoprotein (hdl) oxidative index above or below 1 . In a next step we aimed to determine whether measurement of the hoi has advantages over conventional riskprediction strategies . First, we stratified patients into 4 groups according to combined strata of hoi above and below 1 and ntprobnp above and below 2065 pg / ml, which was the optimal cutoff to predict cardiovascular events calculated with cart analysis . Patients with an hoi 1 and ntprobnp> 2065 pg / ml had a hr of 6.8 (95% ci: 2.916.1, p<0.001) for the combined cardiovascular endpoint compared to patients with an hoi <1 and ntprobnp <2065 pg / ml . The predictive value of hoi 1 was independent of the ntprobnp category (p for interaction 0.282, figure 2). Second, the additional prognostic value beyond that assessable with ntprobnp was confirmed by significant improvements in cstatistic (auc 0.730 [95% ci 6750.786] vs 0.742 [95% ci 0.6860.798], p=0.001), categoryfree nri (32.4% [95% ci 19.9% to 44.9%]), categorical nri (12.8% [95% ci 3.3% to 22.4%], p=0.009) and idi (1.4% [95% ci 0.7% to 2.1%], p=0.04; table 3 and table s3). Third, an hoi 1 provided additional prognostic information even over a comprehensive multivariable model comprising all parameters used in multivariable cox regression analysis (see above) indicated by significant improvements in cstatistic (auc 0.763 [95% ci 0.7130.813] vs 0.768 [95% ci 0.7170.818], p=0.01), and categoryfree nri of 30.4% (95% ci 17.8% to 43.1%; p=0.01; table 3 and table s4). Relative risk of mortality (a) and the combined cardiovascular endpoint (b) according to combined strata of the highdensity lipoprotein oxidative index (hoi) and nterminal probtype (ntprobnp) natriuretic peptide . Metrics of discrimination and reclassification an improvement in individual risk stratification beyond that assessable with nterminal probtype natriuretic peptide and the multivariable model was calculated using harrel cstatistic, categoryfree and categorical net reclassification index, and integrated discrimination improvement . Auc indicates area under the curve; ci, confidence interval; hoi, highdensity lipoprotein oxidative index; nri, net reclassification index; ntprobnp, nterminal probtype natriuretic peptide . The multivariable model includes age, sex, new york heart association functional classification, nterminal probtype natriuretic peptide, left ventricular ejection fraction, estimated glomerular filtration rate, body mass index, highdensity lipoprotein, creactive protein, paraoxonase activity, diabetes mellitus, and atrial fibrillation . Full reclassification tables according to risk categories 0% to 30% (low), 30% to 60% (middle), and> the final study cohort comprised 320 patients with a diagnosis of chf consisting of 147 (46%) patients with ischemic etiology and 173 (54%) patients with nonischemic etiology of chf . Detailed baseline characteristics according to patients with an hoi above or below 1 are shown in table 1 . In brief, patients had a median age of 65 years (iqr 5771 years), 260 (81%) were male, 145 (45.3%) were in nyha class iii / iv, and patients had a median ntprobnp level of 1214 (iqr 4772654) pg / ml . Baseline characteristics baseline characteristics according to patients stratified by a highdensity lipoprotein oxidative index below or above 1 . Comparisons between groups were performed using chisquared test and mannwhitney u test as appropriate . Continuous data are presented as median and interquartile range, categorical data as counts and percentages . Ace indicates angiotensinconverting enzyme; adp, adenosine diphosphate receptor; arb, angiotensin receptor blockers; bpm, beats per minute; chf, chronic heart failure; copd, chronic obstructive pulmonary disease; crp, creactive protein; egfr, estimated glomerular filtration rate; hdl, highdensity lipoprotein; hoi, highdensity lipoprotein oxidative index; ldl, lowdensity lipoprotein; lvef, left ventricular ejection fraction; mi, myocardial infarction; ntprobnp, nterminal probtype natriuretic peptide; nyha, new york heart association functional classification; sbp, systolic blood pressure . With respect to antioxidative capacity of hdl, we found that 223 (69.7%) patients had a preserved antioxidative function with an hoi <1, and 97 (30.3%) patients presented prooxidative hdl serum measurements with an hoi 1 . Patients with an hoi 1 had significantly higher levels of creactive protein (crp; 0.47 [iqr 0.130.62 mg / l], p=0.003), hba1c (6.2% [iqr 5.7% to 7.1%] vs 6.0% [5.6% to 6.7%], p=0.035), ntprobnp (1393 pg / ml [iqr 6633392 pg / ml] vs 1095 pg / ml [iqr 4032229 pg / ml], p=0.025) and were more often in nyha class iii / iv (55% vs 41%, p=0.025). Furthermore, patients with an hoi 1 showed a significantly lower pon1 activity (0.029 u/l [0.0250.036 u/l] vs 0.033 u/l [0.0280.04 u/l], p<0.001). With respect to inflammatory markers, hoi showed weak significant correlations with il6 (r=0.16, p=0.01) and crp (r=0.248, p<0.001). No differences in hoi (p=0.73) or pon1 activity (p=0.409) were found between patients with ischemic and nonischemic etiology of chf . There was no difference in serum hdlcholesterol levels between patients with an hoi 1 or an hoi <1 . However, pon1 activity showed a weak but significant correlation with hdlcholesterol levels (r=0.177, p=0.001). The median followup period was 2.8 years (95% ci 1.84.9 years), corresponding to a total of 1096 patient years . During this observation time, 88 (27.5%) patients reached the combined cardiovascular endpoint, and 116 (36.3%) patients died from all causes . The optimal risk cutoff of the hoi using roc analysis and fisher exact test was 0.995 with a sensitivity of 42.5% and a specificity of 73.9% . An hoi 1 had a significant association with survival free of cardiovascular mortality and htx of patients in unadjusted cox regression analysis with a hr of 2.28 (95% ci 1.483.51, p<0.001; table 2). Corresponding kaplanmeier curves stratified by an hoi 1 or <1 are shown in figure 1 . The association of hoi 1 with cardiovascular events remained significant after multivariable adjustment for age, sex, nyha class, ntprobnp, lvef, estimated glomerular filtration rate, body mass index, hdlcholesterol, crp, il6, pon1 activity, diabetes mellitus, and atrial fibrillation with an adjusted hr of 1.83 (95% ci 1.142.92, p=0.012; table 2). Analysis for subdistribution and causespecific hazard models showed a significant association of an hoi 1 with the cardiovascular endpoint but not with noncardiac death in multivariable analysis (table s1). No difference in the predictive value of an hoi 1 was found between ischemic and nonischemic etiology of chf (p for interaction 0.60). However, we observed a significant interaction between il6 and an hoi 1 versus <1 (p=0.044). Stratification into tertiles of il6 revealed that the hoi 1 versus <1 had increasing hrs with higher levels of il6 and a significant association with the cardiovascular endpoint in the highest il6 tertile (2.09 pg / ml) with a crude hr of 2.91 (95% ci 1.625.24, p<0.001; table s2). Pon1 activity showed a significant inverse association with the cardiovascular endpoint with a hr per 1 sd of 0.72 (95% ci 0.570.9, p=0.004; table 2) in an unadjusted cox regression, but this association turned nonsignificant after multivariable adjustment with an adjusted hr per 1 sd of 0.95 (95% ci 0.371.21, p=0.68; table 2). Unadjusted and multivariable cox proportional hazard models were used to estimate the effect of highdensity lipoprotein oxidative index on survival of patients . Ci indicates confidence interval; hdl, highdensity lipoprotein; hr, hazard ratio; htx, heart transplantation . Adjusted for age, sex, new york heart association functional classification, nterminal probtype natriuretic peptide, left ventricular ejection fraction, estimated glomerular filtration rate, body mass index, highdensity lipoprotein, creactive protein (crp), interleukin6 (il6), paraoxonase activity, type 2 diabetes mellitus, and atrial fibrillation . Results for paraoxonase activity are represented as hazard ratio per increase of 1 standard deviation . Kaplanmeier plots showing the crude cumulative survival free from allcause mortality (a and b) and cardiovascular mortality (c and d) according to the highdensity lipoprotein (hdl) oxidative index above or below 1 . In a next step we aimed to determine whether measurement of the hoi has advantages over conventional riskprediction strategies . First, we stratified patients into 4 groups according to combined strata of hoi above and below 1 and ntprobnp above and below 2065 pg / ml, which was the optimal cutoff to predict cardiovascular events calculated with cart analysis . Patients with an hoi 1 and ntprobnp> 2065 pg / ml had a hr of 6.8 (95% ci: 2.916.1, p<0.001) for the combined cardiovascular endpoint compared to patients with an hoi <1 and ntprobnp <2065 pg / ml . The predictive value of hoi 1 was independent of the ntprobnp category (p for interaction 0.282, figure 2). Second, the additional prognostic value beyond that assessable with ntprobnp was confirmed by significant improvements in cstatistic (auc 0.730 [95% ci 6750.786] vs 0.742 [95% ci 0.6860.798], p=0.001), categoryfree nri (32.4% [95% ci 19.9% to 44.9%]), categorical nri (12.8% [95% ci 3.3% to 22.4%], p=0.009) and idi (1.4% [95% ci 0.7% to 2.1%], p=0.04; table 3 and table s3). Third, an hoi 1 provided additional prognostic information even over a comprehensive multivariable model comprising all parameters used in multivariable cox regression analysis (see above) indicated by significant improvements in cstatistic (auc 0.763 [95% ci 0.7130.813] vs 0.768 [95% ci 0.7170.818], p=0.01), and categoryfree nri of 30.4% (95% ci 17.8% to 43.1%; p=0.01; table 3 and table s4). Relative risk of mortality (a) and the combined cardiovascular endpoint (b) according to combined strata of the highdensity lipoprotein oxidative index (hoi) and nterminal probtype (ntprobnp) natriuretic peptide . Metrics of discrimination and reclassification an improvement in individual risk stratification beyond that assessable with nterminal probtype natriuretic peptide and the multivariable model was calculated using harrel cstatistic, categoryfree and categorical net reclassification index, and integrated discrimination improvement . Auc indicates area under the curve; ci, confidence interval; hoi, highdensity lipoprotein oxidative index; nri, net reclassification index; ntprobnp, nterminal probtype natriuretic peptide . The multivariable model includes age, sex, new york heart association functional classification, nterminal probtype natriuretic peptide, left ventricular ejection fraction, estimated glomerular filtration rate, body mass index, highdensity lipoprotein, creactive protein, paraoxonase activity, diabetes mellitus, and atrial fibrillation . Full reclassification tables according to risk categories 0% to 30% (low), 30% to 60% (middle), and> the current study demonstrates a strong association between impaired antioxidative capacity of serum hdl and clinical outcome in heart failure patients . Survival free of the combined cardiovascular endpoint of cardiovascular mortality and htx was significantly lower in patients with prooxidative hdl serum measurements reflected by a hoi 1 and remained significant after adjustment for potential confounders . Furthermore, determination of the hoi led to considerable improvements of conventional risk prediction in chf patients . Inverse correlations between hdlcholesterol levels and the risk for development of cardiovascular diseases are well investigated.27 however, hdlcholesterolraising therapies failed to improve outcome in patients with coronary artery disease: a large percentage of individuals develop cardiovascular diseases despite normal or even high hdlcholesterol levels, and the prognostic validity of hdl in patients with already existing cardiovascular diseases seems to be diminished.28, 29, 30, 31 this supposed failure of the hdl hypothesis encouraged more indepth investigation of underlying mechanisms, and recent studies suggest that not merely quantity but rather functionality of hdl may be more relevant for the protection against cardiovascular disease.10, 12, 32 accordingly, we observed that an impairment of antioxidative function of hdl determined by an hoi 1 was independent of hdlcholesterol serum levels in our cohort and was a significant predictor of cardiovascular events even though hdl itself was not associated with outcome in these patients . So far, antioxidative properties of hdl have been found impaired in common chfrelated comorbidities such as chronic kidney disease and type 2 diabetes mellitus, and determination of the hoi turned out to be a useful predictor of cardiovascular events and mortality in patients with acute coronary syndrome.13, 15, 33, 34 with respect to chf, a previously conducted casecontrol study has reported reduced hdl antioxidative capacity in patients with ischemic chf.16 the present study extends this initial observation, showing that an impaired hdl antioxidative capacity is associated with higher mortality in chf patients independent of traditional cardiovascular risk factors and irrespective of the underlying etiology . Besides promoting reverse cholesterol transport, hdl particles have been shown to possess antioxidative, antiinflammatory, antithrombotic, and antiapoptotic properties.35 in light of the pivotal role of oxidative stress and inflammation in the development and progression of chf, the assumption that an impaired antioxidative capacity of hdl may affect clinical outcome in patients with chf seems likely.2, 3 the pathophysiological mechanisms for the loss of antioxidative function of hdl have not been fully elucidated yet, but there is increasing evidence that alterations in hdl protein composition and protein quality are crucially involved.36, 37 conditions of infection, inflammation, or tissue injury are thought to trigger structural alterations of hdl particles.38 accordingly, the association of an hoi 1 with cardiovascular events was more pronounced in patients with high levels of the proinflammatory cytokine il6 . These results further support the assumption of inflammatory processes being crucially involved in impairment of the antioxidative capacity of hdl . Interestingly, hoi 1 was still associated with cardiovascular outcome after multivariable adjustment for the inflammatory biomarkers crp and il6 . As a result, the effects represented by the hoi as a global measure of oxidative capacity seem to be beyond the inflammatory pathways indicated by the measured biomarkers . Oxidative stress itself is considered to render hdl dysfunctional by provoking posttranslational modifications of hdlassociated proteins.37, 38 myeloperoxidase (mpo), which is mainly secreted by activated neutrophils, has been demonstrated to mediate these structural and functional alterations of hdl.39 furthermore, the presence of serum amyloid a, a protein family of major acutephase reactants on hdl, has been reported to impair hdl's antioxidative functionality.40 in contrast, hdl loading with the protein sphingosine1phosphate (s1p), has recently been demonstrated to correct for hdl dysfunction.41 as an important finding of our study, no differences in the predictive value were observed between patients with ischemic and nonischemic etiology of chf . This suggests the assumption that the link between impaired hdl antioxidative capacity and poor outcome in chf cannot be solely attributed to atherosclerosis and coronary vascular events, as shown by previous studies, although these may be important confounders.9, 12 in this respect, several lines of evidence indicate a direct involvement of oxidative stress and excess ros production in left ventricular dysfunction and cardiac remodeling by inducing rather unspecific damage on cellular membranes, proteins, and dna.2 these alterations result in multifaceted maladaptive changes including extracellular matrix remodeling, hypertrophy of cardiomyocytes, and impaired excitationcontraction coupling.7 it is therefore tempting to speculate that a preserved antioxidative function of hdl can prevent the various maladaptive responses to excessive ros production involved in left ventricular remodeling . Hdl particles are composed of lipids and more than 100 different proteins and exert their protective functions by the collaboration of essential apolipoproteins and associated enzymes.38 the hdlbinding glycoprotein pon1 is shown to be critical for the antioxidative capacity of hdl.42 a previously published association of low pon1 activity and poor longterm prognosis in patients with chf was visible in our cohort in unadjusted cox regression analysis but turned nonsignificant after adjustment for potential confounders including implementation of hoi in our multivariable model.17 although we saw a significant association between pon1 activity and the measured antioxidative capacity in the unadjusted model, this relationship was no longer observed after multivariable adjustment . Pon1 is thought to be mainly responsible for preventing oxidative modifications, but hoi appeared to be a more robust risk predictor, sustaining adjustment for ntprobnp and other potential confounders . The stronger performance of hoi to prevent or enhance the formation and inhibit or aggravate the biologic activity of oxidized ldl could be due to additional components attached to hdl . These enzymes include apolipoprotein ai, apolipoprotein j, plateletactivating factor acetylhydrolase (pafah), lcat, and glutathione (gsh) selenoperoxidase.43, 44 thus, hdl particles contain at least 2 apolipoproteins and 4 enzymes that protect ldl from oxidation, demonstrating the complexity of the antioxidative capacity . Determination of the global antioxidative function using hoi may therefore represent a reliable surrogate of the multifaceted alterations in hdl structure and function in response to oxidative stress . Strategies to improve the antioxidative function of hdl may be beneficial for patients with chf . In this respect, exercise training has been shown to significantly improve hdl functionality.45 notably, exercise training represents a proven intervention strategy in patients with chf, leading to enhanced survival rates and quality of life.46 the exercisemediated effect on hdl function is still not elucidated, but it is likely that exercise training positively affects the antioxidative capacity because an exerciseinduced reduction of lipid peroxidation has been observed, which is a general measure of oxidative load.45 one may therefore speculate that strategies to improve the antioxidative capacity of hdl represent a potential treatment target for future therapies in patients with chf . A potential limitation of this study is that antioxidative capacity of hdl was determined in apolipoprotein b depleted serum samples instead of isolated hdl . Although polyethylene glycol precipitation is generally accepted as a reproducible and rapid method to extract hdl from patient serum, it remains unclear if antioxidant effects are influenced by other nonhdlassociated proteins, which might interact with the applied assay.12, 16 however, prior studies showed strong correlations of antioxidant results from hdl samples isolated by different techniques.47, 48 therefore, the use of apolipoprotein b depleted serum samples is applicable as long as this method is consistently applied for all samples.48 naturally, measuring the antioxidative capacity of hdl using an in vitro cellfree assay may be limited by the necessity of an artificial environment . However, the assay harbors the advantage of being applicable to large patient cohorts, with potential clinical importance given by the robust association with cardiovascular outcome . Accurate statistical evaluation of novel biomarkers comprises estimation of additional predictive performance in comparison to established riskprediction strategies.24 however, the generally recommended indices of reclassification are subjected to criticisms regarding their statistical properties, as their values are difficult to interpret . For instance, the categoryfree nri summarizes broad changes in risk models, which may incorporate irrelevant information and overestimate the added usefulness of a marker, whereas the categorical nri strongly depends on clinically meaningful risk categories.49 however, with a full disclosure of the reclassification risk (tables s3 and s4), a careful selection of risk categories as recommended by leening et al,25 and consistent improvements of the hoi in cstatistic, nri and idi may overcome statistical concerns . Because hdl is capable of ameliorating endothelial dysfunction by enhancing endothelial nitric oxide synthesis, this may be a possible link between impaired hdl function and adverse outcome in our patient population.50 however, we did not perform examinations such as flowmediated dilation of the brachial artery to investigate a potential association between the hoi and endothelial dysfunction . A potential limitation of this study is that antioxidative capacity of hdl was determined in apolipoprotein b depleted serum samples instead of isolated hdl . Although polyethylene glycol precipitation is generally accepted as a reproducible and rapid method to extract hdl from patient serum, it remains unclear if antioxidant effects are influenced by other nonhdlassociated proteins, which might interact with the applied assay.12, 16 however, prior studies showed strong correlations of antioxidant results from hdl samples isolated by different techniques.47, 48 therefore, the use of apolipoprotein b depleted serum samples is applicable as long as this method is consistently applied for all samples.48 naturally, measuring the antioxidative capacity of hdl using an in vitro cellfree assay may be limited by the necessity of an artificial environment . However, the assay harbors the advantage of being applicable to large patient cohorts, with potential clinical importance given by the robust association with cardiovascular outcome . Accurate statistical evaluation of novel biomarkers comprises estimation of additional predictive performance in comparison to established riskprediction strategies.24 however, the generally recommended indices of reclassification are subjected to criticisms regarding their statistical properties, as their values are difficult to interpret . For instance, the categoryfree nri summarizes broad changes in risk models, which may incorporate irrelevant information and overestimate the added usefulness of a marker, whereas the categorical nri strongly depends on clinically meaningful risk categories.49 however, with a full disclosure of the reclassification risk (tables s3 and s4), a careful selection of risk categories as recommended by leening et al,25 and consistent improvements of the hoi in cstatistic, nri and idi may overcome statistical concerns . Endothelial dysfunction plays an important role in the pathophysiology and progression of chf . Because hdl is capable of ameliorating endothelial dysfunction by enhancing endothelial nitric oxide synthesis, this may be a possible link between impaired hdl function and adverse outcome in our patient population.50 however, we did not perform examinations such as flowmediated dilation of the brachial artery to investigate a potential association between the hoi and endothelial dysfunction . The identification of impaired antioxidative hdl function as a risk prediction marker for poor clinical outcome beyond the known risk factors complements the knowledge on risk stratification in chf . This suggests that antioxidant properties of hdl rather than total hdl serum levels may be considered for an improved risk assessment . Considering the robust effect of impaired antioxidant hdl function on clinical outcome in chf, this highrisk patient population might benefit from a specific treatment to restore and maintain antioxidant hdl capacity . This work was supported by the association for the promotion of research on arteriosclerosis, thrombosis, and vascular biology (atvb) and the ludwig boltzmann cluster for cardiovascular research . Lorenz koller is recipient of a doc fellowship of the austrian academy of sciences (no . Table s1 . Analysis for subdistribution and causespecific hazard models for the cardiovascular endpoint and noncardiac death table s2 . Subgroup analysis of the predictive value of a highdensity lipoprotein oxidative index 1 stratified by tertiles of interleukin6 in cox regression hazard analysis table s3 . Categorical net reclassification improvement (hoi in addition to ntprobnp) table s4 . Categorical net reclassification improvement (hoi in addition to the multivariable model) figure s1.
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Cell invasion is important in many biological processes, particularly in the invasive stage of cancer and in embryonic development [23, 32]. There has recently been some interest in how to mathematically model cell cell and cell extracellular matrix adhesions in the context of cancer invasion, since the latter is believed to be characterized by a number of processes that include loss of cell cell adhesion and enhanced cell matrix adhesion in addition to active migration, cell proliferation and the secretion of matrix degrading enzymes . Accurate modelling of cell adhesion is, therefore, important, but it is challenging to do so using continuous mathematical models because in such an approach one uses continuous variables for cell densities . Individual cells are not recognized as such and, therefore, there is no representation of cell boundaries . A modelling approach that aims to address these difficulties was proposed by armstrong et al . . The ideas have been taken further and applied to various contexts by, for example, [3, 12, 15, 23, 31]. For a cell density p(x, t) the basic model in one spatial dimension is for x (,), t> 0 with initial condition p(x, 0) = 0(x). Equation (1) incorporates a direct representation of cell - cell contact via a non - local flux term, the integral term in equation (1). Spatially structured models of tumour invasion that incorporate other aspects such as haptotaxis and age structure were considered in [911]; see also [34, 35]. The purpose of the present paper is to formulate and analyse an extension of equation (1) to the case of n spatial dimensions, considering only cell - cell adhesion . Again letting p(x, t) denote the cell density at (x, t), but now with x, t> 0 and b denoting the n - dimensional ball centred at 0 and of radius, we propose the equation with initial condition the function f(p) models cell loss and gain, and for biological reasons we assume that f(0) = 0 and that there is a number p1> 0 such that f(p)> 0 for p (0, p1), and f(p) <0 for p> p1 . These assumptions imply that at lower densities, there is cell gain, but at large enough densities cell loss occurs more rapidly than the generation of new cells via division, due to the effects of crowding . Motion of cells is assumed to be partly due to fickian diffusion, which gives rise to the laplacian term . Note from simulations of their more complex cell matrix model that, in the absence of a fickian term, and unusually for a continuous model, non - invasive growth is possible for some parameter regimes while invasive growth takes place in others . The other component to cell motion is the movement of cells due to adhesion as modelled by the cell adhesion term in equation (2). It is an advective flux term and models motion of cells that is caused by mechanical forces attributable to adhesive bonds that cells have formed with their closer neighbours . Cells are able to sense their surroundings over some radius, which is believed to be of the order of several cell diameters, due to their ability to deform and extend protrusions . These protrusions cause adhesive bonds to form with other cells, and the resulting forces cause cell movement . In fact, the cell adhesion term in equation (2) is an advective flux term of the form (p(x, t)u(x, t)), where u(x, t) is the velocity of the cells at x at time t. if we imagine the cells as tiny spheres moving through a viscous fluid, then stokes' law leads us to suppose that they are subject to a resistive force which is directly proportional to the velocity . This analogy leads us to suppose that the velocity of a cell is proportional to the net adhesive force on it due to the bonds formed with nearby cells . So, the velocity u(x, t) can effectively also be thought of as the adhesive force on a cell due to bonds with these other nearby cells within the sensing radius (the range over which a cell can detect its surroundings). This force is taken to be of the form we think of expression (4) as a force, but it is really a velocity . Expression (4) is assumed to contain dimensional constants such as the viscosity of the medium (perhaps embodied within the functions g and h), to ensure that it has the units of velocity . Viewing expression (4) as a force, we are asserting that the total force on a cell at position x is the sum, over all cells within the sensing radius, of the local forces attributable to bonds formed with the cells at the nearby points x +, b. the function h: describes how the magnitude of the force depends on ||, and is a positive function since adhesive forces are always directed towards cell centres . The vector in front of h(||), which is not present in equation (1), gives direction to the force on cells at x due to bonds with cells at x + . Another formulation would be to make this direction vector a unit vector and write (/||)(||) rather than h(||), as some other authors have done, see for example painter et al . . There is no real distinction; we are simply taking h(||) = (||)/|| . The magnitude of the adhesive forces from cells at the nearby location x + will depend on the number of adhesive attachments made by cells at x + to a cell at x, and hence on the cell density at x + . This explains the term g(p(x +, t)) in the integrand of expression (4), and the function g(p) describes how the forces depend on the local cell density . We anticipate that g(p) should increase linearly with p if cell density is not too great, because in this situation a doubling of the number of cells at x + should roughly double the number of adhesive attachments made to cells at x. however, at higher densities, particularly as close cell packing is approached, the tendency to form attachments drops off and so g(p) in fact decreases once p has risen above a certain threshold, and is zero for all p above a critical cell density p2> 0 corresponding to close cell packing . The shift in balance between cell division and cell loss should occur at an achievable cell density . . There is unquestionably a need for analytic study of equations of the form (2) in spatial domains of dimension n recent interest in this kind of equation has been largely in the areas of cancer cell invasion and wound healing . Gerisch and chaplain and painter et al . Both consider systems containing a term of the same form as the non - local term in equation (2), and with the same interpretation cell velocity due to adhesive bonds with other cells with an emphasis on two dimensions so that b becomes a disk of radius . These studies have been largely numerical, which is time - consuming in the 2d case . In addition to their numerical simulations, gerisch and chaplain examine the possibility of expanding the non - local term, using taylor series in the case when is small . This analytical approach does yield some useful insight, although the approximated equations can allow unrealistic phenomena such as blow up . Two- and three - dimensional studies are extremely important in the cancer modelling context because of the manner in which cancer cells invade tissue and the possibility of phenomena such as fingering at the invasive front (see figure 5 in). The shape of a tumour - host boundary is an important diagnostic indicator (, and the references therein). Straight and/or sharp boundaries tend to imply non - invasive tumours, whereas if a tumour has a diffuse and/or wavy boundary it is likely to be invasive . Another important reason for considering equations of the form (2) in higher space dimensions is that the inclusion of another space dimension can be an important test of the stability and robustness of a one - dimensional pattern, and therefore of the plausibility of a type of mathematical model in a situation where many details are unknown . This was an important issue in armstrong et al . Who considered a system with the same type of adhesive flux term as in equation (2) as a model for somite formation . Segmentation in a number of organisms proceeds through the formation of somites, which will eventually become repeated structures such as the vertebrae . Somitogenesis is, therefore, seen as a one - dimensional process (along the anterior - posterior axis of the presomitic mesoderm) and an important step in the model validation process can be to include a second spatial dimension and investigate whether the model is still capable of forming somites, as was done in . Using fractional powers of the diffusion operator, we establish results about the existence and uniqueness of solutions and their positivity and boundedness in spaces of uniformly continuous functions . We also look at solutions in l spaces and show that under suitable conditions the non - zero uniform state is asymptotically stable in l and that under more stringent conditions all solutions converge to this uniform state . Initially, we will work in the spaces in either case, take the norm ||u||k = ||k ||du||, where for i = 1,, n, di: (di) so di is a closed linear operator . Set d = (d1,, dn), with domain (d) = (d1) (dn). If = (1,, n), d = d1 dn . We define || = 1 + + n and say for n - tuples and if the inequality holds component - wise . Note that from page 33, u buc0() implies that, for || k, du 0 as |x| . We write when results apply to either x or x0 and set x = x. we define: () x x, by for> 0 set a = + i, (a) = () so that the spectrum of a is contained in the open right half plane . We rewrite the problem (2)(3) abstractly as where p(t): [0,) x, t 0, f:, g:, and with ith component ki()(x). Let t(t) be the analytic semigroup in x generated by, so and t(t): x0 x0 . Let tk(t) be the analytic semigroup which is t(t) restricted to the space (k an integer); the corresponding generators k are given by the operator but restricted to different domains . It is easy to see that for x, k> 0, || k so we also have t(t): x x, set ak = k + . We will use the fractional powers ak of ak, and exploit the result that, for suitable, the operator diak: x x is bounded, see . We will write = (ak) in x, 0 = (ak) in the space x0 and = (ak) in the space . Thus the mild form of (2)(3) in x is observe that for (d), g c(), h l(0,), we have k() (), note that in our proofs of existence, we will take f, g: but we will then prove that if 0 0 then p(t) 0 . Thus, provided there is enough smoothness at 0 it is only the properties of f and g on [0,) that are relevant as we can then define f and g appropriately for p <0 . We also have [17, 18, 22, 24] if> 0, then for every t> 0 the operator aktk(t) is a bounded linear operator in and for any> 0 there exists c1> 0 such that if 0 1, then ak: is a bounded linear operator so there exists a constant c2 1 such that for all u x, if <<1, then diak: is a bounded linear operator so there exists a constant c3 1 such that for all u x, the following lemma is proved in very much the same way as lemma 1 in suppose that for some k 0, f c(), g c() and h l(0,), <<1 and f(0) = 0 . Then g: and (a)if ||ak||k r1 there exists a constant k1(r1) such that(b)if ||i||k r2, i = 1, 2, then there exists a constant k2(r2) such that(c)if k 2, and ||||k2 r3 if ||ak||k r1 there exists a constant k1(r1) such that if ||i||k r2, i = 1, 2, then there exists a constant k2(r2) such that if k 2, and ||||k2 r3 suppose that for some k 0, f c(), g c(), f(0) = 0, that h l(0,), and that 0 q 0, where <<1, and either q = 0 or there exists p1> 0 such that f(p1) = 0 and q = p1 . Then the problem (2) and (3) has a unique mild solution p(t) such that p(t) q c([0, t0]; 0), for t0> 0 small enough . In addition p(t) is the classical solution on [0, t0] of the problem (2) and (3) in the sense that and for 0 <t t0, p(t) (ak), and also, under the above conditions, if k 2 m, in particular if k = 2, p(t) q c([0, t0]; x0) and equation (23) also holds at t = 0 . Let [0, 0) be the maximal interval of existence of the solution p(t). Now let pn(t) be the solution of equation (2) with initial data pn(x, 0) = n(x) where n q 0 . Suppose that ||ak(n 0)||k 0 as n . Then, given any t1 [0, 0), pn(t) is defined on [0, t1] for large enough p(t))||k 0 as n, uniformly on [0, t1]. To deal with the case q = p1 we set p(x, t) = w(x, t) + p1 in equations (2) and (3) to get where f(w) = f(w + p1), and g(w) = g(w + p1). If we set the abstract form of equation (25) is if g() = d ((a + p1)k(a)) f(a), then it is easy to see that g satisfies lemma 1 and we can apply the same methods to the resulting equation in w. we can now obtain positivity and boundedness . Suppose that h c [0,], f c(), g c(), f(0) = 0, and that 0 q d0, where <<1 and q = 0 or q = p1> 0 where f(p1) = 0 . Let 0(x) 0 for all x . If p(t) is the unique classical solution of equations (2) and (3) on [0, t0], then p(x, t) 0 for all 0 t t0, x . Proof take m1> 0 and m2 0 such that and, using the mean value theorem, choose c sufficiently large that where n is the surface area of b1, and define u(x, t) = p(x, t) e, so we now prove that p(x, t) 0 on [0, t0]. Suppose not, then u(x, t) will be strictly negative somewhere in this set and hence attains a global minimum at some (x0, t0) (0, t0]. Thus for all i in the ith integral in the second term in the right - hand side of equation (31), we may replace /xi by /i and therefore an integration by parts shows that the sum at the point (x0, t0) equals the above quantity is bounded in absolute value by evaluating equation (31) at (x0, t0), and making use of (32) and the above bound, we obtain using inequality (29), the fact that u(x0, t0) <0 and that c satisfies inequality (30). Suppose that h c[0,], h 0, f c(),g c(), f(0) = 0 and that 0 q d0 where <<1, and q = 0 or q = p1> 0 where f(p1) = 0 . Suppose there exists p2> p1 such that g(p) 0 for p [0, p2], f(p2) <0 and that let 0 0(x) p2 for all x . If p(t) is the unique classical solution of equations (2) and (3) on [0, t0], then 0 p(x, t) suppose that h c [0,], h 0, g bounded, f c(), g c(), f(0) = 0, and that 0 q 0 where <<1, and q = 0 or q = p1> 0 where f(p1) = 0 . Suppose there exists p3> p1 such that for p> p3, f(p) <0 and let 0 0(x) p3 for all x . If p(t) is the unique classical solution of equations (2) and (3) on [0, t0], then 0 p(x, t) the method uses proposition 2 or 3 to show first that p(t) is bounded on bounded intervals of time . Thus by lemma 1(a) ||g(ap(t))|| is bounded linearly, so we can use the gronwall - type inequality in 1.2.1 to show that ||ap(t)|| is suitably bounded . Then we can use lemma 1(c) to get a linear bound on ||g(a2p(t))||2, so, using the gronwall - type inequality again, ||a2p(t))||2 is also bounded as required suppose that h c[0,], h 0, f c(), g c(). 0 where <<1, and q = 0 or q = p1> 0 where f(p1) = 0 and 0(x) 0 for all x . Suppose that either (a)there exists p2> p1 such that 0(x) p2 for all x, g(p) 0 for p [0, p2], f(p2) <0 and inequality (34) holds . P1 such that 0(x) p2 for all x, g(p) 0 for p [0, p2], f(p2) <0 and inequality (34) holds . Or g is bounded and there exists p3> p1 such that 0(x) p3 for all x, and for p> p3, f(p) <0 and inequality (35) holds . P <define the operator, l: (l) l() l() by similarly, we can also define l: (l) l() l(), for a suitable domain (l1). For 1 p <,> 0, set al = l + i, (al) = (l). It is well known that l generates a positive analytic semigroup {tl(t)}t0, with ||tl(t)|| 1 . Further, in the case 1 <p <, from propositions 4.1.7 and 1.1.4 and example 1.3.9, if <<<1, thus, if we define di: (di) l() l(), then inequality (18) holds in l(). To show that inequality (18) also holds in l(), it is sufficient to show that (al) w(). To do this note first that it follows from example 1.3.9, example 1.3.11 and remark 1.3.7 that if 1 <s <2 and 0 <<<1, then now, by proposition 4.1.7, theorem 1.7 and prop 4.8, and prop 1.1.4, for 1 <s <2 (the definition and properties of the besov spaces bp, q, and their relationship to the sobolev - slobodeckii spaces w, can be found in [1, 16, 20].) As before, from, tl(t) satisfies equation (8), hence if x l(), tl(t) = tk(t). Equally (al) (al) implies al = ak. We now have the following local existence theorem in l(): suppose that f c(), g c(), that h l(0,), f(0) = 0 and that 0 q (al), where <<1, and either q = 0 or there exists p1> 0 such that f(p1) = 0 and q = p1 . Then, for n 3, the problem (2) and (3) has a unique mild solution p(t) such that p(t) q c([0, t0]; (al)), for t0> 0 small enough . In addition p(t) is the classical solution on [0, t0] of the problem (2) and (3) in the sense that and for 0 <t t0, p(t) (al), and also there is continuous dependence on the initial data . Let [0, 0) be the maximal interval of existence of the solution p(t). Now let pn(t) be the solution of equation (2) with initial data pn(x, 0) = n(x) where n q (al). Suppose that ||al(n 0)||l 0 as n . Then, given any t1 [0, 0), pn(t) is defined on [0, t1] for large enough p(t))||l 0 as n, uniformly on [0, t1]. Finally, if in addition 0 q (al), then also p(t) q c([0, t0]; (al)). The proof of the first part follows from theorems 3.3.3 and 3.4.1 in provided we can show that g: l() l() and g: l() l() are locally lipschitz . Each product has just one term of the form dial. The rest are functions of al d(al). But, for <<<1, and n 3 (see 7.34) and the local lipschitzness follows . Similarly for g. note further that, also using inequality (18) in l (), g, g: l () l() l () l() and are locally lipschitz continuous in l(). For 0 (al) (al), we set up the iteration: 0 = al 0, then the iteration converges in l() and l() and by considering the restrictions to bounded subsets of, it can be seen that the limits are equal almost everywhere . Similarly for g, and we now consider the local asymptotic stability of the uniform state p = p1 . For simplicity, we will consider the case where f and g are logistic . Each of f and g in equation (2) can be of the form rp(1 p / k) with different r and k. after suitable non - dimensionalization, without loss of generality, they can be taken as f(p) = p(1 p) and g(p) = p(p), with> 1 . The uniform steady state is then p = 1 . To examine its stability, set p = w + 1, 0 = w0 + 1 to obtain the following equation for w: take n 3 . Suppose that h l(0,), and that 0 1 (al), where <<1 . For define then if k() <0 for all the zero solution of equation (40) is uniformly asymptotically stable in (al). Precisely, if> 0 is such that k() <for all, then there exist> 0, m 1 such that if ||alw0||l, then moreover, k() <0 for all holds if conversely if there exists 0 such that k(0)> 0, then the stationary solution is unstable . (al) l(), such that and the operator h: l() l() such that h() = g() bal. Using the same reasoning as in theorem 3 it can be seen that if ||||l r, then there exist constants k(r) and k(r) such that in the case of stability, we note that this implies that h() = o(||||l) as ||||l 0 . We have already seen that g: l() l() is locally lipschitz continuous . Thus theorem 5.1.1 of holds so that if the solutions of the linearized problem are uniformly asymptotically stable then so too are those of the nonlinear problem . The right - hand side of equation (44) is linear and generates an analytic semigroup r(t), say . So, for all u0 l(), equation (44) has a global classical solution u(t) = r(t)u0 . First take u0 l() l(), so that, as in theorem 3, u(t) l() l(), and we may take fourier transforms . If we denote the fourier transform of u by thus, using the plancherel identity, as required . Thus by density ||r(t)u0||l ||u0||l2 exp(t) for all u0 l(), and the result follows . The inequality (42) follows from completing the square in k(), while inequality (43) follows from the fact that |sin(x)| | x| |||x| . For instability, we apply corollary 5.1.6 of . From inequality (45) h() = o(||||l) as ||||l 0, so now we require that there exists in the spectrum of the generator of r(t) such that re> 0 . Suppose not, so that there exists m such that, for all l(), but, using the expression for (, t) in (46), there exists> 0 such that if we now choose u0 such that b|0(+ 0)| d> 0, then using the plancherel identity gives a contradiction to inequality (47). The next result follows in a similar fashion to theorem 3 and theorem 3 . Suppose that f c(), g c(), h c[0,]. Let 0 q 0, where <<1, and either q = 0 or q = p1> 0 with f(p1) = 0 . Suppose also that the hypotheses of proposition 1 and of either proposition 2 or proposition 3 on f, g, h, and 0 hold . Take also 0 q (al). Q c([0,); 0) be the unique classical solution of equations (2) and (3). Then, for each t 0, p(t) q (al) = w(). Furthermore p(t) q c((0,); l()). Let h c[0,] be such that h 0, and let f(p) = p(1 p) and g(p) = p(p) with> 1 . Let 0 1 0 (al(n)), where <<1, and let p(x, t) be the solution of the problem (2) and (3). (a)if 0 0(x) for all x, where satisfiesthen 0 p(x, t) for all x, t 0. (b)if 0(x) for all x, where 0 <p(x, t) for all x, t 0. (c)if 0(x) <for all x, where> 0, satisfies inequalities (48), (49), andthen p(x, t) 1 exponentially as t in the sense that for all t> 0 if 0 0(x) for all x, where satisfies then 0 p(x, t) for all x, t 0 . If 0(x) for all x, where 0 <<, satisfies inequality (48), and then p(x, t) for all x, t 0 . If 0(x) <for all x, where> 0, satisfies inequalities (48), (49), and then p(x, t) 1 exponentially as t in the sense that for all t> 0 first, to prove (a), note that inequality (48) implies inequality (34) with p2 =, f(p) = p(1 p), and thus, solutions remain in the closed interval [0,] for all positive times . Next, to prove (b), suppose that there exists t *> 0 and a corresponding x * with p(x *, t *) =, p(x *, t*)/xi = 0, p(x *, t*)/xi 0, then note that 0 g(p) /4 when p [0,]. Therefore by inequality (49). Hence p(x, t) for t> 0 and (b) is proved . Last, to prove (c), define w(x, t) by p(x, t) = 1 + w(x, t), so that w(x, t) satisfies equation (40) and 1 w 1 . Multiplying equation (40) by w(x, t) and integrating with respect to x over, and then integrating by parts on the laplacian term and the /xi term (this is justified by theorem 5), the integrand of the middle term in the right - hand side will now be estimated by using the inequality a b this bounds the integral by a sum of two integrals, one of which cancels with the first term on the right - hand side to give since> 1, it is easy to show that recalling that 0 1 + w(x, t), so using inequality (54) and theorem iv.15 from, therefore inequality (53) becomes note that w(x, t) (1)w(x, t), so that inequality (56) becomes and (c) follows from inequality (50). We illustrate in figures 14 the results of the previous two sections with simulations that can be applied to in vitro wound closure experiments . In these experiments, cell cultures are scored in a thin line that closes as the cell population proliferates . A review of mathematical models of wound healing is given in and other references are found in [57, 13, 14, 21, 2529, 33, 36]. In previous work, we modelled in vitro wound healing experiments for the model (2) with n = 1, and examined the dependence of solutions on the diffusion parameter and the sensing radius . In numerical simulations demonstrated that for larger values of the diffusion parameter and smaller values of the sensing radius the wound closed completely across the wound opening, but for smaller values of and larger values of complex patterns arose across the wound opening with incomplete closing . Either of the alternative conditions (42) or (43) is sufficient for the stability of the uniform steady state p = 1, and note that both of these conditions are largeness conditions on and smallness conditions on both h() and on the sensing radius . Similarly conditions (48), (49) and (50) are sufficient for convergence to the uniform steady state and conditions (48) and (49) are smallness conditions on h() and while (50) is in addition a largeness condition on . In our simulations here, we examine the dependence of the wound healing model with respect to the parameter, which is the non - dimensionalized cell density corresponding to close cell packing, in equation (2) with g(p) = p(= 0.1, = 0.2, f(p) = p(1.0 p), g(p) = p(p), h(x) = arctan (100.0x)/(10.0x arctan 100.0) and initial data 0(x) = 1.0 0.8 exp((0.1x)). The red plane and lines correspond to = 0.2 and the green plane and lines correspond to 0.0 . (a) graph of p(x, t), 0 t <2.0 . The wound does not close, and instead the solution exhibits an evolving pattern of peaks and valleys which do not converge to 1.0 . (b) graphs of p(x, .0), p(x, .5), p(x, 1.0), p(x, 2.0). Simulation with = 1.0, = 1.5 (a), = 17.0 (b), = 18.0 (c), = 19.0 (d), = 1.0, = 0.2, f(p) = p(1.0 p), g(p) = p(p), h(x) = arctan(100.0x)/(9.0xarctan 100.0), and initial data 0(x) = 1.0 0.8 exp((0.1x)). For the higher values of, the wound healing simulation wave patterns as in figure 2 with adhesion strength parameters (a) = 17.0, (b) = 18.0, and (c) = 19.0 . For = 17.0, the solution is shown at times t = 100.0 (blue), t = 95.0 (red) and t = 90.0 (green). The amplitude envelope of the oscillation packet decreases to a minute value for the times shown . For = 18.0 the solution is shown at times t = 200.0 (blue), t = 150.0 (red) and t = 100.0 (green). For = 19.0, the solution is shown at times t = 150.0 (blue), t = 125.0 (red), t = 100.0 (green), t = 75.0 (yellow) and t = 50.0 (cyan). The simulations indicate convergence to the uniform equilibrium 1.0 in (a) <17.62, but not in (b) and (c),> 17.62 . Wound healing simulations with parameters = 2.0, = 1.0, = 3.0, f(p) = p(1.0 p), h(x) = 9.0 arctan(100.0x)/(4.0x arctan 100.0), and initial data 0(x) = 1.0 0.4 exp((0.1x)) (top left), 0(x) = 1.0 0.95 exp((3x)) (top right), 0(x) = 1.0 0.5 exp((0.2x)) (bottom left) and 0(x) = 1.0 0.95 exp((0.1x)) (bottom right). The wounds top left and top right both close but the wound bottom right which has the same width as the first and the same depth as the second fails to close . The widest wound, bottom left, also closes . In figure 1, n = 1, p), = 0.1, = 0.2, = 1.0, h(x) = arctan(100.0x)/(10.0x arctan 100.0), and 0(x) = 1.0 0.8 exp((0.1x)). In this case, theorem 6 (48) is satisfied if <18.94, (49) is satisfied if <1.457, and (50) is satisfied if <2.823 . We take = 19.0, so that none of the conditions (48), (49), (50) of theorem 6 are satisfied . The simulation in figure 1 shows that for this value of, the solution does not remain bounded below by and does not converge to the equilibrium 1.0 . On the other hand, if condition (48) were satisfied, then the solution p would be bounded (0 p), and theorem 2 would give global existence of the solution . Indeed, if we take = 18, then condition (48) holds, and we find that the solution behaves qualitatively the same as in figure 1 . This provides an example where the theory gives global existence of the solution, but the solution does not converge to the equilibrium but instead forms a series of peaks . In figures 2 and 3, n = 1, p), = 1.0, = 0.2, h(x) = arctan(100.0x)/(9.0x arctan 100.0), and 0(x) = 1.0 0.8 exp((0.1x)). We consider different values of to illustrate the sensitivity of this parameter for the convergence of the solutions to the equilibrium 1.0 . The hypotheses of theorem 6 require that <16.94 (48), <2.683 (49), and <2.597 (50). For <2.597 all three conditions are satisfied, and theorem 6 implies the solution remains in the interval [0.2,] and converges to 1.0 for all initial data such that 0.2 0(x). On the other hand if one looks at the stability condition from theorem 4, then the equilibrium 1.0 is asymptotically stable for <17.62 with instability if> 17.62 . In figure 2 (a) = 1.5 and there is convergence to 1.0 (the solution appears to converge monotonically to 1.0 from below, never rising above 1.0). In figure 2(b)(d) = 17.0, 18.0, 19.0, respectively, and the solutions develop a series of peaks, symmetric to the right and left of 0.0, and rising above and falling below 1.0 . Further examination of this wave behaviour is given in figure 3 for the cases = 17.0 (a), = 18.0 (b), and = 19.0 (c). For = 17, the amplitude of the waves decreases as t increases and the solution appears to converge slowly to 1.0 . For = 18 and = 19, the wave propagates with amplitude dependent on (the greater the value of, the greater the amplitude). In general, for the roles of the model parameters, we postulate that cells concentrate in interior and exterior regions of the wound in a regular pattern depending on the strength of adhesion, the sensing radius, and the diffusion coefficient . In figure 4, we show that the convergence to the equilibrium 1.0 corresponding to the healed wound depends on the initial conditions . In these simulations, = 3.0, f(p) = p(1.0 p), g(p) = p(p), and h(x) = 9.0 arctan(100.0x)/(4.0x arctan 100.0). In this figure, the only change in the simulations is the initial data the parameters are the same for all . For initial conditions corresponding to a shallow, wide wound, and to a narrow, deep wound, closure occurs, but for an initial condition corresponding to a wound with the same width as the first and the same depth as the second, closure does not occur . We note that the function k() defined in equation (41) is negative for all, which by theorem 4 implies that the equilibrium 1.0 is stable in this example . We have chosen to illustrate the behaviour of solutions for the model using the simple case of an equilibrium corresponding to wound healing experiments, in which wound closure corresponds to convergence to a constant function in the x - direction, perpendicular to the direction of the scoring.
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Although ascites has been reported in cases with cervical cancer, it is due to other causes such as ovarian metastasis . A 78-year - old diabetic woman who presented with ascites and abdominopelvic mass was misdiagnosed with ovarian cancer and treated with neoadjuvant chemotherapy followed by radical hysterectomy and adjuvant radiotherapy . However, pathology confirmed locally advanced cervical cancer stage iv in this patient . Considering all signs and symptoms to reach a verdict would reduce such malpractices and consequently lead to select the best management and treatment . The initiating event in cervical cancer is hpv infection and immunosuppression is a risk factor (2). Several studies proposed that diabetes mellitus (dm) could increase the risk of developing cervical cancer (35). The most common symptoms are abnormal vaginal bleeding and discharge; patients with advanced disease may present with pelvic pain and bowel or urinary symptoms (6). Although ascites has been reported in the cases with cervical cancer, it is due to other causes such as ovarian metastasis (7, 8). Many studies were done to compare different treatments of locally advanced cervical cancer . Finally, primary cisplatin - based chemoradiotherapy was recommended as the best current treatment of it (9, 10). Recently, a new study has reported that neoadjuvant chemotherapy followed by radical hysterectomy (nac+rh) improves survival of patients with locally advanced cervical cancer in comparison to concurrent chemoradiotherapy (ccrt) (11). In this study, a case was reported who was consulted by gynecologic oncology service because of pelvic mass and ascites . Our case was a menopausal woman who had diabetes mellitus and was a new case of cirrhosis . Neoadjuvant chemotherapy was suggested for her because she had comorbidity and suffered from cirrhosis and it was presumed that she had ovarian cancer . During laparotomy after chemotherapy, our misdiagnosis was discovered because she had a pelvic mass in upper part of cervix that was fixed to bladder . Therefore, radical hysterectomy was done and she received several courses of radiotherapy after surgery . Our case was a 78-year - old diabetic woman who presented with ascites and abdominopelvic mass and was admitted into firoozgar hospital in tehran, iran on june 2010 . She also complained of weight loss about 8 kg in the last 3 months, nausea, vomiting, anorexia, dysuria, and fever . She had diabetes mellitus since ten years ago and took metformin, glibenclamide, losartan, and atorvastatin . On physical examination, her abdomen was distended with fluid wave and there was a palpable 25 cm 20 cm mass in hypogartric part . During bimanual pelvic examination, a large, solid, irregular, fixed pelvic mass was found which occupied the whole pelvis . Laboratory data revealed a normocytic anemia, pyuria, hematuria, mild hyponatremia, hypoalbu - minemia, increased prothrombin time, and elevated serum level of cancer antigen 125 (ca125). Abdominal ultrasound (us) reported a small - sized liver (4 cm) with increased paranchymal echogenicity, bilateral hydronephrosis, huge amount of free fluid in abdominopelvic area, and a large pelvic mass with irregular border which us was not able to identify its origin . Abdominopelvic computed tomography (ct) scan showed ascites and frozen pelvis with no evidence of metastasis and lymphadenopathy (figures 12). Large mass was shown in pelvic ct scan cirrhosis was revealed by abdominal ct scan our first diagnosis was ovarian cancer; therefore, three cycles of neoadjuvant chemotherapy with taxol 175 mg / m and carboplatin 300 mg / m were performed . Laparotomy was performed 3 weeks after the last chemotherapy cycle . During exploration of pelvis, the disease was at stage 4 and therefore radical hysterectomy was prepared for the patient and performed removing parameters and 1/3 of proximal vagina . She was in menopause and bilateral salpingo - oophorectomy was done and the tissue samples were sent for pathologic evaluations . The histopathology of removed cervix showed localized advanced squamous cell carcinoma, stage iv (figures 34). Pathology: invasion to myometrium pathology: invasion to the bladder staging of ovarian cancer was surgicopathological but staging cervical cancer is clinical . There was a misdiagnosis about our case and after pelvic exploration during laparotomy, correct diagnosis was reached and radical hysterectomy was performed . The patient was discharged three weeks later with no complication . She has received twelve courses of epr (external pelvic radiotherapy) after surgery till now . Our patient was successfully treated with nac+rh+epr (neo adjuvant chemotherapy + radical hysterectomy + external pelvic radiotherapy). She was followed up with serial clinical examination, pap smear, and abdominopelvic ct scan . In this case presentation, an old diabetic and newly - diagnosed cirrhotic woman was reported who presented with abdominopelvic mass, ascites, and elevated ca125 . Findings during laparotomy and pathologic evaluation of specimens confirmed locally advanced cervical cancer, stage iv in this patient . The most common symptoms are abnormal vaginal bleeding, postcoital bleeding, and vaginal discharge . In advanced stages of the disease, it presents with pelvic or lower back pain, and urinary and bowel involvement symptoms (6, 12). While abdulhathi et al . Reported cervical cancer cases presented with ascites, they were due to ovarian metastasis (7, 8). No study could be found to report ascites due to cervical cancer alone or with concomitant cirrhosis . Approximately, 5 percent of ascites cases have more than one cause, such as cirrhosis plus peritoneal carcinomatosis (13). In ultrasonography, the presence of solid non hyperechoic ovarian mass and ascites together could be suggestive of ovarian malignancy but metastases to the ovary should be considered as a differential diagnosis . Metastatic disease accounts for 6 to 9 percent of ovarian malignancies and the uterus is also a common primary site for ovarian metastases (14, 15). Therefore, if there is any doubt in diagnosis, image - guided biopsy of the peritoneum or omental cake may help to exclude a non ovarian malignancy (16). In literature review, zhan et al ., jee et al ., and also kuriki et al . Showed that dm increased the risks for developing cervical cancer but there is no evidence to offer, such an association with ovarian cancer (35). Similarly, immunosuppression is one of the risk factors of cervical cancer that can be caused by cirrhosis and dm in this case . Human papillomavirus (hpv) infection is the most common causal agent of cervical cancer and can be detected in 99.7 percent of cervical cancers (17, 18). It is also increased in adenocarcinoma of the cervix, liver disease and cirrhosis, ascites, diabetes, and in approximately 1 percent of healthy women (19). If the patient was diagnosed correctly with cervical cancer stage iv, she needed to be treated by ccrt rather than nac+rh, especially in this case (9, 10). She presented with ascites and had comorbid disease and accordingly was not a good candidate for initial surgery (20). Although yin has recently reported that nac+rh improve the long - term disease - free survival (dfs) and overall survival (os) of patients with locally advanced cervical cancer compared with ccrt, the patients in his sample were all in stage ib2-iib and this case was in stage iv (11). In conclusion, the combination of abdominopelvic mass, ascites, elevated ca125, and negative human papillomavirus infection in conjunction with lack of attention to epidemiologic hints, dm history, bilateral hydronephrosis, and newly diagnosed cirrhosis misled us to diagnosis of ovarian cancer in this patient . It seems that considering all signs and symptoms to reach a verdict would reduce such malpractices and consequently lead to selecting the best management and treatment . It seems necessary to refine our differential diagnosis using complementary workup and confirm our final diagnosis before choosing the appropriate treatment for each patient.
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Inhalation of particulate matter (pm) from fossil fuel combustion is associated with adverse health effects, including reduced lung function (1) and increased mortality (2). Although the mechanism for pm - induced health effects is not fully defined, animal models and in vitro studies suggest that pro - inflammatory cytokine release from airway cells is an important factor (3). Environmental agents associated with elevated lung cancer risk, such as ambient particulate matter, may damage the lung by inducing chronic inflammation . Lung cancer risk is elevated in individuals with emphysema (4, 5), interstitial lung disease (6), and asthma (7), which could similarly reflect effects of the underlying inflammatory disorders . Induction of pro - inflammatory mediators by alveolar macrophages exposed to ambient air particulate matter has been suggested to be a key factor in the pathogenesis of inflammatory and diseases in the lungs . Different contributing physiological and psychosocial factors have been proposed (8). A few prior studies have examined lung cancer risk in relation to polymorphisms in the genes coding for inflammation pathway signaling molecules, such as interleukin 1 (il-1) (9 - 11), il-1 receptor antagonist (il-1rn) (12, 13), il-6 (10, 14), il-10 (15), cyclooxygenase 2 (14), and tumor necrosis factor- (16). These inflammatory cytokines are regulated by the pro - inflammatory transcription factor, nuclear factor nf-b (8). Given the close interaction between the external environment and the lung, tlrs have been implicated in lung - associated immune responses, including airway hyper responsiveness (ahr) and allergic asthma (17). Dysfunction and unregulated activation of the tlr pathway can contribute to decreased lung function and the pathogenesis of acute and chronic lung inflammatory diseases (18). Tlr activation, can occur via two pathways: 1- the myeloid differentiation primary - response protein 88 (myd88)-dependent pathway, and 2- the myd88-independent pathway . These two pathways correspond to early and late - phase nf-b signaling and pathway - specific induction of pro - inflammatory cytokines and chemokines (19, 20). Regular aerobic exercise results in multiple health benefits, including improvement of cardiorespiratory fitness and quality of life, reduction of obesity and blood pressure, and increased longevity (21, 22). When performed chronically on a regular basis, aerobic exercise also reduces oxidative stress systemically (23) in different diseases, such as heart diseases, type 2 diabetes, rheumatic arthritis, and alzheimer and parkinson diseases (23), as well as in the airway epithelial cells of animals with long - term allergic lung inflammation (24). Chronic practice of regular exercise exerts a marked anti - inflammatory effect in different models of pulmonary diseases, such as in asthma models (25 - 28), acute respiratory distress syndrome (29, 30), and chronic obstructive pulmonary disease (31). Studies that have investigated the effects of exposure to air pollutants during exercise have suggested that people exercising in polluted environments are at increased risk of respiratory and cardiovascular morbidity related to air pollution owing to an exercise - induced amplification in respiratory uptake, lung deposition, and toxicity of inhaled pollutants (32 - 35). Exercise may increase the likelihood of an adverse effect by increasing the dose of pollutants delivered to target sites in the lungs as ventilation increases to meet metabolic demands (36). However, these studies do not take into account the potential anti - inflammatory and health effects of exercising in air pollution (37), which could inhibit the pro - inflammatory events induced by air pollution . Therefore, the aim of this study was to investigate the effects of 4 weeks of aerobic exercise performed in association with carbon black pm10 exposure on lung tissue inflammation and lung cancer . This study was approved by the tarbiat modares university of tehran (code number: 62.2987). Twenty four adult male wistar rats aged 8 weeks were obtained from pasteur institute of iran and randomly divided into the 4 groups: a; control (without exposure carbon black pm10 and aerobic exercise; n = 6), b; aerobic exercise (five times per week for 4 weeks; n = 6), c; exposure to carbon black pm10 (5 mg / m; per rat; n = 6), d; aerobic exercise concomitantly with exposure to carbon black pm10 (n = 6). Rats were housed in cages under controlled environment (23c and 12 hour light - dark cycle) with free access to normal chow and tap water . Figure 1 shows the inhalation chamber at the laboratory of tarbiat modares university (falonak) where inhalation exposure was carried out . Rats in groups of c and d were exposed to carbon black in the inhalation chamber at nominal concentrations of 5 mg / m for 2 h / day, 5 days per week for a total of 4 weeks . The control rats were exposed to clean, filtered air containing no carbon black for the same period . The concentrations, size and shape of cb particle were monitored once time weekly by grimm aerosol technique (gmbh and co. kg . Dorfstrae 9 - 83404 ainring - germany (and light microscope (acc.v - spot magn . 25.0 animals in b and d groups were adapted to the treadmill for rat (will running treadmill, lafayette american) training for 3 days (15 minutes, 20 m / min). On the fourth day, the individual maximal exercise capacity test was performed with a 5-minute warm - up (6 m / min) and followed by an increase in treadmill speed (3 m / min every 3 minutes) until animal exhaustion (i.e. When they were not able to run voluntarily after 3 mechanical stimuli) (24, 27, 28). The maximal exercise capacity (100%) the speed average of each group was calculated, and then the rats were submitted to treadmill training as a mean speed of the group workload . Rats were trained at low intensity, corresponding to 50% of the initial maximal speed obtained in the exercise test, for 60 minutes, five times per week, as previously described (24, 27, 28). After sacrificed rat and total lung tissue, rna was isolated using trizolereagent (qiagen, germany), according to the manufacturer s instructions . The rna samples were subjected to reverse transcription using thermo scientific revert aid first strand cdna synthesis kit (ferementase). In the subsequent step, the cdnas were used as templates to perform real - time pcr using sybr green pcr master mix (sybr green i,) by step one abi system (applied biosystem). The crossing threshold values assessed by the real - time pcr were evaluated for the transcripts and normalized to the results for gapdh mrna . The corresponding primer pairs for tlr4, nf-b, and tnf- and gapdh (housekeeping gene) were listed in table 1 . The threshold cycle (ct) for each specific gene, corresponding housekeeping gene (gapdh) and their differences (ct) were determined and then evaluated gene expression changes using 2 formula . Differences in body weight between pre and post interventions were examined by 2-tailed t - test . In order to determine the significant differences between groups one way anova and lsd post hoc test and kruskal - vallis test after examined normal distribution of data by kolmogorov - smirnov test were used . Twenty four adult male wistar rats aged 8 weeks were obtained from pasteur institute of iran and randomly divided into the 4 groups: a; control (without exposure carbon black pm10 and aerobic exercise; n = 6), b; aerobic exercise (five times per week for 4 weeks; n = 6), c; exposure to carbon black pm10 (5 mg / m; per rat; n = 6), d; aerobic exercise concomitantly with exposure to carbon black pm10 (n = 6). Rats were housed in cages under controlled environment (23c and 12 hour light - dark cycle) with free access to normal chow and tap water . Figure 1 shows the inhalation chamber at the laboratory of tarbiat modares university (falonak) where inhalation exposure was carried out . Rats in groups of c and d were exposed to carbon black in the inhalation chamber at nominal concentrations of 5 mg / m for 2 h / day, 5 days per week for a total of 4 weeks . The control rats were exposed to clean, filtered air containing no carbon black for the same period . The concentrations, size and shape of cb particle were monitored once time weekly by grimm aerosol technique (gmbh and co. kg . Dorfstrae 9 - 83404 ainring - germany (and light microscope (acc.v - spot magn . Animals in b and d groups were adapted to the treadmill for rat (will running treadmill, lafayette american) training for 3 days (15 minutes, 20 m / min). On the fourth day, the individual maximal exercise capacity test was performed with a 5-minute warm - up (6 m / min) and followed by an increase in treadmill speed (3 m / min every 3 minutes) until animal exhaustion (i.e. When they were not able to run voluntarily after 3 mechanical stimuli) (24, 27, 28). The maximal exercise capacity (100%) was defined as the maximum speed reached by each animal . The speed average of each group was calculated, and then the rats were submitted to treadmill training as a mean speed of the group workload . Rats were trained at low intensity, corresponding to 50% of the initial maximal speed obtained in the exercise test, for 60 minutes, five times per week, as previously described (24, 27, 28). After sacrificed rat and total lung tissue, rna was isolated using trizolereagent (qiagen, germany), according to the manufacturer s instructions . The rna samples were subjected to reverse transcription using thermo scientific revert aid first strand cdna synthesis kit (ferementase). In the subsequent step, the cdnas were used as templates to perform real - time pcr using sybr green pcr master mix (sybr green i,) by step one abi system (applied biosystem). The crossing threshold values assessed by the real - time pcr were evaluated for the transcripts and normalized to the results for gapdh mrna . The corresponding primer pairs for tlr4, nf-b, and tnf- and gapdh (housekeeping gene) were listed in table 1 . The threshold cycle (ct) for each specific gene, corresponding housekeeping gene (gapdh) and their differences (ct) were determined and then evaluated gene expression changes using 2 formula . Results are expressed as mean sd . Differences in body weight between pre and post interventions were examined by 2-tailed t - test . In order to determine the significant differences between groups one way anova and lsd post hoc test and kruskal - vallis test after examined normal distribution of data by kolmogorov - smirnov test were used . Body weight: there were significant differences among the a (control) and c (pm10 exposure only) groups in body weight pre and post interventions . The mean of increase in body weight in c group was higher than a, b and d groups (table 2). Data are presented as mean sd . Significantly different at p 0.05 . Presented data in figure 2 demonstrate that pm10 carbon black exposure increased the gene expression tlr4, nf-b and tnf- in lung tissue compared with all of the other groups (p 0.05) and that aerobic exercise in carbon black pm10 exposure decreased the expression of these cytokine compared with the carbon black pm10 exposure group (p 0.05). 0ne way anova also demonstrated that carbon black pm10 exposure presented a significant effect on the tnf- (p = 0.047) and nf-b (p = 0.014). No significant effect was observed on the gene expression tlr4 by kruskal - vallis test (p = 0.325). Asterisk indicates treatments that are significantly different at p 0.05 compared with all of the groups . Presented data in figure 2 demonstrate that pm10 carbon black exposure increased the gene expression tlr4, nf-b and tnf- in lung tissue compared with all of the other groups (p 0.05) and that aerobic exercise in carbon black pm10 exposure decreased the expression of these cytokine compared with the carbon black pm10 exposure group (p 0.05). 0ne way anova also demonstrated that carbon black pm10 exposure presented a significant effect on the tnf- (p = 0.047) and nf-b (p = 0.014). No significant effect was observed on the gene expression tlr4 by kruskal - vallis test (p = 0.325). Asterisk indicates treatments that are significantly different at p 0.05 compared with all of the groups . In the present study, we demonstrated that aerobic exercise inhibits lung inflammation and pro - inflammatory cytokine release in lung tissue in an experimental model of pm10 carbon black - induced lung inflammation . Considerable epidemiological and toxicological studies have established a clear link between exposure to air pollution particles and adverse pulmonary health effects . Several experimental and human studies have demonstrated that increased levels of air pollution associated with pulmonary inflammation (37, 38). The association between ambient air pollution particles exposure and lung cancer risk has been investigated in prospective studies and the results are generally consistent, indicating that long - term exposure to air pollution may cause lung cancer . Gene expression analysis identified the association with the effect of pm10 carbon black on the innate immune response . Pm10 carbon black exposure increased the gene expression tlr4, nf-b and tnf- in lung tissue . Tlr4 activation is corresponded to nf-b signaling and pathway - specific induction of pro - inflammatory cytokines and chemokines or interferon signaling (39). An extensive research in recent years has been indicated that chronic inflammation leads to various chronic disorders associated with cancer (40 - 42). A central role in the induction of chronic inflammation is played by a set of genes encoding pro - inflammatory cytokines such as il-1, il-2, il-6, and tnf- and monocyte chemotactic protein 1 that are regulated by the transcription factor (nf-b) (43 - 45). These conditions are associated with derangements in the interplay between metabolic and immune processes and inflammation (47). The authors observed significant increase in body weight with pm10 carbon black exposure which could explain the increased inflammation can be caused by pm10 exposure . Studies have suggested that potential anti - inflammatory effects of aerobic exercise could inhibit the pro - inflammatory events induced by air pollution . The anti - inflammatory effects of exercise (48 - 50) have focused on three possible mechanisms: the reduction in visceral fat mass; increased production and release of anti - inflammatory cytokines from contracting skeletal muscle (such molecules are termed myokines (48 - 51); and reduced expression of toll - like receptors (tlrs) on monocytes and macrophages (52). In the present study, aerobic exercise in pm10 carbon black exposure decreased the gene expression tlr4, nf-b and tnf- in lung tissue and also body weight of animals . Although this change in gene expression was slight, however this result can be clinically important . We conclude that low - intensity aerobic exercise presents protective effects from pm10 carbon black - induced lung inflammation . Future studies should therefore be directed towards better defining the mechanism involved in the induction of symptoms by inflammatory molecules and risk of cancer in exposure to air pollution particles.
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A 36-year - old japanese man was referred to us for the evaluation of bilateral retinal hemorrhages . He developed an acute vision decrease in his left eye, pain in his right chest area, and headache after he had trekked at an altitude of 4600 m in tibet for 1 week . Because of the illness, he descended 3 days later, and he felt better at that time but the visual impairment remained . Our examination showed that his visual acuity was 20/20 od and 20/200 os with refractive errors of 0.25 diopters (d) od and 0.50 d os measured 3 weeks after the onset of the visual decrease . Funduscopic examination revealed multiple intraretinal hemorrhages bilaterally and a macular hemorrhage in the left eye [fig . 1]. Sd - oct (spectralis, heidelberg engineering, heidelberg, germany) showed that the macular hemorrhage was in the superficial layer of the retina beneath the inner limiting membrane of the left eye [fig the thickness of the choroidal layer at the fovea was 530 m od and 490 m os which was thicker than the mean subfoveal choroidal thickness in normal subjects of approximately 300 m in the enhanced depth images obtained by spectralis sd - oct . Most recent studies report that the average subfoveal choroidal thickness is 287 m in caucasians of an average age of 50.4 years and 272.6 m in the japanese of an average age of 30.5 years obtained by the enhanced depth imaging of the spectralis sd - oct . Fundus photograph of the right eye (a) and the left eye (b) showing multiple intraretinal hemorrhages and a macular hemorrhage in the left eye optical coherence tomographic images at the first visit . The choroidal layers (white arrowheads) are thick in both eyes (a: right eye, b: left eye). A macular hemorrhage located in the superficial retina beneath the inner limiting membrane of the left eye (white arrow) can be seen individuals with acute mountain sickness present with lethargy, nausea, headache, insomnia, anorexia, and disorientation . The proposed mechanism for the symptoms of acute mountain sickness is respiratory alkalosis from hyperventilation and increased cerebral blood flow . In hace, there is a breakdown of the blood however, hace can lead to long - lasting severe neurological and psychiatric disorders and even death in some cases . Wiedman and tabin examined 40 climbers who had ascended to altitudes of over 4870 m whether they developed signs of har, symptoms of acute mountain sickness, and clinically signs of hace . Thus, they concluded that when advanced har is recognized, treatment should be initiated for hace with oxygen, steroids, or diuretics, and immediate descent to prevent further progression of hace . A pronounced increase in retinal blood flow in mountaineers with retinal hemorrhage and dilated epipapillary network has been detected with heidelberg retina flowmeter after acute hypoxic stress at high altitudes . The increase in retinal blood blow and cerebral blood flow under hypoxic conditions may also be associated with an increase in choroidal blood flow resulting in an increase in choroidal thickness . The pathogenesis of the increased choroidal thickness may be similar to that of hace, namely a breakdown of the blood it is difficult to draw strong conclusions from a single case; however, we suggest that measurements of the choroidal thickness may be useful in evaluating the status of hace . Currently, data are not available from population - based studies on the normal values for the choroidal thickness of a large number of caucasians or asians . Further studies are needed to evaluate the choroidal thickness in mountaineers with acute mountain sickness.
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Multiple surveillance systems are used in the united states each year to characterize seasonal influenza epidemics and to detect unusual events such as infections with novel viruses or those with pandemic potential . These systems track a variety of outcomes, including laboratoryconfirmed influenza hospitalizations, outpatient visits for influenzalike illness (ili), pneumonia and influenzacoded deaths for all ages, pediatric laboratoryconfirmed deaths, and positive laboratory samples . Despite the utility of these existing systems, additional data to estimate disease severity and track illness at the state level were needed during the 2009 h1n1 pandemic, as timely and representative information describing 2009 pandemic influenza a (h1n1) (ph1n1) activity the centers for disease control and prevention (cdc) and the council of state and territorial epidemiologists (cste) established the aggregate hospitalization and death reporting activity (ahdra) as part of an overall national surveillance strategy implemented to collect timely and representative data describing ph1n1 infections in the united states . The ahdra was designed to (i) track severe disease within states and territories to better characterize the focal nature of the pandemic, (ii) track disease trends over brief periods of time to facilitate rapid public health responses to changes in ph1n1 epidemiology, and (iii) accommodate variation in local resources by providing a simple, flexible method that allowed reliable reporting by all states and territories without overwhelming health departments during the pandemic response . In this report, we describe the methods and implementation of ahdra and provide preliminary results from this new surveillance activity . From august 30, 2009, through april 6, 2010, cdc requested weekly reporting of influenzaassociated hospitalizations and deaths from all 50 us states, the district of columbia, new york city, and six us territories . States and territories were asked to identify hospitalizations and deaths in their jurisdictions according to either a laboratoryconfirmed or syndromic surveillance definition and could use either definition to report hospitalizations or deaths . Polymerase chain reaction (rtpcr) testing, direct fluorescent antigen testing (dfa), immunofluorescent antigen testing, or viral culture; identification of influenza type or subtype was not required . Syndromic reports included cases of pneumonia and influenza based on clinical syndrome, admission or discharge data, or a combination of data elements that could include diagnostic laboratory test results . Prior to the first reporting period, 33 jurisdictions indicated they intended to submit laboratoryconfirmed hospitalizations, and 20 indicated they would submit syndromic hospitalizations . Thirtysix jurisdictions intended to submit laboratoryconfirmed death reports, and 17 indicated they would submit syndromic deaths; information describing method of reporting was unavailable for one state and four territories . Jurisdictions were instructed to submit aggregate counts each week by age group (04, 518, 1924, 2549, 5064, and 65 years). Aggregate counts were used to calculate agespecific weekly and cumulative rates per 100 000 according to 2008 postcensal us population estimates . Laboratoryconfirmed and syndromic data were analyzed for relative increase or decrease by state each week, and laboratoryconfirmed cumulative rates were used to describe the age distribution of ph1n1 influenzaassociated hospitalizations and deaths . Owing to differences between laboratoryconfirmed and syndromic reporting definitions, we calculated two national incidence estimates of ph1n1 influenzaassociated hospitalizations and deaths: one extrapolating reports from laboratoryconfirmed jurisdictions to the entire country and one extrapolating reports from syndromic jurisdictions to the entire country . Calculation of rates involving laboratoryconfirmed influenzaassociated hospitalizations and deaths used the populations of states reporting laboratoryconfirmed cases as a denominator; calculations involving syndromic influenzaassociated hospitalizations and deaths used the populations of states reporting syndromic cases as a denominator . Laboratoryconfirmed reports from ahdra were used to estimate weekly, age groupspecific national influenzaassociated deathtohospitalization ratios . These ratios were incorporated into a model used to estimate the national illness burden of influenzaassociated cases, hospitalizations and deaths during the pandemic, accounting for variation in medical careseeking, laboratory practice and detection capability, and underreporting of confirmed cases . All data were maintained in a database on a secure server at cdc, and all analyses were performed using microsoft excel and sas v 9.1 (sas institute, cary, nc, usa). This activity was determined by cdc to be part of routine public health practice and was not subject to institutional review board approval for human research protections . The median number of jurisdictions reporting laboratoryconfirmed hospitalizations each week was 36 (range 2938), and the median number of jurisdictions reporting syndromic hospitalizations each week was 18 (range 1219). The median number of jurisdictions reporting laboratoryconfirmed deaths each week was 39 (range 3040), and the median number of jurisdictions reporting syndromic deaths each week was 14 (range 816). With the exception of 12 weeks at the beginning and end of the surveillance period, only two jurisdictions changed their surveillance definition during the reporting period (one from laboratory confirmed to syndromic and one from syndromic to laboratory confirmed), and only two jurisdictions failed to report for more than 1 week during the reporting period . Number of jurisdictions reporting to the aggregate hospitalization and death reporting activity by surveillance definition and by week august 30, 2009 to april 6, 2010 . In 27 of 36 jurisdictions reporting laboratoryconfirmed hospitalizations for which information was available, the median proportion of hospitals under surveillance was 100% of all hospitals within the jurisdiction (range 18100%). For 16 of 18 jurisdictions using a syndromic hospitalization definition, the median proportion of hospitals under surveillance was 45% of all hospitals within the jurisdiction (range 9100%). Information regarding the type of diagnostic test used to identify cases was available for 24 jurisdictions reporting laboratoryconfirmed hospitalizations and 22 jurisdictions reporting laboratoryconfirmed deaths from september 8 to october 6, 2009 . Sixteen of 24 (67%) jurisdictions employed rtpcr, viral culture, or dfa testing to identify the majority of reported hospitalizations (at least 75% of reported cases in each jurisdiction), and 18 of 22 (82%) jurisdictions used one of these methods to identify the majority of reported deaths . Laboratoryconfirmed cases not identified by rtpcr, dfa, or viral culture were identified using rapid antigen testing or an unspecified diagnostic test . A total of 41 689 laboratoryconfirmed hospitalizations and 2096 laboratoryconfirmed deaths were reported from august 30, 2009, through april 6, 2010 . Weekly laboratoryconfirmed hospitalizations peaked at> 5000 during the last week of october 2009 and declined from that date to <200 by the end of march 2010 (figure 2). Weekly laboratoryconfirmed deaths peaked at nearly 200 during the same week as the laboratoryconfirmed hospitalization peak, before declining to <20 per week by the end of march 2010 (figure 2). The highest laboratoryconfirmed hospitalization rate was observed in the 0 to 4yearold age group, which had a rate 2 to 3fold higher than those observed in the other age groups (figure 3). The majority of laboratoryconfirmed hospitalizations (> 70%) reported to ahdra were in patients <50 years of age, and fewer than 10% were in patients 65 years of age or older . The ahdra weekly laboratoryconfirmed death rate peaked in october 2009 at 0078 and fell to <0001 per 100 000 persons by march 2010 . The highest laboratoryconfirmed death rate was seen in the 5064 year old age group, and 69% of laboratoryconfirmed deaths occurred in patients between 25 and 64 years of age (figure 3). Weekly laboratoryconfirmed and syndromic ph1n1 hospitalizations and deaths reported to the aggregate hospitalization and death reporting activity august 30, 2009 to april 6, 2010 . Estimated rates per 100 000 persons of laboratoryconfirmed and syndromic ph1n1 hospitalizations and deaths reported to the aggregate hospitalization and death reporting activity, by age group, august 30, 2009 to april 6, 2010 . A total of 134 441 syndromic hospitalizations and 13 983 syndromic deaths were reported to ahdra . Weekly syndromic hospitalizations peaked at nearly 7000 during the last week of october 2009 and were distributed in a pattern similar to the weekly laboratoryconfirmed hospitalization curve . Weekly syndromic deaths peaked at 605 approximately 1 month later but did not show a pattern resembling the weekly laboratoryconfirmed death curve (figure 2). The highest rates of syndromic hospitalizations were reported in patients 65 years of age (399 per 100 000), and in patients 04 years of age (255 per 100 000). Greater than 80% of all syndromic deaths reported were in patients 65 years of age, and fewer than 2% were in patients <25 years of age (figure 3). Extrapolating ahdra reports to the entire country yielded cumulative counts of hospitalizations and deaths that estimate what may have been observed had all jurisdictions reported using either a laboratoryconfirmed or syndromic surveillance definition (table 1). Observed and extrapolated * estimates of ph1n1associated hospitalizations and deaths in the united states reported to the aggregate hospitalization and death reporting activity from august 30, 2009 to april 6, 2010 * extrapolated counts were calculated using the direct method of standardization and represent the number of hospitalizations and deaths that would have occurred in the united states if all states had used either a laboratoryconfirmed or a syndromic surveillance definition . Laboratoryconfirmed hospitalization and death rates were calculated by dividing the number of cases by the sum of the state populations for states using a laboratoryconfirmed definition (207 654 216 for hospitalizations; 245 351 708 for deaths). Syndromic hospitalization and death rates were calculated by dividing the number of cases by the sum of the state populations for states using a syndromic definition (96 405 508 for hospitalizations; 58 708 016 for deaths). Both laboratoryconfirmed and syndromic hospitalization and death rates were then applied to the standard population (u.s . Census, july 2008; 304 059 724) to derive extrapolated counts . Although the weekly laboratoryconfirmed deathtohospitalization ratio demonstrated considerable variability especially during the latter part of the surveillance period (3081376%), the cumulative ratio quickly stabilized near its mean of 502% in october 2009 and remained within 1% of this value throughout the remainder of the surveillance period (figure 4). The cumulative age groupspecific laboratoryconfirmed deathtohospitalization ratio was substantially lower for 0 to 18yearolds compared to older age groups and the overall ratio for all age groups (figure 4). Weekly deathtohospitalization ratio from laboratoryconfirmed reports submitted to the aggregate hospitalization and death reporting activity august 30, 2009 to april 3, 2010 . Laboratoryconfirmed data collected by ahdra helped characterize the epidemiology of ph1n1associated influenza hospitalizations and deaths in the united states, revealing a time course and illness distribution for ph1n1 that were substantially different from those seen in seasonal influenza epidemics . Aggregate hospitalization and death reporting activity laboratoryconfirmed peak hospitalizations and deaths occurred much earlier than the typical peak for seasonal influenza activity, which most often occurs during january or february each year ., furthermore, the age distribution of laboratoryconfirmed hospitalizations reported to ahdra was markedly different from typical influenza seasons when hospitalizations are more common among persons over 65 years of age .,, other recent studies corroborate this finding, showing that nearly half of all patients in the united states hospitalized with ph1n1 influenza infections were under the age of 25 years, and <10% were over the age of 65 ., overall, the age distribution of laboratoryconfirmed death rates determined from ahdra data was also markedly different from that seen in typical influenza seasons . In contrast to typical influenza seasons, when 90% of deaths occur in the elderly,, 86% of laboratoryconfirmed deaths reported to ahdra were in persons <65 years of age, with the highest rate found in persons aged 5064 years . Laboratoryconfirmed ahdra data were also useful in monitoring trends in the distribution of illness and age groups over time in specific jurisdictions . Aggregate hospitalization and death reporting activity laboratoryconfirmed data helped define the beginning and end of the 20092010 influenza season and accurately depicted the second wave of ph1n1 illness seen in the fall of 2009; similar doublewave patterns have been seen in previous pandemics .,, ahdra was also instrumental in the detection of and response to a minor third wave of ph1n1 activity in the southeast united states in early 2010 . Ahdra reporting by state and local health departments allowed tracking of trends in severe disease with greater geographic representativeness than would have been possible with existing systems alone and informed decisionmaking at the state and national levels . For example, although the emerging infections program (eip) has conducted populationbased surveillance for laboratoryconfirmed influenzaassociated hospitalizations in the united states since 1995, the eip network of hospitals conducts surveillance in certain counties in only 16 states . Laboratoryconfirmed surveillance via ahdra during the pandemic was implemented in more than twice the number of states as in the eip network . Aggregate hospitalization and death reporting activity laboratoryconfirmed hospitalization and death surveillance was also consistent with data from existing influenza surveillance systems . Emerging infections program hospitalizations peaked at approximately the same time in october 2009 as did ahdra laboratoryconfirmed reports, and the age distribution of ph1n1 hospitalizations described by the two systems was similar, with each identifying the highest rate in the 0 to 4yearold age group and a similar distribution of rates in other age groups . Aggregate hospitalization and death reporting activity data also accurately reflected outpatient influenza illness during the pandemic . Outpatient influenzalike illness surveillance network (ilinet) collects data from over 3000 healthcare providers each week on the proportion of patient visits for ili . * the ilinet weekly percentage peak for the 20092010 season (77% of all patient visits) occurred 1 week prior to the ahdra laboratoryconfirmed and syndromic hospitalization peaks in october 2009 . This approximate 1week lag between ili onset and severe outcome (hospitalization or death) has been noted consistently during both annual influenza seasons and during pandemics . Despite its usefulness during the pandemic, ahdra first, jurisdictions were permitted to report according to different surveillance criteria (e.g., use of a laboratoryconfirmed or syndromic case definition, multiple diagnostic testing methods) and results therefore did not measure identical outcomes . This disparity is evident in the age distributions of laboratoryconfirmed and syndromic hospitalization rates although both reporting methods show similar peaks in the hospitalization rate for the youngest age group, laboratoryconfirmed rates thereafter generally decline with increasing age, while syndromic rates initially decline but then show a dramatic increase for the 65yearold age group . Presumably, the difference is largely because of the relatively low specificity of a syndromic compared to a laboratoryconfirmed definition of influenza infection . Syndromic reporting likely captures many hospitalizations associated with noninfluenza respiratory illness, which often occur with greater frequency in young children and the elderly,,,, while laboratoryconfirmed reporting is much more likely to identify only cases of influenza illness . Thus, it is inappropriate to make comparisons between reporting jurisdictions in ahdra without adjusting for differences in reporting methods and practices . Instead, ahdra s best use may have been to track the progression of the epidemic within each state, a goal consistent with the original intent of the system . A more important consequence of the decision to allow two surveillance definitions in ahdra is the inherent limitation of using a syndromic definition to conduct national surveillance for influenzaassociated infections . Although ahdra syndromic data were useful to track trends of disease within those jurisdictions submitting syndromic reports, they may not otherwise accurately reflect the burden or severity of influenzaassociated hospitalizations and deaths in the united states . Because the proportion of syndromic respiratory hospitalizations and deaths attributable to influenza is small, it is unclear whether syndromic findings in ahdra represent influenza infections or hospitalizations and deaths caused by other respiratory illnesses . Interpretation of syndromic data was further complicated by the limited number of hospitals included in syndromic reports (a median of 45% of all hospitals within each reporting jurisdiction). A further limitation of ahdra is that the system does not collect several potentially useful data elements, such as population denominator information and influenza type or subtype (although> 99% of circulating influenza viruses during the surveillance period were ph1n1). Also, the additional effort required of reporting jurisdictions to conduct ahdra surveillance may be high and may exhaust state and local health department resources during a pandemic . Finally, extrapolated burden estimates derived from ahdra data do not account for variations in medical careseeking, laboratory practice and detection capability, degree of underreporting of confirmed cases, and other population differences across jurisdictions . However, ahdra laboratoryconfirmed data were important components of modelbased estimates that do account for these sources of underestimation . Understanding the impact of ph1n1 influenza hospitalizations and deaths was important to guide the pandemic response and will be important to inform preparedness and response plans for future public health crises . The ahdra was an important component of us influenza surveillance efforts during the pandemic and provided a level of geographic representativeness and timeliness for reporting of severe influenzaassociated outcomes that was not available from existing national surveillance systems . Laboratoryconfirmed reporting in ahdra supplied valuable information to public health practitioners during the pandemic and should inform refinements to seasonal surveillance activities in the coming seasons, as well as revisions of pandemic surveillance plans . Although useful in monitoring trends within jurisdictions, ahdra syndromic reports, as a measure of influenzaassociated hospitalizations and deaths, were difficult to interpret . These data were complicated by limited representativeness and a low specificity for detecting influenzaattributable hospitalizations and deaths among events associated with respiratory illness . Using only syndromic surveillance data to monitor epidemic or pandemic influenza is thus not recommended, particularly in a setting like the 2009 h1n1 pandemic when elderly persons were largely immune because of prior exposures to antigenically related influenza viruses . Because ahdra was implemented within a few weeks time, the system may prove particularly useful as a prototype for a pandemic or epidemic respiratory infection surveillance system that needs to be implemented quickly and efficiently on a national scale . The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc.
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Ganglioneuromas are rare benign tumors of neural crest origin; they arise from peripheral sympathetic ganglia or the adrenal glands . These tumors are most commonly discovered in older pediatric patients or adults; they are discovered incidentally, through thoracic or abdominal imaging studies performed for unrelated conditions . Intracranial involvement of ganglioneuromas is extremely rare (1, 2), and there has been only one report of ganglioneuroma arising from the trigeminal nerve . We report a very rare case of trigeminal ganglioneuroma in the prepontine and cerebellopontine angle cistern, in a 44-year - old female and describe the magnetic resonance (mr) imaging characteristics, including diffusion - weighted imaging (dwi) findings . A 44-year - old female was admitted to our hospital because of a 2-year history of facial pain and numbness in the left side . The patient complained of an intermittent stabbing pain and numbness over the left zygomatic region . A brain mr imaging study was performed with a 3 t unit (magnetom triotim, siemens, erlangen, germany), which revealed a 2 1.8 1.5 cm well - circumscribed mass in the prepontine and cerebellopontine angle cistern on the left side (fig . The left fifth cranial nerve was displaced superomedially, and the seventh - eighth nerve complex inferiorly by the mass . The mass showed homogeneous iso - signal intensity on a t1-weighted image relative to the brain parenchyma (fig . 1a) as well as heterogeneous increased signal intensity on a t2-weighted image (fig . The apparent diffusion coefficient (adc) values were obtained using a circular region of interest with a diameter of 6 mm at 4 nonoverlapping locations in the tumor (fig . The mean adc values obtained with b values of 0 and 1000 sec / mm of the tumor were 0.72 10 mm / s, compared to 3.01 10 mm / s for csf (measured in the fourth ventricle) and 0.71 10 mm / s for adjacent brain tissue (measured in the pons). The preoperative diagnosis for this poorly enhancing mass with a dwi high signal intensity lesion in the prepontine and cerebellopontine angle cistern was an epidermoid cyst ., a well - circumscribed solid mass was found to be firmly attached to the trigeminal nerve . Histopathologic examination demonstrated a benign tumor composed of mature ganglion cells and spindle - shaped cells in an abundant collagenous stroma (fig . Postoperatively, facial pain and numbness disappeared, and follow - up mr imaging, obtained 16 months later showed no evidence of tumor recurrence . Ganglioneuromas are typically found within the posterior mediastinum and retroperitoneum, though less common locations include the adrenal medulla, parapharyngeal region, and visceral ganglia . Rarely, ganglioneuromas have been observed in the tongue, prostate, skin, and bone (3). Intracranial ganglioneuroma is extremely rare, and only a few cases have been reported in previous literature (1, 2). Histologically, these tumors are composed of single or clustered mature ganglion cells and mature schwann cells (3). Typical radiologic findings of ganglioneuroma arising from the peripheral sympathetic nerve have been described . At ct, ganglioneuromas are well - demarcated hypoattenuated lesions, with calcifications in 20 - 42% of cases . These tumors are not significantly enhanced after the administration of contrast materials, but delayed ct images can show slight or moderate enhancement (3, 4). At mr, ganglioneuromas are generally hypointense on t1-weighted images and heterogeneously hyperintense on t2-weighted images with respect to adjacent muscle . (1) reported the first case of trigeminal ganglioneuroma and described conventional mr imaging findings, which are similar to those of thoracoabdominal ganglioneuromas . Mr images included in their report demonstrated a well - defined mass with low signal intensity on a t1-weighted image, high signal intensity on a t2-weighted image, and relatively homogenous enhancement on a post - contrast t1-weighted image . In our patient, the lesion was iso - signal intense to the adjacent pons on the t1-weighted image and showed slightly increased signal intensity on the t2-weighted image . It has previously been reported that ganglioneuromas with markedly high signal intensity on a t2-weighted image histologically consist of a large amount of myxoid stroma and relatively few cellular components and collagen fibers . In contrast, tumors with intermediate to subtle high signal intensities on a t2-weighted image, as in our case, consisted of numerous cellular and fibrous components and few myxoid stroma (4, 5). In our case, the mass showed poor enhancement on a postcontrast t1-weighted image . Thoracoabdominal ganglioneuromas can demonstrate gradually increasing enhancement with dynamic mr imaging (3 - 5). Diffusion - weighed imaging findings and adc values with regard to intracranial ganglioneuroma have not been published previously . Our case showed a homogeneous hyperintense signal throughout the tumor, on a dwi, and revealed a mean tumor adc value of 0.72 10 mm / s . (6) recently reported dwi findings of the ganglioneuromas / ganglioneuroblastomas originating from the thoracoabdominal sympathetic nervous system . These tumors showed relatively high adc values (mean adc: 1.60 10 mm / s, range 1.13 - 1.99 10 mm / s). The differences between gahr's cases and our case may be due to the different tumor histopathologies . One might postulate that high cellular density and scanty myxoid stroma, revealed through histopathology, may be attributed to relatively low adc values in our case . (7) reported that focal central hemorrhages were observed in 2 of 17 adrenal ganglioneuromas . Our case showed no intratumoral susceptibility signals on gradient echo images, as well as absence of intratumoral hemorrhage or calcification with histopathology . An epidermoid cyst should be included in the differential diagnosis of t2-increased signal intensity lesions in the prepontine and cerebellopontine angle cisterns . The high signal intensity of epidermoid cysts with dwi is caused by the intrinsic t2 shine - through effects of the lesion . Reported adc values of intracranial epidermoid cysts range from 0.98 10 mm / s up to 1.36 10 mm / s (8); these are higher than the values of trigeminal ganglioneuroma in our case . Other differential diagnoses of masses in the prepontine and cerebellopontine angle cisterns include schwannoma, meningioma, neurofibroma, and metastasis . The majority of these tumors do not show high signal intensity with dwi and demonstrate relatively strong enhancement on post - contrast t1-weighted images . Other rare tumors that have been reported to occur in the trigeminal nerve include benign triton tumor (neuromuscular hamartoma) and ganglioglioma . Neuromuscular hamartomas were reported to be profoundly hypointense on t2-weighted images and demonstrated mild homogenous contrast enhancement (9). A trigeminal ganglioglioma may be difficult to distinguish from a ganglioneuroma like that in our case . This tumor showed a nonspecific homogenous mr signal intensity indistinguishable from the adjacent brain stem with all imaging sequences and no contrast enhancement (10). Dwi findings of neuromuscular hamartoma and ganglioglioma, arising from the trigeminal nerve have not been reported . Trigeminal ganglioneuroma is a very rare tumor; however, this tumor should be considered in the differential diagnosis of tumors with low adc values in the prepontine and cerebellopontine angle cistern . Trigeminal ganglioneuroma is a very rare tumor; however, this tumor should be considered in the differential diagnosis of tumors with low adc values in the prepontine and cerebellopontine angle cistern.
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The baylisascaris genus of nematodes (family: ascarididae) is comprised of nine recognized species, each parasitizing distinct definitive hosts and a vast array of intermediate hosts (bauer, 2013, kazacos, 2001, sorvillo et al ., 2002). The most widely studied of these is b. procyonis, which primarily utilizes the common raccoon (procyon lotor) as its principal definitive host (kazacos, 2001). Embryonated eggs of baylisascaris species are infectious to over 90 species of mammals and birds including humans . Larval infections with baylisascaris species can lead to irreversible neural, optical and visceral damage, in both wild animals as well as humans (gavin et al . The severity of pathology of baylisascaris infection in the intermediate host is known to vary depending on the species of the infecting parasite (tiner, 1953). Understanding which species of baylisascaris a host is infected with may provide valuable information concerning prognosis and treatment, as well as the source of the infection . As with other nematodes, baylisascaris species have been traditionally identified through morphometric data . However, distinguishing larval nematodes to the species level is problematic (graeff - teixeira et al ., 2016). With the current widespread availability of molecular tools, the use of genetic analysis to rapidly and accurately identify organisms to the species level is increasingly standard . However, differentiation of closely related species such as b. procyonis and b. columnaris has proven difficult (dangoudoubiyam et al ., 2009, gatcombe et al ., 2010). Extensive previous research has documented the life - cycle, distribution and prevalence of b. procyonis (graeff - teixeira et al ., 2016, additionally, the complete mt genome of b. procyonis, has been sequenced (xie et al . In contrast to the relative abundance of studies on b. procyonis, little is known of its closest relative, b. columnaris . Previous research using copromicroscopic detection has shown b. columnaris infection prevalence of 25% in a population of eastern spotted skunks (spilogale putorius) (lesmeister et al ., 2008) and 25% prevalence in captive striped skunks in europe (d'ovidio et al ., 2017). To our knowledge, the prevalence of b. columnaris in wild populations of striped skunks has not previously been reported . (2013) reported the cloning and sequencing of partial sequences of b. columnaris for the cox1 (413 bp) and cox2 (483 bp) genes . Additionally, in this work franssen et al . Identified three single nucleotide polymorphisms (snps) in the partial cox1 sequence and one snp found in the partial cox2 sequence which are useful in differentiating b. procyonis from b. columnaris . The partial sequence of these mitochondrial genes has provided a reference which has facilitated the differentiation of b. procyonis from b. columnaris by dna sequencing . In addition to the four snps which allow for the molecular differentiation of b. procyonis and b. columnaris, franssen et al . Also reported a number of intragenic differences in b. columnaris mitochondrial sequences suggesting there is a high degree of genetic diversity in this species . In this study we sought to determine the prevalence of b. columnaris in a wild population of striped skunks (mephitis mephitis) in salt lake county, utah, usa . In addition to determining the prevalence of these parasites in a native skunk population we report the complete sequence of 11 mitochondrial genes (cox1, cox2, nd2 and 8 trna genes) comprising 3638 bp of the b. columnaris mitochondrial genome . Results revealed several novel snps in these genes, which further facilitates and improves the molecular distinction between b. procyonis and b. columnaris . Additionally, several intragenic snps were identified in b. columnaris worms . In summary, these results demonstrate the prevalence of b. columnaris in a wild population of skunks and extend the number of known genetic differences between b. procyonis and b. columnaris . Skunks were acquired through nuisance animal calls from the public in salt lake county, utah, usa during the fall and winter of 2013 . Institutional animal care and use committee (iacuc) or ethics committee approval was not necessary, as animals were not sacrificed for research purposes . Skunks were captured in live traps (tru - catch, belle fourche, south dakota, usa). Skunks were euthanized by chemical immobilization with 5/1 ketamine / xylazine followed by intracardiac injection of potassium chloride . The presence of nematode parasites was determined by emptying the small intestinal contents through manual extrusion using finger / grip pressure and the visual examination of intestinal contents . Species determination of collected worms was performed by extracting dna from one or more worms from each skunk and performing dna sequencing of the cox1 gene . Sequences were aligned with the previously published cox1 to determine worm genus and species (genbank: kc543474.1) (https://blast.ncbi.nlm.nih.gov). A total of 34 worms were collected and the cox1 gene of 22 of these worms was sequenced . A single worm from several animals was then used for more extensive dna sequencing of a number of genes as described below . Pcr primers were designed based on the mitochondrial genome of b. procyonis (ac . No . Primers were designed to flank the gene of interest by 100300 bp from the 5 and 3 ends . This primer design resulted in the amplification of segments of dna which contained the complete gene of interest (cox1, cox2, nd2) as well as several small trna genes contained at the 5 and 3 ends of each amplicon (table 1). Three separate amplicons were generated with amplicon 1 containing the sequences for the trna genes q, r, i, s, and l, as well as the complete nd2 gene . Amplicon 2 contained the complete cox1 gene and amplicon 3 contained the trna genes d, g, and h as well as the complete cox2 gene . Primer sequences for each amplicon are contained in table 1 . Upon collection of parasites (described above), individual specimens were preserved by immediately freezing each worm at 80 c. for genetic analysis, specimens were thawed and a 2.0 mm portion of each worm macerated with a razor blade . The macerated tissue samples were then placed in a 1.5 ml microcentrifuge tube and dna extracted using the qiagen dneasy blood & tissue kit (qiagen inc ., pcr reactions were carried out in a final reaction volume of 50 l containing 2 l of dna, 25 l of onetaq dna polymerase (new england biolabs, ipswich, ma usa), 0.5 l of each primer with 22 l of molecular grade water . Polymerase chain reaction conditions used to amplify the cox1 and cox2 containing amplicons (amplicons 2 and 3), involved a 30 s initial denaturation at 94 c followed by 30 cycles of denaturation at 94 c for 30 s, annealing at 55 c for 30 s, and elongation at 68 c for 80 s, followed by a 10-min final extension at 68 c . The pcr amplification of the nd2 target region (amplicon 1) was identical to the aforementioned profile, with the exception of the annealing temperature being 63 c . The pcr amplified products of interest for each amplicon were then extracted and gel purified using the wizard sv gel and pcr clean - up system (promega madison, wi). The sequencing of each amplicon was performed using primers designed to bind approximately every 500 bp (supplemental fig . 1). Cycle sequencing was performed by the brigham young university dna sequencing center using an abi 3730xl automated sequencer . Primers were designed using the forward dna strand and sequencing was performed on a single strand . Contigs for each sample were assembled by mapping sample reads to the mitochondrial genome of b. procyonis (ac . Sequence reads were determined using geneious software (biomatters limited, san francisco, ca, usa). Snp analysis, multiple alignments, and prediction of transmembrane and cytoplasmic regions were performed using geneious software . We first sought to determine the prevalence of b. columnaris in a wild population of skunks . The intestinal tracts of 16 skunks were collected and the prevalence of nematode infection determined by gross examination of the intestinal contents . Ten of the 16 skunks examined were found to be infected with roundworms, with numbers of worms ranging from 1 to 10 in infected animals (table 2). All worms were identified as b. columnaris based on gross morphology and confirmed by detailed sequence homology to the published partial sequence of b. columnaris cox1 (genbank: kc543474.1). Due to the essential function of cox1, this gene is present in a wide variety of organisms yet has sufficient variation to distinguish closely related species (hebert et al ., 2003). Consequently, the sequence of the cox1 gene is a frequently used marker for population genetic and phylogenetic studies (ai et al ., 2011, xie et al ., 2011b). In this study, the complete cox1 gene (1578 bp) of eight baylisascaris worms isolated from eight different hosts our analysis revealed 11 novel loci, which consistently distinguished b. columnaris from b. procyonis, including the three previously reported by franssen et al . The majority of these species specific snps are found in the 3 portion of the gene with 8 of the 11 being found between nucleotides 1002 and 1506 of the cox1 gene . Previously published work has shown a relatively high degree of intraspecies heterogeneity in the cox1 gene of b. columnaris (franssen et al ., 2013). In our analysis of cox1, 11 total intraspecies snps were identified 10 of these having not previously been reported (fig . 1). Previous sequencing of a partial sequence of the b. columnaris cox2 gene has shown one specific snp which was useful in differentiating b. procyonis from b. columnaris as well as three intragenic snps (franssen et al ., 2013). These previous studies were done in a european population of skunks . In our sequencing analysis of a north american population, we found six intragenic snps within the cox2 gene . Importantly, our data showed that none of these nucleotide variations were species specific, whereas previous analysis had suggested the snp at position 168 was useful in differentiating b. columnaris from b. procyonis (fig . 2). There have been no previous reports of sequencing of the b. columnaris nd2 gene . In our sequencing of this gene and subsequent analysis, in addition, two intraspecies snp were identified in the nd2 gene of b. columnaris (fig . Alignment of eight of the b. columnaris concatenated sequences and homologous regions of b. procyonis were used to determine the sequence of several trna genes from b. columnaris . This analysis revealed two snps and an indel within mt - trna genes which differentiated b. procyonis from b. columnaris . In addition, five intragenic snps were identified (one of them being in the same position as the indel of trna s (fig . 4)). Based on the high degree of similarity between these organisms, we were initially skeptical of this degree of variation in trna genes . Therefore, we next compared the trna genes from all mt - dna genomes of sequenced baylisascaris species . This comparison demonstrated a relatively high degree of sequence variation in the trna genes of several closely related baylisascaris species (fig . This level of heterogeneity in trna sequences from numerous baylisascaris species lends confidence in this unanticipated level of heterogeneity if trna genes . Additionally, concatenated sequences were used to generate maximum likelihood relationships of b. columnaris with other ascarid species . Results of these analyses demonstrated maximum likelihood relationships in agreement with previous findings (franssen et al ., 2013). Both skunks as well as raccoons commonly live in urban areas facilitating human contact and potential ingestion of embryonated eggs which can cause visceral, ocular and neural larval migrans (roussere et al ., 2003, the molecular identification of b. columnaris was not possible due to the lack of any dna sequences in public databases . In 2009, a partial (529 bp) sequence of the b. columnaris cox2 gene was generated (dangoudoubiyam et al ., 2009). In 2013 work by franssen et al . Resulted in partial sequences for b. columnaris cox1 (413 bp) and cox2 (483 bp) genes (franssen et al ., the availability of these sequences has facilitated the molecular identification of these parasites for us as well as other researchers (d'ovidio et al ., 2017). In this study, we utilized gene sequencing to identify roundworms as b. columnaris . In this population of wild skunks we then extended earlier findings by sequencing a total of 3638 bp of the mitochondrial genome of b. columnaris . In so doing several novel snps were identified, facilitating the molecular discrimination of b. columnaris from b. procyonis . These snps provide additional species specific targets for the molecular differentiation of b. columnaris from b. procyonis . Importantly this data demonstrate that three previously reported species specific snps do not accurately differentiate b. procyonis from b. columnaris (168 of cox2 and 804 and 834 of cox1). All of these snps involved transitions between the purines g and a. this unique finding, compared to previously published work, is likely due to the previous study being done in the netherlands, while skunks (and by extension parasites of skunks) are native to the americas . Parasites infecting european skunks are likely from a relatively small founder population of nematodes infecting skunks transported outside of north america . It is logical to assume that the nematodes (as well as the skunks) would have less genetic diversity than a native, free roaming population of animals . A recent study on the prevalence of b. columnaris in captive european skunks found that 25% of skunks tested were infected with b. columnaris (d'ovidio et al . Data indicates that in our study area there is a much higher infection rate of striped skunks than reported in studies of captive striped skunks in europe . This is likely due to wild animals encountering other wild skunks as well as infected intermediate hosts more commonly than captive skunks . As expected, more intragenic variation of parasites was observed from this population of native wild skunks compared to the imported non - native population studied previously (franssen et al ., 2013). The higher prevalence of parasites in our study compared to a previous study of spotted skunks (lesmeister et al ., 2008) is likely due to the difficulty in accurately identifying infected animals through fecal analysis compared to our method of visual inspection of the small intestine . Additionally, several of the animals in our study had very few worms infecting them . Animals infected with a single male worm would not be detected by microscopic analysis of feces but would easily be identified through visual inspection on the intestinal contents . Differences in the susceptibility to baylisascaris infection by these two types of skunks, as well as other environmental factors may also play a role in variations in infection prevalence . In summary, in this study several novel intragenic snps were identified . Additionally, nine novel polymorphisms are identified which aid in the molecular differentiation of b. procyonis from b. columnaris . Three polymorphisms, which were previously thought to differentiate these two species were shown to in fact be intragenic rather than species specific snps.
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The epithelial ovarian carcinoma is one of the most fatal gynecological cancers across the globe . In spite of early recovery by surgical and chemotherapy treatments, the database globcan related to the world health organization (who) has reported incidence of about 192000 cases in the world, in the year of 2000 . 6000 cases of the mentioned cases have occurred in the uk, and 21000 cases in the u.s . For treating the disease, the tumor will be removed by surgical procedures and then chemotherapy would be started with platinum - based chemotherapy (cisplatin and carboplatin), which treating regime includes cisplatin and carboplatin with the drugs such as paclitaxel, docetaxel, cyclophosphamide, and doxorubicin . In some of the patients, the disease relapses after 6 months of chemotherapy; this condition is defined as platinum resistant, in which treatment would be continued with drugs such as topotecan and etoposide . Etoposide, as other chemotherapy agents, has many side effects such as bone marrow suppression, granulocytopenia, thrombocytopenia, mucositis, moderate to severe esophagitis, hepatotoxicity, metabolic acidosis, and anemia . The complications of anticancer drugs have caused scientists to try two approaches to solve the problem: developing new drugs with fewer side effects and application of new drug delivery systems with high specificity to cancerous tissues; the second approach has lower costs and more attention nowadays . Solid lipid nanoparticles (slns) are one of the most important nanosized drug delivery systems that were introduced about two decades ago . Slns that are often considered for intravenous application are colloidal submicron carriers sized 50 to 1000 nm and composed of solid lipids dispersed in water or surfactant aqueous solution . These nanoparticles have particular features like small size, high surface area, and high loading of drug that makes them potent and beneficial carriers for improving drug efficacy [5, 6]. Slns are similar to o / w emulsions used for total parenteral nutrition; the difference is that emulsion liquid lipid has been replaced with a solid lipid . Slns have advantages such as controlled drug release in considered site, excellent biocompatibility, increase in drug stability, high drug content, easy industrialization and sterilization, better control of drug release kinetics, high bioavailability for bioactive drugs, chemical protection of sensitive drugs, easier producing rather than biopolymeric nanoparticles, producible by common emulsification methods, long - time stability, and various applications [4, 7, 8]. For parenteral administration, slns with appropriately small particle size less than 200 nm can be sterilized using filtration . Autoclaving the finished dispersion is not practical as the lipids melt at sterilizing temperatures and the molten lipid droplets coalesce . Therefore just aseptic manufacturing processes following sterilization of the starting materials by gamma irradiation of the final dispersion or exposure to ethylene oxide (eo) gas are applicable for their sterilization . Bacterial endotoxins in raw materials need to be monitored, especially when raw materials are of natural origin . It may be possible to lyophilize the sln dispersions, and this lyophile can be irradiated or exposed to eo . Slns are used in transdermal applications, as gene vector carriers, for topical uses, as cosmeceuticals, as targeted carriers of anticancer drugs to solid tumors, in breast cancer and lymph node metastases and in antitubercular chemotherapy . For example, the study of yadav et al . Was performed in the survey of poly(lactic - coglycolic acid)-monomethoxy - poly(polyethylene glycol) and poly(lactic - coglycolic acid)-pluronic block copolymers and the study of reddy et al . On nanoparticles produced by tripalmitin could be mentioned . Hyaluronan (figure 1), available in the market as sodium hyaluronate (ha), is a high molecular weight glycosaminoglycan present in extracellular matrix and is necessary for cellular growth and structural stability of organs and tissue structure . Ha regulates cell proliferation and movements by interacting with cd44 receptors and receptor for ha mediated motility (rhamm). Because of overexpression of cd44 receptors by cancer cells, interfering in cd44-ha interaction by targeting drugs at cd44 is an effective strategy to treat cancers . Ha bound to nanoparticles, in addition to its targeting role, may act as a protecting agent of nanoparticles against body phagocytosis system [1113]. The mentioned method has been used to deliver agents such as doxorubicin, epirubicin, paclitaxel, mitomycin c, sirna, and dna . To our knowledge there is not any report on the application of the hyaluronate targeted slns in drug delivery of etoposide in sk - ov-3 cells although there are some studies on the hyaluronate targeted slns . This study alongside with thousands of similar ones could help to introduce new clinically applicable drug delivery systems with appropriate physicochemical properties, successful targeting, and enhanced cytotoxicity in the future . This study was performed in order to evaluate cytotoxicity of ha targeted slns containing etoposide, prepared and optimized in our previous study in sk - ov-3 cells . Stearylamine (sa), dodecylamine (dda), cetyl alcohol, dialysis bags with molecular weight cut - off of 12400 da, and thiazolyl blue tetrazolium bromide (mtt) were from sigma - aldrich company (us). Acetone, dichloromethane, and tween 80 were from merck chemical company (germany). Rpmi 1640 culture medium, penicillin - streptomycin, and fetal bovine serum were from paa company, austria . Sodium hyaluronate (mw = 6,400 da) was from lifecore biomedical (us) and sk - ov-3 cells were from pasteur institute (iran). Slns were produced by emulsification - solvent evaporation method . According to the results of our previous study, the lipid phase including 30 mg etoposide, 30 mg cetyl alcohol, and 30 mg sa was dissolved in 1.8 ml of 1: 1 mixture of acetone - dichloromethane . Then the mentioned solution was added during 3 minutes to the 18 ml of tween 80 solution (1% w / v) in deionized water, while stirring in 1200 rpm . Ultimately, produced nanoemulsion was stirred in 600 rpm in room temperature for 75 minutes to evaporate the solution . After 15 minutes of adding organic phase to aqueous phase, ha dissolved in deionized water containing tween 80 (1% w / v) was added to nanoparticles mixture during 5 minutes, while stirring at 600 rpm, in order to produce targeted nanoparticles . Nonbound ha was separated from nanoparticles mixture by dialyzing versus 100 ml deionized water containing tween 80 (1% w / v) using dialysis bag with molecular weight cut - off of 12,400 da for 40 minutes so that the deionized water containing tween 80 (1% w / v) was replaced every 10 minutes . To determine the amount of ha bounded to slns after separation of unbound ha, some part of the targeted nanoparticles mixture was dried under vacuum and subjected to elemental analysis (chn) (chns-932, leco, usa) and, by subtracting the total amount of ha from gaining value, the amount of ha bound on the slns surface was calculated . The particle size, polydispersity index, and zeta potential of nanoparticles were measured by a zetasizer (zetasizer 3000; malvern instruments, malvern, uk), after 1: 10 diluting the samples with deionized water . The loading efficiency percent was determined by centrifugation (eppendorf 5430 centrifuge, germany). The dispersion of nanoparticles was poured in centrifugal filter tubes (amicon ultra, ireland) with a 10 kda molecular weight cutoff to separate the aqueous medium . The concentration of free etoposide in the filtrate was determined by measuring its absorption in 276.4 nm (uv - vis spectrophotometer, shimadzu scientific instruments, japan) and converting the absorbance to concentration using the calibration equation of etoposide in aqueous phase containing 1% w / v of tween 80 . The amount of encapsulated drug was computed indirectly by calculating the difference between the total amounts of drug used in preparation of nanoparticles and the free drug . Ultimately, loading efficiency percent was computed by the following equation: (1)loding efficiency percent = (total drug weightfree drug weight)total drug weight100 . Drug release profiles from the npls were determined in phosphate buffer saline (pbs, 0.01 m, ph 7.4 containing 1% w / v tween 80) at 37c . A total of 2 ml of npls suspension was placed in dialysis bag with molecular weight cut - off of 12,400 da and suspended in a beaker containing 50 ml of pbs on a magnetic stirrer with a speed of 200 rpm . Samples were withdrawn periodically and replaced with the same volume of pbs at the same temperature . The content of etoposide in the samples was determined spectrophotometrically at 268.7 nm . To determine cell proliferation, a total of 180 l of the cell suspension (5 10 cells / ml) were placed in each well of a 96-well plate except for one row for blank that was filled by an equal amount of medium . After a 24 h period of incubation at 37c in a co2 incubator with 5% co2 and 95% humidity, all 4 wells of cells were treated with 20 l of one of the concentrations of etoposide as much as 0.475, 0.95, 1.9, and 3.8 m of etoposide . The ic50 of etoposide for sk - ov-3 cells was determined to be 1.9 m . In order to assure that microorganisms would not be able to contaminate the slns and interfere with cytotoxicity results, preparation of solution of free drug and also preparation and dilution of slns suspensions it should be pointed out that solutions of organic and aqueous phases were presterilized by ultraviolet germicidal irradiation method . Treated groups included either a solution of free drug in 1 w / v% aqueous solution of tween 80 or encapsulated drug in nontargeted and targeted nanoparticles, with blanks of nontargeted and targeted nanoparticles, while culture medium and tween 80 1 w / v% (each one in 8 wells) serve as control groups . The cells were incubated for further 48 h. after the treatment, 20 l / well of the mtt solution (5 mg / ml of pbs) was added to the cells and incubated for 3 h; then the supernatant was removed carefully and the formazan crystals were dissolved by adding 150 l of dmso . Finally, the absorbance of each well was measured at 570 nm by an eliza plate reader (stat fax 2100 microplate reader, awareness technology, inc ., us). The effect of each treatment on cell viability was calculated by comparing the relative absorbance of treated cells against the respective controls, using the following equation: (2)cell survival% = (mean absorbance of each group mean absorbance of blank) (mean absorbance of negative control mean absorbance of blank)1 100 . First, 2700 l of the cellular suspension with the concentration of 10 cells / ml was poured into 10 wells of a 12-well plate containing lamels at the bottom and then incubated for 48 h in co2 incubator . Then the nontargeted and targeted nanoparticles were loaded with sodium fluorescein instead of etoposide by the same method as mentioned above for drug - loaded slns . The final concentration of loaded sodium fluorescein in nanoparticles was 1 mg / ml . Blank nanoparticles were also prepared but without sodium fluorescein . To prepare free sodium fluorescein solution, 10 l of stock solution (100 mg / ml) was diluted to 1 ml to provide the final concentration of 1 mg / ml . Finally, 300 l of each sample was added to 2 wells (one for imaging in the 1st hour and the other for imaging in 4th hour) and was incubated . Lamels were withdrawn and imaging was performed by visible fluorescence microscope (olympus, ix71, japan). All data are the results of three separate experiments, and the results are expressed as the mean standard deviation (n = 3). Statistical analysis was performed using one - way analysis of variance (anova) and an independent student's t - test with the spss software (version 18, us). The particle size of nontargeted and targeted slns was 179.6 16.31 and 416.42 31.85, respectively . Zeta potential of nontargeted slns was 11.82 0.52 that changed to 12.65 0.49 after coating with ha . Drug loading efficiency was about 64.92 3.76% and release efficiency percent in 24 h was 65.47 4.68% which is an acceptable value . Slns have generally long - term stability (about 13 years) as small particle size and density close to unity of slns mean that the gravity has little effect on particles in dispersion and the brownian motion is sufficient to maintain colloidal dispersions without creaming or sedimentation . In the present study the presence of physically bound ha and the negative zeta potential of targeted slns may seem to threaten stability, but our unpublished results showed that properties of the mentioned slns suspension did not change significantly within 10 days . However, as freeze - drying is a suitable method to prevent the ostwald ripening and avoid aggregation of slns, we also dried the nontargeted and targeted slns under vacuum with 5% glycerol serving as cryoprotectant and then recovered them by adding deionized water . The results showed that nontargeted slns only needed 5 minutes of stirring at 800 rpm and targeted slns needed twice the stirring at 800 rpm each time for 3 minutes and then 10 seconds of sonication at a power of 30 w, to retrieve their primary properties . . The observed release rate (64.1% in the first 6 hours and 73.1% in 24 hours) could provide appropriate serum concentrations for routine chemotherapy schedules in which the drug (with an iv half - life of 612 hours) is administered once daily . Also the mean diameter of typically 200400 nm is well below the size of the smallest blood capillaries in the range of 5 - 6 m . Furthermore, because of the heterogeneity of tumors and dynamic status of each tumor, it will be very difficult to assume any maximum single value for particles to exploit the enhanced permeation and retention (epr) effect . However, the study of bae and park suggests that the porosity of the blood vessels in tumors is around 400 nm . A tumor - dependent functional pore cutoff size ranges from 200 nm to 1.2 m, but the pore cutoff size of porous blood vessels in the majority of tumors is known to be 380780 nm . Sterically stabilized liposomes of 400 nm in diameter were able to penetrate into tumor interstitium . Accumulation of hyaluronic acid - coated self - assembled nanoparticles with particle size of 400 nm has been reported in the tumor tissue too . The obtained results of mtt cytotoxicity assay have been illustrated in table 2 and figure 3 . All drug - loaded nanoparticles caused higher cytotoxicity compared to the free etoposide at the same concentration and their respective blank slns . The mechanism of enhanced cytotoxicity of drug - loaded lipid nanoparticles has been previously reported [30, 31]. It is well understood that improvement in the cytotoxicity is because of the elevated drug concentrations within the cells . As we can see in figure 3, nontargeted drug - loaded slns have lower cell survival compared to the free etoposide solution . For example, the observed cell survival after treatment with targeted nanoparticles was 36.08 0.88%, while it was 42.73 1.49% and 48.57 1.61% for nontargeted slns and free drug solution, respectively, at the concentration of 1.9 m (p <0.05). The results verified that targeted and nontargeted slns of etoposide have reduced ic50 to 52% and 83% of free drug, respectively (table 2). In a study, saliou et al . Reported that lipid nanocapsules of etoposide reduced the ic50 of the drug from 100 to 2.5 m in h209 cells these lipid nanocapsules also could reduce the ic50 of etoposide to about 430 times in glioma cell lines . In an experiment conducted by nasti et al . Chitosan / triphosphate nanoparticles coated with ha showed the ic50 of about half of the noncoated nanoparticles on murine fibroblasts of l929 and macrophage cells of j774.2 . . Successfully overcame on drug resistance of mcf-7/adr cells with 4.3-fold reduction in ic50 of doxorubicin by sirna polyamidoamine - hyaluronic acid complex . It could be concluded that the internalization of the drug into cells was enhanced when the drug was encapsulated in slns . This phenomenon might be the result of the high affinity of lipid materials of slns for the cell membrane and the nanoscaled size of slns . The correlation between nanoparticles size and intracellular concentration has been observed in the study performed by zhang et al . And their results indicated that the less the particle size is, the more the intracellular drug concentration and cytotoxicity is . In addition, comparing the targeted and nontargeted nanoparticles determines that the cytotoxicity in the targeted nanoparticles has been increased, probably due to the presence of ha on targeted nanoparticles which could interact with cd44 receptors and make them internalized into cells more easily . Cho et al . Have surveyed npls containing docetaxel targeted by ha upon cancer cell line mcf-7 and showed that they were endocytosed by cd44 receptors . After incubating for 1 hour, only targeted nanoparticles made a slight fluorescence in the cells (figure 4). After 4 hours of incubation, the fluorescence was remarkably higher in the cells which were treated by targeted slns rather than those which were treated by nontargeted nanoparticles, and there was no observable fluorescence in cells incubated with pure sodium fluorescein (figure 4). Therefore, it could be concluded that increased cytotoxicity in results obtained from mtt assay has resulted from special uptake of targeted nanoparticles due to presence of ha as targeting agent . Hyaluronate targeted slns containing etoposide increase the cytotoxicity of etoposide in sk - ov-3 cells and could be a valuable method for reducing the prescribed dose and also systemic side effects.
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Simple enones are known to undergo ni - catalyzed cycloaddition with diynes (eq 2). However, these conditions fail to provide any cycloadduct product with tropone as shown in eq 3 . Thus, we focused our investigation on discovering an alternative ni - catalyzed cycloaddition protocol.2 diyne 1 and tropone a were used as model substrates and were subjected to a catalytic amount of ni(0) and a variety of phosphine and n - heterocyclic carbene (nhc) ligands (eq 4). Reactions run with monodentate and bidentate phosphines mostly afforded dimerization of diyne along with traces or low amounts of the desired cycloadduct (table 1, entry 110). However, reactions run with nhcs resulted in good to excellent yields of cycloadduct 1a, which couples the diyne with a single c further optimization led to our final reaction conditions: diyne (1 equiv), tropone (1.1 equiv), 3 mol% of ni(cod)2, 6 mol% of sipr, thf, 60 c, and 5 h. reaction conditions: 10 mol% of ni(cod)2, 20 mol% of l, diyne (1 equiv, 0.1 m), tropone (1.1 equiv), toluene, 60 c, 5 h. determined by gc using naphthalene as an internal standard . The model substrates afforded the desired product 1a along with another isomer 1a in excellent yield and> 90% selectivity for 1a (eq 5). Similarly, cycloaddition of sulfonamide diyne 2 afforded a mixture of major and minor isomers 2a and 2a respectively, which on subsequent treatment with p - bromobenzoyl chloride / net3/dmap afforded major isomer 2a in 74% yield and p - bromobenzoyl derivative of minor isomer (2b) in 13% yield (eq 6) as crystalline solids (figure 2). Surprisingly, 2b (and, therefore, 2a as well) has [576] ring fusion compared to [567] in the case of the major isomer, 2a.5 ortep diagram of 2a and 2b . With optimized reaction conditions in hand, the substrate scope was explored (table 2). The cycloaddition occurred smoothly with the diyne bearing a sulfone backbone to form 3a along with minor isomer 3a. Notably, this diyne is completely unreactive in several reported ni - catalyzed cycloadditions . Although ni catalysts have been reported to catalyze the cycloaddition of nitriles and diynes to form pyridines, diyne 4, which has a nitrile group in the backbone, selectively reacted with tropone to afford the desired cycloadducts (4a and 4a) in excellent yield . Inspired by carreira s work, we performed the cycloaddition reaction with 5, a diyne with a metabolically stable backbone, to give cycloadduct 5a in good yield along with minor isomer 5a. Aryl substituted internal diynes are one of the most challenging substrates in ni / nhc - catalyzed cycloaddition reactions . Nevertheless, the reaction of aryl substituted symmetrical diynes with tropone afforded 6a and 7a in good yields . Interestingly, no minor cycloadduct (6a or 7a) was obtained in these cases . To investigate the effect of electronics on the regioselectivity, we subjected unsymmetrical diynes 8 and 9 to standard reaction conditions; remarkably, exclusive formation of one regioisomer was detected (8a and 9a). The use of different aryl groups (i.e., 3,4-dimethoxyphenyl and naphthyl) on alkyne terminals is also possible (10a and 11a). Due to recent interest in indole bearing novel compounds, diynes 12 and 13 were investigated . Biaryl cycloadducts (12a, 13a) were formed in very good yield and high regioselectivity . Interestingly, the regioselectivity was higher in the case of 3-substituted indole diyne than 5-substituted indole diyne (13a vs 12a). Cycloaddition of phenyl ethyl diyne 14 and phenyl silyloxymethyl 15 afforded regioisomers 14a and 15a, where the carbonyl resides next to the phenyl ring, exclusively, while a diyne with covalently bound -tocopherol can also be easily clicked together with tropone to afford regioselective cycloadduct 16a . An unsymmetrical diyne bearing an internal gem - dimethyl group reacted with tropone to afford an exclusive regioisomer 17a, which suggests that regioselectivity is highly dependent on the substituents on the alkyne units of a diyne rather than backbone . The cycloaddition of unsymmetrical isopropyl methyl diyne (18) afforded products 18a and 18a, where the bulkier group is next to the carbonyl of tropone . Cycloaddition of phenyl isopropyl diyne 19 affords a product where the isopropyl group is away from the carbonyl group (19a), suggesting that electronic factors override the steric factors (19a). Unfortunately, terminal diynes did not afford any cycloaddition product with tropone due to their high propensity to oligomerize under our reaction conditions . Reaction conditions: diyne (1 equiv, 0.1 m), tropone (1.2 equiv), 3 mol% of ni(cod)2, 6 mol% of sipr, thf, 60 c, 5 h. isolated yields (in black), ratio of major and minor cycloadducts (in blue), ratio of major and minor regioisomers (in red). The ratios were determined by h nmr of crude reaction mixture . The lack of general methods to access troponoids prompted our investigation on the ability to convert the cycloadduct to a fully aromatized product . We found that compound 2a can be consistently converted to tropone 20a by a three - step protocol . The hydrogenation of alkene 2a led to a saturated cycloheptanone, which was then subjected to dibromination . Specifically, we studied the catalytic cycles for [ni(ipr)2]-catalyzed cycloaddition of nona-2,7-diyne and tropone with dft calculations . Homocoupling, where two alkynes undergo initial oxidative coupling, and heterocoupling, where an alkyne and the tropone undergo initial oxidative coupling, were both investigated . The free energy changes for the homocoupling pathway are shown in figure 3 . From [ni(ipr)2] complex 21, the coordination of diyne to form intermediate 22 is endergonic by 7.0 kcal / mol . Subsequent intramolecular oxidative cyclization via ts23 requires a 13.4 kcal / mol barrier with respect to 22, generating the metallacyclopentadiene intermediate 24 . This intermediate undergoes a facile 8 insertion (instead of 2 insertion, vide infra) of tropone via ts25, with a barrier of only 9.8 kcal / mol . The 8 insertion produces the eight - membered ring intermediate 26 with the tropone oxygen coordinated to nickel . The tropone piece of complex 26 can coordinate to nickel in four different fashions, generating the complexes 26 to 29 . The four isomers have similar stabilities, and complex 29 undergoes reductive elimination via ts30 to give the product - coordinated complex 31 . Product extrusion from 31 is exergonic by 2.8 kcal / mol to release the product and regenerate the nickel diyne complex 22 . The tautomerization of intermediate complex 29 to 29-enol is endergonic by 5.8 kcal / mol, and subsequent reductive elimination from 29-enol will form the 576 tricyclic product; however, the irreversible reductive elimination via ts30 suggests that the 576 tricyclic product arises from ni - free tautomerization . Free energies (298 k) with respect to 21 are shown in kcal / mol . The calculations indicate that the resting state of pathway a is [ni(ipr)2] complex 21, and oxidative cyclization through ts23 is the rate - limiting step of the catalytic cycle . The overall reaction barrier is 20.4 kcal / mol, which is consistent with the experimental conditions (60 c, 5 h). In contrast, the heterocoupling pathway of [ni(ipr)]-catalyzed cycloaddition between nona-2,7-diyne and tropone displays higher free energies (figure 4). Specifically, from nickel diyne complex 22, the intermolecular oxidative cyclization between alkyne and tropone can occur via ts34, with the carbonyl group of tropone distal to the forming c c bond . This step requires a barrier of 42.5 kcal / mol with respect to the [ni(ipr)2] complex 21, which is much less favorable as compared to the homocoupling pathway discussed above (figure 4). Alternatively, the intermolecular cyclization can occur with the tropone carbonyl group proximal to the forming c c bond, as in ts36 . Ts36 is 31.6 kcal / mol higher in free energy than the resting state 21, which is also less favorable than the productive homocoupling pathway . Therefore, unlike other ni - catalyzed couplings between alkynes and carbonyls, a heterocoupling mechanism is not operative in the [ni(ipr)]-catalyzed cycloaddition between diyne and tropone . Free energies (298 k) with respect to 21 are shown in kcal / mol . Next, insertion of the tropone was investigated, and two probable pathways emerged: a traditional 2 insertion and a distinctive 8 insertion . The transition states of 8 (ts25) and 2 insertion (ts37) of tropone were both located, and their free energies and structures are shown in figure 5 . Interestingly, the 8 insertion is found to be more favorable by 12.3 kcal / mol . Optimized structures, gibbs free energies, and distortion and interaction energies of transition states of 8 insertion (ts25) and 2 insertion (ts37) of tropone . The gibbs free energy changes (298 k) with respect to 24 are shown in kcal / mol . We studied the origins of this preference by employing the distortion / interaction model on ts25 and ts37 . The distortion energy reflects the structural changes from nickel complex 24 or tropone to the corresponding geometries in the transition states, and the interaction energy is the energy of interactions between the distorted fragments, computed as the difference between the activation energy and the total distortion energy . The difference between the distortion energies of ts25 and ts37 is the major reason for the preference for 8 insertion . The distortion energy is 17.8 kcal / mol energy for 24 and 13.7 kcal / mol energy for tropone to achieve the distorted geometries in ts25, while it requires much larger distortions (24.3 kcal / mol for 24 and 16.6 kcal / mol for tropone) in ts37 . Stronger steric repulsions are generated between the nickelacyclopentadiene moiety and tropone in ts37 as compared to those in ts25 . This difference between the steric repulsions eventually leads to the preference of the unconventional 8 insertion, and this is the highest order of poly- insertion so far . The transition states for the 8 insertion of tropone with unsymmetrical diynes were also studied . For the isopropyl methyl diyne (figure 6), the transition state ts38-c1 that contains the bulky isopropyl group on the diyne next to tropone is more stable than ts38-c2 by 0.7 kcal / mol to avoid steric repulsions between the isopropyl group and ipr ligand . For phenyl methyl diyne, the transition state ts39-c1 is 4.7 kcal / mol more stable than ts39-c2 mainly due to the steric repulsions between the phenyl group and the bulky nhc ligand in ts39-c1 . Also, for phenyl isopropyl diyne, the phenyl group is more sterically demanding, and a 4.0 kcal / mol preference to the ts40-c1 is found . Overall, our data suggest that ni - catalyzed cycloaddition occurs via the mechanism shown in figure 7 . The homo - oxidative coupling of diyne on ni(0) forms ni(ii)cyclopentadiene intermediate i that undergoes 8 insertion of tropone to afford seven - membered ring complex ii . Intermediate ii isomerizes from oxygen coordination to -coordination resulting in intermediate iii, which can subsequently isomerize to an -coordinated - ni(ii) complex v through intermediates iv . Finally intermediate v reductively eliminates to give vi, which releases the tricyclic product, vii, and regenerates the ni(0) catalyst . At this point, compound vii can preferentially aromatize via sigmatropic shifts to afford major product viii . However, a minor pathway involves tautomerization of vii to cycloheptatrienol ix, which undergoes further 6 electrocyclization to afford an interesting bis(divinyl)cyclopropane intermediate, x. intermediate x can either revert to ix or irreversibly rearrange to [576] fused intermediate xi, which undergoes further sigmatropic shifts to yield the observed minor product, xii . This sigmatropic shift could be catalyzed by a trace amount of water through the bridge of one or multiple molecules of water . Due to the uncertainty of the catalyst, we did not perform computational studies on the isomerization of the tricyclic product vii . Proposed mechanism for the ni - catalyzed cycloaddition of diynes and tropone . In conclusion, we have discovered a nickel catalyst that can effectively and selectively incorporate a single c c -bond of tropone in the cycloaddition with diynes . The mechanism of this novel cycloaddition reaction has been investigated using dft calculations . It involves a unique 8 insertion of tropone to form the observed major and minor products . The mechanistic studies to further understand this unique reactivity of tropone and application of this chemistry are underway in our laboratories.
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Work - related injuries in the upper extremities are of the most common work - related disorders which are visited by general practitioners and specialists in occupational medicine (1). In studies conducted in different countries, these injuries account for the most of reported injuries with the occupational settings (2,3).these injuries have a negative impact on the quality of life of these people . Quality of life is the most important index in the evaluation of therapeutic interventions in the life of these people (4); most of treatment programs for patients with upper extremity injuries and even impairment measure the quality of life and rehabilitation based on their individual performance and pay less attention to other aspects of quality of life of these people (5). Upper extremity injuries are responsible for more than one - third of all injuries in the united states and its prevalence is 4.3 percent per 3,000 people . It is also responsible for 30 percent of all injuries and damages resulting to lost workdays (6). Studies have shown that people who suffer from upper extremity injuries experience emotional and psychological stresses due to various reasons such as pain, difficulty in performing daily activities, dependence on others and getting help from them to do their affairs, uncertainty of outcomes in the future and the appearance of their injured limb . These stresses together with the loss of job and the high cost of treatment may have serious effects on their quality of life (7). According to the research conducted by the researcher, no study on quality of life after upper extremity injuries resulting from occupational accidents has been done yet in iran and given the importance of this issue and its economic burden on individuals, families and society and the resulted reduced quality of life, this study evaluates the quality of life after upper extremity injuries resulting from occupational accidents . The aim of this study was to assess quality of life in patients with upper extremity injuries caused by work - related accidents . Rasoul akram hospital is one of tehran's most important centers for doing studies on the work - related diseases and injuries . One of the cases assessed in this center is to determine the level of impairment among employed individuals following traumatic injuries during labor . Given the possibility of examining and interviewing with these patients at this center, people referred to the center for determining their impairment in 2011 were examined and individuals with work - related upper extremity injury included in the study . All patients were examined by a specialist in occupational medicine and the rate of their impairment was assessed based on the american medical association book (ama) and the whole person impairment (wpi) and the results were recorded (8) and those with no upper extremity impairment excluded . In this cross - sectional study, after selecting patients, their demographic characteristics were recorded through asking questions from patients and studying their hospital records . Then their wpi calculated (assessed) and other information, such as return of work, location of injury, work experience and type of injury were all recorded . Then in order to evaluate the quality of life of patients, sf36 questionnaire (short form of health survey) this questionnaire is a tool to assess health - related quality of life developed in the united states (9) and the psychometric analysis of its translated version was used in various populations (10 - 16). The mean duration of working shift in participants was ranged from 8 - 14 hours . The sf-36 questionnaire is consisted of 36 questions with following 8 scales: physical function with 10 items, functional limitations due to the physical health with 4 items, general health with 5, vitality (vt) with four, social function with 2, role limitation due to the emotional problems with 3 and mental health with 5 (9). Another item that showed health changes for one year was also assessed by sf-36 questionnaire which ultimately examined both physical health (physical performance, physical limitations, physical pain, general health) and mental health (vitality, social performance, emotional problems, and mental health). 0 reports the worst and 100 reported the best condition in the relevant scale (17). The questionnaire was translated to persian by montazeri et al . In 2005, and its reliability was also determined (18). The standard deviation were used for descriptive data and the chi - square used to compare qualitative variables and t - test and anova applied to compare the quantitative variables . Also correlation test was used to examine the relationship between wpi and the quality of life of patients . Finally, the main variables affecting quality of life were analyzed using multiple logistic regressions . In this study, the confidence level (=0.05) and the test power (b=% 20) were considered as 95% and 80%, respectively . Principles of this study were approved by the ethics committee of the tehran university of medical sciences and patients included in the study with the consent and the patients personal information kept confidential by the researcher . The standard deviation were used for descriptive data and the chi - square used to compare qualitative variables and t - test and anova applied to compare the quantitative variables . Also correlation test was used to examine the relationship between wpi and the quality of life of patients . Finally, the main variables affecting quality of life were analyzed using multiple logistic regressions . In this study, the confidence level (=0.05) and the test power (b=% 20) were considered as 95% and 80%, respectively . Principles of this study were approved by the ethics committee of the tehran university of medical sciences and patients included in the study with the consent and the patients personal information kept confidential by the researcher . Patients included in this study were 203 individuals in number with their mean age of 32.16 years (sd=8.78). The level of education of these patients, revealed that 5 patients were illiterate (2.5%), 39 primary school education (19.2%), 78 had completed secondary school education (38.4%), 75 diploma degree (36.9%) and associate s degree (3%). 114 (56.2% of patients) dominant hand injury, and 89 (43.8%) did not have such an injury . Wpi percentage of patients ranged from 1 to 77% and its average was 21.87% (sd=14.84). The mean duration between the incidence of event and being visited by the physician was 8.45 months ranged from less than one to 250 months . Also the patients work experience ranged from 1 to 480 months and its average was 45.31 months . Results of quantitative variables are presented in table 2 and the results of assessing the factors affecting quality of life of patients using sf-36 questionnaire are shown in table 3 . Between 8 - 12 years of education in school at least 12 years of education in school the patients were divided into two groups according to median age on understudy population (30 years) into the age of 30 years or less and above 30 years and the various aspects of quality of life in these two age groups were not tatistically significant . The relationship between the level of education and quality of life in anova test was not also statistically significant . The t - test for two groups of high school diploma and lower degree showed, no significant difference in terms of their quality of life . In assessing the location of the injury in upper extremity, no difference observed between injuries of right, left and both hands in terms of quality of life . Also the quality of life of patients with dominant hand injury, based on sf-36 questionnaire, was similar to others with no significant difference . As far as marital status was concerned, no difference was observed between the quality of life in both single and married people after injury . Also, the number of children had no affect on the quality of life . When the quality of life of those who had no children was compared with the quality of life of married ones with children, when the relationship between work experience of patients and their quality of life was assessed, only the patients social performance score showed a direct and weak correlation with their work experience (p=0.016, r=0.168). Comparison of quality of life in sharp and blunt injuries showed that social performance in patients with sharp injury was 42.73% (sd=25.63) and 32.98% (sd=24.23) (p=0.031) for blunt injury and the observed difference was statistically significant . The percentage of pain in people with sharp injuries was 37.89% (sd=27.94) and with blunt injury 29.64% (sd=21.66) (p=0.077). Also, the emotional health of individuals in people with sharp injuries was 48.20% (sd=22.61) and with blunt injuries 40.13% (sd=24.05) (p=0.051). Percentage of patients impairment (wpi) was inversely associated with various aspects of quality of life such as physical performance (p<0.001, r=-0.26), limitations due to physical health of patients (p<0.088, r=-0.12), social performance of patients (p=0.001, r=-0.24), limitations due to the emotional problems of patients (p= 0.059, r=-0.13), pain (p=0.005, r= -0.2), the emotional health of patients (p=0.006, r=-0.29), the amount of energy / fatigue in patients (p<0.001, r = -0.29) and the patient's general health (p<0.001, r=-0.27). The total score of sf-36 was significantly and inversely associated with the wpi (p<0.001, r= -0.319). In regression analysis of factors affecting life, only wpi of patients had significant and adverse correlation in the model (table 4). Upper extremity injuries can affect a person's quality of life to the extent that the person may even decide to commit suicide, thus investigations on these patients is necessary (19). In a study it was shown that the physical and mental health of these individuals requires proper rehabilitation to enable them to return to the normal life . Also, the importance of treatment procedures to improve the functional status of these individuals is very important (20). Patients examined in this study were mostly male (98%) with the mean age of 32 years . It shows that their mean age was higher than the mean age in previous studies and in most studies upper lime injuries were mostly found in patients with 24 years old or less (23 - 21). In studies conducted in other part of the world, given the inexperience of the young people in the early years of working, the rate or possibility of injuries was higher . Given that the average work experience of patients in our study was less than 4 years, the results of our study were consistent with the results obtained by the research conducted in the world . Also in this study, similar to previous studies, the rate of upper extremity injuries were higher in men than women (24). Type of damage in 48.3 percents of patients was sharp, in 23.2 blunt and in 25.6 sharp and blunt and 10 percent had amputation . Average percentage of whole person impairment (wpi), was approximately 22% and patients, on average, examined within 9 months after injury . The highest score associated with physical function in these patients was approximately 64%, while the lowest score related to the physical limitations resulted from physical health (21%). Sf-36 questionnaire has been used in various studies as an index for assessing the quality of life with regard to the health status of the injured people . In a study conducted by briem et al ., among a population considered as both control and healthy groups, the average score was more than 60 percent and physical and social performance constituted about 90 percent which was much higher than the results of the present study (26). In the study conducted by chen et al ., using the sf-36 questionnaire, mean scores of different indices of quality of life in people with work - related upper extremity injuries were also higher than the present study (27). The lower scores of quality of life index in this study was due to several factors, in which one was the difference in population in different studies . Also, more consequences of such injuries on the individual's life, due to the fear of job loss and less support, can affect the quality of life of these people . In a study conducted by chen et al ., like in our study, most patients were male with the mean age of 35 years . Most patients had non - academic education and demographic variables were somewhat similar to the variables of this study, although in the present study the rate of married participants was higher than the rate of married participant in the study of chen et al (27). In a study conducted by spreeuwers et al ., the quality of life of patients with upper extremity injuries was higher than the quality of life of patients in the present study in most items of sf-36 questionnaire . The mean age of participants was 42 years and the percentage of female patients was higher (48%). Also, many of the patients had non - academic education . In the present study, age, gender, education and marital status of patients the work experience of these people was effective only on their social performance (r=0.168) and those with more work experience had higher social performance scores . As far as sharp injuries are concerned, social performance scores were significantly higher than blunt injuries; also the scores of other quality of life items in patients with sharp injuries were higher, although this difference was not statistically significant due to the greater extent of the affected area and because the blunt injuries were more complex . People, who had a dominant hand injury, had a quality of life similar to others . The reason for that was people with this injury had to use both hands to do their tasks and perform their daily activities, because in most occupations, individuals need to use their both hands even for doing common tasks . Therefore while it is expected that a person with dominant hand injury has a lower quality of life because they are employed and need to use both hands, but it is expected no difference in quality of life based on the dominant or non - dominant hand injuries no difference would be observed in them . Also, in this study, the percentage of whole person impairment (wpi) was inversely correlated with various aspects of quality of life mentioned in the sf-36 questionnaire . The mean duration between the incidence of event and being visited by the physician was 8.45 months, this variable was relatively shorter than expected which could be due to limitations of insurance companies laws in iran . Eventually it became clear that the quality of life in the present study was lower than other similar studies and quality of life for patients was not significantly correlated with demographic variables and social status of patients . Also, the quality of life of patients was significantly and inversely related to the degree of disability . This study was the first study conducted using the sf-36 questionnaire in iran to examine the quality of life in patients with upper extremity injuries resulting from work - related accidents . The results could lead to consider ways to improve the quality of life for these patients as far as physical and mental improvements are concerned, because lower quality of life in these patients, not only had negative impact on their rehabilitation process, but also puts a huge burden on other family members and given that most of these people were the only employed person in the family and they were the only one who provide material needs of the family, delay in their proper rehabilitation, could result in serious harms to that individual and his family . Some patients did not fill the questionnaire and this was one of the problems of this study . It is suggested that the future research studies the quality of life of patients suffered from work - related injuries in different occupational groups . The results of this study showed that percentage of wpi was inversely and significantly associated with various aspects of quality of life . In other words, most aspects of quality of life in particular the emotional health and the amount of energy / fatigue in patients were reduced with the increase in the percentage of impairment (wpi). Given that this study was the first study conducted in iran to examine the quality of life after upper extremity injury resulting from work - related accidents, it is suggested that further studies to be done to mitigate the limitations of this study.
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Focal epilepsies account for about two - thirds of all adult epilepsy patients, and temporal lobe epilepsy (tle) is the most common type of focal epilepsy (1,2). Temporal lobe epilepsy is subcategorized as mesial (i.e., amygdalohippocampal) and neocortical (i.e., lateral) temporal . Mesial temporal lobe epilepsy (mtle) with hippocampal sclerosis (hs) is one of the most common types of epilepsy referred for surgical treatment; it is often refractory to antiepileptic drugs (aeds), but responds favorably to surgery (1,4). Patients with mtle - hs and intractable seizures often experience progressive behavioral changes including increasing memory deficit with the passage of time . In addition, surgical outcome may be worse with longer duration of epilepsy or increasing age at surgery, which suggests that mtle - hs is a progressive disorder (5). Therefore, early detection of this syndrome and its distinction from other tle syndromes has important practical implications with regard to planning an optimal treatment strategy for the affected patient . In the current study, we tried to compare the clinical characteristics of patients with mtle - hs with those who had tle due to other etiologies in order to identify potentially differentiating clinical characteristics between these two groups of patients . In this retrospective analytic study all patients with a clinical diagnosis of tle were recruited in an outpatient epilepsy clinic at shiraz university of medical sciences, which is the only referral epilepsy clinic in south iran . The diagnosis of tle was made exclusively by one epileptologist working at this institution (the first author) and based on clinical grounds (semiology), electroencephalographic (eeg) findings and imaging, such as magnetic resonance imaging (mri). All patients were followed up by the epileptologist at our institution for at least one year (i.e., until may 2014). Routine eeg was requested for all patients at the time of referral . In difficult - to - diagnose or difficult - to- treat patients we often order video - eeg monitoring to ascertain the diagnosis and formulate an individualized treatment plan, accordingly . For all patients, a 1.5 tesla brain mri (epilepsy protocol) was performed . We classified patients as having mts (if they had clear signs of mesial temporal sclerosis and/or atrophy in their mri) and those who had any other mri abnormality . Patients with dual pathology (i.e., mesial temporal sclerosis associated with other structural lesions) were excluded from the study . Age, gender, age at seizure onset (i.e., the first afebrile seizure), seizure type(s), epilepsy risk factors (including pregnancy complications, history of febrile seizure, cns infection, significant head trauma, positive family history of epilepsy), eeg findings (the most informative eeg), and mri findings of all patients were registered routinely . Seizure types were categorized as generalized tonic clonic seizures (gtcs), complex partial seizures (cps) and auras . Pearson chi - square, fisher's exact, and mann - whitney u tests were used for statistical analyses . Until may 2014, 2890 patients with epilepsy were registered at our epilepsy clinic . Four hundred twenty - seven patients (14.7%) were diagnosed as having tle . Of these, 174 patients were eligible to enter the study (105 patients with mtle - hs and 69 patients with tle due to other etiologies). Demographic and clinical characteristics of patients with mtle - hs and those who had tle due to other etiologies are shown and compared in table 1 . Frequency of seizure types (i.e., gtcs vs. cps vs. auras) was not significantly different between these two groups (table 1). Specific types of auras were significantly different between these two groups (table 1), however most auras were reported by both groups, similarly . Epilepsy risk factors were reported to be as follows (mtle - hs vs. others): parental consanguinity in 41 / 30 (p= 0.6), family history of epilepsy in 18 / 10 (p= 0.6), significant head trauma in 15 / 7 (p= 0.4), cns infection in 2 / 2 (p= 0.6) and pregnancy complications in 2 / 3 (p= 0.3). Mtle - hs: mesial temporal lobe epilepsy (mtle) with hippocampal sclerosis (hs). Video - eeg monitoring was available in 155 patients; and 19 patients had a routine eeg only . Eleven patients (10.4%) with mtle - hs and four (5.8%) with other etiologies had normal eeg (p= 0.2). Right sided, left sided or bilateral focal epileptiform discharges (i.e., temporal seizures, spikes or sharp waves) were observed in 36, 38, and 20 patients with mtle - hs and 31, 21 and 13 patients with other etiologies, respectively (p= 0.4). Focal polymorphic delta activity or temporal intermittent rhythmic delta activity (tirda) was seen in 60 and 7 patients with mtle - hs, respectively . These figures were 32 and 4 in patients with other etiologies (p= 0.3). Magnetic resonance imaging (mri) in patients with other etiologies (non- mtle - hs group) showed temporal lobe tumors in 27 patients (39.1%), non - specific white matter mri abnormalities in 11 (15.9%), cavernoma in seven (10.1%), sequels of head injury in six (8.7%), arachnoid cysts in five (7.2%) and other etiologies in 13 (18.8%) patients . Twelve patients had dual pathology (mesial temporal sclerosis in addition to another lesion). Mesial temporal lobe epilepsy with hippocampal sclerosis is a common type of epilepsy referred for surgical treatment due to medical refractoriness . As a matter of fact however, it has been suggested that early surgical intervention after seizure onset is an important precondition for achieving seizure - free status after surgery (6). Therefore, early detection of this syndrome has important practical implications with regard to planning an optimal treatment strategy for the affected patient, particularly those with medically - refractory seizures . In the current study, we observed that earlier age at epilepsy onset (i.e., teenagers compared to young adulthood), a past history of febrile seizures and reporting affective auras (e.g., fear, anxiety, depression, joy, and anger) were commonly seen in patients with mtle - hs, while auditory auras were more frequently reported by those with tle due to other etiologies . In previous studies dominantly represented by patients with hs on mri, it has been observed that age at onset is variable (7). The finding that age at onset is a potential clinical variable to distinguish mtle - hs from tle due to other etiologies should be further explored in future studies . Several studies have shown a significant relationship between a history of febrile seizures in early childhood and mesial temporal sclerosis . In one study, it was observed that hippocampal t2 hyperintensity after febrile status epilepticus represents acute injury often evolving to a radiological appearance of hs after one year (8). Having a past history of febrile seizure is a useful historical clue in favor of mtle - hs compared to tle due to other etiologies . As the first ictal symptoms, auras can provide important localizing and lateralizing information useful in determining the location of the epileptogenic zone (9). The observation that, affective auras (e.g., fear, anxiety, depression, joy, and anger) were commonly seen in patients with mtle - hs, while auditory auras were more frequent among those with tle due to other etiologies, has very important clinical implications . In one study, the authors examined the relationship between presence of different types of auras and post - surgical outcomes in 157 patients with medically intractable mesial temporal lobe epilepsy (mtle) with unilateral hippocampal sclerosis (hs). The occurrence of multiple auras was not associated with post - surgical outcome (p = 0.7). But, the presence of extratemporal auras (e.g., somatosensory, visual and dysphasic auras) was significantly higher in patients with poor outcome (10). Designing future studies to investigate the relationship between presence of mesial temporal lobe auras (e.g., psychic symptoms, cognitive and affective auras, autonomic auras such as epigastric sensation, olfactory and gustatory auras) versus lateral temporal lobe auras (e.g., vertiginous and auditory auras) and surgery outcome in patients with mtle - hs is necessary . The mainstay for making a correct diagnosis, when evaluating a patient with seizure, is having a standardized approach, particularly with regard to taking a detailed clinical history . One may find important clues in the clinical history (e.g., age at onset, detailed seizure description and past history) to make a syndromic diagnosis . The syndromic diagnosis forms the basis for the treating physician to decide upon an appropriate management plan . This was a clinic - based series and may not represent the full spectrum of tles . We would like to thank neurosciences research center, shiraz university of medical sciences, shiraz, iran for supporting this study.
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Leukocytospermia is a kind of common disease of male reproductive tract infection, which is usually caused by bacterial infection . An increase of white blood cells in seminal plasma can lead to the decrease of sperm quality and the increasing number of inflammatory cytokines such as interleukin il-6 is the main factor involved in the process . According to current studies, paf is a kind of cytokine which has many kinds of biological activities, such as inflammation factors and sperm cell activation factors . How does the paf change and what role does it play the role during the reproductive tract infection? The purpose of our study was to observe the expression of paf and tnf- in seminal plasma of patients with leukocytospermia . Male patients age from 23 to 40 years old, who came to the urology surgical department of our hospital from april to september 2011, were enrolled . According to who directives, 22 cases with leukocytospermia and 15 normal males were chosen . All the patients did not have any urinary, reproductive, blood, or endocrine diseases . The patients who have ever suffered from mumps, cryptorchidism and sexual dysfunction and ever accepted radiotherapy or chemotherapy were excluded . The study was admitted by the local ethical committee, and written informed consent was obtained from all participants . The semen should be obtained in one time by using masturbation method and be placed into disposable sterile dry plastic container with cover . After the full liquefaction of semen plasma, the computer - assisted semen analysis (casa) carried out a routine analysis . The rest of the seminal plasma was collected and accepted centrifugal test for 10 minutes at 4000 r / min, the upper layer of seminal plasma was placed into eppendorf tube and stored at 20c for reservation . Density count of seminal plasma white blood cell in seminal plasma refer to peroxidase dyeing method, recommended by the who . Human platelet activation factor (paf) enzyme league immune kit and human tumor necrosis factor (tnf-) enzyme league immune kit were bought from jiahui biotechnology company (rd import repacking), beijing weili wjy9000 sperm analyzer, low temperature centrifuge (sigma, usa), 37c constant temperature water - bath box, full - wavelength enzyme standard instrument (finland leibo company). The operator should operate strictly in accordance with the manual of elisa kit for inspecting tnf- and paf . The od value should be read on enzyme standard instrument within 15 minutes at 450 nm wavelength . Normal characteristic test and homogeneity test of variance would be carried out before intragroup comparison of paf and tnf- expression, and t - student test was adopted . There are two kinds of correlation analyses here, straight line related analysis and spearman rank correlation analysis, which one should be adopted, it depends on whether the expression of paf or tnf- on normal distribution or not in the seminal plasma . Significant difference was found in the aspects of sperm survival rate and vigor between leukocytospermia group and normal group, p <0.01, while the difference in sperm density and liquefaction time was not obvious, p> 0.01 (table 1). Paf concentration in leukocytospermia group was (2.14 0.43 ng / ml), which was significantly lower than the normal group (6.21 1.38 ng / ml); the difference was statistically significant (p <0.01). Wbc count in leukocytospermia group was (1.33 0.18) 10/ml, and tnf- concentration was (5.51 1.46 ng / ml), which was significantly higher than that of normal group wbc (0.43 0.19) 10/ml and tnf- (3.48 1.08 ng / ml), and the difference was statistically significant (p <0.01) (table 2). In this group, paf has no significant correlation with sperm density and liquefaction time . And what's more, it has positive correlation with sperm survival rate (r = 0.452, p <0.01) and sperm vigor (r = 0.642, p <0.01). (p> 0.01). While it has negative correlation with sperm survival rate (r = 0.415, p <0.01) and sperm vigor (r = 0.725, p <0.01) (table 3). Linear correlation analysis shows that wbc count has negative correlation to paf concentration in leukocytospermia group (r = 0.62, p <0.01) (figure 1), however, it has positive correlation with tnf- concentration (r = 0.77, p <0.01); paf concentration had significantly negative correlation to tnf- (r = 0.68, p <0.01) (figure 2). Leukocytospermia refers to the increase of white blood cell (wbc) density in seminal plasma which is more than 1 10/l, and it is mainly caused by the reproductive tract infection . The white blood cells in seminal plasma can produce many inflammatory cell factors . With the affection of these cell factors, the sperm quality, density and its vigor get lower; sperm capacitation and acrosomal reaction would be affected as well, which lead to male infertility . Paf is a kind of cell factors found in recent years, and it is involved in various physiological and pathological processes . Sperm movement ability is the precondition of fertilization, wherein paf with certain concentration is considered to be one of the conditions to obtain certain movement ability . When the exogenous paf concentration is between 1 10 and 1 10 mol / l, the sperm's motility is prominently increased after 120 min culture time . In animal experiments, the sperm motility can be immediately improved after exogenous paf treatment on freeze - thaw boar sperm; paf concentration in seminal plasma of squirrel monkeys in the breeding season is significantly higher than that during the nonbreeding season, thereby, it is believed that paf plays an important role in the reproductive process . Paf also participates in sperm capacitation and acrosomal reaction (ar); sperm capacitation can be affected through culturing with sperm together, thereby increasing the acrosomal reaction rate . Ar% is different when paf concentration changes, relatively, high density (1 10 mol / l) can induce ar better . In addition, paf can obviously increase the pregnancy rate of accepting artificial insemination treatment due to unexplained infertility . The seminal plasma of dog stored in low temperature has prominently increased sperm movement performance through treatment with different concentrations of exogenous paf . Research shows that the sperm movement performs best when the concentration is 1 10 m with 120 min of cultivating time, at the same time, atp concentration improves obviously as well . We also found that the mitochondrial function of the sperm through paf processing remains unchanged, and the integrity of sperm has not been damaged either . At the same time, it promotes the platelets and neutrophils to gather together under the pathologic state participate in super oxide formation, and it also promotes the protein phosphorylation process . It can also induce submucosal vasoconstriction, and induce neutrophils and other inflammatory cells to produce a large number of active oxygen and free radicals . It is confirmed that paf involves in gastric ulcer, alcoholic stomach injury, necrotizing enterocolitis, ischemic stomach, and small intestinal mucosal injury . In addition, it also plays an important role in acute pancreatitis and acute lung injury development [9, 10]. As a result, the role paf which plays in reproductive tract infection is worth discussing . Alpha subtype of tumor necrosis factor (tnf-) is a kind of important cell factor involved in the inflammation reaction progress . It is mainly produced by macrophages, and widely involved in inflammation response, immune response, endotoxin sex shock, and other pathologic process . At the same time, tnf- is a kind of cell factor, which has close relations with sterility occurrence and development . A certain amount of tnf- can significantly inhibit the activity of sperm acrosomal enzyme and acrosomal reaction . Tnf- has certain influence on sperm mitochondrial function, it can interfere sperm energy metabolism and lower sperm movement ability . It may also promote the sperm apoptosis through the mitochondria apoptosis way, which causes infertility . Tnf- also can reduce the amount of nitrate in normal sperm and affects no synthesis, which is necessary for sperm movement . Studies have reported that paf and tnf- can mutually cooperate with each other to promote the progress of inflammatory response, such as acute lung injury, monilia infection, and necrotizing enterocolitis . How is the paf expression in the condition of reproductive tract infection? And how does the interaction happens between paf and tnf-? Elisa method was adopted to observe the expression of paf and tnf- in seminal plasma of patients with leukocytospermia . The results show that paf level is lower, while tnf- is higher compared with that in the normal seminal plasma . After analyzing the expressions of paf and tnf-, the relation between them, and the white blood count, we found that the expression of paf and tnf- have negative correlation with each other (r = 0.68, p <0.01figure 1); paf is negatively correlated with white blood cell count (r = 0.62, p <0.01figure 2); tnf- has positive correlation to white blood cell count (r = 0.77, p <0.01). The existing research results showed that paf is favorable for sperm function activities, while tnf- could inhibit sperm function activities . So we believe that, under condition of reproductive tract infection, the organism increase the composition of cell factors which can inhibit sperm activity on one hand, such as tnf-, and decrease the composition of cell factors which can promote sperm on the other hand, such as paf, thereby affect sperm vigor . However paf level is lowered under the state of reproductive tract infection, this phenomenon is of great significance and worth being studied . The adjustment of paf is affected by several factors, wherein it is related to platelet activating factor acetylhydrolase (paf - ah). Paf - ah is specific to paf, its concentration is negatively correlated with paf . Paf - ah level has significant negative correlation with sperm motility in panda seminal plasma and has negative correlation with the state of sperm movement too . The concentration of paf - ah in seminal plasma of male patient, whose spinal column was injured, is prominently higher than normal people; meanwhile, it has negative correlation to sperm motility . In a study investigating the relation between paf - ah level and sperm vigor, the researchers found that when sperm motility was equal to or above 50%, paf - ah density was 442.03 14.37 iu / l, while when the vigor is below 50%, the paf - ah density is 882.16 18.45 this research also shows that paf - ah is an important factor to adjust paf . Whether paf - ah plays an important role in lowering paf level in leukocytospermia or not, it is still worthy of further study . In conclusion, low expression of paf or high expression of tnf- in leukocytospermia affects the sperm quality, both of which contributed to sterility . The reason why paf shows a low expression during reproductive tract infection is still not clear now, and it is of important significance, which deserves further study.
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Focal myositis is an idiopathic inflammatory myopathy and a rare inflammatory pseudotumor of the skeletal muscle with unknown etiology (1). The disease typically disappears spontaneously, does not last more than 4 years, and has no recurrence (1,2). This myositis can be treated successfully with steroids or nonsteroidal anti - inflammatory drugs (nsaids) (1,2). We herein describe the case of a man with recurrent episodes of bilateral focal myositis . A 38-year - old man presented with myalgia of the right gastrocnemius muscle in may 2005 . A needle biopsy of the muscle was performed in another hospital and indicated no malignancy . The patient did not receive any treatment at this time . As his symptom did not improve, he visited our hospital in august 2005 with tenderness of the right gastrocnemius muscle . On physical examination, no significant signs were detected on the skin, cardiovascular, respiratory, or abdominal examinations . Manual muscle testing indicated full scores except in the right gastrocnemius, which demonstrated grade 4 (of 5) because of myalgia . Evaluations of his mental status, sensory, and cerebellar systems were normal . Laboratory data indicated that the patient's serum creatine kinase (ck), aldolase, myoglobin, erythrocyte sedimentation rate (esr), and c - reactive protein (crp) were within normal limits, and anti - jo-1 autoantibody, anti - ars autoantibody, and antinuclear antibody were negative (table). Hepatitis b surface antigen (hbs ag) was positive (cut - off> 2,000), and hb virus dna was negative (<3.7 leg / ml). Human immunodeficiency virus (hiv) and hepatitis c virus antibody tests were negative (table). Magnetic resonance imaging (mri) revealed very high signal intensity in the right gastrocnemius medial head muscle on a short - tau inversion recovery (stir) image . 1a and b). A muscle biopsy of the right gastrocnemius revealed interstitial muscle infiltration by mononuclear inflammatory cells without findings of vasculitis, along with muscle fiber necrosis and regeneration (fig . 2a and b). Immunohistochemical staining of the muscle biopsy showed that the infiltrating cells predominantly consisted of cd4 + t cells, cd68 + macrophages, and cd20 + b cells accompanied by a few cd8 + t cells (fig . Focal myositis was diagnosed . Magnetic resonance imaging (mri) of the lower limbs . A, b: august 2005; c, d, e: june 2006; f, g, h: january 2008 . A, b, e, f, h: axial (a, f) and coronal (b, e, h). A - f: unenhanced mri image and g: gadolinium - enhanced mri image . Stir images show high signal intensity in the affected muscles of the lower legs (white arrowhead). C, d: axial and coronal stir images show high signal intensity in the affected femoral muscles (white arrowhead). F, g: axial gadolinium - enhanced mri (g) shows no enhanced area in the thickened fascia of the right gastrocnemius, which showed high signal intensity on the unenhanced mri image (f) (white arrow). Muscle biopsy . A, b: hematoxylin and eosin staining, 400; c - f: immunohistochemical staining, 400; c: cd4; d: cd8; e: cd68; f: cd20; g: electron microscopy . A muscle biopsy of the right gastrocnemius reveals mononuclear inflammatory cells (a, b: black arrowhead) that infiltrated the interstitial spaces of the muscle without vasculitis findings, along with muscle fiber necrosis and regeneration (a, b: white arrowhead). Infiltrating cells predominantly consisted of cd4+t cells, cd68+macrophages, and cd20+b cells accompanied by a few cd8+t cells (c - f: white arrow). Electron microscopy of the muscle biopsy specimens reveals nemaline rods (g: black arrows). As the patient was hbs ag - positive, lamivudine treatment was initiated for the prevention of de novo hepatitis under immunosuppressive conditions . Prednisolone (psl) (30 mg / day) was administered to treat the focal myositis in september 2005, and his symptoms and mri findings were immediately improved . The clinical course is shown in fig . The patient's muscle pain worsened gradually at around this time, and walking became difficult without a cane in june 2006 . Although his crp level was 0.4 mg / dl, the serum myogenic enzyme level was not re - elevated . Mri revealed multiple high - intensity areas in the lower limbs that included the femoral muscle (both sides of the vastus lateralis, the left vastus intermedius, the right biceps femoris, and the left gracilis) (fig . The psl dosage was increased to 60 mg / day for relapse of myositis, and the patient's symptoms immediately improved . Psl: prednisolone, mtx: methotrexate, aza: azathioprine, ivig: high - dose intravenous immunoglobulin after tapering the psl dosage to 17.5 mg / day, a second myositis relapse occurred in october 2007 . 1f and h), left popliteus muscle, and right semimembranosus on stir images (data not shown), and mild edema was evident in the subcutaneous tissue in both lower limbs in january 2008 . As thickening of the fascia of the right gastrocnemius on the stir image (fig . 1 g, white arrow), it was considered to result from edema . Because the patient was unusually resistant to the treatment for focal myositis, we performed a muscle biopsy of the left gastrocnemius to re - confirm the diagnosis, which showed a similar result to the first biopsy (data not shown). Electron microscopy of the muscle biopsy specimens in february 2008 revealed nemaline rods (fig . The dosage of psl was again increased to 60 mg / day for the third relapse of myositis . Thereafter, we administered azathioprine (max 100 mg / day) for a steroid - sparing effect, but it failed to bring the patient persistent relief from his myalgia and gait disturbance . Therefore, we were forced to continue to administer 15 mg / day or more of the steroid in order to maintain remission . In september 2011, the relapse of myositis involved myalgia of both lower limbs and arthralgia of both foot joints . The patient's psl dosage was increased from 15 mg / day to 30 mg / day, however, the effect was incomplete . As he had a compression fracture of the vertebrae due to steroid - induced osteoporosis, high - dosage psl treatment was avoided . Methotrexate (mtx) (7.5 mg / week) was initiated with psl 20 mg / day in october 2011, as hepatitis b virus (hbv) infection had been controlled by lamivudine . The dosage of mtx was gradually increased to 16 mg / week by august 2012 . . The patient can maintain standing on his heels, but not on his tiptoes . The differential diagnoses of inflammatory myopathy were postulated to be polymyositis, dermatomyositis, inclusion body myositis, eosinophilic myositis, and sarcoidosis . He had no past medical history and no other organ disorders including those affecting the skin and lung . The lesion originated from the distal muscle, with no eosinophilia or increase of ck or crp in the peripheral blood . The pathology of the muscle biopsy revealed myositis and did not indicate granulomas, eosinophilic infiltration, or vacuoles . Focal myositis is a type v idiopathic inflammatory myopathy, classified by bohan and peter as miscellaneous myopathies (3). The levels of acute phase reactants and myogenic enzymes, such as ck, and the site of the involved muscle also vary in myositis (1,2,4 - 10). In this case, the patient's myalgia began in the right gastrocnemius and spread to both lower limbs . Morevoer, sekiguchi et al . Reported that normal serum crp and ck levels might be associated with mild muscle inflammation in patients with focal myositis (11). Although the mechanism of muscle pain is not fully understood, inflammatory muscle pain was related to molecules including interleukin (il)-6, tumor necrosis factor - alpha, il-1beta, and kinin in experiments using animal models (12,13). Dina et al . Reported a novel experimental model of chronic muscle pain induced by mild acute muscle inflammation (14). Psl is presumed to have led to the alleviation of symptoms by suppressing muscle inflammation . Some reports have reported an association between chronic hbv infection and myositis (15 - 17). Lamivudine or entecavir, antiviral therapies for hbv infection, improved symptoms in patients with myositis (18,19). Reported that the investigation of possible viral etiology via polymerase chain reaction was negative in focal myositis (20). The present patient was an hbv carrier, however, antiviral therapy did not affect his myositis . Although electron microscopy of specimens from the second muscle biopsy revealed nemaline rods, we judged the finding to be a non - specific change because his clinical syndrome was not characteristic of nemaline myopathy (24), and there have been several reports concerning the non - specific finding of nemaline rods in inflammatory myopathies (polymyositis and dermatomyositis) (22 - 25). The majority of focal myositis cases involve a limited lesion, and bilateral lesions are rare . In our case, the patient demonstrated myositis progression from the right gastrocnemius to both sides approximately 1 year after onset and recurrent myalgia, and the myositis had temporary femoral muscle involvement . Heffner jr et al . Reported six patients with polymyositis beginning as a focal process that rapidly progressed 3 - 6 months from the onset, and some cases of bilateral focal myositis have been reported (26). Additionally, some reports have discussed the difference between focal myositis and polymyositis (27,28). Conversely, there are also some reports of polymyositis cases with distal muscle involvement or without elevation of serum ck (29 - 34). The muscle biopsy in our case showed mononuclear inflammatory infiltrates that predominantly consisted of cd4 + t cells . Previous studies also reported cd4 + -dominant cell infiltration in the muscle in focal myositis (37,38) and dermatomyositis (35,36), but not in polymyositis (39). In our patient, muscle weakness derived from myalgia was present but not progressive, and his immunohistochemical findings indicated focal myositis rather than polymyositis . It appears that focal myositis and polymyositis have different pathophysiological mechanisms; however, whether focal myositis is a subtype of polymyositis remains to be determined . Focal myositis usually shows a good response to treatment with psl and nsaids (2). It has a good prognosis and might be a self - limiting disease (1 - 3). Furthermore, the response to nsaids is often good even in cases of repeated relapse (41 - 43), and some cases of spontaneous remission have occurred (8,49). Although the present patient demonstrated a good response to steroid treatment, psl dose reduction induced the recurrence of myalgia . Because he experienced repeated myositis relapses when the psl dose was reduced to 15 - 17.5 mg, it became necessary to use immunosuppressive agents for a steroid - sparing effect . Azathioprine and ivig did not affect the patient's myositis . Because his hbv infection was successfully controlled with lamivudine, we used mtx and were able to reduce the psl dose . Reports of immunosuppressive therapy for focal myositis are limited (6,47,51,52), and only one previous case report, in a child, described mtx treatment for this condition (47). Further studies are needed in order to clarify the sparing effect of treatment via mtx in steroid - resistant cases of focal myositis.
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Nod.iapp mice were bred in our colony by backcrossing c57bl6.iapp mice (6) onto the nod background; these mice have been fully backcrossed to the nod mouse (10 generations). All experimental procedures were in accordance with the guidelines of the institutional animal care and use committee, national jewish health . (7) were used as a source of -cell tumors for the preparation of antigen; the enrichment of membrane proteins from -cells isolated from nod.riptag adenomas has been previously described (8). In brief, single - cell suspensions of -cell adenomas were prepared by pressing whole - tumor tissue through a mesh screen, followed by several centrifugation steps to obtain a cellular membrane pellet; this pellet (-mem) is highly antigenic for the t - cell clones and serves as a positive control antigen in t - cell assays as well as the source of antigen for further purification . Membrane protein preparations were solubilized in detergent - containing buffer (20 mmol / l tris, ph 8.0, and 1% octyl--glucoside), followed by centrifugation at 18,400 g (10 min at 4c) to remove insoluble debris . Size - exclusion chromatography (sec) was carried out on a superdex 200 16/60 column (amersham biosciences) at 21c (flow rate: 1 ml / min; fraction size: 1.25 ml; and injection volume: 2.0 ml), eluting with sec buffer (20 mmol / l tris, ph 8.0, and 150 mmol / l nacl). Fractions from sec were assayed for antigenic activity with the t - cell clones, and a peak antigenic fraction (950 l) was acidified with 20 l trifluoroacetic acid before adding 30 l acetonitrile . A total of 800 l of this mixture was then applied to a reverse - phase high - performance liquid chromatography (rp - hplc) mrp - c18 high - recovery protein column (agilent). A buffer gradient was used to elute proteins from the column, and a total of 36 fractions were collected between 0 and 72 min at a flow rate of 0.75 ml / min and a constant column temperature of 80c . The antigenicity of islet cells, cellular and biochemical fractions, or peptides was assessed through the ifn- responses of t - cell clones . In 96-well microtiter plates, assay cultures contained 2 10 responder t cells, 2.5 10 nod peritoneal exudate cells as antigen - presenting cells, and -cell antigen . Ifn- production by the responder t cells was determined by elisa (bd biosciences). Ifn- standards were added to the assay plates at concentrations between 0 and 100 ng / ml, but concentrations of the standard> 50 ng / ml were above the assay detection range . An aliquot of -mem or whole nod islet cells were used as positive controls; test wells contained sec or rp - hplc fractions, peptides, or islet cells . The protein - identification strategy we have previously described (5) was used for mass spectrometry . Proteins were digested with trypsin, and extracted peptides were resolved by chromatography online on a c18 column and a 1200 series hplc system (agilent technologies). Analysis was carried out with a 6340 lcms ion - trap mass spectrometer in the national jewish health facility . Raw data were extracted and used to search the swiss - prot or national center for biotechnology information databases with the spectrum mill search engine (rev a.03.03.038 sr1; agilent technologies). Data were evaluated and protein identifications were considered significant if the following confidence thresholds were met: individual peptide scores of at least 10 and scored percent intensity of at least 70% . A reverse (random) database search was simultaneously conducted, and manual inspection of spectra was used to validate the match of the spectrum to the predicted peptide fragmentation pattern . Cell cultures of bdc-2.5 and bdc-5.2.9 were expanded in interleukin-2, and after harvesting, t cells (1 10) were injected intraperitoneally into age - matched 6- to 14-day - old nod or nod.iapp recipient mice . Mice were monitored on a daily basis for urine glucose using diastix (bayer). The presence of urine glucose was followed by blood glucose monitoring using a onetouch ultra glucometer (lifescan). Mice were considered diabetic when blood glucose levels were> 18 mmol / l (324 mg / dl). For disease - transfer experiments, nod, nod.scid, and nod.rip-tag mice were bred and maintained in the biological resource center at national jewish health . Nod.iapp mice were bred in our colony by backcrossing c57bl6.iapp mice (6) onto the nod background; these mice have been fully backcrossed to the nod mouse (10 generations). All experimental procedures were in accordance with the guidelines of the institutional animal care and use committee, national jewish health . Nod.rip-tag mice (7) were used as a source of -cell tumors for the preparation of antigen; the enrichment of membrane proteins from -cells isolated from nod.riptag adenomas has been previously described (8). In brief, single - cell suspensions of -cell adenomas were prepared by pressing whole - tumor tissue through a mesh screen, followed by several centrifugation steps to obtain a cellular membrane pellet; this pellet (-mem) is highly antigenic for the t - cell clones and serves as a positive control antigen in t - cell assays as well as the source of antigen for further purification . Membrane protein preparations were solubilized in detergent - containing buffer (20 mmol / l tris, ph 8.0, and 1% octyl--glucoside), followed by centrifugation at 18,400 g (10 min at 4c) to remove insoluble debris . Size - exclusion chromatography (sec) was carried out on a superdex 200 16/60 column (amersham biosciences) at 21c (flow rate: 1 ml / min; fraction size: 1.25 ml; and injection volume: 2.0 ml), eluting with sec buffer (20 mmol / l tris, ph 8.0, and 150 mmol / l nacl). Fractions from sec were assayed for antigenic activity with the t - cell clones, and a peak antigenic fraction (950 l) was acidified with 20 l trifluoroacetic acid before adding 30 l acetonitrile . A total of 800 l of this mixture was then applied to a reverse - phase high - performance liquid chromatography (rp - hplc) mrp - c18 high - recovery protein column (agilent). A buffer gradient was used to elute proteins from the column, and a total of 36 fractions were collected between 0 and 72 min at a flow rate of 0.75 ml / min and a constant column temperature of 80c . The antigenicity of islet cells, cellular and biochemical fractions, or peptides was assessed through the ifn- responses of t - cell clones . In 96-well microtiter plates, assay cultures contained 2 10 responder t cells, 2.5 10 nod peritoneal exudate cells as antigen - presenting cells, and -cell antigen . Ifn- production by the responder t cells was determined by elisa (bd biosciences). Ifn- standards were added to the assay plates at concentrations between 0 and 100 ng / ml, but concentrations of the standard> 50 ng / ml were above the assay detection range . An aliquot of -mem or whole nod islet cells were used as positive controls; test wells contained sec or rp - hplc fractions, peptides, or islet cells . The protein - identification strategy we have previously described (5) was used for mass spectrometry . Proteins were digested with trypsin, and extracted peptides were resolved by chromatography online on a c18 column and a 1200 series hplc system (agilent technologies). Analysis was carried out with a 6340 lcms ion - trap mass spectrometer in the national jewish health facility . Raw data were extracted and used to search the swiss - prot or national center for biotechnology information databases with the spectrum mill search engine (rev a.03.03.038 sr1; agilent technologies). Data were evaluated and protein identifications were considered significant if the following confidence thresholds were met: individual peptide scores of at least 10 and scored percent intensity of at least 70% . A reverse (random) database search was simultaneously conducted, and manual inspection of spectra was used to validate the match of the spectrum to the predicted peptide fragmentation pattern . Cell cultures of bdc-2.5 and bdc-5.2.9 were expanded in interleukin-2, and after harvesting, t cells (1 10) were injected intraperitoneally into age - matched 6- to 14-day - old nod or nod.iapp recipient mice . Mice were monitored on a daily basis for urine glucose using diastix (bayer). The presence of urine glucose was followed by blood glucose monitoring using a onetouch ultra glucometer (lifescan). Mice were considered diabetic when blood glucose levels were> 18 mmol / l (324 mg / dl). For disease - transfer experiments, as previously described (5), to identify candidate antigens for the t - cell clones, we carried out biochemical fractionation of -cell tumors and mass spectrometric analysis of peak antigenic fractions . Similar to the other clones from the panel, bdc-5.2.9 responds in vitro to pancreatic islet cells or cell extracts of -cell adenomas presented in the context of i - a (2). Our starting source of antigen was -cell tumor tissue excised from nod.riptag mice (7) and separated by differential centrifugation to yield a highly antigenic -cell membrane pellet (-mem); this preparation also served as the positive control in t - cell activity assays . After lysis, the membrane pellet was separated by sequential chromatography, first through an sec column and then by rp - hplc . 1, the presence of antigen was tracked through the ifn- response of the t - cell clone bdc-5.2.9 (top axis, gray lines) to protein (bottom axis, black lines) in individual fractions . Figure 1a is the chromatogram from sec and shows a single antigenic peak that corresponds to a low molecular weight protein region . The antigenic fractions from sec were further purified by rp - hplc, yielding another single peak of antigen activity (fig . Presence of antigen was determined by measuring the ifn- response (gray lines) of the t - cell clone bdc-5.2.9 to small aliquots of individual fractions in the presence of peritoneal exudate cells used as antigen - presenting cells . The highly enriched and antigenic rp - hplc fractions were subjected to in - solution tryptic digest and were then analyzed on an ion - trap mass spectrometer . Resulting spectra were sequenced and matched to proteins by searching the swiss - prot protein database using the search program spectrum mill . Comparison of the antigen distribution profile with individual protein spectral - intensity values, an approximate indicator for relative protein abundances, singled out iapp as the only candidate . Other proteins did not match the antigen distribution profile; for example, secretogranin 1 was detected only in nonantigenic fractions 11 and 14 . We identified two iapp peptides, representing 28% amino acid coverage of prepro - iapp and 70% coverage of the 37-residue peptide hormone (fig . The peptide lanflvr was matched with high confidence and the peptide ssnnlgpvlpptnvgsnty was matched with medium confidence . Antigenic chromatographic fractions were digested with trypsin, separated by rp - hplc, and analyzed by ion - trap mass spectrometry; the resulting spectra were used to search a protein sequence database . A: proteins identified (right column) in the highly purified antigenic rp - hplc fraction no . 12 and neighboring fractions with lower antigen content (fractions 11, 13, and 14, as shown in fig . The summarized spectral intensities (left) are indicative of the relative abundance of a specific protein in a fraction; darker shading indicates higher intensities . B and c: ion - trap mass spectra matching the iapp peptides lanflvr (b) and ssnnlgpvlpptnvgsnty (c) (swissprot accession no . To investigate the peptide(s) targeted by bdc-5.2.9, we analyzed a series of overlapping iapp peptides, spanning the entire propeptide region (fig . One peptide, ks20, elicited a vigorous response from bdc-5.2.9 and could stimulate the clone at concentrations as low as 10 ng / ml (4.6 nmol / l) (fig . Bdc-5.2.9 did not respond to any other iapp peptide tested or to the insulin peptide b923 . Two neighboring peptides, rt20 and gr20, share either the c- or n - terminus sequence with ks20, but neither of these peptides can stimulate bdc-5.2.9, indicating that the t - cell receptor of this clone recognizes the adjoining sequences . An insulin - reactive clone, pd-12.4.4 (9), the fact that bdc-5.2.9 is stimulated by very low concentrations of the iapp peptide ks20 suggests that this peptide may contain the natural ligand for bdc-5.2.9 . The hormone peptide is highlighted (white lettering on black background) and the region identified by ion - trap mass spectrometry is underlined . B: ifn- responses of bdc-5.2.9 and the insulin - reactive clone pd-12.4.4 to various concentrations of the peptide antigens iapp ks20 (), gr20/rt20 (), ins b923 (), or the -cell membrane (-mem) positive control (). Although bdc-5.2.9 did not respond at all to the insulin peptide b923 or the iapp - peptides gr20 and rt20, it responded to iapp ks20 at concentrations as low as 0.01 g / ml . The insulin - reactive t - cell clone pd-124.4 responded to ins b923 and to the -mem control but not to any iapp peptides . Data are representative of at least three separate experiments . To further validate iapp as the antigen for the bdc-5.2.9 t - cell clone, we analyzed ifn- responses to islet cells from either nod (iapp) or nod.iapp mice . The t - cell clone bdc-2.5 was used as a positive control because it responds to a different -cell antigen, chga (5). As illustrated in fig . 4, both clones responded to antigen present in wild - type islets from nod mice (iapp) (fig . 4a), and the chga - reactive clone bdc-2.5 also responded to islets from nod.iapp mice (fig . In contrast, there was no stimulation of bdc-5.2.9 by iapp - deficient islets (fig . 4b), indicating that islets from iapp mice lack the antigen for this clone . Ifn- response of bdc-2.5 (chga reactive) (a) and bdc-5.2.9 (b) to various numbers of islet cells obtained from nod.iapp () and nod.iapp () mice . Data are representative of three separate experiments, in which each cell concentration was measured in duplicate . The highly aggressive nature of bdc-5.2.9 in disease induction can be demonstrated by adoptive transfer into young nod and nod.scid mice . To investigate the in vivo islet reactivity of bdc-5.2.9 in iapp - deficient mice, adoptive transfers with this clone were made into young (aged <2 weeks) iapp recipients . Bdc-2.5 was again used as a positive control and rapidly induced disease in both iapp and iapp recipients (fig . When bdc-5.2.9 was used in transfers, disease developed in most of the nod.iapp mice within 3 weeks, but there was no diabetes in iapp - deficient mice for the duration of the experiment, up to 45 days . We also conducted histological analysis of pancreatic sections from iapp - deficient mice to assess leukocyte infiltration and granulation of islet -cells (fig . The spontaneous development of insulitis and hyperglycemia in nod.iapp mice is typical and very similar to that in nod.iapp mice . Transfer experiments were terminated when recipients were ~2 months of age and the pancreatic histology of nod.iapp mice receiving bdc-5.2.9 closely resembled that of aged - matched, uninjected iapp controls, with at least 60% of islets being well granulated and no infiltrate apparent . In contrast, histology from iapp mice that received the bdc-2.5 t - cell clone showed complete degranulation of -cells . These results further demonstrate that islets lacking iapp do not contain the antigen for bdc-5.2.9 . A: bdc-2.5 or bdc-5.2.9 t cells (1 10) were injected intraperitoneally into 6- to 14-day - old nod or nod.iapp mice, which were monitored for hyperglycemia . Nod or nod.iapp mice that received the bdc-2.5 t - cell clone became diabetic within 3 weeks of transfer . Bdc-5.2.9 transferred disease in nod mice but not in nod.iapp mice (p = 0.002). Data summarize at least three independent experiments for each clone and indicate that bdc-5.2.9 is diabetogenic upon adoptive transfer in nod mice only when iapp is expressed in the islets . Islets in sections of nod.iapp mice that received bdc-5.2.9 showed no infiltrate and were well granulated; in contrast, islets from mice that received bdc-2.5 were infiltrated and the islets were degranulated . Histology was based on two mice per group for each of the following groups: iapp mice injected with bdc-5.2.9; 3-month - old uninjected iapp mice; and iapp mice injected with bdc-2.5 . The bar graph summarizes the islet granulation scores: 3 = well - granulated islet -cells; 2 = 5075% granulated; 1 = 2050% granulated; and 0 = no granulation . As previously described (5), to identify candidate antigens for the t - cell clones, we carried out biochemical fractionation of -cell tumors and mass spectrometric analysis of peak antigenic fractions . Similar to the other clones from the panel, bdc-5.2.9 responds in vitro to pancreatic islet cells or cell extracts of -cell adenomas presented in the context of i - a (2). Our starting source of antigen was -cell tumor tissue excised from nod.riptag mice (7) and separated by differential centrifugation to yield a highly antigenic -cell membrane pellet (-mem); this preparation also served as the positive control in t - cell activity assays . After lysis, the membrane pellet was separated by sequential chromatography, first through an sec column and then by rp - hplc . 1, the presence of antigen was tracked through the ifn- response of the t - cell clone bdc-5.2.9 (top axis, gray lines) to protein (bottom axis, black lines) in individual fractions . Figure 1a is the chromatogram from sec and shows a single antigenic peak that corresponds to a low molecular weight protein region . The antigenic fractions from sec were further purified by rp - hplc, yielding another single peak of antigen activity (fig . Presence of antigen was determined by measuring the ifn- response (gray lines) of the t - cell clone bdc-5.2.9 to small aliquots of individual fractions in the presence of peritoneal exudate cells used as antigen - presenting cells . The highly enriched and antigenic rp - hplc fractions were subjected to in - solution tryptic digest and were then analyzed on an ion - trap mass spectrometer . Resulting spectra were sequenced and matched to proteins by searching the swiss - prot protein database using the search program spectrum mill . Comparison of the antigen distribution profile with individual protein spectral - intensity values, an approximate indicator for relative protein abundances, singled out iapp as the only candidate . Other proteins did not match the antigen distribution profile; for example, secretogranin 1 was detected only in nonantigenic fractions 11 and 14 . We identified two iapp peptides, representing 28% amino acid coverage of prepro - iapp and 70% coverage of the 37-residue peptide hormone (fig . The peptide lanflvr was matched with high confidence and the peptide ssnnlgpvlpptnvgsnty was matched with medium confidence . Antigenic chromatographic fractions were digested with trypsin, separated by rp - hplc, and analyzed by ion - trap mass spectrometry; the resulting spectra were used to search a protein sequence database . A: proteins identified (right column) in the highly purified antigenic rp - hplc fraction no . 12 and neighboring fractions with lower antigen content (fractions 11, 13, and 14, as shown in fig . The summarized spectral intensities (left) are indicative of the relative abundance of a specific protein in a fraction; darker shading indicates higher intensities . B and c: ion - trap mass spectra matching the iapp peptides lanflvr (b) and ssnnlgpvlpptnvgsnty (c) (swissprot accession no . To investigate the peptide(s) targeted by bdc-5.2.9, we analyzed a series of overlapping iapp peptides, spanning the entire propeptide region (fig . One peptide, ks20, elicited a vigorous response from bdc-5.2.9 and could stimulate the clone at concentrations as low as 10 ng / ml (4.6 nmol / l) (fig . 3b). Bdc-5.2.9 did not respond to any other iapp peptide tested or to the insulin peptide b923 . Two neighboring peptides, rt20 and gr20, share either the c- or n - terminus sequence with ks20, but neither of these peptides can stimulate bdc-5.2.9, indicating that the t - cell receptor of this clone recognizes the adjoining sequences . An insulin - reactive clone, pd-12.4.4 (9), was used as a negative control and showed no response to iapp peptides . The fact that bdc-5.2.9 is stimulated by very low concentrations of the iapp peptide ks20 suggests that this peptide may contain the natural ligand for bdc-5.2.9 . The hormone peptide is highlighted (white lettering on black background) and the region identified by ion - trap mass spectrometry is underlined . B: ifn- responses of bdc-5.2.9 and the insulin - reactive clone pd-12.4.4 to various concentrations of the peptide antigens iapp ks20 (), gr20/rt20 (), ins b923 (), or the -cell membrane (-mem) positive control (). Although bdc-5.2.9 did not respond at all to the insulin peptide b923 or the iapp - peptides gr20 and rt20, it responded to iapp ks20 at concentrations as low as 0.01 g / ml . The insulin - reactive t - cell clone pd-124.4 responded to ins b923 and to the -mem control but not to any iapp peptides . To further validate iapp as the antigen for the bdc-5.2.9 t - cell clone, we analyzed ifn- responses to islet cells from either nod (iapp) or nod.iapp mice . The t - cell clone bdc-2.5 was used as a positive control because it responds to a different -cell antigen, chga (5). As illustrated in fig . 4, both clones responded to antigen present in wild - type islets from nod mice (iapp) (fig . 4a), and the chga - reactive clone bdc-2.5 also responded to islets from nod.iapp mice (fig . 4b), indicating that islets from iapp mice lack the antigen for this clone . Ifn- response of bdc-2.5 (chga reactive) (a) and bdc-5.2.9 (b) to various numbers of islet cells obtained from nod.iapp () and nod.iapp () mice . Data are representative of three separate experiments, in which each cell concentration was measured in duplicate . The highly aggressive nature of bdc-5.2.9 in disease induction can be demonstrated by adoptive transfer into young nod and nod.scid mice . To investigate the in vivo islet reactivity of bdc-5.2.9 in iapp - deficient mice, adoptive transfers with this clone were made into young (aged <2 weeks) iapp recipients . Bdc-2.5 was again used as a positive control and rapidly induced disease in both iapp and iapp recipients (fig . 5a). When bdc-5.2.9 was used in transfers, disease developed in most of the nod.iapp mice within 3 weeks, but there was no diabetes in iapp - deficient mice for the duration of the experiment, up to 45 days . We also conducted histological analysis of pancreatic sections from iapp - deficient mice to assess leukocyte infiltration and granulation of islet -cells (fig . The spontaneous development of insulitis and hyperglycemia in nod.iapp mice is typical and very similar to that in nod.iapp mice . Transfer experiments were terminated when recipients were ~2 months of age and the pancreatic histology of nod.iapp mice receiving bdc-5.2.9 closely resembled that of aged - matched, uninjected iapp controls, with at least 60% of islets being well granulated and no infiltrate apparent . In contrast, histology from iapp mice that received the bdc-2.5 t - cell clone showed complete degranulation of -cells . These results further demonstrate that islets lacking iapp do not contain the antigen for bdc-5.2.9 . A: bdc-2.5 or bdc-5.2.9 t cells (1 10) were injected intraperitoneally into 6- to 14-day - old nod or nod.iapp mice, which were monitored for hyperglycemia . Nod or nod.iapp mice that received the bdc-2.5 t - cell clone became diabetic within 3 weeks of transfer . Bdc-5.2.9 transferred disease in nod mice but not in nod.iapp mice (p = 0.002). Data summarize at least three independent experiments for each clone and indicate that bdc-5.2.9 is diabetogenic upon adoptive transfer in nod mice only when iapp is expressed in the islets . Islets in sections of nod.iapp mice that received bdc-5.2.9 showed no infiltrate and were well granulated; in contrast, islets from mice that received bdc-2.5 were infiltrated and the islets were degranulated . Histology was based on two mice per group for each of the following groups: iapp mice injected with bdc-5.2.9; 3-month - old uninjected iapp mice; and iapp mice injected with bdc-2.5 . The bar graph summarizes the islet granulation scores: 3 = well - granulated islet -cells; 2 = 5075% granulated; 1 = 2050% granulated; and 0 = no granulation . The activation of autoreactive cd4 t cells through t - cell receptor responses to peptide / major histocompatibility complexes is a major event in autoimmune diseases such as type 1 diabetes . The identification of -cell antigens, and the peptide epitopes from these antigens, could have important implications for both diagnosis and therapy of type 1 diabetes . Our results reported here demonstrate that iapp is now the third secretory granule protein in islet -cells, after insulin and chga, to be targeted by pathogenic cd4 t cells, and it is possible that other secretory granule proteins (e.g., other secretogranins) also could be antigenic targets in type 1 diabetes . This peptide hormone is cosecreted with insulin from the -cell granules at a molar ratio of ~1:100 iapp: insulin (10). Similar to insulin and chga, iapp is expressed as a larger precursor molecule (pro - iapp), which is then posttranslationally processed within the secretory granules by the enzymes prohormone convertase 2 and carboxypeptidase e (11). Iapp is well known as a component of the amyloid plaques found in the pancreatic islets of patients with type 2 diabetes (1214). In type 1 diabetes, there has been documentation of autoantibodies to iapp in patients, but there was no correlation to disease (15). To date, there has been little association of iapp with type 1 diabetes . (16) in which it was found that a significant proportion of cd8 t cells from hla - a*0201 patients with recent - onset type 1 diabetes respond to a peptide derived from the leader sequence of prepro - iapp . This group also contributed to a later study in which it was found that a second sequence of prepro - iapp (ppiapp 917) elicited responses from cytotoxic t - lymphocytes of type 1 diabetic patients (17). The other indicator that iapp might have a role in type 1 diabetes, and the first evidence from the nod mouse, was a genetic mapping study in which we reported linkage of the iapp gene with the antigen locus for a cd4 t - cell clone bdc-6.9 (18). Although we have not identified an iapp peptide that stimulates this t - cell clone, strong corroborating evidence for iapp as its antigen target was provided by islets from the nod.iapp mouse . As observed with the bdc-5.2.9 clone, iapp - deficient islets were unable to activate bdc-6.9 (k.h ., we have provided definitive evidence that diabetogenic cd4 t cells in the nod mouse react to iapp as an antigen, including the identification of a peptide containing a natural ligand for one of these t cells . The sequence of secreted iapp is quite similar between mouse and human forms of the molecule, and there is only one amino acid difference between the ks20 mouse and human sequences . It is not unreasonable, therefore, to assume that human cd4 t cells also may react to iapp peptides presented, for example, by hla - dq8 . A major function of secretory granule proteins is to participate in the biogenesis of the secretory granules and in the process, these proteins tend to form aggregates (19,20). Iapp is of particular interest in this regard because of its propensity to form amyloid deposits in type 2 diabetic patients . Such deposits are not found in type 1 diabetic patients, but it has been demonstrated by others that the initial step in amyloid deposition is the formation of toxic iapp oligomers (21). Soluble oligomers of iapp also have been shown to be responsible for activation of the nlrp3 inflammasome that leads to increased interleukin-1 production in type 2 diabetes (22). In type 1 diabetes, similar smaller aggregates of iapp it is quite conceivable that such oligomers would be taken up by antigen - presenting cells and processed in a way that leads to the formation of neoantigenic epitopes.
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Refrain: mosquito s in his crab hole, bidin his time, venezuela virus working up the line, boys in beltsville heard the news, horses in texas got the crab hole blues . Down in maracay back in 36, kubes and rios found a virus doin tricks, horses die, this one s gotta be, new cause of cephalitis, v - e - e . The years roll by, the virus makes a score, in vene, colombo, and ecuador, comes rain to the desert instead of dew, and, vee burns the coast of peru . In trinidad, panama, they say hey, hey, we got this creature like every day, his swampy home, you can always tell, by finding some rats and the culex (mel). Voice - over: mosquito, that is . Back at maru, raggin the brains, we found that the virus had different strains horses convulse with the southern kind, northern virus leaves them feelin fine . Cause virus hits man by aerosol, could be used to cripple us all, in maryland, they grew it in vats, tested it out in monkeys and rats . The other team outside the fence, lookin for the answer that made some sense, got a strain from an old donkey, made a vaccine, tc-83 . The offensive team tried to make some noise, a cloud for the commies, but not our boys, did not work, that is why, nixon said declassify. Voice - over: the pentagon papers, volume 25, page 324, 1970 the new york times, los angeles times, the washington post, and the christian science monitor . Sleepy defense gets a latin call, new outbreak and it ai nt small, took the vaccine off the wall, it really worked to save them all . Down in guatemala, year of 69, horses started dying on the borderline, virus come down from ecuador, nobody knows the reservoir . Virus hopped the mountains, got to the sea, vaccine brought in and given out free, virus kept movin, had to go, all the way from here to mexico . Cause everybody said, gotta stop that bug, gotta use the vaccine because there ai nt no drug, beltsville brethren said gotta wait, afraid that the vaccine was mf-8 . Results today are plain to see, story s hot on the wire of associated p, a million horses all over the west, are living proof the vaccine was best . A) the presentation is a chronologically, scientifically, and factually correct poetic and musical history of venezuelan equine encephalomyelitis (vee) viruses and epidemics through 1971 . The music is a traditional us jug band song, a type of music bluegrass popular in rural areas of the eastern united states . Using a background of a guitar and everyday musical, rhythm, and percussion devices, such as whistling, blowing air across the mouth of a 1-gallon glass jug, scraping of a scrub brush on a metal washboard, humming harmonically into a kazoo, and the hambone (a rhythmic slapping of hands on arms, hands, and legs), the voices and lyrics convey a musical story to the audience . The title reflects the discovery that deinocerites pseudes, the crab hole mosquito, found along the pacific coast of central america was a competent vector of the epidemic and epizootic vee virus that was causing disease and death in equids and humans at the time (1). The poetic dialogue was written and set to music in 1971 by scientists at the middle america research unit (maru), us department of health, education, and welfare (now health and human services), national institutes of health, national institute of allergy and infectious diseases, us public health service, and located in ancon, panama canal zone, during a major western hemispheric outbreak of vee . Maru scientists had a> 15-year history of vee epidemiologic and virologic studies throughout latin america to characterize the antigenic relationships between south american vee virus strains of subtypes (i - a / b and i - c) that caused epizoodemics of equine and associated human disease and the caribbean, central american, floridian, mexican, panamanian, and south american vee virus strains of subtypes (i - d, i - e, i - f, ii, iii - a, iii - b, and iv) that existed in sylvatic, enzootic cycles in the absence of equine disease but with occasional human infections and disease (2). In 1969, the transfer of equine virulent vee virus from a raging epizoodemic in colombia, ecuador, and peru to the frontier area of el salvador, guatemala, and honduras precipitated a human and veterinary medical crisis in central america, mexico, and the united states that lasted until 1972 (3). The crab hole mosquito blues was written as a scientific presentation for the international workshop - symposium on venezuelan encephalitis virus sponsored by the pan american sanitary bureau, pan american health organization, world health organization, in washington, dc, september 1417, 1971 . Although the lyrics of the crab hole music blues were not published in the proceedings, other presentations from that meeting are documented (4). B) douglas f. antczak, vocals, guitar, kazoo; william h. dietz, vocals, jug, kazoo; karl m. johnson, vocals, kazoo; david h. martin, kazoo, recording engineer; and thomas e. walton, washboard and scrub brush, hambone, kazoo, vocals . C) gender - specific error . Only female mosquitoes (and females of other hematophagous insects, e.g., culicoids, phlebotomids) take a blood meal, which is needed to provide protein necessary for ovulation; only female mosquitoes are infected with and transmit vee viruses (and other mosquito - borne arboviruses). D) beltsville, maryland, usa, the headquarters at that time of the regulatory officials in the us department of agriculture (usda) responsible for the decision to recommend application of preventive vaccines to the secretary of agriculture in the face of an epizootic . Hesitation to vaccinate was predicated on lack of evidence that reversion of virulence of the attenuated vaccine virus could not occur, international trade considerations, international practices and agreements, and authorities and responsibilities delegated only to the secretary of agriculture . E) from 1969, the epizoodemic moved southeast, eventually reaching northwestern costa rica in august 1970, and northward, eventually reaching texas in late june 1971 (4). F) the venezuelan agricultural and veterinary research laboratories are located in maracay, aragua state, venezuela . Kubes and rios first isolated, identified, and named vee virus, then sent the isolate to the united states for confirmation that the south american virus was antigenically distinct from the north american eastern and western equine encephalomyelitis viruses (5,6). G) the antigenically related and clinically similar eastern and western equine encephalomyelitis viruses had been isolated and identified early in the 1930s in the united states (7,8). H) periodic outbreaks of vee had occurred in colombia, ecuador, peru, and venezuela since at least the 1920s, with hundreds of thousands of equine illnesses and tens of thousands of deaths; equids are the primary virus amplifier hosts for human infections (9,10). I) throughout the history of vee in northern south america, periodic epizoodemics in tropical dry and tropical thorn forests were often associated with unseasonably heavy rainfall and flooding during the normal dry seasons; during interepizootic periods, epizoodemic virus could not be isolated . The great atacama desert stretches along the pacific coast from northern chile and along coastal peru nearly to the border with ecuador; rare, but occasional, rainfall interrupts the barrenness of this parched, hostile environment permitting infrequent but noteworthy incursions of mosquitoes and epizoodemic vee virus (4). J) in contrast, in swampy or jungle areas (tropical wet forest) where a definable dry season does not occur normally in countries of central america and eastern south america and in panama, mexico, the florida everglades, and several caribbean islands, field studies by scientists at maru (11), the center for disease control (now centers for disease control and prevention, atlanta, ga, usa) (12), the trinidad virus research laboratory (port of spain) (13), rockefeller foundation laboratory (belem, brazil) (14), and the gorgas memorial laboratory (panama city, panama) (15) had demonstrated presence of antigenically related vee virus strains; resident equids in these sylvatic foci had antibody without signs of disease, but incursions by humans into these sylvatic or endemic areas often resulted in infections and disease . Sylvatic cycles were found in swampy areas in which floating species of water lettuce, pistia stratiotes, provided appropriate habitat for mosquito species of the subgenus culex (melanoconion), the vectors of sylvatic subtypes and variants of vee virus . The epidemiologic cycle involves sylvatic virus transmission by species of terrestrial rodents and possibly birds and arboreal rodents (4). K) sylvatic virus subtypes were of low or no virulence to experimentally infected equids . The virulence of epizootic subtypes was high, with fatality rates to> 90% of infected equids (16). L) historically, vee virus has been a pathogen studied for aerosol release as a potential biologic weapon . In the united states at the army research and development command, (now the army medical research institute for infectious diseases, usamriid), fort detrick, md, the former soviet union and perhaps, elsewhere, vee virus was studied as a possible offensive weapon . (for more information about the history of the us biologic warfare program and fort detrick, go to the following websites: www.detrick.army.mil/cutting_edge/index.cfm, www7.nationalacademies.org/archives/cbw.html, and www.bordeninstitute.army.mil/published.html .) M) in the defensive research programs at usamriid, an equine - virulent isolate from a donkey was serially passed in fetal guinea pig heart cell cultures to produce an attenuated vaccine, strain tc-83, for use in at - risk laboratory and military personnel (4). Attenuated strain tc-83 was derived from the trinidad donkey number 1 isolate from a diseased donkey in that country during a 1940s epizootic that spilled over to trinidad and tobago from mainland venezuela (17). Era concerns about military personnel of the former union of soviet socialist republics and their allies . Aerosol releases of biological, chemical, and radioactive weapons are notoriously difficult to control, leading to use of vaccine, if available, and other, more cumbersome measures to protect military personnel . O) recognizing the difficulties in controlling and using biological weapons, unauthorized release of classified documents, and the moral outrage of us citizens and world public opinion against biological weapons, president richard m. nixon cancelled the offensive biological weapons development programs . P) beginning in 1967, a major epizoodemic of vee occurred across northern south america (4). Requests to the us state department and us military authorities resulted in release of attenuated vee virus vaccine strain tc-83 for emergency use in equids to stop equine disease and interrupt human infections . Vaccine was effective, but the silent epizoodemic tongue of virus transmission repeatedly had moved ahead of the vaccination teams through populations of susceptible hosts and competent vectors . Q) in an unprecedented biomedical event in the history of vee, the epizoodemic vee virus subtype was transported from northern south america to central america (3). Empirical and circumstantial evidence, such as discovery by scientific investigators of empty vials of vee virus vaccine labeled by a manufacturer in south america, suggested that a formalin - inactivated vee virus vaccine was imported to vaccinate valuable horses at breeding farms in guatemala by worried ranch owners (k.m . Safety tests of such inactivated vee virus vaccines in laboratory systems, e.g., cell cultures and laboratory animals, are exquisitely less sensitive than susceptible equids to residual active virus . Non inactivated virus has been postulated to replicate in equids to high titers and to be infectious for the local populations of competent and capable mosquito vectors . The reservoir of epizoodemic vee virus during interepizootic periods is unknown, but studies during the 1990s and 2000s suggest an enhanced virulence of certain isolates of sylvatic virus subtypes and strains for equids, which occasionally are replicated to high titers under undetermined favorable conditions, with subsequent selection of an epizoodemic clone from a mixed virus population that causes clinical vee and infects mosquitoes . R) an internationally recognized clinical and field research expert on zoonotic diseases, ronald b. mackenzie was a visionary and astute medical scientist with the rockefeller foundation in cali, colombia . S) vee virus was transported by mosquitoes and possibly through transportation of asymptomatic but infected horses from the disease or danger zones to unaffected zones where susceptible equids were subsequently infected . Disease was documented along the pacific and caribbean coasts in the tropical dry and thorn forest environments that have been the traditional cattle - raising areas of central and south america and in mexico, where tropical wet forest and swampy environments that support sylvatic vee viruses are irregularly located and noncontiguous or do not occur . T) because of the lack of understanding of the epidemiologic cycle and virus - vector incubation requirements, the epizoodemic wave of infected mosquitoes and equids incubating the virus had not been anticipated to precede vaccination teams routinely into new areas of susceptible equids by 23 weeks . The disease moved to the southeast along the pacific coast to northwestern costa rica, where the advance finally was stopped, protecting panama, probably because of a combination of vaccination, presence of larger sylvatic foci in which larger numbers of resident equids were already immune, and a belt of lowland and montane rain forests where there are fewer cattle and horses and that stretches along the pacific coast of southwestern costa rica to northwestern panama; antibody to sylvatic virus strains provides cross - protection against epizoodemic virus strains . The virus crossed the isthmus of tehuantepec in mexico and moved up both the caribbean and pacific coasts, finally reaching texas in late june 1971 (detection of vee in texas before july 4 was predicted months earlier by k.m . U) mf-8 is an isolate of the epizoodemic vee virus subtype i - a / b from honduras isolated by miguel figueroa, a honduran scientist working at maru . Despite proven efficacy and safety of the attenuated vee virus vaccine in south and central america and mexico, usda authorities delayed application of the vaccine until the first cases were diagnosed in texas . Official usda emergency response policies and regulations at that time did not include the option to use vaccines to interdict threats of foreign animal diseases in the absence of documented disease within the united states (application of vaccines in the usda emergency response plan to incursions of foreign animal diseases was authorized in 2000). Because of bilateral and international agreements and policies regarding emergency disease responses, the adverse impact of applying foreign animal disease vaccines on the exportation of us livestock and agricultural products internationally and the politically charged decision to change existing policy, an authority delegated only to the us secretary of agriculture, vaccine was acquired and stockpiled along the mexico us border but could not be applied until vee was diagnosed . W) attenuated vee virus vaccine was safe, effective, and stable, and reversion to virulence did not occur (18,19). A potentially catastrophic disaster was marginalized and ultimately stopped in the western hemisphere by application of vaccine and other control techniques . Hundreds of thousands of equids and thousands of humans were saved by the emergency responses of veterinary and medical officials in every country from colombia - ecuador - peru - venezuela to mexico and the united states; among the 13 at - risk nations, only panama (and, in addition the caribbean islands and other countries of south america) was spared from this crisis . The successful application to veterinary use of a vaccine developed by the us military as a defensive tool for use in troops and at - risk laboratory personnel was an unforeseen and unanticipated benefit of the us department of defense research program . President franklin d. roosevelt authorized the us army through a civilian agency to develop the us biological warfare program with offensive and defensive objectives in 1942 . Laboratories and pilot plants were constructed at camp (later fort) detrick, maryland; the special procedures program from which the special immunizations program evolved was one of the earliest operations to open . The special immunizations program is responsible for the investigational vaccines, including strain tc-83, which were developed and are used under the investigational new drug authority and guidelines of the food and drug administration . Seed stock of strain tc-83 virus was made available to the biologics industries of western hemisphere countries . Strain tc-83 and other next - generation iterations of the original vaccine, including an inactivated strain tc-83 product (c-84), are used or available in many countries of the western hemisphere . Refrain: mosquito s in his crab hole, bidin his time, venezuela virus working up the line, boys in beltsville heard the news, horses in texas got the crab hole blues . Down in maracay back in 36, kubes and rios found a virus doin tricks, horses die, this one s gotta be, new cause of cephalitis, v - e - e . The years roll by, the virus makes a score, in vene, colombo, and ecuador, comes rain to the desert instead of dew, and, vee burns the coast of peru . In trinidad, panama, they say hey, hey, we got this creature like every day, his swampy home, you can always tell, by finding some rats and the culex (mel). Voice - over: mosquito, that is . Back at maru, raggin the brains, we found that the virus had different strains horses convulse with the southern kind, northern virus leaves them feelin fine . Cause virus hits man by aerosol, could be used to cripple us all, in maryland, they grew it in vats, tested it out in monkeys and rats . The other team outside the fence, lookin for the answer that made some sense, got a strain from an old donkey, made a vaccine, tc-83 . The offensive team tried to make some noise, a cloud for the commies, but not our boys, did not work, that is why, nixon said declassify. Voice - over: the pentagon papers, volume 25, page 324, 1970 the new york times, los angeles times, the washington post, and the christian science monitor . Sleepy defense gets a latin call, new outbreak and it ai nt small, took the vaccine off the wall, it really worked to save them all . Down in guatemala, year of 69, horses started dying on the borderline, virus come down from ecuador, nobody knows the reservoir . Virus hopped the mountains, got to the sea, vaccine brought in and given out free, virus kept movin, had to go, all the way from here to mexico . Cause everybody said, gotta stop that bug, gotta use the vaccine because there ai nt no drug, beltsville brethren said gotta wait, afraid that the vaccine was mf-8 . Results today are plain to see, story s hot on the wire of associated p, a million horses all over the west, are living proof the vaccine was best . A) the presentation is a chronologically, scientifically, and factually correct poetic and musical history of venezuelan equine encephalomyelitis (vee) viruses and epidemics through 1971 . The music is a traditional us jug band song, a type of music bluegrass popular in rural areas of the eastern united states . Using a background of a guitar and everyday musical, rhythm, and percussion devices, such as whistling, blowing air across the mouth of a 1-gallon glass jug, scraping of a scrub brush on a metal washboard, humming harmonically into a kazoo, and the hambone (a rhythmic slapping of hands on arms, hands, and legs), the voices and lyrics convey a musical story to the audience . The title reflects the discovery that deinocerites pseudes, the crab hole mosquito, found along the pacific coast of central america was a competent vector of the epidemic and epizootic vee virus that was causing disease and death in equids and humans at the time (1). The poetic dialogue was written and set to music in 1971 by scientists at the middle america research unit (maru), us department of health, education, and welfare (now health and human services), national institutes of health, national institute of allergy and infectious diseases, us public health service, and located in ancon, panama canal zone, during a major western hemispheric outbreak of vee . Maru scientists had a> 15-year history of vee epidemiologic and virologic studies throughout latin america to characterize the antigenic relationships between south american vee virus strains of subtypes (i - a / b and i - c) that caused epizoodemics of equine and associated human disease and the caribbean, central american, floridian, mexican, panamanian, and south american vee virus strains of subtypes (i - d, i - e, i - f, ii, iii - a, iii - b, and iv) that existed in sylvatic, enzootic cycles in the absence of equine disease but with occasional human infections and disease (2). In 1969, the transfer of equine virulent vee virus from a raging epizoodemic in colombia, ecuador, and peru to the frontier area of el salvador, guatemala, and honduras precipitated a human and veterinary medical crisis in central america, mexico, and the united states that lasted until 1972 (3). The crab hole mosquito blues was written as a scientific presentation for the international workshop - symposium on venezuelan encephalitis virus sponsored by the pan american sanitary bureau, pan american health organization, world health organization, in washington, dc, september 1417, 1971 . Although the lyrics of the crab hole music blues were not published in the proceedings, other presentations from that meeting are documented (4). B) douglas f. antczak, vocals, guitar, kazoo; william h. dietz, vocals, jug, kazoo; karl m. johnson, vocals, kazoo; david h. martin, kazoo, recording engineer; and thomas e. walton, washboard and scrub brush, hambone, kazoo, vocals . C) gender - specific error . Only female mosquitoes (and females of other hematophagous insects, e.g., culicoids, phlebotomids) take a blood meal, which is needed to provide protein necessary for ovulation; only female mosquitoes are infected with and transmit vee viruses (and other mosquito - borne arboviruses). D) beltsville, maryland, usa, the headquarters at that time of the regulatory officials in the us department of agriculture (usda) responsible for the decision to recommend application of preventive vaccines to the secretary of agriculture in the face of an epizootic . Hesitation to vaccinate was predicated on lack of evidence that reversion of virulence of the attenuated vaccine virus could not occur, international trade considerations, international practices and agreements, and authorities and responsibilities delegated only to the secretary of agriculture . E) from 1969, the epizoodemic moved southeast, eventually reaching northwestern costa rica in august 1970, and northward, eventually reaching texas in late june 1971 (4). F) the venezuelan agricultural and veterinary research laboratories are located in maracay, aragua state, venezuela . Kubes and rios first isolated, identified, and named vee virus, then sent the isolate to the united states for confirmation that the south american virus was antigenically distinct from the north american eastern and western equine encephalomyelitis viruses (5,6). G) the antigenically related and clinically similar eastern and western equine encephalomyelitis viruses had been isolated and identified early in the 1930s in the united states (7,8). H) periodic outbreaks of vee had occurred in colombia, ecuador, peru, and venezuela since at least the 1920s, with hundreds of thousands of equine illnesses and tens of thousands of deaths; equids are the primary virus amplifier hosts for human infections (9,10). I) throughout the history of vee in northern south america, periodic epizoodemics in tropical dry and tropical thorn forests were often associated with unseasonably heavy rainfall and flooding during the normal dry seasons; during interepizootic periods, epizoodemic virus could not be isolated . The great atacama desert stretches along the pacific coast from northern chile and along coastal peru nearly to the border with ecuador; rare, but occasional, rainfall interrupts the barrenness of this parched, hostile environment permitting infrequent but noteworthy incursions of mosquitoes and epizoodemic vee virus (4). J) in contrast, in swampy or jungle areas (tropical wet forest) where a definable dry season does not occur normally in countries of central america and eastern south america and in panama, mexico, the florida everglades, and several caribbean islands, field studies by scientists at maru (11), the center for disease control (now centers for disease control and prevention, atlanta, ga, usa) (12), the trinidad virus research laboratory (port of spain) (13), rockefeller foundation laboratory (belem, brazil) (14), and the gorgas memorial laboratory (panama city, panama) (15) had demonstrated presence of antigenically related vee virus strains; resident equids in these sylvatic foci had antibody without signs of disease, but incursions by humans into these sylvatic or endemic areas often resulted in infections and disease . Sylvatic cycles were found in swampy areas in which floating species of water lettuce, pistia stratiotes, provided appropriate habitat for mosquito species of the subgenus culex (melanoconion), the vectors of sylvatic subtypes and variants of vee virus . The epidemiologic cycle involves sylvatic virus transmission by species of terrestrial rodents and possibly birds and arboreal rodents (4). K) sylvatic virus subtypes were of low or no virulence to experimentally infected equids . The virulence of epizootic subtypes was high, with fatality rates to> 90% of infected equids (16). L) historically, vee virus has been a pathogen studied for aerosol release as a potential biologic weapon . In the united states at the army research and development command, (now the army medical research institute for infectious diseases, usamriid), fort detrick, md, the former soviet union and perhaps, elsewhere, vee virus was studied as a possible offensive weapon . (for more information about the history of the us biologic warfare program and fort detrick, go to the following websites: www.detrick.army.mil/cutting_edge/index.cfm, www7.nationalacademies.org/archives/cbw.html, and www.bordeninstitute.army.mil/published.html .) M) in the defensive research programs at usamriid, an equine - virulent isolate from a donkey was serially passed in fetal guinea pig heart cell cultures to produce an attenuated vaccine, strain tc-83, for use in at - risk laboratory and military personnel (4). Attenuated strain tc-83 was derived from the trinidad donkey number 1 isolate from a diseased donkey in that country during a 1940s epizootic that spilled over to trinidad and tobago from mainland venezuela (17). Era concerns about military personnel of the former union of soviet socialist republics and their allies . Aerosol releases of biological, chemical, and radioactive weapons are notoriously difficult to control, leading to use of vaccine, if available, and other, more cumbersome measures to protect military personnel . O) recognizing the difficulties in controlling and using biological weapons, unauthorized release of classified documents, and the moral outrage of us citizens and world public opinion against biological weapons, president richard m. nixon cancelled the offensive biological weapons development programs . P) beginning in 1967, a major epizoodemic of vee occurred across northern south america (4). Requests to the us state department and us military authorities resulted in release of attenuated vee virus vaccine strain tc-83 for emergency use in equids to stop equine disease and interrupt human infections . Vaccine was effective, but the silent epizoodemic tongue of virus transmission repeatedly had moved ahead of the vaccination teams through populations of susceptible hosts and competent vectors . Q) in an unprecedented biomedical event in the history of vee, the epizoodemic vee virus subtype was transported from northern south america to central america (3). Empirical and circumstantial evidence, such as discovery by scientific investigators of empty vials of vee virus vaccine labeled by a manufacturer in south america, suggested that a formalin - inactivated vee virus vaccine was imported to vaccinate valuable horses at breeding farms in guatemala by worried ranch owners (k.m . Safety tests of such inactivated vee virus vaccines in laboratory systems, e.g., cell cultures and laboratory animals, are exquisitely less sensitive than susceptible equids to residual active virus . Non inactivated virus has been postulated to replicate in equids to high titers and to be infectious for the local populations of competent and capable mosquito vectors . The reservoir of epizoodemic vee virus during interepizootic periods is unknown, but studies during the 1990s and 2000s suggest an enhanced virulence of certain isolates of sylvatic virus subtypes and strains for equids, which occasionally are replicated to high titers under undetermined favorable conditions, with subsequent selection of an epizoodemic clone from a mixed virus population that causes clinical vee and infects mosquitoes . R) an internationally recognized clinical and field research expert on zoonotic diseases, ronald b. mackenzie was a visionary and astute medical scientist with the rockefeller foundation in cali, colombia . S) vee virus was transported by mosquitoes and possibly through transportation of asymptomatic but infected horses from the disease or danger zones to unaffected zones where susceptible equids were subsequently infected . Disease was documented along the pacific and caribbean coasts in the tropical dry and thorn forest environments that have been the traditional cattle - raising areas of central and south america and in mexico, where tropical wet forest and swampy environments that support sylvatic vee viruses are irregularly located and noncontiguous or do not occur . T) because of the lack of understanding of the epidemiologic cycle and virus - vector incubation requirements, the epizoodemic wave of infected mosquitoes and equids incubating the virus had not been anticipated to precede vaccination teams routinely into new areas of susceptible equids by 23 weeks . The disease moved to the southeast along the pacific coast to northwestern costa rica, where the advance finally was stopped, protecting panama, probably because of a combination of vaccination, presence of larger sylvatic foci in which larger numbers of resident equids were already immune, and a belt of lowland and montane rain forests where there are fewer cattle and horses and that stretches along the pacific coast of southwestern costa rica to northwestern panama; antibody to sylvatic virus strains provides cross - protection against epizoodemic virus strains . The virus crossed the isthmus of tehuantepec in mexico and moved up both the caribbean and pacific coasts, finally reaching texas in late june 1971 (detection of vee in texas before july 4 was predicted months earlier by k.m . U) mf-8 is an isolate of the epizoodemic vee virus subtype i - a / b from honduras isolated by miguel figueroa, a honduran scientist working at maru . Despite proven efficacy and safety of the attenuated vee virus vaccine in south and central america and mexico, usda authorities delayed application of the vaccine until the first cases were diagnosed in texas . Official usda emergency response policies and regulations at that time did not include the option to use vaccines to interdict threats of foreign animal diseases in the absence of documented disease within the united states (application of vaccines in the usda emergency response plan to incursions of foreign animal diseases was authorized in 2000). Because of bilateral and international agreements and policies regarding emergency disease responses, the adverse impact of applying foreign animal disease vaccines on the exportation of us livestock and agricultural products internationally and the politically charged decision to change existing policy, an authority delegated only to the us secretary of agriculture, vaccine was acquired and stockpiled along the mexico us border but could not be applied until vee was diagnosed . W) attenuated vee virus vaccine was safe, effective, and stable, and reversion to virulence did not occur (18,19). A potentially catastrophic disaster was marginalized and ultimately stopped in the western hemisphere by application of vaccine and other control techniques . Hundreds of thousands of equids and thousands of humans were saved by the emergency responses of veterinary and medical officials in every country from colombia - ecuador - peru - venezuela to mexico and the united states; among the 13 at - risk nations, only panama (and, in addition the caribbean islands and other countries of south america) was spared from this crisis . The successful application to veterinary use of a vaccine developed by the us military as a defensive tool for use in troops and at - risk laboratory personnel was an unforeseen and unanticipated benefit of the us department of defense research program . President franklin d. roosevelt authorized the us army through a civilian agency to develop the us biological warfare program with offensive and defensive objectives in 1942 . Laboratories and pilot plants were constructed at camp (later fort) detrick, maryland; the special procedures program from which the special immunizations program evolved was one of the earliest operations to open . The special immunizations program is responsible for the investigational vaccines, including strain tc-83, which were developed and are used under the investigational new drug authority and guidelines of the food and drug administration . Seed stock of strain tc-83 virus was made available to the biologics industries of western hemisphere countries . Strain tc-83 and other next - generation iterations of the original vaccine, including an inactivated strain tc-83 product (c-84), are used or available in many countries of the western hemisphere.
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Juvenile idiopathic arthritis (jia) is the most common inflammatory rheumatic disease in childhood, affecting one in 1000 children . Jia is characterized by severe joint inflammation in one or more joints, which persists for at least six weeks, with disease onset before the age of 16 . This heterogeneous group of diseases can be divided into several subtypes on the basis of clinical symptoms, medical history, and abnormalities in laboratory measures . A biomarker is a small component which is easily measurable in accessible patient material, e.g. Blood, urine or saliva, and is ideally obtained using a relatively non - invasive approach . Ideally, the component used as a biomarker should be stable over time within the sample, and would be able to be measured by an accurate, reproducible assay, at a relatively affordable cost to health service providers . In addition, the ideal biomarker for paediatric use would not be affected by age - related development of children, avoiding the need for age - specific normal - range data sets . Biomarkers are already used in many areas of clinical practice, but most biomarker studies focus on adults rather than children . Data from these studies are sometimes extrapolated to children without considering differences in disease pathogenesis, age - dependent changes in reference ranges for biological laboratory measures, growth and development of children over time, effect of ontogeny on disease evolution and response to treatment, and changes in phenotypic gene expression [3, 4]. Despite the huge potential of paediatric biomarkers, for jia there are currently no validated paediatric biomarkers available to help in setting up a tailored or a more tailored approach would be beneficial for patients because it could facilitate disease remission at an earlier disease stage, which would reduce burden of disease, limit side effects, and improve quality of life . In this review we will discuss recent developments in the potential use of biomarkers for jia, for predicting the type, severity, and progression of disease after onset and the development of complications linked to jia, and their effect on our ability to predict response to treatment and determine when stable disease remission has been reached . Jia is a heterogeneous group of disorders, and its classification relies on both clinical findings and a small number of biomarkers used to divide cases into relatively homogeneous subtypes . For example, the two polyarticular forms of jia, involving five or more joints in the first six months of disease, are distinguished by the absence or presence of serum autoantibody, known as rheumatoid factor (rf), on two occasions three months or more apart . These two clinical subgroups of jia are distinct in their genetics, age of onset, and prognosis [6, 7]. Similarly, the presence of positive serum anti - nuclear antibody (ana) has been revealed in several studies to be associated with an increased risk of chronic anterior uveitis in jia . This is a serious comorbidity of jia, involving painless but potentially very damaging inflammation of the anterior chamber of the eye, which requires assiduous screening to prevent permanent visual loss [8, 9]. A recent large cohort study has confirmed earlier studies demonstrating that ana positivity is a risk factor for developing jia - associated uveitis; current uk clinical guidelines for how long screening should be continued include the use of ana positivity to direct clinical practice . Thus, both of these antibody biomarkers (rf and ana), which are stable proteins and are easily measured in a small volume of serum, form part of routine clinical care and treatment pathway decisions for jia . At the mild end of the jia clinical spectrum, oligoarticular jia, which presents with involvement of four or fewer joints in the first six months of disease, can lead to widely divergent outcomes, ranging from complete remission off medication, to a more severe, extended form of jia which spreads to involve many joints . Extended oligoarticular jia can be highly erosive and destructive and may be difficult to control with conventional disease - modifying antirheumatic drugs (dmards), frequently requiring long - term treatment with biological therapy . Several studies have revealed that immunological differences between these two outcomes (persistent versus extended oligoarticular jia) can be observed in the inflamed joint . For example, t cell types (e.g. Regulatory t cells, or highly proinflammatory th17 cells) and their frequencies differ significantly between these two clinical types [13, 14]. In addition, differences in cell frequencies, inflammatory protein levels, and gene expression can be detected in children who will develop more severe disease, before extension occurs: for example, in the so - called extended - to - be group of cases, the cd4:cd8 ratio in synovial fluid is lower and the levels of the chemokine ccl5 are higher in extended - to - be oligoarticular jia compared with persistent oligoarticular jia [15]. In this study, analysis of differentially expressed genes at mrna level also provided novel insights into the pathological mechanisms involved in severity, indicating the importance of the complement pathway and of activated monocytes in extension to more severe disease . Studies analysing the proteome within synovial fluid have also revealed differences between these subtypes: gibson et al . Demonstrated that the proteome and post - translational modifications of proteins differed between those whose disease remained mild and those who went on to develop severe disease . Validation of these findings will be required before development of a predictive biomarker test, but this field may well yield valuable biomarkers by which to identify children with a poor prognosis . Assessment of disease activity in jia typically includes measurement of inflammatory markers in peripheral blood, including either the erythrocyte sedimentation rate (esr) or c - reactive protein (crp) [17, 18]. Both of these inflammatory indices have low sensitivity and specificity, because they can be raised for many reasons other than jia activity and in some children do not closely mirror disease activity . Recently, the pro - inflammatory s100 proteins, s100a8/9 (also known as calprotectin or myeloid - related protein (mrp) 8/14) and s100a12, have been described to be sensitive measures for disease activity in jia, and both correlate well with physicians assessment of disease or with actively inflamed joints [5, 19]. S100 proteins can be measured in serum by enzyme - linked immune - sorbent assay (elisa). However, the standardisation of the measurements and the detailed procedures for assay and dilution of serum samples from a wide range of patients, with varying disease activity, are non - trivial, complicating implementation of such a test in routine clinical care . The s100 proteins are released into serum at highly elevated levels in the most severe form of jia, systemic onset jia (sjia), where they correlate well with disease activity as assessed by physicians global assessment of disease activity (r = 0.62), childhood health assessment questionnaire (r = 0.56), and active joint count (r = 0.46)and with crp (r = 0.71) and esr (r = 0.72) (for all p <0.001). A recent study which included measurement of serum s100 proteins has proposed a biomarker panel for predicting flare in sjia, compared with quiescent disease . The same group also tested the feasibility of measuring such biomarkers in urine for sjia, which could have many advantages for the paediatric population, especially if testing kits could be designed for use in the clinic office or even at home [22]. One of the most severe complications of sjia, known as macrophage activation syndrome (mas) or secondary hemophagocytic lymphohistiocytosis (hlh), remains a cause of mortality in jia and may be difficult to distinguish from infection [23, 24]. A gene expression profiling study, of sjia patients with or without mas compared with control patients, identified clusters of genes that correlated with sjia activity and with mas . If validated, these gene clusters could lead to an mrna - expression biomarker panel for predicting this potentially life - threatening complication, or to distinguish it from other complications . Interestingly, some of the differentially expressed mrna species identified, which differed between children with mas and those without, were transcripts of genes known to be involved in other causes of hlh, for example rab27a and sh2d1a . Thus, several autoantibodies are already in widespread use as biomarkers in routine care of jia, and some newer biomarkers (protein, cellular, or mrna) are under development, for use in defining disease subtype, probable disease course, or comorbidity . Recommendations for treatment of jia are made by the american college of rheumatology (acr), and are regularly reviewed . Depending on the number of joints affected and after failure of monotherapy with non - steroidal anti - inflammatory drugs (nsaids) and/or glucocorticoids joint injection in cases of less severe disease the first - line treatment approach for active disease is administration of methotrexate (mtx). In cases of no or poor response to mtx, biological agents, for example drugs which block tumour necrosis factor alpha (tnf), are added to the treatment strategy . Although many patients respond well to mtx and reach stable disease remission, approximately 3050% [2729] of patients treated with mtx either do not respond or respond poorly . Many studies analysing response to mtx used the core set variables defined by giannini et al ., in which levels of response in terms of acr30, 50, or 70 are calculated . Those patients who fail to respond to mtx are first exposed to mtx, and experience side effects associated with non - response to mtx, before more effective biological agents can be offered . A biomarker (or set of biomarkers) predicting which patients will respond to treatment would be beneficial for the patient and prevent side effects caused by ineffective drugs . The pro - inflammatory s100 proteins have been shown to correlate well with disease activity [5, 19, 20, 30], and recently the level of s100 proteins in serum has been shown to also be correlated with response to treatment . Six months after starting treatment, mrp8/14 levels were lower in sjia patients responding to mtx treatment (defined as reaching at least acr70), whereas mrp8/14 levels in sjia patients not responding to mtx treatment (acr30) were increased or slightly decreased . Furthermore, sjia patients treated with anti - il1 or anti - tnf had reduced mrp8/14 levels and disease activity . Patients who did respond to mtx had more active joints, higher crp levels, and higher serum cytokine levels before the start of mtx, which suggests that higher disease activity is positively correlated with good response to mtx . In addition, jia patients with high levels of mrp8/14 before starting mtx treatment had a higher chance of good response to mtx and had better disease outcomes (at least acr50) after six months of treatment than non - responders (acr30 or below). Differences between patients and response to treatment could be genetic, and therefore genetic factors could be potential biomarkers . One study identified two single nucleotide polymorphisms (snps) in the 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (atic) gene and one snp in the inosine triphosphate pyrophosphatase (itpa) gene which were found to be associated with a higher risk of poor response to mtx treatment . Using a validation cohort, one of the snps in the atic gene had a trend towards association with response to mtx . Genotyping of snps in genes involved in the polyglutamylation process of mtx and in cellular uptake and efflux of mtx have also shown association with mtx response . One snp in the adenosine - triphosphate - binding cassette transporter b1 (abcb1) and one snp in abcc3 were found to be associated with good response to mtx, whereas an snp in the solute carrier 19a1 was associated with poor response to mtx . In another approach, snp genotyping of genes found to be differentially expressed in a gene expression profiling study, of a uk cohort of jia patients before and after treatment with mtx, identified three snps in the solute carrier family 16 member 7 (slc16a7) gene associated with response to mtx . Validation of these snps in a validation cohort revealed significant association of one of the snps with non - response to mtx . A prediction model was recently developed combining clinical and genetic variables to predict non - response of jia patients to mtx . In this study, response was defined as reaching acr70 in at least two out of three visits during the first year of treatment . Such a model is important, because it could help in preventing unnecessary treatment of patients who will benefit from monotherapy with mtx and do not need additional treatment with biologicals, which are expensive and might have side effects . The clinical variable included in the model is the esr . Genetic variables are snps in the genes coding for methionine synthase reductase, multidrug resistance 1 (mdr-1/abcb1), multidrug resistance protein 1 (mrp-1/abcc1), and proton - coupled folate transporter (pcft). The model had a moderate predictive power of 65% for the validation cohort, which might be because of the small number of patients included in the validation cohort [34]. Thus, evidence suggests that the pro - inflammatory s100 proteins have the potential to serve as biomarkers for predicting response to treatment . Furthermore, several snps have been found to be associated with response to treatment, indicating that genetic factors can also provide such biomarkers . It is probable that a combination of genetic, biological, and clinical variables will be required to develop accurate and robust predictive algorithms with which to predict response to drug treatment of jia, and these may need to be disease - subtype specific . Clinical remission in jia can be reached with the use of medication, and is ideally maintained after treatment is stopped (criteria for clinical remission on and off treatment are described by wallace et al . ). However, after discontinuation of mtx treatment, approximately 3050% of patients relapse [2729], suggesting the presence of subclinical disease activity . This subclinical disease activity is not detectable using clinical and standard laboratory tests, and these patients will not reach stable remission off medication [37]. Subclinical disease activity makes it difficult to determine when jia patients have reached stable remission and treatment can be stopped . Therefore, a biomarker identifying patients at risk of disease relapse on the basis of the inflammatory status of their disease would be extremely valuable for improving paediatric medical care . It has been suggested that continuation of mtx treatment for a longer period of time after clinical remission has been reached might reduce the risk of relapse after stopping treatment . Therefore, a randomised clinical trial was conducted to investigate whether longer treatment with mtx, for those who achieved clinical remission, could reduce relapse after mtx withdrawal, and which biomarkers could identify patients at higher risk of relapse after withdrawal . Patients were continuously treated with mtx for either six or 12 months after clinical remission was reached, and then followed after withdrawal of mtx . However, there was no significant difference in relapse between the two groups [37]. On the basis of previously published results from this group, which revealed that mrp8/14 is a marker for subclinical disease activity [38, 39], they determined mrp8/14 levels in serum in both groups . Interestingly, patients with a high relapse rate after discontinuation of mtx had higher levels of mrp8/14 before stopping mtx than patients who did not flare . Thus, serum mrp8/14 levels could be a potential biomarker for predicting which patients will achieve stable remission after withdrawal of mtx, on the basis of inflammatory disease status [37]. Similar results regarding the potential of mrp8/14 as a biomarker to identify patients at risk of relapse were described specifically for sjia patients . Patients with new onset of active disease or with relapse had higher levels of serum mrp8/14, compared with patients who had reached stable remission . Measurements of mrp8/14 levels at time of stopping treatment revealed that, within up to six months, mrp8/14 levels were higher in patients with disease relapse than patients with no disease relapse . Prediction of patients at risk of relapse was highly accurate, with a sensitivity of 92% and specificity of 88% using mrp8/14 levels> 740 ng ml as a cut - off . Another study investigated whether s100a12 and high sensitivity (hs) crp could identify patients who have reached clinical remission on treatment, and are at risk of disease relapse after withdrawal of medication . High s100a12 and mrp8/14 levels were observed in patients who suffered disease relapse within six months of discontinuation of treatment . Hscrp levels did not differ between patients with disease relapse and those in remission, but this could be caused by the use of inclusion criteria based on normal hscrp levels, because patients with high hscrp levels are more prone to disease relapse . The patient group which relapsed within three months of stopping treatment had higher median levels of s100a12 and mrp8/14 compared with the group which relapsed later . S100a12, mrp8/14, and hscrp were separately tested for their performance as biomarkers, and s100a12 was the best single biomarker for predicting disease relapse . Treatment strategies for jia have the objective of full clinical remission, but only a very small number of patients remain in clinical remission, off medication, for long periods of time . A recent study compared the transcriptional profiles of patients with active disease or those in clinical remission on medication (achieved by treatment with mtx or mtx plus tnf blockade) with the profiles of healthy children . Differences in transcriptional profile were observed between patients with active disease and those in clinical remission on medication, and also between those in remission off treatment and healthy children . Interestingly, the same group also demonstrated differences in transcriptional profile between patients in clinical remission induced by treatment with mtx or with mtx plus tnf inhibitor, and healthy individuals . This study confirmed previous findings from the group that the inflammatory status of patients in remission does not return to normal . Network analysis suggested a function for hepatocyte nuclear factor 4 alpha (hnf4), which is expressed by t cells and granulocytes, in controlling genes associated with remission . Such tests need to be reliable, simple to perform, economically feasible, and robust . In addition, they need to be tested and validated for large cohorts of children with jia . Where proved reliable, biomarkers may assist with predicting disease type, course or severity, with predicting response to medication and therefore aiding treatment choices, and with accurate identification of children who can safely stop medication once apparent clinical remission is reached . The accurate choice of medicines most likely to work for each child, to enable the achievement of rapid remission for all, while avoiding unnecessary exposure to toxic drugs for those who do not need them and reducing unopposed inflammation to a minimum, is now the ultimate objective of modern management of arthritis . To move rapidly towards such ambitious objectives, multi - centre and international collaborations are needed to give every child with jia the opportunity to take part in biomarker and cohort studies.
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A 29-year - old man (contact 1) transported 8 dromedaries from oman to united arab emirates on may 7, 2015 (table 1). The same day, as part of a national policy for controlling mers, samples were collected from the dromedaries at a screening center located at the united arab emirates border . The samples were tested by reverse transcription pcr (rt - pcr) on may 10 and found to be positive for the mers - cov open reading frame (orf) 1a and upstream of e genes (10). This finding led local public health authorities to conduct active surveillance on humans who had contact with the infected dromedaries . * contacts 1 and 2, humans who had direct physical contact with infected dromedaries; mers - cov, middle east respiratory syndrome; rt - pcr, reverse transcription pcr . A sputum sample collected from contact 1 on may 10, 2015, was tested by rt - pcr on may 12 and found to be positive for mers - cov; the man was admitted to a hospital the same day . Follow - up respiratory samples obtained on may 13 and 14 were still rt - pcr positive, but a sample obtained on may 18 was negative . The patient was asymptomatic at hospital admission and throughout his hospital stay (technical appendix). Contact 2 was a 33-year - old man who worked at the screening center mentioned above . He had direct contact with the same group of infected dromedaries during the sampling procedures . A nasal aspirate sample was obtained from the man on may 14, 2015, and found to be rt - pcr positive for mers - cov . A follow - up sample obtained on may 18 was rt - pcr negative for mers - cov . Samples from 32 other persons were also tested by rt - pcr (technical appendix). Seven days later (may 14), follow - up nasal swab samples from 5 dromedaries were still positive by rt - pcr (table 2); the animals also had mucopurulent nasal discharge . The animals were tested for the presence of mers - cov specific neutralizing antibodies (11); all were seropositive . Two 4-month - old calves (adfca - hku1 and adfca - hku2) had the highest virus loads by real - time rt - pcr and the lowest neutralizing antibody titers (table 2). Positive by rapid antigen testing (12), which suggests the calves were still shedding virus 7 days after the first detection of virus . The 2 calves were rt - pcr negative for mers - cov, and the whole group of camels was released from quarantine . * ct, cycle threshold; i d, identification; mers - cov, middle east respiratory syndrome; neg, negative; pos, positive; rt - pcr, reverse transcription pcr . Results were determined by open reading frame 1a and upstream of e gene rt - pcr assays; ct values were from the open reading frame 1a assay . Results were determined by immunochromatographic tests for mers - cov nucleocapsid protein (12). Respiratory specimens from the 2 infected humans and the 5 dromedaries that were still positive at the second sampling were analyzed by dideoxy sequencing as previously described (13); the nucleocapsid gene sequences of all dromedary samples were found to be identical . Samples from dromedaries adfca - hku13 were selected for further analysis, and a sequence contig encompassing the 3 end of the orf1ab gene through the 3 untranslated region of the mers - cov genome (8,900 nt; sequence coverage 4) was obtained from each sample . Contigs from the 3 samples were identical, with the exception of a v221i (gttatt) mutation in the orf4b protein of the sample from dromedary adfca - hku2 . The viral rna content of the 2 human samples available for analysis was too low to provide long pcr amplicons (cycle threshold 35.5 and 36.9 by upstream of e gene assay). However, partial sequences of mers - cov spike (466 nt, contacts 1 and 2), orf34a (273 nt, contact 1), and nucleocapsid (451 nt, contacts 1 and 2) gene regions could be detected from the samples . The 3 sequence contigs obtained from the dromedary samples were phylogenetically closely related to those of viruses detected in humans in the saudi arabia, china, and south korea in 2015 (figure). All sequences from this cluster, together with the partial orf34a sequence detected in the sample from contact 1, shared 2 cluster - specific mutations, 79s (tcatct) and p86l (cct ctt), in the orf3 protein, suggesting that these viruses may share a common lineage . Apart from the unique v221i mutation, the sequences for viruses from the 3 dromedaries shared a unique orf4a - q102e (gagcag) mutation that was not found in any published mers - cov genomes . Other than those mutations, all of the orfs (nonstructural protein 13, spike, orf3, orf5, envelope, membrane, nucleocapsid, and orf8b) of these virus sequences were unremarkable . Phylogenetic analyses of partial middle east respiratory syndrome coronavirus (mers - cov) genomic sequences for viruses detected in dromedaries imported from oman to united arab emirates, may 2015 . A partial viral rna sequence spanning the 3 end of the open reading frame 1ab gene through the 3 untranslated region of the mers - cov genome (8,900 nt) was used in the analysis . The phylogenetic tree was constructed with mega6 software (http://www.megasoftware.net/) by using the neighbor - joining method . Symbols indicate mers - covs detected from dromedaries s. scale bar indicates the estimated genetic distance of these viruses . We report 2 cases of mers - cov infection in men who had direct contact with the same group of infected dromedaries . Neither man had a concurrent medical condition or a history of exposure to human mers cases in the 14 days before their first mers - cov positive test results . Genomic sequences for the viruses derived from the men and dromedaries and findings from the epidemiologic investigation suggest possible zoonotic transmission of mers - cov from dromedaries to humans . Although it is unlikely, we cannot exclude the possibility that the men and dromedaries were independently infected by other sources . Both infected humans were kept in the hospital for 2 incubation periods and were asymptomatic during this period . Clinical observations and positive rt - pcr results suggest that the men were asymptomatically infected with mers - cov . Our findings provide further evidence that asymptomatic human infections can be caused by zoonotic transmission . Nonetheless, our findings highlight the importance of systematic surveillance of persons who have frequent contact with dromedaries . A recent study demonstrated that persons who have frequent exposure to camels are more likely than the general population to be seropositive for mers - cov (4). The unique border screening program and multisectoral collaborations highlighted in this investigation serve as a model for effective mers - cov surveillance at the animal human interface . We did not test serum samples from the human contacts; such testing would be of interest for follow - up investigation of the patients serologic responses . We also obtained limited rna samples from these persons, which prevented us from conducting more extensive viral sequence analyses . Mers - cov genomic sequences determined in this study are similar to those of viruses detected in 2015 in patients in saudi arabia and south korea with hospital - acquired infections . The infected dromedaries in this study were imported from oman, which suggests that viruses from this clade are widely circulating on the arabian peninsula . Sequence analyses of mers - covs found in south korea and china do not suggest that viruses from this clade are necessarily more transmissible variants (15). However, given that a single introduction of mers - cov from this clade caused> 180 human infections in hospital settings (2) and that viruses of this clade are causing other human infections in saudi arabia, further phenotypic risk assessment of this particular mers - cov clade should be a priority . Additional information regarding 2 persons with asymptomatic mers - cov infection and other persons tested in the study.
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From msm who attended the amsterdam municipal health service sti outpatient clinic in 2000 and 2001, randomly selected stored specimens of c. trachomatis dna positive (as assessed by ligase chain reaction, abbott laboratories, chicago, il, usa) rectal samples were tested for the c. trachomatis variant by real - time pcr (6). From 2002 to 2005, msm with symptomatic proctitis (i.e., purulent discharge, rectal ulceration, bleeding, or edematous mucosa) and msm without symptoms were included . From the san francisco region, 51 lgv positive isolates from symptomatic msm were analyzed (7). The isolates were collected in medical clinics (e.g., ambulatory care, emergency room, screening, acute care) from 1979 to 1985 (table). Lgv was assessed at the time of collection, according to phenotypic properties observed during cell culture . Although the growth characteristics of lgv serovars can be distinguished from serovars d k, cell culture for c. trachomatis is no longer available in most clinical settings . * in 2002 and 2003, 45 lgv l2b variants of 109 isolates have been described in detail (5). C. trachomatis serovar typing was performed as described previously (5). Briefly, amplification of the ompa gene (1.1 kb) was performed in a nested pcr format . The ompa nucleotide sequences were subsequently analyzed by automated dna sequencing on an abi 310 sequencer (pe biosystems, foster city, ca, usa). The sequences obtained from c. trachomatis infected msm in 2000 and 2001 in amsterdam and from msm in san francisco were compared to the recently identified l2b variant to determine if the strain was present earlier . The table presents the results of this analysis . In the amsterdam c. trachomatis dna positive rectal samples, lgv strains were detected by real - time pcr in 2 of 67 samples in 2000 and in 4 of 28 samples in 2001 . Sequencing showed that in all 6 lgv strain positive samples, the l2b variant was present . Also in 2002 and 2003, 109 l2b - positive samples of 403 c. trachomatis dna positive rectal samples were identified, of which 45 were strain l2b, and these have been described in a previous publication (5). All 51 san francisco specimens (from 51 patients) were positive for lgv variants by real - time pcr . By sequencing variable segment 2 of the ompa gene (vs-2), we identified 15 as serovar l1, 18 as serovar l2 prototype, and 18 as the l2b variant . We sequenced the complete ompa gene of 5 of these 18 l2b variants that originated in san francisco; all were identical to the recently described l2b variant circulating in amsterdam . Four nucleotide changes were found when compared to reference serovars l2, l2a, and the variant l2, including 1 change that encoded the previously undescribed change at amino acid 162, aatagt (5). The l2b lgv variant identified as the cause of all the lgv proctitis in the recent outbreak among msm in amsterdam appears to have been circulating in amsterdam in 2000 . Moreover, we showed that this l2b variant was present in the 1980s in san francisco with exactly the same mutations in the complete ompa gene . However, since we only sequenced the ompa gene, and although the sequence was identical in old and new l2b strains, we cannot exclude the possibility that it could involve different strains of c. trachomatis that differ in other parts of the genome, although this is unlikely . Since lgv causes potentially severe infections with possibly irreversible sequelae if adequate treatment is not begun promptly, early and accurate diagnosis is essential . Sequence - based nucleic - acid tests that can discriminate between lgv serovars and less invasive c. trachomatis species can help detect cases and prevent further transmission of lgv . In conclusion, our results suggest that we are dealing with the same lgv variant> 25 years later, and the current lgv outbreak in industrialized countries has most likely been a slowly evolving epidemic with an organism that has gone unnoticed in the community for many years and is now being detected by new technologies . The numbers detected in 2005 in amsterdam suggest that a considerable reservoir exists, which emphasizes the need for ongoing public health awareness.
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Immune (idiopathic) thrombocytopenic purpura (itp) is an autoimmune disorder characterized by persistent thrombocytopenia due to antibody binding to platelet antigen(s) and causing their premature destruction by the reticuloendothelial system, particularly in the spleen . The american society for hematology guidelines define itp as isolated thrombocytopenia with no clinically apparent associated conditions or other causes of thrombocytopenia . Therefore, itp is a condition generally diagnosed by exclusion of the numerous other causes of thrombocytopenia, such as infections, medications, hematological malignancies, disseminated intravascular coagulation, and other autoimmune conditions . In itp, it is estimated that thrombocytopenia (defined as a platelet count less than 150 10/l) occurs in approximately 7% of pregnant women, with 74% of those with low platelet counts having incidental thrombocytopenia of pregnancy that can be managed routinely and in which the platelet count remains more than 70 10/l . Additional causes of thrombocytopenia include complications of hypertensive disorders in pregnancy (21%) and immunological disorders of pregnancy, including itp, systemic lupus erythematosus, and other secondary causes of immune thrombocytopenia (4%). Itp occurs in 1 to 2 of every 1000 pregnancies, which in the united states represents about 3000 to 6000 cases of itp in pregnancy per year . Several studies have examined pregnancy outcomes for women with itp and found that most pregnancies were uneventful, with successful outcomes for both mothers and children [68]. The majority of studies used data from single medical centers [6, 9, 10], by employing retrospective analyses of medical charts [1015]. Most studies have not differentiated between incidental thrombocytopenia, itp, and citp in women who have low platelet counts during pregnancy, and, to our knowledge, there are few published reports regarding the outcomes of pregnant women with citp . The primary objective of this study was to examine the pregnancy and birth outcomes of women with itp and citp identified from medical claims and abstracted medical records from a large health plan in the united states . Our hypothesis was that there would be no significant difference in adverse pregnancy outcomes, including congenital anomalies, between the two groups of women (itp versus citp). The method of data ascertainment enabled the inclusion of data from multiple medical centers, thus increasing the size of the patient cohort and extending the study to a broader population . We studied pregnancy and birth outcomes among women with itp or citp over 16 years of age from 1994 to 2009 inclusive . The data for this study were derived from a large us health insurance database based on eligibility, pharmacy claims, and medical claims data . This study utilized stork (systematic tracking of real kids) to identify pregnancies and link the health experience of mothers with that of their infants within an administrative claims database in order to study the effects of maternal exposures and health conditions on pregnancy outcomes [16, 17]. Further details about stork are provided in supplementary material available online at http://dx.doi.org/10.1155/2016/8297407 . After obtaining institutional review board (irb) approval and waiver of patient authorization from the affiliated privacy board, the study population consisted of women with at least one diagnostic claim for itp (icd-9 code 287.31 or 287.3 if used before 01/12/2005) and at least one diagnostic or procedure code related to pregnancy (live birth, spontaneous abortion, stillbirth, or therapeutic abortion) in the database between 1995 and 2009 . To be eligible for the study, women with at least one claim for itp had to fulfill the following criteria: have complete medical and pharmacy benefit coverage, have at least 280 days of continuous enrollment before the pregnancy outcome date, and have a 9-month baseline period prior to the estimated date of conception . If a woman had more than one pregnancy during the study period, analyses included the first pregnancy after the first itp claim, ignoring any subsequent pregnancies . If a woman's first itp claim was later than the end date of her last pregnancy, she was not eligible for this study . The database was then searched for infants associated with the deliveries and linked them to the mother's claims . Linkage between mother's and newborn's claims could not be performed if the infant was enrolled in another health insurance plan, as may happen if the partner has a different health insurer . Patients were classified as having citp if they met at least one of these requirements: two or more claims for itp separated by at least 6 months; a claim for itp and treatment occurring at least 6 months later with one or more of these medications: corticosteroids, anti - d antibody, rituximab, danazol, colchicine, and dapsone; and/or a claim for splenectomy after the first diagnostic itp claim . Indicators for the timing of the itp or citp diagnosis relative to pregnancy were based on the estimated date of conception and the end of pregnancy . Patients were classified as having itp or citp before pregnancy if they met the full diagnostic criteria before the estimated date of conception . Patients were classified as having itp or citp during pregnancy if they met the full diagnostic criteria before the end of the pregnancy . Women who had an initial claim for itp prior to delivery or termination of pregnancy but met the full diagnostic criteria for citp after pregnancy were considered to have itp during pregnancy, but not citp, since they did not meet the full criteria until after the pregnancy ended . The primary outcome of interest was the prevalence of major congenital anomalies (mcas) at birth (i.e., anomalies that cause significant functional or cosmetic impairment, require surgery, or are life - limiting). Other birth outcomes evaluated included 3 or more minor congenital anomalies, preterm birth (<37 weeks of gestation), low birthweight (<2,500 grams), and measurements consistent with small for gestational age (sga) status (weight, length, or head circumference below the 10th percentile for sex and gestational age) among infants born to mothers with itp or citp . Pregnancy outcomes in this study included live born infant, spontaneous abortion, elective termination, and stillbirth . If the claims search did not identify a pregnancy outcome, the pregnancy outcome was classified as unknown . Claims were reviewed to identify medical providers and facilities to query from them medical records to confirm pregnancy outcomes and to collect additional data on pregnancy characteristics and infant follow - up for the first year of life . Each patient's pregnancy medical records were abstracted to obtain relevant covariate data from their prenatal and pregnancy outcome history, including estimated date of conception and date and type of pregnancy outcome . For infants with evidence of a congenital anomaly diagnosed within the first 12 months after birth, as identified through icd-9 diagnosis or procedure codes in the medical claims, the medical record was sought from the physician or hospital where the anomaly was diagnosed . A standardized medical record abstraction form was used to record elements from each mother's chart, including the reported estimated date of conception, type of pregnancy outcome, and other pregnancy characteristics from relevant clinician notes, inpatient records, and/or hospital discharge summaries . Patient information drawn directly (without abstraction) from deidentified medical records of infants with claims - based congenital anomalies was combined with claims data and reviewed by a dysmorphologist to validate the diagnosis . Following medical record review, all pregnancies were classified according to covariates describing maternal characteristics, pregnancy characteristics, and maternal comorbidities and according to whether they were identified through claims or medical chart abstraction . Maternal characteristics included age and year at conception, geographic region of health plan, and ethnicity . Pregnancy characteristics included use of prenatal vitamins, amniocentesis, chorionic villus sampling, fetal monitoring, ultrasonography, alpha - fetoprotein (afp) testing, obstetric panel, multiple gestation, high - risk pregnancy supervision, antepartum hemorrhage, abruptio placentae, placenta previa, excessive vomiting, early or threatened labor, late pregnancy, measurements consistent with sga, disorders relating to short gestation and unspecified low birthweight, and total cost of care for up to 280 days before delivery . Maternal comorbidities such as diabetes, hypertension, and infections, along with substance abuse and receipt of teratogenic drugs during pregnancy, were identified using diagnostic codes and medication claims . Frequency analyses were carried out for categorical variables and means and standard deviations were calculated for continuous variables, such as the number of physician visits . Data were analyzed separately for women who fulfilled the criteria for itp or citp prior to pregnancy and those who fulfilled the criteria during pregnancy . For infants with claims of congenital anomalies, data were categorized according to maternal age at conception as follows: 14 years and under, 5-year intervals from 15 years through 49 years, and over 50 years . We identified 585 women with at least one claim of itp and claims indicating pregnancy during the study period (january 1, 1994, through december 31, 2009). The remaining 446 women made up the claims - based itp cohort for this study . Medical records were sought to provide more detailed information on the patients' pregnancy characteristics not captured in the claims data, and charts for 311 of 446 women (69.7%) were obtained . Figure 1 provides a schematic view of the claims - based pregnancy outcomes observed in this cohort of 446 women with claims for itp and the results of chart - based review . Among the 311 women with charts, outcome data were unavailable for 42 of them . Of the remaining, 260 charts had a confirmed live birth and 9 (3.3%) indicated a fetal loss with no further details . Among the 446 pregnancies in women with a claims - based diagnosis of itp the claims - based diagnosis was available in 432 cases before or during the pregnancy (table 1). Approximately half of the women reside in the south / southeast regions of the united states and, of those with ethnicity reported in the enrollment data, approximately 70% are caucasian . Of the 446 women, 84 (18.8%) were identified as having citp before or during pregnancy . Of all 446 pregnancies with claims - based diagnosis of itp, 346 (77.6%) indicated a live birth (table 2). When stratified by timing of itp diagnosis, live births were more frequent among women diagnosed with itp during pregnancy (290 of 357 or 81.2%) than in pregnant women diagnosed with itp before pregnancy (56 of 89 or 62.9%). Of those who were diagnosed with itp during pregnancy, 14/357 (3.9%) were fetal losses compared with 10/89 (11.2%) of those diagnosed with itp prior to pregnancy . The magnitude and trend of this difference (7.3%) were not found among women with citp (9/66 or 13.6% of those diagnosed during pregnancy resulting in fetal loss compared to 2/18 or 10.2% among those diagnosed prior to pregnancy). The prevalence of low birthweight was higher among women with a diagnosis of itp before pregnancy (10 of 56 or 17.9%) compared to women with a diagnosis of itp during pregnancy (28 of 290 or 9.7%). The prevalence of low birthweight in women with citp was similar in both groups, but the sample size was too small to reach a conclusion . Table 3 presents the prevalence of major congenital anomalies (mcas) among the 346 infants of mothers with claims for itp . There were 27 infants (n = 27/346; 7.8%) who had a claim for at least one major malformation and charts were available for 17 of them . The most frequent claims were for ostium secundum type atrial septal defects [n = 10/346 (2.9%)], hypospadias [n = 8/151 (5.3%)], patent ductus arteriosus [n = 6/346 (1.7%)], and ventricular septal defect [n = 4/346 (1.2%)]. A total of 4 infants (1.2%) had claims for 3 or more major malformations . Of all infants of mothers with claims for itp that were assumed to have no mca and whose charts were obtained (n = 336), 10 (3.0%) had at least one chart - confirmed major malformation (table 4). The most frequent of these confirmed mcas were hypospadias [n = 4/336 (1.2%)], ventricular septal defect [vsd, n = 3/336 (0.9%)], and patent ductus arteriosus [pda, n = 2 (0.6%)]. Two infants (0.6%) had 3 or more confirmed major malformations . Among the subgroup of 68 infants of women with claims for citp, 7/68 (10.3%) the most frequent claims were for hypospadias [n = 4/30 (13.3%)], ostium secundum type atrial septal defects [n = 2/68 (2.9%)], and patent ductus arteriosus [n = 2/68 (2.9%)]. Two of these 68 infants (2.9%) had claims for 3 or more major malformations . Of all infants of mothers with claims for citp that were assumed to have no mca and whose charts were obtained (n = 66), 4 (6.1%) had at least one chart - confirmed major anomaly (table 4). The most frequent of these major congenital anomalies was hypospadias [n = 3/66 (4.5%)]. There was no statistically significant difference between the prevalences of malformations in the infants of mothers with itp compared to those with citp, regardless of whether the data was claims - based or chart - based (table 4). Although severe maternal or neonatal bleeding is rare when pregnant women with itp are managed by an expert team, a recent questionnaire of women with itp revealed that 14/50 women were advised to avoid becoming pregnant . Our study is unique because of the availability of data on congenital anomalies in infants born to mothers with itp and citp . In addition, the sample size of the population we evaluated is larger than previously published . The findings of this study are consistent with other published reports . In one prior publication, preterm birth was present in 16/58 (27.6%) infants born to mothers with itp diagnosed prior to pregnancy and in 15/75 (20%) infants born to mothers with itp diagnosed during pregnancy, with an overall prevalence of 23.3% . Premature birth affects 510% of newborns in most developed countries and approximately 12% of live births in the united states . Therefore, a prevalence of prematurity of 23.3% among pregnancies complicated by itp represents approximately a 2- to 4-fold increased frequency . In a study by debouverie et al . Of 50 women with citp who had platelet counts below 150 10/l for at least one year, there were no fetal deaths in 62 pregnancies but 9 (14%) were premature, 6 (9%) were small for gestational age, and 2 (3%) demonstrated evidence of hemorrhage . In a group of women treated for severe thrombocytopenia (platelet count <10 10/l) during pregnancy, there were 29 live births from 31 pregnancies, with a mean gestational age of 36.5 weeks (range, 743 weeks). There was one missed abortion at 7 weeks and one termination because of intrauterine death at 16 weeks of gestation . This child was born to a mother who had complications of a bleeding gastric ulcer due to severe thrombocytopenia (platelet count <20 10/l) and who died from acute pulmonary edema following a caesarean section . The remaining 28 newborns had no complications . Thrombocytopenia attributed to aplastic anemia or myelodysplasia was associated with a 53.8% rate of premature birth compared to incidental thrombocytopenia in pregnancy (11.3%) and itp (16.7%). Contrasted the characteristics of infants born to 57 mothers with itp diagnosed before pregnancy to 75 women diagnosed with itp during pregnancy . In the group of mothers with itp diagnosed prior to pregnancy, there were 2 intrauterine fetal deaths at 224 and 247 days of pregnancy and a higher frequency of infants requiring admission to the neonatal intensive care unit (20/57 or 34.48% versus 12/75 or 16%; p = 0.01). Maternal age and platelet count, gestational age at delivery, 5-minute apgar scores <7, rate of caesarean deliveries, mean neonatal birthweight, and the mean neonatal platelet count did not differ between the two groups . There were three neonates (3/84; 2.3%) with platelet counts <50 10/l that were all born to mothers with itp diagnosed prior to pregnancy, but there were no severe bleeding complications and no intracranial hemorrhage in the infants . However, maternal itp refractory to splenectomy has been correlated with a higher risk of intracranial hemorrhage in the infants . Conducted a retrospective study of women with itp in pregnancy, most of whom (83/92) had itp prior to pregnancy . There were 119 pregnancies and two fetal deaths: one stillbirth at 39 weeks of gestation and a stillbirth at 27 weeks of gestation that had extensive hemorrhage throughout the brain, born to a mother with a 4-year history of severe itp (postsplenectomy) and platelets counts <50 10/l . There were no reports of fetal malformations and the authors estimated that the fetal loss rate was approximately 1 - 2% . No case of hydrocephalus was found in the present study . Hydrocephalus can occur as a rare complication of intracranial hemorrhage in fetuses born to mothers with itp . Kim and choi described a neonate with severe thrombocytopenia (platelet count 1 10/l), multiple bruises on the face and scalp, widespread petechiae, cleft palate, and moderate to severe hydrocephalus without evidence of intraventricular hemorrhage . Computerized tomography confirmed severe hydrocephalus without significant compression of the brain parenchyma, diffuse ischemia, and encephalomalacia of both cerebral hemispheres . Ostium secundum, a type of atrial septal defect (asd), is found in less than 1% of newborns in the general population . In our study, this congenital heart defect occurred approximately three times more frequently among infants born to mothers with itp . In infants born to mothers with citp, the incidence of ostium secundum was 2/68 (2.9%) and there were two additional infants with ostium secundum plus other cardiac malformations (table 3). Ventricular septal defect and patent ductus arteriosus were also noted in other babies born to mothers with citp (table 3). However, the numbers in the group of citp pregnancies were small and the significance of this finding is unclear . This study provides a claims - based and chart - based evaluation of pregnancy outcomes among women with itp and citp in the united states . The wide range inclusion criteria and large source population (more than 1.2 million pregnancies) allowed us to obtain results that reflect pregnancy outcomes broadly among women with itp and citp . However, the database has some limitations and valuable information may be missing or misdiagnosed . However, there were a small number of pregnancy cases in women with itp and citp with no linkage to births . In addition, data from women with different medical or pharmacy coverage and from those without medical coverage could not be assessed by this study . Anomalies documented in live newborns and those requiring billable medical intervention were assessed in this study . However, minor or major anomalies that may have occurred in stillborn infants and in fetuses of elective or spontaneous abortions were not available for analysis . In addition, clinical validation of the diagnoses of itp and citp by a specialist was not feasible in this study . While referral to a maternal - fetal specialist is likely for these patients, the documentation from specialists was generally not included in the primary obstetrical records . Finally, the dataset did not include maternal medical conditions other than itp, nor medical treatments performed during the pregnancy for itp or other conditions . Therefore, it was not possible to evaluate their potential effects on the pregnancy and birth outcomes . Based on the evaluation of 446 pregnant women with itp, a diagnosis of itp or citp prior to their estimated date of conception may indicate a higher risk for stillbirth or fetal loss, premature delivery, and infants with specific congenital anomalies than an itp diagnosis during pregnancy . Therefore, the results of this study provide further evidence that the duration of maternal itp may be an important determinant of the outcomes of pregnancy.
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We selected excised salivary gland tumors, excluding biopsy or consultation cases, from the surgical pathology files of the asan medical center from 1990 to 2012 . For retrieval of masc candidates, 196 salivary gland tumors (mec, n=137; acicc, n=43; anos, n=11; cac, n=2; other unusual salivary gland neoplasm, n=3) the selection of study candidates was built on the histological features described by recent literature1,3 - 5 and sufficient amount of tissue available to construct a tissue microarray (tma). The criteria for selection among the above tumors included the presence of glandular formation or secretory activity . The final specimens comprised 30 cases initially diagnosed as acicc (n=16), anos (n=6), mec (n=3), cac (n=2), salivary duct carcinoma (sdc; n=1), squamous cell carcinoma (n=1), and oncocytic carcinoma (n=1). Additionally, 6 conventional acicc cases, morphologically showing unequivocal serous acinar differentiation, and 2 sdc cases were selected for reference . For all 38 cases, tmas were generated using a manual tissue arrayer (pathology devices, westminster, md, usa). Two 1.0- or 1.5-mm cores were taken from donor blocks and arrayed into recipient blocks . Clinical data were obtained through review of medical records and pathologic staging was based on the cancer staging manual of american joint committee.8 disease - free survival was assessed from the time of histological diagnosis to recurrence or death, based on review of the patients' medical records and information from the national health insurance . A t - test was employed to characterize the relationships between quantitative variables and the fisher's exact test was used to characterize the relationship between categorical variables . Disease - free survival periods with 95% confidence intervals (ci) were estimated using the kaplan - meier method, with statistical significance of differences between groups estimated by log - rank test . A p - value of <0.05 was defined as statistically significant . Immunohistochemical stainings were performed using the ventana autostainer and ultra view dab detection kit (ventana, tucson, az, usa) according to manufacturer's instructions . Fish was performed using a break - apart probe for the etv6 gene (abbott molecular, des plaines, il, usa) according to the manufacturer's recommendations . Fish slides were examined with an olympus bx51 fluorescence microscope (olympus, tokyo, japan) using a 100 objective . Through fitc and texas red band pass filters, a single green (or red) signal without a corresponding red (or green) signal in addition to a fused signal was considered negative (non - rearranged) in the present study . Red and green signals that were less than 2 signal diameters apart were considered as a single fused signal . The average percentage of split signal in 6 referential aciccs, showing unequivocal serous acinar differentiation (conventional acicc) was 4.565 and standard deviation (sd) 3.042 . Thus, we considered cases, if more than 15% (mean+3 sd, rounded up) of examined nuclei showed a split signal, as positive for translocation which is the similar cut - off value used in previous literature.1 the slides were independently interpreted by 2 observers . If 10 - 20% of the analyzed cells showed a split signal, more cells were enumerated and the first and second cell count readings by the 2 observers were added together and a percentage was calculated . Valid fish results were obtained in 31 out of 38 cases . The laboratory used breast secretory carcinoma as a positive control . From tma blocks, 4-m sectioned slides were obtained and prepared for immunohistochemistry and fish . Immunohistochemical stainings were performed using the ventana autostainer and ultra view dab detection kit (ventana, tucson, az, usa) according to manufacturer's instructions . Fish was performed using a break - apart probe for the etv6 gene (abbott molecular, des plaines, il, usa) according to the manufacturer's recommendations . Fish slides were examined with an olympus bx51 fluorescence microscope (olympus, tokyo, japan) using a 100 objective . Through fitc and texas red band pass filters, a single green (or red) signal without a corresponding red (or green) signal in addition to a fused signal was considered negative (non - rearranged) in the present study . Red and green signals that were less than 2 signal diameters apart were considered as a single fused signal . The average percentage of split signal in 6 referential aciccs, showing unequivocal serous acinar differentiation (conventional acicc) was 4.565 and standard deviation (sd) 3.042 . Thus, we considered cases, if more than 15% (mean+3 sd, rounded up) of examined nuclei showed a split signal, as positive for translocation which is the similar cut - off value used in previous literature.1 the slides were independently interpreted by 2 observers . If 10 - 20% of the analyzed cells showed a split signal, more cells were enumerated and the first and second cell count readings by the 2 observers were added together and a percentage was calculated . Valid fish results were obtained in 31 out of 38 cases . The laboratory used breast secretory carcinoma as a positive control . Among the 31 technically successful cases, 13 cases showed etv6 translocation, reclassified as masc, and 10 cases showed intact etv6, categorized as mimic masc (fig . The other 8 cases served as a reference, including 6 conventional aciccs and 2 sdcs, and showed intact etv6 . On average, 57.65% of examined cells showed a split signal with etv6 translocation (masc) in 13 cases, while 9.55% of examined cells showed a split signal in 10 cases with intact etv6 (mimic masc). The percentage of cells with a split signal ranged between 20.75% and 78.57% in cases with etv6 translocation, and between 3.10% and 12.96% in cases with intact etv6 . The clinicopathologic features of 23 masc candidates with etv6 translocation (masc, n=13) or with intact etv6 (mimic masc, n=10), are summarized in table 2 . For referential comparison, conventional aciccs (n=15) although no differences in gender distribution were found between masc and mimic masc cases, a noticeable male predilection was observed compared with conventional aciccs, which affected females more frequently than males (f: m=12:3). The average age in the mascs, mimic mascs, and conventional aciccs was in the fifth decade . Both mascs and the parotid gland was the most frequently involved site in mascs, mimic mascs, and conventional aciccs and the tumor size in mascs ranged from 0.7 cm to 2.5 cm (mean, 1.77 cm). Only 4 cases of masc showed extraparenchymal extension, and any lymph node involvement at the time of surgery was not observed . All patients were initially treated by surgery, and 3 masc, 3 mimic masc, and 2 conventional acicc patients received postoperative radiotherapy and all are currently alive . Three masc patients developed local recurrence (3/13, 23.1%) with median recurrence time of 44 months after diagnosis (range, 10 to 101 months) and 2 mimic masc patients developed metastases to neck lymph nodes (2/10, 20.0%) at 62 months and 70 months after diagnosis, while an acicc patient developed local recurrence (1/15, 6.7%) at 39 months after diagnosis . In summary, masc, mimic masc, and conventional acicc showed no statistically significant differences in gender, age, site, pathologic t stage, and disease - free survival . Histologically, the most frequent features in the 13 masc cases were well circumscribed, encapsulated, or multinodular masses, showing papillary - cystic, microcystic, solid, follicular or their combination growth pattern . Some cells were smaller than typical acinar cells appearing to have an increased nuclear to cytoplasmic ratio, and showed eosinophilic or finely granular cytoplasm and ovoid basophilic nuclei, known as intercalated duct - like cells . Other cells showed a more abundant or vacuolated cytoplasm and low - grade wrinkled nuclei . Numerous variable - sized spaces were frequently observed and the spaces were usually clear but sometimes contained pinkish- to grayish blue - colored amorphous materials, which were positive for periodic acid - schiff . As stated in previous literature, the above - mentioned characteristics were predominant features of masc and corresponded to microcystic, papillary - cystic, or follicular type of acicc.1 - 5 these usual morphological masc features are illustrated in fig . One case was macrocystic, measuring up to 2.5 cm, and partially filled with intraluminal papillary proliferation . Nevertheless, epithelial configuration of the cyst lining and papillary projection was similar to the mentioned masc . The tumor showed focal proliferation of the epithelial lining and even cribriform appearance in some relatively small islands . These features were also reminiscent of cac, however, epithelial lining cells were similar to masc rather than the cuboidal, clear, tall columnar, or oncocytic features of cac . The third case showed a distinct morphologic change previously referred to as dedifferentiation or high - grade transformation of acicc.9 the tumor was composed of 2 distinct areas with an obvious transformation zone . Histological features of one area corresponded to microcystic and papillary - cystic masc and the other area showed poor zymogen granule containing - basophilic polymorphous cells, forming solid nests with comedonecrosis (fig . 4). A diversity of morphological features with intact etv6 was also obtained . Among 10 mimic mascs, 3 cases could be interpreted as cac . Though the cytomorphologic features were not uniform, the tumor cells were slightly different from masc, showing cuboidal, tall columnar, clear to oncocytic features . Additionally, the epithelial lining of cysts was more evenly thin in cac than masc . Additionally, when papillary projections were present, they were mostly large papillae covered with columnar or tall cuboidal cells . Others showed nonspecific morphologies, corresponding to anos . Infiltrating nests of clear cells, glandular growth in the prominent sclerosing background, or solid nests surrounded by very thin fibrovascular septa with reticular and abundant cytoplasm were other examples of mimic masc . Two cases of mecs lacking mucous and epidermoid cells were initially similar to masc, but careful examination can help in distinguishing mec from masc . Most staining patterns were strong and diffuse, except for a case showing dedifferentiation (fig . Three out of 10 mimic mascs expressed s100 protein and no expression was observed in 6 conventional aciccs . Gross cystic disease fluid protein 15 (gcdfp-15) immunostains were focally positive for 2 out of 13 mascs, 3 out of 10 mimic mascs, and none of the conventional aciccs . Dog1 was expressed in 3 conventional aciccs and 1 masc, with weak apical or apicoluminal membranous staining . Estrogen receptor (er) and progesterone receptor (pr) were all negative for masc, mimic masc and conventional aciccs (table 3). Among the 31 technically successful cases, 13 cases showed etv6 translocation, reclassified as masc, and 10 cases showed intact etv6, categorized as mimic masc (fig . The other 8 cases served as a reference, including 6 conventional aciccs and 2 sdcs, and showed intact etv6 . On average, 57.65% of examined cells showed a split signal with etv6 translocation (masc) in 13 cases, while 9.55% of examined cells showed a split signal in 10 cases with intact etv6 (mimic masc). The percentage of cells with a split signal ranged between 20.75% and 78.57% in cases with etv6 translocation, and between 3.10% and 12.96% in cases with intact etv6 . The clinicopathologic features of 23 masc candidates with etv6 translocation (masc, n=13) or with intact etv6 (mimic masc, n=10), are summarized in table 2 . For referential comparison, conventional aciccs (n=15) although no differences in gender distribution were found between masc and mimic masc cases, a noticeable male predilection was observed compared with conventional aciccs, which affected females more frequently than males (f: m=12:3). The average age in the mascs, mimic mascs, and conventional aciccs was in the fifth decade . Both mascs and the parotid gland was the most frequently involved site in mascs, mimic mascs, and conventional aciccs and the tumor size in mascs ranged from 0.7 cm to 2.5 cm (mean, 1.77 cm). Only 4 cases of masc showed extraparenchymal extension, and any lymph node involvement at the time of surgery was not observed . All patients were initially treated by surgery, and 3 masc, 3 mimic masc, and 2 conventional acicc patients received postoperative radiotherapy and all are currently alive . Three masc patients developed local recurrence (3/13, 23.1%) with median recurrence time of 44 months after diagnosis (range, 10 to 101 months) and 2 mimic masc patients developed metastases to neck lymph nodes (2/10, 20.0%) at 62 months and 70 months after diagnosis, while an acicc patient developed local recurrence (1/15, 6.7%) at 39 months after diagnosis . In summary, masc, mimic masc, and conventional acicc showed no statistically significant differences in gender, age, site, pathologic t stage, and disease - free survival . Histologically, the most frequent features in the 13 masc cases were well circumscribed, encapsulated, or multinodular masses, showing papillary - cystic, microcystic, solid, follicular or their combination growth pattern . Some cells were smaller than typical acinar cells appearing to have an increased nuclear to cytoplasmic ratio, and showed eosinophilic or finely granular cytoplasm and ovoid basophilic nuclei, known as intercalated duct - like cells . Other cells showed a more abundant or vacuolated cytoplasm and low - grade wrinkled nuclei . Numerous variable - sized spaces were frequently observed and the spaces were usually clear but sometimes contained pinkish- to grayish blue - colored amorphous materials, which were positive for periodic acid - schiff . As stated in previous literature, the above - mentioned characteristics were predominant features of masc and corresponded to microcystic, papillary - cystic, or follicular type of acicc.1 - 5 these usual morphological masc features are illustrated in fig . One case was macrocystic, measuring up to 2.5 cm, and partially filled with intraluminal papillary proliferation . Nevertheless, epithelial configuration of the cyst lining and papillary projection was similar to the mentioned masc . The tumor showed focal proliferation of the epithelial lining and even cribriform appearance in some relatively small islands . These features were also reminiscent of cac, however, epithelial lining cells were similar to masc rather than the cuboidal, clear, tall columnar, or oncocytic features of cac . The third case showed a distinct morphologic change previously referred to as dedifferentiation or high - grade transformation of acicc.9 the tumor was composed of 2 distinct areas with an obvious transformation zone . Histological features of one area corresponded to microcystic and papillary - cystic masc and the other area showed poor zymogen granule containing - basophilic polymorphous cells, forming solid nests with comedonecrosis (fig . 4). A diversity of morphological features with intact etv6 was also obtained . Among 10 mimic mascs, 3 cases could be interpreted as cac . Though the cytomorphologic features were not uniform, the tumor cells were slightly different from masc, showing cuboidal, tall columnar, clear to oncocytic features . Additionally, the epithelial lining of cysts was more evenly thin in cac than masc . Additionally, when papillary projections were present, they were mostly large papillae covered with columnar or tall cuboidal cells . Infiltrating nests of clear cells, glandular growth in the prominent sclerosing background, or solid nests surrounded by very thin fibrovascular septa with reticular and abundant cytoplasm were other examples of mimic masc . . Two cases of mecs lacking mucous and epidermoid cells were initially similar to masc, but careful examination can help in distinguishing mec from masc . Most staining patterns were strong and diffuse, except for a case showing dedifferentiation (fig . Three out of 10 mimic mascs expressed s100 protein and no expression was observed in 6 conventional aciccs . Gross cystic disease fluid protein 15 (gcdfp-15) immunostains were focally positive for 2 out of 13 mascs, 3 out of 10 mimic mascs, and none of the conventional aciccs . Dog1 was expressed in 3 conventional aciccs and 1 masc, with weak apical or apicoluminal membranous staining . Estrogen receptor (er) and progesterone receptor (pr) were all negative for masc, mimic masc and conventional aciccs (table 3). In this study we investigated the new salivary gland tumor, known as masc, to clarify the clinical, histological, and immunohistochemical characteristics, and to determine any pathognomonic features helpful in diagnosing mascs . The first masc report focused on features reminiscent of breast secretory carcinoma.1 in recent years, there have been several studies extending the clinical, histological, and immunohistochemical properties of mascs.3,5 however, masc clinicopathologic features appear to be indistinctive and the molecular demonstration of etv6 rearrangement is the only diagnostic method . Regardless of the originality of this entity, a salivary gland tumor with etv6 translocation has yet to be documented in korea . The present study compared masc candidate groups with an intact etv6 and etv6 translocation . To evaluate mascs with an emphasis on easy identification of specific characteristics, consideration of the ideal cut - off value to assess the etv6 fish results was necessary . In this study, the negative control cut - off value (mean+3 sd, 13.688) showed a 99.7% ci for intact etv6 . The cut - off value used (15%) was slightly higher than the negative control cut - off value . Moreover, the interpretation of a single colored signal without a corresponding colored signal in addition to a fused signal as negative could decrease the percentage of positive cells for etv6 translocation . The highest negative value for etv6 translocation was 12.96% and the lowest positive value was 20.75% . This result shows the importance of determining an accurate cut - off value in a study . Overall, papillary - cystic, microcystic, or their mixture types were predominantly observed . Aciccs, according to the current world health organization classification, are comprised of heterogeneous tumors . Many cell types, including acinar, intercalated duct type, vacuolated, clear, and nonspecific glandular cells, and variable growth patterns such as solid, microcystic, papillary - cystic, and follicular, are described as constituents of acicc . Anos is also a heterogeneous tumor group, and their diagnosis is based on the lack of features that characterize other salivary gland adenocarcinomas . According to prior studies and our results, a considerable number of anos and zymogen granule - poor aciccs belong to the masc group . The concept that masc is a distinctive entity of salivary gland tumors requires more consideration, however, no zymogen granule - rich acicc or sdc has shown an etv6 rearrangement . Our study revealed previously undescribed histology in etv6-rearranged tumors, such as a solid pattern resembling dedifferentiated acicc and a large cyst with sparse papillary proliferation . Chiosea et al.4 reported that the results of etv6 fish in 4 cases of acicc with high - grade malformation were all negative for etv6 translocation . Apart for a few exceptions, the histological outline of masc appears evident, and mimic masc cases without an etv6 translocation could be differentiated from masc by careful histological examination . Dog1, known as a novel marker of salivary acinar and intercalated duct differentiation, was not helpful.10 gcdfp-15, er, and pr were considered a possibility due to their mammary - like character, but were not expressed in masc . The s100 protein was highly sensitive, but showed low specificity to differentiate masc from mimic masc ., masc could be a molecularly well - defined salivary gland neoplasm, encompassing some portions of acicc and anos, but its histological spectrum and clinical implication need further investigation.
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A 2-year - old girl presented with partial seizures that manifested as twitching of the left angle of the mouth and jerking of the left upper limb but usually did not result in a loss of consciousness . The child s motor and cognitive development were within the normal ranges . At the age of 3 years, she was referred to our center with the aim of achieving control of her seizures . Brain magnetic resonance imaging (mri) showed focal cortical dysplasia in the right centroparietal region (figure 1a), while interictal tc - ethyl cysteinate dimer single - photon emission computed tomography (spect) did not detect any abnormalities (data not shown). A, at the age of 3 years, a t2-weighted mri scan showed focal cortical dysplasia in the right centroparietal region (). B, on day 2 after admission, a t2-weighted mri image showed a high signal intensity lesion () adjacent to the focal cortical dysplasia lesion . C, apparent diffusion coefficient mapping also showed a high signal intensity lesion in the same region . D, the high signal intensity lesion () was located close to the right insular cortex (left). On day 69, the high signal intensity lesion had diminished in size, and the patient s cluster seizures had improved (right). Dimer spect detected hyperperfusion in a region containing the high signal intensity lesion seen on mri, which was located close to the right insular cortex . Interictal tc - ethyl cysteinate dimer spect performed on day 68 did not detect any abnormalities . Mri indicates magnetic resonance imaging; spect, single - photon emission computed tomography . At the age of 10 years and 5 months, the patient was admitted because of a sudden worsening of her seizures . On admission, she was taking phenytoin (250 mg / d) in combination with valproate (400 mg / d). An ictal eeg showed rhythmic fast waves arising from the right central region, which sometimes evolved into diffuse slow waves corresponding to asymmetric bilateral tonic seizures (left> right). The patient s seizures did not respond to the intravenous administration of midazolam, lidocaine, and phenobarbital . On the second day after admission, t2-weighted mri and apparent diffusion coefficient mapping revealed a high signal intensity lesion adjacent to the focal cortical dysplasia lesion (figure 1b - d, left). Diffusion - weighted imaging also revealed a high signal intensity lesion in the same region (data not shown). An eeg performed at the onset of the seizure showed rhythmic fast waves arising from the right central region . Ten seconds after the onset of the seizure, when the epileptic activity evolved into diffuse slow waves, the patient s baseline sinus heart rate of about 100 beats / min suddenly dropped to 40 to 50 beats / min, and she started to exhibit an idioventricular rhythm . Immediately after the seizure activity disappeared, the patient s cardiac rhythm returned to a normal sinus rhythm (figure 2). Oxygen desaturation (an oxygen saturation value of <92%) due to apnea (defined as breath arrest lasting> 10 seconds) also occurred after the appearance of ictal bradycardia . Cardiac assessments, including continuous 24-hour ambulatory electrocardiography (ecg) monitoring and echocardiography, did not detect any abnormalities between the seizures . A, an eeg performed at seizure onset showed rhythmic fast waves arising from the right central region . B, about 10 seconds after seizure onset, when the patient s epileptic activity evolved into diffuse slow waves, her baseline sinus heart rate of about 100 beats / min suddenly dropped to 40 to 50 beats / min and then exhibited an idioventricular rhythm . C, after the seizure activity disappeared (68 seconds after onset), the patient s cardiac rhythm returned to a normal sinus rhythm . Between 3 and 5 days after the patient s admission, the frequency of her seizures increased to 5 to 10 an hour . The patient s ictal bradycardia and apnea were usually accompanied by seizures, despite the prompt discontinuation of phenytoin and lidocaine . The idioventricular rhythm was observed in about half of the seizures . On the sixth day after admission, when a brief period of ictal asystole occurred, barbiturate (sodium thiopental) therapy was started while the patient was under mechanical ventilation . The frequency of the patient s seizures decreased to once or twice an hour after the initiation of the barbiturate therapy (5 mg / kg / h), and her ictal bradycardia subsided . After the continuous intravenous infusion of sodium thiopental for 48 hours, the dose was gradually reduced . However, the patient s bradycardia was less severe than it had been previously, her heart rate only fell to around 60 to 70 beats / min, and she did not develop cardiac arrhythmia . On the 25th day after admission, ictal spect performed on day 27 showed hyperperfusion in the area containing both the focal cortical dysplasia lesion and the high signal intensity lesion seen on mri (figure 1e). The frequency of the patient s seizures was reduced via the continuous infusion of midazolam and high - dose phenobarbital, and hence, her ictal bradycardia subsided again . Interictal spect performed on day 68 did not reveal any abnormalities (figure 1e). On day 69, the high - intensity lesion adjacent to the focal cortical dysplasia lesion was found to have diminished in size on follow - up mri (figure 1d, right). Interictal f - fluorodeoxyglucose positron emission tomography / computed tomography was performed 2 months after discharge and detected hypometabolism in the right centroparietal region but not in the right insular cortex (data not shown). She underwent surgery at the age of 13 years and 1 month, and the lesion was partially resected . After surgery, the patient had infrequent partial seizures that were similar to those observed before surgery but no longer developed cluster seizures . Ictal bradycardia is considered to be rare in children because there have only been a few reported pediatric cases . However, a recent study found that ictal bradycardia occurred in 8.2% of the pediatric patients with epilepsy admitted to a pediatric epilepsy monitoring unit . Another comparative study reported that ictal bradycardia occurred more often in children (23.8% of pediatric seizures) than in adults (7.3% of adult seizures). In adults, ictal bradycardia was found to be more strongly associated with seizures originating from the temporal lobe, while in children it was more prevalent in patients having seizures of extratemporal origin . The risk factors for ictal bradycardia include seizure clustering, having suffered with epilepsy for a long period, taking multiple antiepileptic drugs, a younger age at seizure onset, and refractory epilepsy . The propagation of seizure activity into the autonomic centers that regulate the cardiovascular system is assumed to be the leading mechanism of ictal bradycardia . In 1990, oppenheimer and cechetto demonstrated that stimulation of the left insular cortex evoked bradycardia in rats . The same authors later showed that perioperative stimulation of the insular cortex tended to result in heart rate changes (left insular cortex: bradycardia and right insular cortex: tachycardia) in patients with epilepsy . Although conflicting results have been obtained regarding the left- or right - sided predominance of ictal bradycardia, several other studies have suggested that the insular cortex plays an important role in cardiac regulation in humans . However, to the best of our knowledge, there has only been 1 case report of ictal bradycardia due to an insular cortex lesion, which involved a 3-year - old girl with focal cortical dysplasia in her right insular cortex . Some antiepileptic drugs, such as carbamazepine, lamotrigine, phenytoin, and lidocaine, are known to act as sodium channel blockers and can have side effects that affect the cardiac conduction system . Our patient was receiving phenytoin and lidocaine for her cluster seizures when the first episode of ictal bradycardia occurred . However, phenytoin and lidocaine are unlikely to have caused the bradycardia experienced in the present case due to (1) the prolonged (> 2 weeks) persistence of the bradycardia, despite the prompt discontinuation of the above - mentioned drugs and (2) the absence of cardiac arrhythmia between seizures according to continuous 24-hour ambulatory ecg monitoring . Focal cortical dysplasia constitutes the most important cause of intractable localization - related epilepsy in childhood . A recent study reported that ictal spect is useful for defining the epileptic zone in a high proportion of children with focal cortical dysplasia who undergo surgical evaluation . In our patient, the high signal intensity lesion seen on t2-weighted mri image appeared when the patient had cluster seizures and diminished in size as her seizures improved, suggesting that these mri changes were related to seizure clustering . Ictal spect detected hyperperfusion in the area containing the high signal intensity lesion depicted on mri, which was located very close to the right insular cortex . The ictal bradycardia experienced by our patient might have been caused by the following mechanism: repetitive seizure activity might have expanded from the hyperperfusion zone into the right insular cortex, which controls cardiac rhythm, resulting in ictal bradycardia . The autonomic alterations and heart rate changes seen during epilepsy are complex and are not fully understood . Although the presence of hyperperfusion on ictal spect might represent zones of seizure propagation, there have not been any reports about the ictal spect findings of patients with ictal bradycardia . In our patient, ictal spect demonstrated hyperperfusion in an area that was close to the insular cortex but did not involve the insular cortex itself . Subtraction ictal spect coregistered to mri analysis also failed to detect ictal hyperperfusion in the right insular cortex (data not shown). One possible reason for this is that the patient s bradycardia was less severe at the time of the ictal spect study than it had been previously . Although the seizures experienced by the patient after the injection of the radiotracer used for the spect produced similar symptoms and paroxysmal eeg discharges to the previous seizures, the bradycardia induced was mild (the patient s heart rate only fell to 60 - 70 beats / min) and did not lead to cardiac arrhythmia . Thus, seizures involving more severe ictal bradycardia might have been found to involve the insular cortex on spect . In this study, ictal and interictal spect were performed during the patient s cluster seizures (day 27), when ictal bradycardia was observed, and on day 68, when the ictal bradycardia had gone into remission . However, no ictal spect scans were performed after the patient s ictal bradycardia went into remission because she did not experience seizures often during the follow - up period . Comparing the 2 ictal spect scans might have helped us to clarify the origin of the patient s ictal bradycardia and why her ictal bradycardia was transient . Further studies with ictal spect are necessary to elucidate the pathophysiology of ictal bradycardia in patients with epilepsy, providing that clinical conditions allow it.
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Selecting an optimal stride length in distance running affects metabolic cost demonstrated by increases found in oxygen uptake when non - ideal stride lengths are used (5). Well - trained runners naturally select stride rates and lengths that minimize oxygen uptake at distance running speeds (1, 5, 12, 13, 16). A recent study found a lack of optimization of stride frequency among novice and trained runners (6). This was unexpected due to the multiple previous studies showing the ability to self - optimize stride frequency for economy . Running can be represented as a spring - mass system . While variations exist in the stiffness of this spring between people and across different speeds, for any given situation, a spring constant can be determined (2, 8, 14). The natural oscillation of a spring may be what determines preferred stride rate for a given person, running surface, and speed . The stiffness of the leg spring is primarily determined by the reactions of muscles, tendons, and bones to the ground reaction force (7). Assuming the leg spring adjusts to its natural frequency, inexperienced runners should also have the ability to self - economize stride length . Along with previous studies showing self - economizing of stride length in a non - fatigued state (1, 5, 12, 13, 16), hunter and smith showed stride length is self - optimized at the end of a one - hour high intensity run (12). Many of their subjects had changes in preferred stride length (psl) from the early to late stages of the run . These changes in psl matched changes in the most economical stride length (esl). So, whether rested or fatigued, these runners were capable of self - optimizing stride length for economy even when a change is required during the course of the run . Since experienced runners self - regulate stride length to optimize economy, changing stride lengths will likely result in a greater metabolic cost . If self - economization of stride length is based upon musculoskeletal factors, inexperienced runners should also be able to select their own preferred stride length without modification . On the other hand, self - economization may be something that is learned through running experience . This study aims to determine whether stride self - economization is different between experienced and inexperienced runners (figure 1). We expect that if preferred stride length is determined by musculoskeletal parameters, inexperienced runners will also self - economize stride length . Preferred and economical stride length was measured in 33 subjects (19 experienced and 14 inexperienced). An experienced runner averaged at least 20 miles per week for the past two years . Ten of the experienced runners were intercollegiate distance runners with the same coach, the rest were taken from elsewhere in the university population . Inexperienced runners were defined as never having run more than five miles in one week throughout their lives . An informed consent document was signed by each subject before he / she was allowed to begin ., preferred stride length was calculated by timing 30 strides multiplied by treadmill speed, then dividing by 30 . This stride length along with plus and minus 8 and 16% of this length were used during the preliminary run to practice running at these non - preferred lengths . These percentages of modified stride lengths were used as a results of pilot testing and matching a previous study that used similar methodologies (12). The pace averaged 3.66 m / s for the experienced group and 3.04 m / s for the inexperienced group . The data collection run was completed at the pace determined during the preliminary run . A metabolic cart (parvo medics, salt lake city, ut) reported average oxygen uptake for every 15 seconds during the entire run . Preferred stride length was measured during the fourth minute of the data collection run with the help of a customized computer program (microsoft visual basic, microsoft corp, seattle, wa). The investigator would click the mouse button every time the right foot came in contact with the treadmill for twenty strides . The computer would then divide 20 strides by the amount of time needed to complete 20 strides to get the stride rate . Dividing treadmill speed by stride rate provided stride length . Following the data collection run, after the fourth minute of the data collection run, subjects ran the five different stride lengths for two minutes each in random order . Following the first five minutes of running, economical stride length was measured by having subjects run with five different stride lengths (preferred and plus and minus 8 and 16% of preferred). The minimum value of a best - fit second - degree polynomial with oxygen uptake (taken from the final minute of each stride length) as a function of stride length was used to determine esl (figure 1). An independent t - test was used to compare differences between experienced and inexperienced runners in how much more oxygen was required to maintain preferred stride length over economical stride length . The resulting means, standard deviations, and samples sizes were used to determine statistical power . Preferred and economical stride length was measured in 33 subjects (19 experienced and 14 inexperienced). An experienced runner averaged at least 20 miles per week for the past two years . Ten of the experienced runners were intercollegiate distance runners with the same coach, the rest were taken from elsewhere in the university population . Inexperienced runners were defined as never having run more than five miles in one week throughout their lives . An informed consent document was signed by each subject before he / she was allowed to begin . They self - selected this preferred pace . During this preliminary run, preferred stride length was calculated by timing 30 strides multiplied by treadmill speed, then dividing by 30 . This stride length along with plus and minus 8 and 16% of this length were used during the preliminary run to practice running at these non - preferred lengths . These percentages of modified stride lengths were used as a results of pilot testing and matching a previous study that used similar methodologies (12). The pace averaged 3.66 m / s for the experienced group and 3.04 m / s for the inexperienced group . The data collection run was completed at the pace determined during the preliminary run . A metabolic cart (parvo medics, salt lake city, ut) reported average oxygen uptake for every 15 seconds during the entire run . Preferred stride length was measured during the fourth minute of the data collection run with the help of a customized computer program (microsoft visual basic, microsoft corp, seattle, wa). The investigator would click the mouse button every time the right foot came in contact with the treadmill for twenty strides . The computer would then divide 20 strides by the amount of time needed to complete 20 strides to get the stride rate . Dividing treadmill speed by stride rate provided stride length . Following the data collection run, after the fourth minute of the data collection run, subjects ran the five different stride lengths for two minutes each in random order . Following the first five minutes of running, economical stride length was measured by having subjects run with five different stride lengths (preferred and plus and minus 8 and 16% of preferred). The minimum value of a best - fit second - degree polynomial with oxygen uptake (taken from the final minute of each stride length) as a function of stride length was used to determine esl (figure 1). An independent t - test was used to compare differences between experienced and inexperienced runners in how much more oxygen was required to maintain preferred stride length over economical stride length . The resulting means, standard deviations, and samples sizes were used to determine statistical power . No difference was found between experienced and inexperienced groups for the extra oxygen required for non - optimization of stride length (table 1, t=1.49, p=0.23). The average percent increase in oxygen uptake required for non - optimization are reported in table 1 . Statistical power of 0.80 existed for the ability to detect a difference of 2.73% . The average oxygen uptakes while running at preferred stride length for inexperienced and experienced runners were 33.6 ml / kg / min and 39.4 ml / kg / min respectively (figure 2). Although all subjects were considered optimized for economy in terms of stride length, the steepness and shape of each curve varied from subject to subject . The most commonly observed curves showed equally increased oxygen uptake for shorter or longer strides . Other curves had small increases in oxygen uptake with shorter strides, but large increases with longer strides (figure 3). Inexperienced runners are equally capable of optimizing stride length for minimal oxygen uptake as experienced runners . The small non - significant average difference and the sufficient power to detect a difference of 2.73% provided confidence that if any difference exists between experienced and inexperienced runners, it is quite small . Different running paces might alter how impactful a slight deviation between psl and esl will be . Our study included metronome use during the psl testing whereas de ruiter s did not . We chose to use the metronome during psl in case there is an effect on metabolic cost due to focusing on the cadence . Taking both studies into consideration, there is still a need to determine the specific conditions where inexperienced runners differ from experienced . One thing that is not conflicted between the studies is that psl may change with experience of running . Many factors may lead to this including changes in: stride length, vertical oscillation, leg stiffness, body segment parameters, preferred joint angles, and preferred body positions (15). While no relationship between leg stiffness and running economy was investigated, this study supports the idea of the body acting as a mass - spring system (7, 8, 11). Stride frequency is closely connected to leg stiffness (8). Springs have a resonant frequency at which they will oscillate . This resonant frequency is determined by the stiffness and the applied mass with the equation: where fn is the resonant frequency, k is the stiffness of the spring, and m is the mass . The stiffness depends upon the properties and reactions of the tissues that act as a spring, while the average applied force depends upon the flight time and ground contact time . Heise and martin (11) showed that the less economical runners possess a more compliant running style during ground contact . Although the stiffness may be different for a trained versus untrained group, assuming the untrained is less economical, the current stride a runner uses appears to be the most economical for their conditioning . Over time it is reasonable to assume the stiffness and the mass may change through training, but at any given time the stiffness should lead to a preferred stride rate and length that is also the most economical . Some were capable of using stride lengths substantially different than preferred without large increases in oxygen uptake . In a few cases, shorter strides than preferred were less detrimental than longer strides within a given subject (figure 3). For most subjects, however, the further away from preferred a subject would go, oxygen uptake increased dramatically . Shorter strides result in smaller peak forces and range of motion at the hip and knee (10). If decreased forces and range of motion are desired for someone, they may be able to accomplish that with minimal increase in effort . However, there is still a greater perceived exertion when choosing shorter strides (10). When using a shorter stride, more repetitions will be required if a run of equal length is completed . However, the extra repetitions may not be detrimental since patellofemoral load is considerably less when using a shorter stride (17). However, whenever running technique is modified, stresses on the body are altered and care should be taken if one has a desire to make this kind of modification . As an inexperienced runner becomes experienced, changes may occur in endurance capacities, running economy, body weight, body composition, muscle strength, and muscle power (3, 4, 9). However, at any point of the spectrum from inexperienced to experienced, the chosen stride length is probably ideal for economy . Inexperienced and experienced runners may not need to manipulate stride length to economize their running technique . When maximizing running speed over short distances is the main concern, runners may choose to sacrifice some economy to increase top speed . More consideration and experimentation should be completed to determine how to optimize technique for maximizing running speed.
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Attention has widespread effects on brain activity . From thalamus and colliculus to many regions of cortex, for example, responses to visual input are enhanced when this input is relevant to behavior (oconnor et al ., 2002; ignashchenkova et al ., 2004; roelfsema et al ., 1998; moran and desimone, 1985). Often, attentional modulations grow over time from stimulus onset as the appropriate attentional focus is established (roelfsema et al ., 1998; schall et al ., 1995). In some cases, inputs appear to compete for control of neuronal activity, for example, when two stimuli fall within the receptive field of a visual cell (chelazzi et al . In such cases, directing attention to one or the other stimulus determines how closely neural activity resembles the response to that stimulus presented alone (moran and desimone, 1985; reynolds et al ., 1999; see also reynolds and heeger, 2009). Such competition for control of neural activity resembles classic attentional models, in which concurrent stimuli or cognitive events compete for processing resources (e.g., broadbent, 1958; kahneman, 1973). This form of competition is best established in early visual areas, where it is predominantly local . When two stimuli fall within a cell s spatial receptive field, moving attention from one to the other determines which of the two drives activity . Competition and attentional modulation are much weaker when stimuli are widely separated (moran and desimone, 1985; lee and maunsell, 2010). In behavior, however, there are global limits on attentional capacity, such that even very dissimilar tasks can be hard to carry out together (bourke et al ., 1996; neurophysiologically, attentional modulations are strong in prefrontal cortex (rainer et al ., 1998; lennert and martinez - trujillo, 2011), even with stimuli in opposite visual hemifields (everling et al ., 2002), and it is commonly proposed that prefrontal cortex plays a central role in attentional competition and control (norman and shallice, 1980; dehaene et al ., 1998; botvinick et al ., 2001; miller and cohen, 2001 according to adaptive coding proposals (duncan, 2001; duncan and miller, 2002), prefrontal neurons have highly flexible response properties, allocated to coding different information in different task contexts . Functional brain imaging shows that similar regions of prefrontal cortex are active during many different kinds of cognitive activity (duncan and owen, 2000; miller and cohen, 2001), providing a plausible basis for global limits on attentional capacity (e.g., dehaene et al ., 1998; marois and ivanoff, 2005; bourke et al ., 1996). On such a view, processing activity in prefrontal cortex would be flexible but limited, allocated to a currently attended stimulus or task, and providing a critical prefrontal mechanism for attentional competition and its resolution . Here we examined the dynamics of attentional allocation in prefrontal cortex with widely separated visual stimuli . In the behaving monkey, we used time - resolved measures of neural population activity (e.g., buschman et al ., 2012; kaping et al ., 2011; stokes et al ., 2013) to track development of the attentional focus under varying levels of attentional competition . Attentional competition was manipulated using a simple form of visual search, in which the animal detected and later responded to a cued target object (t). In some trials, t was presented alone, while in others, competition was introduced by an additional nontarget (n) in the opposite visual hemifield . It is well known from human search experiments that processing conflict in such a task is determined by training history, with strong competition from a nontarget that has often previously been experienced as a target (inconsistent nontarget or ni), but much less from a nontarget that can always be ignored (consistent nontarget or nc) (schneider and shiffrin, 1977; schneider and fisk, 1982). Analogous effects of training history have been shown in the frontal eye field, with a relative enhancement of response to stimuli previously trained as targets (bichot et al ., 1996; bichot and schall, 1999). In our task (figure 1a), each trial began with a central cue indicating this trial s target object . Based on preexperimental training,, there followed a choice display containing either a single object, to left or right of fixation, or one object to either side . For single - object displays, the choice stimulus could be either the cued target (t), the stimulus associated with the other cue and thus serving as a target on other trials (ni), or a fixed nontarget object never serving as a target (nc). In the two - object case, a target t could be accompanied by either ni or nc, or ni and nc could appear together . Following the 500 ms choice display and a subsequent brief delay, the monkey was rewarded for a saccade to the t location, or if no t had been presented, maintained fixation (no - go response) for later reward (see experimental procedures). In line with the proposal of adaptive coding, our results show how, in a prefrontal cell population, activity resembles the limited processing resource of classic attentional models . As previously described (e.g., freedman et al ., 2001; kusunoki et al ., 2010), many cells were devoted to making task - relevant stimulus discriminations . With attentional competition, processing capacity was initially divided between competing display objects, with different neurons responding to different objects . Specifically, neural events in each hemisphere were initially dominated by response to the display item in the contralateral visual field, whether t or n. the result was that neurons failed to make critical stimulus discriminations in the ipsilateral visual field, resembling poor information coding when processing resources are diverted in classic attentional models (broadbent, 1958; kahneman, 1973). Subsequently, this initial, incoherent state was replaced by transition to a global focus on the behaviorally critical target, with this target now controlling neural activity in both hemispheres . The speed and extent of transition between these states reflected the strength of attentional competition, being more rapid and complete for t + nc than for t + ni displays . The results track dynamic allocation of processing resources in prefrontal cortex, with gradual establishment of a coherent and global attentional focus . Behavioral data (figure 1b) showed high accuracy for singly presented t and nc stimuli . The comparative difficulty of ignoring ni was confirmed by much reduced accuracy for this stimulus presented alone, with the majority of errors (72.2%) being saccades to the stimulus location at the time of the go signal . Saccades to the ni location were also common (59.2% of errors) for t + ni displays, while for t + nc displays, the most common form of error (79.1%) was a no - go response . During performance of the task, responses of 461 single neurons were recorded on the lateral frontal surface of three hemispheres, two in monkey a (n = 192 right, 113 left) and one in monkey b (n = 156, right). Pooled results are presented here, as similar patterns were seen in all three recorded hemispheres . Recording locations (figure 1c; figure s1 available online) were located in dorsal and ventral regions of the posterior lateral prefrontal cortex, including the posterior third of the principal sulcus . To ask how prefrontal cortex represents task events, we examined responses to the 12 possible single - object displays (3 stimulus categories [t, ni, nc] 2 visual fields [contralateral or ipsilateral to recording location] 2 cues). For each neuron, data were examined by anova with factors stimulus category, visual field, and cue . Analyses were separately conducted on firing rates from early (50250 ms from display onset) and late (300500 ms) response periods . In both analysis periods, many cells (79/461 early, 118/461 late) showed significant (p <0.05) main effects of stimulus category . Two examples are shown on the left of figure 2a . In the first cell (top row), responses were strongest to t and weakest to nc, this pattern arising much earlier for contralateral stimuli . A complementary pattern is illustrated by the second cell (figure 2a left, bottom row), with a late, selective response to nontargets, especially nc . In both analysis periods, there were also many cells (155/461 early, 114/461 late) showing significant main effects of visual field . An early preference for contralateral stimuli and a late preference for ipsilateral stimuli are illustrated by the two cells on the right of figure 2a . Main effects of cue were less common (47/461 cells early, 43/461 late). Numbers of cells showing different patterns of interaction are listed in table s1 . As shown in figure s1, both category- and location - selective cells were broadly distributed across recording locations, including dorsolateral and ventrolateral surfaces, as well as the posterior recording area lying between arcuate and principal sulci . Though these results suggest many cells coding the behavioral category of stimuli, the data suggested little direct role in the saccadic response . Of 58 cells showing an interaction of stimulus category and visual field in the late period (see table s1), there were 33 with a strong, sustained response for targets in one location (figure s2). Though such a pattern might plausibly reflect oculomotor preparation, even these cells showed little evidence of activity linked to the saccadic response (figure s2). These results match prior findings from similar tasks, suggesting prefrontal activity linked largely to behavioral categorization rather than motor output (everling et al ., 2002; kusunoki et al ., 2009). To examine stimulus coding across the whole cell population, neural activity at each time point from stimulus onset (see experimental procedures) was represented as a vector of firing rates across the full sample of 461 recorded cells . Twelve such vectors were obtained for each of the 12 separate single - object displays, and to measure separation of population activity for any two displays, we used euclidean distance between their activity vectors . A two - dimensional representation of the resulting similarity space, derived using multidimensional scaling (mds), is shown in figure 2b . Separate similarity spaces are shown for early (175 ms from stimulus onset) and late (450 ms) stages of processing . Even early in processing, the prefrontal representation already showed discrimination of both stimulus category and hemifield . In particular there was clear separation of t, ni, and nc categories, especially in the contralateral hemifield . Representations of a given behavioral category were similar for the two cues, despite actual stimuli exchanging roles as t or ni . It is noteworthy that hemifield coding was strong for both targets and nontargets, though for the latter it had no behavioral significance . At both stages of processing, neural representations for ni were intermediate between those for t and nc, in agreement with previous data (kusunoki et al ., 2010) and the example cells on the left side of figure 2a . As usual in lateral prefrontal cortex, these data show many cells coding current task events, with strong but not exclusive emphasis on behaviorally relevant stimulus categorizations . To examine attentional competition, we turned to two - object displays and the dynamics of information coding as the choice display is processed . On a resource model of prefrontal activity, a plausible hypothesis is that, just as attentional competition impairs behavioral accuracy, it might impair neural discrimination . Such results would match classic attentional models, in which division of attentional resource reduces processing efficiency (broadbent, 1958; kahneman, 1973). To address this question, we used euclidean distance to measure discrimination between critical stimulus pairs, t versus ni and t versus nc . In each case, we measured discrimination either for the critical stimuli presented alone (no - competition case), or in the presence of an additional nontarget in the opposite hemifield (attentional competition). The design of our displays (figure 1) allowed us to examine four such cases (figure 3): discrimination of t versus ni in the hemifield contralateral to the recording location (tcon versus nicon), either presented alone or with a concurrent stimulus (nc) in the ipsilateral hemifield; discrimination of tcon versus nccon, either presented alone or acompanied by niips; and similarly for discrimination of tips / niips, presented alone or with nccon, and discrimination of tips / ncips, presented alone or with nicon . In each case, we selected for analysis a population of cells that were most relevant to the critical discrimination (e.g., tcon versus nicon), ensuring that this selection was unbiased for comparison of no - competition and competition cases (see experimental procedures). We predicted that critical t / n discriminations might be impaired when an additional stimulus is added in the opposite hemifield . The pattern of results was strikingly different for contralateral and ipsilateral discriminations (figure 3). For single stimuli in the contralateral hemifield, t / n discrimination became significant at around 100 ms from onset and remained throughout the duration of the choice stimulus (figure 3, left panels; cf . Data for two - object displays closely tracked those for single objects, suggesting little change in contralateral t / n discrimination with attentional competition . Ipsilateral discriminations, in contrast, showed evidence of impairment by competing contralateral stimuli (figure 3, right panels). Again, t / n discrimination began at around 100 ms for single - object displays . With addition of nccon, discrimination of tips from niips remained close to zero until around 200 ms and then rapidly increased toward single - object values (figure 3, top right). This delay led to a period of strongly significant difference between discrimination strength in one- and two - object displays (figure 3, top right, dark gray bars). A larger and more extended impairment in discriminating tips from ncips was created by addition of nicon, lasting throughout most of the stimulus duration (figure 3, bottom right). The results confirm that critical neural discriminations can be impaired by competing stimuli, especially early in stimulus processing, and when training history makes a competing stimulus (ni) hard to ignore . The impairment, however, takes an intriguing form impairment of ipsilateral discrimination by a contralateral competitor, but not vice versa . How might the idea of flexible resource allocation, in particular competition of inputs to drive neural responses, explain these discrimination data? The results suggest a critical role of visual field, with a tendency at the start of processing the choice display to ignore the ipsilateral field . Anatomical connections from visual cortex to frontal lobe are much stronger within than between hemispheres (ungerleider et al ., 1989), and if competing stimuli are presented in opposite hemifields, neural activity in inferotemporal cortex is dominated by the contralateral input (chelazzi et al ., 1998). At least early in processing a choice display, prefrontal activity may show a similar contralateral dominance, meaning that in each hemisphere, there is little information concerning a competing ipsilateral event . When a target is present, however, an accompanying nontarget has no relevance to behavior . We considered the hypothesis that, across time of processing a choice display, activity evolves to a coherent attentional state, with responses in both hemispheres controlled by the behaviorally critical target . A neural mechanism of this sort would be analogous to progressive focus of processing resources in classical attention modes (kahneman, 1973). On this hypothesis, responses to a t + n display should be predictable from the separate responses produced by the component t and n presented alone . For tcon + nips, activity should follow response to tcon alone throughout choice stimulus processing . Early in processing, tcon dominates because it is contralateral, while late in processing, the same stimulus dominates because it is the target, but in either case, response to tcon + nips follows response to tcon alone . For tips now, an early response to ncon should be replaced by later activity based on tips . The top left panel shows responses to tcon + nips displays and their component single stimuli for a cell with very different tcon and nips responses . Throughout choice stimulus processing, the bottom left panel shows a complementary pattern, where again, response to the two - object display resembled the suppression produced by tcon alone . The right panels show very different dynamics for tips + ncon displays . In the top panel is a cell showing a strong, sustained response to tips alone . In the two - object display, this strong tips response was initially suppressed by the accompanying ncon . Later, suppression tended to be released but earlier and more rapidly when the suppressing stimulus was nc . The bottom right shows a complementary example, with strong response to a single ncon but little response to tips . In the two - object display, again, the early response resembled that to ncon alone, but, especially when the nontarget was nc, later activity was dominated by tips . To confirm this pattern at the population level in the first, we examined mean responses in selected cell groups (figure 4b) with clear differences in response to the two component stimuli of a two - object display . To examine responses to tcon + nips displays, we selected all cells with significantly different responses to tcon versus nips (see experimental procedures). For tcon> nips cells (figure 4b, top left), strong responses to tcon alone were matched by closely similar responses to both tcon + niips and tcon + ncips displays . <nips cells, with similar suppression to tcon presented alone or with an accompanying nontarget (figure 4b, bottom left). A contrasting pattern was seen for tips> ncon cells responding to the tips + ncon display (figure 4b, top right). Early responses to the tips + ncon display resembled the suppression produced by ncon alone, then, beginning before 200 ms, departed to approach tips responses . For tips + nccon, activity rapidly approached the response to tips alone, while for tips + nicon, some suppression compared to tips alone remained throughout stimulus processing . A corresponding pattern of results was seen for tips <ncon cells (figure 4b, bottom right). In a second analysis, we turned to the whole recorded cell population and asked how well single neuron responses to each two - object display were predicted by responses to the two component objects individually . Results for three different analysis windows early (100200 ms from choice stimulus onset), middle (250350 ms) and late (400500 ms)are shown in figure 5 . Responses of each neuron to the different stimulus displays were first normalized (division by mean response to all displays; see experimental procedures), and responses to two - object displays were then plotted against responses to each of the two component single stimuli . For tcon + nips displays, results were similar at all time points from stimulus onset . Across neurons, there was a strong tendency for response to the two - object display to match the response given by tcon rather than nips . For tips + ncon displays dynamics were more complex . In the early window, response to the two - object display tended to follow response to ncon alone, with response to tips unpredictive . In the middle window, the ncon response retained some influence for the case of ni, though this influence had already disappeared for nc . In contrast, response to the two - object display was increasingly predicted by response to tips . By the late window finally, to ask how closely population responses to a two - object display approached those to component single stimuli, we again used a euclidean distance measure calculated across the whole recorded sample of 461 cells . Across the time course of choice stimulus processing, we measured population discrimination of each two - object display (e.g., tcon + niips; figure 6, top left) from its component single stimuli (tcon, niips). A simple pattern of results emerged when t was contralateral . Throughout stimulus processing, the population response to the two - object display was barely discriminable from response to the contralateral t presented alone (figure 6, left panels). Beginning <100 ms from stimulus onset, in contrast, response to the two - object display diverged rapidly from response to the component ipsilateral n (figure 6, left panels). When t was ipsilateral, however, events followed a more complex time course, in particular for the highest - competition case (tips + nicon; figure 6, top right). In the first phase of processing, the two - object display was strongly discriminated from tips presented alone, but not from nicon . Only toward the end of the stimulus presentation did the curves cross, indicating a response to the two - object display that more closely resembled response to its component target . For the lower - competition case (tips + nccon), strong discrimination from the component target was short lived, with a correspondingly rapid increase in discrimination from the component nontarget (figure 6, bottom right). These results show how, as prefrontal processing evolves, there is large - scale reallocation of processing resources . Early in choice stimulus processing, neural activity in each hemisphere is dominated by response to the contralateral stimulus . In different groups of neurons (figure 4), this response can be either an increase or decrease from baseline; in either case, the response to a competition display resembles response to the contralateral stimulus alone . Later, this separation between hemispheres resolves to a coherent state of activity based on the critical t stimulus, especially when the accompanying stimulus is nc . Across both hemispheres, the final, global state is close to the state produced by the t stimulus alone . In further analyses, we examined the generality of attentional reallocation across recording locations and cell types . To produce an index of reallocation for each cell, we defined dt as the absolute difference between firing rates for a tips + ncon display and for tips alone and, similarly, dn as the absolute difference between firing rates for the tips + ncon display and its component ncon alone . In an early analysis window (100200 ms from stimulus onset), we defined a dominance index as(dtdn)(dt+dn). Following the results shown in figure 6, this early index was generally positive, reflecting response to tips + ncon that was closer to ncon than to tips alone . The same dominance index calculated for a late analysis window (400500 ms) was generally negative, indicating response to tips + ncon that was closer to tips than to ncon alone . Subtracting the late index from the early index gave us a final reallocation index, separately calculated for each cell and for tips + nicon and tips + nccon displays . Across the whole recorded cell population, the mean reallocation index for tips + nicon displays was 0.147 . By t test, this value was significantly greater than zero (p <0.001). For tips + nccon displays, the mean reallocation index was 0.124, again significantly greater than zero (p <0.001). To examine generality across anatomical regions, we divided the full recorded cell sample into three groups, a smaller group (n = 42) recorded in a posterior region between the principle and arcuate sulci (see figure s1) and larger groups recorded in more anterior dorsolateral (n = 145) and ventrolateral (n = 274) regions, divided by the fundus of the principal sulcus . Both for tips + nicon and tips + nccon displays, the mean reallocation index was positive for all three cell groups . Anova showed no difference between cell groups, for tips + nicon f(2, 456) = 1.17, for tips + nccon f(2, 455) = 1.29 (missing data for cases in which dt = dn = 0). In a second analysis, we examined generality across cell types, defined by coding of stimulus category and/or location in single - stimulus displays . To examine category - selective cells, we took all those cells (n = 162) with a main effect of stimulus category in either early- or late - period anovas on single - stimulus activity (see earlier section, coding of single choice stimuli). To examine location - selective cells, we took all cells (n = 210) with a main effect of location in either analysis period . Again, the mean reallocation index was significantly positive in both groups (category - selective cells: mean index = 0.222, p <0.005 for tips + nicon, mean index = 0.226, p <0.001 for tips + nccon; location - selective cells: mean index = 0.233, p <0.001 for tips + nicon, mean index = 0.096, p <0.06 for tips + nccon). These results show substantial generality in the overall pattern of attentional reallocation for tips + ncon displays . For all regions in our recording area, and whatever stimulus feature a cell coded, early response was determined largely by the contralateral n, while later response was determined largely by t. finally, we found no evidence for attentional reallocation on error trials . Combining data for all cells, and for both major error types (saccade to wrong location, no - go), mean reallocation index on error trials was 0.006, t test against zero p = 0.56 for tips + nicon displays, and mean reallocation index 0.014, p = 0.49 for tips + nccon displays . Only correct trials, evidently, were associated with reallocation of prefrontal processing resources from contralateral nontarget to ipsilateral target . When stimuli or other cognitive events compete for attention, processing resources must be allocated to the most important (broadbent, 1958; kahneman, 1973). Our results track development of an attentional focus in the population activity of prefrontal cortex . In line with the proposal of adaptive prefrontal coding of task - relevant information, we found that many prefrontal cells discriminated task - critical stimulus categories and locations . When two stimuli were present in the display, attentional competition resolved through widespread reallocation of neural resources . Early processing lacked attentional coherence, with different neurons responding to different items in the display . Specifically, neural activity in each hemisphere was dominated by the contralateral display item a pattern (figure 2) coding both hemifield and behavioral category of that stimulus . Accordingly, critical stimulus discriminations within one visual field were impaired in the ipsilateral hemisphere, matching the classical proposal of reduced processing efficiency when processing resources are withheld (broadbent, 1958; kahneman, 1973). Construction of this global attentional focus resembles the classical proposal that processing resources are allocated to the most important cognitive events (broadbent, 1958; kahneman, 1973). We found that the time course of transition depends on the attentional weight of nontargets . For nc, a stimulus never serving as a target, control of the contralateral hemisphere was released quickly and easily . For ni, a target stimulus on other trials, release was slow and incomplete . Again, these results match comparable findings from human studies, showing how processing resources are rapidly allocated to targets when nontargets have been extensively practiced as task irrelevant (schneider and shiffrin, 1977; schneider and fisk, 1982). It is commonly proposed that, in early visual areas, stimuli within or close to the receptive field of a cell compete to control its activity (moran and desimone, 1985; reynolds et al ., 1999; reynolds and heeger, 2009). Accordingly, moving attention from one stimulus to another can have large effects when the two are close together; with widely separated stimuli, the effect is much smaller, with only modest enhancement of response to the attended input (moran and desimone, 1985; lee and maunsell, 2010). In prefrontal cortex, instead, we found widespread target dominance by the end of display processing, reflecting global allocation of processing resources to the behaviorally critical stimulus . Global division of prefrontal processing capacity is a plausible neural basis for many cases of attentional competition, including interference between widely separated visual stimuli and even between very dissimilar tasks (marois and ivanoff, 2005; dehaene et al ., 1998; . In prefrontal cortex, global processing competition, and its dynamic resolution, probably reflect the breadth of inputs from other brain regions (pandya and yeterian, 1996) and the strong interconnectivity between one prefrontal region and another (pucak et al ., 1996). Our finding of early activity dominated by the contralateral visual field resembles results previously reported for inferotemporal cortex (chelazzi et al ., 1998). In that study, pairs of stimuli were presented either within one hemifield or one to each hemifield . As in the current study, animals searched for a prespecified target, responding with an immediate saccade or lever release . When both stimuli fell in the same hemifield, neural activity was dominated by the target, but with stimuli in opposite hemifields, activity was dominated by the contralateral stimulus, whether target or nontarget . Because responses in that study were immediate, data were only available for the brief period before the response was made . It is not known whether, with a longer stimulus presentation, global dominance by the attended target might develop in inferotemporal cortex, as we have shown here for prefrontal cortex . The neural mechanisms of visual search have also been examined in the frontal eye field, with some similarities to the current results . In the frontal eye field, as in early visual areas, there is response enhancement when the stimulus within the receptive field is the current target (schall et al ., 1995). Enhancement reflects training history, with earlier enhancement after long practice in searching for a given target (bichot et al ., 1996) and enhancement for stimuli that share features with a previous target (bichot and schall, 1999). As in early visual areas, however, such target enhancements are far from the widespread reallocation of processing activity we observed in prefrontal cortex . Even when the target is well outside the receptive field, the nontarget within the receptive field drives strong activity up to the time of the response (schall et al ., 1995; thompson et al ., 1997 a likely mechanism is communication between the two frontal lobes (tomita et al ., 1999), allowing processing on both sides to be dominated by the same, critical stimulus event . On this model, target information from one hemisphere displaces nontarget information in the other; this happens rapidly and relatively completely when nontarget status is fixed throughout training but only more slowly and partially when the nontarget to be displaced is a target on other trials . Evidently, the competitive mechanisms allowing nontarget displacement must be influenced both by current behavioral status allowing t to dominate even ni but also by long - term learning . On the current trial, some context signal initiated by the cue (stokes et al ., 2013) must determine which stimulus is t and which is ni, thus directing the outcome of competition for control of population activity . Across learning, in contrast, nc is always irrelevant, resulting in long - term reduction of competitive weight . An even stronger separation of competitive weights may be obtained with spatial cues, directly indicating which visual field should be attended in a subsequent visual display . With advance spatial cueing, information from the attended field may dominate some cells even from the outset of visual processing (everling et al ., 2002), though even in this case, there is some response to the unattended side (everling et al ., again, the strength of such attentional modulations reflects the variable strength of spatial cues (lennert and martinez - trujillo, 2011). An enduring debate in the search literature rests on the distinction between serial and parallel processing . Behavioral (egeth and dagenbach, 1991; kyllingsbaek and bundesen, 2007) and neurophysiological (buschman and miller, 2009) arguments can be assembled on both sides of this debate and, in the present case, data are not easily explained by a simple serial model . Instead, processing begins with parallel coding of both display inputs, one dominating each hemisphere, then resolves over several hundred milliseconds (figures 5 and 6) to a global state of target dominance . It is an open question how this conclusion relates to other kinds of task and search display, e.g., displays containing larger numbers of stimuli (buschman and miller, 2009). A second enduring question in the cognitive literature is the extent to which the two hemispheres act as separate pools of processing capacity (pashler and obrien, 1993; alvarez and cavanagh, 2005; see also buschman et al ., 2011). Our data suggest that the answer may be dynamic, evolving with construction of the attentional focus . Early in processing a two - object display, we indeed found that the two hemispheres were focused on different stimuli, like parallel processing pools . Later, we found coherence, with both hemispheres focused on the same, behaviorally critical stimulus . Though here we examined attentional competition between visual fields, more generally, similar processing principles may apply to many different cases of processing competition . In many such cases, prefrontal activity may move from an early, unfocused state to attentional coherence . Attentional coherence is critical to organized cognition, as multiple brain systems must converge to process the stimuli, responses, reward, etc . Of current behavior . Through feedback to multiple brain systems (dehaene et al ., 1998; miller and cohen, 2001; desimone and duncan, 1995; moore and armstrong, 2003), the construction of globally consistent prefrontal activity patterns may be critical in assembly of distributed yet coherent attentional episodes . Subjects were two male rhesus monkeys (macaca mulatta) weighing 11 (monkey a) and 10 (monkey b) kg . All experimental procedures were approved by the uk home office and were in compliance with the guidelines of the european community for the care and use of laboratory animals (euvd, european union directive 86/609/eec). Task events were controlled by a pentium pc running cortex software, with displays presented on a 19 inch led screen placed in front of the monkey s chair . Each trial began with onset of a red dot at screen center, which the animal was required to fixate (window 5 5 for monkey a, 4 4 for monkey b) until the final saccadic response at the end of the trial . A premature saccade away from screen center immediately terminated the trial without reward (trials discarded from all data analyses). Once fixation had been held for 1,000 ms, a central cue stimulus (500 ms) indicated the target for the current trial . Based on preexperimental training, each of two alternative cue stimuli was associated with a different target (see figure 1a inset for cue - target pairs for monkey a; different cue, target, and nontarget images were used for monkey b). A randomly varying delay of 400600 (monkey a) or 400800 (monkey b) ms was followed by a 500 ms choice display . The display contained either a single object, centered on the horizontal meridian randomly 6 to left or right of fixation, or two objects, one to either side . For single - object displays, the stimulus object was either the cued target t, the object associated with the alternative cue (inconsistent nontarget, ni), or a third object never used as a target (consistent nontarget, nc). For two - object displays, major trial types were t + ni, t + nc, and ni + nc (in randomly varying left - right or right - left configuration), though in some sessions, small numbers of ni + ni trials were also included (data not shown). To avoid response biases, we adjusted frequencies of individual trial types to ensure that t was present in half of all single - object and half of all two - object displays; otherwise, frequencies of all major trial types were the same . Following choice stimulus offset, there was a further random delay of 100150 (monkey a) or 300500 (monkey b) ms, after which the fixation point turned green to indicate the monkey s response interval . For go trials (t present in choice display), the monkey was immediately rewarded with a drop of liquid for a saccade to the remembered t location (target window 6 6 for monkey a, 3.5 3.5 for monkey b). For no - go trials (t absent), the monkey was required to hold fixation for the whole 1,000 ms response interval and was then either given immediate reward (monkey b) or rewarded for a further saccadic response (monkey a). For monkey a, some sessions had cues randomly varying between trials, while others had alternating brief (1520 trials) blocks of fixed cues . Physiological data were very similar in the two cases and were combined . For monkey b, cues always varied randomly between trials . Each monkey was implanted with a custom - designed titanium head holder and recording chamber(s) (max planck institute for biological cybernetics), fixed on the skull with stainless steel screws . Chambers were placed over the lateral prefrontal cortex of the left (ap = 25.3, ml = 20.0; ap, anterior - posterior; ml, medio - lateral) and right (ap = 31.5, ml = 22.5) hemispheres for monkey a and the right hemisphere (ap = 30.0, ml = 24.0) for monkey b. recording locations for each animal are shown in figure s1 . We used arrays of tungsten microelectrodes (fhc) mounted on a grid (crist instrument) with 1 mm spacing between adjacent locations inside the recording chamber . The electrodes were independently controlled by a hydraulic, digitally controlled microdrive (electrodes drive, nan for monkey a; multidrive 8 channel system, fhc for monkey b). Neural activity was amplified, filtered, and stored for offline cluster separation and analysis with the plexon map system (plexon). Eye position was sampled using an infrared eye tracking system (120 hz, asl for monkey a; 60 hz, iscan for monkey b) and stored for offline analysis . We did not preselect neurons for task - related responses; instead, we advanced microelectrodes until we could isolate neuronal activity before starting the task . At the end of the experiments, animals were deeply anaesthetized with barbiturate and then perfused through the heart with heparinized saline followed by 10% formaldehyde in saline . The brains were removed for histology and recording locations confirmed on dorsal and ventral frontal convexities and within the principal sulcus . Physiological data were analyzed just from successfully completed trials, on average including 17 repetitions for each combination of cue, choice stimulus type, and hemifield / spatial arrangement . For all analyses, spike data were smoothed with a gaussian kernel of sd 20 ms, cutoffs 1.5 sd . To measure neural discrimination between any two choice stimuli x and y for each cell in the population, we measured the difference in absolute firing rate for x and y. as is standard, euclidean distance was defined as the square root of the sum of these squared differences . For mds (figure 2b), we used raw euclidean distances . For quantitative analysis (figures 3 and 6), we used a correction for the fact that euclidean distance must always be positive and scales with absolute firing rate . For each distance measure, we calculated the expected chance value by randomly permuting x, y labels across trials, then subtracted the median permuted value (across 1,000 permutations) from the obtained raw value . Analyses were repeated at each 1 ms time point from 100 ms to 600 ms from choice stimulus onset . To measure discrimination between two - object displays and their component single stimuli (figure 6), we used activity vectors based on the full cell sample (n = 461). To compare discrimination of the same stimulus pairs (x, y) in single- versus two - object displays (figure 3), we selected just those cells most sensitive to the critical discrimination, giving equal weight to single- and two - object data . For this purpose we used anova on activity of each cell over the full (0500 ms) period of choice stimulus presentation, with factors critical stimulus (x, y) accompanying stimulus (absent, present) cue, and selected just those cells with a significant (p <0.05) effect of critical stimulus . Permutation testing was used to compare distances in single- and two - object cases (figure 3). On each permutation, for each cell we randomly maintained or switched single- and two - object labels; when labels were switched, they were switched for all of that cell s data . After this permutation of labels the true difference in distances (figure 3) was compared with the distribution of permuted values across 1,000 permutations . For comparison of mean neural activities for two - object displays and their component single stimuli (figure 4b), we selected all cells with a significant difference between responses to singly presented targets on one side and nontargets on the other . Significance (p <0.05) was again determined by anova on activity across the full period of choice stimulus presentation, with factors stimulus (e.g., tcon, nips) cue . <nips; tips> ncon; tips <ncon) as shown in figure 4b . For calculation of mean activity across cells (figure 4b, figure s2), responses of each cell were first normalized by dividing by mean firing rate across all choice displays, calculated across the full 0500 ms display period . The same normalization was used to create scatterplots of response to each two - object display as a function of response to component single stimuli (figure 5).
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Irrational and inappropriate use of drugs can lead to suboptimal clinical benefit and possible adverse drug reactions (adrs). Availability of clinically relevant, contemporary, and unbiased drug information goes a long way in promoting rational use of drugs . There are various sources of information which are utilized by treating physicians for accessing relevant drug information such as their indications, adrs, contraindications, and special precautions . Drug information is usually sourced from national formularies (e.g. National formulary of india (nfi), british national formulary), package inserts (pis) of drugs, drug compendia such as monthly index of medical specialities (mims), current index of medical specialities (cims), and medical textbooks . The drug information available in various sources should be uniform, reliable, and conforming to the regulatory label of the drug . Every drug is approved for a specific indication(s) by drug regulator of the country, and these indication(s) is / are known as approved indication(s) of the drug . If the drug is used for any indication other than the approved indication, it is known as off - label use of the drug . Approved indications of all the approved drugs are available with central drugs and standards control organization (cdsco), which is the national drug regulator in india . It has been observed that there is variation in the quantity and quality of information mentioned in different drug information sources and a single credible benchmark is lacking . Such variation not only deprives the medical fraternity from accessing reliable drug information but can also promote off - label and irrational drug use leading to increased incidence of adverse reactions and possible treatment failure . We planned the present study to assess this variation in a sample of randomly selected drugs with respect to their indications given in various sources of drug information . We identified 50 commonly used drugs belonging to different groups (e.g. Antimicrobials, antihypertensives, analgesics, antiulcer and antiemetics, anticancers, antidiabetics, and antiobesity drugs and also lifestyle drugs such as sildenafil) used in various specialty and superspecialty centers of our hospital . Two senior residents (d.m . Clinical pharmacology students) and one phd student collected and analyzed the pis of the selected drugs . These drugs were then analyzed for any variations in the information on a number of indications as mentioned in commonly used drug information sources (cdsco web site, nfi, mims, and pis). The number of drugs out of the selected 50, whose indication information was missing in different sources.total number of indications given in different sources, in respect of these 50 drugs.average number of indications per drug mentioned in different sources.after doing the above assessments, we did a subset analysis in respect of: only those drugs whose indications were given in cdsco website list.in the next step, we compared only those drugs whose indications were mentioned in all the four sources . We also looked upon gross qualitative differences existing across various sources of drug information used in this study . The number of drugs out of the selected 50, whose indication information was missing in different sources . Total number of indications given in different sources, in respect of these 50 drugs . Average number of indications per drug mentioned in different sources . After doing the above assessments, we did a subset analysis in respect of: only those drugs whose indications were given in cdsco website list.in the next step, we compared only those drugs whose indications were mentioned in all the four sources . In the next step, we compared only those drugs whose indications were mentioned in all the four sources . We also looked upon gross qualitative differences existing across various sources of drug information used in this study . The data were represented as mean standard error of mean and median (range). To find the difference between different sources, data were statistically analyzed by applying friedman test using graph pad instat (trial version) software . The data were represented as mean standard error of mean and median (range). To find the difference between different sources, data were statistically analyzed by applying friedman test using graph pad instat (trial version) software . Pis of all the 50 selected drugs were collected, and they had information about indications which was included in the analysis for comparison . Indication information about the selected drugs a subset analysis was done in respect of only those 40 drugs which were available in cdsco . Nfi was excluded from this analysis as this source had information of only about 24 of these 40 drugs . Hence, this analysis included only three sources (cdsco, mims, and pi). In respect of these 40 drugs, the pi had listed maximum number of indications (2.95 indications / drug), followed by mims (2.70 indications / drug) and cdsco website (2.20 indications / drug). We found that the difference in a number of indications given in these three sources was not statistically significant (p = 0.07) [table 2]. Indication information available in cdso, mims, and pi to include nfi as well, a subset analysis was done in respect of those 24 drugs information about which were available in all the four sources including nfi . We found that nfi had listed maximum number of indications (3.79 indications / drug), followed by pi (3.08 indications / drug), mims (3.04 indications / drug), and cdsco website (2.66 indications / drug). This difference in the number of indications was statistically significant (p = 0.02) [table 3]. Indication information available in all four sources after the quantitative comparison, we identified any gross qualitative mismatch in information across these four sources . Following gross discrepancies are observed: of the 40 drugs mentioned in cdsco, only broad single indication is mentioned in respect of some drugs (e.g. Amphotericin b - febrile neutropenia in cancer patients; sodium valproate: all forms of epilepsy; torsemide: diuretic; fluoxetine: for treatment of depression). It is apparent that such abridged information only reflects the broad use of the drug without providing more specific and relevant information to the prescribing physicians.labetalol is one of the preferred drugs for treatment of pregnancy induced hypertension, and its oral administration is considered as safe and effective as methyldopa . However, as per the cdsco website, it is indicated for the treatment of all forms of hypertension except hypertension of pregnancy, whereas mims and pi mentioned hypertension in pregnancy as one of the indications of labetalol.the cdsco site mentions the treatment of depression as the only indication for fluoxetine, whereas mims and pi mention other indications such as obsessive compulsive disorder, bulimia nervosa also . Surprisingly, nfi lists out the maximum indications for fluoxetine which includes premenstrual disorder, anorexia nervosa, and parkinson's disease as well over and above the indications given in other three sources.in respect of tablet levofloxacin, cdsco mention prostatitis as the only indication, whereas mims and pi includes more indications.for tablet topiramate, the cdsco site mentions it only as an antiepileptic, whereas other sources go on to describe the type of epileptic disorders for which it is indicated . In addition, pi mentions prophylaxis of migraine as one of its indication.besides above variations, some minor typographical errors were also noticed in information provided in cdsco, e.g., gabapentin being indicated for naturopathic pain instead of neuropathic pain; febuxostat for treatment of chronic hyperuricemia in conditions where urate depression has already occurred instead of urate deposition; sildenafil for pulmonary osterial hypertension and not for pulmonary arterial hypertension . Of the 40 drugs mentioned in cdsco, only broad single indication is mentioned in respect of some drugs (e.g. Amphotericin b - febrile neutropenia in cancer patients; sodium valproate: all forms of epilepsy; torsemide: diuretic; fluoxetine: for treatment of depression). It is apparent that such abridged information only reflects the broad use of the drug without providing more specific and relevant information to the prescribing physicians . Labetalol is one of the preferred drugs for treatment of pregnancy induced hypertension, and its oral administration is considered as safe and effective as methyldopa . However, as per the cdsco website, it is indicated for the treatment of all forms of hypertension except hypertension of pregnancy, whereas mims and pi mentioned hypertension in pregnancy as one of the indications of labetalol . The cdsco site mentions the treatment of depression as the only indication for fluoxetine, whereas mims and pi mention other indications such as obsessive compulsive disorder, bulimia nervosa also . Surprisingly, nfi lists out the maximum indications for fluoxetine which includes premenstrual disorder, anorexia nervosa, and parkinson's disease as well over and above the indications given in other three sources . In respect of tablet levofloxacin, cdsco mention prostatitis as the only indication, whereas mims and pi includes more indications . For tablet topiramate, the cdsco site mentions it only as an antiepileptic, whereas other sources go on to describe the type of epileptic disorders for which it is indicated . In addition, pi mentions prophylaxis of migraine as one of its indication . Besides above variations, some minor typographical errors were also noticed in information provided in cdsco, e.g., gabapentin being indicated for naturopathic pain instead of neuropathic pain; febuxostat for treatment of chronic hyperuricemia in conditions where urate depression has already occurred instead of urate deposition; sildenafil for pulmonary osterial hypertension and not for pulmonary arterial hypertension . After the quantitative comparison, we identified any gross qualitative mismatch in information across these four sources . Following gross discrepancies are observed: of the 40 drugs mentioned in cdsco, only broad single indication is mentioned in respect of some drugs (e.g. Amphotericin b - febrile neutropenia in cancer patients; sodium valproate: all forms of epilepsy; torsemide: diuretic; fluoxetine: for treatment of depression). It is apparent that such abridged information only reflects the broad use of the drug without providing more specific and relevant information to the prescribing physicians.labetalol is one of the preferred drugs for treatment of pregnancy induced hypertension, and its oral administration is considered as safe and effective as methyldopa . However, as per the cdsco website, it is indicated for the treatment of all forms of hypertension except hypertension of pregnancy, whereas mims and pi mentioned hypertension in pregnancy as one of the indications of labetalol.the cdsco site mentions the treatment of depression as the only indication for fluoxetine, whereas mims and pi mention other indications such as obsessive compulsive disorder, bulimia nervosa also . Surprisingly, nfi lists out the maximum indications for fluoxetine which includes premenstrual disorder, anorexia nervosa, and parkinson's disease as well over and above the indications given in other three sources.in respect of tablet levofloxacin, cdsco mention prostatitis as the only indication, whereas mims and pi includes more indications.for tablet topiramate, the cdsco site mentions it only as an antiepileptic, whereas other sources go on to describe the type of epileptic disorders for which it is indicated . In addition, pi mentions prophylaxis of migraine as one of its indication.besides above variations, some minor typographical errors were also noticed in information provided in cdsco, e.g., gabapentin being indicated for naturopathic pain instead of neuropathic pain; febuxostat for treatment of chronic hyperuricemia in conditions where urate depression has already occurred instead of urate deposition; sildenafil for pulmonary osterial hypertension and not for pulmonary arterial hypertension . Of the 40 drugs mentioned in cdsco, only broad single indication is mentioned in respect of some drugs (e.g. Amphotericin b - febrile neutropenia in cancer patients; sodium valproate: all forms of epilepsy; torsemide: diuretic; fluoxetine: for treatment of depression). It is apparent that such abridged information only reflects the broad use of the drug without providing more specific and relevant information to the prescribing physicians . Labetalol is one of the preferred drugs for treatment of pregnancy induced hypertension, and its oral administration is considered as safe and effective as methyldopa . However, as per the cdsco website, it is indicated for the treatment of all forms of hypertension except hypertension of pregnancy, whereas mims and pi mentioned hypertension in pregnancy as one of the indications of labetalol . The cdsco site mentions the treatment of depression as the only indication for fluoxetine, whereas mims and pi mention other indications such as obsessive compulsive disorder, bulimia nervosa also . Surprisingly, nfi lists out the maximum indications for fluoxetine which includes premenstrual disorder, anorexia nervosa, and parkinson's disease as well over and above the indications given in other three sources . In respect of tablet levofloxacin, cdsco mention prostatitis as the only indication, whereas mims and pi includes more indications . For tablet topiramate, the cdsco site mentions it only as an antiepileptic, whereas other sources go on to describe the type of epileptic disorders for which it is indicated . In addition, pi mentions prophylaxis of migraine as one of its indication . Besides above variations, some minor typographical errors were also noticed in information provided in cdsco, e.g., gabapentin being indicated for naturopathic pain instead of neuropathic pain; febuxostat for treatment of chronic hyperuricemia in conditions where urate depression has already occurred instead of urate deposition; sildenafil for pulmonary osterial hypertension and not for pulmonary arterial hypertension . In india, drugs controller general of india is the drug regulatory authority who is responsible for granting approval and marketing permission of drugs in our country . The drugs are approved by drug regulator of any country for specific indications in specified dosage, which is known as the labeling information of that particular drug . However, the actual use of the drug in clinical practice may vary and may not be according to its labeling information at times . Treating physicians can and do use drugs for any indication based on his / her clinical judgment . For example, metformin is used for the treatment of polycystic ovarian disease which is not its approved indication . Off - label use of a drug is not illegal, however, it may be irrational or unscientific . In order to promote the rational and scientific use of drugs, it is important that relevant information about any drug, namely its indications, contraindications, and dosage are readily available to the prescribing physicians . Some common sources which are relied upon by the prescribing physicians to access drug information are nfi, pi, commercially published drug compendia such as mims and cims . The information available in such sources should confirm with the labeling information approved by the drug regulator of that country so that there is uniformity in decision making irrespective of the source(s) of information . We undertook this study to assess the quantity and quality of drug information available in various sources and compared it with the labeling information of the drug as provided in the cdsco website, which is the regulatory benchmark . The information about indications was taken as the sole benchmark of overall drug information, and various sources were compared on the basis of this parameter . It was observed that no information about indications of 10 out of the selected 50 drugs was available on the cdsco website . Of these 10 drugs, 8 (tablet metformin, tablet acetazolamide, tablet verapamil, tablet carbamazepine, tablet spironolactone, tablet nitrofurantoin, tablet methotrexate, and injection isoprenaline) drugs were not even mentioned and the rest 2 (tablet propranolol and tablet prazosin) were enumerated without any mention of indications . This can be explained on the basis of the fact that cdsco website only contains information from 1971 onward, and all these missing drugs are fairly old . Nfi is mandated to include all the drugs in national list of essential medicines and some other commonly used drugs . The most of the missing drugs (such as risedronate, erythropoietin, and faropenem) are used only in specialized centers and hence shall not be a part of nfi . However, the indication information about some commonly used drugs such as prazosin, levofloxacin, and chlorthalidone was also missing in nfi . Mims is a commercially available drug information compendium and was found to contain more number of indications as compared to the regulatory benchmark . Since this source is commonly utilized for seeking drug information, such a discrepancy may encourage off - label, and sometimes irrational, use of drugs . Pis were also found to contain maximum indications over and above the ones mentioned in regulator's website . In our country, pis are to be provided to the regulator at the time of registration . However, providing and publishing pis along with drug packages in neither regulated nor mandatory . In fact, all the pharmaceutical companies do not provide pis . However, when available, listing any indication in pi which is not in consonance with the regulator's website may again lead to off - label and possible irrational drug use . On the basis of aforementioned, it can be safely inferred that the cdsco website does not contain information about some commonly used drugs even while these drugs are marketed in india . These 10 drugs are part of 50 commonly prescribed drugs as identified for this study . Such a deficiency has the portent to compromise the robustness of regulatory benchmark and may lead to confusion regarding the appropriate use of drugs . This study highlights the discrepancies in drug information available in various sources by taking a representative sample of commonly used drugs . To the best of our knowledge, this study, though parsimonious in design, these 50 drugs were identified on the basis of the prescription pattern in our hospital which is a tertiary care center . One limitation of this study, is that, this prescription pattern may vary from one hospital to other, and a more broad based selection criteria for identifying drugs may be desirable . Variation exists in the quantity and quality of information on indications about drugs available in various sources . Cdsco website is the regulatory benchmark and requires updating so as to provide a sound and reliable reference regarding drug information for all the stakeholders . Further studies involving large sample of drugs and more variables (such as side effect profile, dosage information, information on drug interactions, and special precautions and contraindications) are required to further elucidate the issue of variation in drug information.
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Subclassification and study of cancer patients based on mutational status presents opportunities to learn the significance of genomic alterations (and their combinations) and to develop additional therapies (and combinations of therapies). However, the large number of mutations known to be important in cancer development and the presence of multiple mutations in any individual patient combines to create a great diversity in populations of patients with a specific tumor type . Shrager and tenenbaum note that cancer is in effect, a large number of rare diseases occupying a very high dimensional space with very few opportunities for action and observation in each subtype . To efficiently search a space of this nature, one needs to capture the learnings from as many patients and treatment experiments as possible in a continuously updated knowledge base.1 the stratification of cancer patients by mutational status and resultant decrease in the proportion of patients available for study enrollment presents serious problems for observational and interventional research . The low prevalence (12%) of many driver mutations in solid tumors precludes recruitment of sufficient numbers of subjects from traditionally sized research consortia and has led to new models of clinical research based on interinstitution collaboration, community outreach, and broad data sharing, with the goal of learning from every treatment encounter . This article reviews the challenges and opportunities of such collaborations from the perspective of the department of veterans affairs healthcare system, the largest integrated healthcare system in the united states . Creating generalizable knowledge as well as informing current individual patient care is therefore enabled by learning from all available previous treatment experiences of every relevant patient in the entire system, aggregating data across many medical centers . Familiar challenges to this approach include technical issues, such as data element provenance, data quality, and database variability across institutions, ensuring patient protections in data sharing related to informed consent, privacy / confidentiality, and health insurance portability and accountability act (hipaa) authorization, scalability and sustainability of aggregated databases, and cultural and financial barriers to data sharing in a research community . The ability to commoditize healthcare data has created opportunities for new consortium models that enable data sharing and patient access (for clinical trials). Data sharing is an important element of the collaborations exemplified by orien,2 medc,3 tapur,4 and apollo5 (see table 1). The national cancer institute (nci) has created the genomic data commons, a unified data repository6 that enables data sharing across these and other cancer genomic studies, in support of precision medicine . The repository houses clinical health record and genomic data (fastq file format) and complements the cancer imaging archive,7 another ncisponsored repository that contains radiographic and pathology images for cancer patients . Sharing with the research community the department of veterans affairs (va) has begun to move consented and hipaaauthorized patient data from the va electronic medical records to these nci data repositories for subsequent sharing with the research community (see table 1). This approach replaces the need for cancer patients to sign multiple forms consenting to data sharing with a single broad consent that satisfies the requirements of the various project institutional review boards (irbs). It also provides data collection and curation that meets requirements of the individual project aims . Is a joint effort by the va and the nci to provide these data to early career scientists to develop analytics and other tools in support of clinical and research objectives.8 as discussed above, traditional recruitment approaches from single or limited groups of institutions do not provide sufficient numbers of eligible study subjects to fulfill inclusion criteria with specific tumor mutation combination requirements . Furthermore, thirdparty payers seldom reimburse for mutational analysis required for patient screening prior to entry into research, thus shifting to the research enterprise the cost of screening large numbers of patients, of whom only a small fraction will be found eligible . The lack of data on the mutational status of patients is the major bottleneck for clinical trial execution and slows progress in precision oncology . In the era of precision oncology treatment, the new standard of care requires clinical reimbursement for expanded panel testing in cancer patients, with subsequent recruitment in clinical trials when appropriate for the individual patient . That standard then leads to sharing and reuse of patient data for clinical trials and observational research . Major efforts underway to solve these and related problems are exemplified by the medc and orien initiatives . The medc program offers insurance coverage of testing for institutions and patients who agree to contribute clinical data to the n1 registry for subsequent analysis . The oncology research information exchange network (orien) is a research collaboration founded by the moffitt cancer center in tampa and the ohio state university comprehensive cancer center to match patients to targeted treatments and promote datasharing activities . The group engages industry partners on sponsored projects across the clinical trials continuum and serves as a broker between research and healthcare communities . These and other programs (particularly ncisponsored clinical trial consortia) that foster collaboration between clinical care and research communities represent new models to advance precision oncology . Membership of va sites in nci consortia and programs such as medc, orien, and tapur makes data sharing and clinical trial participation opportunities available for veterans at participating va medical centers but leaves behind patients at facilities that lack oncology research programs and infrastructure . This structural problem is not unique to the va, as opportunities for patients to participate in cancer clinical trials are similarly limited in communities not located near cancer centers . If participation in clinical trials is considered a new standard of care, then this phenomenon exposes a new and important access disparity in a healthcare system . The distributed enrollment program under development at the va cooperative studies program presents a model to move clinical trials to patients remote from va cancer centers by seeking preapproval of cancer protocols by a central irb, reduction of researchspecific training requirements imposed on participating clinicians, and centralized trial management (such as data collection and submission). Indeed, much of the administrative overhead built into the clinical research apparatus in the name of quality assurance, while highly appropriate for research designed primarily to benefit the broader community (such as registration studies for drugs where effective alternatives exist), may have reduced relevance in a precision oncology setting, where the patient's motivation to participate is first and foremost to obtain study drug, whether or not alternatives exist . By its nature, precision oncology is a biomarkerdriven field critically dependent on acquisition of clinical biosamples and electronic medical record data for discovery and validation with lack of access to both resources as a central limitation to the pace of discovery . Collaborations to costshare for data and tissue procurement between biotech, pharma, and healthcare systems, in a precompetitive fashion, are emerging.4 to reach full potential, such collaborations require more complete integration within healthcare systems, as exemplified in the apollo program, a partnership between the nci, dod, and va . In apollo, tumor tissue is made available for biomarker analysis (proteomics in this case) and the results transmitted back to healthcare providers if they are determined to offer incremental value to patient care, beyond mutational analysis . While observational data is useful in this regard, randomization accelerates learning, and results in more certain knowledge . For example, demonstration that patients randomized to genomic augmented with proteomic analysis had superior treatment outcomes to those randomized to genomic analysis alone supports a compelling argument to adopt this new biomarker of response to targeted therapy . Problematically, the introduction of randomization (to biomarkers) into clinical care using traditional clinical trial methods is costprohibitive . The department of veterans affairs continues to make progress in this area through the pointofcare research9 and precision oncology10 programs whereby patients are randomized to minimal risk alternatives with relaxed regulatory requirements appropriate with the degree of risk (riskbased monitoring). Data generated from these embedded studies are derived exclusively from the ehr (realworld evidence) and require fda acceptance if used for registration of a new companion diagnostic.
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Pluripotent embryonic stem cells (escs) have the potential to differentiate into any cell in the adult body, making them an ideal source of cells for tissue regeneration when other options are absent . Since escs display robust teratoma forming potential in vivo, differentiated products are thought to be a better option for cellular replacement of diseased or damaged tissues . However, differentiated esc derivatives are often short - lived and are undetectable after transplantation in vivo, leading us to question the developmental compatibility and possible immune rejection of esc derivatives in the adult host [24]. Syngeneic hematopoietic stem cells (hscs) from the bone marrow (bm) or umbilical cord blood have been used therapeutically to treat blood diseases and allogeneic bm transplantation has been used to induce tolerance to other nonhematopoietic tissues . Embryonic stem cell - derived hematopoietic progenitors (eshps), as well as a variety of terminally differentiated hematopoietic cells, can be cultured in vitro [6, 7], and eshps express markers commonly found on natural adult and embryonic hematopoietic stem and progenitor cell populations [8, 9]. However, eshps often fail to engraft at high levels after transplantation in vivo, even in immunodeficient mouse models [8, 1012]. Investigation of the adult host immune response to escs and their derivatives has resulted in some controversy . Several groups have described eshps as immune - privileged [13, 14], while others have described their ability to induce responses by t cells and natural killer (nk) cells [3, 12, 1517]. Macrophages have been observed to respond and phagocytose cells of the embryonic inner cell mass, escs, and cultured esc derivatives [1821]. We previously showed that macrophages from the 129 and balb / c mouse strains preferentially phagocytosed eshps in vitro . Here, we extend those findings and present evidence which supports that host macrophages are an innate immune barrier to eshp engraftment in vivo . D3 esc lines (derived from 129 mice, h-2) were purchased from atcc (manassas, va, usa). Escs were maintained in an undifferentiated state on mitomycin - c treated sto cells (atcc) in dmem supplemented with 15% fbs (atlanta biologicals, norcross, ga), 0.15 mm monothioglycerol (sigma - aldrich, st . Louis, mo), 1x penicillin - streptomycin (pen / strep) (invitrogen, san diego, ca), and 1000 u / ml leukemia inhibitory factor (lif). Escs were passaged every two to three days by trypsinization (0.25% trypsin - edta (invitrogen)). Prior to differentiation, escs were transferred to 0.1% gelatin - coated plates to wean them from the feeder layer in imdm (invitrogen) media supplemented as described above . Cells were incubated in a humidified incubator with 5% co2 at 37c [21, 22]. Eshps were differentiated from escs using coculture on op9 stromal cell monolayers (atcc), as published . Briefly, op9 cells were cultured in alpha - mem media (invitrogen) supplemented with 20% fbs and 1x pen / strep . One hundred thirty thousand escs were plated on op9 monolayers at 80% confluency in 150 mm tissue culture dishes in the presence of 5 ng / ml flt3l and il-7 (peprotech, rocky hill, nh) in 20 ml of media . At days 4 and 11, 10 ml of media was added with 10 ng / ml flt3l and il-7 . At day 7, all cells were harvested using cell lifters, filtered through 64 m nylon mesh (small parts, inc ., miami lakes, fl), and replated onto fresh monolayers in the presence of 5 ng / ml flt3l and il-7 . Eshps were harvested and digested in medium 199 (invitrogen) containing 0.125% w / v collagenase d and 0.1% v / v dnase i (both from roche, south san francisco, ca), for 60 minutes at 37c, followed by dissociation by vigorous pipetting . M199 + media containing 2% fbs in medium 199 and centrifuged at 2000 rpm for 10 minutes . The bm from tibiae and femora from adult mice were also collected as described as a source of adult hematopoietic progenitors . To obtain lineage - negative (lin) cells from the adult bm, cells were stained with a biotinylated anti - lineage (lin) cocktail (anti - cd3, cd4, cd8, cd11b, cd19, nk1.1, gr1, and ter119), and lin cells were positively selected from the whole bm using the easysep biotin positive selection kit (stem cell technologies, vancouver, canada) and two rounds of magnetic selection per the manufacturer's instructors . Lin bm cells were used in phagocytosis assays and transplantation assays . For sorting eshps, cultured cells were harvested and then blocked with 2.4g2 (anti - cd16/cd32) hybridoma supernatant to block fc receptors . Then, one of two strategies was used for preenrichment and sorting of eshps . In the first strategy, all harvested cells were stained with biotinylated anti - lineage (lin) cocktail (anti - cd3, cd4, cd8, cd11b, cd19, nk1.1, gr1, and ter119), pe - anti - cd41 (clone mwreg30, biolegend), and apc - anti - cd45 (clone 30f11, biolegend) for 30 minutes and then washed . In a second incubation, the cells were then stained with streptavidin - pacific blue (invitrogen) to develop the anti - lineage cocktail and dapi (as a viability marker). Sorting was then performed to obtain cd41 cd45 and cd41 cd45 cells together and/or cd41 cd45 cells on a bd facs aria ii or aria iii flow cytometer in two steps . First, sorting was performed on yield mode, which allowed for rapid enrichment of the target cells, and then the enriched cells were resorted in purity, cd41 progenitors were preenriched by staining with cd41-biotin and easysep - streptavidin coated magnetic beads (stem cell technologies, inc ., three rounds of positive selection on the magnet were performed to collect cd41 cells per the manufacturer's instructions, and the final fraction was then costained with streptavidin - pe, apc - anti - cd45, and dapi as a viability marker . These cells were then sorted on the flow cytometer using purity mode . Sorted progenitors with either strategy were 8595% pure after sorting (supplemental figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/2414906). The uc merced institutional animal care and use committee approved all animal procedures . Mice of the 129 (h-2, cd45.2, stock #002448) and nsg (h-2, cd45.1, stock #005557) strains were purchased from the jackson laboratory (west sacramento, ca). Mice were housed in specific pathogen - free conditions with autoclaved food and sterile water . Mice were given 200 rads of irradiation (a sublethal dose in nsg mice) using a cs - source (jl shepherd and associates, san fernando, ca). After irradiation, mice received 2 mg / ml neomycin sulfate in their drinking water for 2 weeks (sigma). Control mice were transplanted with 5 10 lin bm progenitors or 510 10 whole adult bm cells . Recipients were analyzed at beginning at day 17 after transplantation to assess chimerism in the bm, spleen, and thymus by flow cytometry . Cells were stained at 4c for 30 minutes in 2.4g2 (anti - cd16/cd32) hybridoma supernatant to block fc receptors . Cells were then stained with specific antibody cocktails for 30 minutes in a total volume of 100 l in facs buffer, using antibodies to the lineage markers cd3 (clone 2c11), cd4 (gk1.5), cd8 (2.43), b220 (ra3 - 6b2), igm (rmm-1), gr-1 (rb6 - 8c5), cd11b (m1/70), and f4/80 (bm8). Cells were further costained with cd45.1 (a20) and cd45.2 (104) to mark host and donor hematopoietic cells, respectively . Cells were analyzed by gating on live, singlet cell populations on bd facs aria ii or aria iii flow cytometers and data were analyzed using flowjo software . For histological analysis, half of the spleens from eshp and bm recipients were frozen in tissue - tek optimal cutting temperature compound (sakura finetek, inc ., seven m thick sections were cut using a cryostat, flash fixed in acetone for 1015 seconds, and followed by fixation in 4% paraformaldehyde for 10 minutes . F4/80 or rat igg2a isotype control antibodies at 1: 50 dilution (biolegend) were used to stain the tissue section . Macrophages were enriched by adherence to plastic tissue culture dishes overnight in a humidified incubator at 37c with 5% co2 . Adherent cells were removed by trypsinization for 5 minutes at 37c followed by mechanical lifting using a cell lifter . Fifty thousand macrophages were plated per well with 1 10 cd41 eshps or lin bm target cells labeled with cfse (molecular probes). To label target cells, eshps or lin bm cells were washed twice with 1x pbs prewarmed to 37c and then resuspended at 1 10/ml in pbs . One l / ml of 5 mm cfse was added to the cells, which were then incubated at 37c for 10 minutes, and then washed twice with m199 + . Phagocytosis assay cultures were then harvested and stained with anti - f4/80 apc (biolegend) and dapi . Nsg mice were depleted of macrophages by treatment with clodronate - loaded liposomes (cll), whereas control mice were treated with phosphate - buffered saline - loaded liposomes (pll), obtained through clodlip bv (http://clodronateliposomes.com/). At day 3, mice were treated with 0.04 mg of liposomes per gram of mouse weight, and on days 1, + 5, + 10, and + 15 mice were treated with 0.02 mg per gram of mouse weight via intraperitoneal injection, with day 0 representing the day that mice received irradiation and hematopoietic transplant . Spleens and bm cells were analyzed by flow cytometry after animals were sacrificed . To determine the group sizes for transplantation, we utilized data from previous studies in which eshps were derived in vitro and transplanted into similar genetic backgrounds to the nsg mice [8, 11] to estimate the average expected engraftment success rate in vivo and the minimum number of animals per group required to achieve meaningful and statistically sound results . Two - tailed unpaired t - tests were performed to test differences in the means between groups using graph pad prism (san diego, ca). Transplantation of in vitro - derived eshps into adult mouse hosts has not led to high levels of donor chimerism or long - term engraftment without transgenesis [8, 12]. Our previous work suggested that immune responses to eshps might be partly responsible for their poor engraftment . To test this further, we utilized mice on the nsg background which lack t, b, and nk lymphocytes but still develop myeloid lineage cells, such as granulocytes, dendritic cells, and, importantly, macrophages . Previous studies have shown the definitive mouse hematopoietic progenitors in the embryo express cd41 and transition through cd41 cd45, then cd41 cd45, and cd41 cd45 stages of maturation [25, 26], and we previously showed that eshps with these phenotypes could be generated using a coculture system on the op9 bone marrow stromal cell line . Eshps were sorted based on cd41 (least mature) or cd45 (most mature) expression after 16 days of in vitro differentiation, as shown in figure 1(a), in order to compare their levels of engraftment in vivo . One hundred thousand to 5 10 purified eshps were injected into sublethally irradiated nsg hosts . Since 129 and nsg mice differ at the cd45 locus, cd45.1 (expressed by the nsg host strain) and cd45.2 (expressed by 129 donor strain) specific antibodies were used to determine relative levels of donor engraftment (figure 1(b)). The frequency of host cd45.2 cells was low in eshp nsg recipients, as compared to 129 bm nsg recipients . Controls to distinguish nonspecific background staining of anti - cd45.1 versus anti - cd45.2 antibodies were performed using tissues from untransplanted nsg mice (which do not express cd45.2) and untransplanted 129 mice . These results showed that the cd45.2 signal observed in eshp nsg recipients was clearly distinguishable from that in untransplanted nsg controls . Donor hematopoietic chimerism averaged 5% or less in the spleen and bone marrow in eshp recipients, which was low compared to whole adult bmt controls, which averaged about 90% (figure 1(c)). No significant difference in the level of donor chimerism in recipients of cd41 eshps and cd41 cd45 eshps was observed . The low level of donor chimerism in eshp recipients is consistent with the results from other groups [8, 10]. Eshps were capable of multilineage differentiation, as shown by myeloid differentiation in vitro and lymphoid and myeloid differentiation in the bone marrow and spleen in vivo (supplemental figure 1). Donor chimerism was also evident in the thymus (figure 1(b)), with signs of t cell development into cd4 cd8 and cd4 cd8 thymocytes in 50% of eshp recipients (supplemental figure 2). Donor chimerism in eshp recipients was not observed in any tissues after 34 days after transplant . Based on our previous findings, we tested the hypothesis that eshps were actively rejected by the host innate immune cells . Enlarged spleens in eshp recipients were consistently observed compared to both untransplanted nsg and adult whole bmt controls (figure 2(a)). To quantify this observation, the spleens in adult bm - transplanted controls displayed a 2.62-fold increase in mean weight compared to untransplanted nsg mice (figure 2(b)), consistent with their increased donor hematopoietic chimerism (figure 1(c)). Similarly, the mean spleen weights in eshp recipients were increased 3.71-fold compared to untransplanted nsg controls (figure 2(b)). Although some donor eshp - derived cells were observed in the spleen (figure 2(c)), a higher absolute number of host - derived cells in eshp recipients were present (figure 2(d)). This number of host - derived cells in the spleen was significantly higher than that of adult bm recipients (figure 2(d)). Since nsg mice lack nk, t, and b lymphocytes, we reasoned that only host myeloid cell populations (which include cd11b, gr-1, and f4/80 macrophages could be increased in the eshp recipients). Indeed, a significant increase in host - derived cd11b or gr-1 cells was observed between eshp recipients versus adult bmt controls (figures 3(a) and 3(b)); but the numbers of cd11b and gr-1 cells did not account for the observed enlargement of spleen size (figures 2(a), 3(a), and 3(b)). Remarkably and in contrast, eshp recipients displayed a statistically significant increase in host f4/80 macrophages compared to both untransplanted nsg and bmt controls (figure 3(c)), and f4/80 cells were significantly increased in both percentage and absolute number in eshp nsg mice compared to 129 bm nsg (supplemental figure 3). The prevalence of f4/80 macrophages was also visible by immunohistochemical staining (figure 3(d)). We hypothesized that eshps were actively phagocytosed by host f4/80 macrophages, but the low levels of donor - derived cells in eshp recipients precluded our ability to test this hypothesis directly in vivo . Instead, we used an in vitro phagocytosis assay developed in our laboratory, in which phagocytosis of labeled targets can be quantified by flow cytometry . Macrophages from the 129 mouse strain, which is syngeneic to the eshps, and macrophages from nsg mice, which are allogeneic to the eshps, were used . Regardless of their source, macrophages phagocytosed eshp targets at a higher rate than control adult lin bm targets isolated from the respective strains (11.81- and 24.09-fold higher by syngeneic 129 and allogeneic nsg macrophages, resp . In addition, nsg macrophages were 1.65-fold more efficient in eshp phagocytosis than 129 macrophages (figure 4), suggesting that allogeneic macrophages may react more robustly toward eshps than their syngeneic counterparts . These in vitro data corroborate the increase in host f4/80 splenic macrophages in the spleens and the low donor hematopoietic chimerism in eshp recipients observed in vivo . Furthermore, flow cytometric measures of forward scatter properties of f4/80 macrophages indicated that host f4/80 macrophages in eshp nsg mice were larger in size than f4/80 macrophages in 129 bm nsg controls, consistent with phagocytosis of eshps (supplementary figure 4). The increased phagocytosis of eshps by allogeneic nsg macrophages provided further support to our hypothesis that macrophages were indeed responsible for poor engraftment of eshps after transplantation in vivo . To directly test if macrophages were a barrier to eshp engraftment in vivo, clodronate - loaded liposomes (cll) [24, 28] were used to deplete nsg mice of macrophages prior to and on days 5, 10, and 15 after transplant . Cll treatment specifically depleted f4/80 macrophages subsets in the spleens and bm (supplemental figure 5(a)). Donor hematopoietic chimerism was 6.4-fold higher in the spleens of eshp recipient mice treated with cll compared to control eshp - transplanted mice treated with pbs - loaded liposomes (pll + eshp) (p = 0.0020, figures 5(a) and 5(b) and supplementary figure 5(d)), even though the spleen size of cll + eshp - treated mice was smaller (figure 5(c) and supplementary figure 5(c)). In contrast, in the bm, no significant differences in donor chimerism were observed between pll + eshp and cll + eshp groups (figure 5(b) and supplementary figure 5(e)). Cll treatment did not affect the high level of donor engraftment attained in mice transplanted with whole adult bm cells or enriched lin bm progenitors (supplemental figure 5(b)). Our previous work demonstrated indirect recognition of eshps by macrophages could stimulate t cell proliferation in vitro . In this report, we extend this work to demonstrate that depletion of host macrophages can improve the tissue - specific engraftment of eshps in vivo . Taken together, we conclude that f4/80 macrophages are a specific immune barrier for eshps after transplantation . A working model that summarizes our results we observed a specific increase in host f4/80 cd11b macrophages in eshp recipients and have strong evidence that these macrophages phagocytose eshps in vitro . Although it is possible that local inflammation in the host could result in the macrophage increases in vivo, we have not observed any evidence of contamination in our eshp cultures or pathogenic infection in our eshp or control transplanted mice . F4/80cd11b red pulp macrophages have roles in both filtering the blood and removing damaged erythrocytes [29, 30], so we favor the possibility that host red pulp macrophages are induced to expand specifically in response to eshps in vivo . We hypothesize that macrophage recruitment and phagocytosis of eshps may be related to three aspects of their maturation state: (1) eshps secrete products that recruit monocytes and macrophages or induce differentiation or proliferation of macrophages, (2) eshps express activating ligands [31, 32], and/or (3) eshps lack macrophage inhibitory ligands [3337]. With regard to the latter point, major histocompatibility complex class i (mhc - i) has been characterized as a macrophage inhibitory ligand [29, 38], and eshps express mhc - i at moderate levels compared to adult hematopoietic cells . In addition, in preliminary studies, we observed that eshps express minimal levels of the macrophage inhibitory ligands cd47 and cd200 (supplemental figure 6). Whether induced expression of macrophage inhibitory ligands can improve eshp engraftment, and whether the host macrophage response is directed to a particular cell subtype within eshps, will require further experimentation . The observation of higher eshp numbers in the host spleen versus the bm naturally leads to the question of why this is the case . One explanation could be that the spleen is the natural niche for eshps . In support of this idea, extramedullary hematopoiesis is common in the red pulp region of the spleen in fetal and neonatal mice and decreases in adults as hematopoiesis moves to the bm . Therefore, we speculate that poor engraftment of eshps in vivo may be caused by a developmental incompatibility between the adult spleen microenvironment and the eshps . This is supported by recent evidence that embryonic hscs demonstrate a propensity to engraft better in neonatal recipients than adult recipients and also populate the niches that best match their developmental stage . Another possibility is that eshps may harbor defects in homing that prevents their sufficient migration to the bm cavity and these homing defects reduce their engraftment [10, 41]. Further experimentation to test different routes of eshp transfer, such as intrafemoral injection [12, 42], is necessary to test this hypothesis . Furthermore, macrophages have been implicated as niche cells that promote retention of hscs in the bm and our data showed that cll treatment increased donor hematopoietic chimerism in the spleen but not in the bm of eshp recipients . If f4/80 host macrophages serve as a bm niche cell population for eshps, we posit that their depletion may prevent engraftment of eshps in the bm . There are some caveats of the clodronate liposome system that should be taken into consideration when interpreting our results . Since there is evidence in the literature of toxicity of clodronate liposomes and the liposomes alone can have some nonspecific effect on macrophages, we empirically determined the appropriate dosage of pll and cll in untransplanted nsg animals to find the optimal dose of liposomes that would deplete the macrophages but not kill the animals . Similar levels of donor chimerism in the eshp only group and the pll + eshp group were expected . However, donor chimerism in the pll + eshp group (figure 5) was observed to be lower than the eshp only groups (figure 1(c)), suggesting nonspecific detrimental effects of the control empty since we cannot rule out this possibility, we are of the opinion that comparison of donor chimerism in pll + eshp versus cll + eshp - treated animals is appropriate and better than comparison of donor chimerism in cll + eshp - treated mice versus mice that received eshp without liposomes . We conclude that albeit low, there is clearly a higher level of donor chimerism in the cll + eshp - treated mice compared to the pll + eshp - treated mice . Furthermore, there is evidence that cll can deplete cell types other than macrophages, including dendritic cells, osteoclasts, and neutrophils . Additional studies to target specific subsets of phagocytes are necessary to ascertain their roles on eshp engraftment . The derivation and transplantation of eshps that resemble adult hscs are an important goal for the field of hematopoietic stem cell biology . Although transplanting eshps using the adult definition of hsc seems straightforward (lineage - negative, ckit, and sca-1), it is well documented in the literature that embryonic and adult hematopoietic progenitors expressed different surface markers in vivo and in vitro . In particular, cd41 is expressed on definitive embryonic hematopoietic progenitors in vivo, before the expression of the classic cd45 hematopoietic cell marker [25, 26]. Subfractionated different eshp populations from their esc cultures and found superior engraftment by cd41 eshps after transplantation in vivo (regardless of ckit or sca-1 expression) and more recently demonstrated that these cells are not definitive hscs by transcriptional analysis . In line with these previous studies, our results do not support the assumption that eshps share the same markers and behavior as their adult hsc counterparts . We have only observed short - term hematopoietic engraftment with multilineage differentiation from eshps transplanted into immunodeficient mice . Eshp engraftment was not expected to reach the same level of bm engraftment, as previous work showed that esc - derived hematopoietic progenitors with a similar surface phenotype to our eshps achieved a wide range of donor chimerism levels in lethally irradiated immunodeficient hosts, but reconstitution was not achieved in 100% of the animals . Since it is technically difficult to obtain sufficient eshps in culture for in vivo transplantation and there was a high probability of mouse deaths due to lack of reconstitution, we opted to use sublethal irradiation in our studies and optimized the cll dosage to reduce nonspecific toxicity in the animals . The observation that mice treated with eshps do not achieve the same levels of donor chimerism as bmt controls demonstrates that eshps may not compete well with host nsg bm progenitors, whereas bm cells from 129 adult donors are able to do so . Treatment with cll increased the level of donor chimerism in mice that received eshps compared to pll - treated mice . Cll + eshp - treated mice also showed decreased spleen weights compared to pll + eshp - treated mice (supplemental figure 3(c)), which could be attributed to the presence of fewer host macrophages in the former, delaying the innate immune rejection of eshps . Pll or cll treatment in bmt recipients did not prevent high levels of engraftment in nsg hosts (data not shown). Taken together, we interpret these data as a demonstration that low engraftment from eshps is, in part, due to an innate immune response by host macrophages . Several groups have reported strong recruitment of macrophages after transplantation of esc or esc derivatives undergoing active rejection [19, 20]. Observed that adult macrophages could destroy the inner cell mass (which contain escs) from early blastocysts but that the trophoblast repelled these macrophages, suggesting a possible biological mechanism to protect the inner cell mass cells from the maternal (host) immune system macrophages during early embryonic development . It is possible that this natural embryonic trophoblast mechanism, which prevents adult macrophages from rejecting embryonic cells, might be leveraged to protect in vitro esc - derived tissues after transplantation in adult hosts . Current treatments for hemophagocytic lymphohistiocytosis and macrophage activation syndromes include broad immunosuppressive therapy [4951] which could accompany any eshp transplantation in humans . Recently, the janus kinase inhibitor ruxolitinib was shown to improve the symptoms of hlh in murine models and this is another possible direction for future human eshp transplants . Strategies to skew polarization of macrophages toward proinflammatory m1 versus regulatory m2 phenotypes are currently being explored for clinical applications (although further studies are required to determine the m1 versus m2 phenotype of the f4/80 macrophages in the eshp nsg chimeras). To our knowledge, a comparison of the expression levels and functional abilities of macrophage inhibitory ligands and in the nsg versus 129 strains has not been performed . However, there is evidence of mouse strain differences in the binding of the ability of the nod, balb / s, and b6 forms of sirp to bind to the human macrophage inhibitor receptor cd47 and prevent phagocytosis of xenogeneic cells in mice . Our observations that eshps are phagocytosed by host macrophages in vitro and that clodronate treatment promotes higher donor chimerism from eshps in mice in vivo strongly suggest that eshps stimulate innate immune responses and that control of macrophage - induced immune rejection should be considered in the field as new hematopoietic derivatives are produced from escs or esc - like induced pluripotent stem cells for in vivo transplantation.
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Although spinal cord abnormalities in patients with acquired immunodeficiency syndrome (aids) have been infrequently reported in the literature, myelitis is a known complication of aids and is occasionally the initial complaint . The incidence of myelopathy may be as high as 20%, with 50% of the cases reported post - mortem [3, 5]. Toxoplasmosis is the most common cause of intracranial lesions responsible for neurological deficits in aids patients, occurring in 310% of patients in the united states and in up to 50% of aids patients in europe, latin america, and africa [6, 10]. A review of existing literature suggests that although toxoplasmic myelitis is uncommon, it should be suspected in immunocompromised patients who present with symptoms of acute or sub - acute myelopathy . The initial evaluation should aid in differentiating between other reported causes of myelopathy (such as vacuolar myelopathy, lymphoma, tuberculosis, and viral infections including cytomegalovirus infection, herpes zoster, and herpes simplex) in aids patients [2, 4]. Since 1986, 18 cases of apparent toxoplasmosis of the spinal cord have been described [4, 7, 11, 12]. A case report and pertinent literature were reviewed, leading to the diagnosis and management options discussed below . A 40-year - old hispanic man was admitted to the hospital after being found unconscious . He had a 2-day history of disorientation that manifested itself as his being unable to recognize family members . Upon admission he regained consciousness, becoming alert and oriented, but developed urinary retention and was unable to move or feel his lower extremities . He had no history of systemic illness, but mentioned having been treated for herpes zoster approximately 3 years prior . He denied having a history of fever, chills, nausea, vomiting, rash, seizures, or other constitutional symptoms . He had not traveled recently, and his hiv status was unknown at the time of admission . Physical examination revealed fever, nuchal rigidity, and a distended bladder that required catheterization . A neurological examination confirmed that he had intact cranial nerves and normal upper extremity strength . Both lower extremities exhibited flaccid paralysis and reduced response to pain, touch, and temperature (bilaterally from l1). Cerebellar examination was normal in the upper extremities, with an adequate finger - to - nose exam . The patient s wbc, hemoglobin, platelets, electrolytes, bun, and creatinine were within normal ranges on admission . The initial brain ct (without contrast) revealed a focal area of vasogenic edema in the left frontal lobe (without significant mass effect) and abnormal white matter hypo - density seen along the lateral aspect of the right frontal horn of the internal capsule . A brain mri (with and without gadolinium enhancement) revealed multiple bilateral ring - enhancing intra - axial brain lesions, and an ill - defined cortical and sub - cortical enhancement, with diffuse edema in the frontal lobe . Spinal mri (with and without gadolinium) showed the spinal cord to be abnormally diffuse, with swelling and edema in the cervicothoracic region . Along with the diffuse abnormal hyperintense swelling, the cauda equina had an edematous appearance, and signal intensity was abnormally increased, which is compatible with transverse myelitis (figs . 1 and 2). Finally, imaging also revealed a focal area of abnormal signal intensity in the ventral inferior pons . 1initial mri: notice the abnormal spinal cord swelling cervical and at cauda equinafig . 2normal f / u study initial mri: notice the abnormal spinal cord swelling cervical and at cauda equina he was initially given broad spectrum antibiotics (ceftriaxone, vancomycin, and ampicillin) and acyclovir at admission for a presumptive cns infection . Hiv serology was recommended, though the patient denied ever having engaged in high - risk behavior or having had a blood transfusion at any time in his past . Analysis of csf showed abnormal values including a wbc count of 11/mm and a protein level of 184.5 mg / dl, with a glucose concentration of 51 mg / dl . Stain and cultures for bacteria, fungi, and acid - fast bacilli were negative . A csf cryptococcal antigen titer and htlv i an anti - toxoplasma immunoglobulin (igg) immune titer was positive at 8.99 (<0.9 = negative; 1.10 = positive). At this point, the patient disclosed having engaged in homosexual behavior and accepted hiv testing, which was reactive by elisa . Empiric treatment for toxoplasmosis with sulfadiazine, pyrimethamine, folinic acid, and haart therapy with lamivudine / zidovudine and indinavir were started . Intravenous dexamethasone was administered for 7 days, and oral methylprednisolone for another week . Biopsy was deferred due to possible complications and the evidence of improvement with therapy . Within 4 days of empiric treatment for toxoplasmosis, the patient s neck rigidity resolved . He progressively developed discrimination to touch and his vibratory sensation improved, as did his bilateral strength (1/5) of the lower extremities . Follow - up consisted of a brain and spinal cord mri (with and without gadolinium), 14 days after treatment with anti - toxoplasmosis therapy combined with steroids, which demonstrated resolved swelling and residual t2 hyper - intensity in the mid - thoracic spinal cord, without an interval change in the brain mri . Aids - related spinal cord disorders include neoplasms, infections (including hiv itself), vascular disease, and other undefined etiologies . Toxoplasmosis and lymphoma are the two most common intracranial lesions, and both have been reported in increasing frequency in the spinal cord . Myelopathy is usually under - diagnosed, probably because of the occurrence of coexisting conditions such as aids dementia complex (adc), cerebral lesions of varying etiologies, vacuolar myelopathy, lumbosacral myelopathy, or peripheral neuropathy that may mask the clinical signs suggestive of myelopathy [5, 9, 10]. Other causes of myelopathy appear to be less common than toxoplasmosis and include tuberculoma, cytomegalovirus, varicella - zoster virus, and, possibly, lymphoma [4, 10]. Postmortem histopathology examination has resulted in a definitive diagnosis of myelopathy in the majority of patients . Spinal cord lesions often manifest with a variety of symptoms, such as leg weakness, progressive paraparesis with spasticity, absent reflexes, ataxia, incontinence, and paresthesias . In the 14 toxoplasmosis cases with spinal cord involvement reviewed by vyas and ebright, they found the most common presentations of acute or sub - acute symptoms were paraparesis, urine retention, sensory level deficits, fever, and local pain . Csf cytology showed increased protein - level elevation and a cd4 count of less than 50/mm . In the patients evaluated by spinal cord mri imaging, localized intramedullary lesions or spinal cord edema were found in more than 90% of cases associated with positive t. gondii igg antibody . No cases of associated transverse myelitis, as in this case, were described . In our case, evaluation in a symptomatic patient should include serum and csf cytology and antibody (immunological) studies as they remain the gold standard for identifying an infectious agent . Complete radiographic imaging of the entire neuroaxis [3, 15] is key in clearly defining inflammatory lesions of the brain and spine, and in the visualization of typical lesions that allow for rapid diagnosis . This helps determine whether the lesion is extramedullary or an intrinsic spinal cord disease, which in turn provides information to determine whether surgical or medical therapy is needed . In opportunistic diseases, the imaging studies also have a crucial role in diagnosis and monitoring of the therapeutic response . Neuroimaging findings, along with other clues, help to narrow the differential diagnosis . On an mri, findings of a normal - sized spinal cord but with an abnormal signal should signal the possibility of vacuolar myelopathy and adc - related hiv myelitis . Lesions of viral myelitis can potentially cause spinal cord enlargement; however, laboratory data are required for confirmation . If spinal cord enlargement is present, toxoplasma myelitis and lymphoma should be strongly considered . Lymphoma has been associated with positive csf polymerase chain reaction (pcr) for epstein - barr virus . If both spinal cord enlargement and an abnormal signal are associated with meningeal enhancement, then cmv, m. tuberculosis, lymphoma, and toxoplasmosis should be considered along with other less likely infectious causes . Mri (plain and contrast - enhanced) is currently considered appropriately sensitive for detecting brain and spinal cord lesions; however, equal sensitivity has been reported using delayed - contrast ct scanning . The current guidelines for diagnosis of intracranial lesions state that t1 spect is an option when available . When positive (marked uptake in contrast to toxoplasma), it appears to be highly specific for primary cns lymphoma [6, 8]. These guidelines have been described as helpful in spinal cord lesions and suspected lymphoma, although specific guidelines for spinal cord lesions are not presently available . The newer and more available mri techniques of diffusion - weighted imaging (dwi) may be of help in the differentiation of lymphoma, showing no restriction in water diffusion . If a diagnosis of solitary or atypical lesions cannot be made with noninvasive methods, a biopsy (open or stereotactic) may be warranted in patients with concomitant negative toxoplasmosis serology since a negative serology does not exclude diagnosis of toxoplasmosis nor differentiate from lymphoma . A biopsy may also be considered necessary if a patient experiences a rapid decline in function or, alternately, fails to improve despite therapy . Brain biopsies have been associated with hemorrhage risk and an increased mortality (2%) and morbidity rate (12%) in patients with hiv / aids . Empiric treatment for toxoplasmosis with oral pyrimethamine and sulfadiazine (with folinic acid) has been recommended in all cases of intracranial mass lesions in patients with hiv / aids (except in a solitary mass with negative toxoplasma serology). Steroids promote radiological improvement in about 80% of patients, and improvement can be seen in about 1 week, supporting the diagnosis . Patients are usually monitored clinically and radiographically for response to treatment over a 10- to 14-day period following empiric therapy . If responsive, anti - toxoplasmosis therapy is continued indefinitely, and reevaluation in the absence of steroid treatment is mandated . If the lesions remain unchanged or progress, the diagnosis has to be reconsidered and the therapeutic strategy reevaluated . In cases of atypical large solitary toxoplasma lesions resembling lymphoma (that also present with spinal cord enlargement), additional diagnostic modalities should be performed [3, 6]. Due to the immunosuppressant effects of steroids, the optimal dosage and period of treatment for spinal cord lesions must be addressed in future studies . In this case, the brain and spinal cord lesions were found in an hiv - positive man who had not previously been diagnosed . For this reason, other causes of cns infection and lymphoma were first considered . After the positive serology, empiric treatment for cerebral toxoplasmosis was initiated . The patient s clinical and radiographic improvement led to the final diagnosis of toxoplasmic myelitis and encephalitis, similar to other cases documented in the literature by vyas et al . In 1996 . Furthermore, toxoplasmic myelopathy has been described in patients without hiv, albeit with severe immunosuppression [2, 3, 11, 12], and in patients with isolated spinal cord lesions [1, 2, 11]. New neurological deficit in any patient should raise a high index of suspicion of hiv infection . Appropriate diagnostic methods and management that are practical for all settings, including those with limited technologies, should be sought . Newer methods of diagnosis and management for all neurological complications of hiv should be addressed . We established ours based on the clinical manifestations, history, and general improvement of the patient's condition . Although spinal cord toxoplasmosis is uncommon, it has been suggested that most patients with aids that present with evolving myelopathy, characterized by extremity weakness, sensory involvement, spinal cord enlargement, and enhancing lesions in brain or spinal cord ct or mri, have toxoplasmic myelitis [3, 5]. The likelihood of diagnosis increases if serum titers for toxoplasma antibodies are positive or where the initiation of early treatment with empiric anti - toxoplasmosis therapy and steroids improves both the patient s clinical and radiographic manifestations.
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Recently, the world health organization indicated that drug - resistant a. baumannii is defined as the first priority pathogen, in which researches and developments for new antibiotics are urgently needed . The bacteria has been revealed to persist on dry surfaces for a month and presented several drug - resistant mechanisms including drug efflux pumps, drug - inactivating enzymes, and drug target mutations . Infected patients have many serious diseases including septicemia, pneumonia, and urinary tract infections . The number of global drug - resistant a. baumannii was vary in estimation; therefore, the high prevalence accounted to be approximately 54% and 77% of a. baumannii isolates have been revealed in italy and india, respectively . In thailand, baumannii collected from clinical specimens was approximately 59% . Regarding the limit of antibiotic treatment, several studies revealed the effectiveness of extracted herbs on drug - resistant pathogens including methicillin - resistant staphylococcus aureus, vancomycin - resistant enterococci, and mdr - a . Herein, 10 volatile oils extracted from various medicinal plants were determined for their inhibitory effect on the growth of the most common human pathogens and mdr - a . S. aureus atcc 25923, escherichia coli atcc 25922, pseudomonas aeruginosa atcc 27853, and a. baumannii atcc 19606 were purchased from the department of medical sciences, ministry of public health, thailand . Baumannii were collected from the diagnostic laboratory, maharaj nakorn chiang mai hospital, chiang mai, thailand, during february - april, 2012 . Both biochemical tests followed by constantiniu et al . And molecular biology test using amplified ribosomal dna - restriction enzyme analysis were performed for identification of a. baumannii . Primers used for 16s rdna gene amplification were designed as followed by the previous report . The antimicrobial susceptibility testing was performed using disk diffusion method following the clinical and laboratory standards institute (clsi) guidelines . The mdr was defined according to the unsusceptible of at least one in three agents of antimicrobial classes . All 30 clinical isolates resisted to eight antibiotics in six antimicrobial classes consisting of amikacin, piperacillin / tazobactam, ciprofloxacin, cefoperazone / sulbactam, ceftazidime, trimethoprim / sulfamethoxazole, imipenem, and meropenem . Galangal, ginger, plai, lime, kaffir lime, sweet basil, tree basil, lemongrass, clove, and cinnamon were selected in this study [table 1]. The material was subjected to hydrodistillation using a clevenger - type glass apparatus for 3 - 5 h . Yields of the volatile oils obtained from the plants were calculated as the percent yield . Medicinal plants used in this study the antimicrobial activity testing was modified from prabuseenivasan et al . . 0.5 (1 10 cfu / ml) and spread over the mueller - hinton agar (mha) plates using a sterile cotton swab . Each volatile oil was dissolved in 10% aqueous dimethyl sulfoxide (dmso) with 0.5% v / v tween 80 and sterilized by filtration . Sterilized disks (whatman no . 5, 6 mm diameter) were impregnated with 20 l of volatile oils and placed on the surface of mha . The volatile dissolving buffer (10% aqueous dmso, 0.5% v / v tween 80) and tea tree oil were used as negative and positive control, respectively . After incubation at 37c for 16 - 18 h, the inhibition zone was measured . All experiments were performed independently in triplicate and mean value was calculated . Based on clsi guidelines, the preparation of water - insoluble volatile oils was slightly modified from the recommended clsi guidelines . Each volatile oil was dissolved with 50% dmso and serial 2-fold diluted in a 96-well microtiter plate ranging from 0.125 to 8 mg / ml . The bacterial suspension was diluted into approximately 1 10 cfu / ml, and 100 l of bacterial suspension was applied to each well . The inoculum with 2.5% dmso and media without inoculum were used as cell and media control, respectively . The microplates were incubated at 35c for 20 h. due to the turbidity of volatile oil suspensions, iodonitrotetrazolium chloride (int) (biochemica) was used as color indicator to visualize the bacterial growth . The mic was detected after added 50 l of 0.2 mg / ml int and further incubated at 35c for 30 min . To determine the mbc, 10 l of bacterial inoculums were taken aseptically from the wells with no color change and plated onto mha plate and incubated at 35c for 20 - 24 h. all experiments were separately performed in triplicate and calculated as mode, median, and 90 percentile . Median mic value (mic50) represented the mic value of one - half of the tested population . The 90 percentile (mic90) represented the mic value of 90% of the tested population . Likewise, mbc50 and mbc90 were the mbc values at which 50% or 90% of isolates in a tested population were killed, respectively . In this study, the inhibition zone of each volatile oil was compared with tea tree oil and statistically analyzed using independent student s t - test (spss version 22). The mic and mbc values in each of the tested volatile oils and tea tree oil were statistically analyzed by mann s. aureus atcc 25923, escherichia coli atcc 25922, pseudomonas aeruginosa atcc 27853, and a. baumannii atcc 19606 were purchased from the department of medical sciences, ministry of public health, thailand . Baumannii were collected from the diagnostic laboratory, maharaj nakorn chiang mai hospital, chiang mai, thailand, during february - april, 2012 . Both biochemical tests followed by constantiniu et al . And molecular biology test using amplified ribosomal dna - restriction enzyme analysis were performed for identification of a. baumannii . Primers used for 16s rdna gene amplification were designed as followed by the previous report . The antimicrobial susceptibility testing was performed using disk diffusion method following the clinical and laboratory standards institute (clsi) guidelines . The mdr was defined according to the unsusceptible of at least one in three agents of antimicrobial classes . All 30 clinical isolates resisted to eight antibiotics in six antimicrobial classes consisting of amikacin, piperacillin / tazobactam, ciprofloxacin, cefoperazone / sulbactam, ceftazidime, trimethoprim / sulfamethoxazole, imipenem, and meropenem . Galangal, ginger, plai, lime, kaffir lime, sweet basil, tree basil, lemongrass, clove, and cinnamon were selected in this study [table 1]. The material was subjected to hydrodistillation using a clevenger - type glass apparatus for 3 - 5 h . Yields of the volatile oils obtained from the plants were calculated as the percent yield . 0.5 (1 10 cfu / ml) and spread over the mueller - hinton agar (mha) plates using a sterile cotton swab . Each volatile oil was dissolved in 10% aqueous dimethyl sulfoxide (dmso) with 0.5% v / v tween 80 and sterilized by filtration . Sterilized disks (whatman no . 5, 6 mm diameter) were impregnated with 20 l of volatile oils and placed on the surface of mha . The volatile dissolving buffer (10% aqueous dmso, 0.5% v / v tween 80) and tea tree oil were used as negative and positive control, respectively . After incubation at 37c for 16 - 18 h, the inhibition zone was measured . Based on clsi guidelines, mics were determined by using broth microdilution method . The preparation of water - insoluble volatile oils was slightly modified from the recommended clsi guidelines . Each volatile oil was dissolved with 50% dmso and serial 2-fold diluted in a 96-well microtiter plate ranging from 0.125 to 8 mg / ml . The bacterial suspension was diluted into approximately 1 10 cfu / ml, and 100 l of bacterial suspension was applied to each well . The inoculum with 2.5% dmso and media without inoculum were used as cell and media control, respectively . The microplates were incubated at 35c for 20 h. due to the turbidity of volatile oil suspensions, iodonitrotetrazolium chloride (int) (biochemica) was used as color indicator to visualize the bacterial growth . The mic was detected after added 50 l of 0.2 mg / ml int and further incubated at 35c for 30 min . To determine the mbc, 10 l of bacterial inoculums were taken aseptically from the wells with no color change and plated onto mha plate and incubated at 35c for 20 - 24 h. all experiments were separately performed in triplicate and calculated as mode, median, and 90 percentile . Median mic value (mic50) represented the mic value of one - half of the tested population . The 90 percentile (mic90) represented the mic value of 90% of the tested population . Likewise, mbc50 and mbc90 were the mbc values at which 50% or 90% of isolates in a tested population were killed, respectively . In this study, the inhibition zone of each volatile oil was compared with tea tree oil and statistically analyzed using independent student s t - test (spss version 22). The mic and mbc values in each of the tested volatile oils and tea tree oil were statistically analyzed by mann the yields ranged from 0.1% to 4.3% w / w ginger (0.1), lemongrass (0.2), tree basil (0.2), galangal (0.3), sweet basil (0.3), cinnamon (0.9), lime (1.0), plai (1.1), kaffir lime (2.1), and clove (4.3). A disk diffusion method was performed to preliminarily evaluate the antibacterial activity of the volatile oils against four reference bacterial strains (s. aureus, e. coli, p. aeruginosa, and a. baumannii). Except p. aeruginosa, the difference in the inhibition zones between tea tree oil and each volatile oil was analyzed using independent student s t - test . The results indicated that cinnamon oil exhibited a high potency of antibacterial activity against all bacterial strains tested (p <0.01). Sweet basil and lemon grass were highly active against s. aureus and e. coli; however, these volatile oils showed no significant activity when tested with both non - fermentative gram - negative bacilli, a. baumannii, and p. aeruginosa . The volatile oils of clove, tree basil, lime, and ginger were moderately active against some bacterial strains (p <0.05). The antibacterial activity of plai and kaffir lime was rather inactive compared to tea tree oil . The inhibition zones of various volatile oils against s. aureus, e. coli, p. aeruginosa, and a. baumannii standard strains were shown in figures 1 - 4, respectively . However, the mean of the inhibition zones of the cinnamon and clove oils was significantly higher than tea tree oil (p <0.01). Both standard strains of a. baumannii atcc 19606 and mdr - a . The mic and mbc of the positive control tea tree oil against a. baumannii atcc 19606 were 2 and 4 mg / ml, respectively . Cinnamon oil was highly active, with mic and mbc values of 0.25 mg / ml . The mics and mbcs of the volatile oils tested against a. baumannii atcc 19606 were shown in table 2 . Baumannii activity with mic90 and mbc90 of 2 and 4 mg / ml, respectively . The mean mics of four volatile oils, cinnamon, clove, tree basil, and kaffir lime were significantly lower than the positive control tea tree oil with the mic90 of 0.25, 0.5, 1, and 1 mg / ml, respectively (p <0.05). The mic and mbc of the volatile oils against 30 clinical strains of mdr - a . Inhibition zones of 11 volatile oils against staphylococcus aureus atcc 25923 . * indicated a significant difference at p <0.05, and * * indicated a highly significant difference at p <0.01 inhibition zones of 11 volatile oils against escherichia coli atcc 25922 . * * indicated a highly significant difference at p <0.01 inhibition zones of 11 volatile oils against pseudomonas aeruginosa atcc 27853 . * * indicated a highly significant difference at p <0.01 inhibition zones of 11 volatile oils against acinetobacter baumannii atcc 19606 . * indicated a significant difference at p <0.05, and * * indicated a highly significant difference at p <0.01 average inhibition zone in diameter obtained from various volatile oils against 30 multidrug - resistant - acinetobacter baumannii isolates . * * indicated a highly significant difference at p <0.01 mics and mbcs of volatile oils against standard strain a. baumannii atcc 19606 the mics and mbcs of volatile oils against mdr - a . The most problematic of a. baumannii infections nowadays are the mdr and it becomes a serious issue, in which most antibiotics drug therapy are unable to cure the diseases . Baumannii is urgent; volatile oils are one such compound worth screening . In this study, 10 volatile oils were determined for antibacterial activity against s. aureus, e. coli, p. aeruginosa, a. baumannii, and 30 isolates of mdr - a . The antimicrobial activity of tea tree oil against aerobic bacteria has previously been published; the compounds involved in its antibacterial activity such as terpinen-4-ol, a - terpinene and g - terpinene have been characterized . Similarly to carson and riley s study, an inactive effect of tea tree oil against p. aeruginosa was observed in this study . Baumannii isolates could be inhibited by tea tree oil with mic90 and mbc90 concentrations of 2 and 4 mg / ml, respectively . The extracted volatile oils were preliminarily screened for antibacterial activity by disc diffusion method . Among the medicinal plants tested, cinnamon oil exerted the highest activity to inhibit the growth of all bacteria while sweet basil and lemon grass strongly inhibited in some bacteria . Standard broth microdilution method was performed and revealed that the volatile oils of cinnamon, clove, tree basil, and kaffir lime showed strong antibacterial activity against mdr - a . The antimicrobial activity of cinnamon oil against s. aureus, e. coli, acinetobacter lwoffii, and p. aeruginosa has previously been demonstrated . Recently, rath and padhy indicated that the mic and mbc of methanolic extract of both clove and cinnamon against mdr - a . The inhibition zones of tree basil and tea tree oil were indifferent; the major constituents of tree basil volatile oil have previously been identified including thymol, g - terpinene, eugenol, and r - cymene . The mode of antibacterial action of thymol still unknown but it has been proposed to involve in outer and inner membrane disruption . Cinnamon oil possessed the highest inhibition effect against all bacterial strains and mdr - a . Mass spectrometry analysis was performed in this study to identify the active ingredients with antimicrobial activity . Thirteen peaks were observed and interpreted based on specific retention time compared to a reference database . The major ingredients in cinnamon oil were cinnamaldehyde (75.89%), trans - cinnamyl acetate (7.07%), hydrocinnamaldehyde (2.39%), and 1,8-cineole (2.17%) (data not shown). Cinnamaldehyde has previously been reported to inhibit in both gram - positive and gram - negative bacteria . Noteworthy, aldehyde groups might be associated with the antimicrobial activity of cinnamon oil since these chemicals have an ability to covalently cross - link with the amine groups of dna and proteins and interfere their functions in the cells . Although the mode of action of cinnamaldehyde is inconclusive, gill and holley demonstrated that cinnamaldehyde at a concentration of 30 mm could kill l. monocytogenese through its effect on the energy generation and membrane permeability of the bacteria . In addition, the interaction of cinnamaldehyde with essential enzymes and bacterial cell wall damage at high concentration has been investigated . Although cinnamaldehyde possesses potent antimicrobial activity against mdr pathogen, its cellular and in vivo cytotoxicity have been reported . In addition, it has been reviewed to be a cause of allergic reaction in toothpaste . Consequently, a dosage level at which no adverse effects is indispensable determined before use in the future application . Our study indicated the antibacterial activity of volatile oils extracted from herbs against several bacteria, including mdr - a . These plant extracts would be promising antimicrobial agents for further treating of human pathogens, including drug - resistant bacteria.
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Mirabegron is the first of a new class of drugs licensed for the management of overactive bladder syndrome (oab) in over 30 years . Oab is defined as urinary urgency, usually with urinary frequency and nocturia, with or without urgency urinary incontinence, in the absence of any other pathology.1 oab occurs in both men and women and it has a significant impact on the quality of life . Treatment focuses on lifestyle modification initially, such as weight loss, fluid management, avoiding or reducing caffeine, alcohol, and other bladder irritants, and bladder training . Mirabegron has been gaining popularity in the last few years for treating patients with oab as an alternative to antimuscarinics . It was licensed for the treatment of oab and has been approved for use in japan in 2011 (betanis), usa and canada in 2012 (myrbetriq), and europe in 2013 (betmiga). This article will look at mirabegron in the literature and review its pharmacokinetics, adverse effects, and effectiveness in treating oab . It causes increased cyclic adenosine monophosphate concentrations in the rat bladder tissue and shows a bladder relaxant effect.2,3 it also results in relaxation of bladder smooth muscle in rat and human isolated tissue . The chemical name of mirabegron is 2-(2-amino-1,3-thiazol-4-yl)-n-[4-(2-{[(2r)-2-hydroxy-2-phenylethyl]amino}ethyl)phenyl]acetamide and the chemical formula is c21h24n4o2s (figure 1). Mirabegron is rapidly absorbed after oral administration, with the time to maximum plasma concentration (tmax) being approximately 3 hours and the terminal plasma half - life being 50 hours . About 70% of the compound is bound to plasma proteins, mainly to albumin and then to 1-acid glycoprotein.35 it has an oral bioavailability between 24% and 53%, which differs with dose and gender . The absolute bioavailability increases from 29% at a dose of 25 mg to 35% at a dose of 50 mg . Mean maximum plasma concentration (cmax) and area under the plasma concentration time curve (auc) increased more than dose proportionally over the dose range.3,4 the cmax and auc of mirabegron are not affected by the age of the patient . Gender differences in cmax and auc are mainly the result of differences in body weight and bioavailability . Mirabegron is metabolized in the liver mainly by cytochrome p450 (cyp) 3a4, and cyp2d6 plays a minor role in its metabolism . Appropriate monitoring and dose adjustment may be necessary, especially with narrow therapeutic index drugs metabolized by cyp2d6.3,6,7 it has a biological half - life of 50 hours, is cleared by multiple mechanisms with no single predominating clearance pathway, and is excreted in both urine (55%) and feces (34%).5 the clinical efficacy and safety of mirabegron have been assessed in multiple phase ii and phase iii clinical trials . This includes the phase iia and iib trials by chapple et al . In 2013, they reported the results of the blossom trial.8 this was a proof - of - concept, randomized, double - blind, parallel - group, phase iia dose - ranging trial with mirabegron in patients with oab . The trial showed a statistically significant reduction in the mean micturition frequency from baseline by 17% with 100 mg and 18% with 150 mg of mirabegron, when compared with placebo, which resulted in a 9% reduction, and tolterodine, which resulted in an 11% reduction . Mirabegron was also significantly superior in reducing the mean volume voided per micturition, mean number of incontinence episodes, nocturia episodes, urgency incontinence episodes, and urgency episodes per 24 hours.3 this was followed up in the dragon trial, which enrolled 919 patients.9 the patients were randomized to five groups: placebo, and once - daily mirabegron 25, 50, 100, and 200 mg for 12 weeks . This showed statistically significant dose - dependent reduction in the mean micturition frequency with 50, 100, and 200 mg of mirabegron, compared with placebo . This trial also showed that mirabegron significantly increased the mean volume voided per micturition, decreased the mean number of incontinence episodes, urgency incontinence episodes, and urgency episodes per 24 hours . Nitti et al also looked at the urodynamic parameters in men with lower urinary tract symptoms and bladder outflow obstruction treated with mirabegron.10 two hundred male patients were enrolled . Seventy patients received mirabegron 50 mg, 65 received mirabegron 100 mg, and 65 received placebo . Urodynamic parameters assessed were the change from baseline to the end of treatment in maximum urinary flow rate (qmax) and detrusor pressure at qmax (pdetqmax). The study showed that mirabegron did not adversely affect the voiding urodynamic parameters such as the bladder contractility index and the bladder voiding efficiency after 12 weeks of treatment, compared with placebo . Clinical trial evidence for mirabegron is based on four primary phase iii randomized controlled trials (rcts): study to test the efficacy and safety of the beta-3 agonist mirabegron (ym178) in patients with symptoms of overactive bladder (scorpio), study to test the efficacy and safety of the beta-3 agonist mirabegron (ym178) in patients with symptoms of overactive bladder (aries), a study to test the efficacy and safety of the beta-3 agonist mirabegron (ym178) in patients with symptoms of overactive bladder (capricorn), and yamaguchi et al . Aries, capricorn, scorpio, and the yamaguchi et al rcts were all multiple - arm trials in patients with symptoms of oab . Scorpio and aries evaluated the clinical effectiveness of mirabegron (50 and 100 mg) compared to placebo . Yamaguchi et al evaluated the efficacy and safety of mirabegron 50 mg in a japanese population with oab against tolterodine and placebo over 16 weeks . Scorpio trial was a multicenter trial conducted by khullar et al that included europe and australia.11 it was a randomized, double - blind, parallel - group, placebo- and active - controlled phase iii trial in which 1,978 patients were enrolled . All patients had placebo for 2 weeks and then they were randomized . Of them, 493 patients received mirabegron 50 mg, 496 received mirabegron 100 mg, 495 received tolterodine, and 494 received a placebo for a total of 12 weeks . The trial showed a statistically significant decrease in the mean number of micturitions from baseline with 50 (1.93) and 100 mg (1.77) of mirabegron and in the mean number of incontinence episodes (1.57 with 50 mg and 1.46 with 100 mg) per 24 hours, compared to placebo . A post hoc analysis of the scorpio trial showed that both mirabegron doses were effective in improving the coprimary efficacy endpoints versus placebo in antimuscarinic treatment - nave patients and in patients whose condition failed to respond to previous antimuscarinic therapy, regardless of the reason for discontinuation . The aries trial was a north american, multicenter, randomized, double - blind, parallel - group, placebo - controlled, phase iii trial carried out by nitti et al.12 in this, 1,328 patients were enrolled and randomized to receive either placebo, mirabegron 50 mg, or mirabegron 100 mg for 12 weeks . The trial showed a statistically significant reduction in the number of incontinence episodes from baseline with both doses of mirabegron (50 mg [1.47] and 100 mg [1.63]), compared to placebo (1.13) (p<0.05). There was also a significant reduction in the number of micturitions per 24 hours in both mirabegron 50 mg (1.66) and 100 mg (1.75) groups, compared to placebo (1.05) (p<0.05). The capricorn trial was a phase iii, randomized, double - blind, placebo - controlled trial carried out by herschorn et al in europe and north america.13 the objective was to assess the efficacy and tolerability of mirabegron 25 and 50 mg once daily versus placebo in patients with oab . In this trial, 1,306 patients were enrolled and randomized . Of these, 433 received mirabegron 25 mg, 440 received mirabegron 50 mg, and 433 received placebo for 12 weeks . The primary endpoints were changes in the mean number of incontinence episodes and micturitions per 24 hours at the final visit . Both mirabegron 25 and 50 mg groups showed a reduction in incontinence episodes in 24 hours (1.36 and 1.38, respectively), when compared to placebo (0.96) (p<0.05). There was also a reduction in the number of micturition episodes in 24 hours (1.65 for mirabegron 25 mg and 1.60 for mirabegron 50 mg), in comparison to placebo (1.18) (p<0.05). The results showed that both mirabegron groups demonstrated statistically significant improvements when compared to placebo . However, mirabegron 50 mg showed greater improvement in the mean volume voided per micturition and the mean number of incontinence episodes per 24 hours . Yamaguchi et al conducted a phase iii rct evaluating the efficacy and safety of mirabegron 50 mg in a japanese population with oab.14 a total of 1,139 patients were enrolled and randomized to receive placebo, mirabegron 50 mg, or tolterodine 4 mg for 16 weeks . The primary efficacy endpoint was the change in the mean number of micturitions per 24 hours from baseline to final visit . In this trial, mirabegron 50 mg once daily showed a statistically significant improvement (p<0.05) in the mean number of micturitions per 24 hours (1.67 vs 0.86) urgency episodes per 24 hours (1.85 vs 1.37), incontinence episodes per 24 hours (1.12 vs 0.66), urgency incontinence episodes per 24 hours (1.01 vs 0.6), and volume voided in each micturition (24.3 vs 9.71), when compared to placebo . Abrams et al conducted the symphony trial.15 this was a phase ii, factorial design, randomized, double - blind, parallel - group, placebo- and monotherapy - controlled trial conducted at 141 sites in 20 european countries . It included both male and female patients aged 18 years or above with symptoms of oab for at least 3 months . In this trial, 1,306 patients were randomized to 12 weeks of treatment in one of 12 groups: six combination groups (solifenacin 2.5, 5, or 10 mg plus mirabegron 25 or 50 mg), five monotherapy groups (solifenacin 2.5, 5, or 10 mg, or mirabegron 25 or 50 mg), or placebo . The trial showed that combination therapy with solifenacin and mirabegron significantly improved the mean voided volume, micturition frequency, and urgency, compared with solifenacin 5 mg monotherapy . All combinations were well tolerated, with no important additional safety findings compared with monotherapy or placebo . Recently, results from the beside study were published.16 macdiarmid et al conducted a randomized, double - blind, parallel - group, multicenter, phase iiib study . A total of 2,174 patients were randomized to a combination of mirabegron 50 mg and solifenacin 5 mg (n=727), or solifenacin 5 mg (n=728), or solifenacin 10 mg (n=719). The study showed that more patients on the combination achieved clinically meaningful improvements in incontinence and micturition frequency . The improvements were accompanied by similar improvements in patient perception of bladder condition, symptom bother, and health - related quality of life . It causes increased cyclic adenosine monophosphate concentrations in the rat bladder tissue and shows a bladder relaxant effect.2,3 it also results in relaxation of bladder smooth muscle in rat and human isolated tissue . The chemical name of mirabegron is 2-(2-amino-1,3-thiazol-4-yl)-n-[4-(2-{[(2r)-2-hydroxy-2-phenylethyl]amino}ethyl)phenyl]acetamide and the chemical formula is c21h24n4o2s (figure 1). Mirabegron is rapidly absorbed after oral administration, with the time to maximum plasma concentration (tmax) being approximately 3 hours and the terminal plasma half - life being 50 hours . About 70% of the compound is bound to plasma proteins, mainly to albumin and then to 1-acid glycoprotein.35 it has an oral bioavailability between 24% and 53%, which differs with dose and gender . The absolute bioavailability increases from 29% at a dose of 25 mg mean maximum plasma concentration (cmax) and area under the plasma concentration time curve (auc) increased more than dose proportionally over the dose range.3,4 the cmax and auc of mirabegron are not affected by the age of the patient . Gender differences in cmax and auc are mainly the result of differences in body weight and bioavailability . Mirabegron is metabolized in the liver mainly by cytochrome p450 (cyp) 3a4, and cyp2d6 plays a minor role in its metabolism . Appropriate monitoring and dose adjustment may be necessary, especially with narrow therapeutic index drugs metabolized by cyp2d6.3,6,7 it has a biological half - life of 50 hours, is cleared by multiple mechanisms with no single predominating clearance pathway, and is excreted in both urine (55%) and feces (34%).5 the clinical efficacy and safety of mirabegron have been assessed in multiple phase ii and phase iii clinical trials . This includes the phase iia and iib trials by chapple et al . In 2013, they reported the results of the blossom trial.8 this was a proof - of - concept, randomized, double - blind, parallel - group, phase iia dose - ranging trial with mirabegron in patients with oab . Two hundred and sixty patients were enrolled from 31 european sites . The trial showed a statistically significant reduction in the mean micturition frequency from baseline by 17% with 100 mg and 18% with 150 mg of mirabegron, when compared with placebo, which resulted in a 9% reduction, and tolterodine, which resulted in an 11% reduction . Mirabegron was also significantly superior in reducing the mean volume voided per micturition, mean number of incontinence episodes, nocturia episodes, urgency incontinence episodes, and urgency episodes per 24 hours.3 this was followed up in the dragon trial, which enrolled 919 patients.9 the patients were randomized to five groups: placebo, and once - daily mirabegron 25, 50, 100, and 200 mg for 12 weeks . This showed statistically significant dose - dependent reduction in the mean micturition frequency with 50, 100, and 200 mg of mirabegron, compared with placebo . This trial also showed that mirabegron significantly increased the mean volume voided per micturition, decreased the mean number of incontinence episodes, urgency incontinence episodes, and urgency episodes per 24 hours . Nitti et al also looked at the urodynamic parameters in men with lower urinary tract symptoms and bladder outflow obstruction treated with mirabegron.10 two hundred male patients were enrolled . Seventy patients received mirabegron 50 mg, 65 received mirabegron 100 mg, and 65 received placebo . Urodynamic parameters assessed were the change from baseline to the end of treatment in maximum urinary flow rate (qmax) and detrusor pressure at qmax (pdetqmax). The study showed that mirabegron did not adversely affect the voiding urodynamic parameters such as the bladder contractility index and the bladder voiding efficiency after 12 weeks of treatment, compared with placebo . Clinical trial evidence for mirabegron is based on four primary phase iii randomized controlled trials (rcts): study to test the efficacy and safety of the beta-3 agonist mirabegron (ym178) in patients with symptoms of overactive bladder (scorpio), study to test the efficacy and safety of the beta-3 agonist mirabegron (ym178) in patients with symptoms of overactive bladder (aries), a study to test the efficacy and safety of the beta-3 agonist mirabegron (ym178) in patients with symptoms of overactive bladder (capricorn), and yamaguchi et al . Aries, capricorn, scorpio, and the yamaguchi et al rcts were all multiple - arm trials in patients with symptoms of oab . Scorpio and aries evaluated the clinical effectiveness of mirabegron (50 and 100 mg) compared to placebo . Yamaguchi et al evaluated the efficacy and safety of mirabegron 50 mg in a japanese population with oab against tolterodine and placebo over 16 weeks . Scorpio trial was a multicenter trial conducted by khullar et al that included europe and australia.11 it was a randomized, double - blind, parallel - group, placebo- and active - controlled phase iii trial in which 1,978 patients were enrolled . All patients had placebo for 2 weeks and then they were randomized . Of them, 493 patients received mirabegron 50 mg, 496 received mirabegron 100 mg, 495 received tolterodine, and 494 received a placebo for a total of 12 weeks . The trial showed a statistically significant decrease in the mean number of micturitions from baseline with 50 (1.93) and 100 mg (1.77) of mirabegron and in the mean number of incontinence episodes (1.57 with 50 mg and 1.46 with 100 mg) per 24 hours, compared to placebo . A post hoc analysis of the scorpio trial showed that both mirabegron doses were effective in improving the coprimary efficacy endpoints versus placebo in antimuscarinic treatment - nave patients and in patients whose condition failed to respond to previous antimuscarinic therapy, regardless of the reason for discontinuation . The aries trial was a north american, multicenter, randomized, double - blind, parallel - group, placebo - controlled, phase iii trial carried out by nitti et al.12 in this, 1,328 patients were enrolled and randomized to receive either placebo, mirabegron 50 mg, or mirabegron 100 mg for 12 weeks . The trial showed a statistically significant reduction in the number of incontinence episodes from baseline with both doses of mirabegron (50 mg [1.47] and 100 mg [1.63]), compared to placebo (1.13) (p<0.05). There was also a significant reduction in the number of micturitions per 24 hours in both mirabegron 50 mg (1.66) and 100 mg (1.75) groups, compared to placebo (1.05) (p<0.05). The capricorn trial was a phase iii, randomized, double - blind, placebo - controlled trial carried out by herschorn et al in europe and north america.13 the objective was to assess the efficacy and tolerability of mirabegron 25 and 50 mg once daily versus placebo in patients with oab . In this trial, 1,306 patients were enrolled and randomized . Of these, 433 received mirabegron 25 mg, 440 received mirabegron 50 mg, and 433 received placebo for 12 weeks . The primary endpoints were changes in the mean number of incontinence episodes and micturitions per 24 hours at the final visit . Both mirabegron 25 and 50 mg groups showed a reduction in incontinence episodes in 24 hours (1.36 and 1.38, respectively), when compared to placebo (0.96) (p<0.05). There was also a reduction in the number of micturition episodes in 24 hours (1.65 for mirabegron 25 mg and 1.60 for mirabegron 50 mg), in comparison to placebo (1.18) (p<0.05). The results showed that both mirabegron groups demonstrated statistically significant improvements when compared to placebo . However, mirabegron 50 mg showed greater improvement in the mean volume voided per micturition and the mean number of incontinence episodes per 24 hours . Yamaguchi et al conducted a phase iii rct evaluating the efficacy and safety of mirabegron 50 mg in a japanese population with oab.14 a total of 1,139 patients were enrolled and randomized to receive placebo, mirabegron 50 mg, or tolterodine 4 mg for 16 weeks . The primary efficacy endpoint was the change in the mean number of micturitions per 24 hours from baseline to final visit . In this trial, mirabegron 50 mg once daily showed a statistically significant improvement (p<0.05) in the mean number of micturitions per 24 hours (1.67 vs 0.86) urgency episodes per 24 hours (1.85 vs 1.37), incontinence episodes per 24 hours (1.12 vs 0.66), urgency incontinence episodes per 24 hours (1.01 vs 0.6), and volume voided in each micturition (24.3 vs 9.71), when compared to placebo . Abrams et al conducted the symphony trial.15 this was a phase ii, factorial design, randomized, double - blind, parallel - group, placebo- and monotherapy - controlled trial conducted at 141 sites in 20 european countries . It included both male and female patients aged 18 years or above with symptoms of oab for at least 3 months . In this trial, 1,306 patients were randomized to 12 weeks of treatment in one of 12 groups: six combination groups (solifenacin 2.5, 5, or 10 mg plus mirabegron 25 or 50 mg), five monotherapy groups (solifenacin 2.5, 5, or 10 mg, or mirabegron 25 or 50 mg), or placebo . The trial showed that combination therapy with solifenacin and mirabegron significantly improved the mean voided volume, micturition frequency, and urgency, compared with solifenacin 5 mg monotherapy . All combinations were well tolerated, with no important additional safety findings compared with monotherapy or placebo . Recently, results from the beside study were published.16 macdiarmid et al conducted a randomized, double - blind, parallel - group, multicenter, phase iiib study . A total of 2,174 patients were randomized to a combination of mirabegron 50 mg and solifenacin 5 mg (n=727), or solifenacin 5 mg (n=728), or solifenacin 10 mg (n=719). The study showed that more patients on the combination achieved clinically meaningful improvements in incontinence and micturition frequency . The improvements were accompanied by similar improvements in patient perception of bladder condition, symptom bother, and health - related quality of life . Mirabegron does not have the same adverse effects as anti - cholinergic agents and, thus, may be more tolerable in some individuals who experience side effects with anticholinergics . Mirabegron 50 mg caused dry mouth with an incidence similar to placebo and significantly lower than antimuscarinics . However, patients who use mirabegron could suffer from side effects such as raised blood pressure, tachycardia, urinary tract infections, constipation, headache, back pain, and dizziness.17 less common side effects include palpitations, atrial fibrillation, urticarial, and joint pain and swelling.12,14,17 in 2015, the medicines and healthcare products regulatory agency issued a warning stating that mirabegron is contraindicated in patients with severe uncontrolled hypertension, and advice about regular monitoring is being introduced because of cases of severe hypertension . It stated that mirabegron is contraindicated in patients with severe uncontrolled hypertension (systolic blood pressure $180 mmhg or diastolic blood pressure $110 mmhg, or both). It also recommended that blood pressure should be checked before starting treatment and monitored regularly during treatment, especially in patients with hypertension . There are quite a number of rcts looking at mirabegron, where> 27,000 patients have been included.18 the majority of these trials show that mirabegron 50 mg was efficacious in reducing the frequency of micturition and urgency urinary incontinence episodes, as well as more tolerable in individuals who cannot tolerate antimuscarinics . Currently, a number of ongoing trials further assess the safety and efficacy of mirabegron in combination with other drugs, such as the plus trial which looks at the safety and efficacy of mirabegron when taken with tamsulosin.
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Diabetes is one of the most common chronic diseases among women of reproductive age, observed in about 10% of pregnancies in the us and approximately 0.20.5% of these are in women with type 1 diabetes (t1d). T1d pregnancies are associated with an increased rate of complications, including late intrauterine death or major congenital malformations, which can lead to increased fetal morbidity and mortality compared to non - diabetic pregnancies . Maternal complications are also more frequent, with increased rates of preeclampsia, cesarean section and maternal mortality . Poor glycemic control at the time of conception and organogenesis during the first trimester is a major cause for an increased risk of birth defects and pregnancy complications . It has been recognized that a positive correlation exists between hemoglobin a1c (hba1c) levels during early pregnancy and the incidence of fetal malformations . Therefore, good glycemic control could lead to a reduction of congenital abnormality rates to almost non - diabetic levels . Preconception counseling and strict glycemic control have improved pregnancy outcomes in women with t1d, as evident from reduced rates of congenital malformations, preterm delivery and decreased neonatal morbidity, manifested by reduced macrosomia and admissions to neonatal care units . This is further exemplified by a case study from our clinic (table 1). Patients receiving prenatal care have been shown to maintain better hba1c levels, resulting in a reduction of infant mortality from 20% in the 1950s to less than 3% in the 1980s . This would not have been possible without the significant evolution in glucose monitoring methods, the introduction of insulin pumps and the development of insulin analogs.table 1.prenatal care for a pregnant t1d patient a case study.patienta 31-year - old female with t1d presented to the office for evaluation . She had just relocated and found out that she was pregnant and expressed a wish for things [to] go better this time.historyshe developed diabetes at age 14 and always had poor glycemic control . Her diabetes was complicated by retinopathy with laser surgery, peripheral neuropathy, autonomic neuropathy in the form of hypoglycemic unawareness and diabetic cystopathy with frequent urinary tract infections and incontinence . An analysis showed an unspecified developmental defect . With her second pregnancy she developed preeclampsia at 31 weeks of gestation, requiring antihypertensive medications . This patient had never seen an endocrinologist or maternal fetal medicine specialist, as these specialists were not available where she lived . Her diabetes had always been treated with two injections per day of nph and regular insulin.treatmentafter meeting with a certified diabetes educator her insulin regimen was intensified to four injections per day, with a variable amount of aspart at meals, as determined by carbohydrate counting . . The insulin regimen was adjusted multiple times, and her hba1c declined to 6.9% . Following a dilated retinal exam, an ophthalmologist treated her with panretinal photocoagulation laser therapy.outcomethe patient developed preeclampsia at 36 weeks of gestation, and was treated with antihypertensives . Both the mother and her neonate were discharged to home . Prenatal care for a pregnant t1d patient a case study . One of the main challenges in the care for pregnant women with diabetes is the proper control of blood glucose . Metabolic changes occurring as a result of the pregnancy complicate this task . During the first trimester, increased insulin sensitivity combined with the constant attempts to achieve normoglycemia through insulin therapy, raise the risk of hypoglycemia . The second and third trimesters of pregnancy are characterized by an enhanced secretion of placental hormones, growth factors and cytokines, leading to an increased insulin resistance and hyperglycemia . Hyperglycemia results in the transport of increased amounts of glucose across the placenta, causing fetal hyperinsulinemia and macrosomia . Macrosomia can cause maternal and fetal complications and is observed in about 2762% of infants of mothers with diabetes . Careful monitoring of glucose levels and constant adjustment of insulin therapy are needed to prevent hyperglycemia during pregnancy . T1d diabetic ketoacidosis (dka), common in t1d patients, develops faster during pregnancy due to decreased insulin sensitivity in the second and third trimesters . Dka remains a major cause of fetal loss, affecting 13% of patients with pregestational diabetes . Gestational hypertension is a common complication and a major risk factor for cardiovascular events, retinopathy and nephropathy . Furthermore, rates of preeclampsia are 2- to 4-times higher in pregnant women with t1d, leading to infant complications, including poor growth and premature birth . Preeclampsia can also be associated with serious maternal problems such as eclampsia and hemolysis, elevated liver enzymes, low platelet count (hellp) hellp syndrome, a life - threatening complication, has been linked to severe hypoglycemia attacks during pregnancy . T1d women who develop preeclampsia tend to have significantly higher hba1c values before and during pregnancy . Severe hypoglycemia, a major challenge in the management of t1d, has been reported in 1944% of pregnant diabetes patients treated with intensive insulin therapy, especially in the first trimester . Severe hypoglycemia is dangerous for the mother and can lead to loss of consciousness, seizures and death . Repeated hypoglycemic episodes can lead to hypoglycemia unawareness, causing further loss of symptoms associated with the autonomic response to hypoglycemia . Additionally, symptoms of hypoglycemia (nausea, anxiety, etc .) Might be mistaken for normal pregnancy symptoms, increasing the danger of severe hypoglycemia . A major goal in the management of t1d during pregnancy is the prevention of hypoglycemic episodes . Table 2 summarizes recommendations from the american diabetes association (ada) and the american congress of obstetricians and gynecologists (acog) for glycemic goals, glucose monitoring and prevention of severe hypoglycemia . The patient might, in fact, need to reduce the insulin dose during the first trimester in order to prevent hypoglycemic episodes . Several studies suggest that the use of insulin analogs instead of human insulin may lower the risk of severe hypoglycemia in women with diabetes [911]. In addition, real - time continuous glucose monitoring (cgm) with set alarms for low glucose values, might be useful for pregnant women with hypoglycemia unawareness.table 2.selected ada and acog recommendations .adaacogglycemic goals pre - meal values of 3.35.5 mmol / l (6099 mg / dl). Peak postprandial glucose of 5.57.2 mmol / l (100129 mg / dl). Bedtime and overnight glucose of 3.35.5 mmol / l (6099 mg / dl). Mean daily glucose of <6.1 mmol / l (<110 mg / dl) and hba1c <6.0%. Fasting glucose level of <5.3 mmol / l (<95 mg / dl). Pre - meal values of <5.5 mmol / l (<100 mg / dl). 1-h postprandial levels <7.8 mmol / l (<140 mg / dl), and 2-h postprandial values of <6.7 mmol / l (<120 mg / dl). During the night, glucose levels should not decrease to <3.3 mmol / l (<60 mg / dl). Mean capillary glucose levels should be maintained at an average of 5.5 mmol / l (100 mg / dl); hba1c 6.0%.glucose monitoring daily self - monitoring both before and after meals, at bedtime and occasionally at 2:00 am4:00 am . Hba1c test at the initial visit during pregnancy, monthly tests until target levels <6.0% are achieved, followed by testing every 23 months thereafter. Daily self - monitoring in the fasting state, before and 1 or 2 h after each meal and before bed . In selected patients, especially those on insulin pumps, glucose determinations at 2:00 am3:00 hba1c measurement provides an indication of glycemic control over the past 23 months and should be performed during each trimester.prevention of severe hypoglycemia assess the presence of clinically diminished counter - regulatory responses to hypoglycemia and educate patients to minimize its occurrences . Cgm may be a supplemental tool to self - monitoring for selected patients with t1d, especially those with hypoglycemia unawareness. Patients should be questioned to determine if they can recognize when their glucose levels decrease to <3.3 mmol / l (<60 mg / dl). Patients and their families should be taught how to respond quickly and appropriately to hypoglycemia . Selected ada and acog recommendations . For patients with t1d, the therapeutic insulin dose is adjusted to the patient s glucose profile . Daily monitoring is essential for proper insulin dosing, as insulin requirements vary during pregnancy . Ada and acog recommend self - monitoring several times throughout the day and occasionally at night in order to detect nocturnal hypoglycemia (table 2). Ada and acog also recommend maintaining mean daily glucose levels between 5.2 and 6.1 mmol / l (95110 mg / dl) and hba1c <6.0%, with specific goals for preprandial, postprandial and bedtime glucose . The goal during labor is to avoid maternal hyperglycemia in order to prevent subsequent neonatal hypoglycemia (by maintaining blood glucose levels <6.1 mmol / l [<110 mg / dl], as assessed by hourly blood glucose readings). Currently, the treatment of pregnant women with t1d is influenced by the fact that patients are better informed about the disease . A higher percentage of pregnancies are planned, providing an opportunity for adequate preparation of the patient in anticipation of potential complications from t1d . Glucose levels are controlled through the administration of long - acting (basal) insulin analogs and rapid - acting (bolus) insulin analogs . Patients follow a multiple daily injection regimen (mdi) that involves four, and even up to seven, insulin injections administered before meals and pre - bedtime . Compared with regular human insulin, bolus insulin analogs have a more rapid onset and a shorter duration of action (table 3), resulting in a more effective reduction of postprandial hyperglycemia and avoidance of hypoglycemic events between meals .table 3.characteristics of insulin and insulin analogs .insulin or insulin analogonset of action (minutes)time to peak concentration (minutes)maximum duration of action (hours)insulin regular insulin306090120512 nph insulin601202404801020bolus insulin analogs insulin lispro1015306034 insulin aspart1015405035 insulin glulisine10155535basal insulin analogs insulin glargine60120none24 insulin detemir60120none2024adapted from trujillo ai two rapid - acting insulin analogs, lispro and aspart, are currently classified as pregnancy risk category b, based on reports demonstrating fetal, perinatal and maternal outcomes similar to regular human insulin [1315]. Lispro and aspart are at least as effective as regular human insulin in achieving glucose control, as evident from hba1c levels . The main benefit of insulin analogs is the reduction of severe hypoglycemic events in pregnant t1d patients . Glulisine is another rapid - acting insulin analog available for use in the general diabetic population . However, there are no controlled studies addressing its safety in pregnancy, and glulisine is not recommended for pregnant women with t1d . Basal insulin is required for the maintenance of glycemic control between meals . Until recently, neutral protamine hagedorn (nph), an intermediate - acting insulin, was the only insulin approved for use as basal therapy in pregnant t1d patients and was considered as the standard of care for diabetes in pregnancy . However, nph use can be associated with a peak in concentration 48 h post - injection (table 3) and high intrasubject variability that may lead to an increased risk of hypoglycemic events between meals and at night . In recent years, glargine and detemir have become the basal insulin analogs of choice in the general t1d population . Glargine shows a relatively flat action profile with a near 24-h duration (table 3). Glargine also shows lower intrasubject variability compared to nph, as measured by the coefficient of variation for the 24-h glucose infusion rate glargine used in the general population leads to glycemic control that is at least comparable to that of nph, but with significantly lower risk for hypoglycemia . While there are numerous reports on the off - label use of glargine in pregnant women, there are no randomized clinical trials assessing its safety during pregnancy . Therefore, insulin glargine is currently classified as pregnancy risk category c and is not approved for use in pregnant women . Detemir also shows a flatter action profile with a longer duration of action than nph, approaching 24 h at clinically relevant doses . These characteristics suggest that detemir has the potential to be an improvement over nph for the control of t1d . A significant advance in the care for pregnant women with t1d was marked in 2012 with the reclassification of detemir as pregnancy risk category b, based on results from a randomized, active - controlled study by mathiesen et al . . This study evaluated the efficacy and safety of detemir in 310 pregnant t1d patients, randomized 1:1 to receive either detemir or nph in a basal - bolus regimen with aspart . Treatment was initiated up to 1 year before pregnancy (48%) or at 8 to 12 weeks during pregnancy (52%). Additional endpoints included maternal safety . In terms of efficacy, detemir demonstrated non - inferiority to nph with respect to the primary endpoint of the study . A significant number of women in both groups (41% in the detemir group; 32% in the nph group) reached the target of hba1c 6.0% at 24 and 36 weeks of gestation . Interestingly, fasting plasma glucose was significantly lower in the detemir group at 24 and 36 weeks of gestation, especially in patients initiating treatment with detemir before pregnancy . The incidence of preeclampsia was slightly higher in the detemir group, but within expected rates for pregnancies complicated by diabetes . No significant differences in early fetal death or the health of the fetus and newborn were seen with detemir . Insulin premixes have proven somewhat useful for the treatment of the general population with diabetes, especially patients who need simplified dosing regimens . However, premixes are not useful for the treatment of pregnant t1d patients, as premixed formulations cannot provide the required dosing flexibility during the different periods of pregnancy . Currently, the treatment of pregnant women with t1d is influenced by the fact that patients are better informed about the disease . A higher percentage of pregnancies are planned, providing an opportunity for adequate preparation of the patient in anticipation of potential complications from t1d . Glucose levels are controlled through the administration of long - acting (basal) insulin analogs and rapid - acting (bolus) insulin analogs . Patients follow a multiple daily injection regimen (mdi) that involves four, and even up to seven, insulin injections administered before meals and pre - bedtime . Compared with regular human insulin, bolus insulin analogs have a more rapid onset and a shorter duration of action (table 3), resulting in a more effective reduction of postprandial hyperglycemia and avoidance of hypoglycemic events between meals .table 3.characteristics of insulin and insulin analogs .insulin or insulin analogonset of action (minutes)time to peak concentration (minutes)maximum duration of action (hours)insulin regular insulin306090120512 nph insulin601202404801020bolus insulin analogs insulin lispro1015306034 insulin aspart1015405035 insulin glulisine10155535basal insulin analogs insulin glargine60120none24 insulin detemir60120none2024adapted from trujillo ai two rapid - acting insulin analogs, lispro and aspart, are currently classified as pregnancy risk category b, based on reports demonstrating fetal, perinatal and maternal outcomes similar to regular human insulin [1315]. Lispro and aspart are at least as effective as regular human insulin in achieving glucose control, as evident from hba1c levels . The main benefit of insulin analogs is the reduction of severe hypoglycemic events in pregnant t1d patients . Glulisine is another rapid - acting insulin analog available for use in the general diabetic population . However, there are no controlled studies addressing its safety in pregnancy, and glulisine is not recommended for pregnant women with t1d . Basal insulin is required for the maintenance of glycemic control between meals . Until recently, neutral protamine hagedorn (nph), an intermediate - acting insulin, was the only insulin approved for use as basal therapy in pregnant t1d patients and was considered as the standard of care for diabetes in pregnancy . However, nph use can be associated with a peak in concentration 48 h post - injection (table 3) and high intrasubject variability that may lead to an increased risk of hypoglycemic events between meals and at night . In recent years, glargine and detemir have become the basal insulin analogs of choice in the general t1d population . Glargine shows a relatively flat action profile with a near 24-h duration (table 3). Glargine also shows lower intrasubject variability compared to nph, as measured by the coefficient of variation for the 24-h glucose infusion rate glargine used in the general population leads to glycemic control that is at least comparable to that of nph, but with significantly lower risk for hypoglycemia . While there are numerous reports on the off - label use of glargine in pregnant women, there are no randomized clinical trials assessing its safety during pregnancy . Therefore, insulin glargine is currently classified as pregnancy risk category c and is not approved for use in pregnant women . Detemir also shows a flatter action profile with a longer duration of action than nph, approaching 24 h at clinically relevant doses . These characteristics suggest that detemir has the potential to be an improvement over nph for the control of t1d . A significant advance in the care for pregnant women with t1d was marked in 2012 with the reclassification of detemir as pregnancy risk category b, based on results from a randomized, active - controlled study by mathiesen et al . . This study evaluated the efficacy and safety of detemir in 310 pregnant t1d patients, randomized 1:1 to receive either detemir or nph in a basal - bolus regimen with aspart . Treatment was initiated up to 1 year before pregnancy (48%) or at 8 to 12 weeks during pregnancy (52%). Additional endpoints included maternal safety . In terms of efficacy, detemir demonstrated non - inferiority to nph with respect to the primary endpoint of the study . A significant number of women in both groups (41% in the detemir group; 32% in the nph group) reached the target of hba1c 6.0% at 24 and 36 weeks of gestation . Interestingly, fasting plasma glucose was significantly lower in the detemir group at 24 and 36 weeks of gestation, especially in patients initiating treatment with detemir before pregnancy . The incidence of preeclampsia was slightly higher in the detemir group, but within expected rates for pregnancies complicated by diabetes . No significant differences in early fetal death or the health of the fetus and newborn were seen with detemir . Insulin premixes have proven somewhat useful for the treatment of the general population with diabetes, especially patients who need simplified dosing regimens . However, premixes are not useful for the treatment of pregnant t1d patients, as premixed formulations cannot provide the required dosing flexibility during the different periods of pregnancy . Accurate determination of blood glucose levels is important for the proper control of diabetes . Until recently, this was achieved solely by the self - monitoring of capillary blood glucose using glucose meters . The development of continuous glucose monitoring (cgm) devices represents an advance that can provide real - time measurements and warnings when patients face hypoglycemia . Several studies have examined the benefits of cgm for the general population, but only a few studies have been performed in pregnant women with t1d . A study by yogev et al . Showed that cgm use during pregnancy can detect hyperglycemic and nocturnal hypoglycemic events that might have gone unnoticed with intermittent blood glucose monitoring . A small, randomized trial performed by murphy et al . Showed that cgm improved glycemic control in women with t1d and t2d, led to lower birth weight and reduced risk of macrosomia . Based on these results, the american association of clinical endocrinologists (aace) recommended the use of cgm for all pregnant patients with t1d . A recent randomized trial investigated the intermittent use of real - time cgm during pregnancy (study arm) in addition to self - monitored plasma glucose levels seven times daily (control arm). Hba1c, self - monitored plasma glucose, severe hypoglycemia events and prevalence of large - for - gestational - age infants for women using cgm were comparable to controls . These results suggest that the intermittent use of cgm in pregnant patients with well - controlled diabetes does not further improve disease management and pregnancy outcomes . Additional large controlled studies examining maternal and neonatal outcomes with cgm use are still needed to confirm its benefit . Patients can choose between mdi using a syringe or insulin pen and continuous subcutaneous insulin infusion (csii) via an insulin pump . Several observational studies have attempted to evaluate the effectiveness of insulin pumps versus mdi in pregnant women with t1d . In 2012, the agency for healthcare research and quality (ahrq) published a systematic review summarizing the results of these studies . The ahrq found that hba1c values improved with either mdi or csii and there was no statistically significant difference in outcomes between the two delivery methods . Because no randomized controlled trials had been performed, the strength of evidence was insufficient to show statistically significant differences in any other maternal or fetal outcomes; therefore, the risk of bias was high . A newer, retrospective, observational study by wender - ozegowska et al ., confirmed a reduction in hba1c to similar levels with both csii and mdi use during pregnancy, although there were fewer hypoglycemic and hyperglycemic episodes in the csii group . Insulin pump utilization is high (about 40%) in the general t1d us population due to improvement in the quality of life and observed decrease in hba1c . Further studies focused on pregnant women with t1d are necessary to confirm any superiority of csii use . Csii insulin delivery has drawbacks, such as higher cost of the pump and pump supplies, difficulty of use requiring the patient to be motivated and compliant and willingness to test glucose multiple times daily, that might limit its usefulness in pregnant t1d patients . In addition, there is a risk of hypoglycemia or even dka in case of a pump malfunction or infection at the infusion site . Sensor - augmented pump (sap) therapy (combinations of cgm and csii) might improve the treatment of t1d patients even further . Use of open - loop sap (patient reads the cgm values and manually adjusts the pump) in non - pregnant patients with inadequately controlled t1d results in a significant improvement of hba1c levels . The use of sap in pregnant women has been tested only on a small scale and did not show a significant benefit, except during the first trimester . However, the study was too small to be adequately powered . Closed - loop sap therapy uses a control algorithm to guide insulin delivery based on real - time cgm measurements . Closed - loop systems could be of great benefit to pregnant women with t1d as the algorithm should, in theory, be capable of maintaining optimal glucose levels . T1d in pregnancy is a high - risk clinical situation associated with significant chances of complications for the mother and the fetus . However, clinical practice and research have shown that pregnancy planning, preconception counseling and maintaining optimal glucose levels have a positive effect on pregnancy outcomes . The main goal in caring for the pregnant patient with t1d should be achieving near - normal blood glucose levels while minimizing the risk of hypoglycemia . The development of insulin analogs with improved safety and efficacy profiles has facilitated the achievement of normoglycemia . Evidence supports the use of the bolus insulins, lispro and aspart, as the mealtime component of mdi or as used in csii . The recent reclassification of detemir to pregnancy risk category b provides pregnant t1d women with a basal insulin analog, further expanding treatment options for this patient population . Evidence supports the use of insulin detemir, or nph, as the basal component of an mdi regimen . Women with t1d should attend pre - pregnancy services and antenatal clinics led by professionals with endocrine and obstetrical expertise, in order to maximize the likelihood of positive pregnancy outcomes . The objective is to optimize glycemic control prior to pregnancy (hba1c <6.5%), to start folic acid supplementation (recommended 4 mg daily), to alter medications unsuitable for pregnancy and to ensure that medical issues are addressed before conception and during pregnancy.
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Genetics and environment have both been implicated in the exponential rise in the prevalence of diabetes mellitus and impaired glucose tolerance that affects 65.1 million people and leads to the mortality of 1 million / year in 2013 in india . In addition to these diabetics, there is a larger number of prediabetics, who will eventually contribute to the disease burden . The thrifty genotype postulated by neel, suggests an evolutionary advantage for individuals with the ability to store a high proportion of energy as fat . This enables the storage of fats when food is plentiful so that survival is better in periods of shortage . However, this has been implicated in the escalation of the prevalence of diabetes and obesity worldwide . Environmental factors such as rapid urbanization, availability of calorie - dense processed foods, pervasiveness of smoking and the heavy consumption of alcohol, high intake of refined carbohydrates and dramatically decreased physical activity levels have been implicated in the burgeoning epidemic of diabetes . However, even though they are exposed to these environmental risk factors, some individuals do not develop diabetes . Genome - wide association studies have begun to explain the inter - individual variation in susceptibility to diabetes . Studies on the role of epigenetic factors in the complex interplay between genes and the environment in the occurrence of diabetes are going on . Diabetes inflicts an enormous burden on the healthcare system and economics, and primordial prevention, health education, communication of behavior change and lifestyle modifications are being utilized to reduce this burden . India has been termed as the diabetes capital of the world owing to the very high prevalence of metabolic syndrome including diabetes, hypertension, hypercholesterolemia, and obesity . India had 65.1 million diabetics and had a mortality of one million as a result of diabetes in 2013 . The socioeconomic distribution of the disease has also changed, and people in low and middle - income countries are now contributing to the largest burden of the disease . Relative malnutrition in utero and the abundance of nutrition in early and middle adulthood may explain the epidemic in indians . The westernization of diet and lifestyle, synergistic with the genetic background has also been postulated as an explanation for the rapidly growing prevalence in this population . Large - scale screening to identify asymptomatic individuals who are likely to have diabetes is of particular value because of the long lead time of diabetes, the minimal observable symptoms, the large number of undetected individuals owing to the iceberg phenomenon in the community, and the advantage of early diagnosis . Both mass screening and high - risk screening offer their own advantages and disadvantages . In a country with a high burden such as india, the yield of mass screening could be equivalent to high risk screening, as high - risk conditions for diabetes include a family history of diabetes and belonging to the asian race, criteria that a large proportion of the population satisfies . Various screening tests for diabetes, including fasting blood glucose and oral glucose tolerance test, are available . While fasting blood glucose test is the preferred one for screening, random blood glucose confers the advantage of time and convenience to the study participants . The current cross - sectional study was formulated to screen individuals for diabetes and to obtain the trends of distribution of blood glucose by age and gender . Awareness of the recognition of symptoms with a special focus on gestational diabetes was generated among people . Through information education communication (iec) campaigns, people in the rural areas of a north indian state were made aware of screening tests and when to seek healthcare . A cross - sectional study was conducted in a northern state of india, uttar pradesh . Five districts out of the 72 districts in the state were selected based on concerns about programming including the population in the districts, grant amount, and the location of existing infrastructure . The cumulative population of the five districts, namely, lalitpur, maharajganj, sitapur, shajahanpur, and varanasi was 15,073,666 . The sex ratio in these districts was 904 females to 1000 males . In these districts, the selection criterion was based on the affiliation with the implementing organization and project grant . These eight centers carried out screening activities in the villages of the five districts over a period of 2 years from april 2012 to march 2014 . Iec and screening activities were conducted with the support of a grant provided by the world diabetes foundation (wdf 11647). A camp - based approach was followed . During the study period, 899,960 people were made aware by means of iec activities of the recognition of symptoms of diabetes, screening tests and the search for healthcare through 399 camps . Random blood sugar (rbs) samples were obtained from a convenient sample of 52,633 (5.8%) participants attending these camps, who fulfilled the age (more than 18 years) and study criteria (located in the project area, attended the camp and were willing to provide verbal informed consent). The procedure was explained, and verbal permission was obtained from the respondents before the samples were taken . Age, gender, and rbs values were obtained from the people who underwent screening . Subjects who were known diabetics or aged <18 years were excluded from the blood glucose measurement . A 1-day training was conducted for 8 physicians, 7 nurses, and 25 paraprofessionals by diabetologists in the nodal centers on the study protocol, the use of the glucometer instrument, and procedures to be followed to obtain consent and blood samples so that measurements in all centers are standardized . Measurements were obtained by trained staff using a reflectance photometer instrument (one call plus) with a finger prick to obtain fresh capillary whole blood . American diabetes association criteria were used to classify subjects into normal, prediabetic, and diabetic according to the random blood glucose values . A blood glucose level between 79 and 140 mg / dl was considered normal, between 140 and 200 mg / dl was prediabetes, and more than 200 mg / dl considered diabetes . Subjects with an rbs> 200 mg / dl were referred to the nodal centers for further investigation and management . Statistical analysis was performed using r statistical software (foundation for statistical computing, vienna, austria) version 3.1.1 . All numerical data was entered as such, and the statistical measures obtained included descriptives, box plots, kernel density plots, normal distribution curves, log transformed curves, skew and kurtosis, mean and standard deviations, proportions and test for bimodality including hartigan's dip test . For analysis, data were available at the end of the study period for 45,318 subjects, 44.4% of whom were male and 55.6% female . Ages ranged from 18 to 98 years with mean age of 39.9 14.44 years; 59.2% of the respondents were below the age of 40, 32.2% between 41 and 60 years and 8.6% above the age of 60 years . Age and gender distribution of the study sample in the sample, 39,220 (86.5%) were normal (rbs <140 mg / dl), 4806 (10.6%) were prediabetic (rbs 140200 mg / dl) and 1292 (2.9%) were diabetic (rbs> 200 mg / dl). Of the women, 2647 (10.5%) were prediabetic and 621 (2.5%) diabetic . Of the men, 2159 (10.7%) were prediabetic and 671 (3.3%) diabetic . Chi - square test was performed across gender and diabetes status and the results were significant (= 31.57, p <0.001). Of the subjects aged below 40 years, 1995 (4.4%) were prediabetic and 334 (0.7%) diabetic . Among persons aged 4160 years, 2049 (4.5%) were prediabetic and 736 (1.6%) were diabetic, whereas, of the subjects aged above 60 years, 762 (1.7%) were prediabetic and 222 (0.5%) were diabetic . Chi - square test was performed across age, and diabetes status and the results were significant (= 1491.15, p <0.001). The prediabetic to diabetic ratios for the three age groups, rounded off to the nearest whole number were 6:1, 3:1 and 3:1, respectively [table 2]. Distribution of diabetes and prediabetes among sampled population by age the mean rbs of the sample was 115.49 37.19 mg / dl . The mean rbs of the females was 115.13 35.34 mg / dl and 115.95 39.38 the median blood glucose level steadily rose with increasing age and the medians for the age groups 40 years, 4160 years and 61 years were 105 mg / dl, 111 mg / dl, and 117 mg / dl, respectively [figure 1]. Boxplot of random blood sugar levels versus age showing rising trend of blood glucose levels with age the distribution curve of rbs showed a right skewed distribution with skewness of 3.24 and a kurtosis of 15.69 . Bimodality was tested using the hartigan's dip test, and the dip statistic (d) was 0.0162 with a simulated p a bimodal curve was obtained with the log - transformed rbs with a skewness of 1.38 and kurtosis of 3.71 . The kernel density plots of rbs and log transformed rbs are depicted in figure 2 . Kernel density plots of random blood sugar and log transformed random blood sugar showing bimodal distribution of random blood sugar values after log transformation this cross - sectional study approached a convenient sample in the rural areas of uttar pradesh, a northern state of india . With a view to providing information, education and enhancing awareness on diabetes with a special focus on gestational diabetes, 899,960 people in rural areas this number 52,633 underwent random blood glucose testing and 45,318 subjects were included in the final analysis . The number of women included in the sample exceeded the number of males, which was in conflict with the sex ratio of the area . This can be explained by the special focus on iec activities among pregnant women for gestational diabetes . Besides, women in certain areas are more likely to participate in their decisions on health and participate in the camp activities because of the constraints of work for men . Most respondents were aged between 1840 and 4160 years; 9% of the respondents were above 60 years . There was also a clear upward shift with increasing age of the median values of blood glucose . The overall prediabetes to diabetes ratio was 4:1 in the sample, with an alarming 6:1 ratio among people aged below 40 years, and 3:1 among subjects aged 41 and above . Since a high prediabetes to diabetes ratio is indicative of an impending epidemic of diabetes, this shows a hidden burden of impaired glucose tolerance and prediabetes in rural areas, which is likely to emerge and further contribute to the current immense burden of diabetes . The prevalence of undiagnosed prediabetes in the current study was 10.6% and that of undiagnosed diabetes 2.9% . The diabetes atlas of the international diabetes federation puts the prevalence of diabetes in india in 2013 at 9.1%, with a pooled prevalence of rural diabetes in low and middle - income countries at 5.6% . In a large - scale urban study in india, ramachandran et al . Mohan et al . Observed a self - reported physician diagnosed prevalence of diabetes at 3.1% in rural areas and 7.3% in urban areas . The prevalence of diabetes in the current study is within the range for similar geographical (rural areas in north india) areas, and variations can be explained by the differing definitions of diabetes, measurement used (rbs) and geographical variations . This can be explained by the use of rbs, which is more likely to reveal impaired glucose tolerance after a glucose load . The distribution of blood glucose was right skewed, and a bimodal distribution was obtained after a log transformation . This is consistent with other similar studies conducted in large populations to estimate the prevalence of diabetes . In areas with a high prevalence of diabetes, a log transformation can be applied to reduce the skewness, and typically, in such populations, a bimodal distribution is obtained after the application of the log transformation . This indicates the presence of two discrete sub - groups in the population; one group with a predisposition to developing diabetes . The current study supports two hypotheses with regard to diabetes: that impaired glucose tolerance is a natural sequel of increasing age, and that there are two naturally occurring groups with a genetic dichotomy toward the development of diabetes . Mass screening for diabetes not only provides benefits from a clinical standpoint, but also helps to estimate the prevalence and the hidden burden of the disease, demonstrates the exponential rise in diabetes and prompts epidemiologists to explore the related etiological mechanisms . Selective screening of people aged above 40 years can have a higher yield of diabetic cases as evidenced in the study . Screening programs can strengthen healthcare system initiatives and reduce the growing burden of diabetes in india . The sampling of the subjects and selection of geographical areas was influenced by programming concerns and so may not be representative of trends in the country . For the same reason, rbs grant from world diabetes foundation (wdf11 - 647) there are no conflicts of interest.
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In 1988, reaven introduced the term syndrome x, which consisted a cluster of syndromes including hyperinsulinemia, hypertension, dyslipidemia, hyperglycemia, and resistance to insulin - stimulated glucose uptake (1). He claimed that insulin resistance (ir) plays a central role in the etiology and clinical course of patients with diabetes mellitus, high blood pressure (bp), and coronary heart disease (2). In 1998, the world health organization recognized the importance of this clustering and further defined the components of metabolic syndrome (mets) (3). In 2001, the national cholesterol education program adult treatment panel iii (atp iii) provided criteria for this syndrome that are now often used in general practice (4). The components proposed in the atp iii guidelines include obesity (high waist circumference) (5), high serum triglyceride (tg) level (6), high bp (7), high fasting plasma glucose (fpg) level (8), and low level of high - density lipoprotein cholesterol (hdl - c) (9). In addition, subjects with mets are considered to have ir and an increased risk of cardiovascular disease (cvd) (10). Identifying patients with ir is clinically important because ir is associated with increased cvd - related mortality and incidence of diabetes mellitus (11). However, few data show whether the atp iii criteria are suitable for detecting ir . Therefore, the purpose of this study was to explore the relationship between ir and the atp iii criteria for mets and to evaluate the ability to detect ir from the method of insulin suppression test (ist) in subjects fulfilling those criteria . We enrolled 511 subjects (218 men and 293 women, aged 20 - 75 with mean 45.82.2 yr) during routine health checkups at our hospital . Subjects with a history of hypertension or other clinically significant medical or surgical diseases were excluded . We excluded the diabetic subjects because ir is the major patho physiological defect of type 2 diabetes and in most of the diabetic patients fulfilling the criteria of mets . Thus, this might lessen the impact of the results when interpreting the data to explore our major purpose in the present study . The nature, purpose, and potential risks of the study were explained to the patients, and we obtained their written informed consent to participate in the study . All patients underwent complete routine physical examination to rule out cardiovascular, respiratory, renal, or endocrine disorders . No patient had received any medication known to affect glucose metabolism for at least 3 weeks before the study . Three days before the study, patients were prescribed a stable diet . The patients' ability to process a glucose load after an overnight fast, an intravenous catheter was placed in each of the patients' arms . One arm was used to administer a 180-min infusion of somatostatin at a rate of 250 g / hr, insulin at 25 mu / m / min, and glucose at 240 mg / m / min . The other arm was used to collect blood samples . Blood was initially sampled every 30 min and then at 10-min intervals from minutes 150 to 180 of the infusion . These samples were used to determine the steady - state plasma insulin (sspi) and steady - state plasma glucose (sspg) concentrations for each subject . The sspi concentrations were comparable in all individuals; therefore, the sspg concentrations were used to measure the efficacy of insulin in processing the infused glucose load . Because reaven suggested that the top 25% of sspg values in the general population indicated ir (1), we divided our subjects into four groups by sspg quartiles . Their height and weight were measured to the nearest 0.5 cm and 0.1 kg, respectively . Body mass index (bmi) was calculated as weight divided by [height]. Because waist circumstances were not available for all subjects, we substituted a bmi 27 kg / m as an index of obesity; this was the level the taiwanese department of health defined in 2002 as indicating obesity . Other four specific criteria included tg 1.7 mm / l, bp 130/85 mmhg, fpg concentration 6.1 mm / l, and hdl - c 1.04 mm / l for men or 1.30 subjects who fulfilled at least three out of five criteria met the definition of mets (4). Bp was measured according to the criteria of the seventh joint national committee on prevention, detection, evaluation, and treatment of high blood pressure . Plasma was separated from the blood samples within 1 hr of their collection and stored at -30 until analyzed . The samples obtained at -5 and 0 min were analyzed for fpg, fasting plasma insulin, and lipid levels . Plasma glucose values were determined by using a glucose oxidase method and analyzer (ysi 203 glucose analyzer; scientific division, yellow spring instrument company, inc . Insulin was measured by a commercial solid phase radioimmunoassay kit (coat - a - count insulin kit, diagnostic products corporation, los angeles, ca, u.s.a . ). Serum tg values were measured by using the dry, multilayer analytical slide method (dri - chem 3000 analyzer; fuji photo film corporation, minato - ku, tokyo, japan). Serum hdl - c concentrations were determined by means of an enzymatic cholesterol assay after dextran sulfate precipitation . The sensitivity is the proportion of mets cases of all ir cases in the study population . The specificity is the proportion of cases not fulfilled the mets criteria of all of the cases without ir in the study population . Finally, the positive predictive value means the proportion of ir case of all mets cases in the study population . Analysis was performed by using software (spss version 10.0 statistical package for windows; spss, chicago, il, u.s.a . ). Data were tested for normal distribution with the kolmogorov - smirnov test and for homogeneity of variances with the levene test . An independent student t - test was used to evaluate anthropometric differences between men and women . A test was used to test differences in prevalence in categorical variables for both sexes . Associations of sspg with the criteria for mets were assessed by using spearman correlation analysis . All statistical tests were two - sided, and p values<0.05 were considered to indicate a statistical significance . We enrolled 511 subjects (218 men and 293 women, aged 20 - 75 with mean 45.82.2 yr) during routine health checkups at our hospital . Subjects with a history of hypertension or other clinically significant medical or surgical diseases were excluded . We excluded the diabetic subjects because ir is the major patho physiological defect of type 2 diabetes and in most of the diabetic patients fulfilling the criteria of mets . Thus, this might lessen the impact of the results when interpreting the data to explore our major purpose in the present study . The nature, purpose, and potential risks of the study were explained to the patients, and we obtained their written informed consent to participate in the study . All subjects were tested at our clinical research center . During enrollment, all patients underwent complete routine physical examination to rule out cardiovascular, respiratory, renal, or endocrine disorders . No patient had received any medication known to affect glucose metabolism for at least 3 weeks before the study . Three days before the study, patients were prescribed a stable diet . The patients' ability to process a glucose load after an overnight fast, an intravenous catheter was placed in each of the patients' arms . One arm was used to administer a 180-min infusion of somatostatin at a rate of 250 g / hr, insulin at 25 mu / m / min, and glucose at 240 mg / m / min . The other arm was used to collect blood samples . Blood was initially sampled every 30 min and then at 10-min intervals from minutes 150 to 180 of the infusion . These samples were used to determine the steady - state plasma insulin (sspi) and steady - state plasma glucose (sspg) concentrations for each subject . The sspi concentrations were comparable in all individuals; therefore, the sspg concentrations were used to measure the efficacy of insulin in processing the infused glucose load . Because reaven suggested that the top 25% of sspg values in the general population indicated ir (1), we divided our subjects into four groups by sspg quartiles . Their height and weight were measured to the nearest 0.5 cm and 0.1 kg, respectively . Body mass index (bmi) was calculated as weight divided by [height]. Because waist circumstances were not available for all subjects, we substituted a bmi 27 kg / m as an index of obesity; this was the level the taiwanese department of health defined in 2002 as indicating obesity . Other four specific criteria included tg 1.7 mm / l, bp 130/85 mmhg, fpg concentration 6.1 mm / l, and hdl - c 1.04 mm / l for men or 1.30 mm / l for women (4). Subjects who fulfilled at least three out of five criteria met the definition of mets (4). Bp was measured according to the criteria of the seventh joint national committee on prevention, detection, evaluation, and treatment of high blood pressure . Plasma was separated from the blood samples within 1 hr of their collection and stored at -30 until analyzed . The samples obtained at -5 and 0 min were analyzed for fpg, fasting plasma insulin, and lipid levels . Plasma glucose values were determined by using a glucose oxidase method and analyzer (ysi 203 glucose analyzer; scientific division, yellow spring instrument company, inc ., yellow spring, oh, u.s.a . ). Insulin was measured by a commercial solid phase radioimmunoassay kit (coat - a - count insulin kit, diagnostic products corporation, los angeles, ca, u.s.a . ). Serum tg values were measured by using the dry, multilayer analytical slide method (dri - chem 3000 analyzer; fuji photo film corporation, minato - ku, tokyo, japan). Serum hdl - c concentrations were determined by means of an enzymatic cholesterol assay after dextran sulfate precipitation . The sensitivity is the proportion of mets cases of all ir cases in the study population . The specificity is the proportion of cases not fulfilled the mets criteria of all of the cases without ir in the study population . Finally, the positive predictive value means the proportion of ir case of all mets cases in the study population . Analysis was performed by using software (spss version 10.0 statistical package for windows; spss, chicago, il, u.s.a . ). Data were tested for normal distribution with the kolmogorov - smirnov test and for homogeneity of variances with the levene test . An independent student t - test was used to evaluate anthropometric differences between men and women . A test was used to test differences in prevalence in categorical variables for both sexes . Associations of sspg with the criteria for mets were assessed by using spearman correlation analysis . All statistical tests were two - sided, and p values<0.05 were considered to indicate a statistical significance . In general, men had higher bps and bmis and lower hdl - c levels than the women . The prevalence of various components of mets did not differ between the sexes, except that bmi was higher in men than in women . The commonest abnormality related to mets was a low hdl - c value, followed by high bp, and then the other components (table 2). The sensitivity to detect ir by using the criteria for mets was about 47% overall; the sensitivity was slightly higher in men than in women (53% vs. 41%) (table 3). The positive predictive value was higher in men (70%) than in women (60%), with an overall value of 64.8% . Table 4 presents the predictive value to detect ir in subjects who fulfilled the criteria for mets . In general, the predictive value for ir increased as increasing numbers of components of mets were combined . The most common combination in subjects with both mets and ir were obesity, high bp, and low hdl - c level . A positive correlation was seen in each component of mets with sspg except for hdl - c values, which had a negative correlation . The strongest factor correlated with reaven proposed that ir has a central role in mets, and study data have confirmed that a finding of baseline ir is associated with subsequent cvd (11). The purpose of the atp iii criteria for mets is to identity individuals at high risk for cvd . Our data showed that men had high bmis and bps and low hdl - c levels compared with women, a finding that might have accounted for the differences in prevalence . Traditional risk factors for cvd include abnormal lipid profiles, hypertension, obesity, and diabetes . Individuals with mets are believed have an increased risk of diabetes mellitus (14). This is also true for individuals with ir, who have an increased risk for cvd and diabetes . The ist has a very high correlation (r=0.93) with the gold standard - hyperinsulinemic euglycemic clamp for evaluating ir (15). Assessment for hyperinsulinemia, a surrogate measurement of ir, or the homeostatic model assessment of ir is usually used in epidemiologic studies . The demonstration that the criteria for mets can be used to identify subjects with ir is clinically important . The criteria of mets proposed by atp iii are more clinically applicable than the world health organization criteria proposed in 1999 . Our data revealed that the sensitivity to detect ir by using the atp iii criteria was less than 50%, with a slightly higher rate in men than in women . The sex - related difference may be attributed to the different prevalences of the individual components . Therefore, the criteria do not seem to be useful for evaluating subjects with mets for detecting ir . The criteria for mets included at least three component combinations and individual components associated with ir; therefore, subjects who fulfill the criteria for mets might reasonably be thought to have a high probability of ir . However, the relationship among these five components that can influence each other may explain the lower sensitivity to predict ir . Our results were similar to those of a previous study in which about two - thirds of the subjects with diagnosed with mets had ir (16). Our correlation value (r=0.139~-0.48) was relatively low because the association between sspg and each component of the mets was weaker than previously reported (r=0.39 - 0.59). However, a similar finding was that the two components most strongly associated with ir were obesity and high tg levels . However, the sensitivity to detect ir in subjects fulfilling the criteria for mets was low (table 4). In general, the predictive value increased as more components were included . Of interest, subjects with four of the criteria (all of the criteria excluding tg) had a relatively low positive predictive value of 33% . This finding may imply that subjects with mets and having the high tg concentrations may tend to have ir . Furthermore, in subjects fulfilled the mets criteria with three - component combinations, the predictive values of three - component combinations were 67 to 88% if bmi and hdl - c components were included . Oppositely, the rate of mets or ir and positive predictive rate were low if three - component combinations included any two of bmi, hdl - c, and bmi plus one of bp or fpg component . Furthermore, our data suggest that the prevalence of ir based on individual components of the mets was highest for bmi and tg (60% and 52%, respectively, data not shown). When combinations of two components were evaluated, the lowest positive predictive value for ir was for bp and fpg (0%); this supports the aforementioned findings . Further supportive data was that the association to ir was lower in bp and fpg than that of tg and bmi . However, the highest predictive value for ir was obtained by the combination of bmi and low hdl - c (64%, data not shown), though the weakest association was observed between ir and hdl - c . The different prevalence (67% vs. 14%) may explain why hdl - c had a higher predictive value than that of tg . Second, the relatively higher prevalence of low hdl - c levels in both sexes may need to be clarified . Last, we used bmi as an index of obesity with the same cut - off levels for both genders; this application might have influenced our results . Furthermore, the criteria of bmi might have contributed to the lower prevalence of mets in this study, and thus, the interpretation of results should be accomplished cautiously with the consideration of the atp iii criteria that include the criteria of waist circumstance . In conclusion, obesity and a high tg level were associated with ir and may be important markers for ir in subjects with mets . However, therefore, the criteria for mets may not be practical for determining whether subjects have ir . Meanwhile, in terms of the prevention of cvd, obesity and high tg levels might play an important role than other criteria in subjects with mets.
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Ocular complications of atopic dermatitis (ad) include blepharitis, keratoconjunctivitis, keratoconus, iritis, cataract, and retinal detachment.1 among these, retinal detachment is the severest complication that affects the younger population and significantly impairs their quality of life . Previous single center2 or multicenter3,4 retrospective studies declared the presence of a large number of retinal detachment patients with ad and increase in retinal detachment patients with ad until 1993.2,4 the increase in retinal detachment patients with ad was attributed to the marked increase in severely involved ad patients in late 1980s.57 however, no further detailed studies for retinal detachment with ad has been reported after 2000s . The prevalence of adolescent ad markedly increased from 1967 to 1996 in japan.8 after 2000, ad therapies improved by introduction of new therapeutic tools including the topical and systemic calcineurin inhibitors, and new therapeutic strategy: proactive therapies with topical calcineurin inhibitors and/or corticosteroids . Improvement of ad therapies decreased the prevalence of ad in 2006 compared with that in 1993.9 in japan, topical tacrolimus therapy is approved as an effective and well - tolerated treatment option for patients with ad.10 these recent changes in ad therapies might influence the prevalence of retinal detachment with ad . Therefore, ophthalmologists as well as dermatologists have great interest to the new trend in retinal detachment associated with ad . Herein, we report changes in surgically treated retinal detachment in mie university hospital, mie, japan, for these 20 years . The present study adhered to the tenets of the declaration of helsinki, and was approved by the institutional review board of the mie university graduate school of medicine (#2619). We retrospectively reviewed the records of 1,533 consecutive patients including ad patients with rhegmatogenous retinal detachment, who underwent retinal detachment surgery at mie university hospital during a 20-year period that ranged from january 1992 to december 2011 . The data set examined included patient s age, sex, affected eye, preoperative and postoperative best - corrected visual acuity, type of breaks, preoperative lens status, macula and the ciliary epithelium status, and the surgical results . We also reviewed the records of new ad outpatients consulted to the dermatology clinic at mie university hospital from 1993 to 2011 except 1998 . Cases with retinal detachments were divided into two groups: retinal detachments with ad (ad group) and without ad (non - ad group). Subsequently, the ad group was divided into two subgroups: patients group surgically treated former 10 years of the study period: from 1992 to 2001 (former ad group) and another group treated in recent 10 years from 2002 to 2011 (recent ad group). We also analyzed the annual changes in the number of retinal detachments with ad and the total retinal detachments (figure 1a and b). The number of the first visit ad outpatients to the dermatology clinic was also described in figure 1c . Data were analyzed between the ad and non - ad groups, or between the former ad and recent ad groups . With the exception of patient s age, all preoperative characteristics and reattachment rate were statistically analyzed using either a chi - square test or a fisher s exact probability test . A mann whitney u - test was used for analyses of the patient s age and visual acuity . Differences were considered as significant when the probability of their occurrence by chance was less than 5% . The characteristic differences between the two groups (ad group and non - ad group) are as shown in table 1 . The ratio of the bilateral detachment cases was significantly higher in the ad group than that in the non - ad group (p=0.0011). Patients in the ad group were significantly younger than those in the non - ad group (p<0.0001). The breaks were more frequently observed in the peripheral ocular fundus in the ad group compared with the non - ad group (p<0.0001). A greater number of eyes had macular detachments before surgery in the non - ad vs the ad group (p=0.0052). Ciliary epithelium detachment developed more frequently in the ad vs the non - ad group (p<0.0001). Although the reattachment rate after the first surgery in the ad group (76.2%, 77/101) appeared to be a little worse than that in the non - ad group (81.5%, 1,257/1,542), the difference in the rate of anatomical success was not statistically significant (p=0.2364). Final reattachment rate in the ad group (98.0%, 99/101) was almost the same as that in the non - ad group (98.1%, 1,513/1,542) (p=0.7157). In spite of no significant difference in the preoperative best - corrected visual acuity between the two groups, the postoperative best - corrected visual acuity was significantly better in the ad group compared with the non - ad group (p=0.0002; table 2). In the ad group, 51 eyes had cataract before retinal detachment surgery, and the 23 eyes out of the 51 eyes underwent cataract surgery during retinal detachment surgery . The characteristic differences between the two groups (former ad group and recent ad group) are as shown in table 3 . The bilateral detachment percentage was significantly higher in the former ad group than that in the recent ad group (p=0.0002). Patients in the recent ad group were significantly older than those in the former ad group (p=0.0084). No significant difference was identified between the groups for the type of break, lens status, macular status, or rate of ciliary epithelial detachment . The ratio of the retinal detachment with ad for the total retinal detachment was significantly lower in the recent ad group compared with the former ad group (p=0.0038). Although the reattachment rate after the first surgery in the former ad group (73.0%, 46/63) appeared to be a little worse than that in the recent ad group (81.6%, 31/38), the difference in the rate of anatomical success was not statistically significant (p=0.4589). Final reattachment rate in the former ad group (98.4%, 62/63) was almost the same as that in the recent ad group (97.4%, 37/38) (p>0.9999). The functional outcome, including preoperative best - corrected visual acuity and postoperative best - corrected visual acuity, showed no statistical difference between the former and recent ad groups (table 4). The number of retinal detachments with ad and the total number of retinal detachments are described in figure 1 . No apparent change was observed in the annual number of the total retinal detachments for these 20 years (figure 1b). In contrast, the number of retinal detachments with ad rapidly increased from 1992 to 1995 and continued until 2001 (figure 1a). Because of no apparent change in total retinal detachments, the rate of retinal detachment with ad in the total retinal detachment also increased from 1992, and it tapered in 2000s (data not shown). The number of new ad outpatients in our dermatology clinic linearly decreased in these 19 years (153 cases in 1993 and 65 cases in 2011, figure 1c). Their average age gradually increased during the study period (mean age of 19.41 years old in 1993 and 24.94 years old in 2011). The characteristic differences between the two groups (ad group and non - ad group) are as shown in table 1 . The ratio of the bilateral detachment cases was significantly higher in the ad group than that in the non - ad group (p=0.0011). Patients in the ad group were significantly younger than those in the non - ad group (p<0.0001). The breaks were more frequently observed in the peripheral ocular fundus in the ad group compared with the non - ad group (p<0.0001). A greater number of eyes had macular detachments before surgery in the non - ad vs the ad group (p=0.0052). Ciliary epithelium detachment developed more frequently in the ad vs the non - ad group (p<0.0001). Although the reattachment rate after the first surgery in the ad group (76.2%, 77/101) appeared to be a little worse than that in the non - ad group (81.5%, 1,257/1,542), the difference in the rate of anatomical success was not statistically significant (p=0.2364). Final reattachment rate in the ad group (98.0%, 99/101) was almost the same as that in the non - ad group (98.1%, 1,513/1,542) (p=0.7157). In spite of no significant difference in the preoperative best - corrected visual acuity between the two groups, the postoperative best - corrected visual acuity was significantly better in the ad group compared with the non - ad group (p=0.0002; table 2). In the ad group, 51 eyes had cataract before retinal detachment surgery, and the 23 eyes out of the 51 eyes underwent cataract surgery during retinal detachment surgery . The characteristic differences between the two groups (former ad group and recent ad group) are as shown in table 3 . The bilateral detachment percentage was significantly higher in the former ad group than that in the recent ad group (p=0.0002). Patients in the recent ad group were significantly older than those in the former ad group (p=0.0084). No significant difference was identified between the groups for the type of break, lens status, macular status, or rate of ciliary epithelial detachment . The ratio of the retinal detachment with ad for the total retinal detachment was significantly lower in the recent ad group compared with the former ad group (p=0.0038). Although the reattachment rate after the first surgery in the former ad group (73.0%, 46/63) appeared to be a little worse than that in the recent ad group (81.6%, 31/38), the difference in the rate of anatomical success was not statistically significant (p=0.4589). Final reattachment rate in the former ad group (98.4%, 62/63) was almost the same as that in the recent ad group (97.4%, 37/38) (p>0.9999). The functional outcome, including preoperative best - corrected visual acuity and postoperative best - corrected visual acuity, showed no statistical difference between the former and recent ad groups (table 4). The number of retinal detachments with ad and the total number of retinal detachments are described in figure 1 . No apparent change was observed in the annual number of the total retinal detachments for these 20 years (figure 1b). In contrast, the number of retinal detachments with ad rapidly increased from 1992 to 1995 and continued until 2001 (figure 1a). Because of no apparent change in total retinal detachments, the rate of retinal detachment with ad in the total retinal detachment also increased from 1992, and it tapered in 2000s (data not shown). The number of new ad outpatients in our dermatology clinic linearly decreased in these 19 years (153 cases in 1993 and 65 cases in 2011, figure 1c). Their average age gradually increased during the study period (mean age of 19.41 years old in 1993 and 24.94 years old in 2011). The number of new ad outpatients in our dermatology clinic linearly decreased in these 19 years (153 cases in 1993 and 65 cases in 2011, figure 1c). Their average age gradually increased during the study period (mean age of 19.41 years old in 1993 and 24.94 years old in 2011). Retinal detachment is a serious eye disease that impairs quality of life of the patients, and a significant increase in cases with retinal detachment in ad patients has been reported in 1990s . By advance in understanding of ad pathogenesis and improvement of therapies, prognosis of the severely involved ad patients has been improved . To know recent changes in prevalence of retinal detachment including with / without ad, we investigated the surgically treated patients with retinal detachment during these 20 years in our hospital . Reported retinal detachment cases associated with ad are characterized with common bilateral involvement and development in the younger age compared with the cases without ad . The causative break is frequently located in the peripheral ocular fundus, with the ciliary epithelium found to be detached in many of the cases.24,11 these characteristics are also found in the present study . In addition, the present results also demonstrated that postoperative visual functions in ad patients were better than those found in the non - ad patients (table 2). These findings might be resulted from younger age and higher ratio of preoperative macular attachment in the affected eyes in ad patients compared with non - ad patients . In the present data, there was no significant difference in the preoperative best - corrected visual acuity between the ad and non - ad groups in spite of higher ratio of preoperative macular attachment in the ad group . The discrepancy might be resulted from high ratio of preoperative cataract in ad patients . In the present study, the number of retinal detachments with ad markedly varied in these 20 years . It rapidly increased from 1992 to 1993, remained same levels until 2001, and gradually decreased year by year after 2001 . The annual number of the total retinal detachments showed neither particular increase nor decrease for 20 years with minimum yearly changes . Then, the rate of retinal detachments with ad in the total retinal detachments was evaluated . It increased in early 1990s and decreased in 2000s as well as the number of retinal detachments with ad . Thus, we concluded that the prevalence of retinal detachments with ad rapidly increased in early 1990s and decreased in 2000s . Cases with bilateral retinal detachment decreased in the recent 10 years, and this contributed to the decrease in the number of retinal detachments with ad in part . It is easy to suspect that bilateral involvement has resulted from severe ad inflammation on the face . Unfortunately, the disease activities of ad in these surgically treated cases have not been fully recorded . A decrease in ad patients is a suspicious factor for the noted decreases in retinal detachment with ad . In our present data as well as in a previous study,9 it is also noteworthy that these ad outpatients were referred to our clinic mainly because of uncontrolled ad . In addition, mie university hospital is the only core hospital in mie prefecture, and most refractory cases in this area have been referred to mie university hospital in these 20 years . Therefore, we think that the present data indicate gradual decrease in uncontrolled ad patients in mie prefecture . Thus, the long - lasting dermatitis results in resistant and uncontrolled ad.9 unfortunately, patients with steroid phobia significantly increased from early 1990s as a social phenomenon in japan . Around 2000, the japanese dermatological association declared the definition of ad, guideline for ad diagnosis and treatment and standard therapy campaign.12 simultaneously, topical calcineurin inhibitor tacrolimus has been introduced in japan as a new effective non - steroid and less - rosacea forming topical therapy in the end of 1999.13 additionally, a recently introduced new therapeutic strategy proactive therapy characterized with intermittent topical corticosteroid and/or calcineurin inhibitors associated with good skin care successfully controlled the relapse of ad.14,15 in 2008, systemic cyclosporine was approved for ad in japan, and it has made resistant ad cases more controllable.16 the decrease in retinal detachment with ad after 2001 and rapid fall off after 2008 might have relations to these new therapies in part . From these results, frequent rubbing of the eyes in the setting of ad might be one of the causative factors explaining a higher incidence of retinal detachment in ad patients . Another striking change noted during the 20-year observation period was the change in age for retinal detachment with ad . During the recent 10 years, patients were found to be significantly older than those observed during the former 10 years (table 3). Both the previous study9 and our current data for the new ad outpatients in the dermatology clinic have shown that the age of ad patients has become older year by year . We speculate that there has been an increased onset of ad in older subjects, with this subsequently leading to the onset of retinal detachment with ad . The present data for retinal detachment with ad during the 20-year observation period revealed no particular changes in ophthalmological manifestations of the affected eye itself, including for the type of breaks, lens status, macular status, or ciliary epithelial detachment . The present study failed to detect any improvement in the anatomical or functional results of the retinal detachment surgeries in these 20-year periods . We hope that the recent refinements in the vitrectomy system will contribute to the surgical results in future . The present study revealed increased retinal detachment with ad in 1990s followed by a gradual decrease in 2000s . Various factors including standard therapy for ad campaign, proactive therapy, and topical and systemic calcineurin inhibitor might contribute to good control of ad disease activities and consequent decrease in retinal detachment with ad . It is clear that further studies for ophthalmological and dermatological analysis for retinal detachment with ad in a larger scale are required.
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The stage 1 samples for the gwa scan included 1,999 t2d case subjects and 1,976 nondiabetic control subjects drawn from the nutrition and health of aging population in china (nhapc) study (312 case and 815 control subjects), the gut microbiota and obesity study (gmos) (82 case and 163 control subjects), the fudan - huashan study (807 case and 339 control subjects), and the beijing diabetes survey (798 case and 659 control subjects). The stage 2 samples for replication testing of 96 single nucleotide polymorphisms (snps) consisted of 13,517 unrelated chinese hans (6,570 t2d case and 6,947 nondiabetic control subjects) from the guizhou - bijie type 2 diabetes study (gbtds), the beijing type 2 diabetes studies (btds), and the hubei type 2 diabetes studies (htds). The stage 3 replication testing of 10 snps is carried out by de novo genotyping in 3,410 t2d patients and 3,412 nondiabetic control subjects from the shanghai diabetes inpatient database (sdiid) and the shanghai diabetes study (sds) and in silico replication in the agen - t2d gwas datasets (6,952 t2d case and 11,865 control subjects). There was no overlap of participants among stage 1, 2, and 3 samples . Studies of t2d - related quantitative traits were performed among 3,614 participants from the nhapc and gmos, the two population - based studies . All studies were approved by local ethics committees of each participating institution, and informed written consent was obtained from all participants . A summary of the contributing studies, criteria for t2d case and nondiabetic control subjects in each study, and sample characteristics can be found in the supplementary materials and methods and supplementary table 1 . The stage 1 dna samples were genotyped using the illumina human660w - quad beadchip (illumina, inc ., san diego, ca). Quality control (qc) filters were applied at the individual and snp levels . At the individual level, we removed the samples that met any of the following criteria: 1) call rates <97% (n = 20); 2) with excessive heterozygosity; 3) with sex mismatches between the reported and genetically inferred (n = 69); and 4) with unexpected duplicates (n = 19) or cryptic relatives (n = 149). We also removed population outliers (n = 6) detected by using principle component analysis (13) (supplementary fig . We excluded the copy number variation related snps, the snps in y and mitochondrial chromosomes, and the snps if they had: 1) call rate <95% (n = 1,351); 2) minor allele frequency <05% (n = 63,263); and 3) hardy - weinberg equilibrium (hwe) p <10 in control groups (n = 1,047). The samples that passed all qc criteria were then used to impute for the ungenotyped or missing snps from the phase 2 hapmap chb+jpt (release number 22) reference panel using impute (version 2.1.2; http://mathgen.stats.ox.ac.uk/impute/impute.html) software (14). We removed all imputed snps with an estimated call rate <99%, minor allele frequency <1%, hwe p <10, or the info measure 0.5 . For stage 2 replication, we genotyped 96 snps selected from stage 1 in three independent chinese han populations with 6,570 t2d case subjects and 6,947 control subjects from the gbtds, btds, and htds studies using taqman snp genotyping assays (applied biosystems, foster city, ca) in the fludigm ep1 platform . We excluded the samples with call rate <93% and the snps with call rate <95% or deviation from hwe at p <5.2 10 (bonferroni corrected p value for 96 tests) in control groups . The overall call rates were 99.7, 99.4, and 99.4% for samples from the gbtds, btds, and htds studies, respectively . We genotyped> 5% duplicate samples for each study to assess genotyping reproducibility, and the concordance rates were 99.9% for 189 duplicate samples from the gbtds and 99.5% for 387 duplicate samples from btds and for 166 duplicate samples from htds . We also performed cross - platform validation by genotyping the 96 snps in 135 stage 1 samples that had been run on the illumina assay using taqman snp genotyping assays in fludigm ep1 platform, and the concordance rate was 99.9% . Finally, 10 snps, selected based on results of meta - analysis that combined stage 1 and 2 data, were used for stage 3 replication, including a de novo genotyping in 3,410 t2d case and 3,412 control subjects from the sdiid / sds study and an in silico replication in 8 independent gwas datasets of 6,952 case and 11,865 control subjects from the agen consortium . The de novo genotyping was performed using sequenom massarray (sequenom, san diego, ca) in the sdiid / sds study . After quality control, 3,257 t2d case and 3,262 control subjects with genotyping call rate> 90% were used for association analyses . The in silico replication results were obtained by directly searching the meta - analysis results of 8 independent gwas datasets with 6,952 case and 11,865 control subjects in total that participated in the discovery stage of the agen - t2d consortium . For stage 1 gwas, the genotyped snps (495,686) and imputed snps (1,738,508) were tested for their associations with t2d using plink (version 1.07; http://pngu.mgh.harvard.edu/~purcell/plink/) (15), and snptest (version 2.2.0; https://mathgen.stats.ox.ac.uk/genetics_software/snptest/snptest.html) (14), respectively . The odds ratios (ors) and 95% cis were estimated by logistic regression under an additive genetic model, adjusting for age, sex, and the first two principal components in model 1 . For the x - chromosome snps, males were treated as homozygotes, in that genotypes were coded as 2 for the coded allele (16). The initial association analyses were performed by pooling the data from the beijing and shanghai participants and then stratified by geographical regions (beijing and shanghai) to test whether the pooling would introduce a bias due to potential population stratification . The p values were adjusted for genomic control inflation factor (gc). To find more consistent evidence for associations between each snps and t2d, we further stratified the association analyses by sex or tested the associations between the case subjects with family history and the nondiabetic control subjects . The snp selection for stage 2 replication was largely based on the p values in the discovery stage, and one or two snps with the smallest p values were chosen for each significant genomic region, defined as that contained a set of snps in linkage disequilibrium (ld) at r 0.1 with the most associated snp . For stage 2 and 3 replication analyses, logistic regression analysis was applied to test association of each snp with t2d, assuming an additive genetic effect and adjusting for age and sex . The effect sizes across studies or stages were combined using fixed - effect inverse variance weighted meta - analysis, and the p values were calculated using a fixed effects meta - analysis with sample size weighted z - score . We also examined the effect of t2d - associated snps on t2d - related quantitative traits in participants from the nhapc and gmos studies, of which the individuals receiving glucose - lowering treatment and gmos participants with bmi 28 were excluded from analyses (supplementary table 10). The nhapc samples were genotyped using the illumina human660w - quad beadchip (illumina, inc .) Using the stage 1 qc criteria, and the gmos samples were genotyped using taqman snp genotyping assays in fludigm ep1 platform . Finally, a total of 3,614 samples (2,229 from shanghai and 1,385 from beijing) that passed all qc criteria were used for the association analyses, which were performed in the shanghai and beijing samples separately using linear regression analysis assuming an additive genetic model, adjusting for age, sex, and study covariates (nhapc and gmos for the shanghai samples only). Values of insulin and homeostasis model assessment of -cell function (homa - b) and insulin sensitivity (homa - s) were natural log - transformed before analysis, and results of each study were combined by fixed - effect inverse variance weighted meta - analysis . Homa - s and homa - b were estimated by the homa model using levy s computer model (18). For cis - eqtl analysis, we first searched rs10814916, rs10886471, rs7403531, and their proxy snps (r> 0.5) in the genevar eqtl database, including 166 adipose tissues, 156 lymphoblastoid cell lines, and 160 skin tissues derived from a subset of healthy female twins of the muther resource (19). Associations between each snp and mrna expression level of nearby genes were obtained by using spearman rank correlation for <10,000 permutations as the default settings of genevar (version 3.1.1; http://www.sanger.ac.uk/resources/software/genevar/). We performed gkr5 mrna expression analysis in blood samples of 64 unrelated chinese hans recruited from the peking union medical college hospital, chinese academy of medical sciences, in december 2011 . All participants provided written informed consent, and study protocol was approved by the local ethics committee . Genomic dna and rna samples were extracted from peripheral blood by using a qiagen kit (qiagen) and rna pure blood kit (cowin biotech co.), respectively . The genotype of g - protein coupled receptor (gpcr) kinase 5 (grk5)-rs10886471 was determined by sequencing, using the following primers: 5-tcctacactggaacaagcc-3 and 5-acggactaatacagacggg-3. Quantitative real - time rt - pcr for grk5-rs10886471 was performed using the goldstar taqman mixture kit (cowin biotech co.) on a bio - rad iq5 system (bio - rad). Grk5 we carried out statistical analysis for expression data using linear regressions and an unpaired student t test . P value from linear regression was calculated by assuming a dominant genetic effect of minor allele of rs10886471 and adjusted for the status of t2d . The genomic regions for the grk5 and rasgrp1 loci were selected based on their regional ld plots (supplementary figs . 6 and 7). The varld algorithm is used to compare difference in the regional ld patterns among phase 2 hapmap chb samples (45 chinese hans), ceu samples (60 white europeans), and jpt samples (45 japanese), and results were presented as monte carlo p values (20). The stage 1 samples for the gwa scan included 1,999 t2d case subjects and 1,976 nondiabetic control subjects drawn from the nutrition and health of aging population in china (nhapc) study (312 case and 815 control subjects), the gut microbiota and obesity study (gmos) (82 case and 163 control subjects), the fudan - huashan study (807 case and 339 control subjects), and the beijing diabetes survey (798 case and 659 control subjects). The stage 2 samples for replication testing of 96 single nucleotide polymorphisms (snps) consisted of 13,517 unrelated chinese hans (6,570 t2d case and 6,947 nondiabetic control subjects) from the guizhou - bijie type 2 diabetes study (gbtds), the beijing type 2 diabetes studies (btds), and the hubei type 2 diabetes studies (htds). The stage 3 replication testing of 10 snps is carried out by de novo genotyping in 3,410 t2d patients and 3,412 nondiabetic control subjects from the shanghai diabetes inpatient database (sdiid) and the shanghai diabetes study (sds) and in silico replication in the agen - t2d gwas datasets (6,952 t2d case and 11,865 control subjects). There was no overlap of participants among stage 1, 2, and 3 samples . Studies of t2d - related quantitative traits were performed among 3,614 participants from the nhapc and gmos, the two population - based studies . All studies were approved by local ethics committees of each participating institution, and informed written consent was obtained from all participants . A summary of the contributing studies, criteria for t2d case and nondiabetic control subjects in each study, and sample characteristics can be found in the supplementary materials and methods and supplementary table 1 . The stage 1 dna samples were genotyped using the illumina human660w - quad beadchip (illumina, inc ., san diego, ca). Quality control (qc) filters were applied at the individual and snp levels . At the individual level, we removed the samples that met any of the following criteria: 1) call rates <97% (n = 20); 2) with excessive heterozygosity; 3) with sex mismatches between the reported and genetically inferred (n = 69); and 4) with unexpected duplicates (n = 19) or cryptic relatives (n = 149). We also removed population outliers (n = 6) detected by using principle component analysis (13) (supplementary fig . 1). At the snp level, we excluded the copy number variation related snps, the snps in y and mitochondrial chromosomes, and the snps if they had: 1) call rate <95% (n = 1,351); 2) minor allele frequency <05% (n = 63,263); and 3) hardy - weinberg equilibrium (hwe) p <10 in control groups (n = 1,047). The samples that passed all qc criteria were then used to impute for the ungenotyped or missing snps from the phase 2 hapmap chb+jpt (release number 22) reference panel using impute (version 2.1.2; http://mathgen.stats.ox.ac.uk/impute/impute.html) software (14). We removed all imputed snps with an estimated call rate <99%, minor allele frequency <1%, hwe p <10, or the info measure 0.5 . For stage 2 replication, we genotyped 96 snps selected from stage 1 in three independent chinese han populations with 6,570 t2d case subjects and 6,947 control subjects from the gbtds, btds, and htds studies using taqman snp genotyping assays (applied biosystems, foster city, ca) in the fludigm ep1 platform . We excluded the samples with call rate <93% and the snps with call rate <95% or deviation from hwe at p <5.2 10 (bonferroni corrected p value for 96 tests) in control groups . The overall call rates were 99.7, 99.4, and 99.4% for samples from the gbtds, btds, and htds studies, respectively . We genotyped> 5% duplicate samples for each study to assess genotyping reproducibility, and the concordance rates were 99.9% for 189 duplicate samples from the gbtds and 99.5% for 387 duplicate samples from btds and for 166 duplicate samples from htds . We also performed cross - platform validation by genotyping the 96 snps in 135 stage 1 samples that had been run on the illumina assay using taqman snp genotyping assays in fludigm ep1 platform, and the concordance rate was 99.9% . Finally, 10 snps, selected based on results of meta - analysis that combined stage 1 and 2 data, were used for stage 3 replication, including a de novo genotyping in 3,410 t2d case and 3,412 control subjects from the sdiid / sds study and an in silico replication in 8 independent gwas datasets of 6,952 case and 11,865 control subjects from the agen consortium . The de novo genotyping was performed using sequenom massarray (sequenom, san diego, ca) in the sdiid / sds study . After quality control, 3,257 t2d case and 3,262 control subjects with genotyping call rate> 90% were used for association analyses . The in silico replication results were obtained by directly searching the meta - analysis results of 8 independent gwas datasets with 6,952 case and 11,865 control subjects in total that participated in the discovery stage of the agen - t2d consortium . For stage 1 gwas, the genotyped snps (495,686) and imputed snps (1,738,508) were tested for their associations with t2d using plink (version 1.07; http://pngu.mgh.harvard.edu/~purcell/plink/) (15), and snptest (version 2.2.0; https://mathgen.stats.ox.ac.uk/genetics_software/snptest/snptest.html) (14), respectively . The odds ratios (ors) and 95% cis were estimated by logistic regression under an additive genetic model, adjusting for age, sex, and the first two principal components in model 1 . Bmi was further included as a covariate in model 2 . For the x - chromosome snps, males were treated as homozygotes, in that genotypes were coded as 2 for the coded allele (16). The initial association analyses were performed by pooling the data from the beijing and shanghai participants and then stratified by geographical regions (beijing and shanghai) to test whether the pooling would introduce a bias due to potential population stratification . The p values were adjusted for genomic control inflation factor (gc). To find more consistent evidence for associations between each snps and t2d, we further stratified the association analyses by sex or tested the associations between the case subjects with family history and the nondiabetic control subjects . The snp selection for stage 2 replication was largely based on the p values in the discovery stage, and one or two snps with the smallest p values were chosen for each significant genomic region, defined as that contained a set of snps in linkage disequilibrium (ld) at r 0.1 with the most associated snp . For stage 2 and 3 replication analyses, logistic regression analysis was applied to test association of each snp with t2d, assuming an additive genetic effect and adjusting for age and sex . The effect sizes across studies or stages were combined using fixed - effect inverse variance weighted meta - analysis, and the p values were calculated using a fixed effects meta - analysis with sample size weighted z - score . We also examined the effect of t2d - associated snps on t2d - related quantitative traits in participants from the nhapc and gmos studies, of which the individuals receiving glucose - lowering treatment and gmos participants with bmi 28 were excluded from analyses (supplementary table 10). The nhapc samples were genotyped using the illumina human660w - quad beadchip (illumina, inc .) Using the stage 1 qc criteria, and the gmos samples were genotyped using taqman snp genotyping assays in fludigm ep1 platform . Finally, a total of 3,614 samples (2,229 from shanghai and 1,385 from beijing) that passed all qc criteria were used for the association analyses, which were performed in the shanghai and beijing samples separately using linear regression analysis assuming an additive genetic model, adjusting for age, sex, and study covariates (nhapc and gmos for the shanghai samples only). Values of insulin and homeostasis model assessment of -cell function (homa - b) and insulin sensitivity (homa - s) were natural log - transformed before analysis, and results of each study were combined by fixed - effect inverse variance weighted meta - analysis . Homa - s and homa - b were estimated by the homa model using levy s computer model (18). For cis - eqtl analysis, we first searched rs10814916, rs10886471, rs7403531, and their proxy snps (r> 0.5) in the genevar eqtl database, including 166 adipose tissues, 156 lymphoblastoid cell lines, and 160 skin tissues derived from a subset of healthy female twins of the muther resource (19). Associations between each snp and mrna expression level of nearby genes were obtained by using spearman rank correlation for <10,000 permutations as the default settings of genevar (version 3.1.1; http://www.sanger.ac.uk/resources/software/genevar/). We performed gkr5 mrna expression analysis in blood samples of 64 unrelated chinese hans recruited from the peking union medical college hospital, chinese academy of medical sciences, in december 2011 . All participants provided written informed consent, and study protocol was approved by the local ethics committee . Genomic dna and rna samples were extracted from peripheral blood by using a qiagen kit (qiagen) and rna pure blood kit (cowin biotech co.), respectively . The genotype of g - protein coupled receptor (gpcr) kinase 5 (grk5)-rs10886471 was determined by sequencing, using the following primers: 5-tcctacactggaacaagcc-3 and 5-acggactaatacagacggg-3. Quantitative real - time rt - pcr for grk5-rs10886471 was performed using the goldstar taqman mixture kit (cowin biotech co.) on a bio - rad iq5 system (bio - rad). Grk5 we carried out statistical analysis for expression data using linear regressions and an unpaired student t test . P value from linear regression was calculated by assuming a dominant genetic effect of minor allele of rs10886471 and adjusted for the status of t2d . The genomic regions for the grk5 and rasgrp1 loci were selected based on their regional ld plots (supplementary figs . 6 and 7). The varld algorithm is used to compare difference in the regional ld patterns among phase 2 hapmap chb samples (45 chinese hans), ceu samples (60 white europeans), and jpt samples (45 japanese), and results were presented as monte carlo p values (20). In stage 1, we tested the association with t2d of 2,234,194 genotyped and imputed snps that passed all qc criteria in 3,712 chinese hans from beijing and shanghai, of which 1,839 were case subjects, and 1,873 were control subjects . No significant population stratification was observed between case and control subjects using principle component analysis (supplementary fig . 2), and the genomic control factor was also quite close to 1.0 (= 1.03), suggesting there is no genome - wide inflation due to population stratification for stage 1 samples . The quantile quantile plot showed substantial deviation from the expected at the lower p values, suggesting that some associations were more significant than expected by chance (supplementary fig . 3). Snps in four previously reported t2d loci (cdkal1, cdkn2a / b, kcnq1, and dusp9) reached genome - wide significance (p <5 10) (fig . 2 and supplementary fig . 4), with directional consistency as previously reported (2125). For the other previously reported t2d loci, apart from three loci that were monomorphic in chinese hans, 48 of 51 loci showed directionally consistent association with t2d (binomial test, p = 9.8 10), of which 19 were associated with t2d at p <0.05 (supplementary table 3). Of these, only the rs7754840 in cdkal1 showed a significantly larger effect on t2d in chinese hans than in white europeans (p for heterogeneity = 3.33 10), consistent with our previous findings (26). Manhattan plot for genome - wide association analysis of 495,686 genotyped snps in stage 1 . The log10 p values were from pooled analysis, adjusting for age, sex, and the first two principle components . The red dots at each locus indicate the signals with p <10 . For follow - up, we chose one or two snps per locus that had the smallest p values in stage 1, except for the two strongest association signals at cdkal1 and kcnq1 (p 4.24 10, or = 1.37), for which association with t2d has been repeatedly confirmed in east asian populations . As such, the 96 most significant snps representing 84 independent loci were selected for stage 2 replication (supplementary tables 2 and 4), which consisted of de novo genotyping of the snps in three independent chinese han populations with 6,570 t2d case and 6,947 control subjects (supplementary materials and methods and supplementary table 1). All snps, except for slc30a8-rs13266634 that showed significant deviation from hwe, passed the qc criteria and were included in the replication analyses . A total of eight snps in or near rasgrp1, glis3, cdkn2b, cdc123, hhex, hnf1b, fam58a, and dusp9 were significantly associated with t2d after bonferroni correction for multiple tests in the stage 2 samples, and the p value cutoff for bonferroni correction was 5.26 10 (0.05/95) (supplementary table 5). In meta - analyses combining stage 1 and 2 data, the snps in or near rasgrp1, cdkn2b, cdc123, hhex, hnf1b, fam58a, and dusp9 reached the genome - wide significance (supplementary table 5), of which cdkn2b (24,25,27), hhex (24,25,27), cdc123 (28), hnf1b (29), and dusp9 (29) are established t2d loci . Rs10814916 (p = 5.29 10) at glis3 and rs10886471 (p = 5.92 10) at grk5 were close to the genome - wide significance threshold (supplementary table 5). The association between glis3 variant (rs7034200) and t2d risk had been observed in a previous candidate gene association study (30), but the evidence was inconclusive . The snp rs12010175 in fam58a is in modest ld with rs5945326 near dusp9 (r = 0.35). Conditional analyses by including the two snps in one logistic regression model revealed that these two snps represent the same locus (p adjusted for rs5945326 = 0.02). Apart from the snps that represent previously established t2d loci, 10 snps (each representing independent loci) showed association with t2d at p <5 10 in a meta - analysis that combined stage 1 and 2 data, and no additional novel signal (p <5 10) was observed when the association analyses were stratified by sex (supplementary table 6). These 10 snps were taken forward for replication in stage 3, which included a de novo genotyping replication in 3,410 t2d case and 3,412 control subjects of chinese hans and an in silico replication in a previously published meta - analysis of gwas of 6,952 t2d case and 11,865 control subjects (including 4,026 case and 4,654 control subjects from chinese hans and the remaining 2,926 case and 7,211 control subjects from korean, japanese, malay, and filipino populations) of the agen - t2d consortium (5). A total of three snps at glis3, grk5, and rasgrp1, respectively, exceeded the genome - wide significance threshold (combined p values: 7.1 10 to 6.0 10; or: 1.101.12) when we combined the data across all three stages (table 1 and supplementary table 7). We found no evidence of heterogeneity for the associations at these three loci across all study populations (p for heterogeneity 0.26) or between chinese hans and other east asians (p for heterogeneity 0.14) (supplementary table 8). While our in silico replication was ongoing, glis3 was reported as a t2d locus by the agen - t2d consortium (5). The association signal (rs10814916) at glis3 in our study is in high ld (r = 0.93) with the snp (rs7041847) identified in the agen - t2d meta - analysis . As such, we only consider grk5 and rasgrp1 as the novel t2d loci identified by our study . Snps reaching genome - wide significance in a meta - analysis of combined stages 1, 2, and 3 the association between rasgrp1-rs7403531 and t2d was confirmed by results from diagram plus gwas meta - analysis (22) (8,130 t2d case and 38,987 control subjects of european origin), with comparable effect size and consistent direction (p = 0.023; or = 1.06; p for heterogeneity between east asians and europeans = 0.22), and two imputed snps (rs8043085 and rs12593201, r 0.8 with rs7403531) at this locus even showed a stronger association (p 3.82 10; or = 1.07) in european populations from the diagram consortium (supplementary tables 8 and 13). However, the t2d association for the grk5 locus was not replicated in the diagram plus gwas datasets (rs10886471: p = 0.352, or = 0.98; p for heterogeneity between east asians and europeans = 4.42 10) (supplementary tables 8 and 13). To examine whether adiposity was a potential mediator in the association of the two novel loci and t2d, we tested the association with further adjustment for bmi in populations across all stages . The effect sizes for each snp remained largely unchanged (p adjusted for bmi = 1.73 10 to 2.87 10; or = 1.101.12) (supplementary table 9), suggesting that their associations with t2d were not mediated through adiposity . We further examined associations for t2d - related quantitative traits in 3,614 participants (normal fasting glucose: 2,142; impaired fasting glucose: 1,211; and t2d: 261) from the nhapc and the gmos studies, two population - based studies of chinese hans . Risk allele of the grk5-rs10886471 was also associated with higher fasting plasma insulin levels (p = 0.0204), but not with fasting plasma glucose, whereas risk allele of the rasgrp1-rs7403531 was also significantly associated higher fasting plasma glucose levels (p = 0.0213) and lower homa - b (p = 0.0027) (supplementary table 11). Similar results were observed when the association analyses were performed in normal fasting glucose individuals only (supplementary table 11). The cis - eqtl (cis - expression quantitative loci) analysis using the genevar eqtl database (19) (http://www.sanger.ac.uk/resources/software/genevar/) showed suggestive evidence of association between an intronic snp (rs4752300, r = 0.79 with rs10886471) in grk5 and grk5 mrna expression levels at p = 6 10 (r = 0.361 between the risk allele and grk5 expression levels) in adipose tissue . However, no evidence was found for associations between snps at rasgrp1 and mrna expression levels of their nearby genes . To further confirm whether rs10886471 genotypes modify grk5 mrna expression, the grk5 expression levels were estimated using quantitative real - time rt - pcr in peripheral blood mrna samples from 64 unrelated chinese hans, including 30 t2d case subjects and 34 nondiabetic control subjects . The t2d risk - increasing c - allele of rs10886471 was significantly associated with increased mrna expression levels of grk5 (p[dom] = 0.02) with the same direction of effect in both t2d case subjects and control subjects, consistent with the finding in the genevar eqtl database . Moreover, the grk5 expression levels in t2d case subjects increased by 40% compared with their nondiabetic counterparts (p = 0.0048) (figs . 3 and 4 and supplementary table 12). These results suggested that rs10886471 risk c - allele might contribute to t2d risk through increased grk5 expression, but should be interpreted cautiously because that blood is not a very likely tissue to be involved in t2d . Regional plots of two novel t2d loci . A and b: imputed snps were estimated by mach software (http://www.sph.umich.edu/csg/abecasis/mach/) using ld information from 194 asians (including 68 chb, 25 chs, 84 jpt, and 17 mxl) in 1000 genome 201008 release as references . P values were from pooled analysis, adjusting for age, sex, and the first two principle components in stage 1 samples . The regional plots for the 500-kb region centered on index snps were generated by using locuszoom (http://csg.sph.umich.edu/locuszoom/). The log10 p values of snps were plotted against their genomic position (national center for biotechnology information build 37). The positions of genes were annotated from the university of california santa cruz genome browser by using grch37 assembly . Other snps are colored according to their ld (r) with the index snp from 1000 genome asn . The recombination rate is shown as a light blue line to reflect the local ld structure . The relative expression levels of grk5 were measured in blood samples from 30 t2d case subjects (rs10886471 genotype: tt+tc / n = 11; cc / n = 19) and 34 nondiabetic control subjects (rs10886471 genotype: tt+tc / n = 13; cc / we compared the ld structure of the two novel t2d loci between chinese hans and europeans and japanese (supplementary figs . 6 and 7a), respectively . Different pairwise ld patterns were observed between chinese hans and europeans at both grk5 (p = 0.0432) and rasgrp1 (p = 0.0043) loci, but not between chinese hans and japanese (p 0.101) (supplementary table 14). The risk allele frequencies of both grk5-rs10886471 and rasgrp1-rs7403531 in east asians (0.79 and 0.33 in the hapmap chb population and 0.74 and 0.45 in the hapmap jpt population for rs10886471 and rs7403531, respectively) were also higher than those in europeans (0.48 and 0.28 for rs10886471 and rs7403531 in hapmap ceu population, respectively) (supplementary table 13). In this study, we not only confirmed more than 20 previously reported loci, but also identified two novel t2d loci: grk5 (rs10886471) and rasgrp1 (rs7403531). The snp rs10886471 maps to an ld block within intron 3 of the grk5 gene, the only gene in this ld block (supplementary fig . Regional plots showed that rs10886471 represents the strongest association signals at the grk5 locus (fig . Grk5 belongs to the gpcr kinase family and plays a crucial role in phosphorylation of multiple gpcrs and non - gpcr substrates, such as glucagon receptor (31), 2-adrenergic receptor (32,33), hsp70-interacting protein (34), and nuclear factor-b1/p105 (35), which are either key regulators of glucose homeostasis or inflammation . Disruption of grk5 leads to decreased production of multiple inflammatory cytokines / chemokines and decreased nuclear factor-b activation both in vivo and in vitro (36). The t2d risk - increasing allele of rs10886471 was also associated with higher grk5 mrna expression levels, higher fasting insulin, but not with fasting glucose, suggesting that it might impair insulin sensitivity by increasing inflammatory response and consequently contributes to t2d risk in chinese hans or east asians . Notably, grk5 is a population - specific t2d locus and the t2d association of grk5-rs10886471 was not replicated in populations of european origin . The risk allele frequency of grk5-rs10886471 in chinese hans was also much higher than in europeans (0.79 and 0.48 in hapmap chb and ceu populations, respectively), and the corresponding population attributable risk in chinese hans was estimated to be 8.66% . Moreover, there are significant different ld structure for grk5 (p = 0.0432) locus between chinese hans and europeans . The second novel association signal (rs7403531) is located at chr15q14 and in intron 2 of the rasgrp1 gene (supplementary fig . 7a), which has also been replicated in europeans from the diagram consortium (supplementary tables 8 and 13). Although no heterogeneity of effect was observed between chinese hans and europeans, the risk allele frequency of rs7403531 in chinese hans was moderately higher than in europeans (0.33 and 0.28 in hapmap chb and ceu populations, respectively). Regional plots showed that the strongest association signal at this locus was observed with an imputed snp rs12593201 (r = 0.81 with rs7403531) (fig . 3b and supplementary fig . 5), which also showed a stronger t2d association in the diagram plus gwas meta - analysis (p = 2.4 10) (supplementary table 13). Conditional analysis in stage 1 samples suggested that neither of the snps were likely to be the snp driving the association (p 0.11). Notably, rs7403531 was in modest ld (r = 0.46 in hapmap ceu population) with a variant (rs7171171) previously shown to be associated with type 1 diabetes (t1d) in populations of european ancestry (37), but they are not in ld in chinese hans (r = 0.03 in hapmap chb population) (supplementary fig . 7b), suggesting that the t2d association at rasgrp1 is unlikely to be driven by the t1d - associated snp . Rasgrp1 encodes the ras guanyl releasing protein 1 (rasgrp1) that functions as a guanine nucleotide exchange factor, which is required for the activation of ras / mitogen - activated protein kinase pathways (38) and critically mediates the development and function of both t and b lymphocytes (3942). Rasgrp1-deficient mice exhibit defects in lymphocyte proliferation (40,43,44), inflammatory cytokine production (43,45,46), and apoptosis (38). Rasgrp1 is highly expressed in lymphocytes but also in various other cells, including pancreatic -cells (47,48). Its dysfunction in -cells may lead to islet inflammation and impaired -cell function, which are considered as major factors involved in t2d pathogenesis (49,50). In accordance with this, the t2d risk - increasing allele of rasgrp1-rs7403531 was also associated higher plasma glucose and lower homa - b, suggesting that the t2d risk conferred by rs7403531 is likely mediated through an impaired -cell function . However, more functional studies are required to draw a firm conclusion . To test possible misclassification of t1d in our study, we also examined associations of all known t1d - associated variants with t2d in our stage 1 samples . In contrast to the multiple replicated t2d loci, very few of the t1d - associated variants showed a trend toward association with t2d (p values: 0.010.05, p for binomial test = 0.24) (supplementary table 15), suggesting a low misclassification rate in our stage 1 sample . Moreover, we observed a similar or (combined p = 1 10; or = 1.09) when we performed secondary association analyses for rasgrp1-rs7403531 in 5,678 t2d case and 8,438 control subjects from several hospital - based case - control studies, in which all t2d case subjects were negative for glutamic acid decarboxylase and insulin autoantibody tests . All of these results, together with the facts that all genotyped t2d case subjects in our study were> 30 years of age and had no prior history of t1d, suggested that the observed t2d association signals at rasgrp1 and grk5 were unlikely to be driven by t1d - associated variants or by case misclassification in our study . Taken together, this study is the largest gwas of t2d performed in chinese hans thus far . We have identified two novel loci (grk5 and rasgrp1) that are associated with t2d at genome - wide significant levels . In particular, the association signal at grk5 seems to be specific to east asians, but this finding needs to be confirmed in further studies . We have also confirmed the known t2d loci at kcnq1, cdkal1, cdkn2b, cdc123, hnf1b, glis3, and dusp9 at genome - wide significant levels . Our findings not only provide new insights into the pathophysiology of t2d, but may also shed light on the ethnic differences in t2d susceptibility.
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Primary chest wall chondrosarcoma is a relatively rare malignant tumor [1, 2, 3]. When a patient with a history of long - term asbestos exposure has a pleural tumor with multiple plaques on chest computed tomography (ct), malignant pleural mesothelioma is usually suspected . A 62-year - old man with a history of long - term asbestos exposure as a construction worker was found to have a chest wall tumor on chest ct and referred to our hospital . Chest ct showed a pleural tumor invading the sixth rib and multiple pleural plaques (fig . Additional wide resection of the chest wall was performed, that is, en bloc resection, including the fifth, sixth, and seventh ribs, with a 5-cm safety margin from the tumor along the sixth rib . The chest wall defect was 25 10 cm in size, and chest wall reconstruction was performed with a polypropylene mesh . Microscopic examination revealed a lobulated growth pattern of cartilaginous cells of histological grade 1 (fig . Although the margins are free of tumor cells, the tumor widely spread into the sixth rib, to 17 cm long . Chest wall chondrosarcoma is relatively rare [1, 2, 3]. To the best of our knowledge, this is the first reported case of chondrosarcoma of the rib mimicking malignant pleural mesothelioma . Two cases of extraskeletal mesenchymal chondrosarcoma mimicking malignant pleural mesothelioma have been reported [4, 5]. Goetz et al . Reported a case of myxoid chondrosarcoma with a history of asbestos exposure . In our case, the patient had a history of long - term asbestos exposure and multiple pleural plaques on chest ct . Additionally, diagnosis of malignant pleural mesothelioma by using fnac is insufficient because it cannot differentiate malignant pleural mesothelioma from adenocarcinoma . Chondrosarcoma is relatively radio insensitive, and no effective chemotherapy has been established so far . Thus, complete surgical resection is the only curative option for chondrosarcoma [1, 2]. Reported that local recurrence rate was highly dependent on surgical margins . Therefore, wide resection with adequate safety margin is recommended . In our case, the tumor appeared as small as 3 cm on chest ct, but the tumor cells widely spread into the sixth rib, to 17 cm long . Because it is difficult to diagnose the free margin of the rib in intraoperative frozen sections, preoperative planning for wide resection of the chest wall is important . Chest wall reconstruction is recommended when at least three ribs are resected or when the resected size of the chest wall defect is more than 10 cm in order to avoid pulmonary complications . Our case suggests that it is difficult to diagnose chondrosarcoma in preoperative imaging diagnosis, and wide resection is necessary for curative treatment.
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Soft tissue recessions frequently cause esthetic disharmony and dissatisfaction . Compared with soft tissue coverage around a tooth, the coverage of an implant site is obviously unpredictable . Particularly in the cases of thin mucosa, a significant greater amount of recession takes place compared to thick mucosa . To overcome this problem, this case report demonstrates a two - step mucosal dehiscence coverage technique for an endosseous implant . A 33-year - old female visited us with the chief complaint of dissatisfaction with the esthetics of an exposed implant in the maxillary left cental incisor region . A subepithelial connective tissue graft was positioned in the apical site of the implant and covered by a mucosal flap with normal tension . At 12 months after surgery, the recipient site was partially covered by keratinized mucosa . However, the buccal interdental papilla between implant on maxillary left central incisor region and adjacent lateral incisor was concave in shape . To resolve the mucosal recession after the first graft an esthetically satisfactory result was achieved and the marginal soft tissue level was stable 9 months after the second graft . This two - step approach has the potential to improve the certainty of esthetic results . Soft tissue recessions around dental implants have frequently been observed . A recession during the early phase after implant crown placement originates from the process of modeling of the peri - implant mucosa . Reported that bone resorption and soft tissue recession were manifested as 0.7 and 0.6 mm at the buccal aspect of the implants during the period between implant placement and abutment connection . Muller et al . Has suggested that thin mucosa is friable and recesses more readily following mechanical stress and surgical procedures than does thick mucosa . Furthermore, the quantity, quality, and position of the existing peri - implant bone also affects soft tissue recession . Burkhardt et al . Surgically covered soft tissue recessions using a coronally advanced flap (caf) in combination with a free connective tissue graft although the implant sites revealed a substantial, clinically significant improvement following coronal mucosal displacement, clinically significant soft tissue shrinkage was observed after one month of healing . We hypothesized that a two - step approach is useful in acquisition of esthetic results because the first graft can increase the thickness of the peri - implant mucosa . The present case demonstrates a two - step mucosal dehiscence coverage technique in an endosseous implant . Sufficient soft tissue regeneration was achieved and the marginal soft tissue level was stable with an esthetically satisfactory result . The patient was a 33-year - old female who visited us on october 2008 with a chief complaint of esthetic dissatisfaction in the exposed implant of the maxillary left cental incisor region . Periodontal examination revealed a healthy peri - implant condition with a probing depth ranging from 2 to 3 mm and satisfactory oral hygiene was observed . There was 3 mm abutment exposure in the buccal mucosa around the implant (fig . An intraoral periapical radiograph and cone beam computed tomography were taken to evaluate peri - implant bone resorption . Because only 1 mm of marginal peri - implant bone resorption was observed (fig . 2) and the implant was a bit buccally angulated, no bone graft or removal of the implant was performed, but it was decided to carry out only soft tissue augmentation to restore esthetics . Following local anesthesia, a circumferential partial - thickness incision was performed using a ck3 stainless steel blade (blade round tip angled 10 deg, swan analytical usa inc ., wheeling, il, usa) to achieve a 17 mm wide12 mm height pouch around the dehiscence (fig . 3). A 106 mm subepithelial connective tissue graft (sctg) was harvested from the palate in the right second premolar to second molar region . The graft tissue was trimmed to fit the formerly prepared recipient bed . A horizontal incision to the bone was made 5 mm from the palatal gingival margin and the blade (razor blade, feather safety razor co., osaka, japan) was subsequently placed parallel to the long axis of the roots . Another horizontal incision was made 2 mm coronal to the first incision and the periosteum was dissected before removing the wedge of soft tissue . Then, the sctg was inserted into the recipient pouch and sutured with 7.0-nylon (nicho kogyo ltd ., the graft was positioned and fixed by a 7.0-nylon suture in apical site of the implant . After covering the sctg graft with a mucosal flap, the flap was coronally stretched with a 7.0-nylon suture (coronally positioned flap, cpf). The donor site was sutured with 5 - 0-nylon (look suture 774b, angiotech, vancouver, bc, canada) (fig . 4). At 12 months after surgery, the recipient site was partially covered by keratinized mucosa (fig . 5a). However, the buccal interdental papilla between implant on maxillary left cental incisor region and adjacent lateral incisor was in a concave shape . To achieve an esthetic result, we planned a second graft . Following local anesthesia, an sctg was harvested from the palate in the left second premolar to second molar region . 5c). Because proper vertical mucosal gain was achieved at 8 months after the second graft, prosthetic treatment was started . After an impression of implant on maxillary left central incisor region and prepared adjacent lateral incisor the all - ceramic crowns (zirconia framework and glass - ceramic veneer material) were fixed with resin cement . The intra - oral picture shows that the peri - implant mucosa is harmonious with the right central incisor at 9 months after graft (fig . Although the interdental papilla between implant and adjacent tooth was more apical than the contralateral side, it was esthetically acceptable to the patient . A two - step split pouch technique using an sctg demonstrated that it was possible to achieve a substantial area of soft tissue dehiscence coverage around the endosseous implant . Burkhardt et al . Evaluated soft tissue dehiscence coverage using a cpf in combination with a free connective tissue graft around the implants . The soft tissue recessions were covered with a coronal overcompensation at a mean of 0.5 mm after the graft . After 1 and 6 months, shrinkage of 75% and 66%, respectively, was observed . Compared with the percentages of soft tissue coverage around the tooth, the implant sites clearly could not be covered consistently . A folding process further hampers vascularization of the graft and could induce extensive shrinkage . To overcome these problems, we considered the anatomical and physiological properties of the buccal peri - implant mucosa . Burkhardt et al . Reported that the preoperative mucosal thickness in the implant site was positively correlated with the height of recession coverage . Nozawa et al . Also reported that marginal soft tissue dimensions indicated that there may be a relationship between the thickness and the height of about 1.5:1 at the platform level . The results may support the approach of acquiring thick mucosa to improve vertical mucosal regeneration . Thick soft tissue, which is obtained from the first graft, can increase the blood supply to the graft; thus, it may prevent necrosis and recession after the second graft . Although pedicle grafts, such as the laterally positioned flap, double pedicle flap, oblique rotational flap and cpf have an advantage over free soft tissue grafts, there is only a limited increase in the tissue thickness and width of the keratinized gingiva . On the other hand, free soft tissue grafts, such as the free gingival graft (fgg) and sctg can increase the width of the keratinized gingiva and tissue thickness . The sctg was statistically superior in achieving root coverage when compared to the fgg, cpf alone, guided tissue regeneration, and allogeneic tissue grafts . The caf with envelope technique has the advantages of increasing keratinized mucosa, a better postoperative course, and a more positive esthetic evaluation than caf with vertical releasing incisions (vris). The longer surgical time to complete the caf with vris furthermore, vris often cause unesthetic visible scars after healing that patients can find unsatisfactory . From a biologic standpoint, the blood supply to soft tissue grafts is critical for the success of the surgery . Site - related factors such as the dimensions of the recession defect, vestibulum depth, and the level of interdental or interimplant papilla should be considered . Miller classified the oral mucosal recession by the marginal soft tissue level and interproximal bone loss . Based on the literature, complete root coverage can be expected in class 1 and 2 . However, there are no reports on the classification of soft tissue recession in dental implants therefore, site related factors, surgical methods, and the patient's expectations should be cautiously taken into account.
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Older adults with hoarding behaviour are often at a high risk of being homeless making aging in place extremely complex . This paper reports on a study that examines the value of a community - based planning approach that responds to the needs of this population, a population that is both increasing in number and that is very seldom studied . It synthesizes new research about the complexities associated with remaining in one's own home when he / she is over 55 and has compulsive hoarding behaviour . And it examines how a collaborative community response promotes successful aging in place for this population . Not only are community - based services necessary to better understand because they are central to all health sectors, but also current research surrounding those with hoarding behaviour is mostly focussed on methods addressing individual - level behavioural characteristics of hoarding through cognitive behavioural therapy (cf ., the extensive work of frost and steketee). Missing are descriptions of community - based planning approaches for health and social service sectors working hard to make aging in place a possibility . For older individuals with hoarding behaviour, aging in place is complex because hoarding behaviour is multifaceted; it touches on social, environmental, familial, and personal issues . Aging in place is also not simple for those with hoarding behavior because they want to remain living in their own homes, neighbourhoods and communities which seem to require that a collection of agencies, often representing different sectors, understand their needs in order to help them stay in the community and age in place . In this paper, aging in place refers to an ideal where people can age in the familiarity of their homes, neighbourhoods, and communities where their quality of life is maximized by the availability and accessibility of supports and services that respond to their needs and capacities [4, 5]. Aging in place, in addition, is about belonging to a community that supports one's many needs, for example, physically, socially, mentally, environmentally, and so forth . To understand the varying aspects of aging in place in late life, older individuals with hoarding behaviour need to be further understood so their aging journey is successful; collaborative approaches by community support agencies can help to make that possible . In our case, social and health related organizations from different sectors, that in some way supported people with hoarding behaviour in edmonton, ab, canada, were brought together in 2007 through the leadership of the social worker of a seniors after having visited a number of individuals with hoarding behaviour (age 55 +) in her professional role, she noticed, as did those members of the imminent community collaborative, that those with hoarding behaviour were at a high risk for being evicted from their homes, and they experienced shame associated with their hoarding resulting in isolation, as well as depression; they were at risk of falling in their own homes, and generally, they were living in unsanitary conditions . To approach this highly vulnerable population, to respond to some of their needs, especially to prevent their potential eviction, a concerted effort by a broad representation of social, health, and other agencies was necessary . The collaborative met together regularly (e.g., once every 2 months over the years and as of june 2011, it continues to meet) to continually plan for and improve the support of older adults with hoarding behaviour in the edmonton area . Although little is still known about the effects of one's neighbourhood on the mental health of older adults, positive mental health in later life may be influenced by the way in which older people feel about their neighbourhood . It is fair to conclude, therefore, that for older adults with hoarding behaviour, place matters and the role of place as locality is key when making meaning at both the individual and the collective levels . In particular, local place is an important factor in identity, in ones sense of community, and attachment; this is crucial to the determinants of attachment, satisfaction, and behaviour . Also important is place and the person - place relationship are very much grounded in a context of fear over loss of place and the obliteration of locality . Aging and compulsive hoarding behaviour need to be examined in concert with one another . As our aging population rapidly increases, the number of older adults with hoarding behaviour will also quickly increase . Although hoarding behaviour usually begins in early adolescents, its severity increases with age . Results from the hopkins epidemiology of personality disorder study found that the odds of hoarding were over two times as great in the oldest compared with the youngest age group, and in a study of hoarding - related complaints to public health departments in massachusetts, 40% of individuals that hoarded were involved with elder service agencies . Referring to frost and hartl when defining hoarding behaviour, there are key characteristics: the acquisition of and failure to discard a large number of possessions that seem to be of little use or limited value; cluttered living spaces that cannot be used as intended; significant distress and/or impairment associated with the clutter . Hoarded possessions clutter living spaces until rooms are difficult or impossible to use for their designed purpose, causing significant stress to the individuals themselves . Possessions may be purchased through compulsive buying and/or through acquiring things like newspapers or discarded items from dumpsters . Household clutter may interfere with and prevent daily activities, like food preparation, moving freely in one's home, and using the bathroom [11, 17]. And as clutter increases, falling, fire, sanitation issues, depression, and isolation are possible, creating significant risk for a number of outcomes but especially eviction and thus homelessness . Community collaboration is one way to respond to social and health needs of a population . In fact, genuine collaborate is say to be the very thing that successfully reforms health systems . Although there are many ways to define collaboration, most definitions emphasize the importance of shared responsibility and a team approach, and using a collaborative approach can significantly increase the available pool of resources from which team members can draw . A response to the multiple challenges of older adults with hoarding behaviour requires a comprehensive and far - reaching approach, more than one single agency can provide alone [22, 23]. Philosophically, collaboration is rooted in systems theory which says that entities in a system are dependent on one another and ecological theory in particular proposes that causes and solutions of health and/or social problems are beyond the individual and are associated with such determinants as the health and social services that we receive . Service recipients can benefit from a collaborative approach to the provision of community health services as can the agencies participating in the collaborative [2628]. Using a collective made up of representative agencies to support this vulnerable population between january 2007 and january 2010, approximately 75 older adults (ages 55 +) with hoarding behaviour in edmonton, ab, canada, were provided with community supports to prevent them from being evicted from their homes . The seniors association of greater edmonton (sage) offers support through a program referred to as this full house . And this full house is a direct outcome of the work of the community collaborative . The aim of this full house for older individuals with hoarding behaviour is to prevent eviction from their home, improve their health and well - being, maintain positive social contacts, and contribute to the building of a healthy community . As the population of potential participants with hoarding behaviour in edmonton is not particularly large, a small - n approach was used in an attempt to create heuristic generalizations which tsoukas defines as opportunities to refine analytic understanding and to make more incisive distinctions than were previously possible . Small - n studies are not designed to support or refute a theory, but rather, to further refine it . As such, the purpose of this study is to further examine the role of a collaborative planning approach in a community setting when seeking to help those over 55 years with issues relevant to having compulsive hoarding behaviour and wanting to age well in one's community . To further understand the value of collaboration and, in particular, its role and value in this community support, interviews and a focus group were conducted with seniors with hoarding issues involved with this full house and with the community collaborative . All interviews were semistructured and conducted by a third party researcher (i.e., research assistant). Ethics approval for this research was received from the university of alberta, research ethics board . (n = 5) individuals with hoarding behaviour involved with this full house, all of whom were over the age of 55 . The semistructured, face to face interviews took place at a location expressed as being most comfortable to the interviewee, for example, at their homes, at a university office, at a nearby coffee shop . These study participants were first contacted by the social worker from sage who directs this full house . The individuals with hoarding behaviour had either referred themselves to this full house or had been referred to it by one of their health care practitioners or family members . At an appointment with the individuals, the social worker informed them about the study asking if they might be interested in being interviewed by a researcher about their experience with this full house . If they agreed (which all five did), she gave them a one - page written description of the study . She then went through the summary of the study ensuring they understood what was being requested from them . With their agreement, their telephone numbers were provided to the researcher (i.e., research assistant) who contacted them and established a location and time to meet for the interview . The aim of the one - hour interviews was to gather information regarding their experience with this full house . In particular, the interview was used to understand their perceptions of the impact of or value of their association with this full house, that is, the aspects of the program they benefitted from most . A community collaborative made up of social and health related agency representatives providing insights into the ongoing development of this full house the focus group interview included ten members (n = 10) (of the possible 11 members in total) and was conducted in a face - to - face manner for approximately 1.5 hours . Members of this collaborative represented a number of expert groups: social workers, home care nurses, geriatric neuropsychologists, geriatric nurses, fire and safety investigators, public health practitioners, and environmental health and safety officers . The focus group questions centered on the nature of the working relationship between and amongst the members and their observations about the value and impact their work may have had on the service users, that is, those with hoarding behaviour . The questions guiding the semistructured interview were generated from key themes highlighted in the literature (i.e., health services, collaboration, community support, etc .) That aligned with the purpose of the study . Grounded theory means that the data analysis essentially, is grounded in the data . Therefore, the concepts and themes we describe in our results have evolved from and are embedded in the data collected and have been mined through a process of conceptual ordering . Grounded theories are said to provide further insight, to enhance understanding, and to be used as a guide to inform action (i.e., acting on the results). As strauss and corbin describe in more detail, our interview data, in the form of pages of the exact words from the interview, was organized into categories that were not predetermined but that evolved after reading and rereading this data many times . And that, mainly described ideas and offered explanations from interviewees that had commonalities to each other . The themes described here are those that were mentioned frequently and carried the same meaning . Because our aim in this study was to further our understanding of how older adults with hoarding behaviour were supported by a particular community - based planning approach, grounded theory provided the most effective means of organizing, reducing, and understanding the data . Suggested below is a picture that describes how the work of the community collaborative, because of its high level of collaboration, resulted in many important benefits for older people with hoarding behaviour that align well with and facilitate the goals and ideals associated with an aging in place model . The picture also describes how the community collaborative members valued their experience on the team . As a result of this group working together to respond to the needs of older adults with hoarding behaviour, several themes evolved from the data demonstrating direct benefits for these individuals: being able to remain in their own homes; reducing their potential for harm, and minimizing their isolation all which allowed them to experience a feeling of empowerment which also helped them to generate insight into issues surrounding their hoarding behaviour . There are considerable challenges associated with aging in one place for older individuals with hoarding behaviour . They can be at significant risk of being evicted from their homes and their behaviour can be a major public health concern leading to eviction as a result of violating building, fire, or property maintenance codes . It may not be until a particular emergency occurs (i.e., water leakage, fire, and pest infestation) that a landlord is notified . One focus group member describes their role, as a member of the community collaborative, in minimizing evictions for this population: our legislation says that we do have the right to go into any public or private place if we believe there may be a public health nuisance and if that means we have to order their suite cleaned out, we'll do it . Um, because you can't get control of bedbugs and cockroaches unless you treat all the suites and if somebody's hoarding, you can't get rid of them so, so they have to clean up . The five interviewees in this study were all in a situation where eviction from their homes was a potential, but they were able to remain at home as a result of a community - based approach that addressed some of their needs . Prior to the existence of this community collective, one focus group member describes how the health inspector had to play all roles and visit clients once a week and nag people into cleaning up, which was mostly unsuccessful . For example, when there is a potential home eviction for individuals with hoarding behaviour, the social worker and a public health worker, together with the client and other members of the collaborative, that is, where necessary such as a fire and safety representative, provide input into the problem - solving process . A professional organizer usually assists with the practical aspects associated with cleaning up including heavy lifting, removing garbage, and reorganizing resulting in [these clients being able to] stay living at home without being on the street and [being] homeless . Being able to age in one's own home, one's neighbourhood, and community fosters independence significantly impacts a more positive relationship in the person - place relationship . The five people with hoarding behaviour that we interviewed spoke about the value of being able to remain living in their own homes . One person found it motivating to have someone help him / her to clean up his / her apartment, it's the motivation of having someone there plus the helper doing the heavy lifting and heavy carrying making 76 thousand trips to the garbage bin its stressful but helpful [and] i certainly would not have been able to hire a company on my own [] if the program had not been in effect [and] i would be in deep do - do with capital health . For another individual, what helped him remain in his own home was having the home care worker put him in touch with the social worker and the cleaning person who helped him find ways to deal with issues of parting with his stuff . And for this gentleman, the social worker supported him by suggesting options: she made life more convenient for me by offering me options . And for another person, the social worker and the cleaner reminded her that the condition of her house, the bugs, the mouse droppings, the make up from years ago, the shiny covered magazines all over, and the infestation that she had, was not her fault and she could probably face organizing and cleaning it with encouragement and the help of a plan . Hoarding behaviour creates a significant safety risk for the individual him / her self and for the community . Harm reduction is a core principle that is essential to address the needs of those with compulsive hoarding behaviour because promoting safety is foremost . In this case, focusing on harm reduction by the community collective ensured that safety was embedded within all the actions, initiatives, and supports they provided . One focus group member talks about the value of taking on this strategic focus: we subsequently learned the value of focusing on a harm reduction approach wherein we address issues of harm first so that the person [with hoarding behaviour], at least, will be safe . Even though they may be living with a significant amount of stuff every day of their lives, but at least they are safe . A harm reduction philosophy considers behaviour change to be incremental and assumes that people will maintain their behaviour change when they have decision - making power to influence their goals and put them into action . This focus on safety and reducing harm or the potential for harm helped the individuals with hoarding behaviour buy into the larger process at hand, that is, to contribute to the building of healthy neighbourhoods, supporting their well - being, and helping them stay in their homes as long as possible . Instead of suddenly or immediately removing the person from a potentially unsafe environment or situation, the aim instead is first to reduce the potential for harm and create a safe place to live . In the case of these five individuals, it meant such things as: hiring a person to help them remove and reorganize their excessive items, getting help to fumigate their apartments, openly talking about their hoarding situation to help them reflect upon it, receiving nonjudgemental support, and establishing a plan to minimize household items . One interviewee describes the value of setting goals and generating a plan, we set a plan and the social worker would come back and generally we would accomplish that goal whatever it was . It also meant helping the person at risk of potential eviction, for example, to respond to requests made by the public health inspector . One person with hoarding behaviour describes the role of this service (i.e., this full house): they sort of mediate [between varying agencies] and rub off the sharp corners . Reducing harm by promoting safety enables an aging in place philosophy and model as both can facilitate positive and long - term aging in one location . The community collaborative, used in this study as a planning approach helped to address the problem of isolation for these older adults . Several of the representative organizations of the community collaborative, that is, a social worker, public health nurse, geriatric nurse, and so forth, offer home visitation to many of their clients and observe their living environment . A lot of these seniors are very lonely, very isolated, and so the fact that they have someone that's coming to their home often, helps . Another member talked about the impact of the support group (which is provided for people with hoarding behaviour on a monthly basis as part of this full house services) on minimizing their sense of loneliness and connecting with people that have similar experiences: [there is] value of that coming together, meeting with other people and seeing and hearing that you're not alone . Two interviewees with hoarding behaviour confirmed this same sentiment, about the importance of feeling connected to a group: the group has helped me and [i realized] you are not the odd one out . As a result of not feeling alone and part of a group, interviewees with hoarding behaviour said that they felt empowered . Empowerment can be an outcome of collaborative relationships; it offers a catalyst for new community programs and other supports, changes in policies, and advancing health practices [35, 36]. The members of the community collaborative intentionally aimed to facilitate empowerment using it as a principle to guide their work that addressed the needs of older individuals with hoarding behaviour in the greater edmonton area . During the focus group interview, one member describes how empowerment as a guiding principle was translated into action benefitting a particular individual with hoarding behaviour: we [the community collaborative] have entertained some really creative approaches in terms of dealing with management [i.e., housing manager] and having the client lead those interventions [] as opposed to, we [the service providers] meet[ing] with management, then meet[ing] with the client we really include the client in those interventions, so that the client really is aware of everyone that's involved, what's being discussed and then they are really empowered to be part of the action plan . When individuals with hoarding behaviour are more involved in directing their own support, they may experience greater control in such decision making which can lead to empowerment . As observed by another community collaborative member, [empowerment provides] a sense of control in a situation that they may feel a lack of control . The most successful reported treatment for those with compulsive hoarding behaviour is the use of behavioural treatment, in other words, a cognitive behavioural model . To be motivated to discard their possessions, insight into their hoarding behaviour members of the community collaborative said that individuals with hoarding behaviour seemed to gain insight into their behaviour as a result of the support provided by this collaborative . This observation was expressed during the focus group interview by the social worker: the insights that come out as a part of the intervention, as you go along, then they [clients with hoarding behaviour] start to reveal some insight as to how did i ever get to this place? And, i can't believe this happened to me and i can't believe that i'm actually, i'm making some decisions now that i was not able to before . During an individual interview, one individual with hoarding behaviour reflected on the changes in her own behaviour of accumulating things: i would still be walking down one little path between the bathroom, bedroom and one side of the kitchen and that would have been it . Another interviewee with hoarding behavior expresses insight into her hoarding behavior: they [the social worker] finally were able to get me to accept this condition that it probably was not my fault and that i could probably face it she also said i need to continue both thinking about and maybe following up [] with counseling [] is there an answer why i have become a person who allows clutter around myself []? . In the one - on - one interviews, individuals reported an improved feeling of independence and a sense of empowerment . This occurred because the collaborative team joined forces, they were united by a common goal of supporting, to the best of their ability, the needs of this population who they noticed to be struggling more and more and who they were being called upon more frequently to try to assist . On individual and collective levels, the members of the community collaborative experienced three significant benefits as a consequence of participating in this group . For example, members of the collaborative were able to access the expertise of other professionals, they maximized the use of their own skills and knowledge and significantly enhanced their understanding of hoarding behaviour . Working on the collaborative team allowed the individual members an opportunity to access a broader range of skills and knowledge than those who were found solely in their own area of expertise or their own organization . In one instance, a professional social worker described how she could now present the risks associated with hoarding behaviour more objectively to a client with greater confidence as she could make reference to and more easily call on the authoritative role of the local fire department . Because the firefighter and social worker were both members of the collaborative when i mention to her [the client with compulsive hoarding behavior] the possibility of having someone from [the fire department] come and just do an assessment to let her know what her risk level is [i.e., of eviction from her home], she was suddenly open to that . As a result of working collaboratively, members also got to know more about the professional resources available to them in the community through their ongoing communication together . As one health professional of the community collaborative said: it is a professional benefit to see and use the expertise around this table for the benefit of the individual clients . Evaluating collaborative planning practices must consider not just the purpose of the collaboration but the value of its relational interactions . For example, asking how social relations are changed can reveal how certain conditions are impacted for the group . Access to new areas of expertise is one descriptor of the quality of social relations . The sharing of expertise between and amongst the members was said to directly benefit the older people with hoarding behaviour using the services of this full house . In the words of one member of the community collaborative, a benefit of the collaborative process was working with everybody, to partner, to ensure that we're getting our clients the best support . Participating in this group allowed team members to maximize the use of their own expertise . One member, a public health inspector, recounted a time when such a collaborative approach was not used to support older adults with hoarding behaviour illustrating the tremendous limitations of working in isolation: before [the community collaborative existed] it was [up to] the health inspector to try and play all roles and just sort of go and visit once a week and try and nag people into cleaning up . Which was mostly unsuccessful and wasn't really our job . I mean we are not social workers, we're not mental health workers, we are public health inspectors . Functioning alone, the health inspector had to operate as the only contact for this client group . Working in isolation took away time from the job he / she was actually trained and hired to do forcing them to work beyond their professional scope of practice . As further evidence of the value of being able to maximize one's own expertise, the professional organizer, who provides hands - on assistance with the cleanup of client homes, can now maximize her cleaning and organizational skills while directing clients' emotional issues to a trained professional . As described by the social worker during the focus group interview: a big portion of her [the professional organizer's] time was addressing the [clients] emotional issues . So we've now learned that when those issues come up, it's a direct link back to me . Members of the community collaborative described how their partnering with one another as professionals helped to enhance their knowledge and understanding of compulsive hoarding behaviour . Gaining new knowledge and a more enlightened understanding of compulsive hoarding behaviour was said to be the result of participating in this ongoing process . One member of the collaborative described the value of the increase of his knowledge stating another professional benefit [of being a member of the community collaborative] is deepening my personal understanding of what hoarding is and what the dynamics are . Certainly, it's helped me in recognizing that it's multi - faceted . Group members said they were then able to take their learning back to their representative organizations: i think working with [the collaborative] has really helped to educate me and hopefully the rest of [name of organization]. Successful aging in place requires that support by community - based organizations exists that it is available and accessible and responsive to a variety of their needs; therefore, knowledge about aging and its long list of associated issues, such as compulsive hoarding behaviour, is imperative . The aim of our discussion is to explain several matters that underpin the major themes of our results . Explaining why such themes evolved and their relationship to the broader phenomenon being studied is what strauss and corbin refer to as the process of theorizing . At the heart of this study is community - based planning as a phenomenon of which a number of related concepts are embedded: aging in place, social support, collaboration, vulnerable populations, and community services . Overall, our research attempted to discover how a collaborative approach to planning for and addressing the needs of older adults with hoarding behaviour, living in the community, provided value . For these vulnerable adults living in edmonton, alberta, canada, a collaborative planning approach that involved multiple agencies (representing varying sectors) that worked continuously to improve their quality of life made a difference . And, the members of this community collaborative also benefitted . From this approach they were able to access the professional expertise of the other group members, maximize the use of their own skills and knowledge, overall, giving them an opportunity to generate new insights into hoarding behaviour which they described as helping them provide the best possible care and support to this population . It is feasible; therefore, to use such results to inform the many ways to age in place more successfully in late life . Currently there is no systemic, long - term process to support older people living in the community that have compulsive hoarding behavior . Nor in canada, is there an overall strategy to plan for our aging population, therefore aging in place, at a national, political scale, is not yet a priority . But, because people with this behavior will increase in numbers, and the complexities associated with their need to live safely in their own communities, a national strategy must also address their specific and unique needs . As emphasized by the canadian health services research foundation, our study also finds a need for enhanced integration, cooperation, and coordination at the system and at the service delivery levels . That is, integration and collaboration between health and social services, between ranges of sectors, between disciplines of front line workers, and between government ministries . Collaboration and integration need to be part of the foundation upon which aging in one's own home and community can be realized . In addition, our study supports the findings of keonig et al . Who found that when having to facilitate ethical dilemmas for this population, older adults with hoarding behavior benefit from the use of teams whose members have a variety of disciplines . Well evidenced in the health services planning literature, applied to an aging population, is the need for improved collaboration, both at a principle and a practice - based level . Not only do our findings align with the literature but it provides further insight into the challenges associated with older individuals that want and deserve to remain in their own homes . Our particular case is specific to older individuals that have hoarding behaviour that were supported through the efforts of a community collaborative planning approach . The current literature supports collaboration and integration at varying levels of the health and social support system . This population will increase in numbers over time and they deserve to remain in their own communities with the support of surrounding agencies and organizations that work togather to best support older individuals with hoarding behaviour to age well in their own homes . At the heart of several concepts and themes arising from interviews with older people with compulsive hoarding behavior and members of a community collaborative working to support this population is an approach founded on collaboration between and amongst service providers . Results demonstrated that when a highly collaborative approach to planning is used, there were quite direct benefits for older adults with hoarding behavior and, at the same time, there were benefits for the members of the community collaborative . This approach to planning for the health and social needs of this population resulted in people with hoarding behavior being able to remain in their own homes when eviction was a potential, enhancing their safety, helping to minimize their isolation, and creating opportunities to increase control in their own decision making . The members of the community collaborative could now access the expertise of other professionals, maximize their own expertise, and they generated new insight and understanding of the experience of older adults living with hoarding behaviour in edmonton . Although our data conveys that this approach to planning has quite positive outcomes, our data is short term and situational . Our use of a single, one - time only interview method only allows us to draw insights and observations about that moment in time and not over an extended trajectory . As well, our study is grounded in five interviews with older adults with compulsive hoarding behavior . And although a collaborative approach to addressing the needs of older people with hoarding behaviour conveyed comprehensive benefits, collaboration as a planning approach is rarely the complete answer or solution to people's social and health needs . Health integrated delivery systems, for example, are far more comprehensive but do view collaborative planning as a core principle . Viewed in this light, aging in place may not always be possible, but it must be realized that community - level social and health related supports maximize the quality of later life while aging at home . And further building on that is the need for a well - coordinated model of care [5, 35] where supports are comprehensive, easily accessible, and well connected.
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Hand - assisted laparoscopic donor nephrectomy (haldn) has become the method of choice for removing living donor kidneys . Similar to open live donor nephrectomy cases, however, most laparoscopic donor nephrectomy cases have been limited to the left side because of the longer renal vein than on the right side, the greater technical ease of transplantation, and reasonable positioning of the transplanted kidney in a recipient . In addition, although there have been debates about this issue, some authors have emphasized the left - side preference by arguing that left - sided haldns are successful even in cases with multiple left renal arteries [1,3 - 5]. Obviously, several indications, including significantly lower function in the right kidney than in the left one [6 - 10] or a woman of child - bearing age, should prompt consideration of the right rather than the left kidney . However, fewer reports have been made on right laparoscopic donor nephrectomy because most right donor nephrectomies have been performed in open surgery owing to a lack of experience and the technical difficulties in performing laparoscopic procedures . Moreover, the surgical outcomes of right laparoscopic donor nephrectomies (especially haldns) have not yet been reported in korea . The aim of this study, therefore, was to report the safety and feasibility of right - sided haldn . Between may 2006 and may 2009, 16 patients underwent right - sided haldn in our institution, by the same operator, who had experienced more than 500 cases of open donor nephrectomies and more than 300 cases of left - sided haldns . We retrospectively analyzed these cases after collecting demographic information on donors including age, sex, relation to a recipient, body mass index (bmi), and indications for the right - sided approach . Preoperative donor evaluation included history taking, physical examination, laboratory tests, renal ultrasonography, intravenous pyelography (ivp), and renal function testing by radionuclide renal scan (99mtc - diethylenetriamine penta - acetic acid; dtpa). Three - dimensional spiral computerized tomography was used to define the renal parenchyma and vasculature . Surgical demographics included intraoperative and postoperative parameters such as operative time, delivery time, warm ischemic time (wit), estimated blood loss (ebl), intraoperative and postoperative complication rates, length of hospital stay (los), and serum creatinine levels of donors (at the time of discharge) and recipients (4 weeks postoperatively). Operative time was defined as the time interval from the initial skin incision to closure of the skin . Delivery time was calculated as the time interval from renal artery stapling to being placed in ice slush, and wit was calculated as the time interval from renal artery stapling to back - table flushing . Under general endotracheal anesthesia, the patient was placed on a flexed table in a left - down partial flank position . An axillary roll was placed beneath the donor's arm, and the right arm was maintained on an armrest . A 6 to 8 cm incision was made for the hand (a) below the umbilicus along the border of the right rectus muscle, and an 11 mm trocar was placed 5 cm above the hand port for the camera (b). A 12 mm trocar (c) was placed 5 cm above the camera port for the hem - o - lok (weck closure systems, research triangle park, usa) or endogia (conmed, new york, usa), and we performed most of our procedures through this port . Additionally, a 5 mm trocar (d) was placed below the xiphoid process for liver retraction . When needed, we placed an additional 10 mm trocar at the right subcostal margin in the right mid - clavicular line (fig ., we incised the gerota's fascia and entered the perirenal space after incising the lateral line of toldt and medially reflecting the ascending colon and duodenum . For complete mobilization of the kidney, the perirenal fat and adjacent tissues were sufficiently dissected . The ureter was dissected to the level of the external iliac vessels and divided, leaving enough margins to ensure blood supplies around it . Then, the ascending colon, right transverse colon, and duodenum were widely mobilized to provide a maximal exposure of the right renal hilum and inferior vena cava (ivc). The renal hilum was skeletonized by meticulous dissection of its adjacent structures with great care to avoid any injuries to the hilar vessels (fig . A harmonic scalpel (ultracision; ethicon endo - surgery, flower mound, usa) was used in all of these procedures for the dissection and coagulation of tiny vessels and other peritoneal structures . After 25% mannitol (250 ml) and diuretics were intravenously administered 20 minutes before arterial clamping, the renal artery was clamped with 2 or 3 hem - o - loks and divided (fig . 2b), and a 30 mm endogia stapler was used to transect the renal vein . To gain a maximal length of the right renal vein, the kidney must be gently retracted laterally with the help of the surgeon's left hand to extend the right renal vein, and the endogia stapler must be positioned at the junction of the ivc and right renal vein (fig . Thereafter, the right kidney was removed by the surgeon's left hand through the hand - port device . The staple lines were excised, and the artery was flushed with cold kidney preservation solution . After the abdomen was carefully reinspected at a reduced intraperitoneal pressure, bleeding was controlled, and a jp drain was inserted . The procedures were successfully performed on all 16 patients, and none of the patients experienced intraoperative complications or required conversion to laparotomy . The mean age of the donors was 38.310.4 years, the ratio of males to females was 1.71:1, and the mean bmi was 238.0 . The reasons for right donor nephrectomy were as follows: 10% difference in split renal function as determined by radionuclide renal scan dtpa (n=15) and right renal stone (n=1). The numbers of right renal arteries of the 16 patients were as follows: single renal artery (n=13), single renal artery with an early branching artery that supplied the upper pole of kidney (n=2), and duplicated renal artery (n=1). The numbers of right renal veins of the patients were as follows: single renal vein (n=15), duplicated renal vein (n=1). The mean operative time in our series was 192.131.6 minutes, with a mean delivery time of 122.222.4 seconds, a mean wit of 191.842.5 seconds, a mean ebl of 199.071.4 ml, and a mean los of 4.180.39 days . The mean serum creatinine level in the donors was 1.170.19 mg / dl at discharge . Concerning graft function, the mean serum creatinine level on day 28 was satisfactory in the recipients (1.160.18 mg / dl). No major or minor complications occurred in any of the 16 patients who underwent our right - sided haldn during the step of clipping and dividing the renal vessels of the donors . As for the recipients, there were no technical problems in the anastomosis of the renal vein, and ureteral anastomoses were also successful without any postoperative sequelae such as ischemic ureteral stricture or leakage of urine . In the case of the donor who had a right renal stone, the stone - bearing kidney was transplanted and the recipient underwent subsequent successful elective shock wave lithotripsy in the third posttransplantation week, and no recurrence of calculi had occurred at the 2-year follow - up . Haldn, which was initially reported by wolf et al, may be the safest option for removing living donor kidneys because the surgeon can use the hand in the surgical field by making an abdominal incision at the start of operation . This digital palpation is a valuable tool that provides surgeons with a tactile sensation that permits them to trace the vascular structures and retract the adjacent structures . Therefore, this technique can minimize intraoperative injury, give immediate management to emergent situations such as bleeding, and especially minimize wit as compared with pure laparoscopic donor nephrectomy . For these reasons, haldn has spread quickly and widely, and it has become the method of choice for living donor nephrectomy . Similar to open live donor nephrectomy, most laparoscopic donor nephrectomy procedures have been limited to the left side because of the longer renal vein than on the right side, the greater technical ease of transplantation, and reasonable positioning of the transplanted kidney in the iliac fossa of a recipient . However, despite all these advantages of left nephrectomy, not all potential donors have the same situation conducive to left nephrectomy ., donors who have significantly lower function in the right kidney than in the left one as determined by dtpa scanning must undergo right nephrectomy to preserve their future renal function [6 - 10]. Smaller right kidneys and undiagnosed lesions within the right donor kidney are also indications for right donor nephrectomy . In addition, some previous studies reported that women at a fertile age who want a future pregnancy must undergo right kidney donation because there is a higher chance of pyelonephritis and hydronephrosis on the right side during the gestational period . Some authors recommended that the right kidney should be chosen in such cases, whereas others report that the presence of renal artery multiplicity does not have a significant impact on the outcomes of the renal donors or recipients when performing ldn [1,3 - 5]. In our institution, multiplicity of the renal artery is not included in the criteria by which we select the side of the kidney for donor nephrectomy . Until recently, most transplantation centers have hesitated to perform right - sided haldn and have continued to perform right donor nephrectomy by the open technique . The reasons for this choice include a lack of experience, concerns that the shorter length of the right renal vein poses technical challenges for transplant surgeons in transplanting the kidney and thereby increases the risk of intraoperative and postoperative vascular complications in the recipient, and concerns about an increase in delivery time and wit . Unlike the general method of left - sided haldn in which a hand port is positioned by making a midline incision just above the umbilicus, our method allows surgeons to stand in the most comfortable position so that they can use both hands most freely by making an abdominal incision for the hand below the umbilicus along the border of the right rectus muscle . We widely mobilized the ascending colon, right transverse colon, and duodenum, and thereby provided a maximal exposure of the right renal hilum and ivc . Furthermore, we gently retracted the right kidney laterally with the help of the surgeon's left hand to extend the right renal vein, and positioned the endogia stapler at the junction of the ivc and right renal vein so that we could achieve a maximal length of the right renal vein . We transected the renal vein by use of the endogia stapler instead of by using satinsky clamps to reduce the operative time by eliminating the need for intracorporeal suturing . Also, because we believed that the use of the endogia stapler would waste a similar length of vein compared with the use of a hem - o - lok clip, we used the endogia stapler instead of a hem - o - lok clip for the renal vein . Ko et al reported an opinion similar to ours regarding this matter . Compared with the surgical outcome of left - sided haldns published previously (we used the results of' the single renal artery group' from choi et al), the 16 right - sided haldns in our study showed satisfactory results in not only fundamental surgical parameters such as operative time, ebl, and intraoperative and postoperative complication rates, but also the postoperative serum creatinine level of the recipients, delivery time, and wit, which are thought to be the most important parameters in kidney transplantation (table 2). Based on the result that there were no technical problems in the anastomosis of the renal vein in the recipients, we suggest that our technique enabled us to harvest renal veins with appropriate lengths . There have been several reports on the surgical outcomes of right laparoscopic donor nephrectomy in international journals . Liu et al reported similar surgical outcomes in 19 left - sided haldns and 6 right - sided haldns . Keller et al reported that 36 right - sided procedures out of 230 ldns showed similar results in ebl, amount of blood transfusion, operative time, los, and delayed graft function compared with left - sided haldn . There have been more reports about the excellent surgical outcomes of right laparoscopic donor nephrectomy compared with left - sided operations [21 - 23]. When we reviewed the surgical parameters, mean delivery time and wit were slightly higher in the right - sided haldn group than in the left - sided haldn group . These results seem to be caused by the differences in the operative procedures between the groups . In left - sided haldn, 2 different ports are simultaneously prepared, 1 for hem - o - lok (for arterial clamping) and 1 for endogia (for vein stapling). By use of these 2 ports, the renal artery is divided with the hem - o - lok after the endogia is positioned at the accurate site for renal vein stapling . This technique enables renal vein stapling immediately after renal artery dividing, thereby reducing delivery time and wit . On the other hand, in right - sided haldn, only 1 port (c) in addition to a port for liver traction is actually available . Thus, after the renal artery is divided by using a hem - o - lock instrument and the instrument is removed, we then insert the endogia into the same port, place it in the appropriate position for renal vein stapling, and fire it . It is conceivable that the anatomically short length of the right renal vein will not always make right - sided haldn difficult . It is possible for surgeons to obtain an additional length of the right renal vein by performing an ex vivo microvascular reconstruction technique (bench surgery) by using the intrarenal vein in right donor nephrectomy . Right - sided haldn is safe and technically feasible in donors and shows a favorable graft outcome . The results of this study suggest that right - sided haldn may be preferable for living donor nephrectomy in patients with significantly lower function in the right kidney than in the left one.
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Pyomyositis is a primary pyogenic infection of skeletal muscle, an uncommon cause of musculoskeletal infection . Although relatively common in tropical areas, it is very rare in temperate areas1 . In contrast, pyomyositis is observed in immunodeficient hosts, particularly in the human immunodeficiency virus (hiv) infected condition2 . In nonhiv infected cases, skeletal muscle tissue is resistant to bacterial infection, even in the severe bacterial septic condition3 . In approximately 80% of cases, the location of the infection is unifocal, and the lower extremities, especially the thigh, are the most common site2 . In the early stages of pyomyositis, there are no typical features of this disease . Because of its rarity, it is often misdiagnosed as muscle hematoma, cellulitis or neoplasm4 . Therefore, prompt imaging procedures, aggressive surgical interventions and appropriate antibiotic therapy are very important to cure this disease without any complications . Here, we report a case of a japanese woman who developed multiple abscesses, which were, surprisingly, disseminated over 30 parts throughout the body, under poorly controlled diabetic conditions accompanied by ketoacidosis, but was successfully treated with prompt and appropriate therapy . A 26yearold woman was referred to kawasaki medical school hospital, kurashiki, japan, because of a high fever and right thigh pain . She had mild pain of the right thigh, a sense of thirst and general fatigue . Furthermore, her bodyweight reduced by 6 kg during 2 weeks, and severe thigh pain, and highgrade fever developed and progressively worsened . She had untreated type 2 diabetes, but did not have any history of trauma or illicit drug use . Her grandmother had adultonset type 2 diabetes, but except for this there was no family history of diabetes including maturityonset diabetes of the young and mitochondrial diabetes . On admission, her body mass index was 23.0 kg / m (height 156 cm and body weight 56 kg). Emergent computed tomography (ct) showed a giant mass in the right adductor muscle (figure 1a, b). The drastic elevation of plasma glucose (470 mg / dl) and glycated hemoglobin levels (15.2%) were shown with severe ketoacidosis . Despite prehospital administration of insulin (total 50 u) and bicarbonate, blood gas analysis still showed metabolic acidosis (ph 7.339, hco3 10.9 meq / l, paco2 20.8 mmhg, pao2 107.5 mmhg, base excess 12.8 meq / l, lactate 1.22 meq / l under oxygen inhalation 2 l / min by nasal cannula). Leukocytosis and an increased creactive protein (28.21 mg / dl) were also observed . Renal and liver function, serum creatinine kinase, and myoglobin levels were not elevated . (a, b) computed tomography with contrast enhancement in the right thigh on admission . (a) frontal slice . (c) disseminated abscesses throughout the patient's body including the kidney and muscle (on day 7). Based on these findings, we diagnosed this patient as primary pyomyositis, and immediately started broadspectrum antibiotics therapy (doripenem 3.0 g / day, clindamycin 1200 mg / day). Aggressive hydration with saline (4.5 l / day) and intravenous continuous insulin infusion was immediately started . Furthermore, we carried out surgical drainage and removed 700 ml of yellowish pus . Surprisingly, the followup ct showed multiple pyomyositis disseminated over 30 parts throughout her body, as well as a kidney abscess (figure 1c). We immediately restarted clindamycin, and changed fulconazole to micafungin with additional percutaneous renal drainage . We used broadspectrum antibiotics and an antifungal drug intravenously for a total of 66 days . There was no clear evidence of osteomyelitis, which is closely associated with pyomyositis, but in consideration of the possibility of osteomyelitis, we decided to carry out longterm antibiotics and antifungal therapy . Indeed, despite a good clinical course, lowgrade fever and persistent mild elevation of crp (approximately 1.5 mg / dl) were observed . Therefore, we continued antibacterial and antifungal agents until the normalization of these infectious markers . In the findings of followup ct with the enhancement, the origin bulky abscess in the adductor muscle and systemic intramuscular abscess completely disappeared (figure 2). On day 66 of the hospitalization, when the value of creactive protein remained within the normal range for 7 days, we stopped all intravenous agents . Complete disappearance of origin and disseminated abscesses . Left panel, computed tomography on admission or day 7; right panel, computed tomography on day 96 . It is known that skeletal muscle is resistant to bacterial infection, but under various immunodeficient conditions, such as severe lymphopenia with hiv infection or diabetic ketoacidosis, the host's defense system against microbiota is destroyed, which easily leads to opportunistic infection . Indeed, there is a close relationship between diabetes itself and the incidence of serious infections5, 6, especially sepsis7 . In addition, because of its rarity, the accurate and timely diagnosis of pyomyositis is challenging . In the present patient, severe hyperglycemic state with diabetic ketoacidosis and severe infectious signs led to the diagnosis of pyomyositis . Furthermore, typical ct imaging of the bulky abscess in the right adductor muscle was helpful for the diagnosis . To the best of our knowledge, there have been no reports showing as many as 30 disseminated abscesses . Because there seemed to be no other additional immunodeficiency cause, we assume that the delay of the visit to the medical institution was the main cause of pyomyositis observed in this patient . Stage 1 is the invasive stage where local symptoms and lowgrade fever are observed, but other physical findings are typically absent . However, only a minority of patients (<2%) visits a medical institution at this stage1 . Stage 2 is the suppurative stage with abscess formation . Around 23 weeks after the initial onset, patients complain about progressive fever and severe pain of the affected sites . Stage 3 is the late stage, where sepsis and dissemination of the infection are observed if the abscess remains untreated in the previous stages1 . This progressive stage can be occasionally associated with lethal conditions, especially septic shock, multiple organ failure and rhabdomyolysis6, 8 . Our patient was very afraid of undergoing all treatment, and hesitated to visit a medical institution . We believe it is likely that such a delay progressed the pyomyositis in this patient to the most severe and lifethreatening condition . Therefore, it would be very difficult to save her life, if the only one piece of the following situation is missing: precise and timely diagnosis, intensive surgical approach, longstanding antibiotics therapy, and general supportive care . As observed in the present patient, staphylococcus aureus is the most common (approximately 70%) causative bacteria in both tropical and temperate conditions4 . Treatment of pyomyositis depends on the stage of the disease . In the invasive stage, a key feature of prognosis of pyomyositis is recurrence, but it is uncommon (<30% of all cases). The mortality rate is <1.5% in the early stage, but it becomes as high as 15% in the late stage10 . Therefore, we have to continue antibiotics and antifungal agent for a long enough period . In fact, the present patient was successfully treated with surgical interventions, in combination with longstanding broadspectrum antibiotics and antifungal therapy . Fortunately, during the longstanding broadspectrum antibiotics and antifungal therapy, there were no adverse events including the appearance of resistant bacteria, pseudomembranous colitis, liver dysfunction and electrolytes abnormalities . Pyomyositis is a rare infectious disease, but physicians should be aware of its possibility in poorly controlled diabetic patients . In addition, the present case strongly suggests that we should continue appropriate therapies until the complete disappearance of abscesses.
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Many cases of implant placement involve insufficient buccolingual width of the edentulous ridge . The methods used to resolve this issue are the following: narrow implant placement, horizontal veneer block bone graft, horizontal guided bone regeneration (gbr), and the ridge splitting procedure1,2,3 . Ridge splitting is applied primarily in cases where the bone height is sufficient, but the width is narrow . The purpose of ridge splitting is to widen the alveolar ridge by taking advantage of the elasticity of bones, and it is frequently performed in the anterior maxillomandibular area . This surgery restores the morphology of the lingual side of the alveolar bone and obtains not only aesthetic results, but also a housing effect exerted by the cortical bone of the buccolingual side, which improves the osseointegration of implants by providing an ample supply of blood circulation4 . Nevertheless, fracture and separation of the buccolingual bone during surgery are potential risks, so it is crucial to minimize these complications . Methods augmenting the alveolar bone to the buccolingual side are often done because ridge splitting involving the use of an osteotome and mallet can induce greenstick fractures of the buccal cortical bone . In the ridge splitting procedure, discomfort to patients is often substantial because of malleting, and there is a risk of buccolingual bone fracture when excessive force is delivered . Therefore, the present report introduces and evaluates the clinical results of a procedure that expands the ridge in a stepwise technique using screws of gradually increasing width . This study was performed after obtaining approval from the institutional review board of the seoul national university bundang hospital (no . B-1007 - 105 - 106). In the department of oral and maxillofacial surgery, curette, seoul, korea), and implants were placed sequentially or simultaneously in 6 patients (1 male and 5 females). The age of the patients ranged from 31 to 69 years with a mean age of 55.1 years . The postsurgical follow - up observation period ranged from 24 to 63 months with an average of 44.7 months . A midcrestal incision was made in the maxillary and mandibular alveolar bone, and the bone was exposed through the creation of a full thickness flap . Initial drillings were performed in the area of implant placement, and the implant was placed at the proper placement depth using a splitmaster no . Gradually, thicker screws were used relative to the final width and length of the implants . Simultaneous implants were placed with self - tapping and sequential implants were placed with an approximately 3- to 4-month healing period in between . In two cases, a groove was created in the alveolar ridge with frios saws (dentsply implants, mannheim, germany) or #15 blades, and alveolar ridge splitting was performed using an osteotome . Then, the implant placement site was expanded to the width of the implant by using expanding screws . In some cases, the space between the implants and the bone was filled with graft materials and, if needed, blocking membranes . In one of the ridge expansions a total of 11 implants were placed: 5 in the maxillary anterior and 3 in the maxillary premolar area. (table 1) complications during surgery, such as buccal cortical plate complete fracture, did not develop with the exception of severe edema and post - surgical ecchymosis in one case . In that case, osseointegration failure was observed 4 months after the first surgery, so the implant was removed and was replaced immediately . Wound dehiscence developed after implant placement, and the implant after approximately 8 weeks after replacement, a fixed partial prosthesis was installed . During the follow - up observation period, averaging 44.7 months, all 6 patients maintained normal function without any specific problems. (table 2) a 31-year - old male patient with a history of right unilateral cleft lip and palate presented to our clinic with a missing right maxillary lateral incisor and alveolar bone loss . Together with orthodontic therapy, a treatment plan was developed that included a symphysis bone graft, oronasal fistula closure surgery, and implant placement . The first implant surgery was performed after a 6-month healing period following the bone graft . To secure an adequate view, a full thickness flap was created . The bone height was sufficient for implant placement and the buccolingual width was approximately 3 mm . Using splitmaster expanding screws, a 4-mm ridge expansion was performed; subsequently, an implant that was 3.5 mm in diameter and 11 mm in length was placed . To prevent buccal bone fracture, on the buccal side, a 2-mm implant thread was exposed, so gbr with xenogeneic bones (biocera; osscotec, cheonan, korea) and barrier membranes (bioarm; ace surgical supply, brockton, ma, usa) was performed in the exposed area . After a 4-month healing period, the second surgery to maintain the buccal volume was performed through creating a labial pouch and grafting the biocera . After 3 months of orthodontic treatment for regaining the space of implant prosthesis, a provisional restoration was placed . After the orthodontic treatment was finished (nine months after the implant surgery), the implant was restored with an all - ceramic crown supported by zirconia abutment post. (figs . 1,2,3,4,5,6) a 69-year - old female patient without any history of systemic diseases presented to our clinic with a chief complaint of missing several teeth . Under local anesthesia, bone height was sufficient for implant placement; the buccolingual width was as narrow as 3 to 4 mm, bone quality was d3, and osteoporosis was suspected . After initial drilling, expansion was performed using splitmaster expanding screws at the left mandibular 2nd premolar and 2nd molar area; the areas were expanded to 3.8 mm and 4.3 mm, respectively . Two gs ii implants (osstem, busan, korea) which were 4 mm in diameter, 11.5 mm in length and 5 mm in diameter, 11.5 mm in length were placed . As measured by the osstell mentor (integration diagnostics, savedalen, sweden), the primary stability of the implants in the left mandibular 2nd premolar and 2nd molar was 76 and 77 implant stability quotient (isq), respectively . Subsequently, a xenogeneic bone graft (biocera) was performed in the space between the bones and the thin area of the buccal side . The surgery was performed using the one - stage method, and after a healing period of approximately 2 months, the implants were restored with a fixed partial prosthesis. (figs . A 31-year - old male patient with a history of right unilateral cleft lip and palate presented to our clinic with a missing right maxillary lateral incisor and alveolar bone loss . Together with orthodontic therapy, a treatment plan was developed that included a symphysis bone graft, oronasal fistula closure surgery, and implant placement . The first implant surgery was performed after a 6-month healing period following the bone graft . To secure an adequate view, the bone height was sufficient for implant placement and the buccolingual width was approximately 3 mm . Using splitmaster expanding screws, a 4-mm ridge expansion was performed; subsequently, an implant that was 3.5 mm in diameter and 11 mm in length was placed . To prevent buccal bone fracture, on the buccal side, a 2-mm implant thread was exposed, so gbr with xenogeneic bones (biocera; osscotec, cheonan, korea) and barrier membranes (bioarm; ace surgical supply, brockton, ma, usa) was performed in the exposed area . After a 4-month healing period, the second surgery to maintain the buccal volume was performed through creating a labial pouch and grafting the biocera . After 3 months of orthodontic treatment for regaining the space of implant prosthesis, a provisional restoration was placed . After the orthodontic treatment was finished (nine months after the implant surgery), the implant was restored with an all - ceramic crown supported by zirconia abutment post. (figs . A 69-year - old female patient without any history of systemic diseases presented to our clinic with a chief complaint of missing several teeth . Under local anesthesia, bone height was sufficient for implant placement; the buccolingual width was as narrow as 3 to 4 mm, bone quality was d3, and osteoporosis was suspected . After initial drilling, expansion was performed using splitmaster expanding screws at the left mandibular 2nd premolar and 2nd molar area; the areas were expanded to 3.8 mm and 4.3 mm, respectively . Two gs ii implants (osstem, busan, korea) which were 4 mm in diameter, 11.5 mm in length and 5 mm in diameter, 11.5 mm in length were placed . As measured by the osstell mentor (integration diagnostics, savedalen, sweden), the primary stability of the implants in the left mandibular 2nd premolar and 2nd molar was 76 and 77 implant stability quotient (isq), respectively . Subsequently, a xenogeneic bone graft (biocera) was performed in the space between the bones and the thin area of the buccal side . The surgery was performed using the one - stage method, and after a healing period of approximately 2 months, the implants were restored with a fixed partial prosthesis. (figs . Since 1990, it was widely used and referred to in the literature with various terms such as ridge widening, the split crest procedure, and staged ridge splitting5 . Generally, this surgery induces a greenstick fracture in the narrow alveolar ridge with an osteotome . The procedure forms an implant bed and facilitates the placement of implants with wide diameter . In addition, bone regeneration is achieved on both sides, so the bone healing capacity is good; therefore, sufficient osseointegration can be achieved with relatively small volumes of bone . Furthermore, a 3-month waiting period is required at minimum for distraction osteogenesis and 6 months is required for gbr in implant placement . On the other hand, after performing ridge splitting, the first placement surgery can be performed on the same day or within the month . It is still controversial as to whether a bone graft is required for the space generated after splitting . In simultaneous implant placements, it has been reported that in cases with bony gaps smaller than 3 mm, bone graft materials other than collagen sponges are not required7 . Also, implants placed in the space between expanded bones are protected from biodynamic external loading . Furthermore, bony compaction effects can occur as a result of the compression of bony trabeculae . Nonetheless, this surgery is a method that uses bone elasticity, so it can only be performed in cases with cancellous bone within the cortical bone on both sides . Therefore the indications of this procedure might be limited when compared with gbr or onlay graft that could be applied in a cancellous bone - deficient area . In cases where the width of the basal bone is too narrow, the primary stability or the slope of edentulous alveolar bone it has been reported that cases with a residual width of more than 4 mm are ideal for the procedure; in cases with a width of 3 mm or less, other bone graft procedures should be additionally performed, and the implant should be placed after a certain healing period6 . Moreover, it has been reported that ridge splitting is a procedure that should be applied primarily to the maxilla, as the outcomes are poor in the mandible due to the abundance of cortical bone7 . However, it has also been reported that if the characteristics of the mandible are well understood and slow expansion is performed manually rather than with a mallet, then successful results can be obtained7 . Chiapasco et al.8 demonstrated gradual bone expansion of 1 mm per day for 4 - 5 days using screw - type tools in the mandible . Chan et al.10 reported that the mean increased ridge width was 0.79 mm in a cadaver study on the ridge width gain through the use of screws . In this study, even in the mandibular molar area, the bone width was expanded to a maximum of 4.3 mm using expanding screws and simultaneously placing implants . Even though some of case reports reported the occurrence of cortical bone greenstick fracture after surgery, the separation of bone fragment caused by the occurrence of complete fracture was not reported . One year after functional loading, good results without the gingival recession or alveolar bone resorption were obtained . In a case with poor d4 bone quality, the compaction effect of the bony trabeculae using screw - type tools was observed and resulted in effective primary stability of the implants . In several reports, a 97.0%-97.6% success rate of ridge splitting was reported; however, cortical bone fracture, resorption of the alveolar ridge, nerve injury, and other complications may occur11,12 . Buccal cortical bones are fractured in many cases, particularly when ridge splitting is performed in the mandible with hard bone quality, and this will induce resorption of the alveolar ridge during the bone remodeling process . The screw - type tools used in this study deliver relatively low amounts of force . The bone was also expanded sequentially in the deep area, and it was considered to be more effective in preventing cortical bone fracture . In addition, the conventional malleting process was minimized, thereby minimizing head echo and the resulting headaches, injury to the temporomandibular joint, and others . Jensen et al.6 reported that mucoperiosteal elevation should be performed minimally in ridge splitting, and that the use of partial thickness flaps show good results . In this study, and in cases involving the maxillary anterior area where esthetics are required, full - thickness flap elevations were performed in the alveolar ridge and slightly below it . The ridge expansion technique mentioned in this case report is slightly different from the existing ridge splitting technique . The ridge splitting technique is a methodology to increase the ridge width by performing the ridge splitting using a chisel after the formation of a groove at the center of ridge crest using a saw or surgical bur and is mainly used in the maxilla . If the ridge splitting technique is performed in the mandible composed of mainly cortical bone, then it runs the risk of cortical bone fractures . Whereas, the ridge expansion technique is a stepwise technique using screws of gradually increasing width, so it has a lower risk of cortical bone fracture than the ridge splitting technique therefore, ridge expansion technique is considered to be an acceptable method to use on the mandible . In this study, one case involving implant placement in the maxillary anterior area developed a complication of failed osseointegration . However, implant replacement was performed immediately after removal, and successful prosthesis treatments were completed . Buccal cortical bone fracture did not occur in any of the cases . Even after the prosthesis were functioning, all adjacent alveolar bones and soft tissues remained stable.
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In recent years, core training has been widely studied since it has been considered a pivotal issue in health, rehabilitation and sports performance (hibbs et al ., 2008). However, the definition of the core varies with the interpretation of the literature (hibbs and thompson, 2008). Anatomically, the core region has been described as the area bounded by the abdominal muscles in the front, by paraspinal and gluteal muscles in the back, by diaphragm on the top and by pelvic floor and girdle musculature at the bottom (richardson et al ., 1999). The core represents the connection between lower and upper limbs and should be considered as a functional unit in which different muscles interact, even if not located in the thoraco - lumbar region (such as shoulders and pelvic muscles). However, literature concerning core training sometimes fails to distinguish between concepts of core stability and core strength . Faries and greenwood (in hibbs and thompson, 2008) formulated the following clear definitions: core stability refers to the ability to stabilize the spine as a result of muscle activity, while core strength refers to the ability of muscles contractions to produce and transfer force as a result of muscle activity . Since strength and motor control are complementary qualities, the core training programmes can target mainly, but not exclusively, at muscle strengthening and/or motor control of core musculature . Motor control training seems to require low intensity stabilization exercises focused on efficient integration of low threshold recruitment of local and global muscle systems . Conversely, core strength training seems to require high intensity and overload training of the global muscle system . Vezina and hubley - kozey (2000) suggested that core stability programmes should include muscle activation below 25% of maximum voluntary contraction (mvc), while core strength training should include activation higher than 60% of mvc to result in strength benefits . The available evidence suggests that to adequately train the core muscles in athletes, strength and conditioning specialists should focus on implementing multi - joint full body exercises, rather than core - specific exercises (martuscello et al ., 2013). Exercises involving the full body linkage such as plank exercises, have been advocated to the capacity of transmitting force through the body linkage (schoenfeld et al ., 2014). Training with labile systems has been documented to offer unique opportunities for linkage training challenges (mcgill et al ., 2015). Several studies examined core muscle activation during the execution of various exercises on stable and unstable surfaces (for a review see: behm et al ., 2010). The use of unstable surfaces contacting the subject s feet or hands is becoming popular in strength training . Instability can be obtained through the use of many devices and techniques including, but not limited to, unstable platforms such as bosu or swiss balls . More recently, suspension training systems have been added to the list of instability training devices . In suspension training, lower or upper limbs many core directed exercises are designed with such a device, creating a wide variety of challenges . These exercises consist of multi - planar and multijoint movements, and are executed with complex techniques . It is important to quantify the muscle contraction intensity since it is a key factor in establishing training effects induced by this sort of exercises . Although considerable research has examined more traditional means of instability training (behm and drinkwater, 2010), little previous research has evaluated the effects of suspension training on muscle activation . In particular, some studies focused on core - directed exercises (atkins, 2014; byrne et al ., 2014; czaprowski et al ., 2014; mok et al ., 2014; snarr and esco, 2014), whereas others investigated the effect of the application of suspension system on core muscle activity in push exercises (calatayud et al ., 2014 further investigation of these exercise approaches is needed to understand their influence on muscle activation and joint load levels . The primary purpose of this study therefore, was to examine differences in core muscle activation across four full - body linkage exercises using a suspension training system . These exercises were chosen from a spectrum of whole body linkage exercises focused on the anterior core musculature executed in instable conditions, including a roll - out, bodysaw, pike, and knee - up . Although the selected exercises were mainly focused on anterior slings, we wanted to provide a comprehensive view of core muscle activation by monitoring rectus abdominis, internal and external oblique, and paraspinal muscles . It was hypothesized that significant differences would be found in core muscles among exercises . The second aim of the study was to determine which of these exercises would reach the threshold of 60% of mvc, expected to be high enough to increase muscle strength . It was hypothesized that the four exercises would elicit muscle activity in excess of 60% of mvc in the rectus abdominis, i.e. The muscle on which the main focus was put considering the selected exercises . Seventeen healthy participants were recruited (age 27.32.4 years, body height 1725 cm, body mass 69.29.3 kg). All participants were physically active, declaring three practice sessions per week of resistance training . Inclusion criteria for study participation were as follows: no past or present neurological or musculoskeletal trunk or limb pathology, no cardiorespiratory disease, no history of abdominal, shoulder or back surgery, and no psychological problems . Participants were instructed to refrain from performing strenuous physical activity in the 24 hours preceding all experimental sessions . The study was previously approved by the research ethics committee of the department of medical sciences, university of turin . The surface electromyographic (emg) signals were obtained from six trunk muscles with concentric bipolar electrodes (code, spes medica, battipaglia, italy). Before the placement of the electrodes, the skin was slightly abraded with adhesive paste and cleaned with water in accordance to seniam recommendation for skin preparation (hermens et al ., 2000). The electrodes were placed according to the instructions described in previous methodological works (beretta piccoli et al ., 2014; boccia and rainoldi, 2014) lower rectus abdominis: on the lower part of the rectus abdominis, 3 cm lateral to the midline; upper rectus abdominis: on the upper part of the rectus abdominis, 3 cm lateral to the midline; external oblique: 14 cm lateral to the umbilicus, above the anterior superior iliac spine (asis); internal oblique: 2 cm lower with respect to the most prominent point of the asis, just medial and superior to the inguinal ligament; lower erector spinae: 2 cm lateral to the l5-s1; upper erector spinae: 6 cm lateral to the l1-l2 . The electrodes were placed only on the left (randomly chosen) side of the body; the reference electrode was positioned on the wrist . The signal of a biaxial electrogoniometer (sg 150, biometrics ltd, gwent, uk) positioned at the level of the shoulders (for the roll - out and bodysaw) or the hips (for the pike and knee - tuck), depending on which joint was more involved during the exercise, was used as a trigger to highlight exercise repetitions . The emg signals were synchronized with the electrogoniometer signal, amplified (emg - usb, ot bioelettronica, torino, italy), sampled at 2048 hz, bandpass filtered (3-db bandwidth, 10 - 450 hz, 12 db / oct slope on each side), and converted to digital data by a 12-bit a / d converter . Samples were visualized during acquisition and then stored in a personal computer using ot biolab software (version 1.8, ot bioelettronica, torino, italy) for further analysis . The participants recruited were instructed with regard to the correct technique of suspension exercise and the mvc procedure during the first experimental session conducted one week before the measurement session . The participants were asked to refrain from physical activity 24 hours before the measurements . During the measurement session, participants performed 4 exercises with the use of suspension straps (trx suspension trainer; fitness anywhere lcc, san francisco, ca, usa) in random order . The exercises were selected based on a previous study (behm and drinkwater, 2010) that indicated them as important in developing core strength . At the beginning of the measurement session, three mvc exercises were performed twice for 5 s, with 2 min rest between them . The following standardized exercises (ng et al ., 2002) were used to activate maximally the trunk muscles (figure 1): upper rectus abdominis (ura) and lower rectus abdominis (lra): body supine with hips and knees flexed 90, with feet locked . Participants flexed the trunk (i.e. Crunch execution) against resistance at the level of the shoulders;external oblique (eo) and internal oblique (io): side - lying with the hip at the edge of the bench and feet locked by a second operator . Participants performed side - bend exercise against resistance at the level of the shoulder;lower erector spinae (les) and upper erector spinae (ues): prone position with asis at the edge of the bench and feet locked by a second operator . Upper rectus abdominis (ura) and lower rectus abdominis (lra): body supine with hips and knees flexed 90, with feet locked . Participants flexed the trunk (i.e. Crunch execution) against resistance at the level of the shoulders; external oblique (eo) and internal oblique (io): side - lying with the hip at the edge of the bench and feet locked by a second operator . Participants performed side - bend exercise against resistance at the level of the shoulder; lower erector spinae (les) and upper erector spinae (ues): prone position with asis at the edge of the bench and feet locked by a second operator . Standardized exercises used to maximally activate trunk muscles: lower rectus abdominis and upper rectus abdominis (left); internal oblique and external oblique (middle); lower erector spinae and upper erector spinae (right). Participants were required to achieve a range of motion with the correct technique execution and to maintain a neutral position of the spine and pelvis in each exercise . A certified strength and conditioning coach monitored the exercise performance to ensure that the exercise was properly executed considering its technique . Each exercise was repeated three times and lasted 6 s. a metronome set at 30 beats per minute was used to ensure proper timing (with 4 beats for each repetition): 2 s from the initial position to the final position (concentric phase); 2 s of maintenance (isometric phase); and 2 s returning to the starting position (eccentric phase). The exercises were performed with 3 min of rest in - between to allow complete recovery . The random order of the exercises allowed to mitigate the effects of cumulative fatigue on emg estimates . The following exercises were used (figure 2): roll - out: participants assumed an inclined standing position while placing each hand on the strap handles, with elbows and wrists placed below the shoulders, arms perpendicular to the floor and shoulders flexed approximately 45; they then performed a shoulder flexion moving the hands forward;bodysaw: participants assumed a prone position, they placed elbows below the shoulders, both forearms touching the floor, while placing each foot on the strap handle; participants then flexed the shoulders and extended the elbows pushing the body backwards;pike: participants assumed a push - up position with the feet in strap handles, then they flexed hips to approximately 90, while keeping the knees fully extended;knee - tuck: participants assumed a push - up position while placing each foot in the strap handle, then they flexed both hips and knees to approximately 90, bringing the knees forward . Roll - out: participants assumed an inclined standing position while placing each hand on the strap handles, with elbows and wrists placed below the shoulders, arms perpendicular to the floor and shoulders flexed approximately 45; they then performed a shoulder flexion moving the hands forward; bodysaw: participants assumed a prone position, they placed elbows below the shoulders, both forearms touching the floor, while placing each foot on the strap handle; participants then flexed the shoulders and extended the elbows pushing the body backwards; pike: participants assumed a push - up position with the feet in strap handles, then they flexed hips to approximately 90, while keeping the knees fully extended; knee - tuck: participants assumed a push - up position while placing each foot in the strap handle, then they flexed both hips and knees to approximately 90, bringing the knees forward . Initial and final positions of each exercise: 1) roll - out; 2) bodysaw; 3) pike; 4) knee - tuck . The average rectified value (arv) of emg signals was computed off - line with numerical algorithms using non - overlapping signal epochs of 0.5 s (hibbs et al ., 2011). The mean value of arv over the two repetitions was calculated for each muscle and normalized with respect to the maximum arv obtained during the correspondent mvc . The normality assumption of the data was evaluated with the shapiro - wilk test; homoscedasticity and autocorrelation of the variables were assessed using the breusch - pagan and durbin - watson tests . The differences between exercises (pike bodysaw knee - tuck roll - out) and between muscles (lra ura eo io les ues) were compared with the 2-way analysis of variance (anova). For the purpose of this report statistical analyses were conducted using the r statistical package (version 3.0.3, r core team, foundation for statistical computing, vienna, austria). All participants managed to complete each exercise trial and thus, were included in the data analysis . Figure 3 shows the box plots of the activation values (% of mvc) of each muscle during the four exercises . Muscle activation (median, ir) figure 3each box plot shows the muscle activation (as percentage of maximum voluntary contraction) during exercise . Table 1muscle activation (median, ir) expressed as percentage values of electromyographic amplitude normalized to maximum voluntary contraction . Results of the two - way anova after tukey multiple comparisons are reported as symbols; p <0.01.lower rectus abdominisupper rectus abdominisexternal obliqueinternal obliquelower erector spinaeupper erector spinaepike57 (36) 41 (48) 55 (21)23 (20)12 (7)9 (4)bodysaw100 (42) 57 (52)59 (33)32 (20)4 (3)8 (6)knee - tuck54 (50) 44 (41) 42 (7) 18 (26)8 (5)6 (5)roll - out140 (89) 67 (78) 71 (44) 40 (31)9 (5)11 (6)indicates statistically significant difference between the indicated exercise (explained in row) with respect to the pikeindicates statistically significant difference between the indicated exercise (explained in row) with respect to the bodysawindicates statistically significant difference between the indicated exercise (explained in row) with respect to the knee - tuckindicates statistically significant difference betweenthe indicated exercise (explained in row) with respect to the roll - out each box plot shows the muscle activation (as percentage of maximum voluntary contraction) during exercise . Muscle activation (median, ir) expressed as percentage values of electromyographic amplitude normalized to maximum voluntary contraction . Results of the two - way anova after tukey multiple comparisons are reported as symbols; p <0.01 . Indicates statistically significant difference between the indicated exercise (explained in row) with respect to the pike indicates statistically significant difference between the indicated exercise (explained in row) with respect to the bodysaw indicates statistically significant difference between the indicated exercise (explained in row) with respect to the knee - tuck indicates statistically significant difference between the indicated exercise (explained in row) with respect to the roll - out the normalized lra activity was 140% (ir, 89%) of mvc during the roll - out, 100% (ir, 42%) of mvc during the bodysaw, 57% (ir, 36%) of mvc during the pike and 54% (ir, 50%) of mvc during the knee - tuck . The normalized lra values were significantly higher (p <0.01) during the roll - out and bodysaw compared to the pike and knee - tuck . The roll - out exercise showed significantly greater activation (p <0.01) than the bodysaw . The normalized ura activity was 67% (ir, 78%) of mvc during the roll - out, 57% (ir, 52%) of mvc during the bodysaw, 41% (ir, 48%) of mvc during the pike and 44% (ir, 41%) of mvc during the knee - tuck . The normalized ura values were significantly higher (p <0.01) during the roll - out compared to the pike and knee - tuck . The normalized eo activity was 71% (ir, 44%) of mvc during the roll - out, 59% (ir, 33%) of mvc during the bodysaw, 55% (ir, 21%) of mvc during the pike and 42% (ir, 7%) of mvc during the knee - tuck . The normalized eo values were significantly higher (p <0.01) during the roll - out compared to the knee - tuck . The normalized io activity was 40% (ir, 31%) of mvc during the roll - out, 32% (ir, 20%) of mvc during the bodysaw, 23% (ir, 20%) of mvc during the pike and 18% (ir, 26%) of mvc during the knee - tuck . During all exercises the normalized io values were not significantly higher (p <0.01). The normalized les activity was 9% (ir, 5%) of mvc during the roll - out, 4% (ir, 3%) of mvc during the bodysaw, 12% (ir, 7%) of mvc during the pike and 8% (ir, 5%) of mvc during the knee - tuck . During all exercises the normalized les values were not significantly higher (p <0.01). The normalized ues activity was 11% (ir, 6%) of mvc during the roll - out, 8% (ir, 6%) of mvc during the bodysaw, 9% (ir, 4%) of mvc during the pike and 6% (ir, 5%) of mvc during the knee - tuck . During all exercises the normalized ues values were not significantly higher (p <0.01). Table 2 shows the estimate (difference of means) at 95% of the confidence interval after tukey multiple comparisons; in this case only exercise factor was considered . Estimate at 95% of the confidence interval after tukey multiple comparisons with the exercise factor considered . The estimate shows the difference of means (% of maximum voluntary contraction). Indicates the statistical significance of the adjusted p - value . The roll - out exercise showed significantly (p <0.01) higher activation compared to the bodysaw (16%, ci 8 - 23%), pike (26%, ci 18 - 33%) and knee - tuck (29%, ci 21 - 37%). Pike and knee - tuck exercises showed significantly higher activation compared to the bodysaw of 10% (28%) and 13% (6 - 21%). Suspension training has become increasingly popular as a training tool . Despite this popularity, relatively little research exists on the effects of such training on muscle activation magnitude . The first objective of the study was to investigate the activation differences of four exercises (roll - out, bodysaw, pike and knee - tuck) to better characterize suspension training . Our findings indicate that suspension exercises could be an effective strategy to reach high to very high activation of abdominal muscles such as the rectus abdominis and external oblique . To facilitate comparisons between exercises and previous studies, we categorized muscle activation into four levels according to previous studies, with <21% as low, 2140% as moderate, 4160% as high, and> 60% as very high (escamilla et al ., 2010). Exercises used in the present study provide a range of medium to high intensity exercises through which participants or athletes can progress during a training or rehabilitation programme (blanchard and glasgow, 2014) (figure 2). Roll - out exercise was the most challenging for core musculature, followed by bodysaw, pike and knee - tuck exercises (table 2). The roll - out showed the highest activation of rectus abdominis and oblique muscles compared to other exercises . Although lra showed much greater activation in roll - out and bodysaw compared to pike and knee - tuck exercises, the other muscles showed smaller differences . These findings could suggest that in the exercises characterized by shoulder flexion (such as roll - out and bodysaw), the increased requirement of core stability was reflected more by the lower rectus abdominis . According to vezina and hubley - kozey (2000), the exercises that generate muscle activity greater than 60% of mvc might be more conducive to developing muscular strength . The rectus abdominis (both parts) and eo reached activation higher than 60% of mvc (or very close to that threshold, 55%) in the roll - out and bodysaw; consequently these exercises can be considered suitable for strength training of these muscles . Although in the knee - tuck and pike, the rectus abdominis and eo did not reach the threshold of 60%, they presented high activation levels (41 - 60% mvc). While strengthening of the core is important, an activation level below 60% might be beneficial in increasing muscle endurance within the core . Since the core muscles are primarily composed of type i fibres (haggmark and thorstensson, 1979), muscular endurance should also be a major concern when designing strength and conditioning programmes (vezina and hubley - kozey, 2000). Due to large demand for muscle activation, all the proposed exercises might be appropriate for extremely fit individuals in the latter stages of a progressive abdominal strengthening or rehabilitation programme . This is an expected result as all exercises focused on anterior abdominal wall muscles . This finding confirms that in the herein selected whole - body linkage exercises, the activation of core muscles can be mainly focused on abdominal muscles while keeping the paraspinal muscles involved with low intensity . Although no direct comparison can be made between the selected suspension exercises compared to previously reported similar exercises, it is possible to highlight the following differences . We can compare only the activation of the rectus abdominis, since for oblique muscles we used a different normalization exercise than the other three studies . Plank exercises are frequently included in spine stabilization programmes as a means of improving motor control for spine stabilization . When plank exercises are performed on stable or unstable support surfaces, the reported activation level of the rectus abdominis and eo ranges from low to moderate (garcia - vaquero et al ., 2012). When executed in suspension condition, rectus abdominis muscles also showed moderate activation (byrne and bishop, 2014). Only when the planks were performed with a similar technique (instability on lower limb and shoulder flexion) was the activation similar to that reported here, which was very high for the rectus abdominis (mcgill and andersen, 2015). Therefore, we can assume that our exercises were more challenging than an isometric plank in a stable condition . In the roll - out, we found very high activation of lra (140%) and ura (67%). These levels were higher than previously reported values obtained during the execution of the roll - out with the swiss - ball (about 50 - 60% for rectus abdominis) (escamilla and lewis, 2010; marshall and desai, 2010) and similar to the values reported with the use of the power wheel, being very high for ura (76%) and lra (81%) (escamilla et al ., 2006). In the pike, we found high activation of lra (57%) and ura (41%). The values reported for the pike executed with the swiss ball (escamilla and lewis, 2010) and power wheel (escamilla and babb, 2006) were similar for ura (swiss ball 47%; power wheel 41%) and lra (swiss ball 55%; power wheel 53%). In the knee - tuck, we observed high activation of lra (54%) and ura (44%). Otherwise, the values reported for the knee - tuck executed with the swiss ball (escamilla and lewis, 2010) and power wheel (escamilla and babb, 2006) were lower for both ura (swiss ball 32%; power wheel 41%) and lra (swiss ball 35%; power wheel 45%). Our findings suggest that the two parts of the rectus abdominis can be activated differently according to the needs of the motor task (kibler et al ., 2006). This finding could be explained by the possibility to (voluntary or involuntary) modulate the activation ratio between rectus abdominis parts in order to achieve the best control of the core region . This could be justified by the metameric innervation of rectus abdominis muscles (duchateau et al ., 1988), although this issue is still controversial (monfort - panego et al ., 2009). However, lra muscles were generally more active than ura because of confounding methodological factors . Mvcs of the lra and ura in fact were estimated by a standardized exercise to activate maximally the trunk muscles: it could be argued that the same exercise fully activated ura whereas it failed to fully activate lra . Hence, the emg amplitude recorded during mvc was not the maximum achievable . Consequently, throughout experimental exercises, lra seemed relatively more active than ura because its reference value of mvc was underestimated ., arv estimates of emg signals exceeded the mvc reference values (arv higher than 100%). This inconsistency might be due to incomplete activation during mvc (as in the case of the lower rectus abdominis) and other confounding factors related to emg technique (relative shift of muscle belly with respect to electrodes occurring in dynamic tasks and different activation between isometric and dynamic tasks, among others). As widely reported, variability of muscular activation between participants was high . This suggests that performing these exercises, some individuals might produce more or less activation than the average activity indicated here . Although 17 individuals participated in this research, the differences in their fitness level and exercise experience could have affected the performance of the exercises and the resulting activation levels . Crosstalk between muscles was minimized by using an innovative detection system based on concentric - ring electrodes which had been reported as having higher spatial selectivity compared to the traditional detection systems and reducing the problem of crosstalk from nearby muscles (farina and cescon, 2001). Findings from this study, based on electromyographic analysis, showed that roll - out exercise was the most challenging . Moreover, roll - out and bodysaw exercises executed in suspension activated the rectus abdominis and external oblique muscles at intensities higher than, or very close to, 60% of the maximum voluntary contraction . Based on these findings, we can assume that roll - out and bodysaw exercises can be used to adequately strengthen the antero - lateral, superficial aspect of the core region, and thus they can be considered core strength exercises . These findings appear to have particular relevance for well - trained individuals given the high demand imposed by these exercises.
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Clinical research in the intensive care unit (icu) setting is essential to ensuring that patients are treated with interventions that are both effective and safe . Unfortunately, lack of clarity as to when research risks are acceptable in relation to anticipated benefits has impeded important clinical trials . Federal regulation governing' exception from informed consent requirements for emergency research' considers research risk on the aggregate, and as a result it imposes considerable restrictions on the conduct of research without consent . Recently, the us office for human research protections investigated three ardsnet clinical trials for purportedly exposing trial participants to undue risk . During the protracted review, enrollment in the fluid and catheters treatment trial if burdensome regulation and unnecessary trial suspension are to be avoided, then clear thinking about research risk is required . A comprehensive and systematic approach to the ethical analysis of research benefits and harms by institutional review boards (irbs), called component analysis, was recently proposed . It was endorsed by the us national bioethics advisory commission in its final report and by a number of commentators [4 - 6]. The present commentary provides the reader with a brief introduction to component analysis and highlights its application to icu research . The central insight of component analysis is that clinical research often contains a mixture of study interventions . Therapeutic procedures, such as a particular ventilation strategy, insertion of a pulmonary artery catheter, or administration of a drug, are given with therapeutic warrant . That is, they are administered on the basis of evidence supporting the expectation that the intervention may benefit the study participant . Nontherapeutic procedures, such as downloading data from monitors, drawing extra blood for pharmacokinetic drug levels, or abstracting information from the patient's chart, are administered without therapeutic warrant and are performed solely to answer the study question . Because therapeutic procedures hold out the prospect of benefit to trial participants and nontherapeutic procedures do not, a separate moral calculus is required for each type of intervention . Clinical equipoise requires in essence that therapeutic procedures in a clinical trial be consistent with competent clinical care . More formally, it requires that at the start of the trial there exist a state of honest, professional disagreement in the community of expert practitioners as to the preferred treatment . The irb ensures that this standard is met by reviewing the justification in the study protocol, the relevant literature and, if necessary, the opinions of impartial experts . Therapeutic procedures are acceptable if the irb certifies that there is sufficient evidence supporting each of the procedures such that, were it widely known, expert practitioners would disagree as to the preferred treatment . Nontherapeutic procedures do not offer the prospect of benefit to trial participants and hence a harm benefit calculus is inappropriate . Risks of nontherapeutic procedures must be minimized consistent with sound scientific design and, furthermore, they must be deemed reasonable in relation to the knowledge to be gained . The irb ensures the first standard is met by asking whether all nontherapeutic procedures are necessary to answer the study question and, if possible, by identifying procedures that might equally well piggyback on routine clinical interventions . The second standard requires that the irb judge the scientific and social value of the study to be sufficient to merit the nontherapeutic risks posed to participants . When clinical research involves members of a vulnerable population, such as pregnant women, prisoners, children, or adults incapable of providing informed consent, additional restrictions may apply . A threshold may limit the amount of nontherapeutic risk to which vulnerable research participants may be exposed legitimately . In the case of children, nontherapeutic risks are limited to a minor increase over minimal risk, that is, a minor increase over the' risks of daily life' . It has been cogently argued that a similar degree of protection ought to be afforded to adults incapable of providing informed consent a vulnerable group comprising a large proportion of participants in icu research . To determine whether risks associated with nontherapeutic procedures meet this standard, the irb reasons by analogy . It asks whether risks posed by nontherapeutic procedures are the same as those ordinarily encountered in daily life or are sufficiently similar to those risks . The irb may deem a study acceptable only if the moral calculi for both therapeutic and nontherapeutic procedures are satisfied . Component analysis allows us to disambiguate this claim, and focus attention on the incremental risk posed to icu patients who enter a clinical study . Clinical equipoise ensures a rough parity in terms of benefit, harm, and uncertainty between the procedures that patients would receive as a part of clinical practice and therapeutic procedures in a clinical trial . Thus, whatever incremental risks are posed to participants stem from nontherapeutic procedures . In icu research, these procedures are commonly limited to downloading data from monitors, abstracting chart information, and a few extra blood tests . In these cases, studies are properly understood as posing only minimal risk a finding with implications for both irb review and the informed consent process . We argued elsewhere that acute care research in which it is not possible to obtain the consent either of the patient or of their proxy decision maker might proceed under a simplified version of the waiver of consent . We argue that this approach offers a superior alternative to the unduly restrictive' exception from informed consent requirements for emergency research' . Provocatively perhaps, component analysis also suggests a novel approach to informed consent . In this approach, the focus is shifted away from the life - threatening complications of the patient's illness, which are present regardless of whether the patient participates in research, to the incremental risks posed by study participation . The consent negotiation is thereby allowed to concentrate on the question,' what difference will it make to me to participate in this study, as opposed to being treated in accordance with routine clinical care?' Professor weijer's research is supported by a canadian institutes of health research investigator award and operating grant.
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Spasticity is a frequent consequence of stroke1, 2 . In the lower leg, this malposition or malalignment causes mechanical stress to foot joints in the lateral forefoot area, and joints under this stress are likely to be inflamed and painful4 . Foot pain causes difficulty in standing and walking, and thus limits daily living activities . The malposition or malalignment of the foot can be managed by the control of spasticity . It was proposed that correcting the malposition or malalignment can reduce abnormal mechanical pressure in specific areas of the foot, which leads to a reduction in foot pain . Of the several options available for the management of spasticity, the clinical usefulness of nerve and motor point blocks with alcohol has been well demonstrated5,6,7 . In the current study, whether a reduction of ankle spasticity using nerve and motor point blocks with 20% ethyl alcohol can manage foot pain was examined . A 58-year - old woman visited the rehabilitation department of a university hospital due to piercing pain (numeric rating scale [nrs]: 8) in the left fifth metatarsal head for two years . Erythema and edema were present on the lateral aspect of the head of the fifth metatarsal bone (fig . 1. (a) an image of the patient s left foot shows erythema and edema in the fifth metatarsal head area . (b) the left foot x - ray showed that the fourth and fifth intermetatarsal angle was 7.7). The patient provided informed signed consent for participation in the study . The study was approved by the research ethics committee of yeungnam university hospital (yuh-16 - 0425-d7). On the foot x - ray, the fourth and fifth intermetatarsal angle of the left foot was 7.7 (fig . She had a history of intracerebral hemorrhage on the right basal ganglia about five years previously . The degree of her spasticity was a modified ashworth scale (mas)8 of 1 + . Additionally, slight motor weakness (medical research council9: 4 +) in the left upper and lower extremities was shown . Other neurological symptoms, including sensory and cognitive deficits, were not present . At first, 0.5 ml of 2% lidocaine with 10 mg triamcinolone acetonide was injected into the tender point of the left fifth metatarsal head area . (a) an image of the patient s left foot shows erythema and edema in the fifth metatarsal head area . (b) the left foot x - ray showed that the fourth and fifth intermetatarsal angle was 7.7 it was proposed that the ankle spasticity in the patient caused repetitive pressure and shearing between the soft tissue and head of the fifth metatarsal bone . Thus, we considered the possibility that controlling spasticity might be helpful in the management of pain from the tailor s bunion, and decided to control spasticity in this patient using a nerve or motor point block with 20% ethyl alcohol . To reduce the spasticity of the left ankle plantar flexor, the medial and lateral motor branches to the gastrocnemius muscle of the left tibial nerve the block of nerve branches was performed based on the method described by jang et al5 . To manage spasticity in the left ankle supinator a motor point block was performed in the left tibialis posterior muscle with 5 ml of 20% ethyl alcohol . Both nerve and motor points were located using the nerve stimulator at the popliteal area and at the mid - calf level, respectively . The cathode stimulating needle (teflon - coated, 23-gauge needle) was slowly advanced in the direction of the nerve and posterior tibialis muscle . The needle hub was connected to injection tubing that bore the syringe containing the 20% ethyl alcohol . Contractions were seen and palpated as the tip came close to the nerve or motor point of the tibialis posterior . At this stage, the intensity of the current was between 3 ma and 5 ma . After the needle was placed close to the targeted nerve or motor point, the tip is assumed to be in contact with the nerve or motor point when maximum contraction is obtained with minimum current . The needle was finally positioned at a current of 1 ma with nerve block and 3.5 ma with motor point block . Additionally, the patient underwent the stretching exercises for the left ankle plantar flexors and ankle supinators for 2 weeks after the alcohol block (monday through friday: 15 mins 1 time / day). To evaluate dynamic foot pressure, the foot pressure measurement system (fpms) (tpscan; biomechanics, goyang, korea) was used10 . Dynamic foot contact pressure data during gait were recorded on the fpms floor mat (40.5 40.5 cm) at the middle of the gait test . The foot pressure was presented as a color: red is the highest pressure followed by orange, yellow, green, and blue . Foot contact pressure was evaluated twice before and one- month after the nerve and motor point block procedure . Before the procedure, contact pressure was highly distributed to the lateral forefoot, however, reduced distribution was observed in the medial forefoot and hindfoot (fig . 2.foot pressure measurement imagethe foot pressure image of a normal foot and the images of the patient s foot pressure showing alterations pre - alcohol block and post - alcohol block . ). After the procedure, the distribution of foot contract pressure was found to be similar to that of a normal control (56-year - old female individual). Foot pressure measurement image the foot pressure image of a normal foot and the images of the patient s foot pressure showing alterations pre - alcohol block and post - alcohol block . At the follow - up evaluation, two weeks after the alcohol block, the spasticity in the left ankle plantar flexor and supinator had disappeared (mas: 0). In addition, ankle clonus was not present . Moreover, the pain in the left fifth metatarsal head was significantly reduced from nrs 8 to nrs 1 . At one, two, and three months after the alcohol block the effects on ankle spasticity and foot pain were sustained . In the present study, foot pain related to tailor s bunion was reduced after spasticity treatment using nerve and motor point block with 20% ethyl alcohol . Although the incidence of foot pain in patients with stroke has not been investigated, patients with stroke are prone to have foot pain due to deteriorated musculoskeletal position or alignment induced by spasticity or motor weakness . The tailor s bunion or bunionette, which was first described by davies in 194911, is a painful bony prominence on the lateral, dorsolateral, or plantar aspect of the head of the fifth metatarsal bone12 . The conflict between the fifth metatarsal head and footwear has been reported to be the cause of tailor s bunions12 . In the patient, the spasticity in the ankle plantar flexor and supinator appeared to cause repeated mechanical stress and chronic irritation in the fifth metatarsal head area, which is thought to induce a tailor s bunion . For the management of foot pain due to tailor s bunion, corticosteroids were injected into the tender point of the fifth metatarsal head area; however, the patient s pain was unresponsive to this injection . It was considered that non - controlled spasticity can lead to continuous mechanical stress or irritation and repeated occurrence of inflammation in the fifth metatarsal head area . Therefore, the spasticity was controlled using a block with 20% ethyl alcohol on the motor branches to the gastrocnemius muscle of the tibial nerve and the motor points of the posterior tibialis muscle . After the block with ethyl alcohol, spasticity in this patient had almost disappeared (from mas 1 + to mas 0). Also, the patient received the stretching exercises for the ankle plantar flexors and the ankle supinators . The stretching exercise is known to effectively reduce spasticity13, 14 by increasing tissue extensibility15 . It was thought that the reduced spasticity in the patient was attributed, at least in part, to the stretching exercises . It is thought that the mechanical stress and irritation in the fifth metatarsal head area during walking were significantly reduced with the disappearance of spasticity in this patient . When the stress and irritation were reduced after the management of spasticity, the pain due to tailor s bunion appeared to be significantly reduced . In addition, the fpms was used for detailed evaluation of the effect of the 20% ethyl alcohol block . Before the alcohol block, after the control of spasticity with the alcohol block, increased contract foot pressure in the hindfoot and medial forefoot were observed . This change in pressure distribution is concurrent with the results of several previous studies10, 16, 17 . Using fpms, it was confirmed that mechanical stress abnormally concentrated on the fifth metatarsal head area was corrected to near normal following the alcohol blocks on the tibial nerve branches and the motor point of the tibialis posterior muscle . The correction of foot contact pressure distribution appears to have contributed to the pain reduction in the fifth metatarsal head area . In conclusion, the case of a patient with chronic hemiparetic stroke whose pain due to tailor s bunion showed relief following the control of spasticity using nerve and motor point blocks with 20% ethyl alcohol was reported . This study showed that deteriorated foot position or alignment induced by spasticity and malposition / malalignment - related foot pain can be successfully managed by nerve and motor point blocks with 20% ethyl alcohol . On the basis of the clinical experience in this study, when clinicians treat foot pain in patients with stroke, they should consider the possibility that spasticity can contribute to the development of foot pain . This is first study to show that management of spasticity can reduce foot pain in a patient with hemiparetic stroke . However, since this study reports a single case, further studies involving a larger number of patients are necessary.
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Alzheimer's disease (ad), the most common form of dementia affecting populations aged over 65 years worldwide, is sporadic, genetically non - obvious, and rarely inherited (1). The main well - known pathological features of the disease include the abnormal extracellular deposition of misfolded amyloid- (a) senile plaques in brain parenchyma and cerebral vessels, the intracellular accumulation of hyperphosphorylated tau () in neurofibrillary tangles (nfts), chronic neuroinflammation, neuronal loss and severe brain atrophy as well as progressive loss of memory (2,3). Consequently, in the last 30 years, the deregulation of a metabolism (oligomerization, aggregation and plaque formation) has been the major target for therapeutic intervention (4), and only marginal effects have been registered (57), suggesting the need for preventive / curative treatments or alternative solutions . Therefore, although the causes of ad remain unknown and cures or universally effective treatments, are not available, most experts have highlighted a broad constellation of contributing risk factors . Among these risk factors, alcohol consumption, associated with extensive cognitive problems (8), including alcoholic dementia (9), has been targeted . Similar features have been denoted between the effects of alcohol on cognition, brain disorder and brain biochemistry with the biological effects of ad, suggesting that the use of alcohol may constitute a risk for aggravating or developing ad (10). In line with this view, ad patients with a habitual drinking history have shown cognitive improvement during the clinical course of abstinence (11). By contrast, this effect was not observed in patients consuming high amounts of alcohol prior to diagnosis of ad (11). In line with these data, there is another emerging body of literature that contends alcohol consumption, particularly red wine, may rather serve as a protective factor for cognitive decline (12,13). Several epidemiological studies have shown that low or moderate wine consumption can be effective in retarding age - related cognitive decline (14), possibly linked to polyphenols present in beverages . However, the studies promoting the benefits of alcohol on ad exclusively focus on moderate alcohol consumption (12), the restriction of alcohol to only an elderly population (13), and broad classification of cognitive decline (15). Relevant issues remained regarding the protection, the aggravating or detrimental effects of alcohol consumption or whether protective effects are simply influenced by the quantity and/or frequency of drinking . Despite the limiting factors identified regarding the beneficial effects of alcohol consumption (16), a single - target therapeutic strategy appears to produce only suboptimal results and a broader neuroprotective approach, at least theoretically, appears more appealing (17). Thus, the aim of the present review was to discuss the association between alcohol consumption and ad . Heavy alcohol consumption impairs cognitive performance with immediate and long - term effects on the brain anatomy and neuropsychological functioning (1821). Cognitive impairment is related to clinical dementia as it accelerates shrinkage and atrophy of the brain, leading to critical determinant of neurodegenerative changes and cognitive decline in aging (22). However, these morphological changes induced by alcohol consumption may be reversible unlike ad or aging (23), as atrophy decreases, with cognitive improvement after abstinence from alcohol (24,25). Other data showed that morphological changes in the brain are associated with the loss of a number of nerve cells occurring in the white matter, which largely comprises nerve fibers that connect neurons (26) and/or cortical cholinergic neurons (24), known to be affected in ad . This link renders plausible that alcohol use may be linked to ad as the cholinergic system plays an important role in memory . This role is confirmed and its deficits are well established in ad (27). Chronic alcohol use causes degeneration of cholinergic neurons (28), or decreases receptors of cholinergic system in ad . These may aggravate the reduction of cholinergic neurons already present in ad patients however, the improvement of cognitive function in alcoholics after abstention from alcohol suggests that cognitive deficits may reflect neurochemical alterations rather than neuronal loss (24,28). Therefore, without appearing as an accelerator of ad process, alcohol may induce its effects on the cholinergic system, independently from the cholinergic deficits caused by ad (29,30). However, alcohol - related brain damage appears to differ in young and older alcohol consumers (24), although data suggest that alcohol abuse may accelerate aging - related changes in the brain at any age and that older adults may be more vulnerable to the effects of alcohol (8). Apart from cholinergic deficit, another negative effect of heavy alcohol consumption on cognitive function may be attributed to nutritional deficiency or vascular change (31,32), which consist of damage that may be relatively irreversible even after abstinence from heavy alcohol consumption (11). Therefore, despite the above evidence showing cognitive detrimental effects of heavy alcohol consumption, to the best of our knowledge, no study has established a clear association between alcohol consumption and ad (33). More consistently, recent genetical study on a japanese population provided evidence that the mitochondrial aldehyde dehydrogenase 2 (aldh2 * 2, metabolizes acetaldehyde into acetate, protecting against oxidative stress and playing an important role in the development of ad), and two functional single - nucleotide polymorphisms (snps) of the dopamine--hydroxylase (dbh) gene, involved in the pathophysiology of alcoholism and whose activity is reduced in the neocortex of ad), did not modify the risk for developing ad, suggesting that the polymorphism of the aldh2 and dbh genes were not associated with ad (34). Nevertheless, future studies must be undertaken on other populations worldwide . Low - to - moderate alcohol intake is considered to protect against neurodegeneration pathology (13,15), dementia (3539) and cognitive deterioration (4043). Of the biologic mechanisms suggested to explain such potential beneficial effects on the brain, there are mainly the antioxidant properties of wine flavonoids (44), the effects against a (45) and the prevention of ischemia or stroke by alcohol (46). Specifically, polyphenols, members of a large family of plant - derived compounds, are molecules containing one or more phenolic group . There are thousands of polyphenols that have been identified thus far including, bioflavonoids (anthocyanins, flavanols, favanols, favones, flavanones, isofavones and proanthocyanins), coumestans, ligans and stilbenoids (47). Polyphenolics are in general antioxidant molecules that reduce the in vitro process aggregation of a, reducing the neuronal death of cortical neurons preventing neurodegeneration (48). The morin for example, a specific flavonoid described in red wine exhibited significant effects in preventing aggregation of a (49). In agreement with these data, red wine cabernet sauvignon significantly reduced the number of a plaque - induced neuropathology and attenuated a spatial memory decrease in an adult tg2576 mouse model of ad (50). Resveratrol is known to protect against cardiovascular diseases (which are risk factors for developing ad) and various types of cancer, together with the promotion of the antiaging effect, the modulation of pathomechanisms of debilitating neurological disorder such as strokes, ischemia and huntington's disease, as well as protection against neuronal degeneration (14,51). Other natural molecules including fulvic acid, altered the aggregation mechanism of proteins, a critical protein involved in the stabilization of microtubule and axonal transport, found to be involved in ad pathogenesis (52). However, whatever the facts around the benefits that may follow the potential benefits of low - to - moderate consumption of alcohol, the importance of drinking patterns and specific beverages consumed remain elusive . The operational definition of low or moderate drinking which may vary greatly across studies (53) and the concept of a moderate drinker, which may also be imprecise, comprising a wide range measure that may include those consuming less than one drink a day (54). Red wine consumption appears to promote far more protective effects than the consumption of other ethanol containing beverages (55). This is in keeping with variations that may be introduced by consumers who sometimes associate the consumption of tobacco, or the possible toxicity effects of chronic exposure of the liver to alcohol (56), which can also contribute to brain alteration in regions involved in memory (57). Taken together, the data provide insufficient evidence to suggest abstainers should initiate alcohol consumption to protect against dementia or ad . Based on the abovementioned research data, no relationship between alcohol consumption and ad exists . In addition, although low - to - moderate consumption of alcohol may protect against ad, leading to benefits on neurodegeneration, a, oxidative stress and neurofibrilary tangle formation suggest bias regarding the definition of low - to - moderate consumption as well as the variability of beverages containing alcohol . In addition, the absence of studies on the possible side effects of chronic exposure to alcohol on peripheral organs such as liver, and kidney lead to the necessity to delineate global and standard protocols for advanced studies . These studies render the benefits associated with low - to - moderate alcohol consumption against ad . However, the results should be considered as controversial and insufficient to suggest abstainers that initiate alcohol consumption in a preventive manner against ad.
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In previous papers, we have shown that heteroatom - substituted chiral [d1]methyllithiums, the smallest organometallic compounds, can be prepared in homochiral form by tin lithium exchange from the respective tributylstannyl derivatives at low temperatures . They differ in their chemical as well as in their macro- and microscopic configurational stability, depending on the heteroatom, the solvent, and the temperature used . The oxygen - substituted chiral methyllithiums 1a(1) and 1b(1) are the macroscopically configurationally most stable ones, followed by 1c(2) and 1d(3) (figure 1). The other oxygen - substituted ones are chemically unstable but microscopically configurationally stable on the time scale of a rearrangement . The silyloxy- and germyloxy - substituted ones 1e and 1f undergo retro - brook rearrangements (1,2-wittig rearrangements) with retention of configuration . The (allyloxy)methyllithiums 1 g isomerize (2,3-wittig rearrangement) with inversion of configuration, and the phosphate1h rearranges (phosphate phosphonate rearrangement) with retention of configuration . The chiral (dibenzylamino)methyllithiums (1i) are the most stable of all nitrogen - substituted ones but not completely even at 95 c . At the same temperature, compound 1j is configurationally less stable than 1i. (5) homochiral isocyanomethyllithium (1k) racemizes completely at 95 c when generated in the presence of benzaldehyde as electrophile . The phosphoric acid amide 1l undergoes a phosphoramidate-aminophosphonate rearrangement with retention of configuration . Chloro[d1]methyllithium 1 m decomposes quickly but is macroscopically configurationally stable for its short lifetime . This paper addresses the configurational stability of chiral thio- and bromo[d1]methyllithiums as compared to oxy- and chloromethyllithiums to study the influence of going from heteroatoms of the second (oxygen) and third (chlorine) row elements to ones of the third (sulfur) and fourth (bromine) one, respectively . The hoffmann test of -(phenylthio)butyllithium and -bromopentyllithium and secondary -thiobenzyllithium established configurational stability of the former at 120 and 110 c, respectively but instability of the latter at 78 c on the time scale of their addition to (s)-n, n - dibenzylphenylalaninal . The configurationally most stable -thioalkyllithiums are tertiary ones with a carbamoyl substituent at the sulfur atom . Theoretical calculations for methyllithiums substituted with a heteroatom being an element of the third or a higher row show that they are configurationally less stable than those of the second row because of the increasing ease of planarization of the carbanionic center . Thus, sulfur- and selenium - substituted alkyllithiums are configurationally less stable than the oxygen - substituted ones . The same is true for phosphorus versus nitrogen and silicon versus carbon - substituted chiral methyllithiums . Investigations of the structure of many -heteroatom - substituted alkyllithiums and mechanistic studies on the enantiomerization of -thioalkyllithiums have been performed . Reich has recently published an excellent perspective of his structural work on organolithium reagents using low - temperature nmr spectroscopy . Anticipating that chiral thio[d1]methyllithiums would not be macroscopically configurationally stable, but only microscopically at best, we decided to intercept them after generation at low temperatures by intramolecular reactions . Two types were used, the thiophosphate -mercaptophosphonate and the thia--wittig rearrangement involving short - lived thioalkyllithiums . The former was found to follow a retentive course with microscopically configurationally stable (from 78 to 0 c) (dialkoxyphosphinyl)thioalkyllithiums as intermediates and the latter an invertive one at the carbanionic center . -allylthioalkyllithiums as intermediates of the thia--wittig rearrangement were found to be configurationally stable, but -benzylthioalkyllithiums were found to be unstable and partially enantiomerized prior to the rearrangement . Furthermore, ikemoto et al . Studied the effect of solvents and additives on the steric course of oxy--wittig rearrangement of the chiral 1,3-diphenyl-1-propenyloxy-2-propen-1-yl carbanion . They found that the marked differences on the enantiomeric ratios obtained with different solvents can be ascribed to the extent of ion separation, which depends on the nature of solvent / additive . The substrates for testing the configurational stability of chiral (diisopropoxyphosphinyl)thiomethyllithiums, the diisopropyl s - tributylstannylmethyl thiophosphates, were prepared according to scheme 1 . Here and in all other cases, reactions were first optimized in the unlabeled series and then applied to the labeled ones . Tributylstannylmethanol (2) was converted via its lithium alkoxide at 78 c to mesylate 3, which was used directly to alkylate the triethylammonium salt of diisopropyl thiophosphate (4). Similarly, the deuterated enantiomers (s)- and (r)-[d1]5 were prepared from (r)- and (s)-tributylstannyl[d1]methanol of 99% ee, respectively, obtained by an improved procedure (see the experimental section). To generate the short - lived dipole - stabilized (diisopropoxyphosphinyl)thiomethyllithium (6), thiophosphate 5 was transmetalated in thf at 78 c with meli added dropwise every 3 s. normally, alkyllithiums were added with a syringe equipped with a needle to vigorously stirred reaction mixtures . In analogy to previous results, we assume tin lithium exchange whenever used here in this paper to generate heteroatom - substituted methyllithiums to follow a retentive course . It was found by reich and phillips and others that tin ate complexes form in quantities detectable by nmr spectroscopy under special conditions, e.g., the presence of hmpa as additive in thf, increasing the number of phenyl substituents at tin, and low temperature (80 c). Furthermore, tributyltin compounds are not favorable for the formation of ate complexes, which are substantially less reactive than lithium reagents . Ate complexes play only a minor role here, if at all, and that salt free species of heteroatom - substituted methyllithiums are produced . The migration of the phosphinyl group from the sulfur to the carbon atom, a thermodynamically driven reaction, gives phosphonate 7 . Addition of acoh 2 min later, workup, and chromatographic purification furnished mercaptomethylphosphonate8 in 60% yield . As no starting material could be detected, transmetalation and the ensuing rearrangement must be rapid reactions . When the experiment was performed at 0 c, it furnished merely 33% of the desired mercaptophosphonate, although no starting material was left . To determine the ee in the labeled series, mercaptophosphonate8 was derivatized with (r)-1-(1-naphthyl)ethyl isocyanate to give thiocarbamates 10 (scheme 2). Its h nmr spectrum displayed well - separated signals for the diastereotopic hydrogens of the sch2p group, allowing evaluation of the ee of the deuterated mercaptomethylphosphonates . With this information in hands, thiophosphates (s)- and (r)-[d1]5 were rearranged at temperatures ranging from 95 to 0 c, plausibly assuming a retentive course as in the case of a secondary phosphinylthioalkyllithium . The yields and the enantiomeric excesses of the mercaptomethylphosphonates [d1]8 are given in table 1 . The rearrangement in et2o at 0 c gave racemic [d1]8; however in thf a product with 23% ee (entries 3 and 1). The reason for the stronger influence of et2o compared to thf on the ee is not clear, but possibly attributable to the differing degree of solvation of lithium . The best result in terms of a combination of yield (68%) and ee (51%) was obtained in thf with n - buli at 95 c (entry 5). In summary, these results show that chiral (phosphinylthio)methyllithiums [d1]6 are microscopically configurationally labile down to 95 c on the time scale of the thiophosphate--mercaptophosphonate rearrangement . Three systems with differing lifetimes for the intermediate thiomethyllithiums amenable to thia--wittig rearrangement were investigated . The required stannanes 12a c were accessed from thiols 11a c, t - buona, and tributylstannylmethyl mesylates3 in yields of 82% to 92% (scheme 3). Lithium exchange in thf at 95 c with n - buli produced thiomethyllithium 13a (scheme 4). The ensuing thia--wittig rearrangement furnished lithium thiolate 14a, which would give on acidic workup and extraction 3-butenethiol . Its boiling point of 7080 c is too low to allow direct isolation of small amounts (<1 mmol). Therefore, a substoichiometric amount of cf3co2h (0.33 equiv relative to n - buli) was added 10 min after the addition of n - buli, followed by excess (r)-1-phenylethyl isocyanate, to convert thiol 15 for determination of yield and evaluation of ee in the labeled series to thiocarbamate 16 in 83% overall yield . The two diastereotopic hydrogens at c-1 formed an ab system in the h nmr spectrum (dmso - d6, 600 mhz), which collapsed to two broadened singlets at 2.79 and 2.82 ppm on decoupling of protons at c-2 . Similarly, deuterated stannanes (r)- and (s)-[d1]12a were rearranged using either thf or et2o as solvent at temperatures ranging from 95 to 0 c (table 2). The data show that the yields and the ee are significantly better for thf than et2o . In analogy to the results for secondary allythioalkyllithiums obtained by brickmann and brckner, we assume that the rearrangement proceeds with inversion of configuration at the carbanionic center . Surprisingly, chiral thiomethylithiums [d1]13a are configurationally stable on the time scale of thia--wittig rearrangement below 95 c in thf and ee of thiol obtained at 0 c was still 71% . It decreased only from 95% to 71% by going from 95 to 0 c in thf as solvent . Next, the microscopic configurational stability of benzylthiomethyllithiums (13b) on time scale of -rearrangement was addressed . Benzylthiomethylstannane 12b was transmetalated with n - buli in thf already at 30 c, as a higher activation energy was expected than for allyl analogue (scheme 5). As before, the rearranged lithium thiolate was converted to thiocarbamate 18 via thiol 17 . When the same reaction was performed with stannane (r)-[d1]12b at 50 c (reaction time: 20 min), the yield of thiocarbamates [d1]18 was 59% . When thiomethyllithium (s)-[d1]13b was generated at 78 c and quenched with cf3co2h after 10 min, only benzyl methyl sulfide was detected besides tetrabutyltin by h nmr spectroscopy (400 mhz) in the crude product . The following conclusions can be drawn from these three experiments . First, benzylthiomethyllithium (13b) undergoes thia--wittig rearrangement down to 50 c . At lower temperatures, the activation energy, at least that for dearomatization corresponding to resonance energy of benzene of 36 kcalmol, cannot be overcome . Second, this high activation energy increased the lifetime of intermediates 13b and their chances to enantiomerize, compared to those for allylthiomethyllithium (13a). Third, chiral benzylthio[d1]methyllithium is microscopically configurationally unstable on the time scale of thia--wittig rearrangement . The chemical stability of benzylthiomethyllithium toward thia--wittig rearrangement at 78 c enabled us to investigate its microscopic and macroscopic configurational stability . First, meli was added to a solution of unlabeled stannane 12b in thf, followed by benzaldehyde as our standard electrophile for chiral [d1]methyllithiums 10 min later . Workup after 5 min and flash chromatography furnished ()--benzylthio alcohol 19 in 77% yield (scheme 6). The (r)-mosher ester derived from (s)--methoxy--(trifluoromethyl)phenylacetyl chloride [(s)-mtpacl, (s)-mosher chloride] was prepared and investigated by h nmr spectroscopy . The sch2 group of each diastereomer displayed a well - separated ab part of an abx system, enabling the determination of ee with an estimated accuracy of 3% at best at deuterium bearing carbon atom in the labeled series . To address macroscopic configurational stability of benzylthio[d1]methyllithium [(s)-13b], this experiment was repeated with (r)-[d1]12b and yielded an alcohol being racemic at c-1 and c-2 as deduced from the h nmr spectrum (400 mhz) of the mixture of (r)-mosher esters . Despite the short time of 1 min between addition of meli for tin lithium exchange and addition of benzaldehyde, the lifetime was long enough to allow complete racemization at deuterium bearing carbon atom . To evaluate the microscopic configurational stability of (s)-13b, meli was added dropwise to a solution of stannane (r)-[d1]12b and benzaldehyde in thf at 78 c . The deuterated alcohol (s)-[2-d1]19 isolated in 24% yield, was derivatized with (s)-mosher chloride and the mixture of esters was investigated by h nmr spectroscopy . The ee at the deuterium bearing stereo center was only 16%, but significant . This time thio[d1]methyllithium (s)-[d1]13b was immediately intercepted by benzaldehyde as the electrophile . Despite the short time between generation and addition to carbonyl group, partial racemization of the (very) labile thio[d1]methyllithium interfered . The yield dropped to 9%, but the ee increased to 26%, when the reaction was performed at 95 c in the presence of 2 equiv of 12-crown-4 . It was hoped that this li complexing agent might influence the c li bond length and thus enantiomerization . When the reaction was performed at 50 c, no thiol [] d1]17 formed by rearrangement could be detected (h nmr) in the crude product besides deuterated alcohol 19 (25%, 8% ee). This experiment shows that addition of benzyl[d1]thiomethyllithium to benzaldehyde is (much) faster than -rearrangement . In the former case, the lifetime of the chiral carbanion is shorter and the chance to retain the configuration higher . Finally, the reaction was performed at 0 c and furnished racemic [d1]19 . The low yields were attributed to addition of meli to benzaldehyde as competing reaction to transmetalation, a general phenomenon in experiments for the determination of the microscopic configurational stability of chiral heteroatom - substituted [d1]methyllithiums . Only part of the starting material was consumed . In summary, benzylthio[d1]methyllithium is microscopically very labile on the time scale of the addition to benzaldehyde . To lower the activation energy for the thia--wittig rearrangement involving an aromatic compound, the phenyl ring was replaced by a naphthyl substituent . A preliminary experiment was performed with stannane 12c, which was transmetalated with n - buli at 78 c in thf and quenched after 10 min (scheme 7). No methyl 1-naphthylmethyl sulfide and only a trace of starting material could be detected in the crude product by h nmr spectroscopy . Thiol 20 was converted to thiocarbamate 21 to test its suitability for the determination of the ee in the labeled series . The sch2 group displayed an ab system in the h nmr spectrum (400 mhz). Analogously, thiomethylstannane (s)-[d1]12c was transmetalated at 50 and 95 c, using either meli (entry 1) or n - buli (entries 2) for tin lithium exchange (table 3). Surprisingly, the ees were similar for the rearrangements at 50 and 95 c . These findings demonstrate that the activation energy for isomerization of naphthyl derivative 13c is significantly lower than that for phenyl derivative 13b . Qualitatively, dearomatization of benzene could require up to 36 kcalmol, that of one benzene ring of naphthalene up to 13 kcalmol less, that is 23 kcalmol (resonance energy of naphthalene is 59 kcalmol, as one benzene ring is retained: 5936 = 23). Consequently, the rearrangement of the naphthyl derivative proceeded even at 95 c, while the phenyl derivative rearranged only at temperatures above 50 c . The reaction rate of the former was (much) higher than of the latter, so that the lifetime of carbanion 13c was (much) shorter than that of 13b . Therefore at 50 c only the benzyl - substituted thio[d1]methyllithium [d1]13b had enough time to racemize completely, while in the case of the naphthylmethyl - substituted one 80% of the molecules did not enantiomerize, resulting in an ee of 60% . -haloalkyllithiums, also termed lithium carbenoids, have nucleophilic and electrophilic properties, depending on the solvent and the reaction temperature . Lithium exchange in,-dihaloalkanes,,-dihalocyclopropanes, or preferably dihalomethanes, at low temperatures (78 to 120 c) and are reacted with a variety of electrophiles, especially aldehydes and ketones to obtain epoxides . Chiral -chloroalkyllithiums and -chloroalkylmagnesium halides have also been prepared elegantly, the latter being the configurationally more stable species (below 20 c). Enantiopure chloro[d1]methyllithium was prepared in our group and found to be micro- and macroscopically configurationally stable at 78 c, but chemically very labile . Here we address the configurational stability of enantiopure bromo[d1]methyllithiums similarly to the chloro analogues . However, the preparation of starting bromo[d1]methylstannanes of ee 99% and the high propensity of brchdli to decomposition were challenging obstacles on the way to success . We reasoned that we could generate bromomethyllithium from bromomethyltributylstannane by tin lithium exchange and trap it in situ with benzaldehyde, resulting in the formation of the lithium salt of the corresponding bromohydrin . To check whether it is chemically stable and whether its (r)-mosher ester is suitable for the determination of the ee in the labeled series, some exploratory experiments were performed (scheme 8). When it was converted to the lithium alkoxide in thf with meli at 78 c and then quenched with cf3co2h after 5 min, it was recovered unchanged in 95% yield . No epoxide 24 could be detected in the h nmr spectrum (400 mhz) of the crude product as evidenced by spiking with an authentic sample . Samples of ()-23 and (s)-23 obtained by enantioselective reduction of phenacyl bromide with (+) -dip - chloride were converted to diastereomeric (r)-mosher esters and investigated by h nmr spectroscopy . Bromomethyltributylstannane was prepared from tributylstannylmethanol (2) in yields of up to 91% using ph3p / nbs (scheme 9). When it was transmetalated at 78 c in thf and the reaction quenched with cf3co2h 30 s after the addition of 1 m meli, the crude product was methyltributylstannane containing no starting material . Anticipating fast decomposition of bromomethyllithium, we decided to do two in situ trappings using 4 equiv of benzaldehyde in admixture with bromomethylstannane in dry thf at 78 c in analogy to the experiments with chloromethyllithium . Four equiv of meli (1 m in cumene / thf / diethoxymethane) were added dropwise and the reaction was quenched with cf3co2h after 5 and 15 min, respectively . In both cases, only 25% of the starting material was transmetalated, of which 80% formed bromohydrin ()-23 in 19% yield for both experiments as evidenced byh nmr spectroscopy of the crude product . Meli underwent two competing reactions, tin lithium exchange and addition to benzaldehyde, which was faster and resulted in ()-1-phenylethanol as major side product . To slow down addition of meli to the electrophile relative to transmetalation, acetophenone was tested as alternative electrophile . The desired bromohydrin ()-27 was obtained in 13% yield along with the tertiary alcohol derived from addition of meli to acetophenone, and recovered starting material . Bromohydrin ()-27 although a tertiary alcohol could be converted to diastereomeric (r)-mosher esters under forcing conditions (50 c, 1,4-dioxane, 8 h). The h nmr spectrum (400 mhz) showed ab systems for the ch2br groups, demonstrating the feasibility to determine the ee in the deuterated series . Chiral bromo[d1]methylstannane (r)-[d1]25 was prepared from (s)-[d1]2 at first by the procedure used for the preparation of the unlabeled compound in 92% yield . Transmetalation and in situ trapping (with retention of configuration) of the intermediate bromo[d1]methyllithium [(s)-[d1]26] with benzaldehyde at 78 c furnished bromohydrin (2s)-[2-d1]23 in 11% yield with an ee of 57% at c-2, determined by h nmr spectroscopy of the corresponding (r)-mosher ester . Being unsure about the ee of the starting bromide, we determined it in the same way as that of the chloride, using a homochiral thiol as derivatizing agent and h nmr spectroscopy and found it to be 57% . This proved that the chiral bromo[d1]methylstannane was not enantiomerically pure and that it seemingly produced microscopically configurationally stable bromo[d1]methyllithium, which we wanted to corroborate by more experiments . Apparently, the starting bromo[d1]methylstannane racemized partly under the reaction conditions for the substitution reaction, under which the chloro compound was configurationally stable . Bromide ions in the reaction mixture replaced bromide of the substrate by a sn2 mechanism, resulting in partial enantiomerization . Then we switched to a modified mitsunobu reaction with ph3p / diad (diisopropyl azodicarboxylate)/ph3phbr in toluene, optimized it, and finally obtained (r)-bromo[d1]methylstannane of 99% ee in 35% yield . Chiral bromo[d1]methylstannanes with ee ranging from 77 to 99% were transmetalated in the presence of benzaldehyde or acetophenone at 78 or 95 c . The data show that the ee of the bromohydrins reflect the ee of the respective starting bromo[d1]methylstannanes being microscopically configurationally stable, irrespective of whether benzaldehyde or acetophenone was used for the in situ trapping (table 4, entries 35). In the case of entry 5, the h nmr spectrum of the crude product revealed the following molar ratios: (s)-[d1]2/phcho / bromohydrin (2r)-[2-d1]23/bu3snch3/phch(oh)ch3 = 2:1.62:0.1:0.35:4.18 . The major portion [85%; 2/(2 + 0.35) = 0.85] of the starting material was recovered and only about one - third (0.1:0.35 = 0.29) of the chiral bromomethyllithium as deduced from the formed tributylmethylstannane gave bromohydrin . Evidently, the other two - thirds decomposed because of low chemical stability . When acetophenone was used as electrophile, the ratios for entry 4 of table 4 in the crude product were for (s)-[d1]2/phc(o)ch3/bromohydrin (1r)-[1-d1]27/bu3snch3/phc(oh)(ch3)2 = 0.0:0.93:0.39:1.0:0.72 . This time transmetalation was complete as addition of meli to acetophenone was slowed down compared to benzaldehyde . Surprisingly, again only about one - third of the bromo[d1]methyllithium generated was intercepted by acetophenone . However, two - thirds decomposed apparently . We expected a higher percentage of decomposition because the lifetime of brchdli will be longer in the presence of acetophenone than benzaldehyde as the addition to the electrophile will be slower . We cannot exclude that some of the bromomethyllithiums reacted with the starting bromomethylstannanes to give finally tributylmethylstannane and ethene as found for -haloalkylstannanes . These results demonstrate that chiral bromo[d1]methyllithiums are microscopically stable on the time scales of the addition to benzaldehyde and acetophenone, but extremely labile . Signals of chdbr groups in the h nmr spectra (400 mhz, toluene - d8) of (r)-mosher esters derived from (a) (2s)-[2-d1]23 of 76% ee and (b) (2r)-[2-d1]23 of 99% ee (d 96%). Four chiral thio[d1]methyllithiums with different substituents at sulfur were prepared by tin lithium exchange and their microscopic configurational stability was determined relative to the thiophosphate--mercaptophosphonate and thia--wittig rearrangements, respectively (figure 3). The configurational stability of chiral thio[d1]methyllithiums is delicately influenced by their lifetime depending on the substitutent at sulfur . The chances for enantiomerization of a thiomethyllithium will increase with its lifetime, which is inversely proportional to the rate of the rearrangement . While 13a is configurationally stable at 95 c, 13b underwent the thia--rearrangement only down to 50 c and racemized completely . In case of 13b, the microscopic configurational stability was also evaluated on the time scale of its addition to benzaldehyde . Unfortunately, the solution structures of the respective methyllithiums and their mechanism of enantiomerization are unknown . Hoffmann et al . And reich and dykstra found for -selenium- and -sulfur - substituted alkyllithiums that rotation about the c heteroatom bond is the rate - determining step of enantiomerization . Lithium exchange (salt free?) Proved to be chemically very labile but microscopically configurationally stable on the time scale of the addition to benzaldehyde and acetophenone at 78 c . In summary, we assume that the rearrangements of 6 and 13a, c follow a retentive and invertive course, respectively, in analogy to chiral, nonracemic organolithiums with the same heteroatom, but an alkyl group instead of the deuterium atom . Analogously, thiomethyllithium 13b and bromomethyllithium 26 add to benzaldehyde with retention of configuration . . Microscopic configurational stability of various thio[d1]methyllithiums on the time scale of rearrangements for 6 and 13a c and on the time scale of addition of 13b and bromo[d1]methyllithiums 26 to benzaldehyde . H / c (j modulated) nmr spectra were measured at 300 k at 400.13, 400.27, 600.13 mhz/100.61, 100.65, 150.92 mhz, respectively . They were referenced either to residual chcl3 (h 7.24)/toluene - d8 (chd2: h 2.09)/cd3od (chd2: h 3.31)/dmso - d6 (chd2: h 2.50) or cdcl3 (c 77.00)/toluene - d8 (cd3: c 21.04)/cd3od (cd3: c 49.00)/dmso - d6 (cd3: c 39.50). Ir spectra of films on a silicon disk were recorded on ft - ir spectrometers or by using atr . Optical rotations were measured at 20 c with a polarimeter in a 1 dm cell . Flash (column) chromatography was performed with silica gel 60 (230400 mesh) and monitored by tlc, carried out on 0.25 mm thick plates, silica gel 60 f254 . Spots were visualized by uv and/or dipping the plate into a solution of (nh4)6mo7o244h2o (23.0 g) and ce(so4)24h2o (1.0 g) in 10% aq h2so4 (500 ml), followed by heating with a heat gun . The two diastereomeric boronates 31 and 32 were reduced with libet3d to (1s)-[1-h1]- and (1r)-[1-h1]34, respectively (compound numbers are taken from the literature). An aqueous solution of naoh (2.27 ml, 7.82 mmol, 3.44 m) and a solution of h2o2 (1.0 ml, 10.33 mmol, 30%) were added at 0 c to (1s)-[1-h1]34 (1.689 g, 3.13 mmol) dissolved in dry thf (15.7 ml). After the biphasic mixture was stirred vigorously for 1 h, more naoh (2.27 ml) and h2o2 (1.0 ml) were added . Stirring was continued for another 1 h, and then water (18 ml) and pentaerythritol (600 mg) were added . The organic phase was separated, and the aqueous phase was extracted with etoac (3 30 ml). The combined organic layers were washed with water (30 ml) and brine (30 ml), dried (mgso4), and concentrated under reduced pressure . The residue was purified by flash chromatography (hexane / etoac, 10:1, rf 0.48) to give (s)-[d1]2 (826 mg, 82%, d 9698%). Similarly, (1r)-[1-h1]34 (1.617 g, 2.99 mmol) was converted to (r)-[d1]2 (731 mg, 76%, d 9698%). A solution of alcohol (0.10 mmol), dry pyridine (0.25 ml), and (s)-mtpacl (0.3 ml, 0.15 mmol, 0.5 m in dry ch2cl2) in dry ch2cl2 (2 ml) was left at rt (for bromohydrins 4 h, for all other alcohols 418 h). Afterward, ch2cl2 (10 ml) and hcl (10 ml, 1 m) were added . The organic phase was separated, washed with saturated aq nahco3, dried (mgso4), and concentrated under reduced pressure . The crude product was purified by flash chromatography to furnish oily (r)-mosher esters . Tmp (432 mg, 3.07 mmol) was dissolved in dry thf (12.4 ml) under argon and the solution cooled to 10 c . N - buli (1.93 ml, 3.07 mmol, 1.6 m in hexane) was added . After 15 min, the solution was cooled to 78 c, tributylstannylmethanol (2) (826 mg, 2.56 mmol) in dry thf (4.2 ml) was added, and the solution was stirred for 15 min . Mscl (258 l, 3.33 mmol) was added, the solution was stirred for another 20 min, and then freshly prepared triethylammonium salt of o, o - diisopropyl thiophosphoric acid (3.84 mmol, 10.68 ml of solution prepared from 10 mmol of phosphite in 25 ml of i - proh) was added . The mixture was concentrated under reduced pressure, and water (26 ml) was added . The combined organic phases were washed with water (30 ml), dried (na2so4), concentrated under reduced pressure, and purified by flash chromatography (hexane / etoac, 7:1, rf 0.38) to yield thiophosphate 5 as a colorless oil (1.014 g, 79% for two steps). Ir (si): 2958, 2927, 1253, 979 cm . H nmr (400.27 mhz, cdcl3): 4.72 (dsept, j = 8.9, 6.2 hz, 2h), 2.04 (d, j = 9.0 hz, j(sn) = 35.4 hz, 2h), 1.601.38 (m, 6h), 1.36 (d, j = 6.3 hz, 6h), 1.33 (d, j = 6.3 hz, 6h), 1.29 (sext, j = 7.3 hz, 6h), 1.040.87 (m, j(sn) = 50.8 hz, 6h), 0.87 (t, j = 7.3 hz, 9h). C nmr (100.61 mhz, cdcl3): 72.1 (d, j = 6.0 hz, 2c), 28.9 (j(sn) = 21.8 hz, 3c), 27.2 (j(sn) = 56.5 hz, 3c), 23.9 (d, j = 4.2 hz, 2c), 23.7 (d, j = 5.6 hz, 2c), 13.6 (3c), 9.7 (j(sn) = 323.4 hz, 3c), 4.5 (d, j = 4.9 hz). Calcd for c19h43o3pssn: c, 45.52; h, 8.65; s, 6.40 . Found: c, 45.93; h, 8.85; s, 6.10 . Similarly, (s)-tributylstannyl[d1]methanol {(s)-[d1]2} (826 mg, 2.56 mmol) and (r)-[d1]2 (731 mg, 2.27 mmol) were converted to thiophosphates (r)-[d1]5 (1.014 g, 79%) and (s)-[d1]5 (947 mg, 83%), respectively . Their h nmr spectra (400.27 mhz, cdcl3) were identical to that for 5 except for 2.04 (br d, j = 9.0 hz, j(sn) = 35.4 hz, 1h). Meli (0.52 ml, 0.52 mmol, 1 m in thf / cumene) was added to a solution of s - stannylmethyl thiophosphate 5 (217 mg, 0.43 mmol) in dry thf (3 ml) under argon at 78 c dropwise every 3 s. after 2 min, acoh (0.5 ml, 2 m, in thf) was added, and the solution was warmed and concentrated under reduced pressure . Water (4 ml) was added, and the aq phase was extracted with etoac (3 10 ml). The residue was purified by flash chromatography (hexane / etoac, 1:1, rf 0.14) to yield mercaptophosphonate 8 as a colorless oil (55 mg, 60%). Similarly, thiophosphate 5 (202 mg, 0.40 mmol) was converted to mercaptophosphonate 8 (28 mg, 33%) except that meli was added dropwise every 1 s and that the reaction was quenched after 1 min with acoh (0.48 ml, 2 m, in thf). Similarly to experiment 1, (s)-[d1]5 (280 mg, 0.56 mmol) was converted to (r)-[d1]8 (48 mg, 41%, ee 23%) except that the reaction was performed at 0 c and quenched with acoh 15 s after the addition of meli, added dropwise every 1 s. the spectroscopic data were identical to that of 8 except as follows . H nmr (400.27 mhz, cdcl3): 2.60 (tdd, j = 13.4, 8.2, 2.1 hz, 1h), 1.79 (dd, j = 8.5, 8.2 hz, 1h). C nmr (100.61 mhz, cdcl3): 18.3 (dt, j = 151.8, 21.2 hz). Similarly to experiment 1, (s)-[d1]5 (229 mg, 0.46 mmol) was converted to (r)-[d1]8 (22 mg, 22%, ee 77%) except that the reaction was performed in et2o at 95 c, using 1.05 equiv of meli . Similarly to experiment 4, (s)-[d1]5 (218 mg, 0.43 mmol) was converted to (r)-[d1]8 (52 mg, 56%, racemic), except that the reaction was performed in dry et2o at 0 c . The reaction was quenched with acoh after 15 s. similarly to experiment 4, thiophosphate (r)-[d1]5 (286 mg, 0.57 mmol) was converted to deuterated (s)-mercaptophosphonate (s)-[d1]8 (31 mg, 25%, ee 61%) except that the reaction was performed in dry thf and quenched with acoh 3 min after the addition of meli . Similarly to experiment 6, (r)-[d1]5 (249 mg, 0.50 mmol) was converted to (s)-[d1]8 (73 mg, 68%, ee 52%) except that meli was replaced by n - buli . A solution of mercaptomethylphoshonate8 (76 mg, 0.36 mmol) and (r)-()-1-(1-naphthyl)ethyl isocyanate (0.62 ml, 0.72 mmol, ee 95%) in dry thf (3 ml) under argon was stirred for 5 h at rt . Afterward, water (0.25 ml) was added, and the mixture was stirred for another 2.5 h and concentrated under reduced pressure . The residue was purified by flash chromatography (hexane / etoac, 1:2, rf 0.61) to yield thiocarbamate 10b (136 mg, 97%) as a colorless oil . Ir (si): 3222, 2980, 2931, 1675, 1533, 1238, 1216, 994 cm . H nmr (400.13 mhz, cdcl3): 8.04 (d, j = 8.3 hz, 1h), 7.857.75 (m, 1h), 7.74 (d, j = 7.9 hz, 1h), 7.557.35 (m, 4h), 6.59 (br s, 1h, nh), 5.85 (br s, 1h), 4.61 (dsept, j = 7.7, 6.3 hz, 2h), 3.18 (ab - syst, jab = 15.0 hz, j = 12.9 hz, 2h), 1.63 (d, j = 6.8 hz, 3h), 1.27 (d, j = 6.0 hz, 3h), 1.26 (d, j = 5.7 hz, 3h), 1.23 (d, j = 6.2 hz, 3h), 1.21 (d, j = 6.2 hz, 3h). C nmr (100.61 mhz, cdcl3): 164.3 (br s), 137.9, 133.8, 130.7, 128.8, 128.8, 128.3, 126.4, 125.7, 125.2, 123.0, 122.6, 71.40 (d, j = 6.7 hz), 71.38 (d, j = 6.7 hz), 60.3, 24.0 (d, j = 153.2 hz), 23.94 (d, j = 4.5 hz), 23.91 (d, j = 4.4 hz), 23.79 (d, j = 4.8 hz), 23.77 (d, j = 5.2 hz), 21.1 (br s). Calcd for c20h28no4ps: c, 58.66; h, 6.89; n, 3.42; s, 7.83 . Found: c, 59.05; h, 7.14; n, 3.80; s, 8.41 . Similarly, (r)-[d1]8 (48 mg, 0.22 mmol) was converted to (r)-[d1]10b (66 mg, 73%, ee 23%). The h nmr spectrum (400.27 mhz, cdcl3) was identical to that of 10b except for 3.22 (d, j = 13.3 hz, 0.38h), 3.17 (d, j = 13.2 hz, 0.61h). Similarly, (s)-[d1]8 (31 mg, 0.14 mmol) was converted to (s)-[d1]10b (50 mg, 86%, ee 51%). The h nmr spectrum (400.27 mhz, cdcl3) was identical to that of (r)-[d1]10b except for the different integration of the two doublets 3.22 (d, j = 13.3 hz, 0.74h), 3.17 (d, j = 13.2 hz, 0.23h). Allylmercaptan (0.20 ml, 2.37 mmol) was added to a solution of t - buona (228 mg, 2.37 mmol) in dry thf (5.0 ml) and stirred under argon for 10 min . The reaction mixture was cooled to 30 c, and tributylstannylmethyl mesylate (3) (632 mg, 1.58 mmol) in dry thf (3 ml) was added . After the mixture was stirred for 15 min, 1 m hcl (8 ml) and hexane (8 ml) were added . The organic phase was separated and washed with brine (20 ml), dried (mgso4), and concentrated under reduced pressure . The residue was purified by flash chromatography (hexane / ch2cl2, 30:1, rf 0.54) to yield tributylstannylmethyl sulfide (12a) (500 mg, 84%) as a colorless oil . Ir (si): 2956, 2926, 2853, 1635, 1464, 1376, 911 cm . H nmr (400.27 mhz, cdcl3): 5.74 (tdd, j = 17.2, 10.0, 7.3 hz, 1h), 5.07 (tdd, j = 10.0, 1.7, 0.8 hz, 1h), 5.03 (tdd, j = 17.2, 1.7, 1.2 hz, 1h), 3.06 (ddd, j = 7.3, 1.2, 0.8 hz, 2h), 1.80 (s, j(sn) = 41.3 hz, 2h), 1.601.38 (m, 6h), 1.30 (sext, j = 7.3 hz, 6h), 1.100.82 (m, j(sn) = 50.6 hz, 6h), 0.87 (t, j = 7.3 hz, 9h). C nmr (100.61 mhz, cdcl3): 133.9, 116.7, 41.0, 29.0 (j(sn) = 20.9 hz, 3c), 27.3 (j(sn) = 55.2 hz, 3c), 13.7 (3c), 9.5 (j(sn) = 334.7, 319.6 hz, 3c), 7.5 . Calcd for c16h34ssn: c, 50.94; h, 9.08 . Found: c, 51.16; h, 9.14 . Similarly, mesylates (r)- and (s)-[d1]3 (690 mg, 1.72 mmol and 635 mg, 1.59 mmol) were converted to sulfides (s)-[d1]12a (537 mg, 83%) and (r)-[d1]12a (375 mg, 63%), respectively . The h nmr spectra (400.27 mhz, cdcl3) were identical to that of 12 except for 1.78 (br s, j(sn) = 40.7 hz, 1h). Benzylmercaptan (0.35 ml, 3.0 mmol) was alkylated with mesylate 3 (798 mg, 2.0 mmol) by the procedure used for the preparation of allylthiomethylstannane 12a . The crude product was first heated (45 c, 0.5 mbar) to remove excess benzylmercaptan and then purified by flash chromatography (hexane / ch2cl2, 30:1, rf 0.33) to yield benzylthiomethylstannane 12b (786 mg, 92%) as a colorless oil . Ir (si): 2955, 2923, 2851, 1493, 1453, 1376, 1071 cm . H nmr (400.27 mhz, cdcl3): 7.357.15 (m, 5h, harom), 3.64 (s, 2h, ch2s), 1.77 (s, j(sn) = 41.6 hz, 2h, ch2sn), 1.571.33 (m, 6h, 3 ch2ch2sn), 1.30 (sext, j = 7.3 hz, 6h, 3 ch2(ch2)2sn), 0.970.80 (m, j(sn) = 49.6 hz, 6h, 3 ch2sn), 0.85 (t, j = 7.3 hz, 9h, 3x ch3(ch2)3sn). C nmr (100.61 mhz, cdcl3): cq not detected, 129.0 (2c, carom), 128.3 (2c, carom), 126.6 (carom), 42.5 (ch2s), 29.0 (j(sn) = 20.6 hz, 3c, 3 ch2(ch2)2sn), 27.2 (j(sn) = 55.2 hz, 3c, 3 ch2-ch2sn), 13.7 (3c, 3 ch3(ch2)3), 9.5 (j(sn) = 334.4 hz, 3c, 3 snch2(ch2)2), 8.3 (t, j(sn) = 223.0 hz, sch2sn). Calcd for c20h36ssn: c, 56.22; h, 8.49; s, 7.50 . Found: c, 56.48; h, 8.45; s, 7.26 . Similarly, mesylates (r)-[d1]3 (1.0 g, 2.49 mmol) and (s)-[d1]3 (1.690 g, 4.22 mmol) were converted to benzylthio-[d1]methylstannanes (s)-[d1]12b (870 mg, 82%) and (r)-[d1]12b (1.535 g, 85%), respectively . Their h nmr spectra (400.27 mhz, cdcl3) were identical to that of 12b except for 1.77 (s, j(sn) = 41.6 hz, 1h). 1-naphthylmethanethiol (745 mg, 4.66 mmol) was alkylated with mesylate 3 (1.240 g, 3.11 mmol) by the procedure used for the preparation of allylthiomethylstannane 12a . The combined organic layers were washed with brine (25 ml) and 2 m naoh (3 25 ml), dried (mgso4), and concentrated under reduced pressure . The crude product was purified by flash chromatography (hexane / ch2cl2, 30:1, rf 0.33) to yield (1-naphthylmethyl)thiomethylstannane 12c (1.219 g, 82%) as a colorless oil . Ir (si): 2954, 2922, 2851, 1510, 1462, 1376, 1074, 1016 cm . H nmr (400.27 mhz, cdcl3): 8.198.11 (d, j = 8.5 hz, 1h), 7.857.68 (m, 2h), 7.557.42 (m, 2h), 7.417.33 (m, 2h), 4.12 (s, 2h), 1.83 (s, j(sn) = 40.1 hz, 2h), 1.491.33 (m, 6h), 1.21 (sext, j = 7.3 hz, 6h), 0.980.83 (m, 6h), 0.81 (t, j = 7.3 hz, 9h). C nmr (100.61 mhz, cdcl3): 134.1, 133.7, 131.6, 128.6, 127.7, 127.2, 125.8, 125.6, 125.0, 124.3, 40.4, 28.9 (j(sn) = 20.9 hz, 3c), 27.2 (j(sn) = 55.1 hz, 3c), 13.6 (3c), 9.5 (j(sn) = 319.6 hz, 3c), 9.1 (j(sn) = 214.4 hz). Calcd for c24h38ssn: c, 60.39; h, 8.02 . Found: c, 60.34; h, 7.76 . Similarly, mesylate (s)-[d1]3 (678 mg, 1.69 mmol) was converted to thiomethylstannane (r)-[d1]12c (642 mg, 79%). H nmr (400.27 mhz, cdcl3): 1.81 (s, j(sn) = 41.0 hz, 1h, chdsn). C nmr (100.61 mhz, cdcl3): 8.8 (t, j = 21.0 hz, 1c, schdsn). N - buli (0.25 ml, 0.39 mmol) was added to the solution of sulfide 12a (131 mg, 0.33 mmol) in dry thf (2.5 ml) at 95 c under argon . After 10 min, 2 m cf3co2h (70 l, 0.13 mmol) was added and the mixture was used immediately for the derivatization as thiol 15 cannot be isolated because of its low boiling point . (r)-(+)-1-phenylethyl isocyanate (0,10 ml, 0.66 mmol, ee 99%) was added at 95 c . After 45 min a saturated aqueous solution of nahco3 (10 ml) was added and the mixture was extracted with etoac (2 20 ml). The combined organic phases were dried (mgso4) and concentrated under reduced pressure . The residue was purified by two flash chromatographies (first: hexane / etoac, 10:1, rf 0.38, side product derived from isocyanate 0.45; second: hexane / ch2cl2, 1:1, rf 0.29, side product derived from isocyanate 0.68) to yield thiocarbamate 16 (64 mg, 83%) as colorless crystals . Ir (si): 3293, 2977, 1652, 1520, 1495, 1217, 699 cm . H nmr (400.27 mhz, cdcl3): 7.377.18 (m, 5h, harom), 5.77 (tdd, j = 17.0, 10.2, 6.7 hz, 1h, ch = ch2), 5.54 (br s, 1h, nh), 5.06 (qd, j = 17.0, 1.6 hz, 1h, ch = ch2), 5.05 (br s, 1h, chch3), 5.01 (tdd, jci = 10.2, 1.6, 1.2 hz, 1h, ch = ch2), 2.95 (ab - sys, jab = 13.4 hz, j = 7.1 hz, 2h, ch2s), 2.35 (tddd, j = 7.1, 6.7, 1.6, 1.2 hz, 2h, ch2ch2s), 1.49 (d, j = 6.9 hz, 3h, ch3ch). C nmr (100.61 mhz, cdcl3): 142.8, 136.3, 128.7 (2c), 127.5 (2c), 126.0, 116.3, 51.1, 34.5, 29.2, 22.0 . Anal . Calcd for c13h17nos: c, 66.34; h, 7.28; n, 5.95; s, 13.62 . Found: c, 66.16; h, 7.25; n, 6.04; s, 13.40 . The h nmr spectra (400.27 mhz, cdcl3) of (r)- and (s)-[d1]16 were identical to that of 16, except for 2.95 (t, j = 7.2 hz, 1h, chd), 2.34 (t, j = 6.9 hz, ch2chd). To determine the ee of derivatives [d1]16 the h nmr experiments were performed in dmso - d6 (600.13 mhz) with irradiation at 2.24 ppm (decoupling of schdch2). Two broad singlets (2.79 and 2.82) were observed for the two diastereotopic protons of the schd group . Allylthio[d1]methylstannane (s)-[d1]12a (174 mg, 0.46 mmol) was converted to thiocarbamate (s)-[d1]16 (90 mg, 83%, ee 95%) by the procedure used for experiment 1 . Stannane (s)-[d1]12a (161 mg, 0.43 mmol) was converted to (s)-[d1]16 (95 mg, 95%, ee 91%) by the procedure used for experiment 1 except that it was performed at 78 c . Stannane (s)-[d1]12a (172 mg, 0.45 mmol) was converted to (s)-[d1]16 (104 mg, 99%, ee 83%) by the procedure used for experiment 1 except that it was performed at 40 c . Stannane (s)-[d1]12a (164 mg, 0.43 mmol) was converted to (s)-[d1]16 (73 mg, 72%, ee 71%) by the procedure used for experiment 1 except that it was performed at 0 c and that the reaction was quenched with cf3co2h 3 min after the addition of n - bul . Stannane (r)-[d1]12a (189 mg, 0.50 mmol) was converted to (r)-[d1]16 (74 mg, 62%, ee 50%) by the procedure used for experiment 3 except that it was performed in dry et2o . Stannane (r)-[d1]12a (184 mg, 0.49 mmol) was converted to (r)-[d1]16 (51 mg, 45%, ee 20%) by the procedure used for experiment 5 except that it was performed in dry et2o . N - buli (0.41 ml, 0.65 mmol) was added to a solution of 12b (231 mg, 0.54 mmol) in dry thf (3.8 ml) at 30 c under argon . After 30 min, 2 m cf3co2h (0.11 ml, 0.22 mmol) was added, and the mixture was used directly for the derivatization . The 2-methylphenylmethanethiol 17 (detectable by tlc: hexane / ch2cl2, 10:1, rf 0.54) was not isolated because of its volatility (bp 97 c/14 mm). After the mixture was stirred for 1.5 h at rt, water (1 ml) was added and the reaction mixture was concentrated under reduced pressure . The residue was purified by flash chromatography (hexane / ch2cl2, 1:1, rf 0.43) to yield thiocarbamate 18 (66 mg, 43%) as colorless crystals; mp 9293 c (i - pr2o). Ir (atr): 3277, 2972, 2926, 1643, 1527, 1493, 1446, 1216 cm . H nmr (400.27 mhz, cdcl3): 7.417.20 (m, 6h, harom), 7.197.05 (m, 3h, harom), 5.52 (br s, 1h, chch3), 5.08 (br s, 1h, nh), 4.16 (ab - sys, jab = 13.7 hz, 2h, ch2s), 2.33 (s, 3h, ch3ph), 1.49 (d, j = 6.9 hz, 3h, ch3ch). C nmr (100.61 mhz, cdcl3): co n. d., 142.7 (cq arom), 136.6 (cq arom), 136.1 (cq arom), 130.4 (carom), 129.9 (carom), 128.7 (2c, carom), 127.6 (2c, carom), 127.5 (carom), 126.2 (carom), 126.0 (carom), 51.2 (chch3), 32.4 (ch2s), 22.0 (br s, ch3ch), 19.4 (ch3ph). Calcd for c17h19nos: c, 71.54; h, 6.71; n, 4.91; s, 11.23 . Found: c, 71.29; h, 6.77; n, 4.92; s, 11.06 . Similarly, (s)-benzylthio[d1]methylstannane (s)-[d1]12b (232 mg, 0.54 mmol) was converted to thiocarbamate [d1]18 (92 mg, 59%) except that the reaction temperature was 50 c and the reaction time 20 min . The h nmr spectrum (400.27 mhz, cdcl3) was identical to that of the unlabeled species 18 except for 4.17 (s, 1h, chd), 4.13 (s, 1h, chd) (thiol [d1]17 was racemic). N - buli (0.17 ml, 0.26 mmol) was added to a solution of benzylthiomethylstannane 12b (92 mg, 0.22 mmol) in dry thf (1.6 ml) at 78 c under argon . After 10 min, benzaldehyde (0.17 ml, 0.33 mmol, 2 m in dry thf) was added, followed by saturated aq nahco3 (3 ml) 5 min later . The combined organic phases were dried (mgso4) and concentrated under reduced pressure . The residue was purified by flash chromatography (hexane / etoac, 10:1, rf 0.26) to yield alcohol19 (42 mg, 77%) as a colorless oil . H nmr (400.27 mhz, cdcl3): 7.407.18 (m, 10h), 4.66 (x - part of abx - syst j = 9.2, 3.7 hz, 1h), 3.71 (s, 2h), 2.78 (a - part of abx - syst, jab = 14.0 hz, j = 3.7 hz, 1h), 2.65 (b - part of abx - syst, jab = 14.0 hz, j = 9.2 hz, 1h), 2.47 (br s, 1h, oh). Similarly, stannane (r)-[d1]12b (233 mg, 0.54 mmol) was transformed into alcohol ()-[2-d1]19 (95 mg, 72%) by the procedure used for experiment 1 except that benzaldehyde (0.54 ml, 1.08 mmol, 2 m in dry thf), was added 1 min after the addition of meli (0.27 ml, 0.82 mmol, 3 m in diethoxymethane). The spectroscopic data of (2s)-[2-d1]19 were identical to that of 19 except for the following . H nmr (400.13 mhz, cdcl3): 4.59 (2 overlapping d, j = 9.2, 3.7 hz, 1h), 2.82 (br s, 1h, oh), 2.68 (br s, 1h, schd), 2.58 (br d, j = 9.2 hz, 1h, schd). C nmr (100.61 mhz, cdcl3): 40.4 (t, j = 21.5 hz, 1c, schd). (r)-benzylthio[d1]methylstannane [(r)-[d1]12b] (213 mg, 0.50 mmol) was transformed into alcohol (s)-[d1]19 (30 mg, 24%, ee 16%) by the procedure used for the preparation of the racemic alcohols except that the benzaldehyde (0.50 ml, 1.0 mmol, 2 m in dry thf) was present in the reaction mixture when meli was added dropwise every 3 s. similarly, (r)-[d1]12b (228 mg, 0.53 mmol) was transformed into (2s)-[2-d1]19 (11 mg, 9%, ee 26%) by the procedure used for experiment 3 except that it was performed at 95 c and that 2 equiv of 12-crown-4 were present in the reaction mixture . Similarly, (r)-[d1]12b (237 mg, 0.55 mmol) was transformed into (s)-[d1]19 (34 mg, 25%, ee 8%) by the procedure used for experiment 3, except that the experiment was performed at 50 c . Similarly, (r)-[d1]12b (228 mg, 0.53 mmol) was transformed into ()-[d1]19 (26 mg, 21%) by the procedure used for experiment 3, except that the experiment was performed at 0 c . Racemic alcohol 19 (mg, mmol) was converted to a 1:1 mixture of diastereomeric (r)-mosher esters according to general procedure a. the crude product was purified by flash chromatography (hexane / etoac, 10:1, rf 0.51) to yield esters 19(r)-mtpa (28 mg, 94%) as a colorless oil . H nmr (600.13 mhz, cdcl3): 7.487.07 (m, 30h), 5.97 (x - part of abx - syst, j = 8.2, 5.7 hz, 1h, diastereomer a), 5.86 (x - part of abx - syst, j = 9.0, 4.8 hz, 1h, diastereomer b), 3.66 (ab - syst, jab = 13.5 hz, 2h), 3.60 (t, j = 1.2 hz, 3h, 3.52 (s, 2h), 3.45 (t, j = 1.2 hz, 3h), 2.87 (a - part of abx - syst, jab = 14.4 hz, j = 9.0 hz, 1h, b), 2.83 (a - part of abx - syst, jab = 14.4 hz, j = 8.2 hz, 1h, a), 2.70 (b - part of abx - syst, jab = 14.4 hz, j = 4.8 hz, 1h, b), 2.69 (b - part of abx - syst, jab = 14.4 hz, j = 5.7 hz, 1h, a). Similarly, alcohol (2s)-[2-d1]19 (11 mg, 0.045 mmol), obtained by determination of microscopic configurational stability of chiral benzylthiomethyllithium by experiment 5 was converted to (r)-mosher esters (s)-[d1]19(r)-mtpa (18 mg, 87%). The h nmr spectrum (600.13 mhz, cdcl3) was identical to that of 19(r)-mtpa except for 2.85 (d, j = 9.0 hz, 0.42h, chd), 2.81 (d, j = 8.2 hz, 0.67h, chd), 2.68 (2 overlapping d, 1h, chd); ee of underlying alcohol 26% . N - buli (0.50 ml, 0.79 mmol) was added to the solution of (1-naphthylmethylthiomethyl)tributylstannane (12c) (314 mg, 0.66 mmol) in dry thf (4.6 ml) at 78 c under argon . After 10 min, cf3co2h (0.43 ml, 0.87 mmol, 2 m in ch2cl2) was added, and the mixture was extracted with etoac (3 10 ml). The combined organic phases were dried (mgso4) and concentrated under reduced pressure . The residue was purified by flash chromatography (hexane / ch2cl2, 10:1, rf 0.32) to yield thiol 20 (57 mg, 45%) as a colorless oil . Ir (si): 3050, 2924, 1597, 1510, 1382, 1264, 1248, 1210, 1060 cm . H nmr (400.27 mhz, cdcl3): 8.04 (d, j = 8.5 hz, 1h), 7.79 (d, j = 8.1 hz, 1h), 7.67 (d, j = 8.4 hz, 1h), 7.557.40 (m, 2h), 7.387.33 (m, 1h), 3.94 (d, j = 7.0 hz, 2h), 2.69 (s, 3h), 1.71 (t, j = 7.0 hz, 1h). C nmr (100.61 mhz, cdcl3): 136.1, 133.1, 132.8, 131.1, 128.5, 127.3, 126.7, 126.1, 125.4, 124.1, 27.7, 14.2 . Calcd for c12h12s: c, 76.55; h, 6.42; s, 17.03 . Similarly, (r)-[d1]12c (206 mg, 0.43 mmol) was rearranged to (r)-[d1]20 (22 mg, 28%, ee 60%) except that transmetalation was performed with meli (3 m, in diethoxymethane) at 50 c . The h nmr spectrum (400.27 mhz, cdcl3) was identical to that of 20 except for 3.92 (dt, j = 7.0, 1.8 hz, 1h, chdsh), 1.69 (d, j = 7.0 hz, 1h, chdsh). Similarly, stannane (r)-[d1]12c (208 mg, 0.43 mmol) was rearranged to thiol (r)-[d1]20 (49 mg, 60%, ee 72%) except that transmetalation was performed with n - buli at 95 c . A solution of (r)-(+)-1-phenylethyl isocyanate (67 l, 0.48 mmol) and thiol 20 (46 mg, 0.24 mmol) in dry thf (1.2 ml) was left for 1 h under argon at rt . Water (1 ml) was added, and the mixture was extracted with etoac (3 15 ml). The combined organic phases were dried (mgso4) and concentrated under reduced pressure . The residue was purified by flash chromatography (hexane / ch2cl2, 1:1, rf 0.37) to yield thiocarbamate 21 (39 mg, 50%) as colorless crystals . Ir (atr): 3284, 2975, 2925, 1640, 1509, 1446, 1204, 1179, 1101 cm . H nmr (400.27 mhz, cdcl3): 8.03 (d, j = 8.5 hz, 1h), 7.78 (d, j = 8.3 hz, 1h), 7.63 (d, j = 8.4 hz, 1h), 7.537.39 (m, 3h), 7.387.19 (m, 5h), 5.46 (br s, 1h), 5.10 (br s, 1h), 4.38 (ab - sys, jab = 13.2 hz, 2h), 2.65 (s, 3h), 1.50 (d, j = 6.9 hz, 3h). C nmr (100.61 mhz, cdcl3): c = o (n. d.), 142.7, 133.0, 132.9, 132.4, 128.8, 128.5, 128.2, 127.6, 126.4, 126.0, 125.4, 124.1, 51.3, 33.3, 22.1 (br s), 14.5 . Calcd for c21h21nos: c, 75.19; h, 6.31; n, 4.18; s, 9.56: found: c, 74.89; h, 6.22; n, 4.05; s, 9.56 . Similarly, thiol (r)-[d1]20 (49 mg, 0.26 mmol, obtained by rearrangement at 95 c with n - buli) was converted to thiocarbamate (r)-[d1]21 (36 mg, 42%). The spectroscopic data were identical to that of 21 except for the following . H nmr (400.27 mhz, cdcl3): 4.38 (br s, 0.86h, chd), 4.35 (br s, 0.14h, chd). C nmr (100.61 mhz, cdcl3): 33.1 (t, j = 21.9 hz, 1c, chds). ()-23: a solution of dibalh (3.98 ml, 5.97 mmol, 1.5 m in toluene) was added dropwise to -bromoacetophenone (995 mg, 5.0 mmol) dissolved in dry et2o (25 ml) at 78 c under argon . The reaction mixture was stirred for 2 h at 78 c and then 1 h at 50 c . The reaction was quenched with meoh (0.5 ml) and water (2.5 ml) and stirred for another 30 min at rt . A solution of hcl (12 ml, 2 m) was added at 0 c . The organic phase was separated, and the aqueous phase was extracted with et2o (3 10 ml). The combined organic layers were washed with water (15 ml) and satd aq nahco3 (15 ml), dried (mgso4), and concentrated under reduced pressure . The crude product was purified by flash chromatography (hexane / etoac, 7:1, rf 0.43) to yield ()-2-bromo-1-phenylethanol [()-23] (591 mg, 59%) as a colorless oil . H nmr (400.13 mhz, cdcl3): 7.397.29 (m, 5h), 4.92 (dd, x - part of an abx - syst, j = 9.1, 3.5 hz, 1h), 3.58 (ab - part of an abx - syst, jab = 10.4 hz, j = 9.1, 3.5 hz, 2h), 2.59 (br s, 1h). (s)-(+)-23: (+) -dip - chloride (1.8 g, 5.61 mmol, dissolved in 5 ml of dry thf) was added to -bromoacetophenone (744 mg, 3.74 mmol, dissolved in 5 ml of dry thf) under argon at 0 c . At 0 c water (1 ml), pentaerythritol (916 mg, 6.73 mmol) and again water (15 ml) were added and the mixture was stirred for another 30 min at rt . The organic phase was separated and the aqueous one was extracted with et2o (3 15 ml). The combined organic layers were washed with water (2 10 ml), dried (mgso4), concentrated under reduced pressure, and purified by flash chromatography (hexane / etoac, 10:1). The still impure product was again dissolved in dry thf (10 ml) and pentaerythritol (680 mg, 5 mmol, dissolved in 5 ml of water) was added . The organic phase was separated and the aqueous one was extracted with et2o (3 15 ml). The combined organic layers were washed with water (2 10 ml), dried (mgso4) and concentrated under reduced pressure . Ten drops of dry pyridine were added and the crude product was purified by flash chromatography (hexane / etoac, 7:1) to yield (s)-(+)-2-bromo-1-phenylethanol [(s)-(+)-23] as a colorless oil (193 mg, 26%), ee 94% (by h nmr of (r)-mosher ester), []d + 39.24 (c 1.975, ch2cl2) [lit . []d + 40.10, (c 1.81, chcl3), ee 92%]. ()-2-bromo-1-phenylethanol [()-23] (20 mg, 0.10 mmol) was converted to (r)-mosher esters using general procedure a. the crude product was purified by flash chromatography (hexane / etoac,10:1, rf 0.66) to yield a mixture of diastereomeric mosher esters ()-23(r)-mtpa (39 mg, 94%). H nmr (400.13 mhz, toluene - d8): 7.597.55 [m, 2h, (r, s)-diastereomer], 7.517.47 [m, 2h, (r, r)], 7.086.91 [m, 14h, 8h of (r, s), 6h of (r, r)], 6.786.75 [m, 2h, (r, r)], 6.04 [x - part of an abx - syst, j = 8.8, 4.2 hz, 1h, (r, s)], 5.90 [x - part of an abx - syst, j = 9.6, 3.3 hz, 1h, (r, r)], 3.58 [q, j = 1.0 hz, 3h, (r, r)], 3.32 [q, j = 1.0 hz, 3h, (r, s)], 3.07 [ab - part of an abx - syst, jab = 11.1 hz, j = 8.8, 4.2 hz, 2h, (r, s)], 3.01 [ab - part of an abx - syst, jab = 11.4 hz, j = 9.6, 3.3 hz, 2h, (r, r)]. Similarly, (s)-(+)-23 [20 mg, 0.10 mmol, []d + 39.24, (c 1.98, ch2cl2)] was converted to (r)-mosher esters (40 mg, 96%); ee of alcohol: 94% . The h nmr spectrum was identical to that of the racemic alcohol, except that the signals of the diastereomers differed in intensity . A solution of meli (0.60 ml, 0.60 mmol, 1 m in cumene / thf) was added quickly at 78 c to the ()-bromohydrin ()-23 (100 mg, 0.5 mmol) dissolved in dry thf (2.5 ml) under argon atmosphere . After the mxiture was stirred for 5 min, cf3co2h (75 mg, 0.65 ml, 0.65 mmol, 1.3 equiv, 1 m in dry ch2cl2) and water (5 ml) 3 min later were added . The organic phase was separated and the aqueous one was extracted with et2o (3 15 ml). The crude product did not contain phenyloxirane as determined by h nmr spectroscopy after spiking with an authentic sample . The crude product was purified by flash chromatography (hexane / etoac, 7:1, rf 0.42) to recover bromohydrin ()-23 (95 mg, 95%). Its rf value and h nmr spectrum were identical to that of the starting material . Using nbs / ph3p: a solution of ph3p (772 mg, 2.94 mmol) in dry ch2cl2 (3 ml) was added dropwise to nbs (523 mg, 2.94 mmol) dissolved in dry ch2cl2 (5 ml) under argon at 78 c . After 10 min tributylstannylmethanol (2) (787 mg, 2.45 mmol) dissolved in dry ch2cl2 (4 ml) was added, and the reaction mixture was stirred rt for 30 min . A few drops of meoh were added, and the mixture was concentrated under reduced pressure . The residue was flash chromatographed (hexane / ch2cl2, 1:1, rf 0.98) to yield bromomethylstannane25 (859 mg, 91%) as a colorless oil . H nmr (400.13 mhz, cdcl3): 2.63 (s, j(sn) = 14.6 hz, 2h), 1.551.46 (m, 6h), 1.30 (sext, j = 7.3 hz, 6h), 1.000.95 (m, j(sn) = 51.4 hz, 6h), 0.88 (t, j = 7.3 hz, 9h). Similarly, (s)-[d1]2 (197 mg, 0.61 mmol, prepared by an improved procedure, see below) was converted to (r)-[d1]25 (205 mg, 92%, ee 60%). The h nmr spectrum (400.13 mhz, cdcl3) was identical to that of 25 except for 2.61 (t, j = 1.5 hz, 1h, chd). When the reaction was performed in the same way as before, except that two instead of 1.2 equiv of ph3p / nbs were used, (r)-[d1]2 (251 mg, 0.78 mmol) were converted to (s)-[d1]25 (230 mg, 77%, ee 18%). Using mitsunobu reaction with ph3phbr: ph3p (109 mg, 0.62 mmol), ph3phbr (845 mg, 2.46 mmol), and stannylmethanol 2 (657 mg, 2.05 mmol) were dissolved in dry toluene (8.2 ml) under argon . After stirring for 5 min at 0 c diad (0.71 ml, 4.47 mmol) was added and the reaction mixture was stirred for another 90 min . The mixture was purified by flash chromatography (hexane / ch2cl2, 1:1, rf 0.98) to give bromomethylstannane 25 (383 mg, 49%) as a colorless oil . Similarly, (r)-[d1]2 (852 mg, 2.65 mmol, prepared by an improved procedure, see below) was converted to (s)-d1]25 (491 mg, 48%, ee 74%) except that the reaction time was 10 min . After the addition of methanol, the reaction mixture was immediately applied to the silica column for flash chromatography . The entire procedure (also flash chromatography) was performed in the cold room (3 c). No bath was used during concentration of bromide - containing solutions under reduced pressure . Similarly, (r)-[d1]2 (958 mg, 2.98 mmol) was converted to (s)-[d1]25 (521 mg, 45%, ee 94%) as before except that the reaction was performed at 10 c for 10 min . Similarly, (r)-[d1]2 (417 mg, 1.30 mmol) was converted to (s)-[d1]25 (179 mg, 35%, ee 99%) as before except that the reaction was performed at 25 c for 10 min . A solution of meli (0.51 ml, 0.51 mmol, 1 m in cumene / thf) was added quickly at 78 c to the (bromomethyl)tributylstannane 25 (163 mg, 0.42 mmol) dissolved in dry thf (2 ml) under argon atmosphere . After 30 s, cf3co2h (64 mg, 0.55 ml, 0.55 mmol, 1.3 equiv, 1 m in dry ch2cl2) was added, followed by water (5 ml) 1 min later . The organic phase was separated, and the aqueous phase was extracted with ch2cl2 (3 10 ml). The combined organic layers were washed with water (15 ml), dried (na2so4), and concentrated under reduced pressure . The crude product was virtually homogeneous tributylmethylstannane as determined by h nmr spectroscopy, which did not contain starting material . A solution of meli (1.85 ml, 1.85 mmol, 1 m in cumene / thf) was quickly added to a solution of bromomethylstannane 25 (178 mg, 0.46 mmol) and benzaldehyde (194 mg, 0.93 ml, 1.85 mmol, freshly distilled, 2 m in thf) in dry thf (2 ml) under argon atmosphere at 78 c . After 5 min, cf3co2h (232 mg, 2 ml, 2 mmol, 1 m in ch2cl2) and water (5 ml) were added . The organic phase was separated, and the aqeous phase was extracted with ch2cl2 (3 10 ml), dried (na2so4), and concentrated under reduced pressure . The residue was purified by flash chromatography (ch2cl2, rf 0.46) to give bromohydrin ()-23 as a colorless oil (17 mg, 19%). The spectroscopic data were identical to those of the authentic sample . A solution of benzaldehyde (198 mg, 0.94 ml, 1.87 mmol, freshly distilled, 2 m in dry thf) was added at 78 c to the (r)-bromo[1-d1]methylstannane (r)-[d1]2 (180 mg, 0.47 mmol, ee 50%) in dry thf (2 ml) under argon atmosphere, followed dropwise by meli (1.87 ml, 1.87 mmol, 1 m in cumene / thf). After 10 min, the reaction was quenched with cf3co2h (240 mg, 2.07 ml, 2.07 mmol, 1 m in thf). Water (5 ml) was added, and the organic phase was separated and the aqueous one was extracted with ch2cl2 (3 5 ml). The combined organic layers were dried (na2so4) and concentrated under reduced pressure . The residue was purified by flash chromatography (ch2cl2, rf 0.43) to give bromohydrin (2s)-[2-d1]23 (7 mg, 11%, ee 57%) as a colorless oil; impure fractions (estimated product about 10 mg) were discarded . (r)-[d1]2 (157 mg, 0.41 mmol, ee 77%) was converted to (2s)-[2-d1]23 (15 mg, 18%, ee 76%) by procedure used for experiment 1 except using 2 equiv of benzaldehyde and meli (1 m solution obtained by dilution of 3 m meli in diethoxymethane with dry thf) which was added dropwise every 5 s. if 3 m meli in diethoxymethane was used or it was added more rapidly, no product or only product traces were obtained . (r)-[d1]2 (161 mg, 0.42 mmol, ee 77%) was converted to (1s)-[1-d1]27 (11 mg, 12%, ee 75%) by the procedure used for experiment 2 except that acetophenone was used as electrophile . The bromhydrine was isolated by flash chromatography (hexane / etoac, 15:1; tlc: hexane / etoac, 10:1, rf 0.34). (s)-[d1]2 (218 mg, 0.57 mmol, ee 94%) was converted to (2r)-[2-d1]23 (10 mg, 9%, ee 94%) by the procedure used for experiment 2 . (s)-[d1]2 (259 mg, 0.67 mmol, ee 94%) was converted to (1r)-[1-d1]27 (45 mg, 31%, ee 93%) by the procedure used for experiment 3 except that the reaction was performed at 95 c . (s)-[d1]2 (179 mg, 0.46 mmol, ee 99%) was converted to (2r)-[2-d1]23 (13 mg, 14%, ee 99%) by procedure used for experiment 2 . They were prepared in quantitative yield from alcohols [1-d1]23 according to general procedure a and they were purified by flash chromatography (hexane / etoac, 10:1, rf 0.78). The two diastereomeric (r)-mosher esters obtained in quantitative yield were separated by preparative tlc chromatography (hexane / ch2cl2, 3:1) in one case . The less polar one (rf 0.56) was derived from (1r,2s)-[2-d1]23 (56% ee) and the more polar one (rf 0.35) from (1s,2s)-[2-d1]23 (57% ee) in quantitative yield . H nmr (400.13 mhz, toluene - d8): 5.89 (br d, j = 2.5 hz, 1h), 2.89 (d, j = 2.5 hz, 1h, chd). H nmr (400.13 mhz, toluene - d8): 5.94 (d, j = 8.8 hz, 1h), 3.05 (d, j = 8.8 hz, 1h, chd). ()-27: h nmr (400.27 mhz, cdcl3): 7.477.42 (m, 2h), 7.397.33 (m, 2h), 7.317.25 (m, 1h), 3.71 (ab - sys, jab = 10.4 hz, 2h), 2.52 (br s, 1h), 1.67 (s, 3h). [1-d1]27: the h nmr spectra (400.27 mhz, cdcl3) were identical to that of ()-27 except for 3.73 (br s, 0.5h, chd), 3.68 (t, j = 1.4 hz, 0.5h, chd). The (r)-mosher esters of ()-27 and [1-d1]27 were prepared by a modified general procedure as exemplified for (1r)-[1-d1]27mtpa-(r): a solution of bromohydrin (1r)-[1-d1]27 (16 mg, 0.074 mmol), (s)-mosher chloride (0.15 mmol, 2 equiv, 0.29 ml of a 0.53 m solution in dry 1,4-dioxane), and dmap (37 mg, 0.30 mmol, 4 equiv) in dry dioxane (1 ml) was heated at 50 c for 8 h (no starting material present). After the solution was cooled to rt, a few drops of water were added and stirring was continued for 5 min . Ch2cl2 (3 ml) and hcl (3 ml, 1 m) were added . The combined organic layers were washed with saturated aq nahco3, dried (na2so4), and concentrated under reduced pressure . The residue was flash chromatographed (hexane / etoac, 15:1, rf 0.56) to yield (1r)-[1-d1]27mtpa-(r) (20 mg, 62%). ()-27mtpa-(r): h nmr (400.27 mhz, cdcl3): 7.597.51 (m, 4h), 7.457.35 (m, 6h), 7.357.26 (m, 8h), 7.237.18 (m, 2h), 3.97 (ab - sys, jab = 11.0 hz, 2h), 3.77 (ab - sys, jab = 11.0 hz, 2h), 3.62 (q, j = 1.3 hz, 3h), 3.57 (q, j = 1.2 hz, 3h), 2.10 (s, 3h), 2.02 (s, 3h). (1r)-[1-d1]27mtpa-(r): the h nmr spectrum (400.27 mhz, cdcl3) was identical to that of ()-27mtpa-(r) except for 3.91 (br s, 0.85h, chd), 3.81 (br s, 0.85h, chd) and 4.00 (br s, 0.02h), 3.71 (br s, 0.03h); 93% ee for bromohydrin at c-1.
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The framingham study s original cohort has been evaluated biennially since 1948 and screened prospectively for incident dementia since 1978 . A sample drawn from the offspring (and spouses of the offspring) of the original participants (framingham offspring cohort) was enrolled in 19701972 and has been examined eight times during the past 37 years, approximately once every 4 years . Participants who attended offspring examination 7 (between 1998 and 2001) were invited to participate in brain mri studies and a neuropsychologic assessment . The study population comprised 2,439 individuals (1,311 women), with a mean age of 62 9 years who agreed to participate and were also free of clinical stroke, dementia, or other neurologic conditions such as a brain tumor that could alter mri and cognitive performance . The methods followed for brain mri have been described previously (4). In brief, subjects underwent neuroimaging on a siemens magnetom (munich, germany) 1-t or 1.5-t field strength machine using a double spin - echo coronal imaging sequence of 4-mm contiguous slices from nasion to occiput . Imaging data were transferred to a central location for processing and analyzed by operators who were blinded to the subject s identity, age, sex, exposure to stroke risk factors, and cognitive performance on neuropsychologic testing . Previously reported semiautomated analyses of pixel distributions based on mathematic modeling of mri pixel intensity histograms for cerebrospinal fluid and white and gray brain matter were used to determine the optimal threshold of pixel intensity to best distinguish cerebrospinal fluid from brain matter (5). All analyses were performed using quanta 6.2, a custom - designed image analysis package, operating on an ultra 5 workstation (sun microsystems, santa clara, ca). Mri measures assessed consisted of total cerebral brain volume (tcbv), hippocampal volume (hv), and white matter hyperintensity volume . Brain volume was determined by manual outlining in coronal images of the intracranial vault above the tentorium to determine the total cranial volume as a measure of head size . Once the skull and other nonbrain tissues were removed from the image, mathematic modeling was performed to determine total parenchymal brain volume above the tentorium (cerebral). Because the medial wall of the temporal horn consists of the hippocampus, atrophy of the hippocampus will result in enlargement of the temporal horn volume . White matter hyperintensity volume was measured according to previously published methods (6), expressed as a proportion of the total intracranial volume, and log - transformed to normalize its distribution . We estimated the equivalency between the mean change in tcbv associated with chronologic aging and that associated with the presence of diabetes by dividing the regression coefficient for diabetes by the regression coefficient for age (7). A comprehensive, well standardized, neuropsychologic test battery was administered to all participants and has been described previously (8). The neuropsychologic evaluation consisted of a 40-min test battery that included tests of verbal memory and abstract reasoning that were previously associated with an increased risk of developing ad in the original cohort (9). Also included were tests of frontal executive function that are sensitive in detecting cognitive impairment in persons with diabetes (10). The cognitive domains that we studied were verbal memory (wechsler memory scale logical memory), visual memory (wechsler memory scale visual reproduction), and executive function (trail making tests a & b). The mri and neuropsychologic testing were performed on the same day for most participants and, in all cases, by evaluators who were blind to the diabetes, hba1c, fasting insulin, and ir data . Fasting glucose, hba1c, and fasting insulin levels were estimated from blood samples drawn at the offspring examination 7 after participants had fasted overnight for at least 8 h. diabetes was determined by use of insulin or other diabetes medication or a fasting glucose concentration 126 mg / dl . Fasting glucose concentrations were measured in fresh specimens with a hexokinase reagent kit (a - gent glucose test; abbott laboratories, inc ., south pasadena, ca); the intra - assay coefficient of variation (cv) was <3% . Hba1c was measured using high - performance liquid chromatography (hplc) assays standardized to diabetes control and complications trial values by the national glycohemoglobin standardization program (11). Assay drift in the hplc method used in framingham offspring study is prevented by the use of long - term stored reference samples . Plasma insulin concentrations were measured using the coat - a - count i labeled radioimmunoassay (diagnostic products, los angeles, ca); this assay has a cross - reactivity with proinsulin at the midcurve of 40%; the intra- and interassay cvs were 5 to 10% . Ir was estimated using a standardized measure of insulin sensitivity: the homeostasis model assessment of ir (homa - ir). The homa - ir (mmol / l u / ml) formula is defined as fasting insulin (u / ml) times fasting glucose (mmol / l)/22.5 . We related presence or absence of diabetes and levels of hba1c, fasting insulin, and homa - ir to the mri and cognitive outcomes listed in research design and methods . We constructed linear regression models adjusted for covariates previously shown to affect brain volume and cognitive function in the framingham cohort including: model a: age, sex, education (for cognitive measures) only;model b: the above with inclusion of nondiabetes cardiovascular risk factors at examination 7: systolic blood pressure, smoking, prevalent cardiovascular disease;model c: additional adjustment for factors previously associated with risk of dementia in the framingham original cohort, namely, apolipoprotein e (apoe)-4 genotype, and serum homocysteine levels, c - reactive protein (crp), and interleukin-6 (il-6) at examination 7.we also adjusted for the time interval between examination 7 and mri or cognitive testing in all analyses . Model a: age, sex, education (for cognitive measures) only; model b: the above with inclusion of nondiabetes cardiovascular risk factors at examination 7: systolic blood pressure, smoking, prevalent cardiovascular disease; model c: additional adjustment for factors previously associated with risk of dementia in the framingham original cohort, namely, apolipoprotein e (apoe)-4 genotype, and serum homocysteine levels, c - reactive protein (crp), and interleukin-6 (il-6) at examination 7 . In secondary analyses, we excluded subjects with clinical diabetes and looked at the effect of the duration of diabetes on the relationship between diabetes and mri measures by stratifying the subjects into three categories: 1) nondiabetic as determined at offspring examination 7 and at the earlier examination 5 (19911995) using the same criteria described earlier (referent); 2) nondiabetic at examination 5 and diabetic at examination 7; 3) diabetic at examinations 5 and 7 . The framingham study s original cohort has been evaluated biennially since 1948 and screened prospectively for incident dementia since 1978 . A sample drawn from the offspring (and spouses of the offspring) of the original participants (framingham offspring cohort) was enrolled in 19701972 and has been examined eight times during the past 37 years, approximately once every 4 years . Participants who attended offspring examination 7 (between 1998 and 2001) were invited to participate in brain mri studies and a neuropsychologic assessment . The study population comprised 2,439 individuals (1,311 women), with a mean age of 62 9 years who agreed to participate and were also free of clinical stroke, dementia, or other neurologic conditions such as a brain tumor that could alter mri and cognitive performance . The methods followed for brain mri have been described previously (4). In brief, subjects underwent neuroimaging on a siemens magnetom (munich, germany) 1-t or 1.5-t field strength machine using a double spin - echo coronal imaging sequence of 4-mm contiguous slices from nasion to occiput . Imaging data were transferred to a central location for processing and analyzed by operators who were blinded to the subject s identity, age, sex, exposure to stroke risk factors, and cognitive performance on neuropsychologic testing . Previously reported semiautomated analyses of pixel distributions based on mathematic modeling of mri pixel intensity histograms for cerebrospinal fluid and white and gray brain matter were used to determine the optimal threshold of pixel intensity to best distinguish cerebrospinal fluid from brain matter (5). All analyses were performed using quanta 6.2, a custom - designed image analysis package, operating on an ultra 5 workstation (sun microsystems, santa clara, ca). Mri measures assessed consisted of total cerebral brain volume (tcbv), hippocampal volume (hv), and white matter hyperintensity volume . Brain volume was determined by manual outlining in coronal images of the intracranial vault above the tentorium to determine the total cranial volume as a measure of head size . Once the skull and other nonbrain tissues were removed from the image, mathematic modeling was performed to determine total parenchymal brain volume above the tentorium (cerebral). Because the medial wall of the temporal horn consists of the hippocampus, atrophy of the hippocampus will result in enlargement of the temporal horn volume . White matter hyperintensity volume was measured according to previously published methods (6), expressed as a proportion of the total intracranial volume, and log - transformed to normalize its distribution . We estimated the equivalency between the mean change in tcbv associated with chronologic aging and that associated with the presence of diabetes by dividing the regression coefficient for diabetes by the regression coefficient for age (7). The methods followed for brain mri have been described previously (4). In brief, subjects underwent neuroimaging on a siemens magnetom (munich, germany) 1-t or 1.5-t field strength machine using a double spin - echo coronal imaging sequence of 4-mm contiguous slices from nasion to occiput . Imaging data were transferred to a central location for processing and analyzed by operators who were blinded to the subject s identity, age, sex, exposure to stroke risk factors, and cognitive performance on neuropsychologic testing . Previously reported semiautomated analyses of pixel distributions based on mathematic modeling of mri pixel intensity histograms for cerebrospinal fluid and white and gray brain matter were used to determine the optimal threshold of pixel intensity to best distinguish cerebrospinal fluid from brain matter (5). All analyses were performed using quanta 6.2, a custom - designed image analysis package, operating on an ultra 5 workstation (sun microsystems, santa clara, ca). Mri measures assessed consisted of total cerebral brain volume (tcbv), hippocampal volume (hv), and white matter hyperintensity volume . Brain volume was determined by manual outlining in coronal images of the intracranial vault above the tentorium to determine the total cranial volume as a measure of head size . Once the skull and other nonbrain tissues were removed from the image, mathematic modeling was performed to determine total parenchymal brain volume above the tentorium (cerebral). Because the medial wall of the temporal horn consists of the hippocampus, atrophy of the hippocampus will result in enlargement of the temporal horn volume . White matter hyperintensity volume was measured according to previously published methods (6), expressed as a proportion of the total intracranial volume, and log - transformed to normalize its distribution . We estimated the equivalency between the mean change in tcbv associated with chronologic aging and that associated with the presence of diabetes by dividing the regression coefficient for diabetes by the regression coefficient for age (7). A comprehensive, well standardized, neuropsychologic test battery was administered to all participants and has been described previously (8). The neuropsychologic evaluation consisted of a 40-min test battery that included tests of verbal memory and abstract reasoning that were previously associated with an increased risk of developing ad in the original cohort (9). Also included were tests of frontal executive function that are sensitive in detecting cognitive impairment in persons with diabetes (10). The cognitive domains that we studied were verbal memory (wechsler memory scale logical memory), visual memory (wechsler memory scale visual reproduction), and executive function (trail making tests a & b). The mri and neuropsychologic testing were performed on the same day for most participants and, in all cases, by evaluators who were blind to the diabetes, hba1c, fasting insulin, and ir data . Fasting glucose, hba1c, and fasting insulin levels were estimated from blood samples drawn at the offspring examination 7 after participants had fasted overnight for at least 8 h. diabetes was determined by use of insulin or other diabetes medication or a fasting glucose concentration 126 mg / dl . Fasting glucose concentrations were measured in fresh specimens with a hexokinase reagent kit (a - gent glucose test; abbott laboratories, inc ., south pasadena, ca); the intra - assay coefficient of variation (cv) was <3% . Hba1c was measured using high - performance liquid chromatography (hplc) assays standardized to diabetes control and complications trial values by the national glycohemoglobin standardization program (11). The hba1c assays have intra- and interassay cvs <3% . Assay drift in the hplc method used in framingham offspring study plasma insulin concentrations were measured using the coat - a - count i labeled radioimmunoassay (diagnostic products, los angeles, ca); this assay has a cross - reactivity with proinsulin at the midcurve of 40%; the intra- and interassay cvs were 5 to 10% . Ir was estimated using a standardized measure of insulin sensitivity: the homeostasis model assessment of ir (homa - ir). The homa - ir (mmol / l u / ml) formula is defined as fasting insulin (u / ml) times fasting glucose (mmol / l)/22.5 . We related presence or absence of diabetes and levels of hba1c, fasting insulin, and homa - ir to the mri and cognitive outcomes listed in research design and methods . We constructed linear regression models adjusted for covariates previously shown to affect brain volume and cognitive function in the framingham cohort including: model a: age, sex, education (for cognitive measures) only;model b: the above with inclusion of nondiabetes cardiovascular risk factors at examination 7: systolic blood pressure, smoking, prevalent cardiovascular disease;model c: additional adjustment for factors previously associated with risk of dementia in the framingham original cohort, namely, apolipoprotein e (apoe)-4 genotype, and serum homocysteine levels, c - reactive protein (crp), and interleukin-6 (il-6) at examination 7.we also adjusted for the time interval between examination 7 and mri or cognitive testing in all analyses . Model a: age, sex, education (for cognitive measures) only; model b: the above with inclusion of nondiabetes cardiovascular risk factors at examination 7: systolic blood pressure, smoking, prevalent cardiovascular disease; model c: additional adjustment for factors previously associated with risk of dementia in the framingham original cohort, namely, apolipoprotein e (apoe)-4 genotype, and serum homocysteine levels, c - reactive protein (crp), and interleukin-6 (il-6) at examination 7 . In secondary analyses, we excluded subjects with clinical diabetes and looked at the effect of the duration of diabetes on the relationship between diabetes and mri measures by stratifying the subjects into three categories: 1) nondiabetic as determined at offspring examination 7 and at the earlier examination 5 (19911995) using the same criteria described earlier (referent); 2) nondiabetic at examination 5 and diabetic at examination 7; 3) diabetic at examinations 5 and 7 . The characteristics of the study population at examination 7 (19982001) are presented in table 1 . We observed an inverse association between all indices (diabetes, hba1c, homa - ir, fasting insulin level) of metabolic dysfunction evaluated at examination 7 and tcbv in all models (table 2). Adjustment for covariates (models b and c; table 2) did not appreciably alter these associations . The observed difference in tcbv of 1.24% between participants with and without diabetes was equivalent to the effect of approximately 6 years of chronologic aging . Of note, the association of ir and hyperinsulinemia with tcbv remained significant when the analysis was restricted to subjects who had not yet developed clinical diabetes (table 3). Characteristics of subjects at examination cycle 7 (19982001) continuous data are presented as mean sd; categoric data as percentages . Association between metabolic dysregulation measures and markers of brain aging model a: adjusted for age, sex, education . Model b: adjusted for age, sex, education, systolic blood pressure, smoking, history of cardiovascular disease . Model c: adjusted for age, sex, education, systolic blood pressure, smoking, history of cardiovascular disease, apoe-4 genotype, serum homocysteine levels, crp, il-6 . Association between metabolic dysregulation measures and markers of brain aging after exclusion of participants with clinical diabetes * values in boldface type are significant . * considering the cognitive measures, we found that examination 7 diabetes and hba1c, homa - ir and fasting insulin were each related to poorer executive function . This inverse relationship between executive function and hba1c, homa - ir, and fasting insulin persisted even after adjustment for covariates (table 2) and the exclusion of participants with clinical diabetes from the analysis (table 3). Homa - ir and fasting insulin, but not clinical diabetes or hba1c, were inversely related to visuospatial memory (table 2). These associations remained significant after adjustment for covariates (models b and c; table 2). A surprising finding was an apparent association between diabetes and better performance in verbal memory tasks, although this effect was attenuated after adjustment for all covariates . There was an interaction with insulin levels, so that this apparent beneficial effect was only seen in subjects who also had elevated plasma insulin levels and was not seen in the subsample of participants who were not receiving insulin . Stratifying participants according to their diabetes status at examination cycles 5 and 7 revealed that the cross - sectional association between diabetes and tcbv was present only in those participants who had diabetes at both examination cycles (diabetes duration 4 years; p <0.001) and not in those who developed diabetes only at examination 7 (p = 0.113). No such differential patterns according to length of time with clinical diabetes were observed for the other indices of metabolic dysfunction and measures of brain aging (supplementary table 1a). In the middle - aged community sample of stroke- and dementia - free framingham offspring participants, we observed that clinical diabetes and various metabolic indices associated with diabetic and prediabetic states were associated with the subclinical changes of smaller brain volumes and poorer cognitive performance, especially on tests of executive function and visual memory . These findings were observed even among participants who did not have clinical diabetes, and the most consistent association was with ir, which is important in the evolution of clinical type 2 diabetes . The relationship between diabetes and cognition was reported> 85 years ago, when miles and root (12) observed that patients with diabetes did not perform as well as control subjects in tests of memory, mental arithmetic, and psychomotor efficiency . Multiple studies since then have shown an adverse effect of baseline diabetes status on concomitant cognitive function and cognitive decline on follow - up . Diabetes has been associated with cognitive changes that affect learning and memory, mental speed, and mental flexibility (13), and population - based studies have also related diabetes to the risk of incident dementia and ad (3). The memory in diabetes (mind) substudy of the action to control cardiovascular risk in diabetes (accord) trial showed an inverse relationship between glycohemoglobin levels and performance on cognitive tests; whereas, fasting plasma glucose, a measure with greater day - to - day variability, was not associated with test performance (14). The cognitive impairment associated with diabetes may begin even before the appearance of clinical disease; compared with women with normal glucose, women with impaired fasting glucose levels had worse baseline cognitive scores and an almost twofold risk of developing cognitive impairment over a 4-year period (15). The pathophysiologic mechanisms underlying the purported relationship between diabetes, prediabetes, and dementia, thus far, remains unclear but could involve direct effects on ad neuropathology or indirectly via diabetic vasculopathy, leading to cerebrovascular brain injury . Hyperglycemia can be harmful by promoting oxidative stress, the formation of toxic advanced glycation end products, and induction for receptors for advanced glycation end products . A direct effect of insulin on cognition is supported by observations that insulin readily crosses the blood - brain barrier and the high concentration of insulin receptors on the hippocampus and medial temporal cortex (16), areas of the brain that are primarily involved in memory and affected by ad neuropathology . At excessively high or low levels, insulin has been shown in vitro to downregulate choline acetyltransferase (17), the enzyme that catalyzes the production of the neurotransmitter acetylcholine, which is deficient in ad . Furthermore, insulin has been implicated in phosphorylation and amyloid deposition, the pathophysiologic mechanisms for ad (18). Diabetes is an established risk factor for stroke and other vascular disorders, leading to an alternate hypothesis that the link between diabetes and dementia is indirect, mediated by vascular pathology (19). In the rotterdam study, however, diabetes was associated with mri markers of ad risk, including smaller hippocampus and amygdala volumes, even after accounting for vascular pathology (20). We found that, in this late adult population with a mean age of 62 years, clinical diabetes and each of the metabolic dysfunction markers of ir, hyperinsulinemia, and hyperglycemia (elevated hba1c) were associated with signs of accelerated brain aging, as measured by mri tcbv, and by poorer performance on tests of executive function . The association with ir and hyperinsulinemia, which are early markers of a compensated prediabetic state, were also observed in a subsample without clinical diabetes . In addition, we found that there appear to be different patterns of association between markers of a hyperglycemic state versus measures of ir when each is related to cognitive markers of aging . Measures of ir (homa - ir and fasting insulin levels) were inversely associated with visual memory and executive function, whereas glycemic indices (diabetes and hba1c) were inversely associated only with performance on executive function . We also found a marginally significant direct association between diabetes and verbal memory, which disappeared after adjusting for all covariates . This finding is contrary to previously published studies and may be a chance artifact given our cohort s younger average age compared with previous studies . Because it was restricted to persons on insulin supplementation, it could reflect a beneficial effect of exogenous insulin on memory (21). Our data strengthen earlier findings relating diabetes status to cognitive function, even in stroke- and dementia - free community - dwelling middle - aged men and women . Taken together, these findings suggest that the insulin resistant states seen in prediabetes and diabetes, as well as hyperglycemia, accelerate brain structural and cognitive aging, albeit in slightly different patterns, with the most robust associations being with ir . In contrast to investigations into the association between glycemia and cognition, fewer studies have explored the possible links between insulin levels and ir, and brain structure and cognitive performance . Although hyperinsulinemia has been directly linked (22) to ad risk, there is also accumulating evidence linking insulin levels and cognitive performance in individuals without dementia . Hyperinsulinemia has been associated with lower scores on the mini - mental state examination in individuals without dementia (23). Furthermore, ir, as measured by the homa - ir, has likewise been associated with poorer cognition and, more recently, with diffusely decreased cortical glucose uptake on fluorodeoxyglucose - positron emission tomography scans (24,25). Our findings of inverse relationships between fasting insulin levels, homa - ir, and brain volume and cognitive performance even after controlling for vascular risk factors appear to support these findings . One limitation of our study is that we only examined a cross - sectional association but did not examine the association of these measures with change . Another limitation is the primarily white ethnicity of the framingham study sample; hence, these associations need to be examined among community - dwelling individuals of other ethnicities . Our results do support ongoing studies to examine whether early detection and management of diabetes and metabolic dysfunction, particularly ir, would be able to delay the clinical onset of cognitive disorders . Diabetic and prediabetic states characterized by ir, hyperinsulinemia, and hyperglycemia, when present in late middle age, are related to decreased brain volume and lower cognitive performance on executive function and memory tasks . These results extend the body of evidence linking metabolic dysfunction to the risk of dementia and ad in late life . These results suggest that clinical trials attempting to delay cognitive and structural brain loss by controlling metabolic dysfunction, even in individuals free of clinical diabetes and as early as the 7th decade, might be warranted.
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Children of parents with a chronic medical condition (cmc) are at an increased risk for developing health - related and social - emotional problems, such as somatic complaints, social isolation, and excessive concern to acquire an illness themselves (compas, 1994; earley and cushway 2002; faulkner and davey 2002; pedersen and revenson 2005). Also more recent evidence suggests that these children show internalizing problems (e.g., anxiety and depressed mood) and externalizing problems, that is, aggressive and rule - breaking behavior (ivarsson et al . 2002; sieh et al . Children with parental cmc also display elevated stress levels (pedersen and revenson 2005; sieh et al . Stress in children presumably acts as a mediator between illness - related factors and child outcomes, and it is therefore an important variable to examine (pakenham and bursnall 2006; pedersen and revenson 2005). We choose adolescents as target group because they seem to be especially vulnerable to developmental problems and may adopt more caregiving responsibilities than latency - aged children (korneluk and lee 1998; sieh et al . Hence, this study will focus on adolescent stress . According to the family systems - illness (fsi) model of rolland (1987, 1999), adolescent stress depends on specific illness - related factors, resulting in a differentiation into illness type depending on the diagnosis (compas et al . The fsi model classifies cmc s as a function of illness type and family functioning . Illness type can be categorized based on the presence of disability (non - disabling versus disabling), its onset (gradual versus acute), its course (progressive, episodic, or constant), its outcome (fatal versus non - fatal), and its time stage (beginning versus terminal). Disability implicates problems with activities of daily living, and communicative and cognitive abilities . Disability of parents appears to be associated with elevated stress levels in the family (pedersen and revenson 2005; rolland 1987, 1999; verhaeghe et al . 2005). Concerning onset, diseases can have an acute onset, forcing the family to adapt in a short period of time, which often causes high stress levels directly after the diagnosis . The course of a chronic illness varies depending on the diagnosis and may be categorized based on the pattern of expected trajectory . A progressive illness increases in severity, inducing cumulative responsibilities for family caretakers over time, which is related to elevated stress levels in family members . An episodic illness is represented by exacerbations and remissions that require flexibility of all family members . Constant illnesses are often marked by an acute onset, implicating that the amount of illness - related stressors remains the same after recovery (pedersen and revenson 2005; rolland 1987, 1999). Finally, the illness outcome is characterized by the possibility of death or shortened life span versus non - fatal conditions . The most important element is the initial doubt whether a disease will cause death (rolland and williams 2005). It is crucial to add that the fsi model considers illness type in relation to the illness stage (crisis, chronic stage, and terminal stage) and, in turn, in relation to family functioning . Accordingly, the stressors associated with the crisis stage of a less severe condition could easily generate more strain on a family system than the chronic phase of a more serious condition . It is well - documented that cmc has a tremendous impact on psychological characteristics of ill parents, especially in terms of an increased risk for depression (visser - meily and meijer 2006). For example, in the chronic stage of stroke, depression is a hidden issue that is linked to disability in parents (van de port et al . Similarly, a longitudinal study on children of stroke patients ascertained that depressive symptoms of ill parents predict long - term stress in children (sieh et al . Parental depression is often accompanied by decreased emotional availability of the parent and may directly and indirectly affect the quality of family relationships (faulkner and davey 2002). This study also takes the level of ill parents depressive symptoms into account because of the strong association with family functioning . The fsi model proposes that adolescent stress is associated with family functioning (rolland 1987, 1999; rolland and williams 2005). In this study, family functioning is conceptualized by means of three interpersonal variables: quality of marital relationship, quality of parent - child interaction, and quality of parent attachment . Two months after discharge from the rehabilitation center, it correlated with long - term stress in children (sieh et al . Researchers are inconclusive whether the relationship between quality of marital relationship and adolescent functioning is direct or bidirectional (compas et al . However, they reached unanimity that the parent - child relationship should be evaluated by both parent and adolescent as the interpretations of this relationship might differ depending on the informant (korneluk and lee 1998; hocking and lochman 2005). For example, children may adopt roles that are inappropriate for their age and feel overwhelmed by the sheer presence of household chores and caregiving tasks . The assumption of an adult role by a child, referred to as parentification, is an example of a possibly modified familial interaction pattern (faulkner and davey 2002; meijer et al . All the same, family relationships can remain a source of strength and should be acknowledged as a protective buffer for adolescent stress (carr and springer 2010). Besides, adolescents gender and age may be associated with adolescent stress (pedersen and revenson 2005), but previous research delivered inconsistent results . Some studies found that girls have higher stress levels than boys (sieh et al . 1996). On the contrary, other studies reported no gender differences in child report of stress (barkmann et al . Similarly, some studies suggested that older adolescents experience more stress than younger adolescents (compas et al . 1994; veach 1999), while other studies concluded that older adolescents experience less stress than younger adolescents (faulkner and davey 2002; welch et al . It can be concluded that adolescents gender and age should be controlled for with regard to child report of stress . Inspired by the fsi model, the relation of adolescent stress to specific parental cmc s can be evaluated . Our sample concerns parents with a medical condition in the chronic stage, so we will examine in which regard the fsi model may be a useful explanatory framework apropos of child adjustment (adolescent stress) to chronic parental illness . To date, this has not been accomplished . According to the fsi model, chronic illnesses exhibit variability and without diagnostic differentiation therefore, the aim of this study is to investigate whether adolescents stress level varies while controlling for parental illness type . The first question is whether adolescent stress is directly influenced by illness type (disability, onset, course, and outcome). Our second question is whether the size and significance of the associations with parental depression, family functioning, and child demographics depend on illness type . We expected that children of parents with a disabling cmc showed more stress than children of parents with a non - disabling cmc . Similarly, parental cmc s with an acute onset were assumed to cause more stress in children than cmc s with a gradual onset . In addition, children of parents with a progressive cmc were supposed to have a higher stress level than children of parents with a constant cmc . Children of parents with a possibly fatal cmc were expected to have a higher stress level than children of parents with a non - fatal cmc . Children of families with high family functioning were assumed to show less stress than children of families with low family functioning (rolland 1987, 1999). We included children between 10 and 20 years of age who lived together with at least one parent with a cmc . Parental cmc was defined as a disease or a traumatic injury impairing health, involving one or more organ systems and lasting 6 months or longer (brown et al . 2007; livneh and antonak 2005). Children with severe somatic or psychiatric disorders, which were assessed by way of parent interviews and child report, only 17 families were not part of the final sample, resulting in a high participation rate (85.3%). The remaining 7 families could not participate because their children were too old, too young, or disabled, or because no parent had a cmc . In one case, no data of the parent with cmc were available and the data for this family were deleted from the analyses, leading to a sample of 99 families including 158 adolescents and 82 healthy parents (table 1). Fifteen families were counted as single parent household, four of which were characterized by a long distance relationship between the parents . In two families, both parents had a cmc and the data for the less disabled parent were excluded from analyses . Parental cmc included multiple sclerosis (29.3%), rheumatoid arthritis (19.2%), brain damage (16.2%), muscle disease (14.1%), spinal cord injury (7.1%), inflammatory bowel disease (6.1%), parkinson disease (5.1%), and diabetes type i with physical complications (3.0%). The mean time since diagnosis was 12.6 years and ranged between 7 months and 49 years.table 1demographic characteristics of adolescents, ill, and healthy parents in families with parental cmcn (%) m (sd)adolescents158 gender (female)81 (51.3) age15.11 (2.32)highest educational level primary education27 (17.1) lower vocational education62 (39.2) intermediate vocational education23 (14.6) high school38 (24.1) (pre-)university education7 (4.4)ill parents99 gender (female)67 (67.7) age47.00 (5.43)highest educational level primary / lower education10 (10.1) intermediate vocational education29 (29.3) high school11 (11.1) pre - university or higher vocational education30 (30.3) university14 (14.1) currently working37 (37.4)healthy parents82 gender (female)32 (31.7) age48.06 (5.74)highest educational level primary / lower education11 (12.2) intermediate vocational education26 (31.7) high school6 (7.3) pre - university or higher vocational education25 (30.5) university14 (14.1) currently working70 (85.4) demographic characteristics of adolescents, ill, and healthy parents in families with parental cmc a medical doctor in our team documented illness type in a spread sheet in accordance with the fsi model (rolland 1987; stehouwer et al . When a parent had more than one cmc, the categorization of illness into illness type was based on the worst possible outcome and disability . Because of the small sample size, we decided to dichotomize the scales of the classification system with the following scores: non - disabling (0) versus disabling (1), gradual onset (0) versus acute onset (1), constant course (0) versus progressive course (1), non - fatal (0) versus potentially fatal (1).table 2number of parents with cmc as a function of illness typenon - disablingdisablinggradual onsetacute onsetgradual onsetacute onsetnon - fatalconstant11023progressive150181potentially fatalconstant1000progressive00039examples of cmc s: multiple sclerosis, cystic fibrosis; rheumatoid arthritis, hereditary motor sensory neuropathy; stroke, spinal cord injury, traumatic brain injury; colitis ulcerosa . Disabling disease: expected problems with activities of daily living, and with communicative or cognitive activities like walking, dressing, and talking . Acute onset: onset of disease less than 1 h, diagnosis easily made . Possibly fatal: possibility of death or shortened life span number of parents with cmc as a function of illness type examples of cmc s: multiple sclerosis, cystic fibrosis; rheumatoid arthritis, hereditary motor sensory neuropathy; stroke, spinal cord injury, traumatic brain injury; colitis ulcerosa . Disabling disease: expected problems with activities of daily living, and with communicative or cognitive activities like walking, dressing, and talking . Acute onset: onset of disease less than 1 h, diagnosis easily made . Possibly fatal: possibility of death or shortened life span adolescents filled in the dutch stress questionnaire for children (hartong et al . 2003), a reliable self - report measure to determine global psychological stress (17 items; 4-point likert scale from 1 = not true for me at all to 4 = completely true for me). In this study, cronbach s alpha was = 0.87 . Depressive symptoms of parents with cmc, also referred to as parental depression, were determined with the beck depression inventory (21 items; 4-point scale from 0 = i do not feel like a failure to 3 = i feel i am a complete failure as a person, = 0.87) (beck et al . The interactional problem solving inventory (ipsi) measuring perceived quality of marital relationship from lange (1983) was filled in by both parents . Where applicable, we calculated dyadic scores reflecting the quality of marital relationship for each family (17 items; 5-point likert scale from 1 = exactly applicable to me / my partner to 5 = absolutely not applicable to me / my partner; = 0.85). To assess the quality of parent - child interaction, both parents completed the parent - child interaction questionnaire revised from lange (2001). Average scores were calculated to generate one parent - child interaction score for each family (21 items; 5-point likert scale; 1 = completely inapplicable, 4 = exactly applicable; = 0.87). In addition, adolescents filled in three scales (communication, confidence, and alienation) of the inventory of parent and peer attachment (nada raja et al . 1992; reitz 2004) about the father and the mother, and average scores were calculated to measure overall parent attachment (12 items; 4-point likert scale from 1 = almost never or never to 4 = almost always or always; = 0.88). Families with parental cmc were recruited through general health practitioners, health organizations, rehabilitation and community centers, hospitals, schools, and public places (e.g., libraries) across the netherlands . Collaborating staff was instructed by the project manager and posted brochures and posters in waiting rooms and public spaces . Some of them also provided additional information and invited potential participants to take part in this study . Families had to contact the researchers by e - mail or phone to show their interest in participation . The ethical commission of the research institute of child development and education of the university of amsterdam approved this study . The research questions were answered by means of multilevel regression analyses which accounted for the fact that children within families have more similarities than children between families (snijders and bosker 1999). The nesting structure of adolescents and chronically ill parents is illustrated in table 3.table 3nesting structure of adolescents per marital status or living conditionnumber of adolescents within families1234totalsingle parent home1041015parents married/ living together41348184total51389199 nesting structure of adolescents per marital status or living condition using g*power (buchner et al . 2009), we found that the power of this study was 0.89, while correcting for nested data with the expectation of equal sample sizes in each group . A power value that large means that it was probable that we would find a statistically significant result when such a result actually exists . Due to a small sample size within many cells, no analysis could be performed including all illness types at once . Four analyses of illness type were conducted separately and included all other predictors in each analysis . In the first analysis (model 1), the influence of disability was examined . We report the akaike information criterion as a measure of the relative goodness of model fit . To illustrate comparable estimates, only 0.9% of the data from children and 3.5% of the data from parents were missing throughout the dataset . Data were missing completely at random (little 1988), so we used expectation maximization to substitute missing values . We included children between 10 and 20 years of age who lived together with at least one parent with a cmc . Parental cmc was defined as a disease or a traumatic injury impairing health, involving one or more organ systems and lasting 6 months or longer (brown et al . 2007; livneh and antonak 2005). Children with severe somatic or psychiatric disorders, which were assessed by way of parent interviews and child report, only 17 families were not part of the final sample, resulting in a high participation rate (85.3%). The remaining 7 families could not participate because their children were too old, too young, or disabled, or because no parent had a cmc . In one case, no data of the parent with cmc were available and the data for this family were deleted from the analyses, leading to a sample of 99 families including 158 adolescents and 82 healthy parents (table 1). Fifteen families were counted as single parent household, four of which were characterized by a long distance relationship between the parents . In two families, both parents had a cmc and the data for the less disabled parent were excluded from analyses . Parental cmc included multiple sclerosis (29.3%), rheumatoid arthritis (19.2%), brain damage (16.2%), muscle disease (14.1%), spinal cord injury (7.1%), inflammatory bowel disease (6.1%), parkinson disease (5.1%), and diabetes type i with physical complications (3.0%). The mean time since diagnosis was 12.6 years and ranged between 7 months and 49 years.table 1demographic characteristics of adolescents, ill, and healthy parents in families with parental cmcn (%) m (sd)adolescents158 gender (female)81 (51.3) age15.11 (2.32)highest educational level primary education27 (17.1) lower vocational education62 (39.2) intermediate vocational education23 (14.6) high school38 (24.1) (pre-)university education7 (4.4)ill parents99 gender (female)67 (67.7) age47.00 (5.43)highest educational level primary / lower education10 (10.1) intermediate vocational education29 (29.3) high school11 (11.1) pre - university or higher vocational education30 (30.3) university14 (14.1) currently working37 (37.4)healthy parents82 gender (female)32 (31.7) age48.06 (5.74)highest educational level primary / lower education11 (12.2) intermediate vocational education26 (31.7) high school6 (7.3) pre - university or higher vocational education25 (30.5) university14 (14.1) currently working70 (85.4) demographic characteristics of adolescents, ill, and healthy parents in families with parental cmc a medical doctor in our team documented illness type in a spread sheet in accordance with the fsi model (rolland 1987; stehouwer et al . When a parent had more than one cmc, the categorization of illness into illness type was based on the worst possible outcome and disability . Because of the small sample size, we decided to dichotomize the scales of the classification system with the following scores: non - disabling (0) versus disabling (1), gradual onset (0) versus acute onset (1), constant course (0) versus progressive course (1), non - fatal (0) versus potentially fatal (1).table 2number of parents with cmc as a function of illness typenon - disablingdisablinggradual onsetacute onsetgradual onsetacute onsetnon - fatalconstant11023progressive150181potentially fatalconstant1000progressive00039examples of cmc s: multiple sclerosis, cystic fibrosis; rheumatoid arthritis, hereditary motor sensory neuropathy; stroke, spinal cord injury, traumatic brain injury; colitis ulcerosa . Disabling disease: expected problems with activities of daily living, and with communicative or cognitive activities like walking, dressing, and talking . Acute onset: onset of disease less than 1 h, diagnosis easily made . Progressive course: disease increasing in severity . Possibly fatal: possibility of death or shortened life span number of parents with cmc as a function of illness type examples of cmc s: multiple sclerosis, cystic fibrosis; rheumatoid arthritis, hereditary motor sensory neuropathy; stroke, spinal cord injury, traumatic brain injury; colitis ulcerosa . Disabling disease: expected problems with activities of daily living, and with communicative or cognitive activities like walking, dressing, and talking . Acute onset: onset of disease less than 1 h, diagnosis easily made . Progressive course: disease increasing in severity . Possibly fatal: possibility of death or shortened life span adolescents filled in the dutch stress questionnaire for children (hartong et al . 2003), a reliable self - report measure to determine global psychological stress (17 items; 4-point likert scale from 1 = not true for me at all to 4 = completely true for me). In this study, cronbach s alpha was = 0.87 . Depressive symptoms of parents with cmc, also referred to as parental depression, were determined with the beck depression inventory (21 items; 4-point scale from 0 = i do not feel like a failure to 3 = i feel i am a complete failure as a person, = 0.87) (beck et al . 1961; visser et al . 2006; yin and fan 2000). The interactional problem solving inventory (ipsi) measuring perceived quality of marital relationship from lange (1983) was filled in by both parents . Where applicable, we calculated dyadic scores reflecting the quality of marital relationship for each family (17 items; 5-point likert scale from 1 = exactly applicable to me / my partner to 5 = absolutely not applicable to me / my partner; = 0.85). To assess the quality of parent - child interaction, both parents completed the parent - child interaction questionnaire revised from lange (2001). Average scores were calculated to generate one parent - child interaction score for each family (21 items; 5-point likert scale; 1 = completely inapplicable, 4 = exactly applicable; = 0.87). In addition, adolescents filled in three scales (communication, confidence, and alienation) of the inventory of parent and peer attachment (nada raja et al . 1992; reitz 2004) about the father and the mother, and average scores were calculated to measure overall parent attachment (12 items; 4-point likert scale from 1 = almost never or never to 4 = almost always or always; = 0.88). Families with parental cmc were recruited through general health practitioners, health organizations, rehabilitation and community centers, hospitals, schools, and public places (e.g., libraries) across the netherlands . Collaborating staff was instructed by the project manager and posted brochures and posters in waiting rooms and public spaces . Some of them also provided additional information and invited potential participants to take part in this study . Families had to contact the researchers by e - mail or phone to show their interest in participation . The ethical commission of the research institute of child development and education of the university of amsterdam approved this study . The research questions were answered by means of multilevel regression analyses which accounted for the fact that children within families have more similarities than children between families (snijders and bosker 1999). The nesting structure of adolescents and chronically ill parents is illustrated in table 3.table 3nesting structure of adolescents per marital status or living conditionnumber of adolescents within families1234totalsingle parent home1041015parents married/ living together41348184total51389199 nesting structure of adolescents per marital status or living condition using g*power (buchner et al . 2009), we found that the power of this study was 0.89, while correcting for nested data with the expectation of equal sample sizes in each group . A power value that large means that it was probable that we would find a statistically significant result when such a result actually exists . Due to a small sample size within many cells, four analyses of illness type were conducted separately and included all other predictors in each analysis . In the first analysis (model 1), the influence of disability was examined . We report the akaike information criterion as a measure of the relative goodness of model fit . To illustrate comparable estimates, only 0.9% of the data from children and 3.5% of the data from parents were missing throughout the dataset . Data were missing completely at random (little 1988), so we used expectation maximization to substitute missing values . Children were distributed among the following categories of parental illness type: 132 cases of disability versus 26 cases of no disability, 117 cases of gradual onset versus 41 cases of acute onset, 44 cases of constant course versus 114 cases of progressive course, and 92 cases of non - fatal outcome versus 66 cases of possibly fatal outcome . The level of adolescent stress was comparable to that of children 3 years after parental stroke (sieh et al . Half of the ill parents (50.5%) displayed scores that indicated mild (29.3%), moderate (10.1%), or severe depression (11.1%). The mean quality of marital relationship was under the cut - off score (68.5 points) and 69.5% of the parents scored below the cut - off score, indicating relatively poor marital functioning . The average scores for quality of parent - child interaction were very close to those of the normal population (mean = 88.82, sd = 6.59; lange 2001), see table 4.table 4descriptive statistics for adolescent stress, parental depression, quality of marital relationship, quality of parent - child interaction and quality of parent attachmentmean (sd)rangeadolescent stress34.66 (8.07)17.0 - 63.0parental depression12.19 (7.70)1.0 - 34.0quality of marital relationship62.01 (9.96)34.0 - 78.0quality of parent - child interaction88.99 (10.69)49.0 - 104.0quality of parent attachment38.89 (5.59)15.5 - 48.0 descriptive statistics for adolescent stress, parental depression, quality of marital relationship, quality of parent - child interaction and quality of parent attachment all independent variables (parental depression; quality of marital relationship, parent - child interaction, and attachment; adolescent gender and age) had a significant correlation with adolescent stress, but only quality of parent attachment showed a large correlation and all other correlations with adolescent stress ranged between small and medium (cohen, 1992), see table 5 . Most correlations between the predictors were significant, one of which was large in size, indicating that higher quality of marital relationship was strongly associated with lower parental depression scores . The size of the other correlations between family functioning variables and parental depression, and within family functioning variables was medium . Adolescents gender and age showed three small correlations, that is, girls and older children comparatively reported more stress and older children reported a lower quality of parent attachment.table 5correlations between adolescent stress, parental depression, family functioning, and adolescents gender and age1234561 adolescent stress-2 parental depression0.24**-3 quality of marital relationship-0.20 * -0.67**-4 quality of parent - child interaction-0.30**-0.32**0.42**-5 quality of parent attachment-0.56**-0.31**0.36**0.29**-6 adolescents gender0.19 * -0.040.010.05 - 0.03 - 7 adolescents age0.25**-0.04 - 0.04 - 0.10 - 0.25**0.01*p <0.05 . * * p <0.01 correlations between adolescent stress, parental depression, family functioning, and adolescents gender and age * p <0.05 . * * p <0.01 an empty model was estimated, with only the random intercept and the family as the grouping variable (table 6). The intra - class correlation coefficient (icc = 0.29) and the deviance ratio [(1) = 4.47, p <0.05] indicated that a model with a random intercept fitted the data better than a model that did not allow for random variability . The icc can be interpreted as such that two random children in the same random family shared 29% of the variability.table 6model specifications of multilevel analyses, including 95% confidence intervals [ci] of fixed effectsempty modelmodel 1model 2model 3model 4estimates.e.estimates.e.estimates.e.estimates.e.estimates.e.intercept34.61***0.7234.47***1.4033.47***0.8132.75***1.2433.00***0.92disability-1.69 [-4.60, 1.21]1.46illness onset-1.44 [-4.03, 1.13]1.30illness course0.46 [-2.12, 3.03]1.29illness outcome0.23 [-2.02, 2.47]1.13parental depression1.17 [-0.31, 2.65]0.741.11 [-38, 2.61]0.751.08 [-0.44, 2.59]0.761.11 [-38, 2.61]0.75quality of marital relationship1.24 [-0.32, 2.80]0.781.25 [-0.32, 2.82]0.791.25 [-0.21, 2.83]0.791.25 [-0.32, 2.82]0.79quality of parent - child interaction-1.38 * [-2.62, -0.14]0.62 - 1.44 * [-2.70, -0.19]0.63 - 1.48 * [-2.76, -0.20]0.64 - 1.44 [-2.69, -0.19]0.63quality of parent attachment-3.97 * * * [-5.13, -2.80]0.59 - 3.97 * * * [-5.13, -2.79]0.59 - 3.96 * * * [-5.13, -2.79]0.59 - 3.97 * * * [-5.13, -2.79]0.59adolescents gender3.13 * * [1.27, 5.16]1.033.06 * * [1.03, 5.11]1.033.08 * * [1.51, 5.42]1.033.06 * * [1.03, 5.11]1.03adolescents age0.94 [-0.12, 2.00]0.540.93 [-0.13, 2.00]0.540.95 * [-0.12, 2.03]0.540.93 [-0.13, 2.00]0.54level 1 variance46.0734.9434.7334.8434.92level 2 variance19.866.356.626.866.78aic1105.741021.501021.831022.971023.33explained variance (level 1)37.4% 37.3% 36.8% 36.8% explained variance (level 2)43.4% 44.7% 43.4% 43.5% aic akaike information criterion . Explained variance at level 2 was calculated assuming that families have two children on average. *p <0.05 . * * p <0.01 . * * * p <0.001 model specifications of multilevel analyses, including 95% confidence intervals [ci] of fixed effects aic akaike information criterion . Explained variance at level 2 was calculated assuming that families have two children on average. *p <0.05 . * * p <0.01 . * * * p <0.001 first, model 1 was tested while controlling for the presence of disability . Quality of parent - child interaction and parent attachment, and adolescents gender were significantly linked to adolescents stress level, see table 6 . Estimates for quality of parent - child interaction and parent attachment, and adolescents gender were statistically significant . Quality of parent - child interaction and parent attachment, and adolescents gender displayed significant associations with adolescent stress . Model 4 included the outcome of the illness (non - fatal versus possibly fatal). Again, quality of parent - child interaction and parent attachment, and adolescents gender had significant relationships with adolescent stress ., the results demonstrated that independent of illness type, higher quality of parent - child relationships and adolescents female gender were connected to elevated adolescent stress . The akaike information criterion of model 1 to model 4 showed that the model fit improved compared to the empty model . The four models explained approximately 37% of the variance in adolescent stress between individuals and 43 - 44% of the variance in adolescent stress between families . This study evaluated the predictive power of illness type, the ill parent s depressive symptoms, family functioning, and adolescents gender and age on child report of stress in a sample of parents with a medical condition in the chronic stage . First, illness type in the chronic stage of parental illness did not appear to affect adolescent stress scores . Second, the size and significance of the estimates for parental depression, family functioning and child demographics did not depend on illness type . Hence, the results cannot confirm the expectation based on the fsi model suggesting a significant role of illness type with respect to child report of stress . Contrary to our hypotheses, not illness type but mainly family functioning and adolescents gender were directly related to adolescent stress . All variances explained by the models were moderate, meaning that we identified important risk and protective factors for adolescent stress at the individual level (i.e., quality of parent attachment and adolescent gender) and at family level (i.e., quality of parent - child interaction). In support of the fsi model, strong evidence was found that the parent - child relationship is a crucial determinant of adolescent stress . Unexpectedly, the relationship between parental depression and adolescent stress was not significant in regression analyses . On the contrary, the empirical and theoretical basis suggests that depressive symptoms of ill parents often emerge over time (sieh et al . In accordance, rolland (1987; 1999) stated that disabling and possibly fatal diseases have the strongest impact on family functioning . We did not find support for this assumption and can only present a small to medium positive correlation between parental depression and adolescent stress . Possibly, other mediating variables (e.g., parenting and coping variables) are involved in the relationship between ill parents depressive symptoms and adolescent stress (donofrio and lahey 2010; sieh et al . Similarly, high quality of marital relationship was merely correlated with elevated adolescent stress . However, no significant relationship was found between marital functioning and adolescent stress when we controlled for illness type, parent - child interaction, parent attachment, and demographics . Research is inconsistent about the influence of quality of marital relationship on adolescents functioning (compas et al . 1994, 1996; faulkner and davey 2002; sieh et al . 2010b; veach 1999). In our sample, marital functioning was poor, resulting in lower variance, which may obstruct significant results . A longitudinal study on families of stroke patients showed that marital functioning progressively decreased under the level of adequate functioning . Marital functioning during 3 years post - stoke was just incidentally associated with long - term stress in adolescents (sieh et al . A possible explication for inconsistent findings in the field is that marital functioning and child adjustment are bidirectional and reinforce each other through a mediating variable such as parenting and parent attachment (donofrio and lahey 2010). High quality of parent - child interaction and especially high quality of parent attachment were associated with lower levels of adolescent stress, pointing to a potential protective mechanism in child adjustment to parental cmc . As yet, previous research has attested the paramount importance of family functioning in child adjustment to parental cmc (carr and springer 2010; hocking and lochman 2005; korneluk and lee 1998; sieh et al . Our study extends previous knowledge, affirming that especially variables evaluating the parent - child relationship show high predictive power for adolescent stress . This suggests that rehabilitation staff can be recommended to give attention to the parenting role, how the parenting role may change as a result of cmc, and how parental cmc affects the parent - child relationship . They could also benefit from advice about how to talk to children about their condition and its impact on family life . In addition, the results substantiate that the parent - child relationship should be evaluated by both parents and adolescents who may evaluate the mutual relationship differently (brumariu and kerns 2010; korneluk and lee 1998; meijer et al . 2007). Moreover, girls appear to be more susceptible to stress than boys, which is in line with most research in the field (sieh et al . The estimate for gender was highly significant, bringing into view that girls have different sensitivities than boys . Lastly, adolescents age was unrelated to child report of stress in the regression analysis and we found a small positive correlation . Previous research is inconsistent about the influence of adolescents age on adolescents well - being (faulkner and davey 2002; pedersen and revenson 2005; sieh et al . More research is needed for distinct conclusions regarding the effects of age on adolescent stress in this specific group . Our sample size was small for the amount of cases per illness type except in model 1 (including disability). A related restraint is that we had to dichotomize the scales of illness type and could not account for progressive illness course or fatal outcomes, potentially altering the results . For example, illness outcome may have been unrelated to adolescent stress due to the omission of illness fatality . Similarly, because of the sample size, we had to ignore the interactive effects of one versus two parents with a disability, and one cmc versus more than one cmc . Moreover, it should be borne in mind that we examined cmc in a sample of western culture . The results could have looked differently using data from a sample of non - western culture because of distinctive cultural views and practices affecting health and health behaviors (carr and springer 2010). An important strength of this study lies in the inspiration from the fsi model in which medical information is combined with information about family functioning of the parent and adolescent . In addition, this study recognized factors that emerged as influential predictors in previous research, including reports of the parent - child relationship from parents and adolescents . As recommended (donofrio and lahey 2010), we took into consideration family characteristics at multiple levels (demographic, psychological, and relational variables from several informants). Finally, the use of multilevel analyses is an advancement as grouping according to families led to a considerable similarity between adolescents within the same family . Our study did not aim to generate specific recommendations for the clinical practice, but possible interventions should not go unmentioned . In an extensive review, weihs et al . (2002) stratified studies by specific cmc s and formulated three general goals for interventions for families affected by cmc . Recommendations include helping families cope with illness - related stressors, mobilizing the family support system, and minimizing interpersonal hostility and adverse effects of illness - related trauma . Carr and springer (2010) reviewed research on families and health between 2000 and 2009 . In line with our findings stressing the importance of family relationships, carr and springer interventions should aim to encourage healthy relationships through parent education, conflict and stress management, communicative training, and health promotion . For example, cognitive - behavioral stress management training has shown to benefit mental health, social interaction, and family functioning in adolescents with parental cmc (keypour et al . Future studies should incorporate all elements of the fsi model, taking into account the stage of an illness consisting of crisis, chronic stage, and terminal stage (rolland 1987, 1999). Stages of individual family members and family life cycles also need to be considered . Our data were collected in the chronic stage, making it impossible to compare the stress level in different illness stages . With a larger and more diverse illness sample, the fsi model can be fully applied and the hypotheses can be formulated in more complex ways . Notably, the transitions between time stages are crucial in relation to the other illness types . For instance, the stressors associated with the crisis stage of a less severe condition could easily generate more strain on a family system than the chronic phase of a more serious condition . Similarly, the hypothesis that acute onset cmc is associated with more stress in adolescents than a gradual onset cmc could depend on the time stage in which families are investigated . In case of parental stroke (acute onset) relative to multiple sclerosis (gradual onset), the hypothesis may initially hold . Yet, if the measurement occurred several years later, families dealing with a stroke would show considerable improvement over time (visser - meily et al ., 2009), potentially decreasing the experience of stress (sieh et al ., multiple sclerosis can be non - disabling in the beginning and cause severe disability a decade later . These complex relationships between illness type and illness stage could lead to interesting results from forthcoming longitudinal studies (rolland 1987, 1999; rolland and williams 2005). Further, studies could focus on the mediating and moderating role of stress and family characteristics in the development of problem behavior in adolescents . Donofrio and lahey (2010) have conducted a decade review on biosocial studies focusing on family processes . They emphasize the need to be thoughtful of bidirectional influences among family members and the wider social and biological system, embodying interactions between environmental and biological factors (e.g., stress hormones like cortisol) over time . Lastly, we investigated medical and psychological factors, whereas theories and models are usually developed from a specific perspective, for example, a medical perspective . To increase our understanding of the complex fsi model, it is necessary to better understand family relationships and the interplay between all components of the model . This can be accomplished by means of transdisciplinary theories and models, for example, the social model of disability (tate and pledger 2003). According to that model, the perceived stressfulness does not necessarily result from the parent s physical disability but rather from interpersonal experiences and environmental mismatches that functional limitations can precipitate . In conclusion, this study increases our understanding of the fsi model and provides insight into protective and risk factors for adolescent stress in the chronic stage of parental medical condition . We found some support for the fsi model in the sense that family functioning is associated with adolescent stress, but this association does not seem to depend on parental illness type in the chronic stage . It is valuable to incorporate interpersonal family variables and adolescents gender in predictive models of adolescent stress . Rehabilitation staff is recommended to consider how parental cmc affects the parent - child relationship and how family relationships can be boosted, offering interventions such as stress and conflict management.
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Since their discovery in 1967, radio pulsars have provided insights into physics on length scales covering the range from 1 m (giant pulses from the crab pulsar) to 10 km (neutron star) to kpc (galactic) to hundreds of mpc (cosmological). Pulsars present an extreme stellar environment, with matter at nuclear densities, magnetic fields of 10 g to nearly 10 g, and spin periods ranging from 1.5 ms to 8.5 s. the regular pulses received from a pulsar each correspond to a single rotation of the neutron star . It is by measuring the deviations from perfect observed regularity that information can be derived about the neutron star itself, the interstellar medium between it and the earth, and effects due to gravitational interaction with binary companion stars . In particular, pulsars have proved to be remarkably successful laboratories for tests of the predictions of general relativity (gr). The tests of gr that are possible through pulsar timing fall into two broad categories: setting limits on the magnitudes of parameters that describe violation of equivalence principles, often using an ensemble of pulsars, and verifying that the measured post - keplerian timing parameters of a given binary system match the predictions of strong - field gr better than those of other theories . Long - term millisecond pulsar timing can also be used to set limits on the stochastic gravitational - wave background (see, e.g., [73, 86, 65]), as can limits on orbital variability in binary pulsars for even lower wave frequencies (see, e.g., [20, 78]). However, these are not tests of the same type of precise prediction of gr and will not be discussed here . This review will present a brief overview of the properties of pulsars and the mechanics of deriving timing models, and will then proceed to describe the various types of tests of gr made possible by both single and binary pulsars . The properties and demographics of pulsars, as well as pulsar search and timing techniques, are thoroughly covered in the article by lorimer in this series . This section will present only an overview of the topics most important to understanding the application of pulsar observations to tests of gr . Radio pulsars were firmly established to be neutron stars by the discovery of the pulsar in the crab nebula; its 33-ms period was too fast for a pulsating or rotating white dwarf, leaving a rotating neutron star as the only surviving model [108, 53]. The 1982 discovery of a 1.5-ms pulsar, psr b1937 + 21, led to the realization that, in addition to the young crab - like pulsars born in recent supernovae, there exists a separate class of older millisecond or recycled pulsars, which have been spun up to faster periods by accretion of matter and angular momentum from an evolving companion star . (see, for example, and for reviews of the evolution of such binary systems .) It is precisely these recycled pulsars that form the most valuable resource for tests of gr . The exact mechanism by which a pulsar radiates the energy observed as radio pulses is still a subject of vigorous debate . The basic picture of a misaligned magnetic dipole, with coherent radiation from charged particles accelerated along the open field lines above the polar cap [55, 128], will serve adequately for the purposes of this article, in which pulsars are treated as a tool to probe other physics . While individual pulses fluctuate severely in both intensity and shape (see figure 1), a profile integrated over several hundred or thousand pulses (i.e., a few minutes) yields a shape a standard profile (there is generally some evolution of pulse profiles with frequency, but this can usually be taken into account .) It is the reproducibility of time - averaged profiles that permits high - precision timing . Figure 1 top: 100 single pulses from the 253-ms pulsar b0950 + 08, demonstrating pulse - to - pulse variability in shape and intensity . Bottom: cumulative profile for this pulsar over 5 minutes (about 1200 pulses); this approaches the reproducible standard profile . Top: 100 single pulses from the 253-ms pulsar b0950 + 08, demonstrating pulse - to - pulse variability in shape and intensity . Bottom: cumulative profile for this pulsar over 5 minutes (about 1200 pulses); this approaches the reproducible standard profile . (stairs, unpublished .) Of some importance later in this article will be models of the pulse beam shape, the envelope function that forms the standard profile . The collection of pulse profile shapes and polarization properties have been used to formulate phenomenological descriptions of the pulse emission regions . At the simplest level (see, e.g., and other papers in that series), the classifications can be broken down into gaussian - shaped regions with little linear polarization and some circular polarization, and double - peaked cone regions with stronger linear polarization and s - shaped position angle swings in accordance with the rotating vector model while these models prove helpful for evaluating observed changes in the profiles of pulsars undergoing geodetic precession, there are ongoing disputes in the literature as to whether the core / cone split is physically meaningful, or whether both types of emission are simply due to the patchy strength of a single emission region (see, e.g.,). A short description of pulsar observing techniques is in order . As pulsars have quite steep radio spectra (see, e.g.,), they are strongest at frequencies f0 of a few hundred mhz . At these frequencies, the propagation of the radio wave through the ionized interstellar medium (ism) can have quite serious effects on the observed pulse . Multipath scattering will cause the profile to be convolved with an exponential tail, blurring the sharp profile edges needed for the best timing . Figure 2 shows an example of scattering; the effect decreases with sky frequency as roughly f0 - 4 (see, e.g.,), and thus affects timing precision less at higher observing frequencies . A related effect is scintillation: interference between the rays traveling along the different paths causes time - and frequency - dependent peaks and valleys in the pulsar s signal strength . The decorrelation bandwidth, across which the signal is observed to have roughly equal strength, is related to the scattering time and scales as f04 (see, e.g.,). There is little any instrument can do to compensate for these effects; wide observing bandwidths at relatively high frequencies and generous observing time allocations are the only ways to combat these problems . Figure 2pulse profile shapes for psr j1740 - 3052 at multiple frequencies, aligned by pulse timing . All observations taken with the green bank telescope (stairs, unpublished), except for the 660-mhz profile which was acquired at the parkes telescope [9, 122]. Pulse profile shapes for psr j1740 - 3052 at multiple frequencies, aligned by pulse timing . All observations taken with the green bank telescope (stairs, unpublished), except for the 660-mhz profile which was acquired at the parkes telescope [9, 122]. Another important effect induced by the ism is the dispersion of the traveling pulses . Acting as a tenuous electron plasma, the ism causes the wavenumber of a propagating wave to become frequency - dependent . By calculating the group velocity of each frequency component, it is easy to show (see, e.g.,) that lower frequencies will arrive at the telescope later in time than the higher - frequency components, following a 1/f law . The magnitude of the delay is completely characterized by the dispersion measure (dm), the integrated electron content along the line of sight between the pulsar and the earth . All low - frequency pulsar observing instrumentation is required to address this dispersion problem if the goal is to obtain profiles suitable for timing . One standard approach is to split the observing bandpass into a multichannel filterbank, to detect the signal in each channel, and then to realign the channels following the 1/f law when integrating the pulse . However, when the ratio of the pulse period to its dm becomes small, much sharper profiles can be obtained by sampling the voltage signals from the telescope prior to detection, then convolving the resulting time series with the inverse of the easily calculated frequency - dependent filter imposed by the ism . As a result, the pulse profile is perfectly aligned in frequency, without any residual dispersive smearing caused by finite channel bandwidths . In addition, full - stokes information can be obtained without significant increase in analysis time, allowing accurate polarization plots to be easily derived . This coherent dedispersion technique is now in widespread use across normal observing bandwidths of several tens of mhz, thanks to the availability of inexpensive fast computing power (see, e.g., [10, 66, 123]). Some of the highest - precision experiments described below have used this approach to collect their data . Figure 3pulse profile of the fastest rotating pulsar, psr b1937 + 21, observed with the 76-m lovell telescope at jodrell bank observatory . The top panel shows the total - intensity profile derived from a filterbank observation (see text); the true profile shape is convolved with the response of the channel filters . The lower panel shows the full - stokes observation with a coherent dedispersion instrument [126, 123]. Total intensity is indicated by black lines, and linear and circular power by red and blue lines, respectively . The coherent dedispersion observation results in a much sharper and more detailed pulse profile, less contaminated by instrumental effects and more closely resembling the pulse emitted by the rotating neutron star . Pulse profile of the fastest rotating pulsar, psr b1937 + 21, observed with the 76-m lovell telescope at jodrell bank observatory . The top panel shows the total - intensity profile derived from a filterbank observation (see text); the true profile shape is convolved with the response of the channel filters . The lower panel shows the full - stokes observation with a coherent dedispersion instrument [126, 123]. Total intensity is indicated by black lines, and linear and circular power by red and blue lines, respectively . The coherent dedispersion observation results in a much sharper and more detailed pulse profile, less contaminated by instrumental effects and more closely resembling the pulse emitted by the rotating neutron star . Much better timing precision can be obtained with these sharper pulses . Once dispersion has been removed, the resultant time series is typically folded modulo the expected pulse period, in order to build up the signal strength over several minutes and to obtain a stable time - averaged profile . The pulse period may not be very easily predicted from the discovery period, especially if the pulsar happens to be in a binary system . The goal of pulsar timing is to develop a model of the pulse phase as a function of time, so that all future pulse arrival times can be predicted with a good degree of accuracy . The profile accumulated over several minutes is compared by cross - correlation with the standard profile for the pulsar at that observing frequency . A particularly efficient version of the cross - correlation algorithm compares the two profiles in the frequency domain . Once the phase shift of the observed profile relative to the standard profile is known, that offset is added to the start time of the observation in order to yield a time of arrival (toa) that is representative of that few - minute integration . In practice, observers frequently use a time- and phase - stamp near the middle of the integration in order to minimize systematic errors due to a poorly known pulse period . As a rule, pulse timing precision is best for bright pulsars with short spin periods, narrow profiles with steep edges, and little if any profile corruption due to interstellar scattering . With a collection of toas in hand, it becomes possible to fit a model of the pulsar s timing behaviour, accounting for every rotation of the neutron star . Based on the magnetic dipole model [108, 53], the pulsar is expected to lose rotational energy and thus spin down . The primary component of the timing model is therefore a taylor expansion of the pulse phase with time t: (1)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi = {\phi _ 0} + \nu (t - {t_0}) + \frac{1}{2}\dot \nu {(t - {t_0})^2} + \ldots, $$\end{document} where 0 and t0 are a reference phase and time, respectively, and the pulse frequency is the time derivative of the pulse phase . Note that the fitted parameters and and the magnetic dipole model can be used to derive an estimate of the surface magnetic field b sin: (2)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$b\sin \alpha = {\left ({\frac {{- 3i\dot \nu {c^3}}}{{8{\pi ^2}{r^6}{\nu ^3}}}} \right)^{1/2}} \approx 3.2 \times {10^{19}}{\left ({\frac {{- \dot \nu}} {{{\nu ^3}}}} \right)^{1/2}}{\rm{g,}} $$\end{document} where is the inclination angle between the pulsar spin axis and the magnetic dipole axis, r is the radius of the neutron star (about 10 cm), and the moment of inertia is i10 g cm . In turn, integration of the energy loss, along with the assumption that the pulsar was born with infinite spin frequency, yields a characteristic age c for the pulsar: (3)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\tau _ c} = - \frac{\nu} {{2\dot \nu}}. $$\end{document} equation (1) refers to pulse frequencies and times in a reference frame that is inertial relative to the pulsar . Toas derived in the rest frame of a telescope on the earth must therefore be translated to such a reference frame before equation (1) can be applied . The best approximation available for an inertial reference frame is that of the solar system barycentre (ssb). Even this is not perfect; many of the tests of gr described below require correcting for the small relative accelerations of the ssb and the centre - of - mass frames of binary pulsar systems . But certainly for the majority of pulsars it is adequate . The required transformation between a toa at the telescope and the emission time t from the pulsar is (4)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t = \tau - d/{f^2} + {\delta _ {{\rm{r}} \odot}} + {\delta _ {{\rm{e}} \odot}} - {\delta _ {{\rm{s}} \odot}} - {\delta _ {\rm{r}}} - {\delta _ {\rm{e}}} - {\delta _ {\rm{s}}}. $$\end{document} here d / f accounts for the dispersive delay in seconds of the observed pulse relative to infinite frequency; the parameter d is derived from the pulsar s dispersion measure by d = dm/2.4110 hz, with dm in units of pc cm and the observing frequency f in mhz . The roemer term r takes out the travel time across the solar system based on the relative positions of the pulsar and the telescope, including, if needed, the proper motion and parallax of the pulsar . The einstein delay e accounts for the time dilation and gravitational redshift due to the sun and other masses in the solar system, while the shapiro delay s expresses the excess delay to the pulsar signal as it travels through the gravitational well of the sun a maximum delay of about 120 s at the limb of the sun; see for a fuller discussion of these terms . The terms r, e, and s in equation (4) account for similar delays within the pulsar binary system, if needed, and will be discussed in section 2.3.2 below . Most observers accomplish the model fitting, accounting for these delay terms, using the program @@page11tempo . The correction of toas to the reference frame of the ssb requires an accurate ephemeris for the solar system . It is also clear that accurate time - keeping is of primary importance in pulsar modeling . General practice is to derive the time - stamp on each observation from the observatory s local time standard typically a hydrogen maser and to apply, retroactively, corrections to well - maintained time standards such as utc(bipm), universal coordinated time as maintained by the bureau international des poids et mesures in paris . The terms r, e, and s in equation (4), describe the roemer, the majority of binary pulsar orbits are adequately described by five keplerian parameters: the orbital period pb, the projected semi - major axis, the eccentricity e, and the longitude and epoch t0 of periastron . The angle is measured from the line of nodes where the pulsar orbit intersects the plane of the sky . In many cases, one or more relativistic corrections to the keplerian parameters early relativistic timing models, developed in the first years after the discovery of psr b1913 + 16, either did not provide a full description of the orbit (see, e.g.,), or else did not define the timing parameters, in a way that allowed deviations from gr to be easily identified (see, e.g., [49, 58]). The best modern timing model [33, 133, 43] incorporates a number of post - keplerian timing parameters which are included in the description of the three delay terms, and which can be fit in a completely phenomenological manner . The delays are defined primarily in terms of the phase of the orbit, defined by the eccentric anomaly u and true anomaly ae(u), as well as, pb, and their possible time derivatives . These are related by (5)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u - e\sin u = 2\pi \left [{\left ({\frac{{t - {t_0}}}{{{p_{\rm{b}}}}}} \right) - \frac{{{{\dot p}_{\rm{b}}}}}{2}{{\left ({\frac{{t - {t_0}}}{{{p_{\rm{b}}}}}} \right)}^2}} \right], $$\end{document} (6)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${a_{\rm{e}}}(u) = 2\arctan \left [{{{\left ({\frac{{1 + e}}{{1 - e}}} \right)}^{1/2}}\tan \frac{u}{2}} \right], $$\end{document} (7)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\omega = {\omega _ 0} + \left ({\frac{{{p_{\rm{b}}}\dot \omega}} {{2\pi}}} \right){a_{\rm{e}}}(u), $$\end{document} where 0 is the reference value of at time t0 . The delay terms then become: (8)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\delta _ {\rm{r}}} = x\sin \omega (\cos u - e(1 + {\delta _ r})) + x{(1 - {e^2}{(1 + {\delta _ \theta}) ^2})^{1/2}}\cos \omega \sin u, $$\end{document} (9)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\delta _ {\rm{e}}} = \gamma \sin u, $$\end{document} (10)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\delta _ {\rm{s}}} = - 2r\ln \left\ {{1 - e\cos u - s\left [{\sin \omega (\cos u - e) + {{(1 - {e^2})}^{1/2}}\cos \omega \sin u} \right]} \right\}. $$\end{document} here represents the combined time dilation and gravitational redshift due to the pulsar s orbit, and r and s are, respectively, the range and shape of the shapiro delay . Together with the orbital period derivative b and the advance of periastron, they make up the post - keplerian timing parameters that can be fit for various pulsar binaries . A fuller description of the timing model also includes the aberration parameters r and, but these parameters are not in general separately measurable . The interpretation of the measured post - keplerian timing parameters will be discussed in the context of double - neutron - star tests of gr in section 4 . Equivalence principles are fundamental to gravitational theory; for full descriptions, see, e.g., or . Newton formulated what may be considered the earliest such principle, now called the weak equivalence principle it states that in an external gravitational field, objects of different compositions and masses will experience the same acceleration . The einstein equivalence principle (eep) includes this concept as well as those of lorentz invariance (non - existence of preferred reference frames) and positional invariance (non - existence of preferred locations) for nongravitational experiments . This principle leads directly to the conclusion that non - gravitational experiments will have the same outcomes in inertial and in freely - falling reference frames . The strong equivalence principle (sep) adds lorentz and positional invariance for gravitational experiments, thus including experiments on objects with strong self - gravitation . As gr incorporates the sep, and other theories of gravity may violate all or parts of it, it is useful to define a formalism that allows immediate identifications of such violations . The parametrized post - newtonian (ppn) formalism was developed to provide a uniform description of the weak - gravitational - field limit, and to facilitate comparisons of rival theories in this limit . This formalism requires 10 parameters (ppn,,, 1, 2, 3, 1, 2, 3, and 4), which are fully described in the article by will in this series, and whose physical meanings are nicely summarized in table 2 of that article . (note that ppn is not the same as the post - keplerian pulsar timing parameter) damour and esposito - farse [38, 36] extended this formalism to include strong - field effects for generalized tensor - multiscalar gravitational theories . This allows a better understanding of limits imposed by systems including pulsars and white dwarfs, for which the amounts of self - gravitation are very different . Here, for instance, 1 becomes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\hat \alpha _ 1} = {\alpha _ 1} + {\alpha' _ 1}({c_1} + {c_2}) + \ldots, $$\end{document}, where ci describes the compactness of mass mi . The compactness can be written (11)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c_i} = - 2\frac{{\partial \ln {m_i}}}{{\partial \ln g}} \simeq - {\left ({\frac{{2{e^{{\rm{grav}}}}}}{{m{c^2}}}} \right)_i}, $$\end{document} where g is newton s constant and eigrav is the gravitational self - energy of mass mi, about -0.2 for a neutron star (ns) and -10 for a white dwarf (wd). Pulsar timing has the ability to set limits on 1, which tests for the existence of preferred - frame effects (violations of lorentz invariance); 3, which, in addition to testing for preferred - frame effects, also implies non - conservation of momentum if non - zero; and 2, which is also a non - conservative parameter . Pulsars can also be used to set limits on other sep - violation effects that constrain combinations of the ppn parameters: the nordtvedt (gravitational stark) effect, dipolar gravitational radiation, and variation of newton s constant . The current pulsar timing limits on each of these will be discussed in the next sections . Table 1 summarizes the ppn and other testable parameters, giving the best pulsar and solar - system limits . Table 1ppn and other testable parameters, with the best solar - system and binary pulsar tests . Physical meanings and most of the solar - system references are taken from the compilations by will . References: ppn, solar system:;, solar system:;, solar system:; 1, solar system:, pulsar:; 2, solar system: [105, 152]; 3, solar system:, pulsar:; 2, pulsar:; 3, solar system: [15, 152];, net, solar system:, pulsar:; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left ({{\alpha _ {{c_1}}} - {\alpha _ 0}} \right)^2}$$\end{document}, pulsar:; / g, solar system: [45, 115, 59], pulsar: .parameterphysical meaningsolar - system testlimitpulsar testlimit ppn space curvature produced by unit rest massvlbi, light deflection; measures |ppn1|310 non - linearity in superposition law for gravityperihelion shift of mercury; measures |1|310 preferred - location effectssolar alignment with ecliptic410 1 preferred - frame effectslunar laser ranging10ensemble of binary pulsars1.410 2 preferred - frame effectssolar alignment with ecliptic410 3 preferred - frame effects and nonconservation of momentumperihelion shift of earth and mercury210ensemble of binary pulsars1.510 1 non - conservation of momentumcombined ppn limits210 2 non - conservation of momentumlimit on p for psr b1913 + 16410 3 non - conservation of momentumlunar acceleration10 4 non - conservation of momentumnot independent, netgravitational stark effectlunar laser ranging10ensemble of binary pulsars910 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left ({{\alpha _ {{c_1}}} - {\alpha _ 0}} \right)^2}$$\end{document} pulsar coupling to scalar fielddipolar gravitational radiation for psr b0655 + 642.710 / g variation of newton s constantlaser ranging to the moon and mars610 yrchanges in chandrasekhar mass4.810 yr ppn and other testable parameters, with the best solar - system and binary pulsar tests . Physical meanings and most of the solar - system references references: ppn, solar system:;, solar system:;, solar system:; 1, solar system:, pulsar:; 2, solar system: [105, 152]; 3, solar system:, pulsar:; 2, pulsar:; 3, solar system: [15, 152];, net, solar system:, pulsar:; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left ({{\alpha _ {{c_1}}} - {\alpha _ 0}} \right)^2}$$\end{document}, pulsar:; / g, solar system: [45, 115, 59], pulsar: . The possibility of direct tests of the sep through lunar laser ranging (llr) experiments was first pointed out by nordtvedt . As the masses of earth and the moon contain different fractional contributions from self - gravitation, a violation of the sep would cause them to fall differently in the sun s gravitational field . This would result in a polarization of the orbit in the direction of the sun . Llr tests have set a limit of ||<0.001 (see, e.g., [45, 147]), where is a combination of ppn parameters: (12)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta = 4\beta - \gamma - 3 - \frac{{10}}{3}\xi - {\alpha _ 1} + \frac{2}{3}{\alpha _ 2} - \frac{2}{3}{\zeta _ 1} - \frac{1}{3}{\zeta _ 2}. $$\end{document} the strong - field formalism instead uses the parameter i, which for object i may be written as (13)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}{l}} {{{\left ({\frac{{{m_{{\rm{grav}}}}}}{{{m_{{\rm{inertial}}}}}}} \right)}_i} = 1 + {\delta _ {\rm{i}}}}\\ {\ \ \ \ \ \ \ \ \ \ \ \ \ \ = 1 + \eta {{\left ({\frac{{{e^{{\rm{grav}}}}}}{{m{c^2}}}} \right)}_i} + \eta' \left ({\frac{{{e^{{\rm{grav}}}}}}{{m{c^2}}}} \right)_i^2 + \ldots .} \end{array} $$\end{document} pulsar - white - dwarf systems then constrain net=pulsarcompanion . If the sep is violated, the equations of motion for such a system will contain an extra acceleration netg, where g is the gravitational field of the galaxy . As the pulsar and the white dwarf fall differently in this field, this netg term will influence the evolution of the orbit of the system . For low - eccentricity orbits, by far the largest effect will be a long - term forcing of the eccentricity toward alignment with the projection of g onto the orbital plane of the system . Thus, the time evolution of the eccentricity vector will not only depend on the usual gr - predicted relativistic advance of periastron (), but will also include a constant term . Damour and schfer write the time - dependent eccentricity vector as (14)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{\mathbf{e}(}}t{\rm {) =}} {{\rm{\mathbf{e}}}_{\rm{f}}} + {{\rm{\mathbf{e}}}_{\rm{r}}}(t), $$\end{document} where er(t) is the -induced rotating eccentricity vector, and ef is the forced component . In terms of net, the magnitude of ef may be written as [41, 145] (15)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left| {{{\rm{\mathbf{e}}}_{\rm{f}}}} \right| = \frac{3}{2}\frac{{{\delta _ {{\rm{net}}}}{{\rm{\mathbf{g}}} _ \bot}}} {{\dot \omega a(2\pi /{p_{\rm{b}}})}}, $$\end{document} where g is the projection of the gravitational field onto the orbital plane, and a = x / sin i is the semi - major axis of the orbit . For small - eccentricity systems, this reduces to (16)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left| {{{\bf{e}}_{\rm{f}}}} \right| = \frac{1}{2}\frac{{{\delta _ {{\rm{net}}}}{{\bf{g}} _ \bot} {c^2}}}{{fgm{{(2\pi /{p_{\rm{b}}})}^2}}}, $$\end{document} where m is the total mass of the system, and, in gr, f=1 and g is newton s constant . Clearly, the primary criterion for selecting pulsars to test the sep is for the orbital system to have a large value of pb2/e, greater than or equal to 10 days . However, as pointed out by damour and schfer and wex, two age - related restrictions are also needed . First of all, the pulsar must be sufficiently old that the -induced rotation of e has completed many turns and er(t) can be assumed to be randomly oriented . This requires that the characteristic age c be 2/, and thus young pulsars cannot be used . Secondly, itself must be larger than the rate of galactic rotation, so that the projection of g onto the orbit can be assumed to be constant . According to wex, this holds true for pulsars with orbital periods of less than about 1000 days . Converting equation (16) to a limit on net requires some statistical arguments to deal with the unknowns in the problem . First is the actual component of the observed eccentricity vector (or upper limit) along a given direction . Damour and schfer assume the worst case of possible cancellation between the two components of e, namely that |ef||er| . With an angle between g and er (see figure 4), they write |ef|e/(2sin(/2)). Wex [145, 146] corrects this slightly and uses the inequality (17)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}{c}} {\left| {{{\rm{\mathbf{e}}}_{\rm{f}}}} \right| \le e{\xi _ 1}\left (\theta \right),}&\ \ \ \ \ {{\xi _ 1}\left (\theta \right) = \left\ {{\begin{array}{*{20}{l}} {1/\sin \theta} & {{\rm{for\}} \theta \in \left [{0,\pi /2} \right),}\\ 1&{{\rm{for\}} \theta \in \left [{\pi /2,3\pi /2} \right],}\\ {- 1/\sin \theta} & {{\rm{for\}} \theta \in \left ({3\pi /2,2\pi} \right),} \end{array}} \right . }, is assumed to have a uniform probability distribution between 0 and 2. figure 4polarization of a nearly circular binary orbit under the influence of a forcing vector g, showing the relation between the forced eccentricity ef, the eccentricity evolving under the general - relativistic advance of periastron er(t), and the angle . (after .) Polarization of a nearly circular binary orbit under the influence of a forcing vector g, showing the relation between the forced eccentricity ef, the eccentricity evolving under the general - relativistic advance of periastron er(t), and the angle . (after .) Next comes the task of estimating the projection of g onto the orbital plane . The projection can be written as (18)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left| {{\mathbf{g} _ \bot}} \right| = \left| \mathbf{g} \right|{\left [{1 - {{\left ({\cos i\cos \lambda + \sin i\sin \lambda \sin \omega} \right)}^2}} \right]^{1/2}}, $$\end{document} where i is the inclination angle of the orbital plane relative to the line of sight, is the line of nodes, and is the angle between the line of sight to the pulsar and g . The values of and |g| can be determined from models of the galactic potential (see, e.g., [83, 1]). The inclination angle i can be estimated if even crude estimates of the neutron star and companion masses are available, from statistics of ns masses (see, e.g.,) and/or a relation between the size of the orbit and the wd companion mass (see, e.g.,). However, the angle is also usually unknown and also must be assumed to be uniformly distributed between 0 and 2. damour and schfer use the psr b1953 + 29 system and integrate over the angles and to determine a 90% confidence upper limit of net<1.110 . Wex uses an ensemble of pulsars, calculating for each system the probability (fractional area in - space) that net is less than a given value, and then deriving a cumulative probability for each value of net . In this way he derives net<510 at 95% confidence however, this method may be vulnerable to selection effects; perhaps the observed systems are not representative of the true population . Given a value of net, an upper limit on || is obtained from equation (17). A monte carlo simulation of the expected pulsar population (assuming a range of masses based on evolutionary models and a random orientation of () then yields a certain fraction of the population that agree with this limit on || . This is slightly weaker than wex s previous limit but derived in a more rigorous manner . Prospects for improving the limits come from the discovery of new suitable pulsars, and from better limits on eccentricity from long - term timing of the current set of pulsars . In principle, measurement of the full orbital orientation (i.e., and i) for certain systems could reduce the dependence on statistical arguments thus, even though the required angles have in fact been measured for the millisecond pulsar j0437 - 4715, its comparatively large observed eccentricity of 210 and short orbital period mean it will not significantly affect the current limits . A non - zero 1 implies that the velocity w of a binary pulsar system (relative to a universal background reference frame given by the cosmic microwave background, or cmb) will affect its orbital evolution . In a manner similar to the effects of a non - zero net, the time evolution of the eccentricity will depend on both and a term that tries to force the semi - major axis of the orbit to align with the projection of the system velocity onto the orbital plane . The analysis proceeds in a similar fashion to that for net, except that the magnitude of ef is now written as [34, 18] (19)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left| {{{\rm{\mathbf{e}}}_{\rm{f}}}} \right| = \frac{1}{{12}}{\hat \alpha _ 1}\left| {\frac{{{m_1} - {m_2}}}{{{m_1} + {m_2}}}} \right|\frac{{\left| {{w _ \bot}} \right|}}{{{{\left [{g({m_1} + {m_2})(2\pi /{p_{\rm{b}}})} \right]}^{1/3}}}}, $$\end{document} where w is the projection of the system velocity onto the orbital plane . The angle, used in determining this projection in a manner similar to that of equation (18), is now the angle between the line of sight to the pulsar and the absolute velocity of the binary system . The figure of merit for systems used to test 1 is pb1/3/e . As for the net test, the systems must be old, so that c2/, and must be larger than the rate of galactic rotation . Examples of suitable systems are psr j2317 + 1439 [27, 18] with a last published value of e<1.210 in 1996, and psr j1012 + 5307, with e<810 . This latter system is especially valuable because observations of its white - dwarf component yield a radial velocity measurement, eliminating the need to find a lower limit on an unknown quantity . This is comparable in magnitude to the weak - field results from lunar laser ranging, but incorporates strong field effects as well . Tests of 3 can be derived from both binary and single pulsars, using slightly different techniques . A non - zero 3, which implies both a violation of local lorentz invariance and non - conservation of momentum, will cause a rotating body to experience a self - acceleration aself in a direction orthogonal to both its spin s and its absolute velocity w: (20)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{\mathbf{a}}}_{{\rm{self}}}} = - \frac{1}{3}{\hat \alpha _ 3}\frac{{{e^{{\rm{grav}}}}}}{{(m{c^2})}}\mathbf{w} \times {\mathbf{\omega} _ {\rm{s}}}. $$\end{document} thus, the self - acceleration depends strongly on the compactness of the object, as discussed in section 3 above . An ensemble of single (isolated) pulsars can be used to set a limit on 3 in the following manner . For any given pulsar, it is likely that some fraction of the self - acceleration will be directed along the line of sight to the earth . Such an acceleration will contribute to the observed period derivative via the doppler effect, by an amount (21)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\dot p}_{{{\hat \alpha} _ 3}}} = \frac{p}{c}\bf{\hat n} \cdot {\bf{a}_{{\rm{self}}}}, $$\end{document} where n is a unit vector in the direction from the pulsar to the earth . The analysis of will assumes random orientations of both the pulsar spin axes and velocities, and finds that, on average, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left| {{{\dot p}_{{{\hat \alpha} _ 3}}}} \right| \simeq 5 \times {10^ {- 5}}\left| {{{\hat \alpha} _ 3}} \right|$$\end{document}, independent of the pulse period . The sign of the 3 contribution to, however, may be positive or negative for any individual pulsar; thus, if there were a large contribution on average, one would expect to observe pulsars with both positive and negative total period derivatives . Young pulsars in the field of the galaxy (pulsars in globular clusters suffer from unknown accelerations from the cluster gravitational potential and do not count toward this analysis) all show positive period derivatives, typically around 10 s / s . Thus, the maximum possible contribution from 3 must also be considered to be of this size, and the limit is given by |3|<210 . Bell applies this test to a set of millisecond pulsars; these have much smaller period derivatives, on the order of 10s / s . Here, it is also necessary to account for the shklovskii effect in which a similar doppler - shift addition to the period derivative results from the transverse motion of the pulsar on the sky: (22)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot p_{{\rm{pm}}}} = p{\mu ^2}\frac{d}{c}, $$\end{document} where is the proper motion of the pulsar and d is the distance between the earth and the pulsar . The distance is usually poorly determined, with uncertainties of typically 30% resulting from models of the dispersive free electron density in the galaxy [132, 30]. Nevertheless, once this correction (which is always positive) is applied to the observed period derivatives for isolated millisecond pulsars, a limit on |3| on the order of 10 results [16, 19]. In the case of a binary - pulsar white - dwarf system, the combined accelerations affect both the velocity of the centre of mass of the system (an effect which may not be readily observable) and the relative motion of the two bodies . The relativemotion effects break down into a term involving the coupling of the spins to the absolute motion of the centre of mass, and a second term which couples the spins to the orbital velocities of the stars . The second term induces only a very small, unobservable correction to pb and . The first term, however, can lead to a significant test of 3 . Both the compactness and the spin of the pulsar will completely dominate those of the white dwarf, making the net acceleration of the two bodies effectively (23)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{\mathbf{a}}}_{{\rm{self}}}} = \frac{1}{6}{\hat \alpha _ 3}{c_{\rm{p}}}{\rm{\mathbf{w}}} \times {\mathbf{\omega} _ {{\rm{sp}}}}, $$\end{document} where cp and sp denote the compactness and spin angular frequency of the pulsar, respectively, and w is the velocity of the system . For evolutionary reasons (see, e.g.,), the spin axis of the pulsar may be assumed to be aligned with the orbital angular momentum of the system, hence the net effect of the acceleration will be to induce a polarization of the eccentricity vector within the orbital plane . The forced eccentricity term may be written as (24)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left| {{{\rm{\mathbf{e}}}_{\rm{f}}}} \right| = {\hat \alpha _ 3}\frac{{{c_{\rm{p}}}\left| \mathbf{w} \right|}}{{24\pi}} \frac{{p_{\rm{b}}^2}}{p}\frac{{{c^2}}}{{g({m_1} + {m_2})}}\sin \beta, $$\end{document} where is the (unknown) angle between w and sp, and p is, as usual, the spin period of the pulsar: p=2/sp . The figure of merit for systems used to test 3 is pb2/(ep). The additional requirements of c2/ and being larger than the rate of galactic rotation also hold . The 95% confidence limit derived by wex for an ensemble of binary pulsars is 3<1.510, much more stringent than for the single - pulsar case . It will contribute, along with 3, to an acceleration of the centre of mass of a binary system [149, 152] (25)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{\mathbf{a}}}_{{\rm{cm}}}} = \left ({{\alpha _ 3} + {\zeta _ 2}} \right)\frac{{\pi {m_1}{m_2}\left ({{m_1} - {m_2}} \right)}}{{{p_{\rm{b}}}{{\left [{\left ({{m_1} + {m_2}} \right)a\left ({1 - {e^2}} \right)} \right]}^{3/2}}}}e\ {{\rm{\mathbf{n}}}_{\rm{p}}}, $$\end{document} where np is a unit vector from the centre of mass to the periastron of m1 . This acceleration produces the same type of doppler - effect contribution to a binary pulsar s as described in section 3.2.2 . In a small - eccentricity system, this contribution would not be separable from the intrinsic to the pulsar . However, in a highly eccentric binary such as psr b1913 + 16, the longitude of periastron advances significantly for psr b1913 + 16, it has advanced nearly 120 since the pulsar s discovery . In this case, the projection of acm along the line of sight to the earth will change considerably over the long term, producing an effective second derivative of the pulse period . This p is given by [149, 152] (26)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ddot p = \frac{p}{2}\left ({{\alpha _ 3} + {\zeta _ 2}} \right){m_2}\sin i{\left ({\frac{{2\pi}} {{{p_{\rm{b}}}}}} \right)^2}\frac{{x\left ({1 - x} \right)}}{{{{\left ({1 + x} \right)}^2}}}\frac{{e\dot \omega \cos \omega}} {{{{\left ({1 - {e^2}} \right)}^{3/2}}}}, $$\end{document} where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x = {m_1}/{m_2}$$\end{document} is the mass ratio of the two stars and an average value of cos is chosen . As of 1992, the 95% confidence upper limit on p was 410 s [133, 149]. As 3 is orders of magnitude smaller than this (see section 3.2.2), this can be interpreted as a limit on 2 alone . Although psr b1913 + 16 is of course still observed, the infrequent campaign nature of the observations makes it difficult to set a much better limit on p (j. taylor, private communication, as cited in). The other well - studied double - neutron - star binary, psr b1534 + 12, yields a weaker test due to its orbital parameters and very similar component masses . A complication for this test is that an observed p could also be interpreted as timing noise (sometimes seen in recycled pulsars) or else a manifestation of profile changes due to geodetic precession [79, 75]. General relativity predicts gravitational radiation from the time - varying mass quadrupole of a binary pulsar system . The spectacular confirmation of this prediction will be discussed in section 4 below . Gr does not, however, predict dipolar gravitational radiation, though many theories that violate the sep do . In these theories, dipolar gravitational radiation results from the difference in gravitational binding energy of the two components of a binary . For this reason, neutron - star white - dwarf binaries are the ideal laboratories to test the strength of such dipolar emission . The expected rate of change of the period of a circular orbit due to dipolar emission can be written as [152, 35] (27)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot p_{{\rm{b \ dipole}}}} = - \frac{{4{\pi ^2}{g_*}}}{{{c^3}{p_{\rm{b}}}}}\frac{{{m_1}{m_2}}}{{{m_1} + {m_2}}}{\left ({{\alpha _ {{c_1}}} - {\alpha _ {{c_2}}}} \right)^2}, $$\end{document} where g*=g in gr, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\alpha _ {{c_i}}}$$\end{document} is the coupling strength of body (similar expressions can be derived when casting b dipole in terms of the parameters of specific tensor - scalar theories, such as brans - dicke theory . Equation (27), however, tests a more general class of theories .) Of course, the best test systems here are pulsar white - dwarf binaries with short orbital periods, such as psr b0655 + 64 and psr j1012 + 5307, where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\alpha _ {{c_1}}} \gg {\alpha _ {{c_2}}}$$\end{document} so that a strong limit can be set on the coupling of the pulsar itself . For psr b0655 + 64, damour and esposito - farse used the observed limit of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot p_{\rm{b}}} = \left ({1 \pm 4} \right) \times {10^ {- 13}} $$\end{document} to derive \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left ({{\alpha _ {{c_1}}} - {\alpha _ 0}} \right)^2} <3 \times {10^ {- 4}}\left ({1 - \sigma} \right)$$\end{document}, where 0 is a reference value of the coupling at infinity . More recently, arzoumanian has set a somewhat tighter 2- upper limit of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left| {{{\dot p}_{\rm{b}}}/{p_{\rm{b}}}} \right| <1 \times {10^ {- 10}}\ {\rm{y}}{{\rm{r}}^ {- 1}}$$\end{document}, or \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left| {{{\dot p}_{\rm{b}}}} \right| <2.7 \times {10^ {- 13}}$$\end{document}, which yields \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left ({{\alpha _ {{c_1}}} - {\alpha _ 0}} \right)^2} <2.7 \times {10^ {- 4}}$$\end{document}. For psr j1012 + 5307, a shklovskii correction (see and section 3.2.2) for the transverse motion of the system and a correction for the (small) predicted amount of quadrupolar radiation must first be subtracted from the observed upper limit to arrive at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot p_{\rm{b}}} = \left ({- 0.6 \pm 1.1} \right) \times {10^ {- 13}}$$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left ({{\alpha _ {{c_1}}} - {\alpha _ 0}} \right)^2} <4 \times {10^ {- 4}}$$\end{document} at 95% confidence . It should be noted that both these limits depend on estimates of the masses of the two stars and do not address the (unknown) equation of state of the neutron stars . Limits may also be derived from double - neutron - star systems (see, e.g., [148, 151]), although here the difference in the coupling constants is small and so the expected amount of dipolar radiation is also small compared to the quadrupole emission . However, certain alternative gravitational theories in which the quadrupolar radiation predicts a positive orbital period derivative independently of the strength of the dipolar term (see, e.g., [117, 99, 85]) are ruled out by the observed decreasing orbital period in these systems . Other pulsar white - dwarf systems with short orbital periods are mostly found in globular clusters, where the cluster potential will also contribute to the observed b, or in interacting systems, where tidal effects or magnetic braking may affect the orbital evolution (see, e.g., [4, 50, 100]). However, one system that offers interesting prospects is the recently discovered psr j1141 - 6545, which is a young pulsar with white - dwarf companion in a 4.75-hour orbit . In this case, though, the pulsar was formed after the white dwarf, instead of being recycled by the white - dwarf progenitor, and so the orbit is still highly eccentric . This system is therefore expected both to emit sizable amounts of quadrupolar radiation b could be measurable as soon as 2004 and to be a good test candidate for dipolar emission . Theories that violate the sep by allowing for preferred locations (in time as well as space) may permit newton s constant g to vary . In general, variations in g are expected to occur on the timescale of the age of the universe, such that \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot g / g \sim {h_0} \sim 0.7 \times {10^ {- 10}}\ {\rm{y}}{{\rm{r}}^ {- 1}}$$\end{document}, where h0 is the hubble constant . Three different pulsar - derived tests can be applied to these predictions, as a sep - violating time - variable g would be expected to alter the properties of neutron stars and white dwarfs, and to affect binary orbits . By affecting the gravitational binding of neutron stars, a non - zero would reasonably be expected to alter the moment of inertia of the star and hence change its spin on the same timescale . Goldman writes (28)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left ({\frac{{\dot p}}{p}} \right)_{\dot g}} = {\left ({\frac{{\partial \ln i}}{{\partial \ln g}}} \right)_n}\frac{{\dot g}}{g}, $$\end{document} where i is the moment of inertia of the neutron star, about 10 g cm, and n is the (conserved) total number of baryons in the star . By assuming that this represents the only contribution to the observed of psr b0655 + 64, in a manner reminiscent of the test of 3 described above, goldman then derives an upper limit of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left| {\dot g / g} \right| \le \left ({2.2 - 5.5} \right) \times {10^ {- 11}}\;{\rm{y}}{{\rm{r}}^ {- 1}}$$\end{document}, depending on the stiffness of the neutron star equation of state . Arzoumanian applies similar reasoning to psr j2019 + 2425, which has a characteristic age of 27 gyr once the shklovskii correction is applied . Again, depending on the equation of state, the upper limits from this pulsar are \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left| {\dot g / g} \right| \le \left ({1.4 - 3.2} \right) \times {10^ {- 11}}\;{\rm{y}}{{\rm{r}}^ {- 1}}$$\end{document}. These values are similar to those obtained by solar - system experiments such as laser ranging to the viking lander on mars (see, e.g., [115, 59]). Shklovskii and galactic - acceleration corrections are taken into account, have similarly large characteristic ages (see, e.g., [28, 137]). The effects on the orbital period of a binary system of a varying g were first considered by damour, gibbons, and taylor, who expected (29)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left ({\frac{{{{\dot p}_{\rm{b}}}}}{{{p_{\rm{b}}}}}} \right)_{\dot g}} = - 2\frac{{\dot g}}{g}. $$\end{document} applying this equation to the limit on the deviation from gr of the b for psr 1913 + 16, they found a value of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot g / g = (1.0 \pm 2.3) \times {10^ {- 11}}\;{\rm{y}}{{\rm{r}}^ {- 1}}$$\end{document}. Nordtvedt took into account the effects of on neutron - star structure, realizing that the total mass and angular momentum of the binary system would also change . The corrected expression for b incorporates the compactness parameter ci and is (30)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left ({\frac{{{{\dot p}_{\rm{b}}}}}{{{p_{\rm{b}}}}}} \right)_{\dot g}} = - \left [{2 - \left ({\frac{{{m_1}{c_1} + {m_2}{c_2}}}{{{m_1} + {m_2}}}} \right) - \frac{3}{2}\left ({\frac{{{m_1}{c_2} + {m_2}{c_1}}}{{{m_1} + {m_2}}}} \right)} \right]\frac{{\dot g}}{g}. $$\end{document} (note that there is a difference of a factor of -2 in nordtvedt s definition of ci versus the damour definition used throughout this article .) Nordtvedt s corrected limit for psr b1913 + 16 is therefore slightly weaker . A better limit actually comes from the neutron - star white - dwarf system psr b1855 + 09, with a measured limit on b of (0:61.2)10 . Using equation (29), this leads to a bound of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot g / g = (- 9 \pm 18) \times {10^ {- 12}}\;{\rm{y}}{{\rm{r}}^ {- 1}}$$\end{document}, which arzoumanian corrects using equation (30) and an estimate of ns compactness to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot g / g = (- 1.3 \pm 2.7) \times {10^ {- 11}}\;{\rm{y}}{{\rm{r}}^ {- 1}}$$\end{document}. Prospects for improvement come directly from improvements to the limit on b . Even though psr j1012 + 5307 has a tighter limit on b, its shorter orbital period means that the limit it sets is a factor of 2 weaker than obtained with psr b1855 + 09 . The chandrasekhar mass, mch, is the maximum mass possible for a white dwarf supported against gravitational collapse by electron degeneracy pressure . Its value about 1.4m comes directly from newton s constant: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${m_{{\rm{ch}}}} \sim {\left ({\hbar c / g} \right)^{3/2}}/m_{\rm{n}}^2$$\end{document}, where is planck s constant and mn is the neutron mass . All measured and constrained pulsar masses are consistent with a narrow distribution centred very close to mch: 1.350.04m . Thus, it is reasonable to assume that mch sets the typical neutron star mass, and to check for any changes in the average neutron star mass over the lifetime of the universe . Thorsett compiles a list of measured and average masses from 5 double - neutron - star binaries with ages ranging from 0.1 gyr to 12 or 13 gyr in the case of the globular - cluster binary b2127 + 11c . Using a bayesian analysis, he finds a limit of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot g / g = (- 0.6 \pm 4.2) \times {10^ {- 12}}\;{\rm{y}}{{\rm{r}}^ {- 1}}$$\end{document} at the 95% confidence level, the strongest limit on record . The solid lines show predicted changes in the average neutron star mass corresponding to hypothetical variations in g, where -12=10 implies \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot g / g = 10 \times {10^ {- 12}}\;{\rm{y}}{{\rm{r}}^ {- 1}}$$\end{document}. (from, used by permission .) Measured neutron star masses as a function of age . The solid lines show predicted changes in the average neutron star mass corresponding to hypothetical variations in g, where -12=10 implies \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot g / g = 10 \times {10^ {- 12}}\;{\rm{y}}{{\rm{r}}^ {- 1}}$$\end{document}. (from, used by permission .) While some cancellation of observed mass changes might be expected from the changes in neutron - star binding energy (cf . Section 3.4.2 above), these will be smaller than the mch changes by a factor of order the compactness and can be neglected . Also, the claimed variations of the fine structure constant of order \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta \alpha /\alpha \simeq - 0.72 \pm 0.18 \times {10^ {- 5}}$$\end{document} over the redshift range 0.5<z<3.5 could introduce a maximum derivative of 1/(c)d(c)/dt of about 510 yr and hence cannot influencee the chandrasekhar mass at the same level as the hypothesized changes in g. one of the five systems used by thorsett has since been shown to have a white - dwarf companion, but as this is one of the youngest systems, this will not change the results appreciably . The recently discovered psr j1811 - 1736, a double - neutron - star binary, has a characteristic age of only c1 gyr and, therefore, will also not significantly strengthen the limit . Ongoing searches for pulsars in globular clusters stand the best chance of discovering old double - neutron - star binaries for which the component masses can eventually be measured . The best - known uses of pulsars for testing the predictions of gravitational theories are those in which the predicted strong - field effects are compared directly against observations . As essentially point - like objects in strong gravitational fields, neutron stars in binary systems provide extraordinarily clean tests of these predictions . This section will cover the relation between the post - keplerian timing parameters and strong - field effects, and then discuss the three binary systems that yield complementary high - precision tests . In any given theory of gravity, the post - keplerian (pk) parameters can be written as functions of the pulsar and companion star masses and the keplerian parameters . As the two stellar masses are the only unknowns in the description of the orbit, it follows that measurement of any two pk parameters will yield the two masses, and that measurement of three or more pk parameters will over - determine the problem and allow for self - consistency checks . It is this test for internal consistency among the pk parameters that forms the basis of the classic tests of strong - field gravity . It should be noted that the basic keplerian orbital parameters are well - measured and can effectively be treated as constants here . In general relativity, the equations describing the pk parameters in terms of the stellar masses are (see [33, 133, 43]): (31)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot \omega = 3{\left ({\frac{{{p_{\rm{b}}}}}{{2\pi}}} \right)^ {- 5/3}}{\left ({{t _ \odot} m} \right)^{2/3}}{\left ({1 - {e^2}} \right)^ {- 1}}, $$\end{document} (32)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\gamma = e{\left ({\frac{{{p_{\rm{b}}}}}{{2\pi}}} \right)^{1/3}}t _ \odot ^{2/3}{m^ {- 4/3}}{m_2}\left ({{m_1} + 2{m_2}} \right), $$\end{document} (33)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\dot p}_{\rm{b}}} = - \frac{{192\pi}} {5}{\left ({\frac{{{p_{\rm{b}}}}}{{2\pi}}} \right)^ {- 5/3}}\left ({1 + \frac{{73}}{{24}}{e^2} + \frac{{37}}{{96}}{e^4}} \right){\left ({1 - {e^2}} \right)^ {- 7/2}}t _ \odot ^{5/3}{m_1}{m_2}{m^ {- 1/3}}, $$\end{document} (34)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$r = {t _ \odot} {m_2}, $$\end{document} (35)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$s = x{\left ({\frac{{{p_{\rm{b}}}}}{{2\pi}}} \right)^ {- 2/3}}t _ \odot ^ {- 1/3}{m^{2/3}}m_2^ {- 1}. $$\end{document} where ssin i, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m = {m_1} + {m_2}$$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t _ \odot} \equiv g{m _ \odot} /{c^3} = 4.925490947\;\mu {\rm{s}}$$\end{document}. Other theories of gravity, such as those with one or more scalar parameters in addition to a tensor component, will have somewhat different mass dependencies for these parameters . The prototypical double - neutron - star binary, psr b1913 + 16, was discovered at the arecibo observatory in 1974 . Over nearly 30 years of timing, its system parameters have shown a remarkable agreement with the predictions of gr, and in 1993 hulse and taylor received the nobel prize in physics for its discovery [61, 131]. In the highly eccentric 7.75-hour orbit, the two neutron stars are separated by only 3.3 light - seconds and have velocities up to 400 km / s . This provides an ideal laboratory for investigating strong - field gravity . For psr b1913 + 16, three pk parameters are well measured: the combined gravitational red - shift and time dilation parameter the advance of periastron, and the derivative of the orbital period, b . The orbital parameters for this pulsar, measured in the theory - independent dd system, are listed in table 2 [133, 144]. Table 2 orbital parameters for psr b1913 + 16 in the dd framework, taken from [144]. Parametervalueorbital period pb (d)0.322997462727(5)projected semi - major axis x (s)2.341774(1)eccentricity e0.6171338(4)longitude of periastron (deg)226.57518(4)epoch of periastron t0 (mjd)46443.99588317(3)advance of periastron (deg yr)4.226607(7)gravitational redshift (ms)4.294(1)orbital period derivative (b) (10)-2.4211(14) orbital parameters for psr b1913 + 16 in the dd framework, taken from [144]. A second useful timing formalism is ddgr [33, 43], which assumes gr to be the true theory of gravity and fits for the total and companion masses in the system, using these quantities to calculate theoretical thus, one can make a direct comparison between the measured dd pk parameters and the values predicted by ddgr using the same data set; the parameters for psr b1913 + 16 agree with their predicted values to better than 0.5% . The classic demonstration of this agreement is shown in figure 6, in which the observed accumulated shift of periastron is compared to the predicted amount . Figure 6 the parabola indicates the predicted accumulated shift in the time of periastron for psr b1913 + 16, caused by the decay of the orbit . The parabola indicates the predicted accumulated shift in the time of periastron for psr b1913 + 16, caused by the decay of the orbit . (from [144], courtesy joel weisberg .) In order to check the self - consistency of the overdetermined set of equations relating the pk parameters to the neutron star masses, it is helpful to plot the allowed m1m2 curves for each parameter and to verify that they intersect at a common point . Figure 7 displays the and curves for psr b1913 + 16; it is clear that the curves do intersect, at the point derived from the ddgr mass predictions . Figure 7mass - mass diagram for the psr b1913 + 16 system, using the and parameters listed in table 2 . The lines intersect at the point given by the masses derived under the ddgr formalism . Mass - mass diagram for the psr b1913 + 16 system, using the and parameters listed in table 2 . The lines intersect at the point given by the masses derived under the ddgr formalism . Clearly, any theory of gravity that does not pass such a self - consistency test can be ruled out . However, it is possible to construct alternate theories of gravity that, while producing very different curves in the m1m2 plane, do pass the psr b1913 + 16 test and possibly weak - field tests as well . Such theories are best dealt with by combining data from multiple pulsars as well as solar - system experiments (see section 4.4). The first is that the unknown radial velocity of the binary system relative to the ssb will necessarily induce a doppler shift in the orbital and neutron - star spin periods . This will change the observed stellar masses by a small fraction but will cancel out of the calculations of the pk parameters . The second is that the measured value of the orbital period derivative b is contaminated by several external contributions . Damour and taylor consider the full range of possible contributions to b and calculate values for the two most important: the acceleration of the pulsar binary centre - of - mass relative to the ssb in the galactic potential, and the shklovskii v / r effect due to the transverse proper motion of the pulsar (cf . Section 3.2.2). Both of these contributions have been subtracted from the measured value of b before it is compared with the gr prediction . It is our current imperfect knowledge of the galactic potential and the resulting models of galactic acceleration (see, e.g., [83, 1]) which now limits the precision of the test of gr resulting from this system . A second double - neutron - star binary, psr b1534 + 12, was discovered during a drift - scan survey at arecibo observatory in 1990 . This system is quite similar to psr b1913 + 16: it also has a short (10.1-hour) orbit, though it is slightly wider and less eccentric . Psr b1534 + 12 does possess some notable advantages relative to its more famous cousin: it is closer to the earth and therefore brighter; its pulse period is shorter and its profile narrower, permitting better timing precision; and, most importantly, it is inclined nearly edge - on to the line of sight from the earth, allowing the measurement of shapiro delay as well as the 3 pk parameters measurable for psr b1913 + 16 . Table 3 orbital parameters for psr b1534 + 12 in the dd framework, taken from [125]. Parametervalueorbital period pb (d)0.420737299122(10)projected semi - major axis x (s)3.729464(2)eccentricity e0.2736775(3)longitude of periastron (deg)274.57679(5)epoch of periastron t0 (mjd)50260.92493075(4)advance of periastron (deg yr)1.755789(9)gravitational redshift (ms)2.070(2)orbital period derivative (b) (10)-0.137(3)shape of shapiro delay s0.975(7)range of shapiro delay r (s)6.7(1.0) orbital parameters for psr b1534 + 12 in the dd framework, taken from [125]. As for psr b1913 + 16, a graphical version of the internal consistency test is a helpful way to understand the agreement of the measured pk parameters with the predictions of gr . It is obvious that the allowed - mass region derived from the observed value of b does not in fact intersect those from the other pk parameters . This is a consequence of the proximity of the binary to the earth, which makes the shklovskii contribution to the observed b much larger than for psr b1913 + 16 . The magnitude of this contribution depends directly on the poorly known distance to the pulsar . At present, the best independent measurement of the distance comes from the pulsar s dispersion measure and a model of the free electron content of the galaxy, which together yield a value of 0.70.2 kpc . If gr is the correct theory of gravity, then the correction derived from this distance is inadequate, and the true distance can be found by inverting the problem [17, 121]. Ne2001 galactic model incorporates the gr - derived distance to this pulsar and hence cannot be used in this case .) It is possible that, in the long term, a timing or interferometric parallax may be found for this pulsar; this would alleviate the b discrepancy . The gr - derived distance is in itself interesting, as it has led to revisions of the predicted merger rate of double - neutron - star systems visible to gravitational - wave detectors such as ligo (see, e.g., [121, 7, 71]) although recent calculations of merger rates determine the most likely merger rates for particular population models and hence are less vulnerable to distance uncertainties in any one system . Figure 8mass - mass diagram for the psr b1534 + 12 system . Labeled curves illustrate 68% confidence ranges of the dd parameters listed in table 3 . The filled circle indicates the component masses according to the ddgr solution . The kinematic correction for assumed distance \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d = {\rm{0}}{\rm{.7}} \pm {\rm{0}}{\rm{.2}}\;{\rm{kpc}}$$\end{document} has been subtracted from the observed value of b; the uncertainty on this kinematic correction dominates the uncertainty of this curve . A slightly larger distance removes the small apparent discrepancy between the observed and predicted values of this parameter . (after .) Mass - mass diagram for the psr b1534 + 12 system . Labeled curves illustrate 68% confidence ranges of the dd parameters listed in table 3 . The filled circle indicates the component masses according to the ddgr solution . The kinematic correction for assumed distance \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d = {\rm{0}}{\rm{.7}} \pm {\rm{0}}{\rm{.2}}\;{\rm{kpc}}$$\end{document} has been subtracted from the observed value of b; the uncertainty on this kinematic correction dominates the uncertainty of this curve . A slightly larger distance removes the small apparent discrepancy between the observed and predicted values of this parameter . (after .) Despite the problematic correction to b, the other pk parameters for psr b1534 + 12 are in excellent agreement with each other and with the values predicted from the ddgr - derived masses . An important point is that the three parameters,, and s (shape of shapiro delay) together yield a test of gr to better than 1%, and that this particular test incorporates only quasi - static strong - field effects . This provides a valuable complement to the mixed quasi - static and radiative test derived from psr b1913 + 16, as it separates the two sectors of the theory . There are three other confirmed double - neutron - star binaries at the time of writing . Psr b2127 + 11c [2, 3] is in the globular cluster m15 . While its orbital period derivative has been measured, this parameter is affected by acceleration in the cluster potential, and the system has not yet proved very useful for tests of gr, though long - term observations may demonstrate otherwise . The two binaries psrs j1518 + 4904 and j1811 - 1736 have such wide orbits that, although is measured in each case, prospects for measuring further pk parameters are dim . In several circular pulsar white - dwarf binaries, one or two pk parameters have been measured typically or the shapiro delay parameters but these do not over - constrain the unknown masses . The existing system that provides the most optimistic outlook is again the pulsar white - dwarf binary psr j1141 - 6545, for which multiple pk parameters should be measurable within a few years although one may need to consider the possibility of classical contributions to the measured from a mass quadrupole of the companion . Because of their different orbital parameters and inclinations, the double - neutron - star systems psrs b1913 + 16 and b1534 + 12 provide somewhat different constraints on alternative theories of gravity . Taken together with some of the limits on sep violation discussed above, and with solar - system experiments, they can be used to disallow certain regions of the parameter space of these alternate theories . This approach was pioneered by taylor et al ., who combined pk - parameter information from psrs b1913 + 16 and b1534 + 12 and the damour and schfer result on sep violation by psr b1855 + 09 to set limits on the parameters and of a class of tensor - biscalar theories discussed in (figure 9). In this class of theories, gravity is mediated by two scalar fields as well as the standard tensor, but the theories can satisfy the weak - field solar - system tests . Strong - field deviations from gr would be expected for non - zero values of and, but the theories approach the limit of gr as the parameters and approach zero . Figure 9portions of the tensor - biscalar - plane permitted by timing observations of psrs b1913 + 16, b1534 + 12, and b1855 + 09 up to 1992 . Values lying above the curve labeled a are incompatible with the measured and parameters for psr b1913 + 16 . The curves labeled b and d give the allowed ranges of and for psrs b1913 + 16 and b1534 + 12, respectively, fitting for the two neutron - star masses as well as and, using data available up to 1992 . The vertical lines labeled c represent limits on from the sep - violation test using psr b1855 + 09 . (reprinted by permission from nature, 1992, macmillan publishers ltd .) Portions of the tensor - biscalar - plane permitted by timing observations of psrs b1913 + 16, b1534 + 12, and b1855 + 09 up to 1992 . Values lying above the curve labeled a are incompatible with the measured and parameters for psr b1913 + 16 . The curves labeled b and d give the allowed ranges of and for psrs b1913 + 16 and b1534 + 12, respectively, fitting for the two neutron - star masses as well as and, using data available up to 1992 . The vertical lines labeled c represent limits on from the sep - violation test using psr b1855 + 09 . (reprinted by permission from nature, 1992, macmillan publishers ltd .) A different class of theories, allowing a non - linear coupling between matter and a scalar field, was later studied by damour and esposito - farse [35, 37]. The function coupling the scalar field to matter is given by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$a\left (\phi \right) = \exp \left ({{\tfrac{1}{2}}{\beta _ 0}{\phi ^2}} \right)$$\end{document}, and the theories are described by the parameters 0 and 0=00, where 0 is the value that approaches at spatial infinity (cf . These theories allow significant strong - field effects when 0 is negative, even if the weak - field limit is small . They are best tested by combining results from psrs b1913 + 16, b1534 + 12 (which contributes little to this test), b0655 + 64 (limits on dipolar gravitational radiation), and solar - system experiments (lunar laser ranging, shapiro delay measured by viking, and the perihelion advance of mercury). Currently, for most neutron - star equations of state, the solar - system tests set a limit on 0 (02<10) that is a few times more stringent than those set by psrs b1913 + 16 and b0655 + 64, although the pulsar observations do eliminate large negative values of 0 . With the limits from the pulsar observations improving only very slowly with time, it appears that solar - system tests will continue to set the strongest limits on 0 in this class of theories, unless a pulsar black - hole system is discovered . If such a system were found with a 10-m black hole and an orbital period similar to that of psr b1913 + 16 (8 hours), the limit on 0 derived from this system would be about 50 times tighter than that set by current solar - system tests, and 10 times better than is likely to be achieved by the gravity probe b experiment . Figure 10the parameter space in the non - linear 0, 0 gravitational theory, for neutron stars described by a polytrope equation of state . The regions below the various curves are allowed by various pulsar timing limits, by solar - system tests (1pn), and by projected ligo / virgo observations of ns - ns and ns - bh inspiral events . (from; used by permission .) The parameter space in the non - linear 0, 0 gravitational theory, for neutron stars described by a polytrope equation of state . The regions below the various curves are allowed by various pulsar timing limits, by solar - system tests (1pn), and by projected ligo / virgo observations of ns - ns and ns - bh inspiral events . The shaded region is allowed by all tests . The plane and limits are symmetric about 0=0 . A different and complementary test of gr has recently been permitted by the millisecond pulsar psr j0437 - 4715 . At a distance of only 140 pc, it is the closest millisecond pulsar to the earth, and is also extremely bright, allowing root - mean - square timing residuals of 35 ns with the 64-m parkes telescope, comparable to or better than the best millisecond pulsars observed with current instruments at the 300-m arecibo telescope . The proximity of this system means that the orbital motion of the earth changes the apparent inclination angle i of the pulsar orbit on the sky, an effect known as the annual - orbital parallax . This results in a periodic change of the projected semi - major axis x of the pulsar s orbit, written as (36)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x\left (t \right) = {x_0}\left [{1 + \frac{{\cot i}}{d}{{\rm{\mathbf{r}}} _ \oplus} \left (t \right) \cdot \mathbf{\omega}'} \right], $$\end{document} where r(t) is the time - dependent vector from the centre of the earth to the ssb, and is a vector on the plane of the sky perpendicular to the line of nodes . A second contribution to the observed i and hence x comes from the pulsar system s transverse motion in the plane of the sky: (37)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\dot x}_{{\rm{pm}}}} = - x\cot i\mu \cdot \mathbf{\omega}', $$\end{document} where is the proper motion vector . By including both these effects in the model of the pulse arrival times, both the inclination angle i and the longitude of the ascending node can be determined . As sin i is equivalent to the shape of the shapiro delay in gr (pk parameter s), the effect of the shapiro delay on the timing residuals can then easily be computed for a range of possible companion masses (equivalent to the pk parameter r in gr). The variations in the timing residuals are well explained by a companion mass of 0.2360.017m (figure 11). The measured value of, together with i, also provide an estimate of the companion mass, 0.230.14m, which is consistent with the shapiro - delay value . Figure 11solid line: predicted value of the shapiro delay in psr j0437 - 4715 as a function of orbital phase, based on the observed inclination angle of 429. for such low - eccentricity binaries, much of the shapiro delay can be absorbed into the orbital roemer delay; what remains is the pb/3 periodicity shown . The points represent the timing residuals for the pulsar, binned in orbital phase, and in clear agreement with the shape predicted from the inclination angle . Solid line: predicted value of the shapiro delay in psr j0437 - 4715 as a function of orbital phase, based on the observed inclination angle of 429. for such low - eccentricity binaries, much of the shapiro delay can be absorbed into the orbital roemer delay; what remains is the pb/3 periodicity shown . The points represent the timing residuals for the pulsar, binned in orbital phase, and in clear agreement with the shape predicted from the inclination angle . (reprinted by permission from nature, 2001, macmillan publishers ltd .) While this result does not include a true self - consistency check in the manner of the double - neutron - star tests, it is nevertheless important, as it represents the only case in which an independent, purely geometric determination of the inclination angle of a binary orbit predicts the shape of the shapiro delay . It can thus be considered to provide an independent test of the predictions of gr . A complete discussion of gr effects in pulsar observations must mention geodetic precession, though these results are somewhat qualitative and do not (yet) provide a model - free test of gr . In standard evolutionary scenarios for double - neutron - star binaries (see, e.g., [21, 109]), both stellar spins are expected to be aligned with the orbital angular momentum just before the second supernova explosion . After this event, however, the observed pulsar s spin is likely to be misaligned with the orbital angular momentum, by an angle of the order of 20 . A similar misalignment may be expected when the observed pulsar is the second - formed degenerate object, as in psr j1141 - 6545 . As a result, both vectors will precess about the total angular momentum of the system (in practice the total angular momentum is completely dominated by the orbital angular momentum). The evolution of the pulsar spin axis s1 can be written as [40, 14] (38)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{d{{\rm{\mathbf{s}}}_1}}}{{dt}} = \mathbf{\omega} _ 1^{{\rm{spin}}} \times {{\rm{\mathbf{s}}}_1}, $$\end{document} where the vector 1spin is aligned with the orbital angular momentum . Its magnitude is given by (39)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\omega _ 1^{{\rm{spin}}} = \frac{1}{2}{\left ({\frac{{{p_{\rm{b}}}}}{{2\pi}}} \right)^ {- 5/3}}\frac{{{m_2}(4{m_1} + 3{m_2})}}{{(1 - {e^2}){{({m_1} + {m_2})}^{4/3}}}}t _ \odot ^{2/3}, $$\end{document} where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t _ \odot} \equiv g{m _ \odot} /{c^3} = 4.905490947\;\mu {\rm{s}}$$\end{document}, as in section 4.1 . This predicted rate of precession is small; the three systems with the highest 1spin values are: psr j1141 - 6545 at 1.35 yr, psr b1913 + 16 at 1.21 yr, psr b1534 + 12 at 0.52 yr . The primary manifestation of this precession is expected to be a slow change in the shape of the pulse profile, as different regions of the pulse emission beam move into the observable region . Psr j1141 - 6545 at 1.35 yr, psr b1913 + 16 at 1.21 yr, psr b1534 + 12 at 0.52 yr . Evidence for long - term profile shape changes is in fact seen in psrs b1913 + 16 and b1534 + 12 . For psr b1913 + 16, profile shape changes were first reported in the 1980s, with a clear change in the relative heights of the two profile peaks over several years (figure 12). Interestingly, although a simple picture of a cone - shaped beam might lead to an expectation of a change in the separation of the peaks with time, no evidence for this was seen until the late 1990s, at the effelsberg 100-m telescope, by which point the two peaks had begun to move closer together at a rather fast rate . Kramer used this changing peak separation, along with the predicted precession rate and a simple conal model of the pulse beam, to estimate a spin - orbit misalignment angle of about 22 and to predict that the pulsar will disappear from view in about 2025 (see figure 13), in good agreement with an earlier prediction by istomin made before the peak separation began to change . Recent results from arecibo confirm the gist of kramer s results, with a misalignment angle of about 21. both sets of authors find there are four degenerate solutions that can fit the profile separation data; two can be discarded as they predict an unreasonably large misalignment angle of 180-22=158, and a third is eliminated because it predicts the wrong direction of the position angle swing under the rotating vector model . The main area of dispute is the actual shape of the emission region; while weisberg and taylor find an hourglass - shaped beam (see figure 14), kramer maintains that a nearly circular cone plus an offset core is adequate (see figure 15). In any event, it is clear that the interpretation of the profile changes requires some kind of model of the beam shape . Kramer [81, 82] lets the rate of precession vary as another free parameter in the pulse - shape fit, and finds a value of 1.20.2. this is consistent with the gr prediction but still depends on the beam - shape model and is therefore not a true test of the precession rate . Figure 12 changes in the observed pulse profile of psr b1913 + 16 throughout the 1980s, due to a changing line - of - sight cut through the emission region of the pulsar . Figure 13 top: change in peak separation of the relativistic double - neutron - star binary psr b1913 + 16, as observed with the arecibo (solid points, [141]) and effelsberg (open circles, [80]) telescopes . Figure 14 hourglass - shaped beam for psr b1913 + 16 derived from the symmetric - component analysis of [143]. Figure 15 alternate proposed beam shape for psr b1913 + 16, consisting of a symmetric cone plus an offset core . The red lines indicate an example cut through the emission region, as well as the predicted pulse peak ratio and separation as functions of time . (after [81], courtesy michael kramer .) Changes in the observed pulse profile of psr b1913 + 16 throughout the 1980s, due to a changing line - of - sight cut through the emission region of the pulsar . Top: change in peak separation of the relativistic double - neutron - star binary psr b1913 + 16, as observed with the arecibo (solid points, [141]) and effelsberg (open circles, [80]) telescopes . Hourglass - shaped beam for psr b1913 + 16 derived from the symmetric - component analysis of [143]. (taken from [143]; used by permission .) Alternate proposed beam shape for psr b1913 + 16, consisting of a symmetric cone plus an offset core . The red lines indicate an example cut through the emission region, as well as the predicted pulse peak ratio and separation as functions of time . Psr b1534 + 12, despite the disadvantages of a more recent discovery and a much longer precession period, also provides clear evidence of long - term profile shape changes . These were first noticed at 1400 mhz by arzoumanian [5, 8] and have become more obvious at this frequency and at 430 mhz in the post - upgrade period at arecibo . The principal effect is a change in the low - level emission near to the main pulse (figure 16), though related changes in polarization are now also seen . As this pulsar shows polarized emission through most of its pulse period, it should be possible to form a better picture of the overall geometry than for psr b1913 + 16; this may make it easier to derive an accurate model of the pulse beam shape . Figure 16 evolution of the low - level emission surrounding the main pulse of psr b1534 + 12, over a period of nearly 10 years, as measured with the arecibo telescope [96]. Evolution of the low - level emission surrounding the main pulse of psr b1534 + 12, over a period of nearly 10 years, as measured with the arecibo telescope [96]. (stairs et al ., unpublished .) As for other tests of gr, the pulsar white - dwarf binary psr j1141 - 6545 promises interesting results . As noted by the discoverers, the region of sky containing this pulsar had been observed at the same frequency in an earlier survey, but the pulsar was not seen, even though it is now very strong . It is possible that interference corrupted that original survey pointing, or that a software error prevented its detection, but it is also plausible that the observed pulsar beam is evolving so rapidly that the visible beam precessed into view during the 1990s . The tremendous success to date of pulsars in testing different aspects of gravitational theory leads naturally to the question of what can be expected in the future . Improvements to the equivalence - principle violation tests will come from both refining the timing parameters of known pulsars (in particular, limits on eccentricities and orbital period derivatives) and the discovery of further pulsar white - dwarf systems . Potentially coalescing pulsar white - dwarf binaries, such as psrs j1141 - 6545, j0751 + 1807, and 1757 - 5322, bear watching from the point of view of limits on dipolar gravitational radiation . Another worthy, though difficult, goal is to attempt to derive the full orbital geometry for ultra - low - eccentricity systems, as has been done for psr j0437 - 4715; this would quickly lead to significant improvements in the eccentricity - dependent tests . The orbital - period - derivative measurements of double - neutron - star binaries are already limited more by systematics (galactic acceleration models for psr b1913 + 16, and poorly known distance for psr b1534 + 12) than by pulsar timing precision . However, with improved galactic modeling and a realistic expectation of an interferometric (vlbi) parallax for psr b1534 + 12, there is still hope for testing more carefully the prediction of quadrupolar gravitational radiation from these systems . The other timing parameters, equally important for tests of the quasi - static regime, can be expected to improve with time and better instrumentation, such as the wider - bandwidth coherent dedispersion systems now being installed at many observatories (see, e.g., [68, 129]). Especially exciting would be a measurement of the elusive shapiro delay in psr b1913 + 16; the longitude of periastron is now precessing into an angular range where it may facilitate such a measurement . In the last few years, surveys of the galactic plane and anking regions, using the 64-m parkes telescope in australia, have discovered several hundred new pulsars (see, e.g., [91, 48]), including several new circular - orbit pulsar white - dwarf systems [46, 47, 26] and the eccentric pulsar a complete reprocessing of the galactic plane survey with improved interference filtering is in progress; thus there is still hope that a truly new system such as a pulsar black - hole binary may emerge from this large survey . Several ongoing smaller surveys of small regions and globular clusters (see, e.g., [25, 113]) are also finding a number of new and exotic binaries, some of which may eventually turn out to be useful for tests of gr . The possible recent appearance of psr j1141 - 6545 and the predicted disappearance of psr b1913 + 16 due to geodetic precession make it worthwhile to periodically revisit previously surveyed parts of the sky in order to check for newly - visible exotic binaries . Over the next several years, large - scale surveys are planned at arecibo and the new 100-m green bank telescope, offering the promise of over 1000 new pulsars including interesting binary systems . The sensitivity of these surveys will of course be dwarfed by the potential of the proposed square kilometre array radio telescope, which will be sensitive to pulsars clear through our galaxy and into neighbouring galaxies such as m31 . The next 10 or 20 years promise to be exciting times for pulsar searchers and for those looking to set ever - more - stringent limits on deviations from general relativity.
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Furthermore, the most frequent indication for surgery is spinal stenosis.1 after decompression (laminectomy or foraminotomy), the structural integrity of the lumbar spine can be weakened, and requires additional support . This is achieved by vertebral fusion, which is the current standard for outcome and the most commonly used procedure . Interbody arthrodesis can be performed through anterior, lateral, posterior, or transforaminal approaches . The vertebral disks are replaced with bone to promote arthrodesis, the sagittal height is restored with cages, and supplemental stabilization is completed by internal fixation using transpedicular instrumentation . The result is a bloc of vertebral segments that are permanently fixed and rigid . This resolves the immediate situation, but in the long term the proximal segment is overloaded, which might aggravate the degeneration, lead to hypermobility and osteophyte formation, become symptomatic, and require further surgery.2 this phenomenon is inherent to the principle of fusion and cannot be avoided, with an annual incidence of 2.5% and an estimated 10-year prevalence of 22.2%.3 the increased stresses on the adjacent disk and facets might be dependent on the rigidity of the stabilization.4 in such circumstances, the need for an implant that will allow for dynamic stabilization is paramount . These were the premises that led to the introduction of the total facet arthroplasty system, aimed at restoring normal segmental kinematics . Dynamic stabilization was not a new idea,5,6 but facet arthroplasty was designed to mimic natural movement of the vertebral segments without overstraining the adjacent vertebral disks, which was not possible before.7,8 we performed a prospective observational study on 14 cases operated on in our department between 2005 and 2008 . The patients were diagnosed with lumbar stenosis due to hypertrophy of the articular facets on one to three levels . Dynamic posterior stabilization was performed using the total facet arthroplasty system (archus orthopedics, redmond, wa, usa) (figure 1). We therefore implanted nine at l4l5 and four at l3l4 (see figure 2 and tables 13). Necessary criteria to consider the patient suitable for this implant were: degenerative spinal stenosis, lateral or central, at l3l4 and/or l4l5 levels, with imaging confirmation through at least one of computed tomography, magnetic resonance imaging, x - ray, and myelography, which can show: compression on cauda equina, dural sac, or roots impingement on nerve roots due to bony or soft - tissue elements hypertrophic facets with lateral or central compression in cases of spondylolisthesis, at the level proposed for this new implant, the grade of severity should be no greater than grade i neurogenic claudication thigh or leg discomfort, pain, paresthesia, muscular weakness, fatigue, sensation of heaviness around the leg, pain in the medial thigh and lumbar region, tingling sensation that can be worsened by walking or by orthostatism and relieved with rest or lying down on a bed age between 40 and 80 years patients who have been referred for laminectomy due to spinal stenosis up to a maximum l3 . Exclusion criteria (patients not suitable for this implant) were: patients under 40 years or over 80 years of age spondylolisthesis of grade ii or more and retrolisthesis at the proposed level for the implant more than three levels proposed for the laminectomy levels other than l3l5 . The implant is manufactured using the most recent technology with a variable geometry, which allows an exact choice of angles and dimensions in order to reproduce the intervertebral movements perfectly . Biomechanical tests performed during our study demonstrated that this implant allows relatively normal flexion and extension of the involved vertebrae, as shown in figures 1 and 2 . The new implant is made up of eleven modular components: two superior curved rods two inferior rods to which the connectors are adapted a transverse rod with two cups at the ends to control tracking tests indicated that the best way to fix the implants was with acrylic cement (figure 3). From biomechanical studies, the technical specifications indicated that this system resists for more than 10 million cycles, which roughly translates to flexion and extension movements for average human performance for over 10 years . The adhesion of the implant to the cement can take two to three times its maximum duty loads without debonding . This new implant can withstand more than twice the load it takes to detach a pedicle - screw fusion system (the current standard of care) from bone without loosening from the spine . A standard posterior median approach is used, with associated laminectomy and foraminectomy for decompression . In nine cases, we performed a laminectomy at two levels, and in four cases laminectomy was performed at one and three levels . This was followed by foraminotomy and inferior - facet resection at l3 or l4 accordingly . The next step was drilling of the 24 mm transpedicular tunnels for application of the probes at the two levels . Because this is a cemented procedure, it is very important to keep the walls intact in order to prevent extravasations of the cement into the medullary canal or along the roots . The device is made up of eleven modular components: two superior curved rods, two inferior rods to which the connectors are attached, a transverse rod with two cups at the ends to control tracking, and two connectors . It is fixed through the pedicles to the vertebral bodies, using poly(methyl methacrylate) cement with increased barium content (same as in vertebroplasty). Connections between all four elements are made with bolts, the same as used for transpedicular instrumentation . It is very important that the two bolts are positioned parallel to the sliding surfaces of the distal stems . The medullary canal and the holes for conjugation were verified again, and the wound was closed with a vacuum drain . The most frequent level of decompression was l4l5 (nine cases), followed by l3l4 (seven cases). In nine cases, laminectomy was performed on two levels, in two cases on three levels and in two cases on only one level (table 4). The average time necessary to perform the surgery was about 60 minutes, but with experience this might be reduced . Without complications, the established protocol for the follow - up period was clinical and radiological exams at 3 and 6 months and then yearly, corroborated with function and pain according to a visual analog scale and oswestry score . The preliminary results of the initial 20 patients have been previously presented.911 of the 13 patients available for long - term follow - up, four showed progression of the degeneration, both at the operated level, as well as the proximal segment, with limited range of motion but no clinical deterioration . This is important, because it allows for immediate recovery without a long period of rest waiting for consolidation, as is the case with fusion . Functional scores, together with dynamic radiographic imaging, confirmed the functional efficacy of this new implant (table 5, figures 4, 5, 6 and 7). The most important aspect of our case series is that it is one of only very few long - term reports of clinical outcomes using total facet arthroplasty . Since the procedure s conception and clinical use, the limited articles studying facet joint replacement focused on kinematic7 motion of the adjacent level,8 disk pressures, and load sharing.12 these experimental cadaver analyses proved conceptual validity, but long - term outcomes were uncertain . We have presented proof of the potential validity of this system, though based on a small number of patients . Nevertheless, all of the 13 patients available for follow - up showed the minimum clinically significant improvement in oswestry and visual analog scales, which persisted throughout the follow - up period.13 adjacent - segment disease does not yet have a precisely determined etiology . It can be attributed to decompression, fusion, and pedicle screw misplacement, as well as preexisting degeneration.1417 therefore, there is still effort being made toward improving sagittal and postural balance with better solutions for lumbar arthrodesis.18 currently, there are several implants on the market boasting dynamic posterior stabilization.5 the dynesys device (zimmer, warsaw, in, usa) has received the most attention . Longitudinal imaging studies have found potential protective effects on adjacent - disk disease, especially for seriously degenerated disks.18 biomechanical research has shown that dynamic stabilization has less overall range of motion than the intact spine.19 in addition, intradiscal pressures were altered compared to the normal disk.20 even though dynamic stabilization distributes mobility more physiologically compared to fusion, over the cranial and caudal adjacent segments21 adjacent - disk degeneration seems to continue.22 with long - term follow - up, almost half of the patients showed some degree of progression of degeneration, but satisfaction remained very high.23 as with our cohort, residual range of motion is reduced, as well as the need for secondary surgery.24 yet with all favorable results, there are also reports that show inferior long - term functional and clinical outcomes compared to fusion, except for older patients, who appear to be more satisfied.25 the cemented fixation of the pedicle screws provides great mechanical strength, especially in osteoporotic bone, and might reduce some of the complications and cyst osteolysis encountered with osteointegration.26,27 in addition, cemented fixation allows immediate movement, support, and rehabilitation, and has not yet demonstrated any adverse events . We can conclude that total facet arthroplasty represents a feasible long - term solution for dynamic stabilization of the lumbar spine after decompression . It also has the potential advantage of preserving normal biomechanics of the lumbar segments, which might reduce the impact of adjacent - disk degeneration.
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Innate immunity mediated through pamp recognition by prrs is the earliest stage of immunity against viral infection . The subsequent modulation of the adaptive immune response by prr signaling has been studied using cells and mice deficient in specific tlrs, rlrs, or their associated signaling adaptor proteins . Recent studies demonstrated, for example, that the absence of specific tlr pathways impairs adaptive immune responses against a variety of viruses (18, 19). Rlr signaling also seems to be critical for the outcome of japanese encephalitis virus (jev), vesicular stomatitis virus (vsv), influenza virus, and encephalomyocarditis virus (emcv) infection (11, 20, 21). In mice lacking rig - i, stimulator 1, jev, vsv, influenza virus, and ecmv were more virulent and replicated to higher levels than in wild - type mice, suggesting that rlr pathways are essential for controlling infection by these viruses (11, 21, 22). For example, irf3 target genes induced by rlr signaling directly control viral replication in infected tissues (23, 24). Thus, depending on the nature of virus infection, tlrs and rlrs may work together or independently to mount an efficient immune response . It is thought that the innate immune system protects the host from infection in a nonspecific way . This, along with differences in cellular location likely serve to distinguish self - rna from nonself pamp rna, thus avoiding type i ifn induction in response to components of host nucleic acids . For example, tlr7 recognizes a specific motif within uridine - rich ribonucleotide sequences (25, 26), which are hypothesized to be unique to rna viruses . Tlr9 recognizes dna pamp ligands and triggers signaling through the myd88 adaptor protein to induce type i ifn production (27). The recognition of nonself dna ligands by tlr9 might also be sequence dependent, and some studies have implicated the sugar - base - backbone sequence of pamp dna as a recognition factor (2830). Unlike tlrs, which are found either on the cell surface or within membrane - bound vesicles, rlrs are found in the cytoplasm where cellular rna is also present (1). Rig - i preferentially recognizes single - stranded rna (ssrna) over dsrna (15, 31). Ssrnas containing a terminal 5 triphosphate (ppp), but not 5oh or a 5-methylguanosine cap, bind to the rig - i repressor domain and promote a conformational change that activates rig - i signaling (10, 1316). Rna ligands of rig - i are longer than 23 nucleotides, have a linear structure, and contain a uridine- or adenosine - rich ribonucleotide sequence (3133). Host mrna, trna, mirna, snrna, and rrna are not ideal rig - i ligands because their length, structure, 5 end modifications, and interactions with ribonucleoproteins limit their recognition . Self - rnas therefore do not typically trigger innate immune programs or type i ifn expression (34). Mda5 triggers innate immune signaling in response to emcv infection and synthetic dsrna, such as poly i: c (11, 20). But ssrna viruses such as dengue virus and west nile virus, as well as reovirus, a dsrna virus, have also been shown to trigger signaling partially via mda5 (12, 35). However, in these studies, mda5 appeared only to amplify type i ifn production as compared with rig - i, whose actions were essential to initiate innate immunity (12). (17) in this issue describes a plausible mechanism of rna ligand recognition by mda5, providing new insights into the differential roles of rlrs . The study reveals that mda5 binding to dsrna does not depend on its 5 end modification, but rather on its length . Dsrnas with lengths of 2, 3, and 4 kb were shown to increasingly activate mda5 signaling, whereas rig - i recognized ssrna and, to a lesser extent, short dsrna motifs . These results help explain how reovirus can trigger both rig - i and mda5 signaling . The genome of reovirus comprises at least 10 segments of dsrna that vary in length, including long (3.9 kbp), medium (2.2 kbp), and short (1.2 kbp) rna segments . Show that rig - i and mda5 recognize the short and long dsrna segments of the reovirus genome, respectively . In the case of emcv infection, the authors show that the viral genome forms long dsrna via its antisense replication intermediate, and this dsrna is recognized by mda5 but not by rig - i . On the other hand, vsv, which triggers rig - i dependent immunity, forms medium - length dsrna replication intermediates that are not recognized by mda5 . Propose a model for pamp recognition by mda5 in which only long dsrna viral genomes or stable long duplex viral rnas formed during virus replication serve as mda ligands . This model might explain why mda5 ignores self - dsrna, which is typically not present as long duplex rnas . The current observations advance our understanding of how mda5 discriminates between potential rna ligands, but several questions about recognition of rna by rig - i and mda5 still remain . It is widely accepted that ssrna viruses generate dsrna during their replication process (36, 37). However, only mda5 recognizes emcv (8 kb), whereas rig - i recognizes other viruses with lengthy rna genomes, such as vsv (11 kb), jev (11 kb), and hepatitis c virus (9.6 kb). Mda5's failure to recognize these latter viruses may be due to their inability to form long or perhaps stable dsrna . It is also possible that recognition by mda5 depends on specific compartmentalization of the pamp rna with mda5, as differences in the subcellular site of viral genome replication may influence the pamp the mechanism by which mda5 discriminates between pamp ligands in terms of their length is also unclear . Demonstrate that mda5 binds long, capped, di- or mono-5 phosphate dsrna, whereas rig - i binds to short dsrna or 5ppp uncapped ssrna . When rig - i binds to 5ppp ssrna, it changes conformation, which disrupts the inhibitory interaction between the rig - i repressor domain and the cards . This alteration results in the initiation of downstream signaling by the cards and exposes a 17- or 30-kd trypsin - resistant fragment corresponding to the rig - i repressor domain, thereby marking the activation of rig - i (10, 15, 16). However, poly i: c binding to rig - i promotes a distinct conformational change that results in a 66-kd trypsin - resistant fragment, indicating that pamp - bound rig - i undergoes a distinct conformational change depending on the nature of its ligand . Structural studies validate that rig - i prefers ssrna ligands, whereas dsrna binds only inefficiently to rig - i, possibly due to constrains imposed by the location of basic residues within the rna binding groove of its repressor domain . The charged residues are proposed to anchor 5ppp ssrna within the pocket, which would be less amenable to binding dsrna ligands (15, 16). These observations suggest that rig - i may have two ways to distinguish different rna species: efficient recognition of 5ppp ssrna through its repressor domain (10, 15, 16) and comparably inefficient recognition and binding of dsrna (10, 15). By extension, these conformational alterations of rig - i might provide clues about how dsrna ligands interact with mda5 to stimulate its activation . Thus, mda5 might become active upon binding long dsrna through a ligand - induced conformation change that places its card into a signaling - active conformation . This might involve dsrna interactions with its helicase domain and c - terminal region in a fashion similar to the binding of poly i: c to rig - i . Others have demonstrated that although very short dsrna (2030 bp) fail to trigger rig - i dependent signaling (15, 17), longer dsrnas ranging from 70 kb to 2 kb can at least weakly engage rig - i . These data suggest that the length of dsrnas may dictate the interaction with the rig - i helicase domain and/or repressor domain in a manner that supports a rig - i dsrna complex . However, the mechanism of length discrimination of dsrna pamps by these rlrs has not yet been revealed . The third rlr family member, lgp2, has been defined as a regulator of rig - i signaling based on its suppression of rig - i function when overexpressed in cultured cells (10, 38, 39). However, recent studies imply that lgp2 may also function as either a positive or negative regulator of rlr signaling by forming a heterodimer with rig - i or mda5 when bound to rna ligand (40, 41). Such interactions between the rlrs may also broaden their pamp recognition profiles to accommodate other ligands, including ssrna or dsrna of varied length and composition, or even dna rna duplexes . Structure and function analyses of rna interactions with rlrs or heteromeric rlr complexes will further define the molecular basis of self- versus nonself discrimination . The rlrs are adenosine triphosphate (atp) binding proteins that hydrolyze atp as a result of binding rna ligand (15). However, the role of the atpase activity in the function of rlrs is not fully understood . Rlrs contain multiple domains, including two domains known as the walker a motif and the walker b (also known as the dexd / h - box) motif, each located within the helicase domain (42). Walker a and b motifs comprise an atp binding region wherein specific amino acid residues contact the -phosphate of the nucleotide and mediate atp hydrolysis upon substrate binding (4345). Mutant rig - i with an inactive walker a motif retains ssrna binding function but fails to trigger downstream signaling (15, 46). Thus, atp hydrolysis is not essential for pamp binding but is required for downstream signaling . Recent studies revealed that the rig - i repressor domain, in addition to binding to the rig - i cards, forms a complex with the helicase domain linker region that likely stabilizes rna ligand binding (10, 15, 16). Moreover, it seems that motifs in the rlr helicase domain could also be involved in rna substrate binding according to structure and function studies of other dexd / h - box rna helicases (42). Such studies indicate that the rna binding sites and the walker a and b motifs mediate specific interactions (47). These observations present a model in which a repressor domain helicase domain internal complex forms an rna binding pocket whose association with pamp rna may serve to initiate the atpase activity of rig - i, which then triggers a conformation change that allows innate immune signaling . The repressor helicase domain interaction of rlrs may thus be the key determinant of pamp discrimination by rig - i and mda5 (15, 17). It also seems that rig - i atpase activity is involved in the unwinding of dsrna (15). The importance of this activity for innate immune function is unclear, but it might support rig - i interaction with dsrna ligands by catalyzing their conversion to ssrnas . The breadth of prrs expressed in different cells and tissues and their distinct intracellular distribution provide the means for pamp detection and immune activation against a variety of microbial pathogens at the local site of infection or in the microbial niche . These include the membrane - associated cytosolic replication sites of rna viruses, extracellular sites of microbe interaction, and endosomal sites of microbial trafficking and metabolism (fig . The recent identification of the dai (dlm-1/zbp1) protein as a cytoplasmic sensor of microbial dna (48) suggests the existence of other prrs that detect cytosolic pamp dna . It is reasonable to speculate that, similar to tlrs and rlrs, the cytoplasmic dna sensor molecules also exhibit ligand specificity to cover a variety of dna pamps in association with their specific microbial niche, thus providing a further basis for self- versus nonself discrimination . Understanding the processes of pamp ligand recognition within each microbial niche will provide a foundation for the design of appropriate vaccines, adjuvants, and immunotherapies.
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Inorganic particulate contamination by carbon dioxide (co2) insufflation apparatus and gas delivery systems for laparoscopy was first identified in 1989 . Creation and maintenance of a pressurized flow of gas to establish and preserve abdominal wall separation for safe endoscopic observation and manipulation is routinely accomplished by co2 through a gas delivery system . The gas is produced by vapor pressure changes of liquid co2 contained in chromium - molybdenum - steel alloy cylinders . A pressure - reducing insufflation (throttling) system delivers the gas to the abdomen . This study examines the co2 laparoscopic insufflation gas delivery system and qualitatively evaluates it for the presence of bacteria and fungi . The gas met us pharmacopoeia standards and fda criteria for commercial production and intra - abdominal medical use . The cylinders are made of materials meeting the department of transportation standards for hydrostatic pressure and safe intrastate transport . Gas flow from cylinders was directed into sterilized insufflators, non - sterile insufflators, with and without 0.1 or 0.3 micron filters . Filter evaluation was performed using a sterile ten foot section of polyvinyl chloride tubing and a 0.1 or 0.3 micron filter connected to the exit port of the pressure regulator device . Under a laminar flow hood, gas flow was directed into a sterile flask of thioglycolate broth media at flow rates of 500 cc, one liter and three liters per minute for a total volume of 50 liters . The media was cultured, plated, and evaluated for colonization and species identification by microscan panels, walkaway-40 system with manual backup . Various laparoscopy co2 insufflators (eder, olympus, solos, storz, weiss, wisap, wolf) were used to deliver co2 gas . The following numbers correspond to culture sites and results listed in table 1: 1) cylinders connected to insufflators using a sterile high pressure hose and a sterile ten foot polyvinyl chloride tube at the outflow port delivering gas into sterile growth media . 2) gas delivery as in 1, using a 0.3 micron filter between the growth media and the insufflator for 60 samples . 3) gas delivery as in 1, using a 0.1 micron filter, for 60 evaluations . 4) gas delivery as in 1 through insufflators sterilized by ethylene oxide for 60 samples . 5) internal tubing and conduits of the 52 insufflators at two separate sites for 104 samples . 6) external ports of the insufflator, front and rear making 52 samples and the pin index portion of the 60 gas cylinders for a total of 164 samples . Therefore, each gas insufflation system had cultures taken at the pin index site, intake port, two separate internal conduit sites and exit ports in 52 separate insufflators and 60 co2 cylinders (figure 1). Organisms cultured from 52 insufflators and 60 cylinders delivering co2 for laparoscopy pneumoperitoneum . (+) indicates species identification and colonization . Growth media consisted of thioglycolate, trypticase soy broth, sabourauds, mycosel (dbl), blood agar plates, emb plates and legionella selective agar . The various circumstances of evaluation showed that fourteen of the sixty (14/60, 23.3%) gas tanks had microbial colonization . External connection sites (52 insufflators and 60 gas cylinders or 112 sites, 27.6%) showed microbes in 31 instances . Gas cylinders had microbial growth in twelve of the sixty cylinders (12/60, 20%). No growth occurred in the sixty evaluations for each of the 0.1 or 0.3 micron filter group . 1) gas delivered through standard laparoscopic insufflators using sterile connectors and sterile tubing grew organisms as noted in table 1, column number 1 . This represents growth from both the insufflator delivery system and the cylinder gas supply (14/60, 23.3% growth).2) gas delivered through standard laparoscopic insufflators with sterile connectors, sterile tubing and a 0.3 micron filter showed no growth . This represents gas delivered with a 0.3 micron filter before the culture media (0/60, 0% colonization).3) gas delivered through standard laparoscopic insufflators with sterile connectors, sterile tubing and a 0.1 micron filter showed no growth . This represents gas delivered with a 0.1 micron filter before the culture media (0/60, 0% colonization).4) gas delivered through pre - sterilized insufflators showed microbial growth as noted in table 1, column number 4 . This represents gas cylinder growth (12/60, 20% growth).5) cultures of the internal mechanisms, tubing and pressure - reducing apparatus grew organisms as noted in table 1, column number 5 . This represents growth of the insufflator internal apparatus only (26/164, 15.9% growth).6) cultures of the external fittings for inflow and egress grew organisms as noted in table 1, column number 6 . 1) gas delivered through standard laparoscopic insufflators using sterile connectors and sterile tubing grew organisms as noted in table 1, column number 1 . This represents growth from both the insufflator delivery system and the cylinder gas supply (14/60, 23.3% growth). 2) gas delivered through standard laparoscopic insufflators with sterile connectors, sterile tubing and a 0.3 micron filter showed no growth . This represents gas delivered with a 0.3 micron filter before the culture media (0/60, 0% colonization). 3) gas delivered through standard laparoscopic insufflators with sterile connectors, sterile tubing and a 0.1 micron filter showed no growth . This represents gas delivered with a 0.1 micron filter before the culture media (0/60, 0% colonization). 4) gas delivered through pre - sterilized insufflators showed microbial growth as noted in table 1, column number 4 . 5) cultures of the internal mechanisms, tubing and pressure - reducing apparatus grew organisms as noted in table 1, column number 5 . This represents growth of the insufflator internal apparatus only (26/164, 15.9% growth). 6) cultures of the external fittings for inflow and egress grew organisms as noted in table 1, column number 6 . Microbial colonization was shown for the inside of gas cylinders, the external connection sites and inside the insufflation apparatus . Microbial contamination at the gas delivery site was eliminated by either a 0.1 or 0.3 micron sterile filter . It is not surprising that the external surfaces of the insufflation apparatus, pin index system, intake and exit ports showed the presence of microbes (column 6). These surfaces are contacted by many people, inside and outside the operating room, of varying levels of skill and understanding regarding clean and aseptic technique . Using a protective sheath over the pin index portion of the cylinder during handling and transport to the operating room would reduce contamination from handling . Wiping the pin index connecting portion of the stem of the gas cylinder with a germicidal cloth before placement to the insufflator intake port microbes can enter the insufflation apparatus, internal tubing and pressure regulation mechanisms by one of three routes or combinations of routes: 1) from connecting points (inflow and outflow); 2) the gas cylinder; or 3) a. growth of organisms from within the insufflator pressure regulation equipment because of ambient operating room contamination resulting from influx during periods of reduced or no pressure in the system or being turned off with a negative pressure generated in the insufflator allowing backflow intake, or b. from contamination via backflow of irrigation or body fluids from previous laparoscopic procedures recognized or unrecognized . Microbial growth requirements of organisms that are human contaminants and pathogens are varied and extensive . The growth range is from narrow, precise and extremely favorable conditions to those being able to grow or maintain the capability for growth in low temperature, reduced oxygen tension and with few nutrient requirements . Cylinders containing gas for clinical applications are not tested or regulated for any microbe or participate standard . These unclean oxidized metal containers are filled under a wide range of varying circumstances and conditions . No regulation addresses inorganic or organic contaminants in gases used for surgery (table 2). Maximum limits of impurities for carbon dioxide, usp these findings demonstrate that microbes are contained within the gas cylinders and apparatus used for laparoscopic gas delivery . This allows transmission of organisms into the abdomen of laparoscopy patients during gas delivery and throughout the procedure . Despite the fact that it appears that few infections occur due to this circumstance, it is wise and prudent to reduce foreign body and microbial contamination as much as possible during intra - abdominal surgery . Longer more complex procedures in patients in stressed or compromised circumstances, being very young or old and with different concomitant medical complications, requires caution and prudence to reduce contamination exposure . The handling of gas cylinders and insufflation apparatus by personnel with little or no training and instruction contributes to the contaminated state of the apparatus, especially at points of intake and outflow attachment . Proper training regarding methods of cylinder handling and attachment to insufflation equipment needs to be addressed . Adequate knowledge and understanding of the hazards of contamination, the consequences of incorrect equipment attachment and improper handling of gas tanks' connections contribute to microbial contamination within the cylinders and associated laparoscopic gas delivery systems . This is shown by growth of microbes on the intake, egress portions of the insufflators and on the pin index systems of the gas cylinders . These areas should be cleaned by surface decontamination methods when handled and attached to any portion of the gas delivery apparatus . The insufflation down pressure regulating throttling devices are exposed to microbes from multiple gas cylinders, contaminated intake and outflow portions of the insufflator due to improper handling and by intermittent patency to the ambient operating room environment when not in use and when gas pressures are reduced incorrectly during surgery . When not in use, the insufflator becomes a growth and culture chamber for the organisms contained within it . The insufflator then becomes a pressurized delivery vehicle of gas contaminants, inorganic debris and microbes from all portions of the gas delivery system into the patient's abdomen . It is difficult to isolate the role of a single pathogenic factor leading to surgical infection . However, the initiating phase of bacterial infection of the surgical wound starts with microbial contamination . It has been shown that 68% of 350 wounds after clean operations had bacterial growth . The risk of infection primarily depends on the contamination of the wound during the procedure . Laparoscopic gas delivery systems contain inorganic debris and foreign bodies . During the latent period pathogenic factors this is the time when bacteria adhere, propagate and are protected from host defenses and antibiotics . There is less than six hours between bacterial insult and antibiotic prophylaxis for therapy to be effective . Efficient prophylaxis requires establishing a level of antibiotic concentration exceeding bactericidal resistance in the wound prior to insult or during the first six hours of surgery and maintaining these levels for an adequate period of time . Microbial multiplication in a surgical wound containing foreign material, even with a small inocula, has a latency period preceded by active microbial multiplication . The importance of the latency period is that microbial pathogenic effects are potentially reversible during this interval . Quantitative reduction of microbes, particulates and foreign bodies for laparoscopic gas is accomplished by a 0.3 micron filter . Its use also avoids the consequences and side - effects of antibiotics or development of resistant organisms . Kinetic studies show that interaction of the organisms with host proteins is rapid and irreversible . There is intense binding of microbes and ligands to fibronectin, fibrinogen, collagen, laminin and other extracellular matrix proteins . Reducing foreign body exposure of the peritoneal cavity from gas cylinder debris by filtration, diminishes host protein reaction with significant impairment of bacterial adhesion and reduces progression of infection . No attempt was made in this study to relate clinical infection to the observed colonization of bacterial and yeast species found within the gas delivery system apparatus and components . Use of sterile or filtered materials (i.e., gases, solids, or liquids) when placed in the human body is an accepted standard of medical care . This study shows that insufflation of co2 into the peritoneal cavity that is not filtered by a 0.3 micron filter contains microbial organisms from the cylinder or insufflator or both . Reduction of microbial exposure from gas insufflation apparatus is accomplished by a 0.3 micron bacterial filter placed before gas is delivered into the patient . Further studies are necessary to determine the contribution of bacterial virulence factors at the surgical site in the presence of foreign bodies (inorganic particulates) to the occurrence of infection, tissue healing and/or adhesion formation from laparoscopic surgeries . Due to longer and more complex laparoscopic procedures being performed on patients compromised by age and surgical disease processes compounded by pre - existing medical conditions, surgical margins of safety can be reduced . These circumstances, coupled with these qualitative findings of bacterial and fungal colonization found within the laparoscopic gas delivery system, are fair warning and should raise our awareness to this circumstance and warrant methods to decrease or eliminate this exposure . Germicidal cleansing of external port connections and gas filtration to pre - condition all gases prior to intra - abdominal instillation are preferred methods to reduce microbe exposure from a laparoscopic gas insufflation system.
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Advances in medical care and neonatal medicine have led to changes in survival pattern of high risk neonates (1). One of the complications that have been found in these high risk neonates is lbw (<2500 grams). According to world health organization (who) statistics, the rate of lbw is 17% worldwide (6% in developed countries and 21% in developing countries). Studies revealed that the rate of lbw is 10% in iran (2); and 8.4% in yazd, a city in the center of iran (3). One of the most common neonatal complications of prematurity and lbw is respiratory failures like respiratory distress syndrome (rds) which often needs using mechanical ventilation (mv) for improving the neonatal survival, especially for premature neonates born less than 30 weeks gestation with immature lung function (4). Studies showed that mv in lbw neonates, and especially extremely low birth weight (elbw), is linked to poor neurodevelopmental outcomes (5). Although the main proportions of lbw neonates are born in developing countries (2), the majority of studies on developmental outcomes of prematurity have been performed in developed countries . Considering the effects of different cultures and socioeconomic status on development and the importance of conducting developmental evaluations in developing countries, close attention should be paid to such kinds of studies in these countries . There has been lack of research evidence about developmental outcomes of more complicated lbw neonates like neonates which used mv . In this study we prospectively investigated the development of gross and fine motor skills in infants with the history of lbw and over 14 days use of mv . The aim of this study was to compare gross and fine motor development of infants with the history of lbw with mv and without mv (wmv) over 14 days and healthy nbw infants with 8 to 12 months of age using peabody developmental motor scale-2 (pdms-2). This descriptive, prospective and cross sectional study was carried out in the occupational therapy clinic of aliasghar hospital, tehran, iran . The study was conducted on three groups of infants aged 8 to 12 months (all mentioned ages for lbw infants are corrected for prematurity) as follows: lbw group, birth weight <2500 g, in form of mv and wmv groups and with normal birth weight (nbw) group . The sample size based on random sampling method was calculated to be 35 infants included 35 infants with the history of lbw using mv over 14 days (n=35), 35 infants with the history of lbw and wmv(n=35) and 40 healthy nbw infants (n=40). Mv group consisted of infants with corrected age of 8 to 12 months admitted to nicu of aliasghar hospital who used mv for 14 days or more . 2 . Using mv for 14 days or more after third days of birth with diagnosis of respiratory failures, pco2 more than 60 mmhg, po2 less than 50 mmhg and ph less than 7.20 . Wmv group consisted of all infants with corrected age of 8 to 12 months who admitted to neonatal intensive care unit (nicu) of aliasghar hospital for 14 days or more . The data for lbw infants were obtained from neonatal files of infants in nicu . For nbw group, all infants aged 8 to 12 months referred to healthcare center of aliasghar hospital for vaccination were screened for eligibility to the study . Any brain injury like intraventricular hemorrhage (ivh) or seizure, genetic disease, degenerative disease and /or other acquired problems which affect the development after discharge of nicu . Any sensory problem like deafness and/or blindness . In this study because of our excluded criteria on any prenatal or perinatal brain complications we included all infants regardless of kind of delivery . Also, because of not having access to all apgar scores we included infants regardless of their apgar scores . Moreover, because of the methodology of our study which was prospective we could not completely monitor any complications that might be happened during intubation . However, a regular suctioning and tube control were mentioned for all mv group infants during intubation in their neonatal files . All infants were visited by a pediatrician for clinical and neurological examinations and if they fulfilled the above criteria were enrolled in the study . After screening and selecting all groups randomly, the informed consent form was given to their family and then the questionnaire containing medical and developmental history and demographic information of infants were completed by examiners . Finally two occupational therapists blinded to the history of infants conducted pdms -2 for each infant . The pdms-2 is one of the most commonly - used assessments for measuring motor skills of infants and toddlers from birth through age 5 . For children with special needs, the pdms-2 is one of the most reliable testing instruments used by many professionals as a diagnostic tool for assessing of gross and fine motor skills . It has been used in a number of follow - up studies investigating motor skills in the preterm population ., most dysfunctions of motor skills will be identified . Using the results of the pdms-2, we can develop a more responsive learning and remediation program for the child with special needs . This test is composed of six subtests that assess related motor abilities that develop early in life: reflexes, stationary (body control and equilibrium), and locomotion, object manipulation, grasping, and visual - motor integration . Results from these subtests are used to generate the three composite scores: gross motor quotient, fine motor quotient, and total motor quotient with a mean of 100 and a standard deviation of 15 (10). After collecting the perinatal variables and gross and fine motor scores, the motor quotients were determined and data were analyzed and compared using spss version 17 . Spearman s correlation coefficient was used to examine the relationship between the mean of gross and fine motor quotients and qualitative variable and pearson s correlation coefficient was used to examine the relationship between the mean of gross and fine motor quotients and quantitative variable . This descriptive, prospective and cross sectional study was carried out in the occupational therapy clinic of aliasghar hospital, tehran, iran . The study was conducted on three groups of infants aged 8 to 12 months (all mentioned ages for lbw infants are corrected for prematurity) as follows: lbw group, birth weight <2500 g, in form of mv and wmv groups and with normal birth weight (nbw) group . The sample size based on random sampling method was calculated to be 35 infants included 35 infants with the history of lbw using mv over 14 days (n=35), 35 infants with the history of lbw and wmv(n=35) and 40 healthy nbw infants (n=40). Mv group consisted of infants with corrected age of 8 to 12 months admitted to nicu of aliasghar hospital who used mv for 14 days or more . 2 . Using mv for 14 days or more after third days of birth with diagnosis of respiratory failures, pco2 more than 60 mmhg, po2 less than 50 mmhg and ph less than 7.20 . Wmv group consisted of all infants with corrected age of 8 to 12 months who admitted to neonatal intensive care unit (nicu) of aliasghar hospital for 14 days or more . The data for lbw infants were obtained from neonatal files of infants in nicu . For nbw group, all infants aged 8 to 12 months referred to healthcare center of aliasghar hospital for vaccination were screened for eligibility to the study . Any brain injury like intraventricular hemorrhage (ivh) or seizure, genetic disease, degenerative disease and /or other acquired problems which affect the development after discharge of nicu . Any sensory problem like deafness and/or blindness . In this study because of our excluded criteria on any prenatal or perinatal brain complications we included all infants regardless of kind of delivery . Also, because of not having access to all apgar scores we included infants regardless of their apgar scores . Moreover, because of the methodology of our study which was prospective we could not completely monitor any complications that might be happened during intubation . However, a regular suctioning and tube control were mentioned for all mv group infants during intubation in their neonatal files . All infants were visited by a pediatrician for clinical and neurological examinations and if they fulfilled the above criteria were enrolled in the study . After screening and selecting all groups randomly, the informed consent form was given to their family and then the questionnaire containing medical and developmental history and demographic information of infants were completed by examiners . Finally two occupational therapists blinded to the history of infants conducted pdms -2 for each infant . The pdms-2 is one of the most commonly - used assessments for measuring motor skills of infants and toddlers from birth through age 5 . For children with special needs, the pdms-2 is one of the most reliable testing instruments used by many professionals as a diagnostic tool for assessing of gross and fine motor skills . It has been used in a number of follow - up studies investigating motor skills in the preterm population ., most dysfunctions of motor skills will be identified . Using the results of the pdms-2, we can develop a more responsive learning and remediation program for the child with special needs . This test is composed of six subtests that assess related motor abilities that develop early in life: reflexes, stationary (body control and equilibrium), and locomotion, object manipulation, grasping, and visual - motor integration . Results from these subtests are used to generate the three composite scores: gross motor quotient, fine motor quotient, and total motor quotient with a mean of 100 and a standard deviation of 15 (10). After collecting the perinatal variables and gross and fine motor scores, the motor quotients were determined and data were analyzed and compared using spss version 17 . Mean motor quotients scores were compared using anova . Spearman s correlation coefficient was used to examine the relationship between the mean of gross and fine motor quotients and qualitative variable and pearson s correlation coefficient was used to examine the relationship between the mean of gross and fine motor quotients and quantitative variable . Study population was 70 lbw infants (wmv group, n=35) and (mv group, n=35) and 40 healthy nbw infants (nbw group, n=40). The study comprised 23 males and 12 females in mv group, 19 males and 16 females in wmv group, and 18 males and 22 females in nbw group . Mean birth weight, gestational age and other perinatal characteristics of each group are summarized in table 1 . In this study we could not find any extremely low birth weight (elbw) infant matching our inclusion criteria . Mv: with mechanical ventilation; wmv: without mechanical ventilation; nbw: normal birth weight in relation to mean fine motor quotient, there were significant differences between mv and wmv in comparison with nbw group . But, the mean fine motor quotient in mv and wmv group showed no significant differences (p<0.05). In relation to mean gross motor quotient, there were significant differences between mv and wmv in comparison with nbw group . In addition, the mean fine motor quotient in mv and wmv group showed significant differences (p<0.05). Finally, in relation to mean total motor quotient, there were significant differences between mv and wmv in comparison with nbw group and there were no significant differences between mv and wmv group, in terms of the mean total motor quotient (p<0.05). Comparison of three groups (nbw and mv and wmv) based on mean gross, fine and total motor quotient in two sub groups (lbw, vlbw) is shown in tables 2 - 4 . Moreover, based on the multiple comparisons by tukey s hsd, statistically significant difference was found just in gross developmental motor quotient (dmq) when the dmqs of mv group compared with that of wmv group (p <0.05). Furthermore according to the guide to interpreting pdms-2 quotient scores in peabody examiner s manual, the mean of all motor quotients in mv sub groups (lbw, vlbw) except for fine motor quotient of vlbw were in the range of 80 - 89 . It means that motor development of all these groups except for fine motor of vlbw were below average (16.12%). The mean of fine motor quotient of vlbw was in the range of 70 - 79 that interpreted as poor (6.87%). On the other hand, in wmv group, the gross motor quotients of all sub groups (lbw, vlbw) like the nbw group, were in the range of 90 - 110 and considered as average (49.51%) and the fine motor development is in the range of 80 - 89 and as a result, considered as below average (16.12%). The results of this study also showed that there was no correlation between motor quotients and gestational age, sex, length of stay in nicu and days of mechanical ventilation in lbw group (p>0.05). This study pursued to characterize the motor quotients and compare the gross and fine motor development of lbw infants with and without using mv with nbw infants at the age of 8 to 12 months . Recent studies showed that lbw infants are prone to abnormal neurological signs in tone, coordination and reflexes due to their neonatal complications which lead to developing motor deficits and delays in these children at the age of 6 months or later (11). In general, the shorter the gestational period or the lower the birth weight is, the greater the risk status for motor deficits in the premature infant (1). It is reported that approximately 10% of the elbw (<1,000 grams) preterm infants will develop cerebral palsy (cp). Also a 32% rate of cp is found in those infants weighting less than 1,500 grams (1). Grantham et al in1998 (12) showed that lbw infants had significantly lower scores in mental and psychomotor development index at 6 months of age and the difference in both of these scores increased by 12 months of age . In a study by datar in 2009 (13), mental and motor development of vlbw and mlbw babies during the first two years of life was compared with those of normal birth weight ones . Lbw had a small adverse effect on mental and motor development in the first two years of life . Elbw infants are rarely admitted in aliasghar nicu . However, other studies evaluated the elbw infants or the infants born less than 29 weeks gestation which was not considered in our study . (9,14) in these studies the development of lbw infants without considering the effects of length of stay or days of mv was investigated and showed relatively poor outcome of growth and psychomotor development in these populations . Moreover,, we reviewed a follow up study in iran in 2008, in which fifty lbw preterm neonates admitted to shahid sadoughi hospital nicu were evaluated for developmental status at 6 and 12 months of age using persian version of ages and stages questionnaires (asq). Lbw and preterm infants admitted to the nicu showed degrees of developmental delay at the ages of 6 and 12 months, especially in the gross motor and personal - social developmental domains on the asq (15). Also, in yazd, iran, nbw and moderately low birth weight (mlbw) children of 60-month evaluated by asq . The results showed that frequency of developmental delay in gross motor, fine motor and problem solving domains were significantly higher in mlbw group and mean score in all developmental domains was statistically significant lower in case group . (16) as previous studies reported, the mv is often required by preterm infants with respiratory failure, and invasive form of this respiratory support can be related to lung injury and adverse neurologic outcomes (17). In the study of mv in preterm infants in 1992, graziani et al . (18), described the relationships of prenatal factors, especially those associated with mechanical ventilation and hypocarbia, to the subsequent occurrence of neurosonographic and neurodevelopmental abnormalities in preterm infants . They suggested that prenatal and neonatal factors including the need for mechanical ventilation beginning on the first day of life and marked hypocarbia during the first 3 postnatal days are associated with an increased risk of damage to the periventricular white matter of some preterm infants and developmental delays . (5)mv in an elbw neonate has been linked to poor neurodevelopmental (nd) outcomes . (20) determined whether mv s effect on nd is a result of its relationship to bpd and other associated neonatal co morbidities, or whether mv alone is an independent risk factor for poor nd outcomes . They compared the potential impact of mv vs. continuous positive airway pressure (cpap) at 24 h of age on nd outcomes at 18 to 22 months corrected gestational age (cga), and analyzed other important neonatal morbidities to ensure that any relationship between mode of ventilation and nd outcome was independent of co - morbid conditions . They found that ventilatory strategy at 24h of age independently predicts long - term neurodevelopmental outcome in elbw infants . The use of different inclusion criteria makes it difficult to compare the results of this study with previously reported outcome studies . Most of these studies reported outcome according to birth weight and only included extremely preterm or elbw infants . Nonetheless, in our study we could not find elbw infants matching our inclusion criteria . It seems that it may occur in the light of more adverse conditions of these infants in iranian nicu . As a result the results indicated that although dmq of both mv and wmv groups is significantly poorer than nbw group, this difference was more in mv group especially in fine motor quotient of vlbw infants in comparison to nbw group . These lower results in fine motor development and especially in vlbw were similar to cohort study by goyen and lui in 2002 . In this study there were no correlations between motor quotients and gestational age, sex, length of stay in nicu and days of mechanical ventilation in lbw group . This may be caused by small sample of infants or other possibilities that should be investigated later . In our study there were some limitations such as low parental cooperation and refusal to attend hospital for tests, fatigue and the children s need to rest frequently and the small sample size that interfere with our study.we just evaluated the motor development and more diagnostic evaluations in form of follow up study and in all aspects of development conduct on lbw infants with and without mv and in more days use of mv are suggested . The result of this study indicates the importance of special attention to developmental follow up of high risk and lbw infants, especially more complicated lbw infants . We hope for further studies in the field of developmental assessment and early rehabilitation of high - risk infants in our country . Our results can be used by practitioners in their evidence based clinical works especially in developing countries . We would like to thank all lbw infants and their families and all who helped us in this study . This study was approved by the ethic committee of iran university of medical sciences, tehran, iran.
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The parainfluenza virus 5 (piv5) belongs to the paramyxoviridae family, which contains significant pathogens to mammals such as measles, mumps, and hendra viruses . Two glycoproteins in the lipid envelope, a receptor - binding protein (hn, h, or g) and a fusion protein (f), are required for membrane fusion . The f protein, similar to the influenza hemagglutinin (ha) and the hiv env protein, is synthesized as a homotrimer and is activated by proteolytic cleavage, which creates a highly hydrophobic n terminus called the fusion peptide (fp) that is essential for membrane fusion . The cleaved protein is anchored to the virus envelope by a hydrophobic c - terminal transmembrane (tm) domain . Two heptad repeats, hra and hrb, lie next to the fp and tm domains, respectively . Crystal structures of the water - soluble portions of a number of viral fusion proteins have been determined and have provided much of the current understanding of the mechanism of protein - mediated virus cell membrane fusion . It is known that fusion proteins undergo multiple conformational changes to provide the necessary energy for membrane fusion . The conformations that have been observed correspond to the prefusion states before and after cleavage, an extended prehairpin state and the postfusion hairpin state . The hairpins are formed between two heptad - repeat domains common in class i fusion proteins and give rise to a six - helix bundle (6hb) that is characteristic of the postfusion state of these trimeric proteins . A consequence of this 6hb is that it enforces close proximity of the neighboring fp and tm domains in the merged membrane, but no direct structural evidence of this close packing in the membrane has yet been reported . For the parainfluenza f protein, the crystal structures of the uncleaved prefusion state, the cleaved prefusion state, and the postfusion state have been determined, and an extended prehairpin structure was observed by electron microscopy . In comparison, structural information about the membrane - bound fp and tm domains is still scarce . Solution and solid - state nmr studies of the influenza and hiv fusion peptides in detergent micelles and lipid bilayers have provided insights into the mechanisms of virus cell fusion . The ha fusion peptide is predominantly -helical, but the exact tertiary structure depends on the peptide length and the membrane - mimetic environment . A 20-residue construct adopts an obliquely inserted boomerang conformation in detergent micelles, but in lipid bilayers at fusogenic ph, it also samples a small population of a helical hairpin conformation . A 23-residue construct that includes the conserved gxxxg and gxxg motifs adopts a helical hairpin conformation already in detergent micelles, with the hairpin stabilized by gly gly to ala mutation at residue 8 results in a mixture of hairpin and boomerang structures . It is -helical in detergent micelles but a -strand in lipid bilayers containing more than 20% cholesterol . Solid - state nmr data indicate that both helical and strand conformations of the hiv fp insert into the lipid membrane but cross - linked trimers insert more deeply than monomers and are also more fusogenic . The conformational polymorphism of these viral fusion peptides indicates the importance of the lipid environment in regulating membrane fusion . However, the lipid environment is important not only for modulating the fp structure but also for directly influencing the membrane curvature and hydration during fusion . A large number of computational analyses and experimental studies have probed the structures of membrane intermediates during fusion; however, few studies have combined or correlated the fp structure with the membrane - intermediate structure . We recently reported the first solid - state nmr structural study of the piv5 fusion peptide in lipid bilayers . We found that the peptide adopted an -helical conformation in the negatively charged popc / popg membrane but a -strand conformation on the surface of neutral popc and dmpc bilayers . In the current study, we have determined the complete backbone conformation of the popc / popg - bound piv5 fp using chemical shift constraints . The dopc / dopg membrane retains the same negative surface charge as the popc / popg membrane but increases the unsaturation and disorder of the lipid chains . Surprisingly, this change did not increase the fp mobility but converted the peptide from an -helical structure to a partial -strand structure . In the dope membrane, the piv5 fusion peptide mainly adopts a -strand conformation, similar to its structure in neutral pc membranes, but the -strand is inserted into the dope membrane rather than surface bound . Moreover, the peptide changes the phase behavior and hydration of the dope membrane . These results suggest the structural roles of the piv5 fusion peptide during membrane fusion . The fusion peptide used in this study corresponds to residues 103129 of the piv5 f protein, with the amino acid sequence of fagvviglaalgvataaqvtaavalvk . To increase the peptide solubility, a lys tag kkkk was appended to the c terminus through a flexible dioxa linker (nh(ch2ch2o)2ch2co). Five c-, n - labeled peptides were synthesized by primm biotech (cambridge, ma): gval - fpk4, igalv - fpk4, gvtaa - fpk4, vlaat - fpk4, and aaqv - fpk4 (table 1). The labeled residues cover all except for four residues at the n and c termini (f103, a104, v128, and k129) of the peptide . Fpk4 was reconstituted into popc / popg (4:1), dopc / dopg (4:1), and dope membranes at a peptide / lipid molar ratio of 1:20 . Briefly, the peptide was dissolved in trifluoroethanol (tfe) and mixed with lipids in chloroform . Hcl, 1 mm edta, 1 mm nan3, ph 7.5) or phosphate buffer (10 mm na2hpo4nah2po4, 1 mm edta, 1 mm nan3, ph 7.5) and dialyzed for a day . The proteoliposomes were centrifuged at 55 000 rpm at 4 c to obtain membrane pellets, which were equilibrated to 3040 wt% water before being transferred to 4 mm magic - angle - spinning (mas) rotors . Mas nmr experiments were carried out on bruker avance-600 (14.1 t) and dsx-400 mhz (9.4 t) spectrometers . C chemical shifts were referenced to the adamantane ch2 signal at 38.48 ppm on the tms scale, and the n chemical shifts were referenced to the n - acetylvaline signal at 122.0 ppm on the liquid ammonia scale . P chemical shifts were referenced to the hydroxyapatite signal at 2.73 ppm on the phosphoric acid scale . Two - dimensional (2d) c c correlation spectra were measured using a h - driven c spin diffusion experiment with h irradiation (darr) during a mixing time of 2060 ms . Experimental temperatures ranged from 233 to 303 k to investigate fpk4 conformation in both the gel and liquid - crystalline (lc) phases of the membrane . N c correlation spectra were measured using a redor - based pulse sequence with a coherence transfer time of 857 s . One - dimensional (1d) static and mas p spectra were measured between 273 and 313 k to probe the membrane morphology and structure . The size of the p chemical shift anisotropy (csa) is characterized by its span, defined as the difference between the 0 edge and the 90 edge of the uniaxial powder pattern, = 0 90. a 2d p the depth of insertion of the fusion peptide was measured using 2d h spin diffusion experiments in either the lc phase or the gel phase . The lc - phase experiment was applied to dopc / dopg - bound peptide with igalv and aaqv labels using a h t2 filter of 0.81.0 ms and a spin diffusion mixing time of 9625 ms . Spin diffusion buildup curves were quantified after correcting for h t1 relaxation and were simulated using diffusion coefficients of 0.012 and 0.30 nm / ms for the lipid and peptide, respectively . The water peptide interfacial diffusion coefficient (dwp) was 0.0020.003 nm / ms while the lipid peptide coefficient (dlp) was 0.00250.005 nm / ms . The gel - phase spin diffusion experiment was carried out on popc / popg- and dopc / dopg - bound fpk4 . The intensity ratios between the water and lipid ch2 cross peaks of each residue were measured to compare residue - specific depths . We recently reported that popc / popg - bound fpk4 exhibited only -helical chemical shifts for nine labeled residues, suggesting that this membrane promotes a single conformation of the peptide . To obtain the complete backbone conformation in this anionic membrane, we labeled additional residues (table 1). Figures 1 and 2 show the 2d c c and n c correlation spectra of fpk4 in gel - phase popc / popg bilayers . Consistent with the previous study, most residues exhibited -helical chemical shifts and a single set of signals . Modest conformational disorder was manifested at residues a118v121 as reduced intensities and peak multiplicity . For example, the q120 cc cross peak is 4-fold weaker than the a118 and a119 peak (figure 1c), suggesting dynamic disorder at q120 . A recent hexamer model of piv5 fusion peptide placed q120 in the interior of the hexamer and postulated that this residue may be involved in intermolecular h - bonding . Since oligomeric assembly and h - bonding should order and immobilize the peptide, our data does not support this model for fpk4 in the popc / popg membrane . 2d c c correlation spectra of piv5 fpk4 in gel - phase popc / popg (4:1) bilayers . Shown at the top is the amino acid sequence with labeled residues color - coded according to samples . (a) gvtaa - fpk4 spectrum, measured at 253 k with 20 ms mixing . (b) vlaat - fpk4 spectrum, coadded from two spectra measured at 243 k with 20 ms mixing and 253 k with 60 ms mixing . (c) aaqv - fpk4 spectrum, measured at 253 k with 20 ms mixing . C correlation spectra of piv5 fpk4 in gel - phase popc / popg (magenta), dopc / dopg (black), and popc (blue) membranes . The peptide shows predominantly -strand chemical shifts in the popc membrane, -helical chemical shifts in the popc / popg membrane, and mixed strand and helix chemical shifts in the dopc / dopg membrane . Most residues in the gvtaa and vlaat samples show two sets of chemical shifts in the dopc / dopg bilayer . The aaqv sample shows nearly identical -helical chemical shifts in the popc / popg and dopc / dopg membranes . The assigned c and n chemical shifts of popc / popg - bound fpk4 (table 2) allow us to obtain a backbone conformational model of the peptide . All 23 residues (g105l127) exhibit -helical chemical shifts as the dominant signals, with positive c and co secondary shifts and negative c secondary shifts (figure 3a). Using talos+, we obtained backbone (,) torsion angles (table 3), which indicate a nearly ideal -helical conformation in the popc / popg membrane . Chemical shifts were measured from 2d spectra at 233253 k. italics indicate the second conformation . C chemical shifts are referenced to tms, and n chemical shifts are referenced to liquid ammonia . From the gvtaa sample (g105, v106, t122, a123, and a124). From the vlaat sample (v107, l110, a111, a116, and t117). From the igalv sample (i108, g109, a112, l113, and v125). From the gval sample (g114, v115, a126, and l127). From the aaqv sample (a118, a119, q120, and v121). C and n secondary chemical shifts of fpk4 in (a) popc / popg and (b) dopc / dopg membranes . Fpk4 shows clear -helical chemical shifts (red) in popc / popg bilayers and mixed helical and strand chemical shifts (blue) in dopc / dopg bilayers . The random coil values of zhang et al . Were used to calculate the secondary shifts . The dopc / dopg values were predicted from the main set of chemical shifts . Fpk4 undergoes intermediate - time scale motion in the lc phase of the popc / popg membrane . The resulting line broadening precludes the lc - phase h spin diffusion experiment for measuring the insertion depth of the peptide . Therefore, we carried out the gel - phase spin diffusion experiment, which resolves the water, lipid, and peptide h signals in the indirect dimension by h homonuclear decoupling . Strong cross peaks between lipid protons and peptide c signals indicate deep insertion of the peptide into the membrane . In addition, well - inserted peptides exhibit similar h intensity patterns as the lipid chain carbons, while surface - bound peptides exhibit different h cross sections, with much higher water cross peaks than lipid cross peaks . Figure 4a shows representative gel - phase h spin diffusion spectra of fpk4 in the popc / popg membrane . By 25 ms, the peptide shows strong cross peaks with both lipids and water, and the peptide c and lipid ch2 cross sections have similar h chemical shifts, linewidths, and intensity distributions (figure 4b), indicating that fpk4 is well inserted into the hydrophobic region of the membrane . With a shorter mixing time of 4 ms, more residue - specific depth information is obtained, since different residues give different relative intensities between the water and lipid cross peaks (figure 4c): terminal residues such as g105 and a126 have higher water / lipid intensity ratios than middle residues such as a112 and l113, indicating that the two termini are in closer contact with water . The water / lipid intensity ratios (figure 4d) are the lowest between a111 and t117 (0.100.13) and higher for both the n and c termini (0.170.35), consistent with a membrane - spanning topology of the peptide . The intensity profile is asymmetric, with the n terminus having higher values than the c terminus, indicating that the n - terminal half of the peptide is more exposed to the membrane surface . Depth of insertion of fpk4 in the popc / popg membrane from gel - phase spin diffusion . (a) representative 2d spectra with 0 and 25 ms spin diffusion mixing at 258 k. (b) h cross sections for the peptide c peaks (red) and the lipid ch2 peak (black). Already at 4 ms, the peptide and lipid h cross sections have similar intensity patterns, indicating that the peptide is well inserted into the membrane . (c) c cross sections extracted from the water (blue) and lipid ch2 (black) h chemical shifts from the 4 ms 2d spectra . The n- and c - terminal residues have higher water / lipid intensity ratios than the middle residues . (d) water / lipid intensity ratios for all labeled sites . Since fpk4 undergoes intermediate - time scale motion in the popc / popg membrane at ambient temperature, we searched for a different lipid membrane that may speed up the helix motion . Fast motion not only gives higher - resolution nmr spectra but may also allow helix orientation to be determined from motional order parameters without requiring macroscopically aligned samples . The most obvious choice is the dopc / dopg (4:1) membrane, since it has the same membrane surface charge as the popc / popg bilayer while having a 18 c lower gel - to - lc phase - transition temperature due to the presence of a double bond in both acyl chains of each lipid . Surprisingly, the increased disorder and dynamics of the dopc / dopg membrane did not speed up motion of the fusion peptide but changed the peptide conformation . 1d c cp - mas spectra (figure 5) show high - intensity -strand signals for various residues at ambient temperature and few -helical signals . When the membrane is cooled to the gel phase, the -helix signals become detectable and comparable in intensity as the -strand signals . Thus, the remaining -helical conformation has similar intermediate - time scale motion between the dopc / dopg and the popc / popg membranes, but the new -strand structure is immobilized in the lc phase . The increased disorder of the lipid chains shifted the conformational equilibrium of the fusion peptide toward -strand, without changing the mobility of the -helical segment . Representative 1d c cp mas spectra of dopc / dopg - bound fpk4 as a function of temperature . The vlaat - fpk4 spectra are shown . At high temperature, mainly -strand chemical shifts (blue dotted lines) are observed, while at low temperature, both -helical (red dashed lines) and -strand chemical shifts are detected . Figure 6 shows 2d c c darr spectra of four labeled peptides in the gel and lc phases of the dopc / dopg membrane . In the gel phase, the exceptions are g105, v106, i108, and g109, which exhibit only -strand signals, and q120 and v125, which display only -helical chemical shifts . Increasing the temperature decreased the intensities of the helix signals while retaining the strand signals . The position of the peptide at which the helix and strand have comparable intensities is t117 . The signals of several ala residues partially overlap in the short - mixing - time spectra but become resolved by inter - residue cross peaks at long mixing times . For example, the 300 ms 2d spectrum (figure s1a, supporting information) shows -strand l110a111 cross peaks and -helical a116t117 cross peaks, indicating that a111 is primarily in the strand conformation while a116 is mostly helical . A118 and a119 show chemical shifts for all three conformations, but the -helix intensity dominates the strand and coil intensities (figure 6 g, h). Finally, the n - terminal half of the peptide underwent a slow conformational change from -helical to -strand in the dopc / dopg membrane: v107, l110, and a111 initially showed -helical chemical shifts, which converted to -strand chemical shifts at equilibrium (figure s1b, supporting information). However, the more c - terminal a116 and t117 in the same vlaat sample remained stably -helical . 2d c c correlation spectra of dopc / dopg - bound fpk4 in the gel phase (233 or 243 k, left column) and the lc phase (303 k, right column). C correlation spectra (figure 2) confirmed the mixed strand / helix conformation of the n- and c - terminal halves of the dopc / dopg - bound peptide . Two sets of chemical shifts were observed for many residues, but residues g105l113 show dominant -strand peaks while residues a118v125 have dominant -helical peaks . Comparison of the peptide spectra for three lipid membranes, popc / popg, dopc / dopg, and popc, highlights the membrane - induced conformational polymorphism of fpk4 . The -helical chemical shifts of the c - terminal half of the peptide are the same between the popc / popg and dopc / dopg membranes, whereas the -strand chemical shifts of the n - terminal residues differ between the popc and dopc / dopg membranes . For example, the chemical shifts of i108, a112, and l113 in the dopc / dopg membrane are intermediate between the corresponding chemical shifts in the popc and popc / popg membranes (figure 2c). On the basis of the cross - peak intensities in the low - temperature 2d c c spectra, we quantified the -helical content of each residue (table s1, supporting information). Residues up to l113 are less than 35% -helical, whereas residues a116v125 are greater than 50% helical . The increasing helicity toward the c terminus was consistently observed for all labeled peptides, independent of minor variations in the hydration and salt content of the samples . The talos+ predicted backbone (,) torsion angles of the major conformer of dopc / dopg - bound fpk4 (table 3) confirm the n - terminal -strand and c - terminal -helical structures of the peptide . For this mixed conformation, oligomerization, if present, is expected to be parallel rather than antiparallel . This is consistent with the cross - peak pattern detected at long mixing times . The labeled residues within gvtaa- and igalv - fpk4 lie at the two ends of the peptide . Thus, if antiparallel packing or a hairpinlike structure were present, we would observe inter - residue cross peaks between the n- and c - terminal residues . The 500 ms 2d spectra (figure s2, supporting information) of these samples show only sequential inter - residue cross peaks such as g105v106, t122a123, i108g109, and a112l113 but no long - range cross peaks, thus ruling out antiparallel packing and the helical hairpin conformation . Since fpk4 adopts a surface - bound -strand structure in neutral pc membranes but an inserted -helical structure in the anionic popc / popg membrane, the topology of the partial -strand peptide in the anionic dopc / dopg membrane is not immediately obvious . We thus measured the depth of the peptide in the dopc / dopg membrane, using both the lc - phase h spin diffusion experiment and the gel - phase experiment . By 100 ms, the 2d h c correlation spectra at 293 k (figure s3, supporting information) showed clear cross peaks between lipid - chain protons and peptide c for both the -strand and -helical residues, indicating that the entire peptide is inserted into the dopc / dopg membrane . This is confirmed by the fast lipid - to - peptide spin diffusion buildup rates for both conformations (figure s3c, f, supporting information). More residue - specific depth information is obtained from the gel - phase spin diffusion spectra obtained at 243 k. by 4 ms, the peptide h cross section is already similar to the lipid h cross section (figure 7a), indicating equilibration of the h magnetization among the peptide, lipid, and water . Similar to the popc / popg case, fpk4 has higher water / lipid cross - peak intensity ratios for the terminal residues than the central residues (figure 7b, c), indicating that the peptide spans the bilayer thickness . But in contrast to the popc / popg - bound fpk4, the c - terminal -helical residues are significantly more exposed to water than the n - terminal -strand residues (figure 7c). The lc - phase spin diffusion spectra (figure s3c, f, supporting information) also exhibit slightly faster lipid peptide spin diffusion buildup rates for the n - terminal residues than the c - terminal residues . It is not fully clear whether it is the backbone conformation (helix versus strand) or the residue position (n or c termini) that causes the different insertion asymmetry between the popc / popg and dopc / dopg membranes . However, the minor -strand conformation of the c - terminal a123 and a124 has lower water / lipid intensity ratios than the -helical counterpart, while the minor -helical conformation of the n - terminal l110 has higher water exposure than -strand l110 (figure 7c), suggesting that conformation may be the more important determinant of depth: the -strand conformation is more deeply inserted than the -helical conformation into the dopc / dopg membrane . Depth of insertion of fpk4 in the dopc / dopg membrane from gel - phase spin diffusion spectra measured at 243 k. (a) h cross sections of the peptide c peaks (red) and lipid ch2 peak (black). By 4 ms, the peptide and lipid signals have equilibrated, indicating that the peptide is well inserted into the membrane . (b) c cross sections from the water (blue) and lipid ch2 (black) h chemical shifts of the 4 ms 2d spectra . The c - terminal -helical residues have higher water cross peaks than the n - terminal -strand residues, and the -helical a123/a124 have higher water cross peaks than the -strand a123/a124 . (c) water / lipid intensity ratios of all labeled residues in the dopc / dopg membrane (blue and red symbols). Open symbols indicate the minor conformation . For comparison, the popc / popg - bound fpk4 data are also shown (black open symbols). To investigate whether fpk4 causes curvature and dehydration to the dopc / dopg membrane, we measured the static and mas p spectra (figure s4a, b, supporting information). Fpk4 displayed little perturbation of the structure of the dopc / dopg membrane: the lamellar - bilayer powder pattern is retained, and the isotropic chemical shift is unchanged . However, the mas isotropic line width is significantly broadened by the peptide (from 30 to 130 hz), and the p transverse relaxation times of dopc and dopg decreased from 18.3 and 19.4 ms, respectively, for the peptide - free membrane to 2.4 and 2.0 ms for the peptide - bound membrane (figure s4c, supporting information). Thus, the apparent p linewidths are largely homogeneous, and the fusion peptide slows down the lipid headgroup motion without changing its average conformation . Finally, the 2d p h correlation spectrum shows clear water lipid cross peaks (figure s4d, supporting information) for both dopc and dopg, indicating that fpk4 retains the hydration of the membrane surface . The p mas spectrum (figure s4b, supporting information) exhibits a small isotropic peak at 2.2 ppm . This peak can be assigned to the phosphate buffer, since samples prepared in tris or hepes buffer did not show this peak (data not shown). We previously observed the same isotropic peak in static and mas p spectra of fpk4-containing popc and dmpc membranes, and the peak intensity increased with the peptide concentration . The latter led to the erroneous conclusion that this peak resulted from a peptide - induced high - curvature isotropic phase . We now attribute the concentration dependence of this p peak to electrostatic attraction between the cationic lys tag and the phosphate ions . Similar cases of phosphate buffer interactions with membrane peptides have been reported in the literature . Thus, the -strand fpk4 that binds to the surface of the popc membrane does not cause curvature on the sub-10 nm scale . However, this does not exclude the possibility that the peptide may cause curvature on larger length scales of 50100 nm, which would not manifest as a narrow peak in the static p spectra . To further investigate whether fpk4 induces membrane curvature, we studied the structure and lipid interactions of dope - bound fpk4 . The small headgroup of dope and its disordered acyl chains create spontaneous negative curvature to the membrane, causing an inverse hexagonal phase (hii) in a wide temperature range . The dope phase diagram has been measured using nmr and x - ray diffraction, and the lamellar (l)hii transition temperature (th) is known to depend on the hydration: above 16 water molecules per lipid, the membrane converts to the hii phase by 283 k. if fpk4 causes membrane curvature, then th will be affected: positive curvature generation by the peptide increases th while negative curvature generation lowers the transition temperature . Figure 8 shows the static p spectra of dope membranes without and with fpk4 from 273 to 313 k. at 273 k, pure dope membrane shows an l-phase powder pattern with a chemical shift anisotropy span of + 44.5 ppm . Above 273 k, the p spectrum shows increasing intensities of a narrower line shape with a span of 21.5 ppm, which is inverted from the l line shape around the isotropic p chemical shift . The pure dope membrane fully converted to the hii phase by 283 k (figure 8a), consistent with the reported th value . Upon fpk4 binding, the lhii transition shifted to higher temperatures and was complete only by 293 k, indicating that fpk4 exerted positive membrane curvature . In addition, an isotropic peak appeared in the spectra (figure 8b). In principle moreover, small - angle x - ray diffraction data of dope containing the ha fusion peptide indicated the presence of inverted bicontinuous cubic phases as well as an increase of the lhii transition temperature, and independent md simulations also predicted the same effect . Thus, the isotropic p peak seen here is most likely due to cubic - phase formation in the dope membrane, which suggests that the piv5 fusion peptide causes both positive and negative curvatures; that is, the peptide generates negative gaussian curvature . Also known as saddle - splay curvature, negative gaussian curvature results from the product of positive and negative principal curvatures and is present at membrane pores and protrusions formed during membrane budding and scission . (a, b) static p spectra of the membrane without (a) and with (b) fpk4 from 273 to 313 k. fpk4 increased the l-to - hii phase transition temperature and caused a small isotropic peak . H correlation spectrum of fpk4-bound dope membrane with a spin diffusion mixing time of 225 ms . (d) h cross sections from the 2d p h spectra of peptide - free and peptide - bound dope membranes, compared with the 1d h single - pulse spectrum (top). The equilibrium fpk4 conformation in the dope membrane is predominantly -strand, after the transient existence of a mixed strand / helix conformation (figure 9a, b). The -strand shows clear cross peaks with lipid ch2 protons in the 2d h c correlation spectra (figure 9c), indicating that the peptide is embedded in the hydrophobic region of the hexagonal - phase cylinders (figure 10e). The 2d p h correlation spectrum of the fpk4-bound dope membrane shows a much weaker water p cross peak than the peptide - free membrane (figure 8c, d), indicating that the -strand fpk4 dehydrates the dope membrane in addition to causing curvature to this membrane . Conformation and depth of fpk4 in the dope membrane . (a) 2d c c correlation spectrum of a fresh gvtaa - fpk4 sample at 243 k. the peptide exhibits both helix and strand signals . (b) c cp - mas spectra of the initial and equilibrated gvtaa - fpk4 at 246 k. at equilibrium, most residues exhibit -strand chemical shifts . (c) 100 ms 2d c h correlation spectrum at 293 k, in the hii phase membrane . Lipid peptide cross peaks are observed, indicating that the -strand peptide is inserted into the hydrophobic region of the dope membrane . Piv5 fusion peptide conformations in lipid membranes from solid - state nmr and outside the membrane from crystal structures . (a) fusion peptide is fully -helical in popc / popg bilayers but adopts a mixed strand / helix conformation in dopc / dopg bilayers . The peptide is inserted into both membranes, but the depicted tilt angle is hypothetical . The structures were built using (,) torsion angles predicted by talos+ . (b) prefusion crystal structures of the piv5 f protein in the uncleaved (green) and cleaved (red) states . The fusion peptide domain has similar conformations before and after cleavage and has a bend near t117 . (c) prefusion crystal structures of the influenza ha in the uncleaved (green) and cleaved (red) states . The n - terminal half of the fusion peptide is rotated around n12 before and after cleavage . Seven residues (t122v128) of the fusion peptide are detected and show -helical structure extended from hra . (e) schematic of the piv5 fusion peptide conformation in the dope membrane . The lipid cylinders and water radius are drawn to scale using 1518 water molecules per lipid based on the dope phase diagram . (f) the hemifusion stalk intermediate showing both negative and positive membrane curvatures and dehydration between two opposing bilayers . The present solid - state nmr data indicate at least four distinct conformations and membrane topologies of the piv5 fusion peptide . In the popc / popg membrane, the peptide adopts a membrane - spanning -helical conformation (figure 10a). The popc / popg membrane has a hydrophobic thickness of 27 at 30 c based on x - ray scattering data . The full -helix has a length of 34 from g105 to l127 c based on the talos+ structural model . Thus, the helix may be tilted by 3540 to achieve optimal hydrophobic match between the popc / popg bilayer thickness and the peptide length . In the dopc / dopg membrane, fpk4 adopts a mixed conformation with an n - terminal -strand (residues 105113) and a c - terminal -helix (residues 116125). No chemical shift constraints were measured for residues g114, a115, a126, and l127 . The current structural model assumed a126l127 to be similarly helical as in the popc / popg - bound fpk4 and g114 and a115 to be random coil due to its position near a likely bend (see below). The overall dimension of the mixed helix / strand conformation is not known without long - range distance constraints . However, since the -strand is much more extended than the -helix, the peptide is likely to be significantly tilted to match the hydrophobic thickness of the dopc / dopg bilayer, which is similar to that of the popc / popg bilayer . In the dope membrane (figure 10e), the chemical shift constraints suggest a predominantly -strand peptide, which is inserted into the hydrophobic region between the lipid cylinders . Finally, fpk4 adopts a surface - bound -strand structure in neutral popc and dmpc membranes, as we showed previously . The four conformations and topologies of fpk4 suggest several principles for the influence of the lipid membrane on the fusion peptide structure . First, while the entire sequence of the fusion peptide is capable of conformational polymorphism, the c terminus has a higher propensity for the -helical structure . Second, anionic membranes promote the -helical conformation, as shown by the difference between the popc / popg membrane and the popc membrane and by the difference between dopc / dopg and dope membranes . Third, more disordered membranes shift the peptide conformational equilibrium toward -strand, as shown by the difference between the popc / popg and dopc / dopg membranes . The third observation, while initially unexpected, can in fact be understood by the fact that lipid unsaturation not only changes membrane dynamics but also membrane curvature . Cone - shaped lipids (with negative intrinsic curvature) such as oleic acids, cis - unsaturated lipids, and phosphatidylethanolamine promote stalk formation, whereas inverted - cone shaped lipids (with positive curvature) such as lysophosphocholine inhibit fusion by preventing stalk formation . Thus, the more unsaturated dopc / dopg lipids change the membrane curvature in addition to membrane dynamics compared to the popc / popg lipids . The higher -strand content of the fusion peptide in the dopc / dopg membrane thus suggests that the -strand structure may be the active form in hemifusion intermediates . Our chemical shift analysis indicates that the piv5 fusion peptide has a higher conformational disorder in the middle of the sequence, near g114t117 . This region is not only the transition point between the strand and helix segments in the dopc / dopg - bound peptide but also has multiple conformations and residual dynamics in the popc / popg membrane . The prefusion crystal structures of several viral fusion proteins and the nmr structures of other fusion peptides suggest that conformational disorder in the middle of fusion peptide domains may be general . For example, in the uncleaved piv5 f protein, the c - terminal part (a118v128) of the buried fusion peptide shows an -helical structure extended from hra, whereas the n - terminal part (f103t117) has a mixed conformation of random coil (f103i108), -helix (g109l113), and -strand (v115a116) (figure 10b). After cleavage, the first four residues of the fp undergo an orientational change while the other residues are mostly unaffected . In the prefusion ha crystal structures, the fp is unstructured in both uncleaved and cleaved states, but the n - terminal segment undergoes a large - amplitude rotation with respect to the rest of the protein after cleavage (figure 10c). Isolated ha fusion peptides bound to dpc micelles exhibit -helical conformations, but the middle of the peptide is disordered and forms the bend of the helical hairpin in the 23-residue construct and the bend of the boomerang structure in the 20-residue construct . The exact significance of this mid - domain disorder for membrane fusion is not clear, but we speculate that the disorder may be useful for controlling oligomerization, the degree of peptide insertion into the membrane, and membrane hydration . For example, if the -sheet conformation is indeed more effective in dehydrating the lipid membrane than the -helical conformation, as suggested by the current dope data and the previous popc and dmpc data, then a mixed strand / helix conformation may be useful for dehydrating one of the two surfaces of the lipid bilayer . Mutagenesis data of the fusion peptide domain of the piv5 f protein indicated a competition between protein transport, surface expression, and membrane fusion, but the n - terminal residues f103v115 appear to be more important for membrane fusion than other functions . Mutations of g105, g109, and g114 to ala reduced protein expression but increased membrane fusion . G109a and g114a mutants showed 25% lower expression levels but 10-fold higher membrane fusion than the wild - type protein . While fusion peptides are generally rich in gly and ala residues, in piv5 fp, all three gly residues are located in the n - terminal region while over half of the ala residues are located in the c - terminal region . In contrast, ha and hiv fusion peptides have a more uniform distribution of gly residues . The high gly content of the n - terminal half of the piv5 fusion peptide may be one of the reasons for the stronger -strand propensity of the n - terminal half . Meanwhile, the helix propensity of the c - terminal segment may be related to the neighboring -helical hra domain, as seen in the postfusion crystal structure of piv5 f, which shows -helical t122v128 in the fp domain . Since the n - terminal domain is more important for membrane fusion and has a stronger propensity for the -strand conformation, the -strand conformation may be more critical for membrane fusion . This is also consistent with the ability of the -strand conformation in causing membrane dehydration, as discussed below . Static p nmr spectra indicate that the piv5 fusion peptide neither causes curvature nor dehydration to the popc / popg and dopc / dopg membranes but causes curvature to the dope membrane . The peptide increased the lhii transition temperature by about 10 k and generated a small amount of an isotropic phase . We attribute this signal to a cubic phase, which would suggest that the fp promotes negative gaussian curvature . Increasing experimental evidence and simulations indicate that generation of negative gaussian curvature may be a common property of viral fusion peptides . Recent small - angle x - ray diffraction data of the ha fusion peptide in methylated dope showed that the peptide shifted the lhii phase transition to higher temperatures and additionally promoted the formation of inverted bicontinuous cubic phases, lm3 m and pn3 m, which possess negative gaussian curvature . This result revises earlier literature that concluded that the ha fusion peptide promoted only negative curvature . These earlier studies were based on differential scanning calorimetry experiments, which may not be able to resolve the cubic phases from the l and hii phases, and on p nmr spectra that showed clear isotropic peaks but poor - sensitivity powder patterns that cannot be definitively assigned to either the l phase or the hii phase . Therefore, these earlier data cannot be interpreted as stabilization of the hii phase by the ha fusion peptide, but they do indicate the generation of an isotropic phase, which is consistent with cubic - phase formation . Molecular dynamics simulations of membranes containing the ha fusion peptide indicate that the peptide systematically shifted the lipid phase diagram toward more positive mean curvature and bicontinuous cubic phases . For the hiv gp41 fusion peptide, p nmr spectra and cryo - tem micrographs of dope - containing lipid membranes showed the presence of an isotropic phase . Thus, all reliable evidence converges to indicate that influenza, hiv, and piv5 fusion peptides cause negative gaussian curvature to pe - rich membranes . On the basis of the intensity of the p isotropic peak, the piv5 fpk4 construct used here has weaker curvature - generating ability than the influenza and hiv fusion peptides . In addition to generating membrane curvature, fpk4 also partially dehydrated the dope membrane (figure 8c, d). For the hii - phase dope, this means a reduction of the water - core diameter of the cylinders . Figure 10e depicts the hexagonal cylinders, where the relative dimensions of the water pore and the hydrophobic chains match the values reported from x - ray diffraction data of dope at 1518 water molecules per lipid, which is the hydration level of the fpk4-containing dope membrane . At this hydration, the water channel radius is 19, while the distance between the centers of two cylinders in adjacent layers (dhex) is 70, whose hydrophobic portion (32) is traversed by the -strand fpk4 . This peptide location is consistent with x - ray scattering data that showed that the hiv fusion peptide increased the dhex value of dope at peptide concentrations above 2 mol% . The increased hexagonal spacing results from a compensatory effect of an increased hydrocarbon volume, which implicates the hiv fusion peptide to be embedded in the hydrocarbon region, and a decreased water volume, which agrees with the dehydration seen in the current 2d p h correlation spectra of the dope membrane . Thus, the piv5 and hiv fusion peptides exert similar changes to the dope membrane . The cross section of the inverse - hexagonal phase dope, in which the opposed lipid chains of different cylinders experience negative curvature (figure 10e), is similar but not identical to the cross section of the hemifusion stalk intermediate (figure 10f), since the latter also contains lipids experiencing positive curvature . The hemifusion stalk is topologically more similar to inverted bicontinuous cubic phases, which are the likely cause of the isotropic peak in the p spectra . The lipids that experience positive curvature in the cubic phase should correspond to lipids in the distal leaflet of the opposing membranes . On the basis of the conformation, topology, and lipid and water interactions of piv5 fpk4 in the four lipid membranes obtained from solid - state nmr, we propose the following relations between the fp structure and viral membrane fusion . When the fp is released from the globular head of the f protein, it inserts into the target cell membrane in an -helical structure . As the protein rearranges its structure and hra forms a coiled - coil trimer in the prehairpin intermediate, the fusion peptides of the three proteins interact in the cell membrane to form a homotrimer . When several trimers cluster in regions of the membrane containing high concentrations of unsaturated lipids with their ensuing negative intrinsic curvature, the gly - rich n - terminal half of the fusion peptide converts to a -strand conformation, which dehydrates the membrane surface and exerts negative gaussian curvature to the membrane . At this point, depending on the local lipid composition, the fusion peptide may be partially -strand (in pc - rich membranes) or fully -strand (in pe - rich membranes), and the peptide is well inserted unless the local membrane composition is predominantly neutral pc . When the water - soluble ectodomain completes its conformational change to a six - helix bundle, the fp and tm domains are forced into close proximity, which may revert the fusion peptide to the -helical conformation, which may in turn reduce membrane curvature and increase membrane hydration . Multiple lines of evidence obtained here suggest the -strand conformation of the fusion peptide to be the most relevant structure in hemifusion intermediates, responsible for remodeling the membrane to acquire the curvature and low hydration necessary for progression to complete fusion.
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Plasma and serum samples are generally acceptable for the detection of various physiologically active substances; however, drawing blood can induce stress in animals . In the instance of collecting repeated blood samples from mice, using the saphenous and lateral tail vein these sites yield a 5% volume of circulating blood from the saphenous veins and 0.10.15 ml from lateral tail vein . However, the small volume of blood sampled limits detection of substances by any assay . Blood sampling by decapitation or cardiac puncture is employed as a collection method to obtain whole blood, gathering approximately 1 ml of whole blood and 0.5 ml of plasma . This volume is adequate to measure multiple substances using an immunoassay . In this case, a large number of mice are required, due to the sacrifice of animals at each time point when exhibiting responses to experimental treatments . In addition to this problem, it is impossible to investigate multiple time points, before and after responses to the treatment in the same mouse . Alternatively, non - invasive samples (including feces, urine and saliva) can be used for the assays . In particular, many studies discuss salivary biomarkers in responses to stress (for reviews [10, 21]). Stress responsive biomarkers, including glucocorticoid, -amylase and chromogranin a, have demonstrated some correlations between salivary and plasma concentration in human and animals [19, 21]. However, there are few reports evaluating saliva for these biomarkers in response to stress using small experimental animals, particularly mice . Human saliva can be easily collected using absorbent materials, such as cotton balls, placed in the mouth . In contrast, it is more difficult to obtain mouse saliva as mice swallow their saliva while they are awake; thus, the mice need an anesthetic for saliva collection [3, 11]. In addition to anesthesia, pilocarpine (muscarinic receptor agonist) is also needed as a salivator [11, 30], since salivary volume is expected to be low without pilocarpine . However, anesthesia itself may induce a stress response [1, 4] in mice during saliva collection . In order to clarify whether the salivary corticosterone is available to evaluate stress response in mice, first we confirmed the transfer of glucocorticoid from the blood to the saliva after administration of exogenous cortisol . Secondly, we compared corticosterone levels between plasma and saliva in response to restraint stress, and finally, we investigated the adequate recovery, at set intervals after anesthesia, for saliva collection between pre - stress and post - stress in mice . Animals: adult male icr mice were purchased from kiwa laboratory animals co., ltd . All mice were allowed ad libitum access to a commercial diet (ef; oriental yeast co., ltd ., tokyo, japan) and tap water . Mice were maintained under controlled conditions: temperature (2325c), relative humidity (4060%) and light (12 hr, 07:0019:00 hr). Nine- to thirteen - week - old animals were used in each experiment; their body weights before the experiment were 42.0 0.3 g mice were treated according to the provisions for animal welfare of the nippon veterinary and life science university, which follows the guidelines for animal experimentation issued by the japanese association for laboratory animal science . Drug preparation: all agents were injected intraperitoneally (ip) at an injection volume of 0.1 ml/10 g body weight / mouse . A mixture of three drugs (medetomidine, midazolam and butorphanol) acting as the anesthetic agents, were used throughout the present study . The injection of atipamezole (an antagonist of medetomidine) allowed mice to undergo a rapid recovery from anesthesia [6, 25]. The mixed anesthetic agents were prepared as a three - drug mix, modifying the dose slightly dose according to naganuma et al . . The preparation involved using sterilized saline (0.9%): 0.3 mg / kg of medetomidine hydrochloride (domitor; nippon zenyaku kogyo co., ltd ., fukushima, japan), 6.0 mg / kg of midazolam (dormicum; astellas pharma inc ., tokyo, japan) and 7.5 mg / kg of butorphanol tartrate (vetorphale; meiji seika pharma co., ltd ., tokyo, japan). Pilocarpine hydrochloride (nacalai tesque, inc ., kyoto, japan), as a salivator to enhance salivary secretion, and atipamezole hydrochloride (antisedan; nippon zenyaku kogyo co., ltd . ), as an antagonist of medetomidine, were dissolved in saline and treated at a dosage of 0.5 and 0.3 mg / kg, respectively . Cortisol (hydrocortisone; nacalai tesque, inc .) Was dissolved in sesame oil (nacalai tesque, inc .) And treated at a dosage of 2.0 mg / kg . Saliva collection procedure: mice were treated with the mixed anesthetic agents, and 10 min later, they were additionally treated with pilocarpine (0.5 mg / kg ip). Saliva was collected for 40 min by absorption onto a cotton roll (of approximately 5 mm in diameter, 10 mm in length) and placed in the oral cavity, exchanging the cotton occasionally . After the end of the saliva collection, atipamezole was injected into mice to allow recovery from the anesthetic . During anesthesia and saliva collection, the body temperature of the mice was maintained at 38c by using a heating plate . To extract saliva from the cotton rolls, they were transferred to 1.5 ml centrifuge tube with a hole (approximately 1.5 mm in diameter) at the bottom onto empty the tube . Effect of cortisol administration on cortisol levels in plasma and saliva: mice were treated simultaneously with the mixed anesthetic agents and 2.0 mg / kg ip of cortisol or by vehicle injection, and 10 min later, they were additionally treated with pilocarpine . After the end of the saliva collection, mice were euthanized by decapitation under anesthesia, and trunk blood was collected in heparinized sampling tubes . Blood samples were centrifuged to obtain the plasma for the determination of cortisol levels by enzyme immunoassay (eia). Comparison of corticosterone levels before and after the restraint stress in plasma without anesthesia: the restraint stress group was immobilized for 60 min by a restrainer that was made of transparent acrylic, forming a cylinder (icn-3; inner diameter, 36 mm; length 100 mm; icm co., ltd ., blood samples were treated as described above, and the plasma was obtained for the determination of corticosterone levels by eia . Comparison of corticosterone levels before and after the restraint stress in plasma and saliva under anesthesia: the restraint stress group was immobilized for 60 min by the restrainer and then treated with the mixed anesthetic agents immediately after release from the restraint . At the end of the 40 min saliva collection period, mice were euthanized by decapitation for blood sample collection . On the other hand, the intact control group without restraint stress was euthanized by decapitation after the saliva collection under anesthesia . Investigation of the adequate recovery period from anesthesia for saliva collection: to investigate the adequate recovery period from anesthesia for saliva collection between pre - stress and post - stress in the same individual, three different recovery times were examined . For the collection of saliva samples in the pre - stress condition, the male mice without restraint stress were treated with the mixed anesthetic agents, and saliva was collected for 40 min under anesthesia . After 1, 3 and 7 days since the first saliva collection, the same individual male mice were immobilized for 60 min by the restrainer and treated with the mixed anesthetic agents immediately after release from the restraint . Saliva was collected for 40 min, and atipamezole was injected into mice for recovery from anesthesia at the end of the saliva collection . Enzyme immunoassay and statistical analysis: plasma samples were extracted by diethyl ether, according to the method described by kanesaka et al . The levels of cortisol and corticosterone in plasma and saliva were measured by competitive enzyme immunoassay, using anti - cortisol-3-cmo - bsa igg (fka 404-e; cosmo bio co., ltd ., tokyo, japan) and anti - corticosterone-3-cmo - bsa igg (fka 420-e; cosmo bio co., ltd .) As the primary antibody . Cortisol-3-cmo - hrp (fka 403; cosmo bio co., ltd .) And corticosterone-3-cmo - hrp (fka 419; cosmo bio co., ltd .) Were used as the labeled steroid . The intra- and inter - assay coefficients of variation were 4.5 and 4.4% for cortisol, and 7.0 and 14.0% for corticosterone, respectively . T - test or paired t - test was performed using jstat software, version 10.0 for windows (http://toukeijstat.web.fc2.com/), for the data analysis of plasma and salivary corticosterone levels . The differences were considered significant at probabilities of less than 5% (p<0.05). Effect of cortisol administration on cortisol levels in plasma and saliva: the results are shown in fig . 1.fig . 1.effect of cortisol injection (2.0 mg / kg ip) on plasma and salivary cortisol levels in adult male icr mice . Nd denotes not detectable. The cortisol levels in the treatment group administered with 2.0 mg / kg ip of cortisol were 633.1 35.9 ng / ml in the plasma and 75.6 4.4 ng / ml in the saliva . Salivary cortisol was not detected in the group treated by vehicle injection, while plasma cortisol was detected by eia; however, it was only represented by very low levels . The total salivary cortisol levels, from 10 to 50 min after the treatment of cortisol, were approximately 12% of the plasma cortisol levels at the time point of 50 min after the treatment . Effect of cortisol injection (2.0 mg / kg ip) on plasma and salivary cortisol levels in adult male icr mice . Nd denotes not detectable. Comparison of corticosterone levels before and after the restraint stress in plasma without anesthesia: figure 2afig . 2.effects of restraint stress (60 min) on corticosterone levels in adult male icr mice treated with or without anesthesia . The restraint group was euthanized by decapitation immediately after the restraint stress (60 min) without anesthesia (a). The intact and restraint groups were euthanized by decapitation after saliva collection, and saliva was collected for 40 min following 10 min anesthesia induction (b). The open bars show intact control (non - stress) group, and the hatched bars show restraint stress groups . Statistically significant corticosterone levels were determined by student s t - test (* * * p<0.001). Indicates the corticosterone levels of plasma obtained immediately after 60 min restraint stress without anesthesia . The levels of the restraint group were significantly higher than the levels of the intact group (403.0 16.5 vs. 97.9 12.1 ng / ml, p<0.001). Effects of restraint stress (60 min) on corticosterone levels in adult male icr mice treated with or without anesthesia . The restraint group was euthanized by decapitation immediately after the restraint stress (60 min) without anesthesia (a). The intact and restraint groups were euthanized by decapitation after saliva collection, and saliva was collected for 40 min following 10 min anesthesia induction (b). The open bars show intact control (non - stress) group, and the hatched bars show restraint stress groups . Statistically significant corticosterone levels were determined by student s t - test (* * * p<0.001). Comparison of corticosterone levels before and after the restraint stress in plasma and saliva under anesthesia: figure 2b indicates the corticosterone levels of plasma and saliva under anesthesia . The plasma was obtained after the end of the 40 min saliva collection following restraint stress . The plasma corticosterone levels of the restraint group were significantly higher than the levels of the intact group (258.5 11.3 vs. 170.4 6.0 ng / ml, p<0.001). The salivary corticosterone levels of the restraint group were also significantly higher than the levels of the intact group (19.7 1.8 vs. 6.3 1.3 ng / ml, p<0.001). Investigation of the adequate recovery period from anesthesia for saliva collection: the results are shown in fig . 3.fig . 3.examination of adequate recovery period from anesthesia for saliva sampling in stress experiment using adult male icr mice tested as individuals . On 1, 3 and 7 days (day 1, day 3 and day 7, respectively) after the prior saliva collections (intact control groups), saliva was collected again following restraint stress (60 min) by the restrainer . The open bars show intact control groups, and the hatched bars show restraint stress groups . Statistically significant corticosterone levels were determined by student s paired t - test (* p<0.05, * * * p<0.001). The salivary corticosterone levels of the 1-, 3- and 7-day recovery group (day 1, day 3 and day 7) treated with restraint stress were significantly higher than the levels of the respective intact control . The numerical results were as follows: day 1, 11.3 0.9 vs. 8.1 1.2 ng / ml, p=0.0429; day 3, 11.7 1.7 vs. 4.4 0.8 ng / ml, p=0.0165; and day 7, 14.1 1.5 vs. 5.4 0.5 ng / ml, p<0.001 . The ratio of salivary corticosterone levels from the recovery groups with restraint stress was compared with the levels of each intact control group . The 7-day recovery group showed a significant increase in salivary corticosterone levels compared to the intact control group, p=0.0036 . On the other hand, 1- and 3-day recovery groups did not show a significant increase in levels when compared with each intact control group, p=0.1247 and p=0.0610 (data not shown as the figure). In addition to this comparison, the increase of salivary corticosterone induced by restraint stress, was significantly suppressed (p<0.01; one - way anova followed by tukey s test) in 1-day or 3-day recovery (repeated anesthesia) group (fig . The increased corticosterone secretion was not suppressed in the 7-day recovery (repeated anesthesia) group (data not shown). Examination of adequate recovery period from anesthesia for saliva sampling in stress experiment using adult male icr mice tested as individuals . On 1, 3 and 7 days (day 1, day 3 and day 7, respectively) after the prior saliva collections (intact control groups), saliva was collected again following restraint stress (60 min) by the restrainer . The open bars show intact control groups, and the hatched bars show restraint stress groups . Statistically significant corticosterone levels were determined by student s paired t - test (* p<0.05, * * * p<0.001). In this paper, cortisol was detected in both the plasma and saliva as a result of the ip administration of cortisol . On the other hand, cortisol was not detected in the saliva, but was detected in the plasma at very low levels, after treatment with vehicle (fig . Rodents, including mice, possess corticosterone as a glucocorticoid and do not retain endogenous cortisol . In the present study, the anti - cortisol showing 2% cross - reactivity with corticosterone was used for eia . The reason why cortisol was detected in vehicle control group, might be accounted for by the cross - reactivity of anti - cortisol . Cortisol unconjugated with protein in the blood circulation has been reported to pass easily through the acinar cells on the secretory end - piece of the salivary glands . The present data showed that the origin of cortisol detected in mouse saliva is exogenous corticosteroid and that the cortisol was considered to spread to the saliva from the blood via the salivary glands, suggesting that glucocorticoids in saliva may reflect the levels present in the blood circulation . The result of the present study indicated that the restraint stress significantly increased plasma and salivary corticosterone levels (fig . 2), and our observations agreed with previous studies, reporting plasma corticosterone levels in mice and rats treated with restraint stress [9, 22, 28, 29]. In humans, saliva, in addition to blood samples, is readily available for the measurement of cortisol in various stress - related studies [8, 13, 26]. However, there is no scientific literature mentioning salivary corticosterone levels in mice treated with stress, with only one study reporting salivary corticosterone in rats treated with isolation stress . The present report indicates that mouse salivary corticosterone is detected and the level is significantly increased following 60 min restraint stress . These results suggest that salivary corticosterone levels reflect changes in plasma corticosterone levels, caused by induced restraint stress in mice . In the experiments investigating the adequate recovery periods from anesthesia, three different time periods (restraint stress significantly increased salivary corticosterone levels in all three groups (fig . 3); even so, the statistical evidence of corticosterone increase is stronger in a 7-day recovery group (p<0.001) than the others (p<0.05). When comparing the ratio of corticosterone levels of intact control group, restraint stress increased the levels significantly in 7-day recovery group, but did not significantly increase the levels in 1- and 3-day recovery groups (data not shown as the figure). Repeated restraint stress after 7 days, increased the levels of plasma corticosterone . In rats, a study shows an increase of heterogeneous nuclear rna of corticotrophin - releasing hormone in the paraventricular nucleus equivalent to levels in single acute stress while the others experiencing repeated restraint stress (daily, alternate days and at 3 day intervals) increased, but to lower equivalent levels of single acute stress . Moreover, rat serum corticosterone levels elevated by repeated nicotine treatment as a chemical stress, decreased to baseline levels after 7 days withdrawal . Nicotine elevated the rat plasma adrenocorticotropic hormone levels in addition to corticosterone, suggesting that this treatment would induce chemical stress in animals . These previous studies also indicate that the recovery period from physical or chemical stress is estimated at one week . Additionally, the previous studies showing that the diethyl ether anesthesia increased the plasma corticosterone levels in rats [7, 27], indicate that the anesthesia itself may induce a stress response . To evaluate corticosterone secretion in response to restraint stress using saliva samples, it could be possible to set a 1-day recovery period from anesthesia, however, 7-day recovery periods would be preferable for using the same individual mice . When using small experimental animals, such as mice and rats, few studies have examined the evaluation of salivary corticosterone as a stress biomarker, while numerous studies have examined salivary cortisol for stress research in humans [8, 13, 26]. The results of present study may contribute to stress - related research using saliva samples of mice . Saliva collection is a less invasive procedure compared to blood sampling and allows for repeat sampling in the same individual mouse . Moreover, the anesthetic mixture of the three drugs (medetomidine, midazolam and butorphanol) may make it easier for repeated salvia collection . Thus, it is easy to investigate the time course of a response to any stimulus using saliva samples from mice using the same individual . In the laboratory animal science field, russell and burch proposed a practical principle of three rs (reduction, refinement and replacement) in 1959 . Reduction is defined as obtaining equivalent levels of information from fewer animals in scientific investigation or to obtain maximum information without rise in discomfort level from a small number of animals maintained long term . Refinement is also defined as performing appropriate experimental procedures to improve animal well - being and minimize or avoid discomfort . Our proposal techniques in the present study, using saliva sampling to measure levels of corticosterone in it, would make it possible to compliment refinement in addition to reduction practices, since the saliva collection is low invasive technique and is performed under anesthetized condition . In conclusion, we suggest that salivary corticosterone levels reflect plasma corticosterone levels, and thus, it will be a useful and less - invasive biomarker of physical stress in mice . Moreover, to evaluate restraint stress by the corticosterone level in saliva collected from mice under anesthesia, an adequate recovery period of one week is preferable . The present study may contribute to the concepts of reduction and refinement as part of the three rs, in small animal experiments.
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The superior semicircular canal dehiscence (sscd) syndrome was described by lloyd b. minor et al1 in 1998 . Third opening or third window between the superior semicircular canal and the middle cranial fossa, secondary to a bony defect in the canal . This abnormal communication can result in vertigo and oscillopsia induced by loud sounds (tullio's phenomenon), by valsalva maneuvers, or by changes in pressure in the external auditory canal (hennebert's sign). The patients may also present autophony, hyperacusis, pulsatile tinnitus and hearing loss . In patients with debilitating symptoms and low health utility scores, a surgical management may be offered, since the manifestations of the disease and the utility score may improve with surgery . The different surgical techniques used to approach and repair the sscd include the middle cranial fossa approach, the transmastoid approach, the endoscopic - assisted middle cranial fossa approach, and the round window tissue reinforcement . In this review of the literature we analyze this clinical syndrome and its diagnosis, as well as the aforementioned surgical approaches and techniques performed to repair the dehiscence . There are two functional windows in the cochleovestibular system, the oval window, which allows the passage of soundwaves into the inner ear's scala vestibuli through the stapes footplate, and the round window, involved in the release of sound and mechanical energy from de scala tympani out of the inner ear . When a dehiscence in the superior semicircular canal is created, the hydro - acoustic waves flowing through the cochlea are inadvertently transmitted throughout the labyrinthine system . In addition, the intracranial pressure may be transmitted through the dehiscent superior canal, also causing the stimulation of vestibular end organs until the round window releases this pressure.2 in a more detailed manner, a sscd alters the hydrodynamic stability of this system, enabling an exaggerated movement of the endolymph . Since a third window is present, the transmission of pressure through the inner ear may result in an outward bulging of the membranous canal at the bony defect to the side of the middle cranial fossa . Hydraulically speaking, it would serve as a pressure release valve and, therefore, less pressure would be distributed in the inner ear, as it would in a closed hydraulic system.3 when this outward bulging of the membranous labyrinth is produced, the disturbed endolymph fluid motion causes the ampullofugal deflection of the cupula of the superior semicircular canal, which the brain perceives as body movement, thus resulting in vestibular symptoms.4 intracranial pressure fluctuations or negative pressure in the external or middle ear may lead to the contrary, an abnormal reversal of energy through the dehiscence into the labyrinth, resulting in an inward bulging of the membranous canal with a consequent ampullopetal deflection of the cupula.3 4 these theories support the noise or pressure - induced vertigo observed in the sscd syndrome . The increased compliance of the inner ear explained earlier also contributes to the conductive hearing loss and the perception of the pulsatile tinnitus observed in these patients.2 the pathologic opening from the sscd may weaken the energy transmission produced by the movement of the stapes footplate, resulting in the reduction of sound transmission to the cochlea and hearing loss . On the other hand, the normal impedance inequality of the oval and round windows may be altered by the third window, which results in bone hyperconduction that the patient perceives as autophony or hyperacusis.5 the diagnosis is suspected by the clinical manifestations mentioned earlier . Nystagmus evoked by sound or pressure, with eye movements oriented in the same plane as the dehiscent semicircular canal, is a classic feature . Audiometric findings typically include supernormal bone thresholds and a low - frequency conductive hearing loss . The tympanometric examination and acoustic reflexes are normal.6 cervical vestibular evoked myogenic potential (cvemp) testing in patients with sscd syndrome will often have responses with lower thresholds (less than 70 decibels [db] to tone burst testing) than in people without the syndrome.7 in 2012, zuniga et al8 showed that cvemp threshold results showed a sensitivity and a specificity ranging from 80% to 100% for the diagnosis of sscd syndrome . In contrast, the ocular cvemp amplitudes demonstrated a sensitivity and a specificity greater than 90% . Finally, when a sscd syndrome is suspected, a temporal bone computed tomography (ct) scan confirms the diagnosis . The best images are seen in a high resolution 0.5 mm collimation, and projections in the plane of the superior canal (pschl views) and in a perpendicular plane to this same canal (stenvers views).9 two types of sscd have been described after a ct examination: dehiscence of the arcuate eminence or dehiscence in the region of the superior petrosal sinus . Studies have found that magnetic resonance imaging (mri) is also very useful for diagnosing sscd syndrome . In 2013, browaeys et al10 described that when diagnosing sscd syndrome, the mri had a sensitivity of 100%, a specificity of 96.5%, a positive predictive value of 61.1%, and a negative predictive value of 100% in comparison with the ct . The first manifestations of the syndrome usually occur during adulthood; nevertheless, a congenital anomaly is suspected, since thinning of the bone overlying the superior semicircular canal occurs bilaterally in almost one - third of the patients, and there are some cases that began having symptoms during childhood.11 12 in contrast to adults, children with sscd syndrome usually present with auditory symptoms first, and conservative management should be carried out as often as possible.12 another possibility for the pathophysiology of this disease is that it may be an acquired condition . Repeated, low intensity cranial trauma from combat sports or diving, for instance, has been related to this disease . A defect in the floor of the middle cranial fossa, at the level of the canal, may also occur secondary to increments in the pressure of the cerebrospinal fluid.11 patients with mild symptoms should be treated with conservatively . For patients with debilitating symptoms and lower health utility scores, both the clinical manifestations of the disease and the so - called health utility scores improve after surgery.13 when surgery is necessary, the bony dehiscence can be resurfaced, plugged or capped by different surgical approaches.14 15 16 17 18 this approach to treat sscd syndrome was first described by minor et al.14 a 4 4 cm craniotomy is drilled . The temporal lobe is retracted, and the arcuate eminence is identified . At this point the canal may also be capped or resurfaced with bone pate, bone wax, hydroxyapatite cement or soft tissue . The advantages of the middle fossa craniotomy include the direct access to the arcuate eminence defect without the need for labyrinthine bone removal and the exposure of the surrounding cranial base if the repair is needed for other associated tegmen defects . Concomitant resurfacing of the tegmen mastoideum and tympani is performed, since the floor of the middle cranial fossa is usually thin . The surgical technique begins with a post auricular incision, as it usually performed for a mastoidectomy . Supra and subperiosteal flaps are made, and then a mastoidectomy is performed using a cutting burr with suction irrigation . The sigmoid sinus, the posterior and middle fossa dura, and the pre - sigmoid area are all skeletonized . The horizontal, posterior and superior semicircular canals are identified and skeletonized with a diamond burr . The area of dehiscence is identified, and the middle fossa dura is carefully elevated from de dehiscent superior semicircular canal . In patients with dehiscence at the superior petrosal sinus, the sinus was exposed at the sinodural angle, posterior to the solid angle, and followed to the superior canal.16 two points of the bony labyrinth are fenestrated with a 1 mm diamond burr, and the endosteum is opened just inferior to the fenestrated apex of the superior canal, on the ampullated and non - ampullated portions of the canal.19 care is taken to avoid suction or manipulation of the membranous labyrinth to prevent hearing loss or chronic disequilibrium after surgery.20 bone dust, bone wax, bone pate, fascia and even bone chips may be used to fill the lumen of the superior canal at the points of fenestration (plugging the canal). If bone wax is used, cheng et al21 recommend that the applications of two wax spheres, 2 mm in size, is sufficient to occlude the canal without risk of damaging the neuroepithelium, which prevents vestibular hypofunction and hearing loss after the repair . Conchal cartilage is harvested and placed in an intracranial extradural position, repairing the middle cranial fossa floor defect, and finally the wound is closed in a regular fashion.19 when the transmastoid middle fossa resurfacing is selected, after dissecting the middle cranial fossa dura from the dehiscent canal, tragal perichondrium is placed in the space between the dura and the dehiscence by folding it over the top of an annulus elevator and inserting it over the superior canal . The dura and overlying temporal lobe or superior petrosal sinus will stabilize the position of the graft, with no tendency to extrude.16 it is well known that the transmastoid approach avoids the risks of performing a craniotomy in the middle fossa, resulting in lower morbidity and a shorter hospital stay . The disadvantage of transmastoid procedures is that the dehiscence is visualized with more difficulty compared to the middle fossa approach.22 another limitation is when the tegmen hangs too low, precluding the safe exposure and manipulation of the dura just lateral to the superior semicircular canal . A preoperative ct scan is very important in order to anticipate this difficulty, and it could precisely identify the size, extension and location of the dehiscence . The endoscopic - assisted middle fossa procedure appears to be advantageous when compared with binocular microscopy, since it provides a high definition view of the middle cranial fossa . In this surgical technique, following the incision, a temporalis fascia graft is harvested . A periosteal flap is made, and a minicraniotomy (3 2 cm) is performed . Under microscopy, the dura is gently elevated off the tegmen mastoideum and tegmen tympani until the arcuate eminence is visualized . Then, a 3 mm wide, 14 cm long, 30 rigid endoscope is introduced in order to visualize the defect . The house - urban middle fossa retractor is used to maintain the retraction of the temporal lobe while working with the endoscope . The dura is dissected from the medial aspect of the defect under endoscopic view . At this point, a gentle occlusion of the ampullated and non - ampullated limbs of the defect is performed using bone wax.15 peng et al23 reported 10 cases managed with the endoscopic - assisted middle fossa technique in which they used hydroxyapatite bone cement to resurface the superior canal with good results . In 2014, carter et al18 reported their experience with 5 patients managed endoscopically . They mentioned that endoscopy enhances the visualization of the superior canal defect, allows for transillumination, and reduces temporal lobe retraction, making it a useful adjunct in craniotomies used to repair sscd defects . This surgical technique is performed using a traditional tympanomeatal flap approach under general or local anesthesia . If necessary, a drill or curettage is used to enlarge the posterior auditory canal wall in order to gain adequate exposure of the round window niche . This niche and the promontory are denuded of mucosa, and the round window is reinforced with temporalis fascia, tragal cartilage, perichondrium, fat or connective tissue.24 the tympanomeatal flap is repositioned and the external auditory canal is packed . The round window reinforcement is a procedure with low risk compared to the middle fossa or transmastoid approaches, and it may be offered as a first procedure in patients with mild symptoms . Complete occlusion of the round window is another technique that has been used for sscd syndrome, but some authors suggest that it should not be recommended, since it has been noticed that symptoms may become worse in the late postoperative period.24 different studies have shown that the combination of plugging and resurfacing achieves better long - term control of the symptoms than resurfacing alone . When only resurfacing is performed, a complete sealing of the defect is not guaranteed, and this area may remain sensitive to pressure changes.25 26 goddard and wilkinson,22 in 2014, showed excellent hearing outcomes and a reduction of the preoperative symptoms in 24 ears in which the plugging technique was used . A meta - analysis of 64 surgical procedures for sscd syndrome indicated that capping and plugging had a significantly higher success rate when compared to resurfacing.27 this approach to treat sscd syndrome was first described by minor et al.14 a 4 4 cm craniotomy is drilled . The temporal lobe is retracted, and the arcuate eminence is identified . At this point the canal may also be capped or resurfaced with bone pate, bone wax, hydroxyapatite cement or soft tissue . The advantages of the middle fossa craniotomy include the direct access to the arcuate eminence defect without the need for labyrinthine bone removal and the exposure of the surrounding cranial base if the repair is needed for other associated tegmen defects . Concomitant resurfacing of the tegmen mastoideum and tympani is performed, since the floor of the middle cranial fossa is usually thin . The surgical technique begins with a post auricular incision, as it usually performed for a mastoidectomy . Supra and subperiosteal flaps are made, and then a mastoidectomy is performed using a cutting burr with suction irrigation . The sigmoid sinus, the posterior and middle fossa dura, and the pre - sigmoid area are all skeletonized . The horizontal, posterior and superior semicircular canals are identified and skeletonized with a diamond burr . The area of dehiscence is identified, and the middle fossa dura is carefully elevated from de dehiscent superior semicircular canal . In patients with dehiscence at the superior petrosal sinus, the sinus was exposed at the sinodural angle, posterior to the solid angle, and followed to the superior canal.16 two points of the bony labyrinth are fenestrated with a 1 mm diamond burr, and the endosteum is opened just inferior to the fenestrated apex of the superior canal, on the ampullated and non - ampullated portions of the canal.19 care is taken to avoid suction or manipulation of the membranous labyrinth to prevent hearing loss or chronic disequilibrium after surgery.20 bone dust, bone wax, bone pate, fascia and even bone chips may be used to fill the lumen of the superior canal at the points of fenestration (plugging the canal). If bone wax is used, cheng et al21 recommend that the applications of two wax spheres, 2 mm in size, is sufficient to occlude the canal without risk of damaging the neuroepithelium, which prevents vestibular hypofunction and hearing loss after the repair . Conchal cartilage is harvested and placed in an intracranial extradural position, repairing the middle cranial fossa floor defect, and finally the wound is closed in a regular fashion.19 when the transmastoid middle fossa resurfacing is selected, after dissecting the middle cranial fossa dura from the dehiscent canal, tragal perichondrium is placed in the space between the dura and the dehiscence by folding it over the top of an annulus elevator and inserting it over the superior canal . The dura and overlying temporal lobe or superior petrosal sinus will stabilize the position of the graft, with no tendency to extrude.16 it is well known that the transmastoid approach avoids the risks of performing a craniotomy in the middle fossa, resulting in lower morbidity and a shorter hospital stay . The disadvantage of transmastoid procedures is that the dehiscence is visualized with more difficulty compared to the middle fossa approach.22 another limitation is when the tegmen hangs too low, precluding the safe exposure and manipulation of the dura just lateral to the superior semicircular canal . A preoperative ct scan is very important in order to anticipate this difficulty, and it could precisely identify the size, extension and location of the dehiscence . The endoscopic - assisted middle fossa procedure appears to be advantageous when compared with binocular microscopy, since it provides a high definition view of the middle cranial fossa . In this surgical technique, following the incision, a periosteal flap is made, and a minicraniotomy (3 2 cm) is performed . Under microscopy, the dura is gently elevated off the tegmen mastoideum and tegmen tympani until the arcuate eminence is visualized . Then, a 3 mm wide, 14 cm long, 30 rigid endoscope is introduced in order to visualize the defect . The house - urban middle fossa retractor is used to maintain the retraction of the temporal lobe while working with the endoscope . The dura is dissected from the medial aspect of the defect under endoscopic view . At this point, a gentle occlusion of the ampullated and non - ampullated limbs of the defect is performed using bone wax.15 peng et al23 reported 10 cases managed with the endoscopic - assisted middle fossa technique in which they used hydroxyapatite bone cement to resurface the superior canal with good results . In 2014, carter et al18 reported their experience with 5 patients managed endoscopically . They mentioned that endoscopy enhances the visualization of the superior canal defect, allows for transillumination, and reduces temporal lobe retraction, making it a useful adjunct in craniotomies used to repair sscd defects . This surgical technique is performed using a traditional tympanomeatal flap approach under general or local anesthesia . If necessary, a drill or curettage is used to enlarge the posterior auditory canal wall in order to gain adequate exposure of the round window niche . This niche and the promontory are denuded of mucosa, and the round window is reinforced with temporalis fascia, tragal cartilage, perichondrium, fat or connective tissue.24 the tympanomeatal flap is repositioned and the external auditory canal is packed . The round window reinforcement is a procedure with low risk compared to the middle fossa or transmastoid approaches, and it may be offered as a first procedure in patients with mild symptoms . Complete occlusion of the round window is another technique that has been used for sscd syndrome, but some authors suggest that it should not be recommended, since it has been noticed that symptoms may become worse in the late postoperative period.24 different studies have shown that the combination of plugging and resurfacing achieves better long - term control of the symptoms than resurfacing alone . When only resurfacing is performed, a complete sealing of the defect is not guaranteed, and this area may remain sensitive to pressure changes.25 26 goddard and wilkinson,22 in 2014, showed excellent hearing outcomes and a reduction of the preoperative symptoms in 24 ears in which the plugging technique was used . A meta - analysis of 64 surgical procedures for sscd syndrome indicated that capping and plugging had a significantly higher success rate when compared to resurfacing.27 even though the conservative management, which avoids the precipitating stimuli and vestibular rehabilitation, is most often indicated, patients with debilitating symptoms may be offered surgical repair of the sscd . The middle fossa approach gives a better view of the dehiscence, which may prove beneficial at the moment of the repair, but comes with a higher morbidity than the transmastoid route . Endoscopic assistance may also improve the middle cranial fossa approach, giving it an advantage . The plugging and capping techniques are associated with higher success rates than resurfacing, with no added risk of hearing loss; thus, those techniques should be recommended.
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Continued improvements in therapeutic medicine have resulted in prolonged survival of patients with cancer, and consequently, physicians must manage the long - term complications of cancer and its treatment . Bone metastases are a common complication in patients with a variety of solid tumors,1 and the skeleton is 1 of the 3 most frequent sites of metastasis (including lung, liver, and bone).2 all cancers have the potential to metastasize to bone; however, tumors of the breast, prostate, lung, kidney, and thyroid do so most frequently.1 these tumors show an intense osteotropism and represent approximately 80% of cases of bone metastases . A devastating complication for patients, the development of bone metastases signals that their disease has become incurable . Furthermore, bone metastases can result in potentially debilitating skeletal - related events (sres) including pathologic fracture, the need for orthopedic surgery to treat or prevent a pathologic fracture, spinal cord compression, severe bone pain requiring radiotherapy, and potentially life - threatening hypercalcemia of malignancy . Treatment of patients with bone metastases should involve a multidisciplinary team of experts, including oncologists, radiation oncologists, and orthopedic surgeons, with the primary goals of relieving pain, improving quality of life, preventing sres, and restoring functional independence, to minimize the impact on patients lives . Management options for patients with bone metastases include pharmaceutical agents (eg, bisphosphonates [bps] and analgesics), radiotherapy, and surgery . These treatments are normally used in combination, depending on the severity of bone destruction and the life expectancy of the patient . Analogues of pyrophosphate, bps have a high affinity for the mineralized surface of bone and were initially recognized for their ability to reduce bone resorption by inhibiting osteoclasts . However, preclinical studies have demonstrated direct and indirect anticancer activities for some bps, such as reducing cancer cell proliferation, inducing cancer cell apoptosis, antiangiogenic effects, and inhibiting cancer cell adhesion and invasion of the extracellular matrix.3 additionally, bps have been shown to reduce bone tumor area in multiple animal models.3 moreover, zoledronic acid (zol) has demonstrated anticancer benefits in some early breast cancer trials and in other settings, including metastatic disease.4 two different types of bps are currently utilized for treating patients with bone metastases from breast cancer those that contain nitrogen and those that do not . Those without nitrogen are known as first - generation bps (eg, clodronate) and inhibit osteoclast- mediated bone resorption mostly via inhibition of mitochondrial atp . Nitrogen - containing bps (eg, pamidronate, risedronate, ibandronate, and zol) prevent bone resorption by inhibiting farnesyl diphosphate synthase . On the basis of systematic review and meta - analyses of published data from clinical trials of bps,5,6 it is clear that bps reduce sre risks in patients with metastatic bone disease from breast cancer . Furthermore, zol has been shown to significantly delay the onset of sres and reduce the ongoing risk of sres, supporting initiation of therapy as soon as bone metastases are diagnosed and continuing until performance status significantly declines.6,7 to date, the majority of evidence supports the use of the intravenous (iv) nitrogen - containing bp zol for preventing sres in patients with multiple myeloma or bone metastases secondary to any solid tumor.6,8 when administered intravenously, bps have been associated with a transient acute - phase reaction including fever, arthralgia, and bone pain (described as flu - like symptoms). These reactions generally occur with the first infusion only, are usually self - limiting, resolve within 1 to 2 days of administration, and can typically be managed with nonprescription analgesics.9 in the majority of patients, reactions are infrequent with subsequent infusions.9 the underlying cause of the characteristic acute - phase reaction with iv zol is believed to be through transient release of cytokines such as tumor necrosis factor alpha and interferon from immune cells and activation of the immune system (eg, v9v2 t cells) against cancer cells.10 we report here a case study documenting a dramatic difference between the safety profiles of a generic zol and the brand - name zol formulations . A 50-year - old hispanic woman presented in january 2007 with cancer in her right breast (stage iiia, t3, n2, m0). A tru - cut biopsy was performed, revealing poorly differentiated, infiltrating, her2/ neu- negative ductal carcinoma with vascular permeation and estrogen- and progesterone - receptor strong positive staining in 10% of cells . The patient received 4 cycles of neoadjuvant 5-fluorouracil, epirubicin, and cyclophosphamide (fec) chemotherapy with good clinical response, followed by right modified radical mastectomy in may 2007 . Postmastectomy histopathology revealed multicentric, poorly differentiated, infiltrating ductal carcinoma, with 5 of 10 nodes positive . She received adjuvant therapy with 4 cycles of docetaxel and radiotherapy, followed by sequential endocrine therapy with tamoxifen for approximately 6 months followed by letrozole, beginning in november 2007 . In march 2009, after 22 months of adjuvant endocrine therapy and surveillance, the patient reported lower back pain . A bone metastasis was detected in the lumbar spine, for which she received external beam radiotherapy to the lumbar spine and began therapy with the brand - name zol (zometa; 4 mg every 34 weeks) plus second - line adjuvant letrozole . She received zometa for the first 3 cycles with good tolerance, and reported no acute adverse events . As of june 2009, our institution s policies dictated that she receive generic zol for continued monthly bp therapy . On infusion of generic zol, the patient experienced extreme weakness, nausea, vomiting (all grade 2, as defined by common terminology criteria for adverse events [ctcae]), and incapacitating bone pain (ctcae grade 3). As a result, the use of weak opioids such as tramadol (50 mg iv every 8 hours) in conjunction with paracetamol (500 mg every 8 hours) was necessary for pain control, and the patient had to be hospitalized for 2 to 3 days of evaluation and monitoring after each of the 2 generic zol infusions received . The severity of adverse events experienced by the patient was the same after both the first and second infusions of generic zol . Because of the noticeable differences in toxicity profiles between generic zol and zometa, the patient (under her physician s care) decided to purchase zometa and assess tolerability . Thereafter, the patient continued therapy with zometa without complications, and experienced a meaningful reduction in bone pain and improved mobility until eventually succumbing to her disease in february 2011 . Written informed consent was obtained from the patient s family for publication of this case report . Infusion of iv bps, including zol, is known to be associated with a transient acute - phase reaction (flu - like symptoms) that is generally mild and manageable with nonprescription analgesics . Here we have reported a case in which generic zol resulted in hospitalization of the patient because of nausea, vomiting, severe bone pain, and weakness . These debilitating symptoms resulted in increased use of medical resources including nursing care, laboratory tests, and pharmaceuticals including opioids, in addition to standard hospitalization fees . Because the patient had already received zometa without experiencing these acute complications, it was very likely that the new generic zol was responsible for the differences in tolerability . This suggests that the safety profile of zometa was better than that of the generic zol used in this patient . In the case reported herein, we observed a clear increase in the severity of adverse events associated with the use of generic zol . The same phenomenon of increased toxicity with generic zol has been observed in numerous other patients at our institution; however, until now it was not possible to make direct comparisons between generic zol and zometa in the same patient . In this case study experience, the generally mild adverse events were amplified so much that the patient required hospitalization after generic zol infusion, which is of great concern . Furthermore, although not observed in this case report, we have observed increases in the frequencies of more severe adverse events such as kidney damage and osteonecrosis of the jaw (onj) in patients receiving generic zol . Interestingly, according to the package inserts, both the active substance (4 mg zol) and inactive ingredients (mannitol, sodium citrate, and sterile water for injection) were the same for the generic zol and zometa formulations . Therefore, the precise reason for the new, acute adverse events experienced after the generic zol infusions remains unknown; however, we cannot rule out that patient awareness of the change from zometa to generic zol may have at least contributed to the perceived severity of her symptoms . We believe strongly that before institutions can ethically require the substitution of generic zol for zometa, it will be important to closely monitor and re - evaluate both the efficacy and safety of generic zol formulations in patients with bone metastases . Our institutional experience suggests that the generic zol used in our patient presents a potential danger to patient safety, perhaps resulting from insufficient manufacturing and/or testing processes . Clearly, more information is needed to validate the safety and efficacy of generic zol formulations, and this will be of direct interest to oncologists, but will also have relevance to all healthcare professionals who are confronted by decisions regarding the increasing number of generic drug choices . Those of us who work in healthcare are entrusted with our patients best interests, and should be highly concerned about the quality and regulation of generic pharmaceuticals such as the emerging formulations of zol . Ideally, in the future it will be possible to have international regulatory bodies with the resources and power to monitor and regulate the day - to - day quality of generic medicines, particularly in emerging or developing countries . However, until that time, it is important that treating physicians monitor patients under their care to ensure their safety when new drug formulations are introduced into clinical practice . Although these may be silent substitutions enacted by pharmacists, unexpected toxicities, such as those observed in our patient treated with generic zol, can alert us to emerging safety concerns . By reporting this case study, we hope to increase vigilance and allow more rapid identification and management of toxicities with substandard zol formulations and allow other patients to benefit from our experience.
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One of the most major public health problems over the world is cardiovascular disease including coronary heart disease (chd), peripheral vascular disease and stroke.1 countries in the middle east bear a heavy burden from chd . The prevalence of chd is due to high rate of its risk factors, particularly obesity, smoking, hypertension (htn), dyslipidemia, diabetes, and sedentary lifestyles . Patients in the middle east present with myocardial infarction at a younger age, on average, compared with patients elsewhere . The projected future burden of mortality from chd in the middle east is set to outstrip that observed in other geographical regions.2 improved socioeconomic conditions in saudi arabia during the past few decades have been accompanied by rapid changes in the lifestyle of the people, particularly unhealthy dietary patterns, and physical inactivity . Consequently, chronic diseases commonly associated with affluent societies, such as chd, diabetes mellitus (dm), obesity have become the dominant illnesses in saudi arabia . Hospital - based studies have shown that smoking, htn, and dm are common risk factors among patients with acute myocardial infarction . A recent study on chd mortality in the eastern province of saudi arabia, using proportionate mortality ratio, showed that 26% of deaths were caused by chd (27.0% of male and 23.5% of female deaths).3 in 2013, there was study on the prevalence of the obesity in saudi arabia which showed that 28.7% were obese (body mass index [bmi]> 30). Obesity consider as an important risk factor of chd.4 one study in the east province of saudi arabia in 2008 showed that 21% was the prevalence of htn in screening campaign and increase with age, lower educational level, and divorced women.5 in 2009, there was study on the prevalence of dm in saudi arabia . The prevalence of dm was 30% and dm increase with obesity and female gender, who more than 50 years compare to male group.6 a national study measured hyperlipidemia and 5-year risk of development of chd among university and school workers in jeddah, saudi arabia . About half of the participants were overweight, 18.8% were current cigarette smokers and 19.9% were hypertensive . Hyperlipidemia was present in 10.1% and was significantly related to older age (40 years and over) and place of work . According to the previous study, the estimated risk of chd in the coming half - decade for those classified in the fifth quintile was 0.068.7 the government of saudi arabia is becoming increasingly concerned about the high prevalence of chd risk factors . Knowledge of the causative factors and methods of prevention of chd are essential to reduce morbidity and mortality from them . The objective of this study is to determine the prevalence of chd risk factors among the faculty and staff members of qassim university and also to assess their knowledge of the risk factors of chd and compare the result with other prevalence of risk factors in other area of saudi arabia . A cross - sectional study was conducted among the faculty and the staff members of qassim university for male and female . All employees were invited to voluntarily participate in the study by advertisement through email and social network . Data were collected through a self - administered health risk assessment questionnaire, as well as through clinical screening that included measurement of blood pressure, fasting blood glucose (fbg) and lipid profile . The employees working under contract for an outsourcing company (for example, the housekeeping staff) were excluded . Participants were recruited through general announcements to staff, as well as among patients visiting the clinic . This sample is expected to provide sufficiently reliable estimates of the most common risk factors among university staff and faculty . A self - administered questionnaire was used to collect information on sociodemographic, biological, and behavioral risk factors . Information was collected on personal, past and family history of chronic diseases, cigarette smoking, and stress experienced in the workplace . We also documented the level of willingness of the participants to engage in a behavior change program . Data were focused on providing point estimates for the risk factors of chd and testing hypotheses regarding expected correlations between these risk factors . The odd ratio and 95% confidence interval were calculated . We present the results of analysis of data from 233 faculty / staff members (157 males, 67%; 76 females, 33%). More than half of the participants were in the 30 - 49 year age group . 53.6% of study participants have family history of any of risk factor of chd and 20.6% of the staff members and faculties were obese 10.3% were diabetic . Sociodemographic data and risk factor status the most common age group for male who are overweight is more than 50 and for obese is between 30 and 50 years . For female gender we can find the most common age group for overweight and obese is between 30 and 50 years (35.7% and 30.2%), respectively (table 2). Prevalence of obesity among male and female there were 16 of the staff members newly diagnosed to have impaired fasting glucose (13 male, 3 female). 20 of the staff members and faculties were found to have newly diagnosed dm . When the age increase in both gender, the chance of development of dm will be increase with no significant p value as shown in table 3 . Regarding the blood pressure, we found the blood pressure was increased with age and there was no significant difference between male and female (table 3). Prevalence of dm and htn according to the gender and age group in table 4, we found that the smoking behavior is among male gender only and there was no smoking behavior among female gender . Relation of gender, age group, and smoking status youngest age group <30 was expected to have increase in high - density lipoprotein (hdl) in both gender (male and female). Low - density lipoprotein (ldl) more than 190 mg / dl in age group more than 30 years for both genders (male and female) as shown in table 5 . In our study, the prevalence of dm, dyslipidemia, and smoking was found to be lower than other studies.8,9 according to the 2011 american diabetic association guidelines, based on fbg, 6.9% of the participants were found prediabetic and 8.6% having diabetes . Fbg values were found significantly higher with increasing age especially among more than 50 years old . No statistically significant difference was found among males or females in the prediabetic category (p = 0.441). The ldl levels were found to increase with age with participants more than 50 years of age having ldl values> 190 mg / dl (p = 0.001). A study carried out among saudi diabetic patients attending primary health - care (phc) service concludes that uncontrolled diabetes was found to be the common risk factor followed by uncontrolled lipid profile, obesity, uncontrolled systolic blood pressure, and smoking . The result of study revealed that seven percent (7%) of male group felled in highest risk group in comparison with 1% in female group (p <0.05), while 31% in male group felled in mild risk group in comparison with 90% in female group (p 62% in male group felled in high - risk group in comparison with 9% in female group (p <0.05).10 the directorate of scientific research at king abdul aziz city for science and technology recognized that there is a major transition going on in the kingdom in terms of lifestyle, featuring a more sedentary lifestyle, increased consumption of fast food and unhealthy diet . These changes were associated with rapid socioeconomic development, urbanization, and increased purchasing power of the population.2 various other studies in the kingdom have similarly indicated a rise in the lifestyle - related risk factors.11,12 moreover, dm and obesity are the leading risk factors associated with chd . Their study done in king faisal specialist hospital in 2011 showed the prevalence of dm was 30%.6 while the prevalence of obesity is 64% and 70% among males and females, respectively.13 our study aimed to estimate the prevalence of risk factors of chd among qassim university s staff / employees . The overall prevalence of these risk factors (dm, dyslipidemia, obesity and overweight, smoking, and sedentary lifestyle) which is found to be 30% in our study was similar to several other studies done elsewhere in similar setups in educational institutions,12 but was significantly lower than the general population in saudi arabia . Chd in saudi arabia study found a high prevalence of htn (26%) and diabetes (22%), together with a prevalence of chd of 6%, once again higher in the urban setting . Urban location, age, male gender, bmi, htn, smoking, high serum cholesterol, and triglycerides were significant risk factors for chd.14 in a similar study carried out in jeddah in 2007 among university employees and school teachers estimated the prevalence of htn, smoking, dyslipidemia, and overweight to be 19.9%, 18.8%, 10.1%, and ~50%, respectively.15 a study at qassim university conducted in 2007 estimated the prevalence of low hdl was 73.6% and increased total cholesterol and triglycerides 60.0% and 46.4%, respectively.16 another study among attendees of a phc center in south - west region of saudi arabia found the prevalence of risk factors as follows: diabetes (28.2%), obesity (37.9%), and physical inactivity (68.3%). Less than half of the study participants knew about these risk factors and their importance as preventive measures for noncommunicable diseases . Knowledge of risk factors and prevention was significantly associated with educational level.17 a study in al - rabhwa phc center, riyadh in 2004 among diabetic patients found increased cholesterol (5.2 high triglyceride (1.7%) was 50% in both genders; 13.4% of males were hypertensive as against 44.3% females . There was no significant difference between risk factors for chd and duration of diabetes except that there were more smokers among those who had diabetes for <10 years . Most of the diabetics with poor glycemic control (fasting blood sugar> 8.3 mmol / l) tended to be smokers, were more obese, had high triglyceride and high total cholesterol.18 the current focus of services at most phc centers and hospitals is curative, not preventive . Health promotion and health education are currently mostly missing from the health - care system of saudi arabia . Phc centers can fill this gap by providing health promotion and education programs, which have been found to play a significant role in improving lifestyle practices . This could significantly reduce the burden of chronic noncommunicable disease.19 our study has a number of limitations . It was conducted in a university setting and so the results may not be generalized to the population . Furthermore, the study sample includes only those employees who opted for medical checkup, further restricting the generalizability of our results . The health risk assessment relied on self - report . In our study, interviewing onsite might have caused limitation in the study as it might have resulted in underestimation in one or overestimation of the figures in other areas . It is emphasized that only those subjects, who were interested in knowing about their health status might have participated in the study resulting in overestimation of the general health status of the university employees . It was conducted in a university setting and so the results may not be generalized to the population . Furthermore, the study sample includes only those employees who opted for medical checkup, further restricting the generalizability of our results . The health risk assessment relied on self - report . In our study, interviewing onsite might have caused limitation in the study as it might have resulted in underestimation in one or overestimation of the figures in other areas . It is emphasized that only those subjects, who were interested in knowing about their health status might have participated in the study resulting in overestimation of the general health status of the university employees . The population risk for the development of chd in the coming decades is not trivial . Short - and long - term strategies are recommended to decrease the risk of chd and improve the quality of life . Health education and disease promotion program as worksite wellness program is now recognized almost universally as an important intervention to reduce the occurrence and severity of diabetic complications, and also in reducing the delays to seek care when the first signs and symptoms of a complication are noticed by the patient . Qassim university s employees worksite wellness program is already in place, although it needs to be up scaled and expanded to other universities in saudi arabia mandatory maintenance of a personal health profile, at within the campus clinic with periodic checkups linked with some incentives and gifts are some of the options that could also be used to combat with the situation . Moreover, the university also has the responsibility to develop interventions to be implemented at the community level . Prevention research is needed to help design health promotion and disease prevention programs to be implemented through phc centers; in this regard, a number of studies are already going on with assistance from qassim university s faculty . Further research studies are required to find out the exact current status and how and what are the best available measures and strategies that can be adapted and could be found useful in combating the situation and improving the health status of the qassim university s staff . Research studies at the community and mass scale level are also required to find out the current status of chronic diseases as risk factors for chd and the best behavioral modification, health educational, and other strategies to address the current situation in the general local population.
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Giving birth is one of the most amazing physiologic events in human life and it can be safe and pleasant in many circumstances, but in some situations it might end with known difficulties and complications for mother and her fetus . It means regular uterine contractions with adequate frequency and intensity resulting in giving birth before 37 completed weeks of pregnancy . Preterm birth is the major reason of the neonatal death and it is responsible for about one third of neonatal mortality in the world [1, 2]. Who publication mentions that each year more than 1 million babies die from complication of preterm delivery and the annual numbers of premature newborns are about 15 millions and this number is rising each year . Based on who publications, un millennium development goal 4 is reduction of children mortality in two - thirds till 2015 by special attention to neonatal deaths in countries where the neonatal mortality is higher than other places . A great number of the global neonatal deaths happen in asia . Finding the ways to prevention of preterm birth is a very important issue for health care providers . There are a lot of modalities to achieve this goal but none of them has been introduced as the best way and the role of different methods in preventing preterm labor varies in different places . Magnesium sulphate (mgso4) as the major tocolytic agent has been used by many medical centers as the first line therapy to inhibition of preterm labor . However, women receiving mgso4 should be closely monitored during treatment; also the severe pain and discomfort in injection site in case of i m administration is a problem with this item . On the other hand, some studies have mentioned that mgso4 cannot be so effective in suppression of preterm labor . Prostaglandin producing by the decidua and fetal membranes might be one of the essential events of parturition that is followed by initiation of the uterine contraction . It seems likely that prevention of producing prostaglandins or suppression of their effects can stop preterm uterine contraction . So in theory inhibitors of prostaglandin synthesis (including cyclooxygenase inhibitors such as celexib) are capable of arresting uterine contractility and they can prevent preterm birth . Celecoxib is a sulfa nonsteroidal anti - inflammatory drug (nsaid) and selective cox-2 inhibitor . It is known under the brand name celebrex or celebra and onsenal used in the treatment of inflammatory diseases and painful menstruation, menstrual symptoms, and so forth . Labor was arrested for 48 h in 42 (81%) and 45 (87%) of the patients in the celecoxib and magnesium sulfate groups, respectively (p-0.298). We aimed to use celecoxib as a cyclgenase inhibitor to suppression of preterm labor instead of magnesium sulfate (mgso4) to prevent preterm labor . It was a single blinded randomized clinical trial study which had been done on 600 pregnant women with gestational age> 24 weeks and <34 weeks; those had referred to ahvaz educational hospitals from march to august 2013 . These criteria were as follows: at least 4th uterine contractions during 20 minutes or 8th uterine contractions during 1 hour followed by progressive changes in the cervical dilatation and effacement, the cervical dilatation of more than 2 cm, the cervical dilatation of more than 80% at the beginning . At least 4th uterine contractions during 20 minutes or 8th uterine contractions during 1 hour followed by progressive changes in the cervical dilatation and effacement, the cervical dilatation of more than 2 cm, the cervical dilatation of more than 80% at the beginning . Informed written consent was obtained from all subjects and they received preterm labor routine and maintenance therapy including bed rest and hydration (bolos infusion of 500 cc ringer lactate during half and over, corticosteroid therapy) and if the uterine preterm contractions did not stop with these modalities, all patients were divided in two groups by simple random sampling; the first group has taken 4 gr of mgso4 intravenously as loading dose and 1 gr per hour as maintenance dose till suppuration of the uterine contractions or maximum 48 hours and in this period the patients were observed for signs of mgso4 toxicity by control of the urine output, deep tendon reflex, and respiratory rate . Conditions of the uterine contractions were checked every 2 hours in 20-minute periods . Also the patients were observed for symptoms of headache; the patients' vital signs were checked as routine protocol . The second group had taken 100 mg celecoxib (one capsule) orally every 12 hours till suppuration of the uterine contractions or for maximum duration of 48 hours . The patients in this group were checked for nausea and vomiting and skin rush and headache . In all subjects if the uterine preterm contractions did not stop with those medications, so immediatry drug has been stopped . Gestational age 24 weeks and 34 weeks with diagnosis of preterm labor based on upper criteria . Gestational age 24 weeks and 34 weeks with diagnosis of preterm labor based on upper criteria . Existing problems that interfere with normal labor process and might need early termination of pregnancy: premature preterm rapture of membrane (pprom), evidences of placental abruption in clinical and ultrasound examination, and ultrasound, maternal fever chorioamnionitis, none reassuring fetal heart rate (fhr) patterns.contraindication for stopping preterm labor uterine contraction: some maternal medical conditions such as preeclampsia, any signs of vaginal bleeding, fetus anomaly, intrauterine fetal death (iufd), and fetal growth restriction (fgr).issues related to mediations: history of being allergic to nonsteroidal anti - inflammatory drugs (nsaids), renal or hepatic dysfunction, concurrent use of other medications except supplements, and history of peptic ulcer.the patients' disagreement to enter the study.stopping the uterine preterm contractions after initial preterm labor maintenance therapy . Existing problems that interfere with normal labor process and might need early termination of pregnancy: premature preterm rapture of membrane (pprom), evidences of placental abruption in clinical and ultrasound examination, and ultrasound, maternal fever chorioamnionitis, none reassuring fetal heart rate (fhr) patterns . Contraindication for stopping preterm labor uterine contraction: some maternal medical conditions such as preeclampsia, any signs of vaginal bleeding, fetus anomaly, intrauterine fetal death (iufd), and fetal growth restriction (fgr). Issues related to mediations: history of being allergic to nonsteroidal anti - inflammatory drugs (nsaids), renal or hepatic dysfunction, concurrent use of other medications except supplements, and history of peptic ulcer . The patients' disagreement to enter the study . Stopping the uterine preterm contractions after initial preterm labor maintenance therapy . The study protocol was approved by ethics committee of ahvaz jundishapur university of medical sciences . All patients provided written informed consent the data were entered and analyzed using spss 11 (spss inc ., chi - square and independent t - test were used for statistical analysis . For all analyses, the finding of this study has shown that mean age of all subjects was 26.03 4.33 in mgso4 group and in celecoxib group was 26.6 4.01, 25.8 4.63, respectively . With using independent t - test, there were no significant differences between mean age subjects in two groups (p = 0.194). Frequencies of gestational age at time of delivery in all subjects were 31.74 1.86 weeks and in mgso4 group and celecoxib group were 32.1 1.82 and 31.38 1.83 weeks, respectively . The lowest gestational age was 27 weeks and the greatest gestational age was 34 weeks . Frequencies of nulliparity in all pregnant women were 350 (58.3%), in mgso4 group were 174 (58%), and in celecoxib group were 176 (58.7%). Frequencies of history of preterm delivery in celexib group were 12 (4%) and in mgso4 group were 12 (4%). There were no significant differences between two groups (mgso4 group and celecoxib group) in frequency of history of preterm labor (p = 1), frequencies of nulliparity (p = 0.99), duration of drug use and arrest contraction (p = 0.29), delivery before 48 hours (p = 0.20), and mean gestational age in lack of response to treatment (p = 0.24) (tables 1 and 2). There was significant association between two groups (mgso4 group and celecoxib group) in frequency of effacement (p <0.0001), gestational age (p <0.0001), and dilation (p = 0.06) (table 3). About two - thirds of neonatal mortality are due to preterm birth, so preterm labor suppression is a big deal for health care providers . On the other hand decreasing childhood mortality . A similar study has been done by borna and saeidi in 2007 and preterm labor was suppressed in 87% of mgso4 group and 81% of celecoxib group so their result was similar to our result . They concluded that celecoxib is as effective as mgso4 in preterm labor suppression [8, 10]. In another study by mcwhorter et al . In 2004 they concluded that there was no difference between oral rofecoxib and intravenous magnesium sulfate in arresting preterm labor . Another study compared safety of indomethacin and celecoxib to arrest preterm labor . They reported that safety of short - term celecoxib in women with preterm labor was superior to indomethacin . In this study 600 pregnant women with preterm labor criteria mgso4 was used in one group for suppression of preterm labor and the other group took celecoxib instead of mgso4 . 80% of mgso4 group and 75% of celecoxib group showed positive response to these tocolytic agents and uterine contraction stopped at least for 48 hours and cervical dilatation and effacement did not progress in this period of time, but in this aspect; statistical calculation did not show any significant differences between two groups . In our study there were not any significant differences between cervical dilatation in two groups, but from the beginning, cervical effacement showed a significant difference between two groups; we found that although celecoxib group already had more cervical effacement, celecoxib effectiveness was as the same as mgso4 group . In this study, cost effectiveness of celecoxib is more reasonable than mgso4 and it can play a role in expenses reduction in health centers . No need for closes monitoring of the patients, and there are not invasive procedures such as injections and same effect of mgso4.
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