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+{"text": "Perineodynia , anal and urinary incontinence are the main symptoms of the pudendal canal syndrome (PCS) or entrapment of the pudendal nerve. The first aim of this study was to evaluate the effect of bilateral pudendal nerve decompression (PND) on the symptoms of the PCS, on three clinical signs  and on two neurophysiological tests: electromyography (EMG) and pudendal nerve terminal motor latencies (PNTML). The second aim was to study the clinical value of the aforementioned clinical signs in the diagnosis of PCS.In this retrospective analysis, the studied sample comprised 74 female patients who underwent a bilateral PND between 1995 and 2002. To accomplish the first aim, the patients sample was compared before and at least one year after surgery by means of descriptive statistics and hypothesis testing. The second aim was achieved by means of a statistical comparison between the patient's group before the operation and a control group of 82 women without any of the following signs: prolapse, anal incontinence, perineodynia, dyschesia and history of pelvi-perineal surgery.When bilateral PND was the only procedure done to treat the symptoms, the cure rates of perineodynia, anal incontinence and urinary incontinence were 8/14, 4/5 and 3/5, respectively. The frequency of the three clinical signs was significantly reduced. There was a significant reduction of anal and perineal PNTML and a significant increase of anal richness on EMG. The Odd Ratio of the three clinical signs in the diagnosis of PCS was 16,97 .This study suggests that bilateral PND can treat perineodynia, anal and urinary incontinence. The three clinical signs of PCS seem to be efficient to suspect this diagnosis. There is a need for further studies to confirm these preliminary results. The objective of perineology is to treat each defect of the perineum with the right procedure -3. PudenBefore going into details of this procedure, it is necessary to remember the anatomy of the pudendal nerve. This anatomy is still controversial.While summarizing the data of the literature and the results of our dissections, the likeliest anatomy of the pudendal nerve presents itself as follows. The pudendal nerve is a mixed nerve carrying motor and sensory fibers. Its fibers are derived from the sacral roots S2, S3 and S4 . Once thThe pudendal canal syndrome (PCS) and its surgical treatment have been described by Shafik in 1991 . This syThe cause of the PCS is not always clear but it is often possible to find a compression  or a stretching  of the pudendal nerve in the Alcock's canal -19 in thThe clinical signs and investigation results proposed by Shafik  to confiThe trans-gluteal approach proposed by Robert to treat pudendal neuralgia aimed to open also the \"clamp\" between the sacro-spinal and the sacro-tuberous ligament by cutting one or two of them .Since Shafik's study in 1991, some questions about the effect of PND on the PCS remained open. No peer reviewed publications confirming the efficacy of this surgery on anal incontinence or on urinary incontinence could be found. Even the existence of a genuine PCS has never been validated.The aim of this study was to answer the following questions:Is there any effect of the bilateral PND according to Shafik on:- three main symptoms of PCS ?\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0- perineodynia  \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0- anal incontinence\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0- urinary incontinence- three clinical signs of PCS ?\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0- abnormal sensibility\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0- painful Alcock's canal\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0- painful \"skin rolling test\" - two neurophysiological tests ?\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0- electromyography (EMG) of the anal sphincter and of the bulbocavernosus muscles.\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0- pudendal nerve terminal motor latencies (PNTML) of the anal and perineal branches.What is the clinical value of the aforementioned three clinical signs in the diagnosis of PCS ?A retrospective analysis to study the effects of a bilateral PND. The studied sample comprised 74 female patients who underwent a bilateral PND between 1995 and 2002 done by the same surgeon. The average age was 56.1 years (range: 28 \u2013 77). All these patients underwent a complete history and clinical examination following the three perineal axis  according to the concept of perineology .The frequency of the 3 main symptoms of the PCS  in the 74 patients before surgery is presented in Figure Associated surgical procedures were performed at the same time as the PND to treat all the defects revealed by the clinical examination, and are presented in Table The following variables were used:For perineodynia, four situations were encountered: no pain, proctalgia, unilateral pain, bilateral pain. The effect of surgery was estimated by the patient using one of the following proposals: cured, improved, unchanged or worsened.For anal incontinence, a four levels ordinal scale was used: no incontinence, gas incontinence, liquid incontinence, solid incontinence. \"Cured\" was defined as \"no incontinence\". The patient was considered \"improved\" if there was a change of at least one level in the scale going from \"solid\" to \"gas\" incontinence. The patient was defined as \"worsened\" if there was a change of at least one level in the opposite direction.For urinary incontinence, a four levels ordinal scale was used: no incontinence, mild incontinence, moderate incontinence and severe incontinence. The two types of urinary incontinence, stress and urge incontinence, were evaluated separately even if both were present in the same patient (mixed incontinence). \"Cured\" was defined as \"no incontinence\". The patient was considered \"improved\" if there was a change of at least one level in the scale going from \"severe\" to \"mild\" incontinence. If the change observed was in the opposite direction the patient was considered \"worsened\".Sensibility was tested with a needle comparing the left and the right sides of the vulva and of the skin 2 cm lateral to the anus. The interpretations of the results were done using a four levels ordinal scale: 0 = total anaesthesia, 1 = reduced sensibility, 2 = normal sensibility, 3 = hypersensibility. 0, 1 and 3 were considered as \"abnormal sensibility\".The pain induced by the palpation of the pudendal canal by rectal examination was evaluated using a seven levels ordinal scale : 0 = no pain, 1 = mild pain, 2 = mild pain with Tinel sign (irradiation of the pain), 3 = moderate pain, 4 = moderate pain with Tinel sign, 5 = severe pain, 6 = severe pain with Tinel sign. The Alcock's canal was considered \"painful\" if the pain was 4 or more.Beginning from 5 cm behind the level of anus the skin was pinched and then rolled to the front until the skin fold was at the level of the clitoris. The skin rolling test was considered \"painful\" if it induced a severe pain at least at one level Figure .Concentric needle EMG was done at rest and during voluntary contraction on both sides of the external anal sphincter and on each bulbocavernosus muscle. The richness of the EMG was grossly evaluated using a six levels scale: 1 = simple, 2 = poor, 3 = intermediate poor, 4 = intermediate, 5 = intermediate rich and 6 = rich.Anal and perineal PNTML were measured before and after the operation using the St Marks electrode to stimulate the pudendal nerve by the rectal route just under the ischial spine. For anal PNTML the electrical potentials induced in the striated anal sphincter were collected using the ring of this electrode. For the perineal PNTML the method described by Kiff  and SnooAt least one of the 3 following symptoms resistant to conservative treatments :a. Anal incontinenceb. Perineodyniac. Urinary incontinenceAssociated with at least two of the five following criteria:a. increased anal or perineal PNTMLb. pathological EMG of the anal sphincter or bulbocavernosus muscles .c. painful Alcock's canal on rectal examination (at least on one side)d. abnormal perineal sensibility (at least at one level)e. painful \"skin rolling test\" (at least on one side).Surgical procedure as described by Shafik in 1991 . The opeThe different steps of the procedure were:- Vertical incision of the skin between the anus and the ischial tuberosity.- Opening of the ischio-rectal fossa with scissors.- The inferior rectal nerve is hooked under the finger and followed to the entrance of the Alcock's canal Figures  and 4.- Opening of this canal .- Control of the haemostasis.- Self draining closing of the skin with nylon.The efficacy on the symptoms, on the clinical signs and on the neurophysiological tests was evaluated during a follow up consultation one year or more after the surgical procedure because the nerve healing can be very slow .Firstly, the efficiency of the PND on the symptoms and clinical signs was studied by means of descriptive statistics. Tests of hypothesis were done to compare the mean values of the neurophysiological tests before versus after PND.Secondly, the diagnostic value of the clinical signs was evaluated in a \"case control\" setting. A subject belongs to the \"controls group\" when PCS is considered to be absent, namely if the patient does not present any of the following symptoms, signs or risk factors for PCS: perineodynia, anal incontinence, prolapse, previous surgery in the area, dyschesia. The clinical signs are not used to decide if a subject belongs to the controls or cases group. The statistical comparison was done between the patient's group before (\"cases group\") and after the operation, and the \"controls group\" of 82 women .Statistical analysis of differences was performed using chi-square testing for categorical variables and t-tests for continuous variables.The effect of PND on the symptoms of PCS is presented in Table In order to treat completely the patient, PND was frequently associated with other procedures which might have an effect on the symptom studied. Therefore, the results for each symptom were presented in two columns: the first corresponds to the entire sample  and the second to the small group of patients in which the symptom was treated by PND only (\"without\").On the 26 patients with pain before the operation, 18 were reviewed 12 months or more after the operation. The pain had disappeared in 11 and was reduced or had another origin  in 3. The cure rate with a mean follow-up of 22,2 months was 61,1 % .As none of the surgical associated procedures used in this study were known to improve or cure perineodynia, the only procedure removed was levatorplasty. Theoretically this operation can reduce the stretching on the pudendal nerves by reducing the sagging of the levator plate. The results were similar in the group without levatorplasty.On the 46 patients with anal incontinence before the operation, 36 were reviewed 12 months or more after the operation. 23 of them were cured, 7 improved, 2 were worse and 4 reported no change. The cure rate with a mean follow-up of 26.4 months was 63.9 % .The results according to the severity of incontinence are presented in Table To study the real impact of PND on anal incontinence, we removed the following cases in which PND was associated with anal sphincteroplasty, levatorplasty or cure of rectocele by fascia and perineal body restoration:- 2 patients had anal sphincteroplasty together with the PND: one was cured and the other improved.- Levatorplasty according to Shafik  was used- 30 cases had a cure of rectocele by fascia and perineal body restoration  and 1 improved (liquid incontinence).Anal ultrasound was done before the operation in 13 of the 36 patients reviewed. Only 4 were normal, 3 showed a rupture of the internal and external sphincters, and 6 presented a disruption of the external sphincter alone.In the 7 patients who had a rupture of the anal sphincter  without anal sphincteroplasty and a follow up of more than 12 months (mean 18.5 months), 4 were cured and 3 were a failure.5 patients who were still incontinent after 1 year follow up (2 incontinences for flatus and 3 for liquid) became continent 2 years after the operation.Five patients presenting urinary incontinence (4 urge and 1 stress incontinence) had PND without any other procedure around the urethra. The mean follow up was 18,5 months for the 4 patients with urge incontinence. In this small group, 3 patients were cured and one was a failure. The patient with stress urinary incontinence was improved (the number of pads used per day was reduced from 9 to 4) one year after surgery.The effect of PND on the three clinical signs is described in Table Because levatorplasty can theoretically reduce the stretching of the pudendal nerve, the evaluation of the effect on the clinical signs has been done for the entire sample  and for the same group without the patients who had a levatorplasty (\"without: levat\").The cure rate of the 3 clinical signs was between 60 and 70 % depending of the sign and of the type of sample studied.38 patients underwent a complete EMG evaluation before and after surgery. A relevant comparison was possible in 35 patients. 3 patients were excluded because one of the two EMG explorations was insufficient for technical reasons. The average follow up was 16,9 months .Left and right values for one parameter correspond to two different nerves. Therefore, these values were considered independent .The effect of PND on EMG and PNTML is presented in Table The \"Anal Richness\" on EMG after surgery was significantly higher than before. The mean \"Bulbocavernosus Richness\" after surgery was slightly higher than before but this difference was not significant. Both anal and perineal PNTML after PND were significantly reduced compared to values before.The box-plots of the 4 studied parameters are presented in Figures We present here the results concerning the evaluation of the three clinical tests: abnormal sensibility, painful Alcock's canal and painful \"skin rollling test\" as diagnostic tests for PCS.The statistical analysis is based on the following contingency tables presented in Tables The proportions of observations in the \"Cases Before Surgery\" and \"Controls\" columns of the contingency table vary significantly from row to row , whereas no significant difference is observed between the proportions of observations between \"Cases After Surgery\" and \"Controls\" .Estimated sensibility, specificity, predictive values (positive and negative) and odd ratio  corresponding to each of the three clinical tests and combinations of all of them are presented in Tables The most sensible test is the \"Painful Alcock's canal\" and the most specific is the \"Skin rolling test\".Using the three signs altogether, the most sensible combination is \"At least 1 positive versus All negative\" and the most specific combination is the \"All positive versus All negative\".During one operation a heavy bleeding coming from the pudendal artery just near the pudendal nerve was very difficult to treat (selective ligature). This patient had a blood transfusion but no long term side effect. Since the operation, one patient has presented sometimes a short lasting clitoridal pain. This patient had also an increase in her anal incontinence (gas incontinence became a liquid incontinence). Three patients had wound healing problems which resolved with simple disinfection.In the literature there is no data available about the prevalence of the PCS. Therefore, it seems to be a rare event. In this study, we evaluated the prevalence of the PCS in an outpatient perineology clinic. By using three different methods during the last 24 months  the estimated prevalence should be around 20%.Before discussing the results of surgery, the first important issue is about the interest of a bilateral decompression. The benefit \u2013 risk ratio must be studied. The results of Shafik were obtained after bilateral decompression ,29,30. FThe treatment of pain starts with a holistic approach of the woman  with exclusion of other causes of pain: piriformis syndrome, coccygodynia, interstitial cystitis, endometriosis... The other neurological causes must be excluded by a complete electrophysiological study of the perineum  and imaging of the spinal cord . If the Even with the transgluteal approach where the \"clamp\" between the sacro-spinal and the sacro-tuberous ligament is opened by sectioning the sacro-spinal ligament, the cure rate remains around 50%. In the 4 cases of proctalgia fugax the results were better (3 cured and 1 improved). Shafik [For Robert early diagnosis appears to be the determining factor in improving results . He usedFor anal incontinence, our results were in the same range as Shafik . In a prMore interesting was the cure rate in the group of patients with a clear rupture of one or the two anal sphincters. The traumatic rupture of the anal sphincter  usually induces an immediate anal incontinence. In the patients who remained continent the power of the broken muscles remain sufficient to avoid flatus or faeces leakage. In the long term the continence is probably maintained with the help of the fibrous tissue located between the two edges of the ruptured muscle which acts like a bridge and therefore enables the sphincter to be efficient during many years. The aging process of the muscle and the pudendal neuropathy reduce the power of the muscle  with time and explain the appearance of an anal incontinence. Therefore it is logical to restore continence by improving the conduction in the pudendal nerve. This fact can also explain why the results of sphincteroplasty decrease with time especially in the non diagnosed or treated pudendal neuropathies .The fact that 5 patients were cured from their anal incontinence only 2 years after surgery emphasized the importance of a long follow up period to obtain relevant cure rates. Surprisingly, the cure rate seems to be not dependant of the degree of anal incontinence but the number of solid incontinences  was too small to validate this impression.The results of the pudendal nerve decompression seem to be equivalent to these of neuromodulation  and the For urinary incontinence the number of cases is too small to give a relevant cure rate but there were enough cases to suggest that this surgery can treat some patients with stress or urge incontinence.In a previous study, 3 of the 7 patients presenting a stress urinary incontinence were cured by bilateral pudendal nerve decompression alone ,37.In Shafik's study 6 patients were cured from their stress urinary incontinence, 5 improved and one was a failure . For thiThis study is the first one dealing with a possible effect of the pudendal nerve decompression on urge incontinence. It is probably due to a better control of the urethral sphincter which can reduce urethral instability  and imprOne weakness of this study is the rough evaluation of the symptoms. We did not use any scoring system, pad test, quality of life questionnaires or \"visual analog pain scale\". Furthermore the number of anal and urethro-vesical manometries done before and after PND was too small to give relevant results.The objective evaluation of PND was done using two neurophysiological tests and the clinical examination. Like Shafik ,30 we foShafik described many clinical signs of the PCS ,29,30. ICompared to patients with negative clinical signs, those having positive clinical signs have a 4,42 ; 5,52 and 6,56 higher likelihood of PCS for \"Abnormal sensibility\", \"Painful Alcock's canal\" and \"Painful skin rolling test\", respectively Table . When paThe most specific sign was the \"Painful skin rolling test\" and the most sensitive was the \"Painful Alcock's canal\". The association of the three positive tests had a very high specificity in the diagnosis of a PCS (89 %). This high specificity was confirmed by the low frequency of this association after the operation (return to the same level as the control group).Therefore, in some cases the clinical examination should be sufficient to prove the existence of a PCS. For example, a patient presenting anal incontinence, an intact sphincter proven by ultrasound and the three clinical signs positive has almost certainly a PCS. Of course, from a scientific point of view it is still interesting to perform a complete electrophysiological study and a precise neurological examination to exclude a central problem  or a polyneuropathy.However, making the diagnosis of PCS is not usually an easy task. Many times, there is a high degree of suspicion but, as in many illness, all the symptoms or signs are not present. In this study, we decided to operate when at least two of the five clinical and neurophysiological signs described in the methods section were associated with one or more of the 3 classical symptoms . At the beginning of this study, it was usually \"increased PNTML\" and \"painful Alcock's canal\". With the introduction of the \"skin rolling test\" and of the \"sensibility test\", clinical examination became more important in the decision. The more symptoms  and signs were present, the more confident we were in the diagnosis of PCS. Further studies are necessary to validate this and to define more precisely the minimal criteria needed for the PCS diagnosis.The pudendal nerve decompression by the perineal route is a blind procedure. The search for the inferior rectal nerve and the opening of the Alcock's canal are done under finger control. In our experience it is not easier with retractors. Therefore it is necessary to have a clear anatomical vision of this area before performing the operation.Maybe the use of a laparoscope would help  but the Despite the blind character of the procedure we only had one heavy bleeding probably coming from the pudendal artery. One patient still presents with a mild intermittent clitoridal pain and a worsening of anal incontinence. Because the nerve of the clitoris leaves the pudendal nerve just before the entrance into the Alcock's canal this problem is probably the result of a too large dissection in the upper part of this canal. The two cases of anal incontinence worsening (gas incontinence becoming liquid incontinence), including the aforementioned patient, are difficult to explain. Maybe the neuropathy increased with the stretching involved in the procedure, the scarring process or a too large dissection. It could also be the result of the changes in the posterior level anatomy induced by concomitant procedures (easier expulsion of gas or faeces). For the 2 patients the EMG data and the clinical examination after the operation did not improve therefore showing that the neuropathy was not healing.Because the roughly estimate prevalence of PCS is around 20%, this \"defect\" seems to be a very frequent problem in Perineology. Therefore it should be logical to search for it in each clinical examination of a patient presenting with prolapse, perineodynia, urinary or anal incontinence.Pudendal neuropathy is probably a frequent \"defect\" in perineology. Pudendal nerve decompression seems to be the defect specific procedure indicated in such a problem. In fact it can treat perineodynia, anal and probably urinary incontinence. Anal incontinence can be cured by pudendal nerve decompression alone even in the presence of a clear disruption of the anal sphincter on anal ultrasound. Anal richness on EMG increases and PNTML decrease significantly after surgery proving an objective effect on the nerve. The frequency of abnormal puncture sensibility, painful Alcock's canal and painful \"skin rolling test\" are significantly reduced by the operation. This study suggests that the three clinical tests could be used in practice to confirm or suspect the diagnosis of pudendal neuropathy in case of pain, urinary and/or anal incontinence. However, further studies are necessary to confirm these preliminary results.PCS = pudendal canal syndromePND = pudendal nerve decompressionEMG = electromyographyPNTML = pudendal nerve terminal motor latencyThe authors declare that they have no competing interests.JB conceived the study, carried out surgery and clinical follow up and drafted the manuscript.DC participated in the design of the study and performed the statistical analysis.MB carried out the neurophysiological examinations.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Molecular Pain, Tang and colleagues use a novel set of approaches to characterize the role of the anterior cingulate cortex (ACC) in the formation of Pavlovian fear memories.An emerging theme in systems neurobiology is that even simple forms of memory depend on activity in a broad network of cortical and subcortical brain regions. One key challenge is to understand how different components of these complex networks contribute to memory. In a new study in  This is adaptive since when these cues are next encountered, an animal can take measures  that help to reduce the likelihood of injury.Because survival may depend upon it, we learn about painful stimuli quickly and efficiently through a process known as Pavolvian fear conditioning . Viewed unpleasantness . Recent studies in humans and experimental animals have established that these different attributes are processed by partially dissociable brain networks [A painful stimulus may have a number of different attributes . These inetworks ,3. For enetworks . Taking networks  first asHaving established that activation of the ACC produces a cluster of behaviors consistent with fear or unpleasantness, Tang and colleagues posed a more ambitious question: Would it be possible to artificially fear condition an animal by replacing a footshock with electrical stimulation of the ACC? In a normal fear conditioning experiment, an otherwise neutral stimulus, such as a tone, is repeatedly paired with an aversive stimulus, such as a shock, in a particular context. Later an animal exhibits a number of species-typical fear responses, including freezing behavior, when presented with the tone or placed back in the original training context. Here, Tang and colleagues conducted the same experiment except that they replaced the footshock with stimulation of the ACC. Sure enough, mice trained in this way froze when placed the mice back in the same context, or when presented with the tone in a different context. These artificially-generated conditioned fear memories were not transient \u2013 they lasted at least 3 days, indicating that mice will condition as readily to ACC stimulation as they might to shock delivery. This experiment, therefore, shows that mice readily associate both the context and the tone with ACC-evoked 'unpleasantness'. The formation of CS-US associations underlying fear memories depends on NMDA-mediated plasticity in the amygdala -8. Consipairing of the tone with ACC stimulation. When this temporal relationship was perturbed, the mice did not freeze to the tone, indicating that the observed learning was associative in nature. A second issue they addressed was the possibility that ACC stimulation produces analgesic effects [Tang and colleagues took care to rule out a number of alternative interpretations. They first showed that the fear conditioning to the tone depended on the  effects . If this effects . These dA third control experiment yielded a particularly interesting dissociation. They considered the possibility that brain stimulation, regardless of site, is aversive. To address this they conducted the same experiment but paired a tone with stimulation of the primary somatosensory cortex. Under these conditions, no fear developed to the tone (nor to the context in which conditioning occurred). This first demonstrates that their effects are specific to ACC stimulation, and secondly, suggests an important dissociation. Whereas the sensory features of a pain stimulus are processed by the primary somatosensory cortex and the affective features by the ACC, only ACC stimulation appears to functionally substitute for a shock during fear conditioning. This suggests that fear conditioning critically depends on the affective qualities  of the US. When the US is stripped down to its sensory properties alone, conditioning proceeds very slowly . This conclusion is further supported in another experiment in which mice were fear conditioned with a tone-shock pairing. Tang and colleagues showed that inactivating the ACC during training blocked the development of conditioned fear. This suggests that disrupting processing of the affective (but not sensory) features of a US is sufficient to disrupt conditioning.This clever series of studies starts to dissociate the contribution of different cortical regions to the formation of even simple memories. Tang and colleagues provide evidence that the ACC specifically processes the affective features of painful stimuli, and that ACC activation can artificially induce Pavlovian fear memories. But how do these findings fit into the broader context of functional studies on the ACC? While other rodent studies have also provided evidence that the ACC processes Pavolvian fear memories, these have emphasized the role of the ACC in memory recall rather than memory formation ,12. Furt"}
+{"text": "In patients with acute respiratory distress syndrome (ARDS), it is well known that only part of the lungs is aerated and surfactant function is impaired, but the extent of lung damage and changes in surfactant turnover remain unclear. The objective of the study was to evaluate surfactant disaturated-phosphatidylcholine turnover in patients with ARDS using stable isotopes.13C-dipalmitoyl-phosphatidylcholine, we measured the 13C enrichment over time of palmitate residues of disaturated-phosphatidylcholine isolated from tracheal aspirates. Data were interpreted using a model with two compartments, alveoli and lung tissue, and kinetic parameters were derived assuming that, in controls, alveolar macrophages may degrade between 5 and 50% of disaturated-phosphatidylcholine, the rest being lost from tissue. In ARDS we assumed that 5\u2013100% of disaturated-phosphatidylcholine is degraded in the alveolar space, due to release of hydrolytic enzymes. Some of the kinetic parameters were uniquely determined, while others were identified as lower and upper bounds.We studied 12 patients with ARDS and 7 subjects with normal lungs. After the tracheal instillation of a trace dose of de novo synthesis of disaturated-phosphatidylcholine were also significantly lower, while mean resident time in lung tissue was significantly higher in ARDS than in controls. Recycling was 16.2 \u00b1 3.5 in ARDS and 31.9 \u00b1 7.3 in controls (p = 0.08).In ARDS, the alveolar pool of disaturated-phosphatidylcholine was significantly lower than in controls . Fluxes between tissue and alveoli and In ARDS the alveolar pool of surfactant is reduced and disaturated-phosphatidylcholine turnover is altered. ARDS is a syndrome of reduced gas exchange due to a diffuse injury to the alveolar capillary barrier and is characterized by filling of the alveoli with proteinaceous fluid, infiltration by inflammatory cells and consolidation . It may Constriction and microembolism of the pulmonary vessels are also present, leading to ventilation perfusion mismatch. Moreover an increase in the alveolar surface tension causes alveolar instability, atelectasis and ventilatory inhomogenieties. In severe ARDS, just a small fraction of parenchyma remains aerated, and the damage can be so widespread that normal parenchyma, as judged by computed tomography, may shrink to 200\u2013500 g .in vitro and in vivo [One of the hallmarks of ARDS is reduced lung compliance and loss of stability of terminal airways at low volumes, suggesting surfactant dysfunction or deficiency. Samples of bronchoalveolar lavage fluid from patients with ARDS have low concentrations of disaturated-phosphatidylcholine, phosphatidylglycerol and surfactant-specific proteins and fail to reduce surface tension both  in vivo ,5. Surfa in vivo . To our Data on surfactant metabolism in ARDS are available from animal studies which showed a faster turnover rate and a decreased alveolar pool of disaturated-phosphatidylcholine, while the tissue pool was increased in some studies and unchanged in others -9. Howev13C-dipalmitoyl-phosphatidylcholine into the trachea and then followed over time the 13C enrichments in disaturated-phosphatidylcholine-palmitate isolated from serial tracheal aspirates.In this paper we studied the turnover of surfactant disaturated-phosphatidylcholine in patients with ARDS and in control subjects. To this end we instilled a trace dose of Available evidence indicates that surfactant dipalmitoyl-phosphatidylcholine is recycled several times before being degraded by alveolar macrophages or within lung parenchyma . There i13C-dipalmitoyl-phosphatidylcholine and 40 mg of surfactant extract  as spreading agent. Both palmitates were uniformly labeled with carbon 13 . The suspension was instilled close to the carina with a 4.5 mm bronchoscope . Patients with ARDS were studied within 72 h from the onset of the acute respiratory failure and ventilator parameters were adjusted to maintain an oxygen saturation > 85% and pH > 7.25. Ventilator and gas exchange parameters were recorded at time 0 and subsequently every 6 h in ARDS patients and at least once in controls.We studied 12 adult patients with ARDS, defined according to Bernard , and 7 sTracheal aspirates, collected by suction below the tip of the endotracheal tube after instilling 5 ml of normal saline, were obtained at baseline, every 6 h until 72 h and then every 12 h for 7 days or until extubation. Aspirates were filtered on gauze, centrifuged at 150-g for 10 minutes and supernatants were stored at -20\u00b0C.13C -disaturated-phosphatidylcholine-palmitate were measured by gas chromatography-mass spectrometry , as previously described [Lipids from tracheal aspirates and from the administered tracer were extracted according to Bligh and Dyer after addition of the internal standard heptadecanoylphosphatidylcholine . One thiescribed .Data were analyzed with the two compartment model shown in figure Tracer model equations are:01 + k21)m1 (t) + k12m2 (t) + u(t)21m1 (t) - (k01 + k12)m2 (t) \u00a0\u00a0\u00a0 (1)1 and m2 are the amount (in mg) of disaturated-phosphatidylcholine-palmitate tracer in compartment 1 and 2 respectively, 21 and k12 (h-1) are inter-conversion rate parameters, k01 and k02 (h-1) are irreversible losses, and u is the labeled disaturated-phosphatidylcholine-palmitate injection into the accessible compartment.where mTracee steady state equations are:01 + K21)M1 + K12M2 = -F01 - F21 + F120 = -(K21M1 - (K01 + K12)M2 + P = F21 - F01 - F12 + P \u00a0\u00a0\u00a0 (2)0 = K1 and M2 (mg) are the steady state tracee disaturated-phosphatidylcholine-palmitate masses in the two compartments, P (mg/h) is disaturated-phosphatidylcholine-palmitate de novo synthesis, F21 = k21M1, F12 = k12M2, F01 = k01M1, F02 = k02M2 (mg/h) are inter-conversion and irreversible loss fluxes.where MMeasured tracer to tracee ratio at time t is the ratio between tracer and tracee masses in the accessible compartment:1, k01, k02, k12, k21 [1 can be uniquely identified, together with some combinations of the original parameters, namely k01+ k21, k02 + k21 and k21 k12. To resolve model nonidentifiability, assumptions on the relative role of the two degradation pathways need to be incorporated into the model. Based on the results of studies in which rabbits or mice received non-degradable analogues of disaturated-phosphatidylcholine into the trachea [01 varies between 5 and 50% of F01+F02). In ARDS, we assumed that the degradation of disaturated-phosphatidylcoline in the airways could vary between 5 and 100% due to the degradative activity of inflammatory cells, bacteria or enzymes released in the alveolar spaces (i.e. F01 varies between 5 and 100% of F01+F02). Using this information, upper and lower bounds for parameters k12, k21, k01and k02 were estimated from tracer to tracee data in each individual [2 and tracee fluxes F21 and F02, while flux F12 was uniquely solved [tot = M1 + M2), the mean residence time of molecules entering the system from alveoli or lung tissue , defined as the sum of the elements in column 1 and 2 of the mean residence time matrix \u0398:The tracer model (equations 1 and 3) is not identifiable, since it is not possible to quantify from the input-output tracer experiment in the alveolar compartment unique values for the unknown parameters of the tracer model, namely Mk12, k21 . Only th trachea ,11, we adividual . Using ty solved . Additioand the percentage R (%) of particles that recycle back after leaving the intracellular pool:tot, MRT1 and MRT2[Upper and lower bound were calculated for M and MRT2, while u21, k12, k01, k02, and M1 of the model , fluxes (F) and synthesis (P) were multiplied by 24 to obtain the respective values per day.Results are presented as mean \u00b1 SEM. Data in Table We studied 12 ARDS patients and 7 controls. No ARDS patient was treated with exogenous surfactant. Eight ARDS patients (67%) died before hospital discharge, 5 for multi-organ failure and 3 for the underlying disease. Patients died within 4 to 18 days of study completion and during the study respiratory and gas exchange parameters were stable. No death occurred in the control group. In the control group, five patients suffered from spinal muscular atrophy, two had polineuropathy and one had encephalopathy secondary to head injury. Clinical characteristics of the 12 ARDS and 7 controls are reported in Table The average time courses of disaturated-phosphatidylcholine-palmitate tracer to tracee ratio in controls and ARDS are shown in figure 1, F12 and R) were uniquely identified, the others are presented as ranges of values included between two extremes, the upper and lower bounds.Individual curves of the tracer to tracee ratio were fitted to the model presented in figure De novo synthesis (P) of disaturated-phosphatidylcholine ranged from 4.25 \u00b1 0.7 to 8.64 \u00b1 1.44 mg/kg/day. The flux from alveoli to tissue (F21) ranged from 3.12 \u00b1 1.49 to 4.80 \u00b1 1.78 mg/kg/day. The flux from tissue to alveoli (F12) was 5.23 \u00b1 1.78 mg/kg/day and recycling (R) was 31.9 \u00b1 7.3%. According to the model, labelled disaturated-phosphatidylcholine is expected to accumulate into the lung parenchyma of control subjects, reaching a maximum concentration between 12 and 24 hours after instillation. Afterwards, tissue isotopic enrichment is expected to decrease, so that 96 hours after the start of the study around 20% of the tracer remains associated with lung tissue (data not shown).In controls, the alveolar pool of disaturated-phosphatidylcholine was 1.31 \u00b1 0.40 mg/kg, far smaller than the tissue pool, which, depending on assumptions about degradation of disaturated-phosphatidylcholine by alveolar macrophages, ranged from 9.64 \u00b1 2.43 to 19.35 \u00b1 3.74 mg/kg. 1) was smaller than in controls: 0.16 \u00b1 0.04 vs 1.31 \u00b1 0.40 mg/kg (p < 0.05). Fluxes between tissue and alveoli (F12 and F21) and de novo synthesis (P) of disaturated-phosphatidylcholine were also smaller than in controls. Fractional rates of transfer between tissue and airways (k21 and k12) and alveolar mean resident time (MRT1) were not different from controls. In ARDS, the tissue mean resident time of disaturated-phosphatidylcholine was significantly longer than in controls .Pulmonary surfactant is essential for normal lung function, and it is well established that surfactant impairment contributes to respiratory failure in ARDS ,5,25-27.Most of our knowledge on surfactant kinetics in acute lung injury derives from animal studies done with radioactive isotopes . In thisThe design of the study assumes that the tracer was administered as a pulse, that there was good mixing between tracer and endogenous surfactant, that the administered material did not perturb endogenous surfactant, that tracheal aspirates were representative of events happening in the most peripheral airways and that patients were at steady state.While in neonatal respiratory disorders the lung parenchyma is relatively homogeneous, this is certainly not the case in patients with ARDS, where areas of atelectasis and over-distension coexist and the tracer might distribute preferentially to aerated sections of the lungs . In thisThe dose of disaturated-phosphatidycholine administered to control subjects (20 \u00b1 2 mg) represented 1.1\u20132.1% of the estimated lung pool , an amou1), the flux from the lung tissue back to the alveolar space (F12) and recycling (R) can be uniquely solved. Only a far more complex experiment, with tracer administered also in the lung tissue compartment, could permit to uniquely identify all kinetic parameters. Since this experiment could not be done, we used existing knowledge on the contribution of alveolar macrophages to surfactant degradation to derive bounds for parameters that could not be uniquely identified. Thus, on the basis of animal experiments done by Gurel and Rider [Data were analysed according to the two compartment model reported in figure nd Rider ,11, we and Rider . In ARDSnd Rider  and applnd Rider ,42 and gnd Rider  kineticsOur estimate of the alveolar and tissue pools of disaturated-phosphatidylcholine in controls agree quite well with measurements taken by Rebello et al. during autopsies of adults without lung disease . In factUsing morphometric methods Young et al. estimated that the alveolar pool of disaturated-phosphatidylcholine is comparable to the lamellar body pool . Thus itde novo synthesis of disaturated-phosphatidylcholine were all smaller than in controls, while the mean residence time in lung tissue was greater than in controls. These differences appear to be robust, since, with the exception of de novo synthesis, they persist even assuming that in controls alveolar macrophages degrade between 5% and 100% of surfactant disaturated-phosphatidylcholine. Thus most of our conclusions remain valid independent of any assumption regarding the site of degradation of surfactant.In patients with ARDS alveolar pool, fluxes between tissue and alveoli and 2) was greater while the rate of recycling tended to be lower than in controls. The greater mean residence time of disaturated-phosphatidylcholine in lung tissue could be due to a number of factors, namely to a decreased ability to degrade surfactant components, to an increased reacylation of lysophosphatidylcholine , to a proliferation of type II cells, to the distribution of tracer to lung structures not pertaining to the surfactant system (i.e. infiltrating inflammatory cells), or to a combination of these phenomena [The present results agree with the view that, in ARDS, only a fraction of the lung is accessible to exogenous surfactant. In fact, the decrease of the alveolar pool of disaturated-phosphatidylcholine, the decrease of fluxes between tissue and alveoli and the decrease in the rate of synthesis can all be interpreted assuming that instilled surfactant reached only aerated lung sections. However, our data do not support the notion that these residual lung sections were normal, since the mean resident time of disaturated-phosphatidylcholine in lung parenchyma  compartment.The fact that the alveolar pool of disaturated-phosphatidylcholine can be estimated unambiguously is an important result of this work. In future studies this approach could be used to relate changes in surfactant turnover with time course and severity of ARDS or to evaluate the effect of different treatments  on surfactant metabolism.ARDS = acute respiratory distress syndrome21 and k12 = disaturated-phosphatidylcholine inter-conversion rate parameters,k01 and k02 = disaturated-phosphatidylcholine irreversible losses,ku = labeled disaturated-phosphatidylcholine-palmitate injection into the accessible compartment.1 = the alveolar pool of disaturated-phosphatidylcholineM2 = the tissue pool of disaturated-phosphatidylcholineMtot= total disaturated-phosphatidylcholine poolM21, F12, F01, F02 = disaturated-phosphatidylcholine inter-conversion and irreversible loss fluxes in compartment 1  and 2 (tissue)FDe novo synthesis of disaturated-phosphatidylcholineP = 1 and MRT2 = mean residence time of disaturated-phosphatidylcholine in compartment 1  and 2 (tissue)MRTThe author(s) declare that they have no competing interests.PEC participated to the design and coordination of the study and drafted the manuscript. GMT, MC, CC performed the data modeling and analysis. CO and AV were responsible of the clinical conduction of the study. AG performed the mass spectrometry analysis. BA and VPC participated in the study design and helped to draft the manuscript."}
+{"text": "Praziquantel (PZQ) is the drug compound of choice in the control and treatment of schistosomiasis. PZQ is administered as a racemate, i. e. 1\u22361 mixture of enantiomers. The schistosomicidal activity arises from one PZQ-enantiomer, whereas the other enantiomer does not contribute to the activity. The WHO's Special Programme for Research and Training in Tropical Diseases (TDR) has assigned the low-cost preparation of pure schistosomicidal (\u2212)-PZQ a key priority for future R&D on PZQ, but so far this transition has not happened. PZQ has two major administration drawbacks, the first being the high dose needed, and its well documented bitter and disgusting taste. Attempts of taste-masking by low-cost means have not been successful. We hypothesized that the non-schistosomicidal component in PZQ would be the main contributor to the unpleasant taste of the drug. If the hypothesis was confirmed, the two major administration drawbacks of PZQ, the high dose needed and its bitter taste, could be addressed in one go by removing the component contributing to the bitter taste.R)-PZQ by X-ray crystallography, and the extent of bitterness was determined for regular racemic PZQ and the schistosomicidal component in a taste study in humans. Finding: The schistosomicidal component alone is significantly less bitter than regular, racemic PZQ.PZQ was separated into its schistosomicidal and the non-schistosomicidal component, the absolute stereochemical configuration of (\u2212)-PZQ was determined to be (R)-PZQ should be specifically suitable for the treatment of school-age children against schistosomiasis. With this finding, we would like to offer an additional incentive to the TDR's recommendation to switch to the pure schistosomicidal (R)-PZQ.Our hypothesis is confirmed. We propose to use only the pure schistosomicidal component of PZQ, offering the advantage of halving the dose and expectedly improving the compliance due to the removal of the bitter taste. Therefore,  is the drug compound of choice in the control and treatment of this disease. Only half of the drug dose currently administered actually has activity against schistosomiasis, whereas the other half has no activity. Therefore, the WHO has assigned the low-cost preparation of the pure active component a key priority for future PZQ research and development. PZQ has two major administration drawbacks, the first being the high dose needed, the second its well documented bitter taste. Attempts of masking the unpleasant taste have not been successful. We hypothesized that the non-active component in PZQ would be the main contributor to the unpleasant taste of the drug. We determined the extent of bitterness for regular PZQ compared to the pure active component in a taste study in humans. We found that the pure active component alone is significantly less bitter than regular PZQ. This new finding should serve as an additional incentive for the PZQ research and development community to provide a low-cost, large-scale preparation route to the pure active component of PZQ. Schistosoma mansoni to PZQ in vitro have been described Praziquantel While the safety and efficacy against all schistosoma species are outstanding, PZQ has two major administration drawbacks, the first being the high dose needed, 40 mg PZQ/kg bodyweight: Dosages in children are determined by measurement of children's heights using tablet poles, and range from one to five 600 mg-tablets for one treatment. Especially young children have been reported not to be able to swallow these 600 mg tablets PZQ is administered as a racemate, i. e. 1\u22361, mixture of two compounds of identical constitution but non-superimposable mirror-image configuration, so called enantiomers. The straightforward and low-cost chemical synthesis has to be assumed as the reason for the use of the racemate, although it has been known for years that the schistosomicidal activity mainly relies in one PZQ-enantiomer, designated (\u2212)-PZQ , whereas the other enantiomer, designated (+)-PZQ , does not contribute to the activity The Synaptic LeapIn the context of the WHO's Global Plan to combat NTDs R-configuration of the molecule by measuring Friedel pairs and the Flack parameter (x\u200a=\u200a\u22120.1(3)) (www.ccdc.cam.ac.uk) quoting the names of the authors and journal citation.Although effective synthetic methods for the enantioselective preparation of PZQ have been reported \u22120.1(3)) . FurtherR)-PZQ were determined according to the European Pharmacopoeia 5. The bitterness value is defined by the European Pharmacopoeia as the reciprocal of the concentration of a solution in a dilution series of a compound, a liquid or an extract that still has a bitter taste. Concentrations of solutions used in the tests ranged from 1.69\u00d710\u22128 to 1.0\u00d710\u22124 g/mL. A test panel consisting of sixteen members was assembled. Although children comprise the treatment target group no children were included in the test panel. All panel members were adults completely untrained in performing sensory tests. To correct for individual differences in tasting bitterness amongst the panel members a correction factor was determined for each panel member by preparing dilutions of quinine hydrochloride. The mouth was rinsed with water before tasting. The dilution with the lowest concentration having a bitter taste was determined by taking 10 mL of the weakest solution into the mouth and passing it from side to side over the back of the tongue for 30 seconds. If the solution was not found to be bitter, the panellist had to spit out and wait for one minute before the mouth was rinsed again with water. After 10 minutes, the next dilution in order of increasing concentration was tasted. The correction factor k for each panel member was calculated according to the European Pharmacopoeia by k\u200a=\u200an/5, where n is the number of millilitres of the stock solution in the dilution of the lowest concentration that is judged to be bitter. One panel member detected bitterness already in pure water, and was therefore excluded from the test panel. Dilutions of the test compounds racemic PZQ and (R)-PZQ were prepared and tasted by the remaining fifteen members of the test panel in the same manner as described for quinine hydrochloride. The bitterness value as experienced by each member was calculated according to the European Pharmacopoeia taking the individual-related correction factor into account by Y\u00d7k/X\u00d70.1, where Y is the dilution factor of the dilution, and X is the number of millilitres of the respective dilution which, when diluted to 10 mL with water, still has a bitter taste. The bitterness value of the test compounds resulted from calculating the average of the individual values.The bitterness values of racemic PZQ and its schistosomicidal component . Nevertheless, it was conducted according to the principles of the WMA Declaration of Helsinki where applicable.R)-PZQ to taste less bitter than racemic PZQ. Although no statistical test is required or proposed by the European Pharmacopoeia, a statistical test  was conducted to investigate the observed difference between the compounds. Considering the small sample size and the nature of the data which does not justify the assumption of a normal distribution, a nonparametric, distribution-free method was chosen. On the 5% level of significance, Wilcoxon's Signed Rank Test (two-sided) resulted in a significant difference between the taste of racemic PZQ and (R)-PZQ (p\u200a=\u200a0.0107). This result was confirmed by the Sign Test .The results of the determination of bitterness values are shown in R)-PZQ the panellists commonly described the sensation of a moderate chemical taste, comparable to that of a polyethylene or a rubber pipe. Although the tastes were not recognized alike across the test panel, for the majority of the test panel we can state that (R)-PZQ had a less unpleasant taste compared to racemic PZQ.In addition to the quantitative determination of the bitterness values, qualitative taste sensations were noted by the members of the test panel for each compound. For racemic PZQ, all panel members commonly observed the sensation of an unpleasant chemical or metallic taste or a taste circumscribed best by old rubber. On the other hand, for (R)-PZQ has a less unpleasant taste compared to racemic PZQ, which was found to be comparably bitter or unpleasant. It can be assumed that the disgusting taste of racemic PZQ stems from the non-schistosomicidal component, (S)-PZQ. Removing the latter from currently used racemic PZQ therefore not only offers the chance to halve the dose, with the potential to decrease the number or size of the tablets, but also addresses the second disadvantage of regular, racemic PZQ-its unpleasant taste. With this finding and its publication we would like to offer an additional incentive to focus work of the PZQ R&D community on further decreasing the cost of production of (R)-PZQ with the goal to switch to pure (R)-PZQ as a replacement for racemic PZQ for the treatment of school-age children against schistosomiasis.The schistosomicidal component of regular PZQ, ("}
+{"text": "Bystander affiliation (post-conflict affiliation from an uninvolved bystander to the conflict victim) may represent an expression of empathy in which the bystander consoles the victim to alleviate the victim's distress (\u201cconsolation\u201d). However, alternative hypotheses for the function of bystander affiliation also exist. Determining whether ravens spontaneously offer consolation to distressed partners may not only help us to understand how animals deal with the costs of aggressive conflict, but may also play an important role in the empathy debate.This study investigates the post-conflict behavior of ravens, applying the predictive framework for the function of bystander affiliation for the first time in a non-ape species. We found weak evidence for reconciliation (post-conflict affiliation between former opponents), but strong evidence for both bystander affiliation and solicited bystander affiliation (post-conflict affiliation from the victim to a bystander). Bystanders involved in both interactions were likely to share a valuable relationship with the victim. Bystander affiliation offered to the victim was more likely to occur after intense conflicts. Renewed aggression was less likely to occur after the victim solicited affiliation from a bystander.Our findings suggest that in ravens, bystanders may console victims with whom they share a valuable relationship, thus alleviating the victims' post-conflict distress. Conversely victims may affiliate with bystanders after a conflict in order to reduce the likelihood of renewed aggression. These results stress the importance of relationship quality in determining the occurrence and function of post-conflict interactions, and show that ravens may be sensitive to the emotions of others. Aggressive conflicts feature regularly in the lives of many group-living animals over matters such as positions in the dominance hierarchy, access to limited resources, or over decisions that have to be made. Such conflicts, however, may be costly, using up valuable energy and time and risking injury. Moreover, aggressive conflicts may damage the opponents' relationship The term consolation implies a distress-alleviating function and a motivation rooted in empathy for the distressed victim but post-conflict affiliation from bystanders to victims , another member of the corvid family famed for their primate-like cognitive abilities Here, we investigated the post-conflict behavior of ravens , it is likely to be provided by valuable partners, as these are more likely to be responsive to each other's distress, and may occur after more intense conflicts, when the victim is more likely to be distressed If bystander affiliation serves a relationship repair function through mediation of a valuable partner, the bystander is likely to share a more valuable relationship with the aggressor than with the victim Bystander affiliation is predicted to serve a self-protection function if victims redirect aggression towards bystanders and the bystander-victim relationship is characterized by a low degree of compatibility and/or security, as those bystanders are most likely to be at risk of redirected aggression Finally, we predicted that if bystander affiliation or solicited bystander affiliation protects the victim from renewed attack from the aggressor, the risk of renewed aggression would be lower following the interaction than in its absence.This study complied with Austrian and local government guidelines and permission was received from the Konrad Lorenz Forschungstelle to observe the ravens for this study.2) along with a nine-year old male and a four-year old female who were unrelated to each other or the nestlings. During the study, two subjects died as a result of predation at the end of 2004 and the two adult subjects were removed from the group in August 2005. The aviary was enriched with trees, branches, stones, tree trunks and shallow pools for bathing. The ravens were fed twice per day with meat, milk products and kitchen leftovers and always had access to water.We used 13 hand-reared ravens  housed at the Konrad Lorenz Forschungstelle, Austria as subjects for this study. Eleven of those subjects were taken as nestlings were taken from four nests  in February 2004. The nestlings were hand-raised in their sibling groups  in artificial nests, with the exception of one subject raised in a single nest with two other unrelated nestlings who were removed from the group prior to the start of this study. After fledging all the nestlings were housed together in a large aviary  were recorded. The identities of the aggressor and the victim  were recorded along with the intensity of the conflict . The post-conflict (PC)-matched control (MC) method A total of 152 PC-MC pairs were collected on 11 conflict victims (58 aggressor-victim dyads). The two adult subjects were never recorded as victims but were included in analyses involving aggressors or bystanders. For the remaining nine individuals, a mean (\u00b1S.D.) of 13.8 (\u00b17.6) PC-MC pairs per individual were collected (range\u200a=\u200a1\u201324).Following de Waal & Yoshihara We investigated the influence of conflict intensity (high or low) and the occurrence of solicited bystander affiliation on the occurrence of bystander affiliation using generalized linear mixed models (GLMMs). A similar model investigating the effect of conflict intensity and bystander affiliation on the occurrence of solicited bystander affiliation was also run. We considered bystander affiliation or solicited bystander affiliation to have occurred when the PC-MC pair was labeled \u2018attracted\u2019 to control for baseline levels of affiliation. The identities of both conflict opponents were entered as random factors, thus controlling for variation in individual contribution to the data set. We used GLMMs with binomial error structures and a logit-link function. Akaike's information criteria (AIC) values were used to select the best (most parsimonious) model for all mixed model analyses 2 tests.To examine the temporal interdependency between bystander affiliation and renewed aggression, we compared the probabilities of bystander affiliation and solicited bystander affiliation occurring after and without renewed aggression and the probabilities of renewed aggression occurring after and without bystander affiliation and solicited bystander affiliation using ChiWe analyzed the effects of the quality of all potential victim-bystander dyads' relationships on the level of bystander affiliation provided and solicited bystander affiliation received to determine whether certain types of partner were more likely to be involved in bystander affiliation or solicited bystander affiliation than others. Following Fraser et al. Measures of each component of relationship quality were previously obtained by entering seven behavioral variables into a principal components analysis and using the three extracted components as composite, quantitative measures of relationship value, compatibility and security To test the hypothesis that bystanders affiliating with victims were acting as proxies for the aggressors All analyses with the exception of GLMMs were run using SPSS v.17. GLMMs were run in R v. 2.1.0 Although post-conflict affiliation between former opponents occurred after 16 of the 152 conflicts, no difference was found between the proportion of attracted (mean \u00b1S.E.\u200a=\u200a0.09\u00b10.12) and dispersed (mean \u00b1S.E.\u200a=\u200a0.01\u00b10.03) PC-MC pairs, indicating the absence of reconciliation at the group level in the study population.2\u200a=\u200a12.198, P<0.001; For bystander affiliation, the proportion of attracted (mean \u00b1S.E.\u200a=\u200a0.38\u00b10.06) PC-MC pairs was significantly higher than the proportion of dispersed (mean \u00b1S.E.\u200a=\u200a0.15\u00b10.04) PC-MC pairs . A survival analysis confirmed the significant tendency for affiliation from a bystander to the conflict victim to occur earlier in the PC than in the MC  PC-MC pairs was also significantly higher than the proportion of dispersed (mean \u00b1S.E.\u200a=\u200a0.14\u00b10.04) PC-MC pairs . A survival analysis confirmed the significant tendency for affiliation from a victim to a bystander to occur earlier in the PC than in the MC  indicating that victims were at risk of renewed aggression from the original aggressor during the post-conflict period.We found no significant difference between the proportion of attracted (mean \u00b1S.E.\u200a=\u200a0.30\u00b10.08) and dispersed pairs (mean \u00b1S.E.\u200a=\u200a0.17\u00b10.04) for redirected aggression , indicating that victims were no more likely to attack bystanders after losing a conflict than during control periods. Conversely, for renewed post-conflict aggression between opponents, the proportion of attracted pairs (mean \u00b1S.E.\u200a=\u200a0.28\u00b10.09) was significantly higher  than the proportion of dispersed pairs (mean \u00b1S.E.\u200a=\u200a0.07\u00b10.03), and renewed aggression was likely to occur earlier in the PC than the MC  and bystander affiliation was not more likely to occur after renewed aggression than alone . In contrast, renewed aggression was less likely to occur after solicited bystander affiliation than alone . However, when baseline levels of affiliation were controlled for using TCT values, only kin were more likely to engage in post-conflict affiliation with the victim .Bystanders who initiated post-conflict affiliation with victims of aggression shared more valuable , more compatible  and more secure  relationships with the victim of the conflict than with the aggressor .The occurrence of reconciliation could not be confirmed in this group of ravens, consistent with findings in rooks In contrast to reconciliation, both bystander affiliation and solicited bystander affiliation were demonstrated as post-conflict interactions in ravens. Bystander affiliation was more likely to occur after more intense conflicts, which, as victims may experience a higher degree of distress following more intense conflicts, suggests that bystander affiliation may indeed serve a distress-alleviating, or consoling, function. Furthermore, bystanders who provided post-conflict affiliation were likely to share a valuable relationship with the victim of aggression, supportive of a distress-alleviating function as such partners are more likely to be responsive to each other's distress Sharing a valuable relationship with the victim does not, however, necessarily rule out the possibility that the bystanders also share a valuable relationship with the aggressor, and thus bystanders may still be acting as proxies for the aggressor in reconciling the opponents. For this to be the case bystanders would be expected to share a more valuable relationship with the aggressor than with the victim The fact that bystanders shared a valuable relationship with the victim, and that their relationship was no less compatible or secure than the victim's relationship with non-affiliating bystanders lead us to reject the hypothesis that bystanders affiliate with the victim of aggression to protect themselves from redirected aggression, as such bystanders are unlikely targets Interestingly, in chimpanzees, the only species in which consolation has been shown, most studies found that solicited bystander affiliation did not occur According to the predictive framework, our findings are consistent with a distress-alleviating function for bystander affiliation and should thus be considered to be consolation. The term \u2018consolation\u2019, however, infers not only the function of the interaction, alleviating the victim's post-conflict distress, but also its mechanism, empathy for the distressed victim. That bystander affiliation was more likely to occur after intense conflicts, when victims were more likely to be distressed, and that it was most likely to be provided by valuable partners, are supportive of both the functional and mechanistic components of consolation. As emotional contagion (when a subject's emotional state reflects the state perceived in a partner Whether the initiator of post-conflict affiliation between a bystander and a victim is the bystander or the victim is a critical differentiation when a consoling function is considered because while both interactions may alleviate the victim's distress, only affiliation initiated by the bystander is likely to require empathy. However, if consolation provided by a bystander is preceded by a vocal or other signal from the victim \u2018requesting\u2019 support, such a cognitive ability may not be necessary. Thus, although we found suggestive evidence for different functions for bystander affiliation and solicited bystander affiliation, caution must always be taken when interpreting the initiator of an interaction, as signals prior to the first physical interaction may go undetected. Notably, vocalizations were not recorded during this study, and are not usually taken into account in studies of post-conflict behavior (exceptions: All studies on consolation thus far have, for methodological reasons, focused on the effect of consolation on the victim rather than on the consoler. In order to fully understand the mechanism behind consolation, however, we really need to understand more about the consequences of offering consolation for potential consolers. Firstly, although bystanders may experience post-conflict distress The patterns of post-conflict behavior observed in ravens match what we would expect from what we know about the structure of their relationships. As a pair-bonded species, adult ravens are likely to share valuable relationships primarily with their mates, and thus patterns of post-conflict behavior among adults are expected to resemble those described in rooks"}
+{"text": "An affiliation was omitted. The additional affiliation, which is for first author Nino K\u00fcnzli, is University of Basel, Basel, Switzerland."}
+{"text": "The case of a 50-year-old patient who had undergone male to female gender reassignment surgery is presented. She presented with mixed incontinence with symptoms of stress incontinence predominating. Initial conservative treatment was unsuccessful and subsequent videourodynamic assessment demonstrated urodynamic stress incontinence in association with a partially open bladder neck at rest. Also noted during the study was cough-induced detrusor overactivity. The option of inserting a pubo-vaginal sling using autologous rectus sheath was chosen. The procedure proved to be straightforward to perform and was uncomplicated. Subsequent follow-up demonstrated a resolution of her stress incontinence. As we are aware, the management of stress urinary incontinence is well standardized in females with the use of pubovaginal sling and now with the less invasive approach of TVT and TOTVTs. In our case TVT or TOT was not used as we were unsure as to whether the neo-vaginal skin would heal over the sling.The use of pubovaginal sling following gender assignment has not been reported previously. A case report on the use of intraurethral collagen injection suggests some degree of improvement in the incontinence.Use of artificial urinary sphincter is well known in cases of incontinence but literature search lacks any reports on its use in gender assigned patients.A 50-year-old patient, who had undergone male to female gender reassignment surgery nine years previously, was referred with a four-year history of urinary incontinence. During the first five years after surgery she had been free of urinary symptoms although in the early postoperative period she had developed a meatal stenosis which was treated by dilatation and meatoplasty. In addition, during that procedure, residual erectile tissue was excised from the area of the urethral meatus.Urinary incontinence was of gradual onset and had been present for four years at the time of referral. Incontinence was noted to occur on coughing and movement but could also occur in association with a sense of urgency or with minimal sensation. She had two hourly day time frequency and nocturia once every night. She also complained of reduction in stream and terminal dribble. Pads were used to contain the urinary incontinence. She was taking oral estrogens in order to promote feminization.On examination there was evidence of a prolapse of the posterior wall of the neo-vagina while the prostatic remnant was small and atrophic.Investigations included a midstream urine sample and ultrasound of the urinary tract; these were normal.2O [Initial management was conservative with a regime of bladder and pelvic floor training along with the use of anticholinergic medication. The response to these measures was disappointing and hence further assessment with videourodynamic investigation was undertaken. This study showed normal bladder compliance but the bladder neck was partially open at rest. Stress incontinence was observed at 100 ml bladder capacity; at 400ml and abdominal leak point pressure of 70 cm of water was observed with evidence of gross urinary leakage. Cough-induced detrusor overactivity was also observed during the study. Voiding took place with complete bladder emptying and a maximum flow rate of 27 ml/sec with a detrusor pressure at maximum flow of 41 cm of H2O .The patient wished to consider surgical treatment for her incontinence and the options of artificial sphincter, pubovaginal sling and urethral bulking agents were considered. A decision was made to undertake placement of a rectus sheath pubovaginal sling. Cystoscopy and examination under anesthesia showed an intact distal urethral sphincter which was situated within 2 cm of the external urethral meatus. The bladder neck was slightly open. The prostate was noted to be freely mobile which was taken to indicate that the puboprostatic ligaments and endopelvic fascia had become stretched or disrupted. A 1.5 \u00d7 15 cm sling was harvested from the rectus sheath and an inverted U incision made on the anterior wall of the neo-vagina. Pelvic dissection was carried out as a neo-vagina was created by dissection between the prostate and rectum. The sheath was secured just distal to the apex of the prostate. Lateral and cephalad dissection into the retropubic space proceeded using an identical technique to that used in a standard pubovaginal sling procedure in a female patient.[On subsequent follow-up, there was no complaint of stress incontinence although very occasional urge incontinence was reported. Her follow-up flow rate showed good emptying with very minimal residual urine. Patient declined pad testing and urodynamic study as she was completely dry.This is believed to be the first reported case of the use of a rectus sheath pubovaginal sling in the management of stress urinary incontinence in a male to female gender reassignment patient. Previous reports describe the use of periurethral collagen injections in this context.Stress urinary incontinence in the male is rare and essentially confined to patients who have undergone prostatic surgery or who have neuropathic disorders of the lower urinary tract. In the gender reassignment patient, the etiology of sphincter weakness might include iatrogenic damage to the external urethral sphincter or its nerve supply.4A retrospective study from Belgium used questionnaires regarding voiding habits and lower urinary tract symptoms. They had 31 MTF and 92 FTM transsexuals with the incontinence rate of 19.3%in MTF and 50% in FTM. Of the six MTF transsexuals who suffered incontinence one had dribbling, two urge incontinence, two stress and one had mixed incontinence. The authors in the study have put forth different hypotheses for the lower urinary tract symptoms, which most likely are the effect of surgery. During the surgery for vaginoplasty a pocket is created between the bladder and the rectum that will contain the neo-vagina. This pocket is created by blunt dissection behind the bladder and prostate. The sphincter complex and the pelvic floor muscles are in the dissected area, so some of the observed stress incontinence could be attributed to the surgery. Also damage to nerves supplying the bladder and the change of position of the bladder itself could lead to incontinence.[Another study from Switzerland aimed to determine if transsexuals have an increased risk of micturition disorders and if so which. In their study they had 25 patients of which 18 were MTF and seven were FTM transsexuals. In MTF transsexuals, a diverted stream, overactive bladder and stress urinary incontinence was a common problem. The authors assumed that the reasons for the development of incontinence might be surgery including pudendal nerve damage, hormonal reasons and ageing.It is also possible that there is a failure of effective pressure transmission to the prostatic and membranous urethra as a result of the failure of the fascial supports to the bladder neck and prostate. In this case the prostate was surprisingly mobile suggesting a possible hypermobility effect while the low abdominal leak point pressure is suggestive of the intrinsic weakness of the external sphincter. Some denervation of the external sphincter may have occurred as a result of the construction of the neo-vagina. The experience with this patient suggests that the use of a pubovaginal sling is both logical and applicable to this unusual clinical situation.The autologous pubovaginal sling is a viable option for management of stress urinary incontinence in male to female gender reassigned patients."}
+{"text": "Objective: Amniotic band syndrome is an uncommon clinical entity with a spectrum of presentation including constriction rings, syndactyly, amputations, and spontaneous abortion. The bands themselves are not present at the time of birth. This has created controversy regarding the pathogenesis of the disorder. Methods: The unusual case of a neonate with a persistent band of fibrous amniotic material and the associated deformity of a constriction ring is evaluated. The significance of this unique case is discussed in the context of existing understanding of the syndrome. Results: The amniotic band was released in the neonatal intensive care unit shortly after birth. Despite removal of the band, the constriction ring persisted. Conclusion: The presence of a fibrous amniotic band at the site of a constriction ring is an extremely unusual finding. This case further demonstrates the importance of amniotic material in the etiology of constriction rings. Amniotic band syndrome is an uncommon clinical entity that has been recognized for centuries.In this report, the unusual case of neonate with postnatal persistence of an amniotic band and associated constriction is reported.A female dichorionic, diamniotic twin born at the 27th week of gestation, was noted at birth to have a constriction ring at the distal aspect of her right leg Fig . The conThe fibrous band was removed in the neonatal intensive care unit. The skin underlying the band was noted to be intact, and the band was easily removed Fig . The tisThe etiology of constriction rings has been a topic of debate since the recognition of the clinical entity. Some have speculated that the clinical findings result from malformations due to defective germ cell lines."}
+{"text": "One mechanism of information storage in neurons is believed to be determined by the strength of synaptic contacts. The strength of an excitatory synapse is partially due to the concentration of a particular type of ionotropic glutamate receptor (AMPAR) in the post-synaptic density (PSD). AMPAR concentration in the PSD has to be plastic, to allow the storage of new memories; but it also has to be stable to preserve important information. Although much is known about the molecular identity of synapses, the biophysical mechanisms by which AMPAR can enter, leave and remain in the synapse are unclear. We used Monte Carlo simulations to determine the influence of PSD structure and activity in maintaining homeostatic concentrations of AMPARs in the synapse. We found that, the high concentration and excluded volume caused by PSD molecules result in molecular crowding. Diffusion of AMPAR in the PSD under such conditions is anomalous. Anomalous diffusion of AMPAR results in retention of these receptors inside the PSD for periods ranging from minutes to several hours in the absence of strong binding of receptors to PSD molecules. Trapping of receptors in the PSD by crowding effects was very sensitive to the concentration of PSD molecules, showing a switch-like behavior for retention of receptors. Non-covalent binding of AMPAR to anchored PSD molecules allowed the synapse to become well-mixed, resulting in normal diffusion of AMPAR. Binding also allowed the exchange of receptors in and out of the PSD. We propose that molecular crowding is an important biophysical mechanism to maintain homeostatic synaptic concentrations of AMPARs in the PSD without the need of energetically expensive biochemical reactions. In this context, binding of AMPAR with PSD molecules could collaborate with crowding to maintain synaptic homeostasis but could also allow synaptic plasticity by increasing the exchange of these receptors with the surrounding extra-synaptic membrane. One of the most accepted theories of information storage in neurons is that it is partially localized in the strength of synaptic contacts. Evidence suggests that at the cellular level, in combination with other cellular mechanisms, this is implemented by increasing or decreasing the concentration of a particular type of membrane molecules. Two opposing mechanisms have to coexist in synapses to allow them to store information. On one hand, synapses have to be flexible, to allow the storage of new memories. On the other hand, synapses have to be stable to preserve previously learned information. Although much is known about the molecular identity of synapses, the biophysical mechanisms by which molecules can enter, leave and remain in the synapse are unclear. Our modeling work uses fundamental biophysical principles to quantify the effects of molecular collisions and biochemical reactions. Our results show that molecular collisions alone, between the diffusing proteins with anchored molecules in the synapse, can replicate known experimental results. Molecular collision in combination with biochemical binding can be fundamental biophysical principles used by synapses for the formation and preservation of memories. Efficient synaptic signaling demands that these receptors be concentrated at high densities in order to optimally respond to rapidly diffusing neurotransmitter molecules. For instance, at excitatory glutamatergic synapses of the central nervous system, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are highly concentrated . We first studied the effects of molecular crowding of PSD molecules on the movement of AMPARs in a membrane in the case where the AMPARs do not bind to PSD molecules. Classical theories of diffusion suggest that the effect of elastic collisions with static obstacles is to reduce the Dfree of AMPAR to a lower constant value known as the apparent diffusion coefficient (Dapp) We simulated the diffusion of receptors within synaptic and extra-synaptic regions over a discrete lattice of 2\u00d72 \u00b5m, with periodic boundary conditions to obviate finite size effects see . The lar of time (7)The vat is not expected from classical theories of diffusion or by assuming tortuosity. Tortuosity is characterized by a constant diffusion coefficient (\u03b1\u200a=\u200a1) that is lower than what would be expected from unobstructed diffusion Further analysis of the dependence of \u03b1 as a function of C showed a switch-like behavior. The sigmoidal shape of the plot shown in app. In this case, Dapp\u200a=\u200a\u0394x2/4\u0394t, where \u0394x is the displacement from the initial position after a set time \u0394t determined by the observer. Under conditions of free diffusion (C\u200a=\u200a0) the histogram of values of Dapp results in a median Dapp equal to that reported experimentally (1\u2212exp(\u221210Dfreet/L2)))). If the assumption is that the molecules are confined to diffuse within compartments, then the calculated diffusion coefficient is equal to Dfree. We fitted Kusumi's model to the MSD plots obtained in The model originally developed by Kusumi et al. Synaptic activity regulates the number of AMPAR and their mobility in and out of the PSD BT BT. We considered the possibility that synaptic activity causes PSD molecules or receptors to be modified so that they can bind to each other at random times. As previously shown in Independent of the precise binding and unbinding probabilities of AMPAR to other proteins or the potential multi-step nature of receptor-scaffold interactions, the range of values for hydrogen bonds for protein-protein interaction motifs such as the PDZ domains is 2\u201313 kBT. The logarithmic analysis shows that although anomalous diffusion is present over a short period of time, AMPAR diffusion returns to a normal process characterized by tortuosity (Dfree>Dapp\u200a=\u200aconstant) (BT). Note that since this behavior resulted when all molecules covering 45% of the PSD bind strongly to AMPAR we consider this result to be an extreme case. As has been previously shown, the strength of the binding increases the cross-over time from anomalous to tortuous diffusion We compared the results shown in onstant) . As bindWe next studied the general principle of how transient alterations in binding of PSD molecules can affect the concentration of AMPAR in the context of molecular crowding. Such reactions can be very complex, such as in long term potentiation (LTP) or depression (LTD) BT. For each combination of molecular crowding and binding energies we ran 1000 different simulations in which an AMPAR was randomly placed in the membrane. After each set of 1000 simulations we calculated the percentage change of AMPARs found inside the PSD before and after the stimulation. Each simulation consisted in 500 ms before the stimulus, 100 ms of stimulation, and 700 ms after the stimulus, we calculated the average concentration in the first and last 400 ms of the simulation. The stimulus randomly activated only 10% of all the PSD molecules. Accumulation of AMPARs in the PSD depended on the amount of molecular crowding and strength of the stimulus. BT more AMPARs left the PSD than were absorbed. As the binding strength increased AMPAR started to accumulate in the PSD. We simulated a large PSD that occupied a square area of 0.5\u00d70.5 \u00b5m over a 2\u00d72 \u00b5m patch of membrane. We explored the influence of a wide range of molecular crowding, from 0.00\u20130.60, and of binding energies, from 0\u201314 kTaken together, our results suggest that, under conditions of molecular crowding and anomalous diffusion, steric interactions can have a significant effect in AMPAR retention inside the PSD. Steric interactions in combination with molecular binding can provide synapses the ability to retain AMPARs for prolonged periods of time and the flexibility to allow stimulus evoked AMPAR trafficking with the surrounding membrane.Experimental evidence suggests that AMPAR diffuse non-linearly in the PSD and our simulations based on fundamental biophysical principles replicates these observations. Our model showed that the diffusion and retention of AMPAR in the PSD could be strongly affected by molecular crowding, which arises as a consequence of the high density of macro-molecules found in the PSD. Our simulations suggest that macro-molecular crowding within the PSD can result in anomalous diffusion of AMPAR, a process fundamentally different from diffusion in viscous or tortuous media. Thus, diffusible AMPARs could be retained inside this structure for long periods of time without the need of strong and prolonged binding. Counter-intuitively, but supported by experimental results, the binding of AMPAR to PSD molecules could serve to increase the net AMPAR mobility within synapses. The increased mobility is a consequence of the fundamental biophysical properties of protein diffusion and reaction on membranes. The functional consequence of the combination of trapping of AMPAR by molecular crowding and mobility by binding to PSD molecules results in the capability to regulate AMPAR transport in and out of the PSD.There are three fundamental physical assumptions common to all membrane protein diffusion studies that apply to the diffusion of AMPAR in and out of the PSD. First, when diffusing in an obstacle-free membrane AMPARs undergo normal diffusion (eq. 1). Second, AMPARs bind non-covalently and reversibly with PSD molecules There is extensive experimental data showing that AMPAR movement in the extra-synaptic membrane can be described as an elastic random walk with elastic collisions The presence of membrane anchored proteins acting as obstacles for membrane protein diffusion has been documented in other cell types \u221215 gr), with a volume of 3.06\u00d7106 nm3\u221221 gr/nm3\u221215 gr. Therefore, the fraction of PSD occupied by macro-molecules is (1.83\u00d710\u221215/4.27\u00d710\u221215) 43% by mass. The volume occupied in a PSD with the same dimension can be calculated by using the assumption that in a PSD there are 10,000 molecules of 100 kD 3 * A)]1/3, with A being Avogadro's number, and M.W. the molecular weight). Although, not all cases of molecular crowding necessarily result in anomalous diffusion Since steric interactions are due to the physical presence of molecules and is not dependent on their identity, the diffusion of AMPAR should be affected by the total concentration of all macro-molecules. Even though a single molecular species might be regularly distributed over the PSD, the collection of all molecules could generate a dense mesh that effectively constitutes a random distribution of macro-molecules An alternative structural mechanism to confine receptor diffusion is the picket-and-fence model BT . Although no data is available to determine the percentage of PSD molecules that might bind to AMPAR or their strength, our simulations predict that changes in only a few of them are necessary to flip the \u2018molecular switch\u2019 from AMPAR retention to AMPA diffusion. These interaction energies may be directly measured using optical trapping techniques, as for the case of diffusing cadherin and transferrin receptors Dissociation constants for binding of AMPAR, or transmembrane AMPA receptor regulatory proteins (TARPs), such as stargazin, to the C-terminal domain binding proteins (CTDBP), such as GRIP, PICK and MAGUK proteins such as PSD-95, are in the range of 1\u201310 \u00b5M rot) rot range from 10\u2013100 \u00b5s. Rotational diffusion in combination with conformational changes can significantly modify the diffusional environment of diffusing molecules in the PSD. Molecular aggregation can modify this rotation to the point of stopping it Although AMPAR-TARP interactions can be disrupted by glutamate Experimental evidence suggests that the PSD is a tight mesh of proteins that can be considered stationary rot will decrease, thus measurements of Drot in time would show changes from high to low values. However, if the receptors are in a crowded environment without binding then we expect to have a similar distribution of the values of Drot inside and outside the synapse.Measuring rotational diffusion of AMPARs or non-interacting molecules, such as NCAM, in the synaptic and extra-synaptic space would provide valuable information. We hypothesize that when AMPAR binds to scaffolding molecules its DBT, assuming a characteristic attempt rate of 1 \u00b5s results in an expected time to unbind of 0.44 ms (1\u00d710\u22126/e\u221213). In the absence of steric interactions these AMPARs could escape the PSD if they are not bound to another anchored molecule. Steric interactions and molecular crowding could improve the retention of AMPARs in the PSD by considerably reducing the diffusion coefficient and thus increasing the probability of binding to another PSD molecule.The notion of synaptic stability requires the retention of AMPARs for long periods of time zero-energy model of AMPAR retention in the PSD requires no extra energy from the synapse to retain a high density of AMPARs over long periods of time.As noted above, the binding time of AMPAR to any PSD scaffold protein is much smaller than the characteristic time expression of LTP or LTD (minutes to hours). Our models suggest that molecular crowding, in conjunction with other cellular mechanism of synaptic homeostasis We hypothesize that plasticity mechanisms work on top of the basic biophysical mechanisms of receptor diffusion in a crowded PSD. For instance, phosphorylation of receptors, may allow a diffusing receptor to bind to PSD molecules via weak PDZ-domain interactions and enter the synapse. Even if the receptor is subsequently dephosphorylated, it can remain trapped in the synapse by crowding. Secondly, while experimental evidence suggests that the PSD is a tight mesh of proteins that can be considered stationary In order to maintain a constant density of AMPARs in the PSD there should be a constant number of PSD molecules ready to bind AMPARs Assuming a well-mixed PSD reduces the computational capacity of the synapse. In a well-mixed PSD all AMPARs have the same probability of binding with any other activate PSD molecule and their position within the PSD is not important. Such a process reduces the capacity of encoding synaptic activity to the total current generated by the activation of the glutamate receptors. If a PSD contains a small number of AMPARs then the encoding of the synaptic signal is inherently noisy and it is assumed to be averaged over multiple trials or multiple synapses freeAnomalous diffusion due to molecular crowding results in an increase in the correlation of the particle position along time, the appearance of long correlations can be measured using fluorescence correlation spectroscopy Overall, our modeling efforts suggest that molecular collisions can keep AMPAR inside the PSD without requiring extra energetic processes free is the diffusion coefficient, and MSD is the average mean square displacement of the particle. The instantaneous MSD can be calculated over an ensemble of multiple independently diffusing particles,i is the relative position of particle i to its initial position at time t, and N is the number of particles.The average spatial displacement of a particle diffusing in a two dimensional membrane is described byfree\u200a=\u200a0.200\u00d710\u22123 \u00b5m2/ms which results in a median Dfree\u200a=\u200a0.138\u00d710\u22123 \u00b5m2/ms; The membrane model consisted of a square mesh with toroidal boundary conditions. Particles crossing over the edge of the diffusing space would appear on the opposite edge in the next time step. The size of the rectangular mesh is specified in each simulation, but it was at least equivalent to 1 \u00b5m in side. The simulations were fully characterized by the time step \u0394t and the diffusion coefficient of AMPAR in the extra-synaptic membrane which we assume is the closest to an unobstructed system .The models were implemented using Matlab  in combination with Star-P . Star-P allowed us to utilize and run the original Matlab model in parallel at the Computational Biology Initiative high performance cluster at UTSA (Text S1Supplementary Information(0.27 MB PDF)Click here for additional data file."}
+{"text": "The 10th author's affiliation is incorrect. The affiliation should be institution 1: Department of Clinical Pharmacology, University Hospital of T\u00fcbingen, T\u00fcbingen, Germany."}
+{"text": "Sickle Cell Disorder is a global health problem with psychosocial implications. Nigeria has the largest population of people with sickle cell disorder, with about 150,000 births annually. This study explored the psychosocial impact of sickle cell disorder in 408 adolescents and adults attending three hospitals in Lagos, Nigeria. A questionnaire was designed for the study, with some of commonly described areas of psychosocial impact including general public perceptions and attitudes, education, employment, and healthcare issues, and emotional responses.The majority of participants thought that society in general had a negative image of SCD, and reported negative perceptions and attitudes. Some issues in education, employment, and healthcare were expressed, however these were in the minority of cases. The results also showed that depressive feelings were experienced in almost half the study population, even though feelings of anxiety or self-hate were uncommon. Clinical implications of these findings are considered. Sickle Cell Disease (SCD) and Thalassaemia are classified as the two main Haemoglobin Disorders, and in recent years have been acknowledged to have a global impact by the World Health Organisation (WHO). SCD comprises a group of inherited red blood cell conditions that result from the synthesis of variant or mutant haemoglobins. Over 300,000 babies are born worldwide with SCD mostly in low and middle income countries, with the majority of these births in Africa . SCD oriGenerally, the prevalence of healthy carriers (sickle cell trait) ranges between 10% and 40% across equatorial Africa and decreases to between 1% and 2% in Northern Africa and less than 1% in Southern Africa. In West African countries such as Ghana and Nigeria, the frequency of carriers is 15% to 30% while in East African countries such as Uganda and Tanzania it shows wide variations of up to 45% in some areas [SCD is a chronic condition with recurrent episodes of pain and symptoms persistent throughout the lives of individuals. The clinical syndromes include anaemia, infection, and the consequences of blood vessel blockage (vaso-occlusion). The latter deprives tissues of oxygen and is suggested as the cause of the acute painful episodes, the hallmark of SCD, and other clinical syndromes such as stroke, chest complications, priapism, leg ulceration and chronic organ failure. The prominence of the pain is the basis upon which SCD has been named in certain cultures in West Africa . These iThere is no universal cure for SCD and treatment options are rather limited, however improved knowledge has greatly advanced medical management over the past four decades. Antibiotic prophylaxis is used to prevent infections especially in children . Other tBlood transfusions may be required for stroke and other complications, and hydroxyurea has also been found to be very effective in reducing the 'sickling' process and consequently the frequency of pain and hospitalisations experienced by patients . Bone maPsychosocial issues for people with SCD and their families mainly result from the impact of pain and symptoms on their daily lives, and society's attitudes to SCD and those affected. In Africa, cultural factors are particularly relevant to these problems because of beliefs and traditional practices. In Nigeria, beliefs are usually influenced by cultural and religious values, which influence health behaviour such as coping strategies. For example, among the Igbo communities, SCD is believed to be the result of malevolent 'Ogbanje' (reincarnation) that is repeated cycles of birth, death and reincarnation . Other sThe object of this study was to explore the psychosocial impact of SCD within a Nigerian population. It is acknowledged that psychosocial interventions for people with SCD are as important as medical treatment for comprehensive management.Pre-study focus groups were conducted earlier to explore and identify issues in general and specific concerns of individuals with SCD in order to develop or obtain appropriate measures to be used in the studies. These groups comprised of adolescents and adults aged thirteen years and over, were non-directive, but semi-structured with open-ended questions, and facilitation towards a goal at the end of each session. There were two group sessions with 6 to 8 participants, and each group talked about the same issues with the same set of questions. A number of open-ended questions were discussed in each group session, and answers were written down. The main areas explored included knowledge and understanding, beliefs, pain experience, coping, health service utilisation, social issues including education and employment, and quality of life. This led to the development of the questionnaire, which was subsequently used in the study. However, the questionnaire was not utilised in the focus group sessions.Psychosocial Impact of Sickle Cell Disorder - a self-complete questionnaire designed for the study  were from a Yoruba ethnic origin. The attitudes and perception about SCD were examined under three broad categories of general public, education and employment, and health services Table . These wThe responses of the adolescents were mostly similar to the adults. The majority of participants thought that society in general had a negative image of SCD through its attitudes and perceptions, although they did not feel that people had a negative approach towards them because of their SCD. Nonetheless, a very large proportion of respondents thought that SCD as an illness is less known in society and needs more awareness, and if society was more aware of people with SCD it would be more positive about those with the condition. Participants had not experienced much teasing and bullying during their education in school, college or university. Discrimination from employers was also not widely reported. Experiences of health services were mostly positive especially among adolescents, as the vast majority of them felt that medical staff had a good understanding of SCD compared to adults. Despite their age group, most participants acknowledged that medical staff offered them fair treatment.Emotional responses to SCD are presented in Table The impact of chronic illness such as SCD on individuals may be grouped into a set of illness-related tasks: adjusting to the symptoms and incapacities; maintaining adequate relationships with health-care providers; and managing the emotional and social consequences of the illness . The extThis study sought to explore the psychosocial impact of SCD in a Nigerian population. The results support the notion that society's attitudes and perceptions had a psychosocial impact on people with SCD. Health beliefs could be influenced by external factors such as advice given by health workers, family support, and work responsibility. Beliefs can also be influenced by their culture. For example, it has been suggested that Nigerians have tried religious healing (prayer) as an alternative approach or in addition to routine medical treatment . This maAbout three-quarters of the study population were students, and over a third were adolescents aged 14 to 18 years. Anecdotal evidence suggests teasing and bullying are common complaints among school going children with SCD. This was reported in 23% of the study population. Other major psychosocial problems experienced by young people with SCD during their school going years have also been described in focus groups [Mood is an important consequence of SCD. People with SCD commonly report low self-esteem and feelings of hopelessness as a result of frequent pain, hospitalisations, and loss of schooling (in children) and employment (in adults). These accounts could indicate depressive symptoms. Some studies have revealed anxiety and depression in children with SCD , and depThere is considerable variability in the ability of people with SCD to cope with their condition. People with SCD experience different levels of health, and such variations can lead to differences in psychosocial functioning. Some people cope relatively well, attend school or work, and are active physically and socially. Others cope inadequately' leading more limited and secluded lives. Nonetheless, this may not necessarily be a consequence of severity of their condition, and the reasons for these should be sought and addressed. Quality of life in people with SCD may therefore be more impaired than that of the general population.There are some limitations to the study. The use of dichotomous questions was mainly to address possible literacy problems in some patients who may have found it difficult to grade their responses on a Likert type scale, thus avoiding confusion. Dichotomous questions are useful in situations where the intention is to direct participants to express a clear opinion between opposing perspectives. However, a major drawback is that questions have only fixed alternatives, that is 'Yes' and 'No' in this case. Closed questions do not allow respondents to qualify or explain their answers, which may lead to bias in the interpretation due to the categories imposed. No standard questionnaires for the psychosocial impact of sickle cell disorder were identified from previous research, nor were there generic psychosocial impact measures in the chronic illness literature that could be employed for the purpose of this study.Furthermore, information obtained was cross-sectional, longitudinal data would have enhanced the results. More information on socio-economic status would have been useful. Also, the opportunistic selection of patients in a clinic setting may have been biased; a community-based study may have yielded different results, given that the views of patients not receiving medical treatment could be different. Nonetheless, the study contributes to our understanding of the psychosocial impact of SCD within the African setting.The findings of this exploratory study indicate that there is a need to develop appropriate psychosocial interventions within a global context. The aim should be more public education about SCD to change beliefs, attitudes, and stigma as a starting point. This should be followed by other interventions that are tailored depending on access to health care at different levels, and in different settings. In developing countries such as Nigeria, the focus would be initiating basic psychosocial interventions by non-specialised health workers in a primary care team at a Community Health Post (Level 1) or Primary Health Centre (Level 2), where psychologists or social workers are not be available. This would be in line with the WHO's current priority of primary care as a hub of coordination for person-centred care .Within the global context, the aims of psychosocial interventions in chronic illnesses such as SCD are to help reduce negative thoughts and feelings about the condition, and encourage the acquisition or maintenance of coping strategies. In the case of SCD, psychological therapies should be offered as standard care in the management of SCD adjunctive to routine medical treatment, where studies of these therapies have shown encouraging results . The oveThe authors declare that they have no competing interests.KA conceived, designed, and participated in the co-ordination of the study. FE participated in the co-ordination, data collection, and performed the statistical analyses. OO was involved in the design and supervision of the study. KA took the lead in the write up with support from FE, and review and editing by OO. All authors read and approved the final manuscript.Ethics Approval was not sought for the study, however each individual had to give their consent for participation in the study.Psychosocial Impact of Sickle Cell Disorder. A 29-item self-complete questionnaire designed for the study.Click here for file"}
+{"text": "Cough in the patients with cough variant asthma is triggered by bronchoconstriction, which responds to bronchodilator therapy. Following airway narrowing induced by inhaled methacholine, deep inspiration (DI) causes dilation of the airways in both asthmatic and non-asthmatic subjects. The aim of the present study was to investigate the relationship between bronchodilator effect of DI and bronchoconstriction-triggered cough.40) was obtained and the volume was used as the reference volume to determine the isovolume flow from the partial curve (PEF40). Coughs were counted for 32 min during and following the inhalation of methacholine at the provocative concentration which produced a 20% fall or more in FEV1from the post-saline value (PC20-FEV1). The bronchodilator effect of DI on bronchoconstriction induced by methacholine at the PC20-FEV1 concentration was expressed as the ratio of (MEF40-PEF40)/PEF40 (DI index).We measured airway responsiveness to methacholine using partial and full flow-volume curves in 28 healthy adults. The expiratory flow at 40% above residual volume from the full forced vital capacity (MEF20-FEV1 concentration of methacholine was 39.3 \u00b1 29.7 (mean \u00b1 SD)/32 min. The number of coughs during and following the inhalation was correlated with DI index , but not with PC20-FEV1 or change in FEV1 or PEF40 by inhalation of the PC20-FEV1 concentration of methacholine.The number of coughs for 32 min during and following the inhalation of PCWe found that methacholine-induced cough was associated with the bronchodilator effect of DI on methacholine induced-bronchoconstriction in normal subjects. Cough is a common and distressing symptom. Eosinophilic airway disorders such as bronchial asthma (BA), cough variant asthma (CVA) , eosinopCapsaicin has achieved widespread use for measurement of cough reflex sensitivity . SeveralIt is well recognized that the response of the airways to deep inspiration (DI) differs between asthmatic and nonasthmatic subjects. Following airway narrowing induced by inhaled methacholine, DI causes dilation of the airways in both asthmatic and nonasthmatic subjects . HoweverThe aim of the present study was to investigate the correlation of cough triggered by methacholine-induced bronchoconstriction to airway smooth muscle (ASM) tone and bronchodilating effect of DI on methacholine-induced bronchoconstriction in normal subjects.Twenty-eight normal subjects  were selected from 30 randomly selected Japanese college students who visited our pulmonary function laboratory for actual training of pulmonary function testing including methacholine challenge test using partial and full flow-volume curves. Subjects with a history of wheezing were excluded (Table 1 (PC20-FEV1) from the post-saline value occurred. After each concentration of methacholine inhalation, partial and full flow-volume curves were measured. Coughs were counted for 2 min during the inhalation and for 30 min following the inhalation of methacholine at PC20-FEV1. After cough count, the bronchoconstriction was subsequently reversed with salbutamol.We measured airway responsiveness to methacholine using partial and full flow-volume curves in 28 normal subjects. Study design is shown in Figure 1 from the baseline value after inhalation of saline solution was 10% or less, inhalation of methacholine was started. Methacholine was inhaled for 2 min by tidal mouth breathing wearing a nose clip, and this was followed immediately by 3 measurements of partial and full flow-volume curves at 1 min intervals and the curve with the largest forced vital capacity (FVC) was retained for analysis. Subjects avoided to deep inspiration (DI) before the each measurements of partial and full flow-volume curves. Increasing concentrations of methacholine were inhaled until PC20-FEV1.Methacholine chloride was dissolved in physiologic saline to make solutions of 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 mg/ml. Physiologic saline solution and methacholine were inhaled from a nebulizer  operated by compressed air at 5 l/min. The nebulizer output was 0.28 ml/2 min. Saline solution was inhaled first for 2 min and partial and full flow volume curves were measured using a computerized spirometer . After confirming that the change in FEV40) was obtained and the volume level was used as the reference volume level to determine the isovolume flow from the partial curve (PEF40). The levels of end-tidal inspirations were similarly adjusted in all partial flow-volume curves. /PEF40 [40-PEF40)/PEF40 after the inhalation of PC20-FEV1 concentration of methacholine was defined as DI index. DI index means bronchodilating effect of deep inspiration following measurements of partial flow-volume curves on PC20-FEV1concentration of methacholine-induced bronchoconstoriction.The bronchodilating effect of DI on methacholine induced-bronchoconstriction was expressed as the ratio of (MEF0)/PEF40   and PEF40(PC35-PEF40) were expressed as geometric means with the geometric standard error of the mean (GSEM) expressed as a factor. Values for FVC, FEV1, MEF40, and PEF40 were reported as arithmetic means and standard deviations of the mean (SD). Wilcoxon signed-ranks test was applied to assess the change in the ratio of (MEF40-PEF40)/PEF40. We constructed simple regression models to evaluate the relationship between any pairs of the number of coughs for 32 min during and following the inhalation of PC20-FEV1 concentration of methacholine, DI index, concentration of inhaled methacholine and changes in FEV1 and PEF40 by the methacholine inhalation. In all analyses, values of p < 0.05 were considered statistically significant.Values of PC1, % predicted value of FEV1, FEV1/FVC ratio, MEF40, PEF40, (MEF40-PEF40)/PEF40 are shown in Table 20-FEV1, PC35-MEF40, and PC35-PEF40, the mean value for percent change in FEV1, MEF40, and PEF40 from the baseline value, DI index and the number of coughs for 32 min during and following the inhalation of PC20-FEV1 concentration of methacholine are shown in Table 1 did not occur at the final concentration of methacholine solution (160 mg/ml), and the PC20-FEV1 value for these subjects was assumed to be 320 mg/ml for statistical analysis. 35% or more fall in MEF40 was not produced by the final concentration of methacholine in 4 (14.2%) of 28 subjects and the PC35-MEF40 value for these subjects was assumed to be 320 mg/ml for statistical analysis. On the other hand, 35% or more fall in PEF40was produced by 160 mg/ml or less of methacholine in all subjects.The mean values for FVC, % predicted value of FVC, FEV40-PEF40)/PEF40 following inhalation of methacholine at individual concentration causing 20% or more fall in FEV1 (DI index) was significantly increased from the baseline value.  [Figure The mean value for the ratio of , or change in FEV1  or PEF40  by inhalation of the PC20-FEV1 concentration of methacholine.The number of coughs for 32 min during and following the inhalation of PC20-FEV1 concentration of methacholine in 28 normal subjects. The bronchodilating effect of DI on methacholine induced-bronchoconstriction was evaluated using the ratio of (MEF40-PEF40)/PEF40. We found that methacholine-induced cough was associated with the bronchodilating effect of DI on methacholine induced-bronchoconstriction, but not with PC20-FEV1 concentration of inhaled methacholine or the provocation-induced decrease in FEV1 in normal subjects. We, therefore, suggest that cough triggered by bronchoconstriction is regulated by protective response against bronchoconstriction, but not by the magnitude of bronchoconstriction. Canning et al. [In this study, we measured airway responsiveness to methacholine using partial and full flow-volume curves and coughs caused by the inhalation of PCg et al.  reportedin vivo preparation of guinea pig airway smooth muscle (ASM). Bronchoprotection was defined in these studies as the reduction in bronchoconstriction resulting from act of DI before the ASM has been stimulated to constrict [in vivo studies [DI can affect acute airway narrowing by following two mechanisms: bronchoprotection and bronchodilation. Scichilone and coworkers  studied onstrict  and brononstrict ,20. The  studies , which sIt has been shown that in asthmatics there is impaired dilatation of the airways in response to stretch ,21. This1, or the methacholine-induced change in FEV1 or PEF40. Irwin and coworkers [In this study, the number of coughs following methacholine inhalation was not correlated with concentration of inhaled methacholine causing a 20% or more fall in FEVoworkers  studied oworkers  to disti20-FEV1 concentration of inhaled methacholine or methacholine-induced decrease in FEV1 in normal subjects. Further studies are needed to investigate the relationship among cough triggered by methacholine-induced bronchoconstriction, ASM tone and bronchodilating effect of DI on the methacholine-induced bronchoconstriction in patients with CVA in comparison with typical asthma, atopic cough and so on.In conclusion, this is the report concerning cough triggered by methacholine-induced bronchoconstriction in humans. We found that bronchoconstriction-triggered cough was associated with the bronchodilating effect of DI on the induced-bronchoconstriction, but not with PCThe authors declare that they have no competing interests.NO recruited the subjects, performed the data collecting and draft the manuscript. MF conceived the study, contributed to its design, data acquisition, data interpretation, and review and correction of the manuscript. AT performed the statistical analysis and data interpretation. JH participated in data acquisition. AH participated in data acquisition. MN participated in data acquisition. MA contributed to data interpretation. NK contributed to data interpretation. All authors have given final approval of the version to be published."}
+{"text": "There was an error in affiliation 2 for authors Ewert Linder and Cecilia Thors. Affiliation 2 should be: 2 Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden"}
+{"text": "Several lines of evidence suggest that sickle cell disease (SCD) is associated with a chronic inflammatory state. In this study of 70 children with SCD at steady state evaluated by a broad panel of biomarkers representing previously examined mechanisms of pathogenicity in SCD, high sensitivity C-reactive protein (hs-CRP), a marker of low-grade, systemic inflammation, emerged as the most significant laboratory correlate of hospitalizations for pain or vaso-occlusive (VOC) events. While markers of increased haemolytic status, endothelial activation and coagulation activation all correlated positively with VOC events by univariate analysis, baseline hs-CRP levels provided the most significant contribution to the association in multiple regression models (22%), and, hs-CRP, along with age, provided the best fit in negative binomial models. These data highlight the clinical relevance of the role of inflammation in paediatric VOC, providing both a rationale for future therapeutic strategies targeting inflammation in microvessel occlusive complications of SCD, and the potential clinical use of hs-CRP as a biomarker in childhood SCD. Microvessel occlusion, the main pathological process underlying the cardinal clinical manifestation of sickle cell disease (SCD), is frequently termed as vaso-occlusive crisis (VOC) or pain crisis . Since tSeveral lines of evidence suggest that SCD is associated with a chronic inflammatory state  aged 2\u201320 years, evaluated in basal steady state at the time of their \u2018routine\u2019 health care maintenance visit to the sickle cell clinic. Patients were considered to be in steady state if they were afebrile, asymptomatic with SCD, and had not been hospitalized for at least 10 d prior to date of blood draw. No patient was on chronic transfusion therapy. No subject was on hydroxycarbamide therapy. Age-matched HbAA controls (3\u201321 years) were also included. This study was reviewed and approved by the Institutional Review Committee for the protection of human subjects at St Christopher\u2019s Hospital for Children/Drexel University and Thomas Jefferson University. In accordance with the Declaration of Helsinki, informed consent was obtained prior to patient blood sampling at the time of enrollment in these studies. For minors, patient assent where appropriate, was obtained in addition to parental permission. A retrospective review was performed on individual patient records for a discharge diagnosis of painful episode or VOC, including those complicated by the occurrence of acute chest syndrome  for Windows version 15 to account for overdispersion and excessive zeros in the dependent variable, i.e. the number of hospitalizations for pain in the designated 3-year period.Biomarkers were assayed by commercially available enzyme-linked immunosorbent assay kits as follows: Those pertinent to inflammation and endothelial activation ; and coagulation activation/fibrinolysis . LDH levels were measured using an LDH assay kit . The enzyme activity in i/u per litre was read from a calibration curve generated using an LDH standard (Sigma Aldrich). Haematological indices  were obtained using a Coulter Counter, Model StK S. Statistics was performed using Sigmastat statistical package . Association between any two variables was tested with Spearman and Kendall Tau Correlation . Multipl0thalassemia n = 48 including n = 5 with HbS\u03b20thalassemia (henceforth referred to as HbSS group), HbSC, n = 22; males = 37, females = 33. Clinical and laboratory data were consistent with previously described haematological parameters in children with SCD  and hospitalizations for pain HbSS > HbSC, P = 0\u00b7001 (versus HbSC with regard to coagulation activation , haemolysis (LDH) (P < 0\u00b7001), and endothelial activation [VCAM-1 (P = 0\u00b7027)], sP-selectin (P < 0\u00b7001)  . These rn = 48, median CRP = 2\u00b78 mg/l ], whereas median hs-CRP in the HbSC group  was similar to the NHANES control values [median CRP = 0\u00b74 mg/l ] as well as the smaller number of age-matched controls from our study . In 25 of the 70 patients, hs-CRP levels were assayed on available plasma samples from at least two different time points (ranging from 3 to 9 months apart). Intra-individual variability for CRP levels in these 25 samples was low, as evidenced by strong association between the pairs of measurements by Kendall Tau rank correlation  and Spearman rank correlation  identified associations only with hs-CRP and WBC which contributed 22% and 5% respectively, to the overall model. When data from HbSS group alone were evaluated in the regression models, only hs-CRP stayed in the models contributing 13% to the overall association. These results were confirmed with a negative binomial regression model that identified age and hs-CRP as the best fit to these count data, predicting a 12% increase in pain frequency for every year of age, and a 6\u00b76% increase in pain frequency for every 1 mg/l increase in hs-CRP . Thus hs0Thal patient group the inverse correlation between CRP and HbF levels fell short of statistical significance in this study .In the SCD group , hs-CRP This study of children and adolescents with SCD showed that, from a broad panel of steady-state biomarkers, the inflammatory marker hs-CRP was the most significant correlate of hospitalizations for painful episodes. While markers of increased haemolytic status, endothelial activation and coagulation activation all correlated positively with VOC events by univariate analysis, only the inflammatory markers hs-CRP and WBC stayed in multiple regression models, and age and hs-CRP provided the best fit in negative binominal models. These data showed that hs-CRP, a well- established inflammatory biomarker , was strOver recent years, the role of hs-CRP as a plasma biomarker for low-grade systemic inflammation has been intensely investigated for its predictive associations with adverse outcomes in vascular diseases, such as cardiovascular . A subsen = 12) (n = 35) . Our cohort was also skewed more toward younger children given our interest in the development of SCD symptomatology from early childhood through adolescence and young adulthood. However, we feel that because of this unique data set, we have been able to document hs-CRP elevations at a relatively young age in the HbSS with potential future clinical consequences.In summary, the present study demonstrated a strong association between the inflammatory biomarker hs-CRP and hospitalization for VOC events in paediatric SCD. We believe these results highlight the clinical relevance of inflammation in microvessel occlusive complications, and provide a basis for the study of hs-CRP as a potential biomarker for predictive modelling of clinical outcomes in paediatric SCD. Perhaps most significantly, these data culled from a moderate-sized cohort of 70 children with SCD, lend support for the clinical reconciliation of strong experimental evidence for the role of inflammation in SCD vasoocclusion, and provide a basis for the future trials of primary or adjuvant anti-inflammatory therapies in SCD."}
+{"text": "The near exclusive use of praziquantel (PZQ) for treatment of human schistosomiasis has raised concerns about the possible emergence of drug-resistant schistosomes.Schistosoma mansoni obtained from patients from Kisumu, Kenya continuously exposed to infection as a consequence of their occupations as car washers or sand harvesters. We used a) an in vitro assay with miracidia, b) an in vivo assay targeting adult worms in mice and c) an in vitro assay targeting adult schistosomes perfused from mice. In the miracidia assay, in which miracidia from human patients were exposed to PZQ in vitro, reduced susceptibility was associated with previous treatment of the patient with PZQ. One isolate (\u201cKCW\u201d) that was less susceptible to PZQ and had been derived from a patient who had never fully cured despite multiple treatments was studied further. In an in vivo assay of adult worms, the KCW isolate was significantly less susceptible to PZQ than two other isolates from natural infections in Kenya and two lab-reared strains of S. mansoni. The in vitro adult assay, based on measuring length changes of adults following exposure to and recovery from PZQ, confirmed that the KCW isolate was less susceptible to PZQ than the other isolates tested. A sub-isolate of KCW maintained separately and tested after three years was susceptible to PZQ, indicative that the trait of reduced sensitivity could be lost if selection was not maintained.We measured susceptibility to PZQ of isolates of S. mansoni from some patients in Kisumu have lower susceptibility to PZQ, including one from a patient who was never fully cured after repeated rounds of treatment administered over several years. As use of PZQ continues, continued selection for worms with diminished susceptibility is possible, and the probability of emergence of resistance will increase as large reservoirs of untreated worms diminish. The potential for rapid emergence of resistance should be an important consideration of treatment programs.Isolates of  The emergence of drug resistant pathogens is a great challenge to the control of infectious diseases. Schistosomiasis is one of the world's greatest neglected tropical diseases, and it is primarily controlled with the drug praziquantel. This drug is often used by repeatedly treating patients to maintain reduced worm burdens, an ideal situation to encourage the evolution of resistant worms. Although drug based control programs are increasing, monitoring efforts for drug resistance remain rare. We measured drug susceptibility of schistosomes from a cohort of patients in Kenya who are enrolled in a longitudinal study in which they are repeatedly treated with praziquantel. We found that schistosomes from previously treated patients were significantly less susceptible than those that were not. Also, schistosomes derived from a single patient who had been treated with praziquantel 18 times showed marked resistance. Although the findings of this study indicated that reduced drug susceptibility occurs in this population of schistosomes, this trait does not seem to be spreading widely or creating clinical levels of resistance. We hypothesize that the trait remains at low frequency because of the large population of schistosomes that are not exposed to the drug and/or potential fitness costs associated with reduced susceptibility. Schistosoma mansoni is one of the most common etiological agents for human schistosomiasis, and is estimated to infect more than 83 million humans in 54 countries Schistosomiasis is one of the most common human parasitic diseases in the world. An estimated total of 207 million persons are infected worldwide, 97% of which are on the African continent Praziquantel (PZQ) is the least expensive, easiest to use and most readily available of all currently available schistosomicides S. mansoni in only 2 generations of repeated exposure to sub-lethal doses of the drug in mice The extensive use of PZQ for over 20 years in some African nations has raised concern regarding the selection of drug resistant worms in vivo tests in laboratory hosts such as mice can address some of these concerns in vitro testing that removes host-induced effects. In vitro tests for PZQ sensitivity have been developed for use on schistosomes Measuring the impact of PZQ on adult worms harbored by human subjects is at best an indirect process in that it relies on the relatively insensitive method of measuring reduction in schistosome egg excretion in treated individuals S. mansoni that were derived from Kenyan car washers and sand harvesters occupationally exposed to schistosome infection in Lake Victoria. These people have been enrolled in a longitudinal study designed to investigate immunologically-based host resistance to S. mansoni re-infection, and many of the participants have been treated with PZQ on multiple occasions throughout the study in vitro assay with miracidia to evaluate PZQ susceptibility of S. mansoni from several patients. Then, an isolate of S. mansoni was established in laboratory hosts from one of the patients, who had never fully cured after initial or several subsequent PZQ treatments. The susceptibility of adult worms of this and other Kenyan isolates and of two laboratory stocks of S. mansoni were compared using both conventional in vivo trials in mice, and with an in vitro assay with adult worms.The aim of this study was to evaluate the level of susceptibility to PZQ among a natural population of S. mansoni isolates were recovered from eggs in the fecal samples of adult males working as car washers or sand harvesters in or near Kisumu, western Kenya. The car washers use the shallow water along the Lake Victoria shoreline to wash cars and trucks and so are repeatedly in contact with water in an area where snails infected with S. mansoni snails have been repeatedly found S. mansoni infection S. mansoni using the modified Kato-Katz technique. For the purposes of the present study, miracidia hatched from S. mansoni eggs were used either directly in the PZQ susceptibility assay for miracidia described below or, in two cases, were used to establish laboratory isolates.The patient-derived B. sudanica and six to eight week old outbred mice:Miracidia obtained from eggs in positive fecal samples from individual patients were used to establish the following isolates in laboratory raised KCW: The KCW isolate was established from a car washer who had received 18 PZQ treatments since the beginning of the study. Following initial and all subsequent treatments, egg counts in this patient had never fallen to zero KSH: The KSH isolate was established from a sand harvester who had never been treated with PZQ prior to this study.KAS: This isolate was established from a human fecal sample collected near a stream (Asao) about 38 Km south east from Kisumu in 2006 .KNY: The KNY isolate was established from cercariae obtained from B. sudanica collected in Nyabera marsh  on the outskirts of Kisumu on the road to Ahero. Both KAS and KNY isolates were from areas not previously included in PZQ treatment campaigns of local school children.In addition to the isolates obtained from Kenya patients, the following isolates were used for comparison:PR1: This laboratory stock is originally of Puerto Rican origin. It has been maintained in the laboratory, including at the University of New Mexico, for more than 20 years.NMRI: This laboratory stock also originated from Puerto Rico and has been maintained at the Biomedical Research Institute, Rockville, Maryland (www.afbr-bri.com).in vitro technique developed by Liang et al. \u22125 M, or 10\u22126 M, in a 40 \u00b5l volume. PZQ was prepared as a stock solution of 10\u22124 M in 1% DMSO, and the final concentration of DMSO was 0.1% in all wells including the control wells. The mean number of groups of miracidia used per patient per concentration of PZQ was 10.2 (range: 4\u201312) . This design was repeated with miracidia from three different fecal samples from each patient. Miracidia were observed with the aid of a dissecting microscope prior to, 10 min and 20 min after addition of PZQ, and the observer had no knowledge of the PZQ concentration of each well. The number of miracidia that were alive and dead in each well was recorded. Miracidia were considered dead if they remained immobile. We present only results using 10\u22125 M at the 20 min observation time to simplify the presentation and because they are representative of the combinations of observation times (10 or 20 minutes) and PZQ concentrations  used. A Generalized Estimating Equations (GEE) approach was used to fit a logistic regression model to the data using the Proc GENMOD procedure in SAS 9.1. Separate models were fit for each concentration of PZQ . The number of previous PZQ treatments the subjects received was included in the model using an indicator variable that was set to \u20181\u2019 if a patient had received no previous treatments, and\u20180\u2019 for patients who had received one or more previous treatments, and the time of exposure to the drug in vitro .To test the idea that miracidia derived directly from different patients may differ in their susceptibility to PZQ, particularly given that some patients (see KCW above) had never fully cured following treatment with PZQ, we employed a modified version of the S. mansoni examined, 10 to 30 outbred mice were infected with 200 cercariae per mouse. Seven and a half weeks after exposure, mice were randomly divided into two groups, one of which was given 1000 mg/kg PZQ dissolved in 2% Cremaphor EL over 4 consecutive days (250 mg/kg per day). This dose was chosen because it will achieve a parasite reduction of at least 95% in mice For each isolate of in vivo assay was performed in which treatment was given at two time points. Mice were exposed to S. mansoni, randomly divided into 3 groups, and treatment was administered at either 7.5 or 10.5 weeks after exposure. The control group received the sham treatment at 7.5 weeks post exposure. Immature worms are not susceptible to PZQ, and studies of the NMRI isolate indicated that susceptibility corresponds to the onset of reproductive development at 6 to 7 weeks post infection To eliminate the possibility that low PZQ susceptibility was due to a longer development time of the KCW worms, an additional in vivo after 3 years of passages in the absence of drug pressure or testing. Mice infected with this subset of KCW were treated at 10 weeks post-exposure, following the above protocol.The sub-isolate of KCW that was kept at the Kenya Medical Research Institute (KEMRI) was tested for PZQ sensitivity The efficacy of treatment (ET) with PZQ as applied to the different isolates was measured as the percent of reduction of worm burdens based on the numbers recovered using the formula S. mansoni isolates with a randomized Generalized Linear Model 2-way ANOVA after log transformation of worm counts with SAS 9.1. The main factors considered were S. mansoni isolate, and treatment; 2-way interactions among the main factors were analyzed.The number of worms recovered from the treated and control groups were compared across in vivo mouse treatment results an in vitro assay was devised to assess susceptibility of adult worms of S. mansoni to PZQ. Adult worms exposed to PZQ immediately contract. Although the mechanism by which PZQ causes this contraction is not fully understood, it is accompanied by a rapid influx of calcium ions, a slower influx of sodium ions and a decreased influx of potassium ions To complement the 2, in RPMI medium supplemented with 20% Fetal Bovine Serum, 100\u2013500 IU Penicillin, and 100 \u00b5g/ml Streptomycin. Overnight incubation allowed for recovery from the stress caused by the perfusion. A single worm was placed in each well of a 96-well plate in 180 \u00b5l of supplemented RPMI medium. Before exposure to PZQ, each worm was photographed with a Nikon Coolpix 4500 camera mounted on a dissecting microscope phototube with a Thales Optem digital camera adapter. Then, 20 \u00b5l of either the control medium or a PZQ solution, of five different concentrations, was added to each well, resulting in final concentrations of 0, 0.8, 8.0, 80.0, 400.0, and 800.0 \u00b5g/ml. The control solution contained 0.1% DMSO, the same concentration as that in the experimental wells. The worms were incubated in the PZQ solution at 37\u00b0C for 3 hours and then photographed. The medium in each well was removed; the worms were washed three times, and were then transferred to a new 96-well plate in fresh RPMI medium, and held at 37\u00b0C. Each worm was re-photographed at the end of the 24 hour recovery period.Adult male worms  were recovered by perfusion of mice and incubated overnight, at 37\u00b0C in an atmosphere of 5% COS. mansoni isolates and PZQ concentrations and analyzed statistically with SAS 9.1 with a randomized ANOVA design; this was followed by Tukey's multiple comparisons to find significant pair-wise differences.The images of the worms before treatment and after recovery were processed with Metamorph v.4.65 software using the \u201cfiber length\u201d option which measures the object's length. The length of the worms was compared across This research project has been reviewed and approved by the University of New Mexico's Institutional Animal Care and Use Committee (IACUC), and the institutional review boards of the University of Georgia and the Centers for Disease Control and Prevention, the Scientific Steering Committee of the Kenya Medical Research Institute, and the KEMRI/National Ethics Review Board of Kenya. All investigators/assistants in this study have attained animal use certification regarding the ethical treatment of animals.in vitro effect of PZQ on the survival odds of miracidia was significantly influenced by drug concentration and time of exposure. To simplify data presentation, we show the results of exposure of miracidia derived from 23 different patients to 10\u22125 M PZQ after 20 minutes ] for comparing survival of miracidia in a previously-treated versus untreated group. In other words, the odds of surviving PZQ exposure were 2.42 times higher for miracidia collected from individuals who were previously treated than for those collected from patients who had never before been treated.As expected, the  minutes . For allMiracidia obtained from the car washer who had never fully cured following treatment and from whom the KCW isolate was derived were among the least susceptible to PZQ # 22, . We thenWith subsequent mouse infections we compared the PZQ susceptibility of adults of the KCW isolate with adults from other Kenyan isolates (KAS and KNY) or from long-maintained laboratory stocks (PR1 and NMRI).S. mansoni from the other 4 sources , using a randomized ANOVA design. It should be noted that while the sex ratio for the worms recovered from mice infected with the KCW strain was male biased, the infections were not predominantly single sexed.The results  are summDue to low infectivity for snails and mice and the inevitable attendant loss of genetic diversity, KAS worms recovered from mice were mostly or only males. The relatively low efficacy of treatment in mice infected with the KAS isolate (67.8%) might be explained by this phenomenon, since worms from single-sex infections are less susceptible to PZQ than those from mixed-sex infections in vivo trial to examine the effects of delayed maturation showed that the efficacy of treatment was 28.1% at 7.5 weeks post-exposure and 26.3% at 10.5 weeks post-exposure. The mean number of worms recovered from control and treated mice did not differ significantly at either 7.5 weeks post-exposure  or 10.5 weeks post-exposure . Worms from untreated control mice at both times were sexually mature, and the livers from the mice harboring these infections contained many granulomas. These results indicate that the reduced susceptibility to PZQ observed in vivo with the KCW isolate was not a result of delayed maturation rate of KCW worms.The results of the in vitro assay to test adult worm PZQ susceptibility  and for all other isolates tested: 87%, 79%, 100%, and 78% for the PR1, NMRI, KAS and KSH isolates, respectively (p<0.05). As shown in in vivo trials as compared to PR1 or KSH, and was significantly more susceptible to PZQ than the New Mexico subset of KCW (labeled KCW (2006) to indicate the year it was tested). The in vitro test of adult worms showed similar results. The difference in mean body length after the 24 hour recovery period between the New Mexico KCW sub-isolate and PR1, KNY and Kenyan KCW (2008) was significant (p\u200a=\u200a0.038). Kenyan KCW (2008), PR1 and KNY did not differ in this regard (p\u200a=\u200a0.211). In combination, these results indicate that the KCW sub-isolate maintained in Kenya lost the trait of diminished sensitivity to PZQ that we saw in miracidia when KCW was first isolated, and in comparison to the New Mexico sub-isolate.After the initial S. mansoni from a high-transmission endemic area in vitro assays involving both miracidia and adult worms, and an in vivo assay measuring drug susceptibility of adult schistosomes. In this population, we found worms with reduced susceptibility to PZQ. Miracidia collected from multiple patients varied significantly in their susceptibility to PZQ, and this susceptibility was associated with whether the patient had a history of exposure to the drug. Miracidia from car washers were more tolerant to PZQ than those from sand harvesters, which is not surprising because the car washer focus had been studied since 1995, while the sand harvesters were just being recruited in the study and had no prior history of exposure to PZQ. These results may suggest population level differences; however, we note that the sites are relatively close (within 5 km of each other) and our ongoing population genetics studies suggest no population subdivision between these groups or any other schistosomes collected throughout the Kenyan portion of Lake Victoria In this study, we investigated PZQ susceptibility of in vivo and in vitro assays of adult worms confirmed this observation and showed that they had significantly lower susceptibility to PZQ than adults from other recent Kenyan isolates or from lab stocks.More in-depth studies were undertaken on an isolate (KCW) established from a patient who yielded miracidia with relatively low susceptibility to PZQ. Both in vitro assays applied here were developed to monitor PZQ susceptibility of schistosomes, and have been validated by demonstrating for a variety of isolates that in vitro susceptibility of eggs, miracidia and cercariae to PZQ correlate with in vivo susceptibility of adult worms in vitro assay is helpful given the inherent problems with measuring PZQ efficacy in humans, which is determined by measuring egg counts in fecal samples before and after treatment. Such an indirect measurement of efficacy can lead to erroneous conclusions about susceptibility of the schistosomes harbored by the treated person: they may harbor immature worms not susceptible to PZQ in vitro assays to determine drug susceptibility can be very useful, but they pose difficulties of their own. Most notably in our study was the difficulty of hatching eggs from field-collected fecal samples which made it hard to perform in vitro assays on populations of miracidia from some patients. Persistent difficulty in getting eggs to hatch ultimately led to the loss of the KCW sub-isolate maintained in New Mexico. Egg hatchability is an understudied phenomenon that could play an important role in the epidemiology of S. mansoni particularly if low hatching rates are an associated cost of drug resistance.Drug resistance has evolved in many different microbial pathogens and parasites in vivo trials as efficacy of treatment was comparable across replicates regardless of intensity. In clinical reports, high intensity sometimes correlates with reduced efficacy of PZQ, but this could be due to high recruitment rates and the presence of immature worms within the host, or merely be a consequence of the fact that complete cures, as measured by cessation of egg production, are harder to obtain in people with high worm burdens even though the efficacy of treatment is the same. Even if reduced efficacy with high intensity infections was a phenomenon relevant to our in vivo trials, it would strengthen our conclusions because our more susceptible worms were present in higher intensities.Inclusion in our study of other Kenyan isolates that proved to be susceptible to PZQ provides evidence of variable susceptibility in natural populations and strengthens our findings of reduced susceptibility because the laboratory stocks potentially could have abnormal responses to praziquantel. Interestingly, the intensity of infection achieved with the Kenyan isolates was lower than that resulting from laboratory stocks. This difference likely is due to host adaptation as the laboratory stocks have been maintained in mice for several generations. It is unlikely that intensity influenced the outcome of the Worm maturation is a potential alternative explanation for our results if the KCW worms develop more slowly in mice such that they were still immature and thus less susceptible to PZQ at standard treatment times. Although we saw no consistent evidence of a slower development rate, and KCW worms recovered upon perfusion at 7.5 weeks post-infection were mature, we nonetheless addressed this possibility in an experiment that allowed three weeks of additional development before treatment was administered. The low PZQ susceptibility of these older, fully mature worms renders any argument invoking slower development times an unlikely explanation for the low PZQ susceptibility of KCW worms.In our study, diminished susceptibility to PZQ was a heritable trait that persisted across multiple generations: the KCW sub-isolate maintained in New Mexico retained reduced PZQ susceptibility over 6 generations, even in the absence of drug pressure. However, for the KCW sub-isolate maintained in Kenya, this trait was lost sometime during 8 generations of laboratory rearing. Loss of diminished susceptibility traits has also been reported for Egyptian field isolates brought into the laboratory S. mansoni population in the Lake Victoria region have lower susceptibility to PZQ. As with most anthelmithic resistance, it is assumed that this trait has occurred naturally within the population even prior to drug treatment in the region. In fact, the patient from whom the KCW isolate was derived was never fully cured, even after the very first treatment. Furthermore, a longitudinal epidemiological study of these patients over the course of 12 years reported an average cure rate of 66%, but no evidence of increased treatment failure or clinically relevant level of resistance over the course of the study S. mansoni in snails, humans and other mammals in the Lake Victoria basin is vast The data from this study suggest that at least some members of the S. haematobium in coastal Kenya, found variability in responsiveness to the drug, but no evidence of progressive emergence of PZQ resistance over an 8 year interval One of the most concerted programs of PZQ treatment, directed against S. mansoni adults more tolerant to PZQ and through recombination of alleles due to sexual reproduction, can lead to enhanced resistance in offspring. The pattern of decreased responsiveness to PZQ of miracidia derived from patients that had received multiple treatments, including KCW, is compatible with such a possibility. Trials to assess the safety and efficacy of higher doses for patients that fail to cure following exposure to standard PZQ dosages, or approval of more widespread availability of alternative treatments, such as oxamniquine, may both be prudent considerations when treatment failures occur. Second, because we lack a fundamental understanding of PZQ's mode of action, we are also ignorant of the natural variability in PZQ's targets in schistosome populations in endemic areas. It will be important to determine if variants inherently less susceptible to PZQ's effects are particularly likely to occur in the large, genetically diverse populations of species like S. mansoniThree aspects pertaining to the potential emergence of PZQ resistance are particularly deserving of additional study. The first is to determine if repeated cycles of infection and treatment of a single patient can favor the accumulation of a subset of We conclude by noting that, ironically, the maintenance of PZQ susceptibility may come at the expense of poor coverage and continued high transmission. Continued monitoring of PZQ susceptibility using assays such as the ones employed here, and new and improved assays, is warranted as increased use of PZQ in control programs becomes a reality."}
+{"text": "This review article discusses the efficacy of various conservative therapies in the management of voiding dysfunction with special reference to urinary incontinence. The article emphasizes the fact that conservative therapies have limited side effects and they do not jeopardize future treatment options. Behaviour therapy, pelvic floor therapy and biofeedback; electrical and magnetic stimulation are discussed here individually. Though there is unanimous agreement that these therapies improve quality of life, complete cure is rare. All therapies work better in conjunction with each other rather than in isolation. The review also highlights the need for randomized controlled trials of better methodology. Often described as a social cancer, urinary incontinence and allied voiding dysfunction continue to show rising prevalence. The main reasons for this are increased awareness and diagnosis as well as the growth of the ageing population. Though the statistics in India are not known, 13 million citizens are believed to have urinary incontinence in the US. out of wAs urinary incontinence is not a life-threatening condition, quality of life takes precedence over other issues when deciding the therapy. According to the \u201cClinical Guideline Panel\u201d of the US Department of Health and Human services, \u201csurgery, except in very specific cases, should be considered only after behavioral and pharmacological interventions have been tried\u201d. Various This article reviews various conservative treatment options for the management of voiding dysfunction and urinary incontinence. Drug therapy and advanced neuromodulation techniques are not covered in this review.Various conservative therapies rely heavily on the motivation of both the patient and the treating physician / nursing staff. Before starting any therapy, a detailed clinical, urological, and in select situations, neurological examination is recommended. However, invasive testing is rarely required before initiating these conservative treatments. All consBehavioral therapies are based on educating the patients to modify their behavior so as to reduce or sometimes even cure the incontinence. Patients suffering from urgency and urge incontinence seem particularly suitable for this form of therapy. The regime starts with maintaining a voiding diary. Very often this reveals excess water drinking as the cause of frequent urination, especially in India. The diary helps in setting the goals of treatment, i.e., decreasing the frequency, prolonging the intervals between urination and avoiding incontinence.et al. describe detailed techniques of behavioral therapy including timed voiding and techniques to stop / delay urination by sitting or standing quietly and repeatedly contracting the pelvic muscles. Distraction techniques such as deep breathing or mathematical problem-solving were also shown to be helpful. and high intensity [maximum tolerated stimulation with no pain.] The latter is usually used once or twice daily for 15-20 minutes at higher frequencies [> 20-50 Hz]. Stimulation can be applied with vaginal, anal or surface electrodes. However, the results for cure or improvements vary. In a platinence). Rapid masymptoms.et al. evaluated magnetic stimulation as a treatment for female urinary incontinence. Both urge and mixed incontinence patients were included in the study. Treatment was given twice a week for eight weeks. Outcome was assessed by pad test, patient satisfaction as well as urodynamics. This revealed objective improvement in 58% of patients. However, the sample size was small (24 patients) with no control group and the follow-up was short.[Chandi as short.Conservative therapies for urinary incontinence are safe with no documented side effects. Literature reports give conflicting opinions regarding their efficacy. Though there seems to be unanimous agreement that these therapies improve the quality of life, a complete cure is uncommon. Various therapies work better when used in conjunction with each other. Systematic reviews of each aspect of conservative therapy point towards a lack of good quality trials. The consensus in such reviews is that there is a need for RCTs of better methodology."}
+{"text": "The effect of age on the bone mineral density and microarchitecture of the equine radius and tibia was investigated. Fifty-six bones from 15 horses aged four to 21 years were used. There were nine geldings and six mares, and none of the horses had any disease influencing bone properties. Xtreme computed tomography was used to evaluate a 9-mm segment of the diaphysis and metaphysis of each bone. The following variables were determined: length of the bone, circumference and diameter in the frontal and sagittal planes in the middle of the bone.Diaphysis: total volume, bone volume, bone volume ratio, slice area, bone area, marrow area, cortical and marrow thickness, bone mineral density, polar moment of inertia of the cortex.Metaphysis: total area, bone area, cortical bone area, cortical thickness, bone mineral density, bone mineral density in the cortex, bone mineral density in the trabecular region, trabecular number, trabecular thickness, trabecular separation, polar moment of inertia of the metaphysis, polar moment of inertia of the cortex of the metaphysis.Bone density and microarchitecture were not affected by breed or gender. However, the microarchitecture varied with the age of the horse; the number of trabeculae decreased significantly and the distance between trabeculae increased significantly with increasing age. There were no significant differences between bones of the left and right limbs or between the radius and tibia.The variables investigated did not differ between geldings and mares. However, there were age-related changes in the microstructure of the bones. Further experimental studies are necessary to determine whether these changes reduce bone strength. Age-related changes in the bones were seen and may explain the higher incidence of fractures and fissures in older horses. In human medicine, determination of these variables is critical for the early detection of osteoporosis and other bone diseases. For years only BMD was determined, but recently three-dimensional microarchitecture was added to the list of criteria -3. This y (pQCT) ,6-9. They (pQCT) . Howevery (pQCT) . The decy (pQCT) ,13; thesy (pQCT) . The cory (pQCT) .Although equine bone diseases cannot be compared directly with osteoporosis in humans, they affect the use and longevity of the horse and therefore require adequate diagnosis and treatment. The most commonly-used diagnostic methods in horses include macroscopic and radiographic evaluation ,17, DEXAPeripheral quantitative computed tomography is another non-invasive and very exact method of bone evaluation, which allows, to a certain extent, separate assessment of cortical and cancellous bone. The results of BMD determined by pQCT are in good agreement with other methods of measurement . CorneliIn the present study, micro-CT (Xtreme CT) was used to obtain detailed data on cortical and trabecular bone, including microstructure, in the radii and tibiae of 15 horses. Special emphasis was given to the effects of age on the BMD and microarchitecture of the bones. These variables are likely to affect the mechanical properties of bones and may influence the fracture tendency in human bones . Our invFifty-six bones, which consisted of 28 radii and 28 tibiae, of 15 horses euthanised at our clinic for various reasons  to a level above the widest part of the epiphysis, and the bones were labeled. The bones were re-wrapped in saline-soaked cloth, placed individually in plastic bags to prevent loss of moisture and stored at 4\u00b0C until further use.Both ends of the bones were embedded in epoxy resin . The following variables were determined:XtremeCT measures a large number of geometric, densitometric and physical variables, which are displayed in Excel data sheets. All BMDs were expressed as mg hydroxyapatite/cm3),  Bone volume (in mm3), Slice Area = Total Area (in mm2), Bone Area (in mm2), Marrow Area (in mm2), Bone Volume Ratio, Cortical Thickness (in mm), Marrow Thickness (in mm), Bone Mineral Density (in mg HA/cm3), Polar moment of inertia of the cortex (in mm4).Capitals for total and bone :  Total volume (in mm2), Bone Area (in mm2), Cortical Bone Area (in mm2) Cortical Thickness, Bone Mineral Density in the metaphysis (in mg HA/cm3), Bone Mineral Density in the cortex (in mg/cm3 HA), Bone Mineral Density in the trabecular region (in mg HA/cm3), Trabecular Number, Trabecular Thickness, Trabecular Separation, Polar moment of inertia of the metaphysis, Polar moment of inertia of the cortex of the metaphysicTotal Area  was also included as an explanatory variable. To allow for possible correlations between observations in the same horse, a random effect was introduced for each horse [A commercial software program was used for all calculations . Statistical regression analysis was done using the software R . Means ach horse . The 5% 2) than in the tibia   Table . There wia Table . The mea tibia 1,99 mm2  determined that the weight-bearing ability of the human vertebrae was approximately 1,000 kg in young adults, but only 150 to 250 kg in the elderly . This chOur study investigated BMD and microarchitecture of the radius and tibia because we have been interested in the frequency and configurations of fractures of the tibia and radius for some time . Other sTo our knowledge, there are no high-resolution imaging studies on the micro-architecture of equine long bones and how it is affected by the gender and age of the horse. The results of the present study show that bone microstructure undergoes age-related changes, which may predispose to fractures. Further investigations are necessary to determine the effect of microarchitectural changes on the strength of bones and their susceptibility to fracture. Such studies should include very old mares to investigate the effect of ovarian senescence on bone micro-architecture.AF conceived the idea for the study, was involved in the analysis of the data and wrote the manuscript. DM collected the data and was involved in analysis of the data. SM made substantial contributions to the design of the study and assisted in the interpretation of the data. AS assisted during examination of the bones using Xtreme-CT. LH carried out the statistical analysis. AL assisted in the analysis and interpretation of the data and was involved in the drafting of the manuscriptList of measurementsClick here for file"}
+{"text": "Pfeiffer syndrome is an autosomal dominant condition classically combining craniosynostosis with digital anomalies of the hands and feet. The majority of cases are caused by heterozygous mutations in the third immunoglobulin-like domain (IgIII) of FGFR2, whilst a small number of cases can be attributed to mutations outside this region of the protein. A mild form of Pfeiffer syndrome can rarely be caused by a specific mutation in FGFR1. We report on the clinical and genetic findings in a three generation British family with Pfeiffer syndrome caused by a heterozygous missense mutation, p.Ala172Phe, located in the IgII domain of FGFR2. This is the first reported case of this particular mutation since Pfeiffer's index case, originally described in a German family in 1964, on which basis the syndrome was eponymously named. Genetic analysis demonstrated the two families to be unrelated. Similarities in phenotypes between the two families are discussed. Independent genetic origins, but phenotypic similarities in the two families add to the evidence supporting the theory of selfish spermatogonial selective advantage for this rare gain-of-function FGFR2 mutation. \u00a9 2013 Wiley Periodicals, Inc. Pfeiffer syndrome classically describes a combination of craniofacial and limb anomalies. Multisuture craniosynostosis, exorbitism, and midface hypoplasia are common craniofacial features. Radially deviated broad thumbs and broad great toes are typical extracranial features and less frequently, partial syndactyly in the hands and feet may be present [Anantheswar and Venkataramana, FGFR2 [Wilkie, Pfeiffer syndrome is an autosomal dominant condition with an incidence of approximately 1 in 120,000 births. It is caused by heterozygous mutations in the fibroblast growth factor receptors types 1 and 2 (FGFR1 and FGFR2) [Johnson and Wilkie,  Wilkie, . Ninety- Wilkie, .FGFR2 encodes a protein involved in cell division and regulation of cell growth and maturation, affecting processes such as embryonic development, formation of blood vessels, and wound healing. Specifically, this protein is a transmembrane receptor tyrosine kinase comprising an extracellular ligand-binding region , a single pass transmembrane region and a split tyrosine kinase domain. Mutations in FGFR2 lead to predominantly missense substitutions in the amino acid sequence resulting in a gain-of-function. Of the mutations that have occurred outside the main hotspot region, only a single instance has been identified in exon 5, which encodes part of the IgII domain. This mutation involved substitution of two consecutive nucleotides  and was previously known only from Pfeiffer's index case, a three-generation German family that he described in 1964 and was associated with an atypical phenotype [Pfeiffer, A 6-month-old boy (proband) was referred to the Oxford Craniofacial Unit at the request of his mother and maternal grandfather, both of whom had previously been told they had Pfeiffer syndrome. He had been born at 38 weeks by forceps assisted delivery, following an uncomplicated pregnancy. Antenatal ultrasound scans had raised the concern of abnormal head shape; however this was not evident at birth and the anomalies were predominantly confined to the hands and feet.On examination, the proband A\u2013H was dThe proband's mother I,J also The proband's maternal grandfather K,L againIn the British family, DNA samples were obtained from the affected child, his affected mother and his affected maternal grandfather. DNA samples from Pfeiffer's original German family were already available to us [Kan et al., FGFR2 in the mother of the British family was performed, demonstrating heterozygosity at two adjacent nucleotides shown in HaeIII demonstrated the same mutation in all three affected individuals, and revealed that the normal sequence (GGCC) was preserved on one allele , revealed that in the German family the disease-causing mutation segregated with a 140 bp allele; in contrast, in the British family the disease-causing mutation segregated with a 142 bp allele, suggesting that the p.Ala172Phe mutation had arisen independently in the two families (data not shown). However, we could not exclude the small possibility of either a length mutation of the microsatellite sequence or recombination between the microsatellite and exon 5 occurring in an ancestral generation. Therefore, to corroborate this result, we used phased haplotype data  obtained from HapMap [The International HapMap 3 Consortium, MboII digestion. This showed that in the German family, the C allele is present in cis with the mutant FGFR2, whereas in the British family, it is the T allele  or independent origins, we typed selected individuals from both families  are both present at measurable frequency in the CEPH-Utah population . By comparison the genome-averaged probability of recombination within a physical distance of 15.7 kb (assuming 1 Mb \u224d 1 cM) would be \u223c1.6 \u00d7 10Two features of the conclusion that the two families have independent mutational origins appear remarkable\u2014first, that a double nucleotide mutation should occur within the exon encoding the IgII domain when no single nucleotide mutation affecting this domain has been recorded; and second, that this identical double nucleotide mutation should occur independently in two different families. However, both observations can be rationalized based on the known biology and pathophysiology of FGFR2 action. Upon binding of the FGF ligand, two adjacent FGF-FGFR complexes dimerize with activation of the tyrosine kinases. The dimer, shown to involve a symmetric and two-ended configuration [Ibrahimi et al., FGFR2 provides a paradigmatic example [Goriely and Wilkie, FGFR2 [Goriely and Wilkie, A seeming paradox raised by our conclusion that the same double nucleotide mutation has arisen independently on two separate occasions, is that a random double nucleotide substitutions arising by chance alone are expected to be present less than once in the entire human population [Kondrashov, In conclusion, Pfeiffer syndrome resulting from the p.Ala172Phe mutation is infrequent\u2014only two families, 500 miles and 45 years apart. However, the independent origin of the two double nucleotide substitutions, and similar phenotypes associated with the resulting missense mutation, lend weight to the exquisite specificity of the functional consequences of this particular mutation."}
+{"text": "Investigation of an outbreak of tuberculosis (TB) in a primary school in Milan, Italy, found 15 schoolchildren had active TB disease and 173 had latent TB infection. TB was also identified in 2 homeless men near the school. Diagnostic delay, particularly in the index case-patient, contributed to the transmission of infection. Incidence in children 0\u201314 years of age was 3.38/100,000 (n = 47 cases). In 2009 in Milan, the largest urban area of Lombardy (1.6 million inhabitants), the incidence was 20.44/100,000 population (www.asl.milano.it/user/download.aspx?FILE=OBJ06171.PDF&TIPO=FLE&NOME=report_prevenzione_2011).Italy has a low incidence of tuberculosis (TB); in 2008, incidence of notified cases was 7.6/100,000 population ; induration of In this investigation, the boy\u2019s family and friends all had negative TST results. His classmates and the children in the 2 adjacent classrooms were screened; results indicated LTBI for 20% of the students in his classroom and 20% and 14% of students in the other 2 classrooms. His teachers and some other school staff members were also screened (n = 43); no cases of active TB were detected among children or school staff.M. tuberculosis from a secondary school pupil who had attended the same primary school as the first case-patient during the previous year. Genotyping confirmed that the isolate was the same Bejing strain. A search of the regional strain database found that this genotype had also been identified in a case of pulmonary TB reported in November 2009 in a homeless person who lived in the city park in front of the school. This man had been lost to therapeutic follow-up but had infected his daughter.In December 2010, pleural TB was confirmed by isolation of M. tuberculosis isolated was of the same Bejing strain as that isolated from the other homeless man and the first 2 infected schoolchildren.After the second school case was identified, health authorities extended TST testing to all children who had attended the school during 2010, including those who had moved to other schools , and to M. tuberculosis isolation. The latter type is defined as clinical evidence of the disease and any of the following: contact with an adult with TB, positive TST results, suggestive appearances for TB on chest x-ray, and favorable response to antituberculous therapy  as found in the previous cases. The collected data suggest that this boy was the index case-patient for this outbreak: 90.9% of his classmates were infected, and a relevant ratio of TB infection was found among pupils of the other classrooms on the same floor . He had been in the primary school for 3 years. He had the same me floor . The sprM. tuberculosis isolates during this investigation enabled the establishment of a connection between the cases of TB among the schoolchildren. However, no contact between the 17-year-old boy and the first homeless man has been identified (Genotyping has proved to be an essential tool in TB contact investigations (entified .The results of this investigation indicate that a diagnostic delay for the index case-patient played a primary role in the transmission of infection inside the school. The main cause of this delay was the low degree of diagnostic suspicion toward the disease; however, TB can also be difficult to diagnose in children because children are less able to produce sputum. Physicians should be aware of the signs and symptoms of early TB infection and should consider this diagnosis accordingly. Furthermore, routine screening for TB could be considered for persons with disabilities or special needs who take part in recreational and educational activities in which they come into close contact with more susceptible groups, such as children.In addition, cases of TB among the homeless, particularly those in close proximity to susceptible groups such as schoolchildren, highlight the problem of therapeutic monitoring among persons who may be lost to follow-up. Careful evaluation of compliance at time of discharge from health care is critical, and social protection programs are needed to improve rates of follow-up care. In particular, in urban areas where risk factors for transmission of TB are highly concentrated, a TB reference center may improve collaboration between local health authority, physicians, and social services."}
+{"text": "Salmonella typhi and typhimurium, respectively, are responsible for significant morbidity and mortality in both developed and developing countries. We model typhoid fever using mice infected with Salmonella typhimurium, which results in a systemic disease, whereby the outcome of infection is variable in different inbred strains of mice. This model recapitulates several clinical aspects of the human disease and allows the study of the host response to Salmonella typhimurium infection in vivo. Previous work in our laboratory has identified three loci  in the wild-derived MOLF/Ei mice influencing survival after infection with Salmonella typhimurium. Fine mapping of the Ity3 locus indicated that two sub-loci contribute collectively to the susceptibility of B6.MOLF-Ity/Ity3 congenic mice to Salmonella infection. In the current paper, we provided further evidence supporting a role for Ncf2 (neutrophil cytosolic factor 2 a subunit of NADPH oxidase) as the gene underlying the Ity3.1 sub-locus. Gene expression profiling indicated that the Ity3.1 sub-locus defined a global gene expression signature with networks articulated around Ncf2. Furthermore, based on differential expression and complementation analysis using Selp (selectin-P) knock-out mice, Selp was identified as a strong candidate gene for the Ity3.2 sub-locus.Typhoid fever and salmonellosis, which are caused by  Salmonella enterica, an intracellular Gram-negative bacterium, is the causative agent for a wide spectrum of clinical diseases with manifestations ranging from asymptomatic carriers, self-limiting gastroenteritis to fatal systemic infection , while the 129 sub-strains are highly resistant  (ogenesis \u20139. As thogenesis \u201313. Clasesistant . The wileptor 4) .Salmonella infection, we have previously used linkage analysis in an F2 panel of (C57BL/6\u2009\u00d7\u2009MOLF/Ei) mice to identify two loci linked to host defense against Salmonella typhimurium, Ity2 (Immunity to Typhimurium locus 2) and Ity3  production, reduced inflammatory cytokine response, and increased bacterial burden. The Ity3.2 sub-locus is characterized by a hyper-responsive inflammatory cytokine phenotype after exposure to Salmonella (Ncf2 (neutrophil cytosolic factor 2 a subunit of NADPH oxidase) as the gene underlying the Ity3.1 sub-locus  as one of the candidate genes underlying Ity3.2 based on expression analysis, coding sequence polymorphism, and functional and allelic complementation studies.In the current study, we used global expression profiling to better understand the genetic networks that are being influenced by the All animals were maintained at the Animal Care Facility of McGill University according to the guidelines of the Canadian Council on Animal Care (CCAC). The animal protocol for this study was approved by the McGill University Animal Care Committee.Ity and B6.MOLF-Ity/Ity3 and sub-congenic mice as described previously , were used for the complementation assay.Classical inbred strain C57BL/6J and wild-derived MOLF/Ei mice were used to generate congenic, B6.MOLF-eviously , 16. TheSalmonella typhimurium strain Keller as described previously  . Three age-matched male mice were used per group. The concentration of RNA was determined using a NanoDrop spectrophotometer . All hybridization and scanning of mice microarrays were carried out at the McGill University and Genome Quebec Innovation Centre, using the Illumina BeadArray technology . The expression data were analyzed using FlexArray and normalized using a Lumi algorithm (Illumnia). Following the normalization, two approaches were used to generate a list of genes differentially expressed across the various strains. First, a Cyber nd Long)  was usedIty strain. This was done by comparing the expression of genes in each strain to the control Ity strain at both day 0 and day 3 post-infection. The gene lists were further refined using the Benjamini Hochberg false discovery rate algorithm. Genes with an FDR p-value of <0.05 was used as a cut-off to characterize genes as significantly differentially regulated as compared to Ity. The gene lists generated using the two approaches were studied using a suite of online tools including DAVID  were ordered from the Jackson Laboratories . These mice were on C57BL/6J background with a mutant Slc11a1 allele. In order to correct for this, we crossed the Selp\u2212/\u2212 mice to Ity as well as to the Ity3 mice. Mice were genotyped for the Selp\u2212/\u2212 allele, and mice carrying the MOLF/Ei Slc11a1 allele along with the Selp\u2212/\u2212 allele were inter-crossed to generate homozygous Selp\u2212/\u2212 mice with a MOLF/Ei allele at the Slc11a1 gene. Furthermore, these mice were crossed with Ity or Ity3 mice to generate mice, which are homozygous wild-type at Slc11a1, but carry a Selp knock-out allele complemented by either a C57BL/6J or MOLF/Ei allele .In order to study the effect of a MOLF/Ei 2 and at the required day post-infection; both organs were removed aseptically, weighed and homogenized using a Polytron . The resulting homogenate was diluted in 0.9% saline and plated on tryptic soy agar to determine organ bacteria burden.For bacterial burden quantification in the spleen and the liver, mice were euthanized using COp-value <0.05 was used to establish significant differences.Statistical analysis was performed using Graph Pad Prism 6 . One-way ANOVA with Dunnet\u2019s multiple correction test was used to analyze the bacterial burden in the spleen. A corrected Ity3 locus is a complex QTL containing at least two sub-loci. We have previously studied the phenotypic impact of these two sub-loci , susceptible B6.MOLF-Ity/Ity3 (Ity3), and Ity3.RecG and Ity3.RecN strains. These two sub-congenic strains were selected because they carry either the MOLF/Ei alleles at Ity3.1 (Ity3.RecG) or at Ity3.2 sub-locus (Ity3.RecN), which result in an intermediate survival phenotype after infection with Salmonella typhimurium , 204 (Ity3), 218 (Ity3.RecG), and 201 (Ity3.RecN) genes were differentially expressed during infection as defined by a cut-off of a fold change >2 and a p-value <0.1 , Ity3 (20 genes), Ity3.RecN (62 genes), and Ity3.RecG (54 genes) . A large number of differentially expressed genes specific to Ity mice  were up-regulated in granulocytes and/or macrophages including S100a8 and S100a9 that are of particular interest as they are involved with expression of inflammatory mediators, phagocytosis, oxidative burst as well as migration of neutrophils and monocytes to the site of infection (Clec7a (dectin 1) was differentially regulated only in Ity. Recent work has linked dectin 1/Syk kinase signaling with autophagy-dependent maturation of phagosomes . Overall, 241 . Interestingly, a large proportion (~40%) of genes specific to the strain Ity3.RecG are known to be down-regulated in B and T cells, as analyzed by BioGPS (Ity3 on the cellular composition of the spleen and/or changes in gene expression in specific cellular populations during infection. Very few genes were similarly regulated between Ity3 and the sub-congenic strains Ity3.RecG (4 genes), Ity3.RecN (7 genes), and Ity3.RecN and Ity3.RecG (4 genes) .In y BioGPS  . These results are consistent with previous observations of reduced inflammatory responses following in vivo Salmonella infection in Ity3.RecG mice  to classify the genes differentially regulated in each strain  into gene ontology (GO) molecular pathways, GO processes, pathways, and process networks, in order to identify the pathways differentially regulated in each strain during infection Figure . The strecG mice .Ncf2 is a strong candidate for the Ity3.1 locus . The wild-derived inbred MOLF/Ei had been separated from the classical inbred trains by over 1 million year of evolution, and as a result they have accumulated significant sequence variability, to the order of 1 SNP every 100\u2009bp  showed that a large percentage of the genes in the list plays a role in cell cycle, DNA binding, cytoskeletal reorganization, and hemopoietic and lymphoid organ development   , 28. Ano kinases  and with kinases , 31.Ity3 as well as the two sub-congenic strains Ity3.RecN and Ity3.RecG. Figures Tor3a and Fam20b as examples of the expression pattern of the list of genes provided in Table S2H in Supplementary Material, which have a similar expression pattern in Ity3, Ity3.RecN, and Ity3.RecG. Only 7 of the 47 genes were within the Ity3 interval, and almost all of them were within the genomic region common to Ity3.RecN and Ity3.RecG. This gene list was classified within functional categories  lie within the genomic region harboring Ity3.2 . The coagulation factor V is synthesized by the liver and is involved in the acceleration of prothrombin to thrombin conversion  modules [or as short consensus repeats (SCRs) functional domains]. We re-sequenced the coding region of Selp in C57BL/6J and MOLF/Ei mice and identified eight SNPs . Ity (B6/B6Selp) and Ity3 (MOLF/MOLFSelp) mice were crossed to Selp\u2212/\u2212 knock-out mice and susceptibility to infection was assessed by survival analysis in F1 progeny with MOLF/Selp\u2212 and B6/Selp\u2212 genotypes. MOLF/Selp\u2212 mice were significantly more susceptible to infection than B6/Selp\u2212 mice and Ity controls , adding further support for the candidacy of Selp as the gene underlying the Ity3.2 locus  subunit of the NADPH complex have reduced expression of VCAM-1, ICAM-1, SELP, and SELE in the vascular cell walls .Recent studies have shown that TNF, as well as other cytokines through NF-\u03baB signaling induces transient increase in ROS level in endothelial cells, which results in cell surface expression of and Selp \u201352. Thisll walls , 54. In  oxidase  on Selp Ity3.1 locus have higher expression of a number of genes playing a role in cell cycle, DNA binding, and cytoskeletal pathways. There is a growing body of evidence discussing the link between ROS, cell-cycle progress and arrest. As discussed by Martindale (We also illustrated that mice carrying a MOLF/Ei allele at the rtindale , ROS canIty3.1 and regroups genes involved in heme biosynthesis. Increased expression of genes within the heme biosynthesis pathway could result in increased free heme within the cells, which can act as a potent cytotoxic pro-oxidant (Another pathway influenced by ROS production, is up-regulated by -oxidant . Free he-oxidant . TherefoSalmonella infection. We highlighted the role of low ROS and cytokine production in reduced survival of mice carrying the Ity3.1 locus, and the importance of the Ity3.2 locus, which synergistically led to increased susceptibly of the Ity3 mice. We have also shown that several pathways identified in strains Ity3, Ity3.RecN, and Ity3.RecG, are influenced by Ncf2. Furthermore, the Ity3.1 sub-locus has additional effects, which have not previously been characterized, in expression of genes involved in cell-cycle arrest and hematopoiesis. Lastly, we propose a hypothesis that the combined effects of low ROS production by the MOLF/Ei Ity3.1 locus together with the impact of Selp MOLF/Ei allele at Ity3.2 influences the host survival after infection with Salmonella typhimurium.In conclusion, our study highlights the complex nature of multi-loci interaction in the wild-derived MOLF/Ei response to The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fimmu.2014.00375/abstractThe Supplementary Material for this article can be found online at Click here for additional data file."}
+{"text": "We previously reported that one-third of HIV-positive adults requiring medical admission to a South African district hospital had laboratory-confirmed tuberculosis (TB) and that almost two-thirds of cases could be rapidly diagnosed using Xpert MTB/RIF-testing of concentrated urine samples obtained on the first day of admission. Implementation of urine-based, routine, point-of-care TB screening is an attractive intervention that might be facilitated by use of a simple, low-cost diagnostic tool, such as the Determine TB-LAM lateral-flow rapid test for HIV-associated TB.Mycobacterium tuberculosis in any sample using Xpert MTB/RIF or liquid culture. The diagnostic yield, accuracy and prognostic value of urine-lipoarabinomannan (LAM) testing were determined, but urine-LAM results did not inform treatment decisions.Sputum, urine and blood samples were systematically obtained from unselected HIV-positive adults within 24\u00a0hours of admission to a South African township hospital. Additional clinical samples were obtained during hospitalization as clinically indicated. TB was defined by the detection of n\u2009=\u2009427) were enrolled regardless of clinical presentation or symptoms. TB was diagnosed in 139 patients . In the first 24\u00a0hours of admission, sputum (spot and/or induced) samples were obtained from 37.0% of patients and urine samples from 99.5% of patients (P\u2009<\u20090.001). The diagnostic yields from these specimens were 19.4% (n\u2009=\u200927/139) for sputum-microscopy, 26.6% (n\u2009=\u200937/139) for sputum-Xpert, 38.1% (n\u2009=\u200953/139) for urine-LAM and 52.5% (n\u2009=\u200973/139) for sputum-Xpert/urine-LAM combined (P\u2009<\u20090.01). Corresponding yields among patients with CD4 counts <100 cells/\u03bcL were 18.9%, 24.3%, 55.4% and 63.5%, respectively (P\u2009<\u20090.01). The diagnostic yield of urine-LAM was unrelated to respiratory symptoms, and LAM assay specificity (using a grade-2 cut-off) was 98.9% . Among TB cases, positive urine-LAM status was strongly associated with mortality at 90\u00a0days .Consecutive HIV-positive adult acute medical admissions not already receiving TB treatment  contains supplementary material, which is available to authorized users. Tuberculosis (TB) is the leading cause of HIV/AIDS-related mortality globally, accounting for an estimated 0.4 million HIV-related deaths worldwide in 2015 \u20135 and th\u00a9  lateral-flow urine assay\u2014a technically much simpler, instrumentation-free, low-cost, point-of-care diagnostic tool [Using an intensive clinical sampling approach, we have previously demonstrated that one in three unselected HIV-infected acute medical adult admissions to a South African township district hospital had TB that could be diagnosed microbiologically . Presenttic tool . We reasThis study forms part of a larger study of rapid urine-based approaches to diagnosis of HIV-associated TB that has previously been reported . The proAdult patients aged \u226518\u00a0years were recruited on 4\u00a0days of the week from medical wards. On recruitment days, the study nurse coordinator ascertained from the ward register all medical admissions in the previous 24-hour period and recorded these in the study register. All patients who either previously tested negative or who had undocumented HIV status were offered HIV testing using two approved rapid tests. All patients who had a current TB diagnosis and/or were receiving treatment for TB at the time of hospital admission were excluded from the present study. Otherwise, all other adult patients with documented positive HIV status were eligible for the study and were invited to participate regardless of presenting symptoms or reason for hospital admission.Demographic and clinical details  were reIn the initial 24-hour period of hospital admission, two sputum samples were requested from each patient with careful instruction and supervision by the study nurse coordinator (an experienced respiratory nurse) as previously described . A spot M. tuberculosis complex with the MTBDRplus assay . Non-respiratory samples were also tested using MGIT liquid culture, with the exception of venous blood, which was tested using Myco F/Lytic culture.Specimens were processed using standardized protocols in centralized accredited laboratories of the South African National Health Laboratory Service as described previously , 15. Decg for 15\u00a0min. Following removal of the supernatant, the pellet was re-suspended in the residual urine volume and 0.75\u00a0mL was tested using Xpert. This second sample derived from centrifugation of a large urine volume was referred to as the \u2018concentrated\u2019 urine sample and these samples were batch processed on a weekly basis due to study-related logistical considerations and laboratory workflow.Urine was tested using Xpert MTB/RIF in two different ways as described previously . Fresh uThe same frozen urine samples (unconcentrated and unprocessed) were also retrospectively tested for the presence of LAM using the Determine TB-LAM test (Alere Inc.) following the manufacturer\u2019s instructions. Samples were thawed to ambient temperature and for each sample, 60\u00a0\u03bcL of unprocessed urine was applied to the sample pad at the bottom of the test strip. After 25\u00a0min, test strips were independently read by two investigators (SDL and MV) blinded to patient status and all other test results. Having checked for the presence of the positive control band on each strip, test results were scored as grade 1, 2, 3, 4 or 5 when compared to the reference card as described elsewhere . For exaResults of all microbiological tests were returned to the clinical team to inform treatment decisions with the exception of the results of the Determine TB-LAM assay, which was not endorsed for clinical use in South Africa or by the WHO at the time of the study.Patient and ward records as well as district, regional and national electronic data systems were used to ascertain deaths during the first 90\u00a0days following enrolment as fully described elsewhere . PatientM. tuberculosis from at least one clinical sample of any type using MGIT culture and/or Xpert MTB/RIF. The total number of confirmed cases (n\u2009=\u2009139) was used as the denominator to calculate the comparative diagnostic yield obtained from different sample types and different assays, including Determine TB-LAM. The yield of TB diagnoses from Determine TB-LAM testing of urine samples obtained during the initial 24\u00a0hours was compared with the yield from sputum samples collected in the same period. We included Xpert results in our diagnostic reference standard. While we calculated the yield of diagnoses made by Xpert in different specimen types compared with other methods, this does not represent a measure of diagnostic sensitivity . Diagnostic yield in our study reflected both the performance of the diagnostic test and the ability to obtain samples for that test in a real-world clinical setting. The associations between the yield of Determine TB-LAM and patient symptoms and other patient characteristics were explored and multivariable logistic regression was used to identify factors independently associated with positive urine-LAM status among patients with confirmed TB. One or more positive reference standard tests on a non-respiratory sample was taken as evidence of extrapulmonary TB (EPTB).The diagnostic reference standard for a confirmed new TB diagnosis was the detection of M. tuberculosis were used as the denominator to calculate the sensitivity of Determine TB-LAM (n\u2009=\u2009136). In contrast, patients whose clinical samples all tested negative for M. tuberculosis were defined as \u2018TB-free\u2019 and were used as the denominator to calculate the specificity of Determine TB-LAM (n\u2009=\u2009277).For the purposes of assessing the diagnostic accuracy (sensitivity and specificity) of Determine TB-LAM, we identified a cohort of patients for whom sufficient microbiological data and the results of Determine TB-LAM urine testing were available. For inclusion in this analysis, patients were required to have the results of reference standard tests (liquid culture and/or Xpert MTB/RIF) from two or more samples obtained from two or more different anatomic sites, such as blood, sputum, urine or other non-respiratory samples. The rationale for this was to reduce the risk of patients with disseminated TB having false-negative reference standard tests and being inappropriately designated as \u2018TB-free\u2019. By doing so, we enhanced the methodological rigour with which the specificity of Determine TB-LAM was assessed . From tht tests. Chi-squared, Fisher\u2019s exact and McNemar\u2019s tests were used as appropriate to compare proportions. Kaplan\u2013Meier plots were used to examine mortality risk among confirmed TB cases stratified by urine-LAM status and a Cox proportional hazards model was used to determine factors associated with mortality. Person-time was accrued from the date of study enrolment until death, loss to follow-up or censorship 90\u00a0days after study entry. All variables in the univariable model meeting a cut-off of P\u2009\u2264\u20090.1 were included in the multivariable model. Statistical tests were two-sided at \u03b1\u2009=\u20090.05.Patients were characterized using simple descriptive statistics. Anaemia severity was defined using WHO criteria: no anaemia , mild anaemia , moderate anaemia  or severe anaemia  . TB prevn\u2009=\u2009158, 27.0%) were excluded, leaving 427 eligible patients who formed the study cohort  unselected new admissions to the adult medical wards was ascertained . Nearly half of all patients had previously received treatment for TB and two-thirds had moderate or severe anaemia  Table\u00a0. Immunodn\u2009=\u200912), which were contaminated, and 4.3% of sputum Xpert tests (n\u2009=\u200912) and 3.1% of urine Xpert tests (n\u2009=\u200926), which had indeterminate results. However, all urine samples yielded interpretable LAM results (n\u2009=\u2009418). TB was diagnosed in 139 patients with a mean of 3.2 positive samples per case diagnosed. Thus, the overall TB prevalence was 32.6% (95% CI 28.1\u201337.2). The mean number of tests done was similar for patients in whom TB was or was not diagnosed . The characteristics of those with and without TB are summarized in Table\u00a0During hospital admission, clinical samples for mycobacteriology were obtained from all 427 eligible study participants. The total of 1745 samples (mean 4.1 samples per patient) were derived from a median of three anatomic locations , who were therefore designated as \u2018TB-free\u2019 and used to assess the specificity of Determine TB-LAM. Among these 277 patients, the mean number of negative reference standard tests per patient was 5.5.Having carefully defined which patients in the cohort had TB and which were TB-free, the diagnostic accuracy of urine-LAM was next assessed using data from the subset of 413 patients (96.7%) for whom reference standard test results on samples obtained from at least two anatomic sites plus urine-LAM results were available and complete . Urine-LAM was negative in 274 of the 277 patients who were TB-free, giving a specificity of 98.9% (95% CI 96.9\u201399.8). Two of three patients with apparent false-positive urine-LAM results had clinico-radiological diagnoses of TB made by the routine medical team, although diagnoses were never confirmed microbiologically. Of note, neither had a full set of investigations available, with sputum and blood culture results missing. One patient had a 3-week history of weakness, diarrhoea and constitutional symptoms, was pancytopaenic (grade 2 LAM-positive), and subsequently died without any diagnosis being made. The other patient had a 3-month history of abdominal pain and weight loss, and an abdominal ultrasound demonstrated splenomegaly and splenic micro-abscesses with para-aortic and mesenteric lymphadenopathy suggestive of abdominal TB (grade 5 LAM-positive). Thus, had a composite reference standard for TB diagnosis  been used in a post hoc analysis, the specificity would have been 279 out of 280\u00a0TB-free patients . Using a grade 1 reference band cut-off resulted in modest improvement in sensitivity with a small decrease in specificity . We therefore next compared the diagnostic yield of urine-LAM, sputum-Xpert and sputum microscopy from samples obtained in the first 24\u00a0hours of admission from all 427 included patients.The potential yield of sputum-based diagnoses was substantially limited by the high proportion of patients who were unable to produce a sputum sample. In the first 24\u00a0hours of admission only 158 of 427 (37.0%) patients were able to produce at least one sputum sample (spot and/or induced samples) despite assistance from an experienced respiratory nurse. Of these sputum samples, 36 (23.1%) were only obtained following sputum induction. The remaining patients who did not produce sputum were typically too sick or uncooperative to tolerate sputum induction or to be taken to the sputum induction room at the hospital, or it was attempted but was simply unsuccessful. In marked contrast, urine samples were readily obtained from 425 of 427 (99.5%) patients, although seven samples were misplaced in transit to the laboratory, leaving 418 urine samples available for testing  at some point during their hospital admission. Of the 161 patients who self-reported sputum production prior to admission, 143 (88.8%) were able to produce a sputum sample for testing. Additionally, of the 201 patients reporting current cough at admission, 159 (79.1%) produced a sputum sample.n\u2009=\u200927/139), 26.6% (n\u2009=\u200937/139) and 38.1% (n\u2009=\u200953/139), respectively (comparison of the proportionate diagnostic yield from sputum microscopy with the yield from sputum-Xpert and urine-LAM: McNemar\u2019s P\u2009<\u20090.01 for each comparison).Figure\u00a0n\u2009=\u200965/139: a 2.4-fold increase; P\u2009<\u20090.001) of all TB cases. The addition of urine-LAM testing to sputum-Xpert increased the diagnostic yield from 26.6% to 52.5% .The incremental diagnostic yield of Determine TB-LAM used in combination with sputum testing is shown in Fig.\u00a0n\u2009=\u200974) for specimens obtained in the first 24\u00a0hours of admission. The diagnostic yield from sputum microscopy, sputum-Xpert and urine-LAM were 18.9% (n\u2009=\u200914/74), 24.3% (n\u2009=\u200918/74) and 55.4% (n\u2009=\u200941/74), respectively . The addition of urine-LAM testing to sputum microscopy increased the diagnostic yield from 18.9% to 60.8% . The addition of urine-LAM testing to sputum-Xpert increased the diagnostic yield from 24.3% to 63.5% . Thus, the incremental diagnostic yield of urine-LAM was greater among patients with lower CD4 counts  observed among those with CD4 counts of <50 cells/\u03bcL Fig.\u00a0. Yield wn\u2009=\u200917) could be diagnosed by urine TB-LAM instead , compared with 39.1% (n\u2009=\u200953) for urine-LAM  were lower CD4 cell counts and more severe anaemia.Comparing the clinical phenotype of patients with confirmed TB stratified according to urine-LAM status revealed that those testing LAM-positive had lower CD4 cell counts and a higher prevalence and severity of anaemia Table\u00a0. In contP\u2009=\u20090.006). Therefore, urine-LAM detected TB in 13 of 19 patients (68.4%) with HIV-associated TB who died within 90\u00a0days of study entry. In unadjusted analyses, a greater mortality risk was also associated with male sex and rifampicin-resistant TB but not with CD4 cell count  [As reported in the parent study, a microbiological diagnosis of TB was made in one in three unselected HIV-positive adult patients admitted to medical wards in this South African district hospital. The prevalence is so high in this vulnerable patient group that a simple, rapid screening test for TB that can be immediately performed at the time of hospital admission is highly desirable. In the present study, we found that routine urine-LAM testing could detect 39% of all HIV-associated TB diagnoses and the large majority of TB diagnoses among patients dying within 90\u00a0days of admission to hospital. We previously showed the remarkable utility of using Xpert MTB/RIF to test urine samples concentrated by centrifugation, with 64% of total diagnoses being made using samples obtained within the first 24\u00a0hours of admission (1.6-fold more diagnoses than LAM and 2.3-fold more than sputum Xpert testing). We are currently conducting a clinical trial in sub-Saharan Africa to assess whether implementation of systematic testing of urine samples using rapid TB assays compared to the current sputum-based strategy results in improved survival among HIV-patients requiring acute medical hospitlization (Rapid Urine-Based 1603869) .Should implementation of such a screening strategy (routine urine Xpert testing) be attempted in the countries of southern Africa worst hit by the HIV and TB epidemics, several potential barriers to the implementation would exist. These include the cost to purchase and maintain the GeneXpert platform, as well as the cost and supply chain of Xpert MTB/RIF cartridges . Our screening strategy employing urine-based testing with Xpert MTB/RIF is also dependent upon the availability of laboratory facilities with sufficient capacity and biosafety equipment to undertake centrifugation of urine samples, including an uninterrupted electricity supply. All of these requirements may potentially undermine routine screening using Xpert testing of concentrated urine. It was for these reasons that we sought to explore the utility of the Determine TB-LAM lateral flow urine assay for LAM. The potential advantages of this diagnostic tool are self-evident: it currently costs approximately $2.66 per test (nearly one-quarter the cost of an Xpert MTB/RIF cartridge) and it requires no laboratory infrastructure, no laboratory personnel or expertise, and no instrumentation or electricity supply.The main perceived limitation to the use of Determine TB-LAM is that it has only modest overall sensitivity , 20. HowPrevious studies of the diagnostic accuracy of urine-LAM have restricted their evaluation to testing patients selected on the basis of their ability to produce sputum samples , 15, 22.Many researchers have previously suggested that urine-LAM assays lack sufficient specificity to be used as a \u2018rule-in\u2019 diagnostic test. Indeed studies have reported specificity to be as low as 75% . In markThe diagnostic yield varied substantially with CD4 cell count, with no useful yield among those with counts >200 cells/\u03bcL, but with a gradually increasing yield within lower CD4 cell count strata, reaching a maximum of 56.6% among those with counts of 0\u201349 cells/\u03bcL. The yield from urine-LAM screening was independent of ART status. This study confirms that assay utility is restricted to HIV-infected patients with CD4 cell counts of <200 cells/\u03bcL as previously shown among HIV-infected medical inpatients , 22 and During prospective follow-up, mortality was also found to be substantially greater among those testing urine-LAM positive in both crude and adjusted analyses. Other studies too have found a strong association between positive LAM status and mortality, including a recent meta-analysis that found a more than two-fold increased odds of mortality associated with LAM-positivity , 25\u201329. The Determine TB-LAM assay underwent expert review by the WHO in 2015. On the basis of a Cochrane review of all available data (that included unpublished data from the present study) , the WHOStrengths of this study include the recruitment of consecutive admissions without selection according to presenting symptoms or other criteria. Patients were very thoroughly investigated, yielding a large number of TB diagnoses and providing a comprehensive reference standard to assess the diagnostic accuracy of urine-LAM. We believe that this has provided the most rigorous assessment of the diagnostic accuracy of Determine TB-LAM to date. Patients were well characterized and are likely to be representative of HIV-infected medical admissions in countries with a high TB burden. The consistency in findings of post-mortem studies reporting on the burden of undiagnosed disseminated TB in HIV-infected inpatient deaths across the African continent  suggestsWe have rigorously evaluated the Determine TB-LAM diagnostic urine assay and demonstrated that its specificity is extremely high and it provides substantial incremental diagnostic yield when used as a routine screening test on unselected HIV-infected adults newly admitted to hospital. The addition of urine-LAM screening doubled the diagnostic yield derived from systematic screening of sputum using Xpert MTB/RIF. Much of the benefit from urine-LAM testing was among TB patients without respiratory symptoms or sputum production. In conjunction with recently published WHO recommendations, this study further supports the implementation of the Determine TB-LAM lateral-flow assay as an initial, routine point-of-care rule-in test for TB among HIV-patients requiring hospitalization in settings with a high TB burden."}
+{"text": "Introduction of GeneXpert MTB/RIF (Xpert) assay has constituted a major breakthrough for tuberculosis (TB) diagnostics. Several patient factors may influence diagnostic performance of Xpert including sputum quality.We carried out a prospective, observational, cross-sectional study to determine the effect of sputum quality on diagnostic performance of Xpert among presumed TB patients in Uganda.We collected clinical and demographic information and two sputum samples from participants. Staff recorded sputum quality and performed LED fluorescence microscopy and mycobacterial culture on each sample. If both smear examinations were negative, Xpert testing was performed. We calculated diagnostic yield, sensitivity, specificity, and other indicators for Xpert for each stratum of sputum quality in reference to a standard of mycobacterial culture.Patients with salivary sputum showed a trend towards a substantially higher proportion of samples that were Xpert-positive  compared with those with all other sputum sample types . Blood-stained sputum produced the lowest sensitivity  and salivary sputum the highest . Specificity didn\u2019t vary meaningfully by sample types. Salivary sputum was significantly more sensitive than mucoid sputum , while blood-stained sputum was significantly less sensitive .Our findings demonstrate the need to exercise caution in collecting sputum for Xpert and in interpreting results because sputum quality may impact test yield and sensitivity. In particular, it may be wise to pursue additional testing should blood-stained sputum test negative while salivary sputum should be readily accepted for Xpert testing given its higher sensitivity and potentially higher yield than other sample types. These findings challenge conventional recommendations against collecting salivary sputum for TB diagnosis and could inform new standards for sputum quality. Introduction of the GeneXpert MTB/RIF (Xpert) assay has constituted a major breakthrough for tuberculosis (TB) diagnostics, providing a rapid and accurate way of identifying TB patients in high TB-burden, low-income countries , 2. NeveA recent systematic review identified no studies describing the effect of sputum quality on Xpert performance , 17. ThiFrom September 2008 through January 2016, we carried out a prospective, observational, cross-sectional study to determine the effect of sputum quality on diagnostic accuracy of Xpert. This study was carried out at Mulago National Referral Hospital, an inpatient tertiary-care facility affiliated with Makerere University in Kampala, Uganda. We enrolled consecutive adults with possible pulmonary TB into the Mulago Inpatient Non-invasive Diagnosis of Pneumonia\u2014International HIV-associated Opportunistic Pneumonia study, as previously described \u201323. PatiFollowing written informed consent, parent study participants provided clinical and demographic information and two expectorated sputum samples collected one hour apart. Trained research staff delivered standardized instructions on proper sputum submission . LaboratWe performed univariate analyses of participant characteristics, and bivariate analyses stratified by sputum quality type. We compared dichotomous variables using chi-squared tests, and continuous variables using the Wilcoxon rank-sum test. We calculated simple diagnostic yield as the proportion of each specimen type that were Xpert-positive. We also calculated sensitivities, specificities, positive and negative predictive values, and positive and negative likelihood ratios for Xpert for each stratum of sputum quality in reference to a gold standard of mycobacterial culture on two sputum samples and, if available, on bronchoalveolar lavage. We compared diagnostic yield by specimen type for our primary analysis. As a secondary analysis, we also compared the sensitivities and specificities of samples of different sputum quality types to confirm that differences in yield reflected differences in true-positive results. We selected the comparisons of diagnostic yield for the primary analysis because this metric reflects how treatment decisions are guided in routine practice. Another reason for this choice was that diagnostic sensitivity, the usual standard metric for comparisons of performance, may have limitations for the current analysis because sputum characteristics may reduce the yield of both the index test and the reference test, sputum culture, leading to uncertain effects on diagnostic accuracy. Finally, we conducted a multivariate analysis adjusting for age, gender, HIV status, CD4 count, cigarette smoking, and alcohol use, in order to assess the extent to which differences in performance reflect differences in patient characteristics versus differences in sputum characteristics. Although sample size was based on convenience, we calculated 95% confidence intervals for all study measures. We performed all analyses using STATA version 14.1 .The Makerere School of Medicine Research Ethics Committee, the Uganda National Council for Science and Technology, the Mulago Hospital Institutional Review Board, the University of California San Francisco Committee on Human Research, and the Yale Human Research Protection approved the study.Of 3572 patients enrolled in the parent study from September 2008 through January 2016, 1782 (50%) were eligible for this analysis . Of thosPatients were generally young, with median age 34 years , with salivary being the next most common sample type . Blood-stained  and purulent  samples were less common. When comparing patients producing salivary sputum with those producing other sputum types, there were few statistically significant differences. Women, however, were significantly more likely to produce salivary sputum than men  1.44 95% Confidence Interval (CI) 1.16\u20131.78, p = 0.001). While there was no significant association between sputum type and HIV status, HIV-infected patients with CD4 counts >200 cells/\u03bcL were significantly less likely to produce salivary sputum than those with CD4 counts \u2264200 cells/\u03bcL .Mycobacterium tuberculosis (MTB) culture results, while 1392 (78%) had negative MTB cultures  had positive cultures . Among MPatients with salivary sputum had a substantially higher proportion of samples that were Xpert-positive  15\u201324) compared with those with all other sputum sample types , yielding 4%  more TB diagnoses. There were no significant differences between the proportions positive for each sample type when compared to mucoid sputum . We saw The overall diagnostic sensitivity of Xpert was 53% (95% CI 48\u201358), and the overall, specificity was 95% , without meaningful changes in the above-reported sensitivity differences. Adjusted sensitivity of salivary sputum remained significantly different from mucoid samples (p = 0.02), as did adjusted sensitivity of blood-stained sputum (p = 0.01).Specimen quality has long been assumed to be as an important predictor of the performance characteristics of microbiologic tests, particularly those used to diagnose lower respiratory-tract infections. Unfortunately, the amount and quality of evidence about how sputum quality affects the performance of TB diagnostic tests is limited. In this prospective cross-sectional study, we found no significant difference in diagnostic yield of Xpert testing between salivary and non-salivary specimens among adults with negative sputum AFB-smear examinations in a low-income country with high burdens of TB and HIV. In fact, we identified a strong trend towards a higher diagnostic yield in salivary than in non-salivary specimens. These differences were confirmed by a secondary comparison of diagnostic accuracy in reference to mycobacterial culture. This analysis showed significantly higher diagnostic sensitivity of Xpert on salivary samples as compared with the referent category, mucoid sputum samples, while blood-stained sputum was associated with significantly lower sensitivity.Macroscopic quality has long been emphasized in guidelines on the use of smear microscopy in TB evaluation. Despite this emphasis, there is only one published study of sputum quality and smear microscopy, which demonstrated substantially higher sensitivity with purulent or bloody sputum as compared with mucoid or salivary sputum among 170 TB patients . HoweverThus, our finding that salivary sputum does not have lower but perhaps higher diagnostic yield when testing for TB with Xpert may have great clinical importance. Salivary sputum has been considered unsuitable for examination by smear microscopy, and therefore laboratory staff have historically been trained to discourage patients from producing and submitting salivary sputum samples in preference for other sample types. Our results suggest that the conventional assumptions that salivary sputum is of lower quality and bloody sputum of higher quality for smear microscopy do not apply to samples tested with Xpert. Given the observational study design, we were not able to explore reasons why salivary sputum may provide greater sensitivity than other samples. Potential reasons could include: a greater bacillary load of MTB DNA in saliva than in other sample types; a greater recovery of MTB DNA from salivary sputum than from more viscous sample types; or more efficient amplification of MTB DNA from saliva than from samples such as sputum that have a more complicated specimen matrix that could include inhibitors of amplification. The last explanation is unlikely because the Xpert assay includes a positive control to detect inhibitors in all samples.Our findings about the enhanced yield of salivary sputum may be of additional importance in high HIV-burden areas because of the inverse association we identified between CD4 count and the likelihood of producing salivary sputum. Because those with lower CD4 counts are also more likely to develop TB and to be smear-negative, it is crucial that they be tested with a diagnostic tool that has high sensitivity and high likelihood of obtaining a true positive result , 26. SinIn contrast, blood-stained sputum appears less desirable for Xpert testing in smear-negative populations. Xpert testing of blood-stained sputum missed twice as many TB cases as it diagnosed. A potential explanation for lower sensitivity could be that blood is a known inhibitor of DNA amplification, although this is less likely because by design Xpert inhibition should be detected by failed amplification of the internal positive control and reported as \u201cInvalid\u201d. NeverthOur study had some limitations. First, a majority of samples were mucoid (73%), resulting in small sample sizes for other sputum types and relatively large confidence intervals for all study measures. In particular, our primary analysis comparing diagnostic yield by specimen type is underpowered, because the 95% confidence intervals for yield differences include clinically important effects. Nevertheless, our sub-analyses were sufficiently powered to detect meaningful differences in sensitivity among three of four sputum types. Second, our sub-analyses could have been biased if the yield of sputum culture is also influenced by specimen quality. However, direct comparisons showed no difference in culture yield, and even if underpowered, the similarities in effect size and direction of our yield and accuracy analyses make this unlikely. Furthermore, we may have misclassified some culture-negative TB patients as not having TB, since we utilized solid rather than liquid culture media . HoweverOur study also had many strengths. First, we carried out our study in a relevant population, possible TB patients in a low-income country with a high TB burden. This helps make our results generalizable to many other populations being tested with Xpert in high-burden, resource-limited settings. Second, our study is the first of sufficient size and power to provide meaningful comparisons of the effects of sputum quality on both Xpert diagnostic accuracy and Xpert diagnostic yield. We therefore believe our study fills a crucial gap in understanding Xpert testing.In conclusion, for patients who are smear-negative, utilizing Xpert may provide a rapid diagnosis that might have otherwise been missed. As it replaces smear microscopy in an increasing number of high-burden countries, it has the potential to reduce the time and number of visits needed to obtain a diagnosis. Our findings, however, demonstrate the need to exercise caution in collecting sputum for Xpert and in interpreting results because sputum quality may impact test yield and sensitivity differently from what has been traditionally taught for smear microscopy. In particular, it may be wise to pursue additional testing should a blood-stained sputum test negative, especially in high TB-burden communities. In addition, laboratory staff should not reject salivary sputum for Xpert testing but accept it readily given its higher sensitivity and potentially higher yield than other sample types. Future studies attempting to replicate these findings and examining additional factors that may impact Xpert diagnostic performance are warranted to help enhance the yield and sensitivity of Xpert testing.S1 FileComplete dataset utilized for data analysis.(CSV)Click here for additional data file."}
+{"text": "In order to modify the hydrophobicity of the membrane , an amphiphilic monomethoxyl poly(ethylene glycol)-b-poly (PEG-PDLLA) was selected to improve its surface hydrophilicity through a simple self-assembly approach. It was found that the contact angles of the modified membrane can be well controlled by variation of PEG-PDLLA concentrations. In vitro cell biological study indicates that optimized cell adhesion can be achieved on the modified membrane with a contact angle of around 50\u00b0 via its self-assembly with an ethanol/water solution of PEG-PDLA (35\u2009mg\u2009ml\u22121). The surface modification of the membrane also changed its biodegradation property in the process of its incubation period up to 240\u2009days. The surface modification method may afford an effective way for adjustment of the surface (interface) of membrane (scaffolds) of different biomaterials, beyond polylactide.Cell functions can be mediated through their interactions with the microenvironments, which highly depend on the surface state of the substrate. However, how to finely adjust the surface of biomaterials is still very challenging. In this study, poly( Generally, cells can sense the signals of the extracellular matrix via mechanotransduction . A lack of cells . Therefo2 and H2O under biological conditions [As a kind of synthetic biocompatible polymer, polylactide (PLA) is produced from 100% renewable resources. PLA can also degrade into COnditions ,8. Therenditions \u201312. Howenditions .Generally, the surface modification of polymeric membranes can be achieved via physical decoration and/or chemical conjugation. For instance, different inorganic compositions  or organd,l-lactide) (PDLLA) with high molecular weight via ring opening polymerization. The resulting polymer was processed into PDLLA membrane through a hot-pressing method. After that, an amphiphilic monomethoxyl poly(ethylene glycol)-b-poly (PEG-PDLLA) was assembled on the membrane under mild conditions (ethanol/water solution). The results indicated that, by controlling the amount of PEG-PDLLA on the PDLLA surface, the resulting modified membrane (PDLA-M) presented controllable contact angles. Cell culture evaluation indicated that PDLA-M with moderate contact angle (around 50\u00b0) enabled optimal cell adhesion and proliferation. PDLA-M membrane also displayed different biodegradability. Compared to the conventional surface modification techniques, our method is expected to have the following advantages: (i) membranes (even scaffolds) with controllable hydrophilicity can be achieved through dipping them in PEG-PDLLA solutions with different concentrations; (ii) the materials which have been introduced onto the membrane have known structure and compositions, which can be more easily approved for biomedical applications; and (iii) the modification process does not involve any toxic organic solvent. Therefore, this simple and environment-friendly approach to modification of hydrophobic membrane may not only offer a good model for investigation of its surface effect on cell functions, but also enlighten a design of novel biomaterials for tissue engineering application.Herein, in this study, we firstly developed poly were purchased from Jinan Daigang Biomaterial Co. Ltd, China. Tin-2-ethylhexanoate (Sn(Oct)2) was obtained from Sigma-Aldrich, China. Anhydrous ether, dichloromethane and ethanol were bought from Shanghai Taitan Chem Co. Ltd, China. 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) and 4,6-diamidino-2-phenylindole (DAPI) were ordered from Life Technology, China. C2C12 cells (mouse myoblast cell line) were obtained from American Type Culture Collection, USA.2.2.2 as catalyst at a reaction temperature of 140\u00b0C for 6\u2009h [PDLLA was synthesized via ring opening polymerization of lactide using Sn(Oct) for 6\u2009h . The obt\u22121) for self-assembly for 24\u2009h. After that, the samples were taken out and dried in an oven at 50\u00b0C for 6\u2009h to get the modified membranes designated as PDLA-M_0, PDLA-M_5, PDLA-M_15, PDLA-M_35, PDLA-M_50, respectively.For preparation of the modified membrane , PEG-PDL\u22121. Polystyrene standards were used for calibration for calculation of the weight average weight (Mw) and number average molecular weight (Mn).The molecular weight and polydispersity index of the polymers were investigated via gel-permeation chromatography with a Shimadzu Prominence HPLC instrument  using tetrahydrofuran as eluent with a flow rate of 1.0\u2009ml\u2009minThe chemical structure of the samples was studied by nuclear magnetic resonance (NMR) spectroscopy with a NMR instrument .\u22121. The curves were scanned from room temperature to 230\u00b0C at a heating rate of 10\u00b0C\u2009min\u22121, followed by maintenance of 230\u00b0C for 3\u2009min, and then cooled down at the same scanning rate to \u221260\u00b0C. After maintaining the samples at \u221260\u00b0C for 5\u2009min, the final curves of the samples were obtained by increasing the temperature to 230\u00b0C at a heating rate of 10\u00b0C\u2009min\u22121.The thermal analysis of the samples was conducted with a differential scanning calorimeter  at a heating rate of 10\u00b0C\u2009minThe hydrophilicity of the self-assembled membrane was evaluated by measurement of the static contact angle with a contact angle meter . Images of the water droplets on the membrane were recorded, and the results were processed by the image acquisition system.w0 is the weight of the original samples used for incubation, tw is the sample weight at the incubation time t. Five parallel specimens were conducted for each group.Sample sheets of 250\u2009mg were incubated in 10\u2009ml phosphate buffer solution  in a heating incubator (37\u00b0C) at shaking rate of 80\u2009rpm. At a specific interval, the samples were washed with ultrapure water and lyophilized until constant weight. The weight of specimens and the pH values of the degradation media were recorded. At each measurement, incubation medium was renewed with 1\u2009ml fresh PBS. Sample degradation was evaluated by the weight loss ratio of the samples calculated according to the following equation:2.3.\u22121 penicillin, 100\u2009mg\u2009ml\u22121 streptomycin at 37\u00b0C in a humidified atmosphere and 5% CO2. The samples were sterilized by 60Co  radiation at 10\u2009kGy before biological analysis.C2C12 cells were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100\u2009U\u2009ml4\u2009cells per well in a 24-well cell plate. After 24\u2009h incubation, 100\u2009\u00b5l MTT (5\u2009mg\u2009ml\u22121) was added into each well and was further incubated for 4\u2009h. After that, cell medium was removed and replaced with 100\u2009\u00b5l dimethyl sulfoxide to dissolve violet formazan crystals at 37\u00b0C in oscillation container. After standing for 10\u2009min at room temperature, the plate was measured at 492\u2009nm by an enzyme-linked immunoadsorbent assay plate reader . Results were presented as the percentage ratio of ODsample/ODcontrol\u2009\u00d7\u2009100% (n\u2009=\u20093).Cell viability and proliferation on the sheets modified was evaluated via the MTT assay. Briefly, the cells were seeded on the polymer membrane at a density of 1.0\u2009\u00d7\u2009104\u2009cells per well in a 24-well plate. After 24\u2009h incubation, cell media were removed from the wells, washed with PBS thrice and fixed with glutaraldehyde solution . Scanning electron microscopy  was employed to observe cell morphology. For fluorescent imaging of the cell morphology and spreading, the fixed cells were treated with FITC-phalloidin  to stain cell cytoskeleton for 45\u2009min at 37\u00b0C, followed by washing with PBS thrice. Cell nuclei were stained with DAPI (5\u2009\u00b5g\u2009ml\u22121) for 10\u2009min at room temperature and washed with PBS five times. The fluorescent images of the cells were obtained using a confocal laser scanning microscope .To investigate the cell morphology, cells were seeded on the sample membranes at a density of 5.0\u2009\u00d7\u2009102.4.The significant differences of the results were analysed via one-way analysis of variance (ANOVA).3.3.1.The chemical structure of PDLLA was firstly characterized by NMR analysis. As can be seen from \u03b4\u2009=\u20093.64 [Although the good biodegradability and biocompatibility of PLA endow it with broad biomedical applications, its strong hydrophobicity results in poor cellular affinity and cell recognition ability, which is a bottleneck for further applications . A simpl\u03b4\u2009=\u20093.64 ,28, suggTg) at around \u221245\u00b0C, a crystallization temperature at \u22125\u00b0C, and a melting temperature at around 40\u00b0C. After self-assembly, all these peaks of pure PEG-PDLLA in the modified membrane disappeared, suggesting that PEG-PDLLA has been well dispersed on the PDLLA surface which may disorder its crystalline structure. The increase of the amount of PEG-PDLLA led to the gradual decrease in the glass transition temperatures, indicating PEG-PDLLA and PDLLA had good miscibility [To get an insight into the thermal properties and miscibility of PDLLA and PEG-PDLLA in the membranes, their crystallization and melting temperatures were investigated by DSC analysis. As shown in cibility \u201332. This\u22121 can effectively decrease the contact angles of the membranes from 74.5\u2009\u00b1\u20092.4 to 50.0\u2009\u00b1\u20092.3\u00b0  during tissue/organ development . BesidesSince the degradability of materials is very important for their biomedical applications, the biodegradation process of the pure PDLLA and modified membranes was studied by investigation of their weight loss as a function of incubation time in PBS (pH 7.4). It can be seen from 4.\u22121 allows for adjustment of the membrane contact angle from 74.5 to 50.0\u00b0. Results indicated that the membrane which was modified with 35\u2009mg\u2009ml\u22121 of PEG-PDLA offered an optimal cell adhesion, spreading and proliferation, where its contact angle is about 50.0\u00b0. The surface modification method can also affect the biodegradation process of the PDLLA membrane. The effectiveness in combinative improvement of the surface condition as well as degradation property of PDLLA may offer a good way for fabrication of biodegradable membranes (scaffolds) with controllable surface (interface) for tissue engineering applications.In summary, we have developed a simple and effective approach to adjust the hydrophilicity of PDLLA by self-assembling amphiphilic PEG-PDLLA onto membranes of PDLLA. Variation of PEG-PDLLA decoration amount from 0 to 50\u2009mg\u2009ml"}
+{"text": "PRESENILIN1 (PSEN1), PRESENILIN2 (PSEN2) and AMYLOID BETA A4 PRECURSOR PROTEIN (APP) have been identified in familial Alzheimer\u2019s disease (AD). The length of mitochondrion-endoplasmic reticulum (M-ER) appositions is increased in Psen1-/-/Psen2-/- double knockout murine embryonic fibroblasts and in fibroblasts from AD-affected individuals. Development of an easily accessible, genetically manipulable, in vivo system for studying M-ER appositions would be valuable so we attempted to manipulate M-ER apposition length in zebrafish (Danio rerio) embryos. We injected fertilized zebrafish eggs with antisense morpholino oligonucleotides (MOs) that inhibit expression of zebrafish familial AD gene orthologues psen1 and psen2. Furthermore, we treated zebrafish embryos with DAPT (a highly specific \u03b3-secretase inhibitor) or with sodium azide . We then analyzed M-ER apposition in an identified, presumably proliferative neural cell type using electron microscopy. Our analysis showed no significant differences in M-ER apposition lengths at 48 hours post fertilization (hpf) between psen1 & psen2 MO co-injected embryos, embryos treated with DAPT, or sodium azide, and control embryos. Instead, the distribution of M-ER apposition lengths into different length classes was close to identical. However, this indicates that it is feasible to reproducibly measure M-ER size distributions in zebrafish embryos. While our observations differ from those of murine and human studies, this may be due to differences in cellular differentiation and metabolic state, cell age, or species-specific responses. In particular, by focusing on a presumably proliferative embryonic cell type, we may have selected a cell heavily already reliant on anaerobic glycolysis and less responsive to factors affecting M-ER apposition. Future examination of more differentiated, more secretory cell types may reveal measurable responses of M-ER apposition to environmental and genetic manipulation.Mutations in the human genes  Alzheimer\u2019s disease (AD) is a neurodegenerative disorder characterized by the occurrence of memory loss in its initial stages, with other effects such as the impairment of speech and motor ability, depression, hallucinations, behaviour disturbances and, ultimately death in more advanced stages of the disease [reviewed in ]. The maThe majority of AD cases are sporadic (sAD), with a small number of cases that are familial (fAD). Familial AD characteristically has an early age of onset (<65 years). Although only accounting for a small percentage of AD cases, most of our understanding of the molecular events underlying the development of AD comes from fAD, since genetic analysis can be used to identify the genes and proteins involved.PRESENILIN genes (PSEN1 and PSEN2) , cholesne (MAM)  and calcne (MAM) . Interesne (MAM)  and is ine (MAM) . The MAMne (MAM) ). Furthene (MAM)  looked ay of MAM . This suDanio rerio, is advantageous as a model organism for the study of human disease. Zebrafish embryos are robust and can undergo experimental manipulations such as the injection of MOs into embryos to modify simultaneously the expression of multiple genes. Embryos are produced in large numbers and develop rapidly -S-phenylglycine t-butyl ester) in DMSO was purchased from Calbiochem . This was added to the aqueous support E3 medium of 4 hpf embryos until they developed to 48 hpf so that the final concentration was 100 \u03bcM with 1% DMSO. We  and othe3, Sigma-Aldrich CHEMIE GmbH, Steinheim, Germany) was performed at 100\u03bcM that we have previously demonstrated is an effective concentration for mimicry of hypoxia [3 was added to the aqueous support medium of 36 hpf embryos until they developed to 48 hpf.Exposure of larvae to sodium azide . Embryos were post-fixed in 2% osmium tetroxide, followed by dehydration through an ethanol series, and rinsed with propylene oxide followed by resin infiltration. Embedded embryos were sectioned laterally past the embryonic yolk ball, at the beginning of the yolk extension using an ultramicrotome to obtain several 85nm thick sections of the spinal cord. The ultrathin sections were stained with uranyl acetate and lead citrate and imaged on an Olympus-SIS Veleta CCD camera in a FEI Tecnai G2 Spirit TEM. Images were obtained from 3 to 6 cells at the midline area of the spinal cord; this was performed for 3 embryos of each treatment. The mitochondria-ER apposition lengths were measured using Image J software and statistically analysed using Fisher\u2019s exact test .PRESENILIN gene function and might prove to be a useful tool for analysis of the role of these genes in communication between the ER and mitochondria. Therefore, we sought to determine the effect of inhibition of psen1 and psen2 activity on M-ER apposition in the zebrafish animal model. Since our broader research focus is Alzheimer\u2019s disease we wished to analyse M-ER apposition in a neural cell type. However, unlike analysis of M-ER apposition in cultured cells, the central nervous system of developing zebrafish embryos contains many cell types with varying energy production and protein secretion characteristics that might affect the extent of M-ER apposition within cells and confound the statistical analysis required to reveal changes in M-ER apposition length caused by experimental treatments. Additionally, it can be difficult to identify particular neural cell types by morphological criteria in sections observed by transmission electron microscopy (TEM). Therefore we chose cells near the midline of the developing spinal cord in the mid-trunk region of the embryo as relatively easily easy to identify reproducibly (by their position) and most likely representing a proliferative progenitor cell type that had not adopted a final differentiated state.A previous study by Area-Gomez et al.  found thpsen1 and psen2 mRNAs , hypoxia mimicry (with 100 \u03bcM sodium azide from 36 hpf), Presenilin activity loss (injected with MoPS1Tln plus MoPS2Tln) and MO-injection negative control (injected with MoCont). Since exposure to hypoxia slows embryo development, the treatment group exposed to sodium azide was allowed to develop until these embryos reached a stage equivalent to that of 48 hpf embryos under normoxia . The effectiveness of the above treatments was confirmed by observation for developmental delay (under mimicry of hypoxia), the loss of melanotic cells (melanophores) in the trunk region (under \u03b3-secretase inhibition), and for loss of melanophores and the occurrence of hydrocephalus (caused by Psen1 and Psen2 protein loss). Only embryos showing the appropriate phenotypes were selected for analysis.Once again, three embryos were examined per treatment and transverse sections were taken through from the yolk extension region of the trunk. In this study, up to 6 cells were examined from each embryo to reduce the possible confounding effects of measuring M-ER apposition lengths in cells that might have different differentiation states.The individual M-ER apposition length measurements from our analyses at 48 hpf are shown in As previously, we examined the M-ER appositions lengths when grouped into three and five classes of range length . Fisher\u2019Psen1 and Psen2 activity. Also, mimicry of hypoxia might be expected to increase M-ER apposition length as cells attempt to increase mitochondrial activity. However, Area-Gomez et al. [The 2012 study by Area-Gomez et al. , observez et al.  did not in vivo analysis) this method is very labour intensive and expensive and limits the scale of research studies that can be performed.During the development of the zebrafish spinal cord, cells divide near the apical \u201csurface\u201d (the midline) and terminally differentiating neurons migrate outwards (e.g. see ). Thus, In mammalian cells, the majority of apposition lengths observed in wild type cells were punctate (< 50 nm)  while in2+ ions from the ER [2+ ion source to form M-ER appositions [2+ to support the activity of a number of enzyme systems (reviewed by [Cells can control the activity of mitochondria via release of Cam the ER . Mitochoositions  and theyiewed by ). Howeveiewed by , 67 and iewed by , 69). OuPsen1 and Psen2 translation at 24 and 48 hours post fertilization. Furthermore, we observed no significant difference in the distribution of M-ER apposition lengths in such cells between embryos treated with DAPT or sodium azide and in untreated embryos at 48 hours post fertilization. The inconsistency between our observations and those performed in mammalian systems may be due to differences in cell type, developmental stage and/or differences in vertebrate species. Future studies should examine other zebrafish cell types and ages.We observed no significant difference in the distribution of M-ER apposition lengths in apically located cells of the developing spinal cord in the trunk of zebrafish embryos between individuals injected with negative control morpholinos and those injected with morpholinos for simultaneous blockage of S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S1 File(ZIP)Click here for additional data file.S2 File(ZIP)Click here for additional data file.S3 File(ZIP)Click here for additional data file.S4 File(ZIP)Click here for additional data file.S5 File(ZIP)Click here for additional data file.S6 File(ZIP)Click here for additional data file.S7 File(ZIP)Click here for additional data file.S8 File(ZIP)Click here for additional data file.S9 File(ZIP)Click here for additional data file.S10 File(ZIP)Click here for additional data file.S11 File(ZIP)Click here for additional data file."}
+{"text": "The last half century of paleornithological research has transformed the way that biologists perceive the evolutionary history of birds. This transformation has been driven, since 1969, by a series of exciting fossil discoveries combined with intense scientific debate over how best to interpret these discoveries. Ideally, as evidence accrues and results accumulate, interpretive scientific agreement forms. But this has not entirely happened in the debate over avian origins: the accumulation of scientific evidence and analyses has had some effect, but not a conclusive one, in terms of resolving the question of avian origins. Although the majority of biologists have come to accept that birds are dinosaurs, there is lingering and, in some quarters, strident opposition to this view. In order to both understand the ongoing disagreement about avian origins and generate a prediction about the future of the debate, here we use a revised model of scientific practice to assess the current and historical state of play surrounding the topic of bird evolutionary origins. Many scientists are familiar with the metascientific scholars Sir Karl Popper and Thomas Kuhn, and these are the primary figures that have been appealed to so far, in prior attempts to assess the dispute. But we demonstrate that a variation of Imre Lakatos\u2019s model of progressive versus degenerative research programmes provides a novel and productive assessment of the debate. We establish that a refurbished Lakatosian account both explains the intractability of the dispute and predicts a likely outcome for the debate about avian origins. In short, here, we offer a metascientific tool for rationally assessing competing theories\u2014one that allows researchers involved in seemingly intractable scientific disputes to advance their debates. Good scientific practice makes for good science! This dictum is not, by itself, exceedingly diagnostic.Why is it so difficult to separate the good scientific wheat from the bad scientific chaff? The answer to this question is two-fold. First, there are many standards to meet when practicing good science. Second, meeting them often requires activities which can be cast in either a positive or negative light. For instance: evidence seeking is a typical scientific activity. But seeking evidence whether or not it turns out to support one\u2019s preferred scientific theory is often indistinguishable, in practice, from seeking evidence in support of one\u2019s preferred scientific theory. The former meets the standards of good science but the latter does not. When a scientist has gathered evidence in support of their preferred scientific theory, observers often cannot tell whether the evidence was gathered in the good evidence-seeking way or the bad. In ambiguous cases, proponents of the theory are liable to view the process as corroborative, while opponents are liable to view it as verificationist. Another example is that of theory evolution. Those who endorse a theory will likely see development and modification of the theory as progressive; but critics may interpret these changes as excessively revisionist. Finally, consider the notion of scientific agreement. When proponents of a debate are happy with the establishment of an agreement, they call it a consensus. When unhappy, they label it dogma. For each of these antagonisms, there is some practice that seems to be associated with good science\u2014something that might help to characterize it, such as evidence gathering or theory evolution or scientific agreement\u2014but then it turns out that it is the No single or simple standard for assessing good science has yet been proposed and validated by widespread, successful application. Sir Karl Popper\u2019s criterion of falsifiability  is a favDeinonychus antirrhopus functional scientific practice, and we develop and apply that account to a historical review of the BADM versus BAND debate in a diagnostic manner. We caution against conflating merely static or even degenerative scientific practice with unscientific practice, and conclude with a prediction generated by the Lakatosian model of scientific practice regarding the likely fates of BADM and BAND. Although the idea that birds are dinosaurs is currently at the core of a progressive Lakatosian research programme, the idea that birds are not dinosaurs has become, at best, the core of a static research programme and is, at worst, a degenerative one. Our ultimate aim is to demonstrate how Lakatos\u2019 model can be applied not just here, but elsewhere\u2014wherever contradictory, contested assessments of research quality are impeding progress and increasing hostility in scientific debate.A significant attitudinal shift in the modern debate about avian origins occurred in 2003, when one disputant  accused Despite how simple and elegant a solution to the problem of demarcating science from nonscience the falsifiability criterion appears to be, there are problems with it. The most significant problem is that only rarely does the practice of science consist in assessing theories by isolating hypotheses and testing them in fortuitous conditions where everything else is fixed. Hypotheses are designed to test scientific theories, and scientific theories are often vague and underdeveloped, especially in their early stages. A scientist with a nascent theory may need to be able to develop speculative hypotheses, test them, and reject the ones that do not pan out without having to reject the entire underlying theory. Or, a scientist might need to call into question another aspect of the experimental framework in response to a failed test, holding onto both that hypothesis and the underlying theory\u2014just in case what they have really discovered is a previously undetected problem with the testing conditions, or an insufficiently examined background assumption. Using falsifiability as a minimal standard for scientific practice fails to accommodate evolution and uncertainty in theory and testing conditions. We need an account of scientific practice that allows for the development of theories and tests via trial and error, but which also allows us to say that at some point, enough is enough\u2014that the pursuit of a once-scientific idea has given way to dogmatism.ad hoc revisionism. The problem is in discriminating between these two\u2014principled and ad hoc revisionism\u2014and Popper himself did not ever manage to craft a compelling solution to this problem. This is where the Hungarian-born Imre Lakatos, another philosopher of science and eventual \u00e9migr\u00e9 to Britain, comes into the picture. Scientists tend to be much more familiar with Thomas Kuhn\u2019s  and its positive heuristic was to look for mechanical explanations of phenomena . We might further want, within the category of bad science, a way of distinguishing merely imprudent practices  from pernicious ones . And within the category of junk science, we might want to discriminate between spurious science  and fake science . One virtue of having a more nuanced framework\u2014one that distinguishes beyond the binary of good science and nonscience\u2014is that having such a framework makes it possible to critique bad science, while nonetheless admitting that at least some of it is still scientific.Given the preexisting need for a new and more exacting framework, and while recalling the remarkably antagonistic character of the recent debate between BADM and BAND, neither is it hard to understand how plausible objections to contentious scientific practice might have been mistakenly expressed as accusations of nonscientific practice ; nor is So, let us grant that scientific practice simply is whatever it is that those who are typically called scientists at what are typically called scientific institutions do that is typically called scientific. Philosophers of science have offered other assessments e.g.,  relevantD. antirrhopus  or dogma (if you are not) was sparked by John Ostrom\u2019s  discoverirrhopus . In a seirrhopus ; that thirrhopus ; and thairrhopus . There wr flight , for exar flight , and thar flight . A spater flight ; featherr flight ; a transr flight ; and prer flight .The Origin of Birds (1926), in which Heilmann argued that the absence of either a clavicle or a furcula in dinosaurs, plus the presence of a furcula in birds, along with Dollo\u2019s law of irreversibility , altogether entailed that birds cannot be descended from dinosaurs. This is why the discovery of dinosaurian furculae (mentioned just above) was especially important to the avian origins debate. Because such fossils were unknown at the time, Heilmann identified pseudosuchians rather than coelosaurians as more likely ancestors of birds. Despite the overwhelming resemblance of birds and small theropod dinosaurs noted by Heilmann, the incompleteness of the fossil record alone forced Heilmann to conclude that \u201cit is evident that all our requirements of a bird-ancestor are met by the Pseudosuchians, and nothing in their structure militates against the view that one of them might have been the ancestor of the birds. This of course does not prove that this ancestor was one of the known Pseudosuchians\u201d (Ornithosuchus woodwardi (Euparkeria capensis (Prior to the (still reigning) ascension of Ostrom\u2019s theropod hypothesis for avian origins, there was an alternative paleontological agreement about avian origins: birds were descended from other, rather more basal archosaurs. The dominance of this view is generally credited to the success of Gerald Heilmann\u2019s uchians\u201d , p. 191.oodwardi  and Eupacapensis  to be esOn the Origin of Species by Means of Natural Selection, the German paleontologist Christian Erich Hermann von Meyer reported the discovery of a single feather of Archaeopteryx lithographica  ascension of Heilmann\u2019s pseudosuchian hypothesis, the state of play is perhaps best described as one of a lack of agreement about avian origins. Just 2 years after the 1859 publication of Charles Darwin\u2019s  macrura  and the iemensii . By 1867f Birds\u201d , and on e Review . In his American Association for the Advancement of Science, for instance, the American paleontologist Othniel Charles Marsh adopted Huxley\u2019s view of a dinosaurian origin for birds , characterized by a lack of paleontological agreement; and intermediate phase (1926\u20131968), characterized by widespread paleontological agreement that birds were reptiles but not dinosaurs; and the latest phase (1969\u2013current), which is characterized by widespread paleontological agreement that birds are in fact descended from dinosaurs. What we need to ascertain now is whether this history is one of mere variation in opinion, or advancement of science\u2014and the Lakatosian framework can help.According to Lakatos, a scientific research program is a healthy one when it produces progressive problem-shifts , 1970. TSince Ostrom began reviving the notion in 1969, the idea that \u201cbirds are dinosaurs\u201d has emerged as the hard core of a progressive research program that has continually expanded the protective belt around its core\u2014and expansion of that belt has occurred via the pursuit of multiple, distinct lines of postulation and corroboration leading to further, related postulations and corroboration. See The BADM research program has added empirical content along multiple lines by, for instance: postulating that birds are not just dinosaurs but, more specifically, maniraptoran theropods ; predictThe Lakatosian account also allows us to rationally characterize unhealthy scientific practice. First, we should note that even a once-progressive research program can become degenerative, and vice versa; the status of a research program is not fixed. An unhealthy research program is just one that, according to Lakatos, does not produce or is no longer producing progressive problem-shifts: it is one that does not expand or is no longer expanding beyond its core. Since Lakatos\u2019 account allows for some scientific speculation and failure, it can be difficult, and take quite some time, to identify when a research program is deteriorating. And because a progressive research program must both continually generate postulates and occasionally corroborate some of these postulates , there are at least two distinct ways in which a program can ail.The first and more obvious mode of deterioration is the sort that occurs when a research program regularly generates speculative postulates, but corroboration of these postulates continually fails to occur, and so a protective belt never grows around the core. Lakatos calls this sort of research program degenerative rather than progressive. Insofar as the BAND research program has not lately grown beyond the claim\u2014initially articulated in 1926 by Heilmann\u2014that birds are descended from basal archosaurs, it looks as if BAND might be a degenerative research program of just this paradigmatically Lakatosian sort. See The lack of a protective belt around the \u201cbirds are not dinosaurs\u201d core does not mean that BAND is not a scientific research program: clearly, the program has an extended recent history of proposing speculative hypotheses , 2016. ITyrannosaurus rex; Microraptor a bird, but by default its feathered relatives, such as Velociraptor mongoliensis, the nonvolant predatory theropod of movie fame, must also be a flightless bird . This is not a hypothesis that has garnered support among neontologists or paleontologists and it is worth noting that, previous to the discovery of vaned feathers in dromaeosaurs and other theropods , proponents of BAND argued vehemently that these taxa were totally unrelated to birds (It is worth noting here that a core commitment to the idea that birds are not dinosaurs was one that Heilmann himself was somewhat uncomfortable with, in that the simple lack of recognition of fossilized furculae in dinosaur remains known in the early 20th century essentially forced Heilmann, who adhered strictly to Dollo\u2019s law of irreversibility, to argue against the otherwise overwhelming evidence that birds were in fact dinosaurs . Nonaviato birds .But there is a second way in which a research program can turn out to be degenerative, in an expansive Lakatosian sense: it is not only that a program can fail to corroborate its speculative hypotheses; it can also cease to offer any such hypotheses at all. We propose this as an addendum to the Lakatosian model, and posit that it is better to call this sort of ailing research program static rather than degenerative. This is because it could be true, as some have suggested , that biThis sort of static research program might best be described as residing in an intermediate, purgatorial territory between progressive and degenerative programmes. Static science is not necessarily bad science: once-progressive research programmes can become static, for instance, when they become so established that they stop offering and corroborating new postulates. And in the absence of means for testing its postulates, a research program can be forced to go into stasis until technological or other developments allow for testing to begin or resume. Having a static research program might be better for a field of study than having a degenerative research program or having no research program at all. But such a program will not fare well in a field in which there is direct competition with a progressive research program. If this is indeed the current, comparative state of the BAND research program\u2014if it is either straightforwardly degenerative, or has to resort to stasis , 2016, iA key component of the currently entrenched dispute between BADM and BAND is the surprising frequency of appeals now being made, on both sides, to various philosophical figures and their ideas about what counts as scientific. David Hume and Arthur Schopenhauer as well as Popper and Kuhn have all made recent appearances in the debate, as have their ideas about skepticism, induction, falsification, paradigm shift, confirmation, and dismissal . Some phFinally, one further and interesting result that follows from our application of the rehabbed Lakatosian model to the BADM versus BAND debate is the generation of the following pair of predictions: it is highly unlikely that either a straightforwardly degenerative or a static version of the BAND research program will flourish in paleornithology, without further generation of empirically confirmable speculative hypotheses, and at least occasional corroboration of those hypotheses. And given the competitive recent history of the BADM research program\u2014with its continued generation of speculative hypotheses, and lately, its more than occasional corroboration\u2014it is far more likely that this progressive research program, which considers birds to be maniraptoran theropod dinosaurs, will flourish instead. But this is a pair of speculative hypotheses designed to test the viability of the refurbished Lakatosian theory of progressive versus degenerative or static research programmes; we eagerly await either the refutation or the corroboration of these empirical postulates."}
+{"text": "Polydopamine can form biocompatible particles that convert light into heat. Recently, a protocol has been optimized to synthesize polydopamine/protein hybrid nanoparticles that retain the biological function of proteins, and combine it with the stimuli-induced heat generation of polydopamine. We have utilized this novel system to form polydopamine particles, containing transferrin (PDA/Tf). Mouse melanoma cells, which strongly express the transferrin receptor, were exposed to PDA/Tf nanoparticles (NPs) and, subsequently, were irradiated with a UV laser. The cell death rate was monitored in real-time. When irradiated, the melanoma cells exposed to PDA/Tf NPs underwent apoptosis, faster than the control cells, pointing towards the ability of PDA/Tf to mediate UV-light-induced cell death. The system was also validated in an organotypic, 3D-printed tumor spheroid model, comprising mouse melanoma cells, and the exposure and subsequent irradiation with UV-light, yielded similar results to the 2D cell culture. The process of apoptosis was found to be targeted and mediated by the lysosomal membrane permeabilization. Therefore, the herein presented polydopamine/protein NPs constitute a versatile and stable system for cancer cell-targeting and photothermal apoptosis induction. Over the last few years, the emerging field of nanomedicine has developed a versatile array of promising new applications, in the fields of therapy and diagnosis. A range of nanomaterials, such as liposomes, polymers, dendrimers, carbon nanotubes, and metallic nanoparticles (NPs), have been designed for drug delivery and cancer therapy . AdvanceA frequently-used approach of exploiting the advanced functionalities of nanomaterials, for cancer treatment, is the stimuli-induced increase of temperature inside cells or tissues, i.e., hyperthermia. Various stimuli can be used to this end, i.e., magnetic fields for superparamagnetic iron oxide nanoparticles (SPIONs), radio waves for silica NPs, and ultrasound or light for gold NPs ,8,9,10. A relatively new approach to inducing hyperthermia is the induction of lysosomal membrane permeabilization (LMP) . LysosomMytilus edulis, which uses the polymer as an adhesive to attach to various, usually non-reactive, surfaces  scanning electron microscope. The NPs were drop-casted and sputter-coated with 3.5 nm of gold, before use.PDA/protein NPs were prepared according to previously published work . BrieflyA previously described lock-in imaging setup was applied to perform thermal measurements ,40. PDA/2 water-saturated atmosphere, in tissue culture flasks 75 . For the sub-culturing of the melanoma cells, the cells were washed with phosphate buffered saline (PBS)  and subsequently incubated for 5 min with trypsin-EDTA , until the majority of the cells detached. For the detachment of the macrophages, a cell scraper  was used.For the cell experiments, mouse macrophage (J774A.1)  and mouse melanoma (B16F10)  cell lines were used. The macrophages were cultured at the Roswell Park Memorial Institute (RPMI) 1640 , supplemented with 1% L-glutamine , 1% penicillin-streptomycin , and 10% heat-inactivated fetal bovine serum (FBS) . For culturing the melanoma cells, Dulbecco\u2019s Modified Eagle Medium (DMEM)  supplemented with 1% L-glutamine, 1% penicillin-streptomycin, and 10% heat-inactivated FBS was used. Both cell lines were incubated at 37 \u00b0C, under a 5% CO2/well)  and incubated at 37 \u00b0C in 5% CO2, for 48 h. The cells were then washed with PBS and different concentrations of the PDA/protein NPs , in a complete cell culture medium were added, followed by a 24 h incubation, at the cell incubator conditions. To measure cell viability, two different approaches were used. (i) Lactate dehydrogenase (LDH) assay to assess the membrane rupture, and (ii) resazurin to assess the amount of viable cells with active metabolism. To this end, the supernatant was collected and an LDH assay was performed, according to the instructions of the provider , and quantified by a spectrophotometer . In addition, after removal of the supernatant, a cell culture medium containing 22 \u03bcg/mL resazurin, was added to the wells, incubated for 3 h, and then the formed resorufin was quantified, fluorometrically, at 590 nm . Based on the interference results, i.e., the same experimental setup and measurements conducted in the absence of cells, only the highest measured concentration of the PDA/Tf and the PDA/HSA NPs, respectively, resulted in increased background absorbance values. In the cell experiments, the used concentrations are significantly lower than the one which resulted in an increased background.Cells were seeded at a number of 20,000 cells/well (in 480 \u03bcL) into an eight-chamber well slide  , 0.1% Triton X-100 in PBS, antibody , DAPI , and rhodamine-phalloidin . The control stainings were performed without antibodies. After a 1 h incubation period in the dark, the plates were washed three times with PBS and finally mounted with Glycergel2, for 24 h. Then, the PDA/protein NPs were added to the wells at a concentration of 20 \u03bcg/mL and the slides were incubated for another 24 h. Subsequently, the supernatant containing the NPs was removed, the wells washed, once, with PBS to remove the weakly-associated NPs, and fresh cell culture media was added. In order to visualize apoptosis, fluorescent-labeled Annexin V  was added to the media, at a ratio of 1:200, allowing the binding of Annexin V to phosphatidylserine, a marker for cellular apoptosis. The slides were examined, using a Zeiss LSM 710 microscope, combined with an incubation chamber, pre-warmed to 37 \u00b0C, with a humidified 5% CO2 atmosphere. The time-lapse videos were recorded with a 63x objective and a pixel size of 0.439 \u03bcm \u00d7 0.439 \u03bcm. Two defined regions (162 px \u00d7 172 px) were continuously exposed to a laser beam, at either 405 nm or 633 nm wavelength. Every 6 sec an image of the whole field of view was taken.Cells were seeded on cover glass bottom microscopy slides  in plastic wells, with a density of 50,000/well, and incubated at 37 \u00b0C and 5% COA transferrin receptor (\u03b1TfR) antibody  was diluted 1:100 and added to the wells, 30 min before exposing the cells to the PDA/Tf NPs. Subsequently, the cells were incubated with the PDA/Tf NPs, and again with the 1:100 \u03b1TfR antibody, throughout the incubation. The experiment was then performed, as described above (2.6).In order to obtain a comparable quantification for the different experiments, an imageJ script was written. The regions exposed to the two different laser beams, i.e., 405 and 633 nm, were analyzed individually. In brief, the area covered by cells was detected on the bright field channel, by a variance filter with radius 1. This yielded a binary image where the area with a high variance  received the value 1, while the rest of the region had the value 0. The binary mask, thereby obtained, was then multiplied by the green fluorescent signal , thus, eliminating all signals not overlapping with the cells, due to the multiplication by 0.\u00ae Red DND-99 , prior to the start of the light-induced cell-killing experiment. The experiment was then continued, as described above. The mean fluorescent intensity of the different regions was analyzed by ImageJ. Additionally, a region of the same size was chosen, arbitrarily, and its M.F.I. was also recorded. The third region was then used as a baseline, as there was no increased irradiation performed, but a decrease of the Lysotracker\u00ae intensity was observed, possibly, due to the inherent dye-leakage or bleaching. After subtraction of the baseline, the rate was normalized and plotted, using the GraphPad PrismMouse melanoma cells (B16F10) were seeded and exposed to the PDA/protein NPs for 24 h, as described above. The cells were incubated for 1 h with 50 nM Lysotracker6 cells per well, (0.69 cm2) for 24 h. The cells were then washed with ice-cold PBS and scraped, after addition of Nonident P-40 lysis buffer , which was completed with a protease inhibitor cocktail [Sigma]. Following the lysis of the cells, the three wells were pooled and left on ice, for 30 min. The cell lysates were centrifuged for 20 min, at 12,000 rpm and 4 \u00b0C, and the supernatant was collected. The protein concentration was measured with NanoDrop [Thermo Fisher Scientific] and the samples were adjusted to the same concentration. The samples were boiled for 5 min, in a reducing Laemmli buffer, and 50 \u03bcg were loaded on to the gradient Mini\u2013Protean\u00ae TGX\u2122 Precast Gel , as well as the Precision Plus Protein\u2122 Dual Color  molecular weight standards. The gel was first run at 80 V, until the samples entered the gel, and then at 120 V, using a Bio Rad electrophoresis system . After the Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred for 1.5 h, at 58 mA  onto the polyvinilidene difluoride (PVDF) membrane, under semi-dry conditions. The membrane was then blocked with 2.5% of bovine serum albumin (BSA) in tween tris buffered saline (TTBS) , for 2 h, at room temperature, while being mixed. The blocked membrane was then treated with a primary antibody, diluted in 0.2% BSA in TTBS (1:1000 for anti-transferrin receptor antibody [Abcam 84036] and 1:2500 for anti-\u03b2-actin antibody [Abcam 8227]), and incubated, overnight, at 4 \u00b0C. The following day, the membrane was incubated with a secondary antibody (Goat Anti-Rabbit IgG HRP [Abcam 97080]) for 2 h, at room temperature, before visualization, using a luminol reagent  as a detection staining. The optical density of the bands was estimated using a GelAnalyzer [GelAnalyzer 2010a] and the values were normalized, based on the \u03b2-actin band.Western blot analysis was performed according to literature . In briew/v) alginic acid  was dissolved in RPMI supplemented with 10% (v/v) FBS, 1% (v/v) L-glutamine, and 1% (v/v) penicillin/streptomycin, and were sterilized before utilization. As divalent cations like Ca2+ are required to cause the gelling of aqueous alginic acid solutions, 75 mM CaCl2  in a complete RPMI, was prepared according to the method used by Rowley et al. and was sterile-filtered  with 150 \u00b5L complete DMEM and 88.5 \u03bcL buffer solution, containing 30 \u03bcL 10\u00d7 PBS, 7.5 \u03bcL 1M NaOH , 6 \u03bcL 7.5% (w/v) NaHCO3 , and 45 \u03bcL 1M HEPES . Thirty microliter of collagen type I solution containing spheroids, was added to each well of a 4-well microscope slide, and placed in the incubator, at 37 \u00b0C, to allow gelling. After 1.5 h, 500 \u03bcL complete DMEM was added to each well, and samples were kept in the incubator, upon utilization. For the stained samples, the B16F10 cells were incubated with the VybrantTM [Thermo Fisher Scientific] cell-labeling DiD dye, in the concentration suggested by the manufacturer. After two washing steps, the spheroids were assembled as described above.Sterile 1.5% . Then the frames were analyzed by the increase of the black area, inside the irradiated area.For illustration purposes and the performance of student\u2019s T tests, GraphPad Prism version 6.00 for Windows,  was used. Imaris  was used for the renderings of the confocal data sets and Huygens  was used for deconvolution.For this study, three different PDA/protein hybrid particles were synthesized, i.e., PDA/HSA, A, PDA/TfThe mean hydrodynamic diameters of the NPs in water, were determined by DLS. Furthermore, the \u03b6-potential was measured in water and both of the media were used in the study. Since the size of the NPs is based on the ratio of the proteins to dopamine, in the reaction mixture, the unspecified proteins in the mixture make a size-controlled synthesis impossible .As reported by Jiang et al., we assumed\u2014due to the stochastic nature of the synthesis\u2014an even distribution of the protein on the NP surface . The sliTwo different mouse cell lines were used in this study\u2014B16F10 melanoma cells and J774A.1 macrophages. The J774A.1 cells were chosen because they are professional phagocytic cells and the B16F10 melanoma cancer cells because they express a high ratio of transferrin receptors on their surface. The expression level of the transferrin receptor, in both cell lines used in this study, was probed, using immunofluorescent staining and western blot analysis. First, the presence of TfR was confirmed by western blot analysis, where the TfR specific band was detected in lysates of both cell lines, as well as in the additionally included non-mitotic control\u2014primary macrophages derived from human buffy-coat-isolated monocytes (MDM) . The MDMThe cellular uptake of the PDA/protein hybrid NPs was analyzed by immunofluorescent staining. The mouse macrophages (J774A.1) and mouse melanoma cells (B16F10) were incubated with the PDA/HSA and the PDA/Tf NPs, for 24 h. cLSM revealed the intracellular localization of fluorescent antibodies, against HSA or Tf, in the NPs . The facThe PDA/Tf-A488 NPs were administered to the J774A.1 mouse macrophages, in a live cell imaging experiment\u2014the dynamics of the NP uptake process were assessed by measuring the fluorescence signal over time. Many particles were detected inside the cells, within hours , which wTo measure the cell viability after exposure to NPs, J774A.1 and B16F10 cells were exposed to the PDA/Tf  and the Lock-in thermography (LIT) has been used to analyze the heat generation of the PDA NPs ,54. ThisIn order to assess the ability of the PDA hybrid NPs to exert their heating potential inside cells and, thereby, induce apoptosis, the following experiment was conducted. Both cell types were exposed to either PDA/HSA or PDA/Tf NPs, at a concentration of 20 \u03bcg/mL, for 24 h, and then observed by cLSM. Two regions of interest were excited, with either 405 nm , indicating the presence of the PDA/Tf NPs inside lysosomes , in vitro. The NPs retained the protein functionality at the surface, as shown by the indirect antibody labeling and the receptor-blocking experiments, and the combination of the PDA with Tf could be used to actively target the B16F10 mouse melanoma cells in vitro, because of the upregulated Tf receptors on the surface of cancer cells. Through LIT analyses, PDA/Tf NPs were shown to convert the near-UV-light into heat, more efficiently, than the PDA/HSA. Both NPs were taken up by the J774A.1 mouse macrophages and the B16F10 cells. However, in the latter cell type, more PDA/Tf hybrid NPs were observed, intracellularly, than the PDA/HSA particles. This could be attributed to the fact that B16F10 cells are cancer cells. After exposure of the NPs to the B16F10 and J77A4.1 cells, we were able to induce apoptosis by irradiation of a defined region, with UV-light. The effect was more pronounced when irradiating with a UV-light source than when using a 633 nm laser, which was in line with the absorption spectra of the NPs. Additionally, the PDA hybrid, core-shell NPs were tested in an organotypic melanoma model, i.e., a 3D-printed spheroid, showing, again, a more pronounced induction of apoptosis, in the presence of the PDA/Tf NPs, when irradiated with 405 nm. Evidence suggests that LMP is the prevalent mechanism by which the PDA NPs can elicit an apoptosis response. We, thus, provide a new, highly adaptable system to target cancer cells and to induce apoptosis, via LMP, which is also capable of being effective in densely-packed cellular structures, like spheroids. By the combination of PDA with proteins, which are both inherently biocompatible, the system is unlikely to cause problems in organisms but this still needs in vivo experimental validation. If the results follow the expectations, the system has the potential to be used as a new cancer treatment. The inexpensive and straight-forward combination of the PDA with protein-based-targeting functionality, provides a suitable system for the further exploration of phototherapy, as the NPs can easily be modified and tailored to a specific task.By targeting a basic cellular mechanism, namely the lysosomal degradation pathway, the NPs presented in this study, potentially, circumvent the development of resistances. However, a number of challenges need to be addressed to effect successful translation. For instance, the required UV stimulus has a very limited penetration depth, although the experiments with the spheroids showed a pronounced induction of apoptosis, also in the central layers. Systems that are capable of being stimulated by a longer wavelength radiation, might become accessible with a further investigation into how deep-cell-killing can be induced in the various progression states of the melanoma tumor tissues, as similar NPs have already been shown to undergo a stimulation with infrared light . In conc"}
+{"text": "N\u2009=\u2009110  included 18 individuals suffering from MDD and 19 individuals suffering from DSM-IV anxiety disorders. We found that glutamine and glutamine-to-glutamate ratio were correlated with neuroticism in the whole sample , even when controlling for depression and anxiety disorder diagnoses . Glutamate and GABA were not significantly correlated with neuroticism . Lack of self-confidence and emotional instability were the clinical correlates of glutamate\u2013glutamine dysfunction. In conclusion, this study suggests that prefrontal glutamine is increased in early phases of mood and anxiety disorders. Further understanding of glutamate\u2013glutamine dysfunction in stress-related disorders may lead to new therapeutic strategies to prevent and treat these disorders.There is growing evidence for GABA and glutamate\u2013glutamine dysfunction in the pathogenesis of mood and anxiety disorders. It is important to study this pathology in the early phases of the illness in order to develop new approaches to secondary prevention. New magnetic resonance spectroscopy (MRS) measures allow determining glutamine, the principal metabolite of synaptic glutamate that is directly related to glutamate levels in the synaptic cleft, as well as glutamate and GABA. In contrast to previous investigations, this study used community-based recruitment methods and a combined categorical and dimensional approach to psychopathology. In the study protocol, neuroticism was defined as the primary outcome. Neuroticism shares a large proportion of its genetic variance with mood and anxiety disorders. We examined young adult participants recruited from the general population in a cross-sectional study using 3-T 1H-MRS with one voxel in the left dorsolateral prefrontal cortex (DLPFC). The total sample of  The rapid and robust antidepressant and anxiolytic effects of ketamine, which acts on the glutamate system, suggest that dysfunction of glutamate neurotransmission may be an early and primary pathology of stress-related disorders3. Similarly, alterations in GABAergic neurotransmission have been implicated in depression and anxiety disorders, and positive GABA modulators have also been reported to have anxiolytic effects4.Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the human brain, respectively. There is growing evidence that the central glutamate system plays a major role in the pathogenesis of mood and anxiety disorders6 and reduced Glx (glutamate+glutamine) in the prefrontal cortex of acute, severe, and chronic forms of major depressive disorder (MDD)8, but normal Glx in milder and earlier stages of depression11 and in panic disorder12. Increased Glx in the anterior cingulate cortex (ACC) has also been found in healthy subjects with high scores on the Spielberger trait anxiety scale13. However, Glx and glutamate concentration are not indicators of glutamate release but reflect glutamate\u2013glutamine cycling that is indistinguishable from overall glutamate metabolism14.Magnetic resonance spectroscopy (MRS) provides a noninvasive tool for studying both the glutamate and GABA systems in living humans. A series of MRS studies demonstrated a highly consistent pattern of reduced occipital GABA7. In addition, increased glutamine relative to glutamate levels were found to be relevant for excitotoxicity15. Finally, antidepressants appear to decrease glutamine levels16. Taken together, glutamine levels may be relevant in the vulnerability, pathogenesis, and treatment of mood and anxiety disorders.More recent MRS studies have used methods to decompose Glx into glutamate and glutamine concentrations. Glutamine levels are of particular interest because they have been associated with glutamatergic neurotransmitter activity in animals and humans17. Abdallah et al. found increased prefrontal glutamine levels in unmedicated MDD patients; in the patient group, they identified a positive correlation between glutamine levels and depressive symptoms18. These findings are in line with a study that found increased glutamine concentration in the cerebrospinal fluid (CSF) of subjects with MDD19. There is also preliminary evidence that glutamine is increased in anxiety disorders20, although other MRS studies did not find increased prefrontal glutamine in MDD23.There is also preliminary evidence for increased glutamine levels in MDD. Using MRS, Godlewska et al. found increased glutamine levels in the putamen in unmedicated individuals with MDD at an average age of 31 years24. Neuroticism, mood, and anxiety disorders share many genetic risk factors26, and levels of neuroticism strongly predict the risk for both lifetime and new-onset MDD27. There are some reports suggesting that the association between neuroticism and depression is stronger in women than in men28. In addition to neuroticism, we also examined trait anxiety as a secondary end point as a dimensional construct related to anxiety disorders.However, many previous MRS studies were conducted in chronically ill patients, where the effects of progression and long-term medication use can present major confounds. To address these shortcomings, we used a community-based sampling method in a relatively large sample of the general population, focusing on young adults, thereby reducing confounds associated with neuroprogression, long-term medication, and long-term interactions with disease-related environmental factors. We adopted a dimensional approach in addition to the categorical approach to psychopathology that has been used in previous neuroimaging studies. Neuroticism was selected as the main outcome measure because while it used to be considered as a general dimension of internalizing psychiatric conditions, more recent studies have provided strong evidence that neuroticism is associated with the etiology and pathogenesis of MDD29. We hypothesized that left DLPFC glutamate levels, reflecting reduced glial cell density and size8, would not be related to neuroticism because such pathology appears to develop in later stages of chronic psychiatric conditions. However, we expected glutamine levels to be positively related to neuroticism, reflecting increased glutamatergic neurotransmission as an early biomarker of MDD and other stress-related internalizing psychiatric conditions. We further expected GABA levels to be negatively correlated with neuroticism, in keeping with the decreased GABA levels previously reported in MDD30 and decreased GABA levels as a response to acute stress31.We examined the link between neuroticism and glutamate, glutamine, and GABA levels in the left DLPFC since this region has been found to be relevant for the pathogenesis of mood and anxiety disordersSubjects were recruited using advertisements in local newspapers as well as university blackboard webpages as part of a large ongoing neuroimaging study. The recruitment in the student environment provided us with a sample of predominantly young adults. Subjects were included into the study only after full explanation of the goals of the study and the risks of the study procedures. The study and the written consent were approved by the local ethics committee . Individuals with severe psychiatric disorders such as schizophrenia and acute eating disorders were excluded.32). Additional two subjects were excluded because they had mild abnormalities that could potentially influence the results . From these 180 datasets, only 114 had an estimated fit for glutamine that was lower than the quality criterion . In addition, four outliers in glutamine concentration, whose estimate was at least two standard deviations higher than the mean, were excluded, resulting in a sample size of 110. . In the whole sample, 81.7% did not smoke (less than one cigarette per day). Out of the 110 subjects, 18 were diagnosed with MDD, including 12 in partial or full remission. Two of the MDD cases were on psychotropic medication . Nineteen subjects had an DSM-IV anxiety disorder . Out of these individuals with a psychiatric disorder, 11 suffered from comorbid MDD/anxiety disorder. Table Of 192 subjects who underwent MRI scanning, ten were excluded for poor spectral quality (manifested as a Cramer\u2013Rao lower bound (CRLB) of greater than 10% for the N-acetyl-aspartate (NAA) fit, see also33 and the trait section of the state trait anxiety inventory (STAI)34. We used carefully validated German versions of these instruments36 and double checked the items indicated in Table Subjects underwent an extensive survey online, including the neuroticism extraversion openness five-factor inventory (NEO-FFI)34, Beck\u2019s depression inventory (BDI)37, and Beck\u2019s anxiety inventory (BAI)38. Psychiatric diagnoses were made based on the structured clinical interview for DSM-IV39.STAI and NEO-FFI were administered online some days before the MRI session. On the day of the MRI session, subjects filled out self-report questionnaires on acute psychiatric symptoms: the state section of the STAI40, with TE\u2009=\u200969\u2009ms, TR\u2009=\u20092000\u2009ms, 320 averages (160 pairs), and an eight-step phase cycle. However, since the assessment of GABA with MEGA-PRESS is confounded by the coediting of a macromolecule, which contributes to the edited peak at 3ppm, the GABA findings described represent GABA+ rather than pure GABA values41.All participants were scanned with a 3.0\u2009T GE Discovery MR750, using an 8-channel receive-only head coil. The scanning session included a localizer and a 3D T1-weighted SPGR-sequence , used for localization of the MR spectra. As displayed in Fig. 42. In addition, the NAA linewidth was below 8\u2009Hz for 102/110 subjects in the Gln group .All spectra were processed with LCModel version 6.3\u20131\u2009H and visually inspected for the quality of the glutamate/glutamine and GABA fit Fig. . Glutamahttp://www.fil.ion.ucl.ac.uk/spm/software/spm12). The segmented gray matter, white matter, and CSF maps were written out in native space, and the MRS concentrations were corrected for partial volume CSF contamination, following the method described in43.T1-weighted images were segmented and warped into standard MNI space using the \u2018new segment\u2019 procedure in SPM12 for Matlab . In addition, glutamine, glutamate, and GABA concentrations were intercorrelated. As a result, testing for GABA, glutamate, and STAI trait anxiety was not independent. Group comparisons of MRS measures and diagnostic groups  were performed with a Mann\u2013Whitney U test. Because we considered these analyses as exploratory, we did not apply methods to correct for multiple comparisons. However, applying a Bonferroni correction factor of 6 to our results  would result in a corrected r\u2009=\u20090.263, p\u2009=\u20090.005, and n\u2009=\u2009110, Fig. r\u2009=\u20090.201, p\u2009=\u20090.035, n\u2009=\u2009110). Note that these two variables were significantly correlated . There were no sex differences in these relationships  and negatively correlated with glutamine . The significant association between neuroticism and glutamine did not change if we used glutamine-to-glutamate ratio instead of glutamine concentration , or if the gray matter fraction within the voxel was included as a covariate . When controlling for MDD diagnosis, DMS-IV anxiety disorder diagnosis, smoking, age, and sex, the association between glutamine concentration and neuroticism remained statistically significant .We found relationships between left prefrontal glutamine concentration and both neuroticism  than STAI trait was able to reduce the correlation of neuroticism with glutamine concentration . State anxiety  did not correlate with left prefrontal glutamine  or any of the other concentrations . There was no correlation between glutamine and BDI scores  and BAI scores .To estimate the associations of both STAI trait anxiety and neuroticism with glutamine concentrations, we conducted a partial correlation analysis in which neuroticism as a mediator variable was able to reduce the correlation of STAI trait with glutamine concentration more strongly (from n\u2009=\u200992) on all psychiatric scales (p\u2009<\u20090.001). MDD had a tendency toward higher glutamine concentrations in the left DLPFC voxel . There were no significant differences in MRS measures between partly/fully remitted MDD (N\u2009=\u20097) and acute MDD (N\u2009=\u200911). Removing the three medicated subjects did not change these findings. The subjects with anxiety disorders (n\u2009=\u200919) similarly scored higher in psychiatric scales than the subjects without anxiety disorder (n\u2009=\u200991). They had higher glutamine concentrations in a left prefrontal voxel . Smoking and age did not influence any of these associations.Although our focus in this study was the dimensional approach to psychopathology, we also analysed the data in terms of DSM-IV diagnoses. As expected, the 18 subjects with MDD . In contrast to our study, they did not examine the DLPFC, but examined the ACC, the occipital cortex, and the putamen. Only in the putamen, they found increased glutamine concentrations in the MDD sample. As in our study, they did not find group differences in glutamate concentrations in any of the three voxels examined. Abdallah et al.18 included 16 MDD subjects and 23 healthy controls in an MRS at 4\u2009T study. Their subjects were older than ours (with an average age of 36 years), and they only examined the ACC, not the DLFPC. Consistent with our study, they found increased glutamine levels in subjects with MDD. In contrast to our study, MDD was also associated with increased glutamate levels. Levine et al.19 demonstrated a 17% increase in glutamine concentrations in the CSF of 18 hospitalized patients with acute unmedicated severe depression (mean age 58 years) relative to 22 healthy controls. This finding is consistent with our MRS results in a younger sample with less severe MDD.We are not aware of previous studies on neuroticism and glutamine. However, our main findings are in line with recent MRS studies in MDD. Godlewska et al.44 found increased glutamine concentrations in the right DLPFC in 25 unmedicated depressed patients, including 15 with a history of suicidal behavior relative to 33 healthy controls. However, this result did not survive Bonferroni\u2019s correction or covariation with age. In 18 patients with treatment-resistant depression, Baeken et al.21 found decreased rather than increased glutamine levels in the left DLPFC. In 14 medicated depressed children, Caetano et al.22 found normal glutamine levels in the left DLPFC. Taken together, previous MRS studies on DLPFC glutamine concentration are heterogenous. They might suggest that medication, disease stage, and age are relevant factors that should be studied in larger samples.Consistently with our results, Jollant et al.10. Glx reduction was positively associated with depression severity and age. This finding is consistent with our finding of normal glutamate and Glx levels in young MDD subjects in remitted or mild depressive states. Our findings are also in agreement with a recent meta-analysis including 49 studies and a total of 1180 MDD patients and 1066 controls on glutamatergic metabolite levels that reported normal Glx levels in unmedicated patients23. Further, our results are consistent with recent large-scale genetic studies on neuroticism that point to central glutamate-related genes to be involved in neuroticism45.A relatively recent meta-analysis on 17 reports found reduced prefrontal Glx in MDD; the results were mainly driven by glutamate46. The study of dimensional constructs related to mood and anxiety disorders, such as anxious personality, and early phases of MDD may add important information to investigations in advanced stages of the illness.The strengths of this study include the relatively large total sample compared to previous MRS studies. We used a dimensional approach to psychopathology that is consistent with novel concepts of psychiatric diagnostics such as NIMH\u2019s Research Domain Criteriar\u2009=\u20090.321, p\u2009=\u20090.001, and n\u2009=\u2009110) and negatively correlated with glutamine .The following limitations merit comment: Our MDD and anxiety disorder samples were relatively small. We used a 3\u2009T scanner; higher field strengths may have yielded better peak separation, particularly for glutamine measurement. In addition, we used a voxel which contains a higher proportion of white matter than other  frontal voxels, but which offers high spectral quality and reliability, since high quality spectra are a prerequisite for reliable separation of glutamine from glutamate at 3\u2009T. Two of our MDD subjects (one of them with comorbid anxiety disorder) and 1 non-MDD subject (without anxiety disorder) were on medication; we did not exclude them to maintain the advantages of our epidemiological sampling methods, making findings more representative. In addition, no formal correction for multiple comparisons was performed because the MRS measures of interest  were intercorrelated and therefore not statistically independent. In particular, since glutamate acts as a precursor to GABA, which is cycled between astrocytes and neurons via glutamine, glutamate in our sample was positively correlated with GABA 18 in the PFC as response to increased glutamine concentrations is consistent with this interpretation. The increased glutamine-to-glutamate ratio associated with neuroticism and anxiety disorders may also reflect early impairment of neuron\u2013astrocyte integrity in the development of mood and anxiety disorders48.Early in the pathogenesis of depression, glutamate-related excitotoxicity may lead to reductions of gray matter and finally reduced brain metabolism in the DLPFC49, and other glutamate-related drugs such as lamotrigine and memantine50, in subjects with high prefrontal glutamine levels in early phases of psychiatric disorders.This study suggests that glutamatergic abnormality is related to depression as well as to anxiety disorders. This is consistent with our dimensional approach showing association between increased prefrontal glutamine levels and neuroticism. Increased glutamatergic activity may be a promising target for pharmaceutical approaches to stop neurotrophic processes, such as gray matter volume reduction and glia cell pathology, in the early course of mood and anxiety disorders. Finally, our findings might encourage the testing of antidepressants such as phenelzine that attenuate glutamate neurotransmission"}
+{"text": "This study reports on the crude oil-sensing using carbon nano structures (CNSs). A mixture of CNSs was obtained by a simple method of preparation using palm cellulose ash and nitric acid as precursors, the powder was characterized by x-ray diffraction and infrared spectroscopy. The optical density of crude oil from Rhoud El-Baguel area (Southeast of Algeria) studied using UV-Vis spectroscopy, before and after adding an amount of CNSs powder to view the CNSs crude oil sensing and therefore a new method to determine the quality of crude oils and the comparison between them. Results show that CNSs prepared from palm cellulose ash have a good crystallinity and it is formed mainly from carbon nano dots (CNDs) with 4.32\u2009\u00c5 in layers spacing and 7.4\u2009\u00c5 in crystallite size, indicate that CNSs can be used as an excellent crude oil sensor. The compounds in oil, responsible on optical response named: Fluorophores  are an ultra-small photoluminescent (PL) nanomaterial (<10\u2009nm), It has significant optical properties, disposable surface functions, chemical inactivity, high photoresist, simple and inexpensive methods of preparation, and an abundance of raw materials,\u20268. The preparation of CNSs is a process of mix between two precursor forms, one of which installs the main carbon frame and the other within the structure elements. In this regard, the most prominent synthesis was cellulose ash as a carbon source, while nitrogen acid contains activating molecules9.Nano carbon structures have a wide range of interest because of their use in applications such as energy storage, tribology, electronics, medicine, catalysis and sensors10. These molecules contain non-bonding electrons that form a cloud around the molecule and are usually prone to excitement and shine in response to the light energy projected on them11.CNSs are crystals which can act as a sensor by sparkling at the desired wavelength or color. We emphasize that fluorescent organic molecules are often aromatic or contain multiple bonds, which are alternating single and double bonds, responsible for the high-octane number and therefore the quality of petroleum13.The Phoenix Dactylifera L. leaves were crushed and screened to ensure that the particle size was distributed from 8 meshes to 30 meshes. Leaves were immersed in 5\u2009wt% sodium hydroxide solution at ambient temperature for 12\u2009h. Then they were washed with water for several times and dried in the oven at 80\u2009\u00b0C for 24\u2009h. To remove the wax, the debris of leaves were immersed in the solution of methylbenzene and ethyl alcohol (volume ratio of 1:1), and kept boiled for 8\u2009h. The residues were washed with ethyl alcohol several times and then dried in the oven at 80\u2009\u00b0C for 24\u2009hours. And to remove lignin, leaves were soaked in hydrogen peroxide (30\u2009vol%) and acetic acid solution (volume ratio of 1:1), and boiled with magnetic stirring at 60\u2009\u00b0C for 7\u2009h. Water was used to wash the residue and then filtered until the filter was neutral. The fibers obtained were boiled in 5\u2009wt% of sodium hydroxide solution at 80\u2009\u00b0C for 2\u2009h, then, washed with water to neutral and dried in the oven at 80\u2009\u00b0C for 24\u2009h. The cellulose fibers from Phoenix Dactylifera L. leaves were obtained4. About 5\u2009g of fine ashes obtained from cellulose furnace and mixed with concentrated nitric acid (60%) and stay in agitation for 24\u2009hours. The mixture was separated by centrifugation at 12,000\u2009rpm for an hour to separate the residue and supernatant. The latter was heated in a vacuum oven at 200\u2009\u00b0C14.In order to obtain C-nanostructures, the cellulose extracted previously was carbonized in a muffle furnace directly at 240\u2009\u00b0C for 2\u2009h16. Six samples of oil diluted in cyclohexane at different concentrations were used for the measurements. Table\u00a0The oils were excited by ultraviolet rays (300\u2013400\u2009nm) which fluoresce in the visible wavelength range of 400 to 600\u2009nm. The crude oil sample was obtained from Rhoud El-Baguel, close Hassi-Messaoud region, city of Ouargla south eastern of Algeria. To perform optical density measurements, it was required to dilute the sample to obtain a transparent solution to transmit the light. Cyclohexane was chosen as solvent that can optically respond in the range of 350\u2009nm\u2013500\u2009nm, wavelengths used to excite crude oilK\u03b11Source . Fourier transform infrared spectra were obtained on a SHIMADZU 8400\u2009s (FT-IR) spectrometer whose extent is between 400 and 4000\u2009cm\u22121. UV/visible absorption spectra were recorded with a UV/VIS 6305 spectrophotometer (JENWAY Company).The type of the carbon Nano structure was analyzed by X-ray powder diffraction (XRD) using a BENCHTOP PROTO AXRD diffractometer in the range 2\u03b8:10\u201380\u00b0(step: 0.1\u00b0) and Cu17 or shows a shift down; what is indicates an increase in sp2 layer spacing18.Figure\u00a020. The spacing between the layers was calculated by applying the Bragg equation and found at approximately 4.23\u2009\u00c5. As long as the average crystallite size, Lc, can be determined using the Scherrer equation:Crystal planes and a small broad peak to about 2\u03b8\u2009=\u200944.22\u00b0 and 77.5\u00b0 correspond to the set (100) and (110) reflectionsor:\u03bb: the wavelength of X-rays (1.54\u2009\u00c5),\u03b2: the width at half height (in radians),\u03b8: the diffusion angle21.and K is the Scherrer constant (0.9)The Lc has been estimated at 7.0\u2009\u00c5.\u22121. The presence of oxygen-containing carbon structures has been confirmed. The peak at 1528\u2009cm\u22121 can be attributed to the C=C stretching vibrations. The \u03b4 (C=O) vibration band is found at approximately 680\u2009cm\u22121\u200923. The bands at 1900, 2098.172 and 2334.892\u2009cm\u22121 can been attributed to inorganic \u028b3CO3, manganese carbonyl stretching frequency and water molecule under strongly hydrogen-bonded conditions26.As shown in Fig.\u00a0In Fig.\u00a0The analysis also shows that the samples have a better optical density for a minimum value of wavelength (350\u2009nm) and this for concentrations less than or equal to 0.4\u2009ml/l. While it is less intense for other wavelengths throughout the UV-Vis domain Fig.\u00a0. For comIn order to determine clearly the effect of carbon nanostructures on the optical properties of the oil sample, we study the optical density changes in terms of concentration of samples under a constant wave length of 400\u2009nm before and after adding an amount of CNSs powder Fig.\u00a0.Figure 5And we study the optical density changes in terms of UV-Vis wavelengths for 0.4\u2009ml/l concentration of sample before and after adding an amount of CNSs powder Fig.\u00a0. It is cCarbon Nanostructures (CNSs) can be synthetized simply with an ash of palm cellulose available locally and maybe used as a very effective tool for sensing and estimating the quality of crude oil and comparing between them."}
+{"text": "There is limited evidence on how to implement shared decision-making (SDM) interventions in routine practice. We conducted a qualitative study, embedded within a 2\u2009\u00d7\u20092 factorial cluster randomized controlled trial, to assess the acceptability and feasibility of two interventions for facilitating SDM about contraceptive methods in primary care and family planning clinics. The two SDM interventions comprised a patient-targeted intervention (video and prompt card) and a provider-targeted intervention (encounter decision aids and training).Participants were clinical and administrative staff aged 18\u00a0years or older who worked in one of the 12 clinics in the intervention arm, had email access, and consented to being audio-recorded. Semi-structured telephone interviews were conducted upon completion of the trial. Audio recordings were transcribed verbatim. Data collection and thematic analysis were informed by the 14 domains of the Theoretical Domains Framework, which are relevant to the successful implementation of provider behaviour change interventions.n\u2009=\u200929) indicated that the interventions were not systematically implemented in the majority of clinics. Participants felt the interventions were aligned with their role and they had confidence in their skills to use the decision aids. However, the novelty of the interventions, especially a need to modify workflows and change behavior to use them with patients, were implementation challenges. The interventions were not deeply embedded in clinic routines and their use was threatened by lack of understanding of their purpose and effect, and staff absence or turnover. Participants from clinics that had an enthusiastic study champion or team-based organizational culture found these social supports had a positive role in implementing the interventions.Interviews (Variation in capabilities and motivation among clinical and administrative staff, coupled with inconsistent use of the interventions in routine workflow contributed to suboptimal implementation of the interventions. Future trials may benefit by using implementation strategies that embed SDM in the organizational culture of clinical settings. Contributions to the literatureShared decision-making is widely advocated in several nations to promote patient-centered care for contraceptive choices. There is limited evidence, however, on how shared decision-making interventions are used by health care professionals in contraceptive care.Through semi-structured interviews guided by an implementation theory, the Theoretical Domains Framework, we identified that staff members\u2019 use of shared decision-making interventions was a complex process. Implementation required individual and organizational capability, opportunity, and motivation over a sustained period of time.Our theory-driven qualitative results fill a critical gap in the literature on the feasibility and usability of shared decision-making interventions.Shared decision-making (SDM) is a process in which providers and patients exchange information, deliberate about available options together, identify the patient\u2019s preferences, and incorporate those preferences in choosing the option or treatment plan . In contSDM is also a model for operationalizing World Health Organization recommendations to offer \u201cevidence-based, comprehensive information, education and counselling to ensure informed choice,\u201d so\u00a0that \u201cevery individual is ensured an opportunity for their own use of modern contraception\u2026without discrimination\u201d .Patients who reported that \u201cthe provider and me together\u201d decided what contraceptive method they would use were more satisfied with the decision-making process than those who reported other roles in decision-making . Other rImplementation researchers have attempted to identify systematically the behaviors that may influence SDM adoption at the individual, organizational, and policy level \u201314. For The aim of this qualitative study was to explore the feasibility and acceptability of two interventions for facilitating SDM about contraceptive methods with a particular focus on factors that influenced their implementation by clinical and administrative staff. The study was embedded within a 2\u2009\u00d7\u20092 factorial cluster randomized controlled trial (RCT), the Right For Me study, and conducted in 16 primary care and reproductive healthcare clinics in the Northeast United States .ClinicalTrials.gov Identifier: NCT02759939). Methods for the trial are published elsewhere , so like physically and mentally. So rearranging the rooms in a way so that we have those sort of stackable file holders, and putting all of the tear-off sheets in those in an order and in a way that made sense. Clinics implemented the interventions in a way aligned with their work style, contextual needs, and team dynamics Handing the interventions directly to patients and explaining their purpose was more acceptable and feasible than relying on the patient to use them if interested. In contrast, in the context of Clinic 5, participants used a passive strategy for the video and prompt card because staff perceived it was the patient\u2019s responsibility to engage with them:Clinic staff were motivated to implement the interventions if they felt that they were aligned with their existing roles and responsibilities  and added value to their work (\u201cReinforcement\u201d) .Administrative staff did not know what took place between patients and clinical staff after the patient had watched the video, while clinical staff reflected that they did not know which patients had watched the video in the waiting room: \u201cI\u2019d be curious to know what the study shows as far as patients who took the survey who also said they watch the video. It\u2019s just a tool that was much more hands off for me\u201d . Staff who watched the video had some criticisms of the content, namely that the featured patient was not representative of their patients and sounded \u201crehearsed,\u201d negatively impacting their motivation to implement it. In contrast, clinical staff involved in contraceptive counseling illustrated the motivating value of directly observing or experiencing a positive effect as a result of using the decision aids (\u201cReinforcement\u201d). For instance, some staff shared that, after using a decision aid, patients chose a method of contraception that seemed best aligned with their preferences.The physical context of implementation  influenced staff members\u2019 motivation and plans to use the interventions. The implementation process was perceived to be easiest in clinics with a self-described \u201csmall team\u201d and low caseload, and/or where staff felt they had flexible procedures and infrastructure to adapt the interventions to fit existing routines and their clinic environment . However, these staff typically found the longer visits easy to adjust to . Staff that received both interventions did not perceive that having multiple components added to their implementation \u201cload,\u201d in part because of the division of labor between clinical (decision aids + training) and administrative (video + prompt card) staff. Rather, comments about time pressure and workload were more common among staff who were already experiencing environmental stressors, such as participating in other research studies or having unexpected staff turnover.Staff from clinics with existing approved counseling materials used these and the decision aids together, so that one supplemented the other . Few staff felt that using multiple resources was cumbersome in counseling, because the resources were so similar. As described above, a minority of staff at reproductive health care clinics perceived that they already do SDM (\u201cCompetence\u201d) and this attitude led them to believe that the decision aids and accompanying training were redundant (\u201cMotivation\u201d). No staff suggested that there were any existing materials that were replaced by or supplemented the video or prompt card.Finally, one of the core domains that influenced implementation behavior was the \u201cSocial Influence\u201d of patients on staff members\u2019 routine use of the interventions. While staff perceived that patients found the decision aids acceptable, they felt that patients had minimal interest in the video and prompt cards, potentially because using them was inconsistent with typical waiting room behavior. Nonetheless, staff felt that both interventions were appropriate for their patient population, in particular for those with lower education and literacy, and those making their own health care decisions for the first time.When asked if they would want to continue using the interventions now that the trial was complete, staff reported that they would like to continue using the decision aids but had mixed feelings about the video and prompt card. Without the tablets provided by the Right For Me study, most felt that their clinic would be unlikely to continue use of the video in its current format. While staff were keen to continue using the decision aids, those affiliated with a larger organization or network also felt that future implementation decisions would have to be made \u201chigh up,\u201d for consistency of content and branding across the organization.Our findings suggest that the decision aids were more acceptable, feasible, and sustainable than the video and prompt cards. Awareness of these interventions, knowing how to use them correctly and competently, integrating the interventions into regular workflow, and having a professional role and organizational culture that supported using the interventions appeared to facilitate intervention implementation. Clinic environments, workflow, and physical space supported implementation of the decision aids, but did not facilitate use of the video and prompt card. While some facilitators are context-specific, our findings suggest that introducing interventions will not be successful without the resources required to modify existing routines and to monitor and sustain behavior change.In clinics where implementation was explained as relatively weak, it appears that the interventions were not considered an essential professional responsibility. While integrating a video via tablet into a busy waiting room may not be a feasible strategy for facilitating SDM, integrating paper-based decision aids into clinical routines may prove more successful. However, it requires negotiating and planning as to what the task is, who does it, how it gets done, and whether it adds any real value.Elwyn and colleagues conducted a thematic analysis of qualitative interviews embedded in the intervention phase of a trial of similar clinical encounter decision aids for treatment of knee osteoarthritis . Before Our findings suggest that participants in clinics that implemented both interventions did not experience implementation overload in comparison with those that were exposed to one only. Similar to a study investigating implementation of a clinical encounter decision aid for circumcision, we observed that gaining the skills to use the decision aid through practice (a learning curve) was necessary .The research team overestimated some clinics\u2019 capacity to self-organize in designing and preparing for implementation even when the interventions were conceptually aligned. However, the solution for bridging this capacity gap is unclear. The MAGIC (making good decisions in collaboration) program, which sought to implement SDM into routine primary and secondary care, similarly observed that clinical teams feel they already involve patients in decisions about their care . In thatAll clinics had a strong perception that SDM was already a part of their organizational culture, and was facilitated in some clinics by use of existing educational resources. The high acceptability of the decision aids may stem from our extensive provider consultation about their content . ImplemeClinics exposed to the video and prompt cards had limited awareness of the cards, and perceived that the videos were difficult to integrate into routine workflow and were of limited interest to patients. The implementation process was seen to be easiest in smaller clinics, or where staff felt they had flexible procedures and infrastructure to adapt the interventions to fit routine practice. The success of implementing these new routines was also dependent on the actions of clinic patients, who may either accept or decline to use the Right For Me interventions. Survey responses from patient participants in the trial will provide further insight into the number of patients who reported using the Right For Me interventions, and what proportion would recommend them to a friend . Participants felt that both interventions were most appropriate for their low health literacy patients. However, these attitudes may not be supported by emerging literature. Recent investigations from Australia suggest that generic question sets alone, like those used in our video and prompt card, are not sufficient to support shared decision-making among adults with low literacy  and addiThe video and prompt card used in this study were adapted from the \u201cAsk, Share, Know\u201d program previously tested and implemented in an Australian primary care setting . A systeOur study findings also suggest that there may be differences in implementation practices for patient-targeted interventions implemented by administrative staff (video + prompt card) versus those that are intended for the provider to use with the patient (decision aids). The organizational or institutional context may also play an important role. Sexual and reproductive health clinics and organizations have well-established norms, such as organization-wide counseling protocols and branding. These norms may represent a double-edged sword\u2014they provide the capability, opportunity, and motivation for staff to engage in SDM, but may be inflexible to change.Limitations of this study include lack of accompanying observational strategies for assessing implementation success. This meant we were unable to investigate the relationships between staff perceptions and actual implementation. We also took measures to minimize social desirability bias, but some participants may have over-reported positive and under-reported negative perceptions, attitudes, or experiences. In spite of our partnerships with and recruitment support from clinic staff at each study clinic, we received limited interest in participation from some clinicians, leading to no data for Clinic 4 and only one participating clinic staff each for Clinics 8 and 11. Findings from those clinics should be interpreted in relationship to the other settings, not individually.The strengths of this study include our systematic application of a theoretical framework for behavior change  to develOur results suggest that clinical and administrative staff perceived the clinical encounter decision aids to be more acceptable and feasible to implement than the patient video and prompt card questions. Implementation of interventions that align with existing roles, tasks, and workflow may have greater acceptability, feasibility, and sustainability than those that require new procedures and infrastructure. We demonstrated how use of the Theoretical Domains Framework can be used to understand the factors that influence implementation of SDM, and to create interventions that are theoretically and behaviorally informed. Future studies could build on our findings of the factors that influence implementation of SDM and use the Behavior Change Wheel and COM-B frameworks to characterize and design strategies for implementing our study interventions in different settings .Additional file 1. Orientation materials for clinics implementing the Right For Me interventions."}
+{"text": "In animal studies, the effects of THC are highly dose-dependent, and biphasic effects of cannabinoids on anxiety-related responses have been extensively documented. A more precise assessment is required of both the anxiolytic and anxiogenic potentials of phytocannabinoids, with an aim towards the development of the \u2018holy grail\u2019 in cannabis research, a medicinally-active formulation which may\u00a0assist in the treatment of anxiety\u00a0or mood disorders without eliciting any anxiogenic effects.Cannabis has been documented for use in alleviating anxiety. However, certain research has also shown that it can produce feelings of anxiety, panic, paranoia and psychosis. In humans, \u0394To systematically review studies assessing cannabinoid interventions (e.g. THC or CBD\u00a0or whole cannabis interventions) both in animals and humans, as well as recent epidemiological studies reporting on anxiolytic or anxiogenic effects from cannabis consumption.The articles selected for this review were identified up to January 2020 through searches in the electronic databases OVID MEDLINE, Cochrane Central Register of Controlled Trials, PubMed, and PsycINFO.Acute doses of CBD were found to reduce anxiety both in animals and humans, without having an anxiogenic effect at higher doses. Epidemiological studies tend to support an anxiolytic effect from the consumption of either\u00a0 CBD or\u00a0THC, as well as whole plant cannabis. Conversely, the available human clinical studies demonstrate a common anxiogenic response to THC\u00a0.Based on current data, cannabinoid therapies (containing primarily CBD) may provide a more suitable treatment for people with pre-existing anxiety or as a potential adjunctive role in managing anxiety or stress-related disorders. However, further research is needed to explore other cannabinoids and phytochemical constituents present in cannabis (e.g.\u00a0terpenes) as anxiolytic interventions. Future clinical trials involving patients with anxiety disorders are warranted due to the small number of available human studies. Cannabis spp. have over 500 phytochemicals documented, including well over 100 cannabinoids, which are unique to the genus , preMcLendon et al. 1976)  used pai  used paAs detailed in Table\u00a0Of the initial 1095 articles detected in our initial search, 26 full text articles were assessed for eligibility. Of these, 17 met our initial inclusion criteria and an additional five were identified through handsearching of references. Of these, eight were found to meet inclusion criteria and are included in this review.The anxiogenic properties of isolated THC has have been firmly established in humans and as demonstrated in Table\u00a0Evidence of THC\u2019s potential anxiolytic effects in humans, was first published in 2004. The study sample size consisted of 22 healthy individuals who had\u00a0previously used cannabis, but had never been diagnosed with a cannabis abuse disorder . In a 3-In a follow up US study, the same methodology was applied to people who were frequent users of cannabis . The resConverse to the anxiogenic effects of THC, CBD appears to have the opposite effect. In Bergamaschi et al. (2011) , particiAnother two studies utilised Spielberger\u2019s State-Trait Anxiety Inventory (STAI) to measure anxiety , 84. In Another similar study, used a differing assessment, the Subjective Drug Effects Questionnaire (SDEQ) . Ten freThe overall pattern of human clinical data supports consistent anxiogenic effects from THC, while CBD shows a consistent anxiolytic effect. In combination with CBD, the anxiogenic effect of THC has been shown to be decreased. However, further investigation is needed to categorically affirm this effect. Based on this data, it would imply that cannabis preparations higher in CBD and lower in THC cannabis would be most successful in treating anxiety. However, some survey data does not support this, with a preference for high THC cannabis being of greater interest to consumers for addressing affective symptoms. Further to this, only a very small percentage in surveys reported severe or intolerable side effects of using cannabis for their symptoms ; and in These discrepancies could be due to the fact that while a substantial number of patients cross-sectionally report using cannabis and related products to treat anxiety symptoms or disorders, it has not been firmly established whether this anxiety occurred before or as a result of the cannabis usage , 85. As In respect to the animal model research, there is strong evidence suggesting that an anxiolytic effect occurs after the administration of a small acute dose of CBD , 61, 63.As the present data indicates, no clear conclusion can be drawn from the preclinical studies of acute administration of THC. This could in part be due to the types of animal model being utilised. For example, Onaivi et al. 1990) [90 [19] fIn humans, research has also shown that the anxiogenic effects of THC are greater among infrequent or non-users relative to frequent users , and \ufeffhiDifferences in methodologies and limitations of data provided across the studies reviewed, further reduces our ability to draw strong conclusions. This includes the irregularities in doses given, where some studies used mg/kg and others mg only, and different administration methods being used. Most acute studies using THC employ an oral or inhalation route of administration . Oral adIn summary, the human clinical studies using acute THC consistently produced an anxiogenic effect, while studies using CBD and epidemiological studies of whole plant cannabis in anxiety disorders showed an anxiolytic effect. This is surprising as the doses of CBD that have been shown to have therapeutic effects are far lower than what is commonly found in cannabis plant matter, such as that which is being used by the majority of participants surveyed in the epidemiological studies . FurtherPharmacological treatment of anxiety relies on our understanding of the neurobiological interactions responsible . While t1\u00a0receptor activators which elicit pronounced psychotropic effects, something which has seen them recently revoked across most Western countries. THC on the other hand, is a partial agonist at the CB1 receptor [1\u00a0and CB2 receptors in the endocannabinoid system, is therefore a prime substance for investigation.Though several classes of synthetic CB receptor agonists have been developed, these alternatives are high-potency CBreceptor , 90, whireceptor . CannabiHowever, research has been limited given the controversial legal history surrounding cannabis. Policies are rapidly evolving, and access to cannabis and cannabinoid products is increasing worldwide , with itWith ongoing clinical research into the use of cannabis for anxiety, it is likely that optimised cannabinoid ratios of THC and CBD will eventually be better understood. Various software programs in use by the general public  may also be of value to researchers tackling this challenge. Apps such as these are able to track patient symptoms and collect data on the specific cannabis dosage form, cannabinoid ratios and particular cannabis products used for certain diseases, conditions or symptomatic relief.Cannabis genus, it is plausible that many phytochemicals could be contributing to anxiolytic activity, likely interacting with numerous receptor types. \ufeff Further, as previously mentioned, some research has shown that the adverse effects of THC, may be dose dependent and are potentially decreased by low doses of CBD [These two constituents may only be part of the story,\u00a0and continuing research into the pharmacological activity of the cannabinoids themselves may reveal that THC and CBD are not the only cannabinoids of clinical interest in anxiety. Notwithstanding the academic appetite for cannabinoid research, an often-overlooked phytochemical class, such as the terpenes/terpenoids, has also shown significant anxiolytic action. D-limonene and linalool, whilst not exclusively found in cannabis, have demonstrated anxiolytic activity; the former via the 5HT1A receptor , 94, 95.s of CBD . Hence, Lastly, each individual using cannabis is also unique, making the study of pharmacogenomics an important aspect of ongoing cannabis research . VariabiThe results of this review suggest that there is tentative support based on epidemiological surveys and clinical studies showing that whole cannabis and CBD may have a beneficial\u00a0role in anxiety disorders (for certain candidates in this population). In contrast, for isolated THC, acute human studies consistently show an anxiogenic effect. However, animal studies show that there may be potential for THC to be used as an anxiolytic, if given at the right dose for the patient, and that this may require gradual titration to ameliorate initial anxiogenic effects. Further to this, such an approach may be assisted via the co-administration of CBD, other cannabinoids or terpenes found in the cannabis plant which have yet to be studied substantially.Further\u00a0human studies are needed to establish consistency in the results, therapeutic thresholds, and dosage required for cannabinoid therapies to produce an anxiolytic effect in humans, and\u00a0further research on cannabinoids and terpenes may yield a more optimised anxiolytic formulation."}
+{"text": "The COVID-19 pandemic, caused by the spread of a novel coronavirus, has onceagain drawn the attention of the entire globe to the threat posed by emerginginfectious diseases (EIDs). In recent months, so too has monkeypox \u2013 adisease long known in West Africa \u2013 but only now in other parts of the world.What is the history of the concept of \u201cemerging infectious diseases\u201d?What are the critical approaches regarding the invention and circulation of thisconcept? And how have historians examined the appearance of \u201cnew\u201ddiseases before and after the emergence of the EID concept? This essay is intendedfirstly as a short, guided introduction to the recent history of this concept and tocritical approaches to it. Secondly, it offers a reflective enquiry, examining howhistorians have discussed disease emergence. We argue that humanities scholars havecritically examined the EID concept and invoked it to converse with scientists, orto comment on its contemporary realities. Simultaneously, they have demonstrated howthe burgeoning interest in EIDs highlights the need for research into theontological and epistemological factors that have posited \u201cnew\u201ddiseases and transformed them into epidemics and pandemics.2 the development of the EID concept is relatively short: itachieved mainstream traction only after the 1990s.3 In the previous two decades, many American and West Europeanscientists believed that the West was witnessing a linear epidemiologicaltransition. According to this assumption, infectious diseases had been pacifiedthanks to vaccination, sanitation, and antibiotics. Thus, deaths would be primarilycaused by age-related problems such as cancer, degenerative, or cardiovasculardiseases.4 This triumphantvision was partly engendered by the successful control of malaria and theeradication of smallpox. Another reason, according to Frank Snowden, was theprevailing notion of \u201cmicrobial fixity.\u201d According to this idea,future disease threats would come solely from illnesses that were already known toscientists. Coupled with this, was the idea that diseases would decline invirulence, through a process of natural selection, and would eventually come tocoexist with humans.5 The firstoutbreaks of Ebola in the 1970s, the reappearance of cholera in Latin America, andplague in India, and most importantly the emergence of HIV/AIDS delivered shocks tothis scientific consensus and demonstrated that infectious diseases were still athreat.6Although scientific debates on how new diseases appear can be traced to theearliest developments in biomedicine, in the years following the\u201cSpanish\u201d Flu Pandemic,7 Thepresentations and conclusions of the conference were published in 1993 under thetitle Emerging Viruses.8 One year later, the Institute of Medicine and National Academyof Sciences published the findings of its Committee on Emerging MicrobialThreats to Health.9Finally, in 1995, the journal Emerging Infectious Diseases, editedby the Centres for Disease Control, was launched.10 Together, these publications put together the concept ofEIDs, and transformed it into a public health tool in the US. 11To address the threat posed by such diseases, a conference was convened in1989 by epidemiologist Stephen S. Morse and molecular biologist Joshua Lederberg.This conference, called \u201cEmerging Viruses: The Evolution of Viruses and ViralDiseases,\u201d was sponsored by the National Institute of Allergy and InfectiousDiseases, the Fogarty International Center of the National Institutes of Health, andthe Rockefeller University.12 Morse listed a plethora of factors which produce emergingor reemerging diseases, including: \u201cecological changes, such as those due toagricultural or economic development or to anomalies in climate; human demographicchanges and behaviour; travel and commerce; technology and industry; microbialadaptation and change; and breakdown of public health systems.\u201d13In a foundational paper published in 1995, Morse defined EID as\u201cinfections that have newly appeared in a population, or have existed but arerapidly increasing in incidence or geographic range\u201d\u2014diseases likeHIV/AIDS. Morse categorized other infections like cholera in South America as\u201cre-emerging diseases,\u201d because they \u201cwere once decreasing butare now rapidly increasing again.\u201dThe Coming Plague(1994), and the Hollywood movie Outbreak (1995).14 It has since been deployed toencompass a myriad of diseases: from once \u201cdefeated\u201d resurgingdiseases such as tuberculosis in the USA,15 to new diseases like SARS or COVID-19, to pathogens, likeanthrax, that might be used by bioterrorists.16 EIDs also brought renewed attention to the need for rapidinternational responses to emergence events. Thus, the WHO developed strategies inthe 1990s to respond to outbreaks in as little as twenty-four hours.17 In 1997, the WHO established theGOARN , a network of 120 partners whichenabled the rapid global dissemination of information.18The concept gained increased traction in the late 1990s, with the help ofpopular media, such as Laurie Garrett's 19 Thethreatening nature of such diseases ushered in a new era of what Andrew Lakoff calls\u201cglobal health security.\u201d20 No longer was it a question whether new diseases would bedetected, but how best to predict and contain them.21 This framing of EIDs as global threats to the USsparked initial criticism of the concept as Americentric.22 However, the concept was also adapted andrecreated as it circulated around the world.23 For instance, various African scientists have describeddiseases like Ebola and Lassa fever as EIDS, though they are not a problem indistant and exotic places, but are threats to the local African population.24 The recent monkeypoxpandemic is perhaps an ideal example of both points: a largely neglected diseaseknown in West Africa since 1970, became an \u2018emerging\u2019 global concernafter its appearance in the UK in 2022.25 Simultaneously, its spread has provoked alarm in other parts ofthe African continent, such as South Africa, where it was described as\u201canother emergent virus.\u201d26Despite such lofty, global goals, EIDs were mainly framed as threats to theWest: problems \u201cout there\u201d that thanks to globalization could becomelocal problems. According to Nicholas King, emerging infectious disease researchgained both traction and funding by \u201creframing \u2018international'problems in language palatable to American interests,\u201d such as bordersecurity and terrorism.Aside from its successes as a concept in the field of public health, broaderintellectual questions posed by disease emergence have interested humanitiesscholars long before the 1990s. In what follows, we highlight firstly how scholarshave taken the EID concept as the object of their enquiry or employed it as acritical tool. Secondly, we examine how historians have tracked the emergence ofdiseases through synthesizing scientific knowledge and history. Finally, we discusssome of the approaches taken within the historiography of \u201cframed\u201ddiseases: the epistemological appearances of new diseases, or the transformation ofold scourges, by emphasizing changes in science and society.27 Grmek'scategories stressed biological processes but also highlighted how social andscientific changes could make visible phenomena that had until then been ignored.This is a point that is sometimes missed by scientists.28The first means by which scholars have engaged with the concept of EIDs is indefining it as their object of enquiry. In this section, we analyze the criticalexaminations given by historians, anthropologists, and literary critics. One earlyexample of the historical engagement with the EID concept is found in the work ofmedical historian Mirko Grmek. In a 1993 essay, Grmek proposed five conditionsaccording to which a disease could be considered as emerging in the present or inthe past.29 Jon Arrizabalaga has interpreted the profusion ofemerging diseases in the last few decades as proof of the risks brought bymodernity. He argues that many well-known emerging diseases were created by thepharmaceutical industry \u2013 bacteria resistant to antibiotics, among others\u2013 or by the development of agriculture. To him, the pessimistic view thatmodernity itself is a source of disease emergence has been proven by the ongoingCOVID-19 pandemic.30More recently, historians have used the concept of EIDs as a critical tool tointervene in current debates both in public health, and history itself. BrianDolan's work reveals that underlying governmental models of pandemicpreparedness were based on the assumption that the \u201cnext pandemic\u201dwould be a mutation of influenza. This assumption left governments unprepared, inearly 2020, to deal with a coronavirus pandemic.31 Indeed, both Joshua Lederberg and the virologistRobert Shope stressed that epidemics did not \u201cstrike societies randomly or inaccord with the caprices of angry gods,\u201d but instead reflected\u201crelationships that human beings establish with one another and with thenatural and built environments,\u201d spreading across \u201cfault lines createdby demography, poverty, environmental degradation, warfare, mass transportation, andsocietal neglect.\u201d32 ForWarwick Anderson, such analyses signal a recognition that the developed world had\u201cfinally read the ecological lesson\u201d provided by disease ecologists incolonial settings decades earlier.33 Pierre Olivier-M\u00e9thot and Bernardino Fantini'swork has complemented such arguments, emphasizing that EIDs drew attention to theimportance of disease ecology because they did not necessarily emerge from\u201cparticularly virulent germs,\u201d but rather from \u201csignificantecological changes within an ecosystem\u201d and through \u201ccultural andsocio-economic factors\u201d in human populations.34 Grmek, likewise, interpreted AIDS as \u201ctheprice we pay for having radically perturbed millenary ecologicalequilibria.\u201d35According to Mark Honigsbaum and Olivier-M\u00e9thot, histories of disease ecologyin conversation with histories of EIDs allow us both to interpret the past, and to\u201cilluminate current scientific debates around emerging infectious diseases,and the interaction between biological, economic, and cultural factors in currentpandemic emergencies.\u201d36While these historians have drawn upon the past to critique the present, afew others have demonstrated how the concept of EIDs is indebted to the past. Early-to mid-twentieth century disease ecological thinking developed in colonial settingswas often overlooked in twentieth century Western public health campaigns, but tookcentre stage after the emergence of HIV/AIDS and Ebola.37According to Nash, from the days of Richard Preston's 1994 sensationalisticnonfiction thriller, The Hot Zone, emerging diseases have oftenbeen attributed to environmental degradation.38 Yet despite this recognition, scientific analyses of the\u201crelevance of the environment to disease emergence\u201d have been treatedin a \u201cvery generic way.\u201d39 To address this problem, historians should write environmentalhistories which chart the \u201cspecific social, economic, and environmentalcontexts that have produced the conditions conducive to outbreaks of infectiousdisease.\u201d40An ecological perspective on disease can help us understand contemporaryproblems with EIDs. Some have even argued that EID's ecology should bebrought to the forefront of environmental history. According to Linda Nash, EIDsprovoke historians to investigate the relationships between environments and health.Emerging diseases, she argues, make \u201ccompelling political reasons for tellingthe history of disease as an environmental and social story, rather than simply as amedical and personal one.\u201d41In 2003, Anne Hardy's pioneering article, \u201cAnimals, Disease, and Man:Making Connections\u201d drew attention to the relationship between animal andhuman health since the mid nineteenth century, both in Europe and in Europeancolonies. One particularly important point posed by Hardy was that human intimacieswith animals have historically been identified with outbreaks of disease, oftenthrough the lens of colonial racial and class prejudices.42 Subsequent studies of SARS and influenza, such asthose penned by Lyell Fearnley and Fr\u00e9d\u00e9ric Keck, have continued todraw attention to how scientists have interpreted human relationships with birds,and how these have led to the positing of new diseases and their transformation intoepidemics and pandemics.43Similarly, Karen Brown's study of resurgent rabies in South Africademonstrates the need to analyze human/animal relations within environmental,medical, and social history.44 Thecoronavirus pandemic has also underlined the importance of treating animals asagents of historical change on account of their epidemiological significance.Writing on the history of pangolins, Sujit Sivasundaram has argued that historically\u201czoonotic transfer occurs where relations between humans and animals havebeen unstable or where they are entering a new phase of contact.\u201d45 Given climate change and theemergence of zoonotic disease, he suggests that scholars write \u201cmulti-speciesand even trans-species history that is about the assembly of various life forms andthings generative of historical change.\u201d46Histories of human and animal relations may be of particular importance inaddressing Nash's concerns. Given the importance of animal to humanspillover, human and animal relations have emerged as an important, if relativelysmall, subfield of EID studies.47 Some have pointed out that despite issues with the framingof the concept, it has been \u201cimmensely successful\u201d in gathering\u201cinternational resources to fuel the development of new collaborativeresearch\u2026on infectious diseases\u201d that threaten \u201cboth the Northand the South.\u201d48 Lorna Weirand Eric Mykhalovskiy consider the EID a concept that has been successful inaltering \u201cunderstandings of infectious disease in ways that mobilizedwidespread public health concern over new microbial threats and drove significantinstitutional change in the scope and form of global public health surveillance andfield response,\u201d49While historical analyses of the emergence of the EID concept and itssociopolitical realities are relatively scarce, scholars of the medical humanitieshave examined this concept in more detail.50 This hascompounded preexisting medical funding problems within the South.51 Critical medical anthropologistsand specialists in global health in the 1990s and 2000s have likewise drawnattention to geopolitical inequalities and EIDs. For them, emerging diseases werenot simply an unavoidable consequence of globalization as their clinical definitionimplies. Rather, global capitalism played a critical role in the emergence ofdiseases such as HIV/AIDS. Since the 1990s, anthropologists have produced studies onthe \u201cstructural violence\u201d that generates vulnerability to diseasessuch as \u201cpoverty and economic exploitation, gender power, sexual oppression,racism and social exclusion.\u201d52 To name one prominent example, in 1996, anthropologist andphysician Paul Farmer critiqued the definition of EIDs, claiming that it paidinsufficient attention to social inequalities, and treated emergence as a somewhatrandom biological event. To move beyond this problematic approach, Farmer called fora critical epistemology of emerging infectious diseases, and argued that historical,sociological, and anthropological expertise needed to be mobilized to study howinequalities of class, race, gender, and sexuality, facilitated diseaseemergence.53 RichardParker, a medical anthropologist who was instrumental in the founding of theGlobal Public Health journal, also blamed inequalities in theIMF's programs for the breakdown of public health systems in many parts ofthe world.54 According toM\u00e9thot and Fantini, these problems persist to this day. Despite theindebtedness of the EID concept to disease ecological research, many experts stillfocus \u201con purely epidemiological and clinical aspects of emergingevents\u201d at the cost of ecological and sociological factors.55 Perhaps it would be better,speculate M\u00e9thot and Fantini, to refer to EIDs instead as emerging epidemics,because of \u201cenhanced diffusion of pre-existing microorganisms thanks toseveral factors such as migrations, wars, travels, and trade.\u201d56On the other hand, the successes of this concept have simultaneously exposedits shortcomings. According to Weir and Mykhalovskiy, because the idea of diseaseemergence has become so popular, global-health funding bodies have prioritizedattention to EIDs over endemic diseases that continue to plague parts of Africa,Asia, and Latin America.57 Under this cosmology, medicalprofessionals view nature as \u201calready one step ahead\u201d: they areperpetually \u201cstruggling to keep up with nature's relentlessevolution.\u201d58Expertise is reduced to ignorance: nature produces new viruses before they are evennamed, and once they have been given a name, they continue to mutate adinfinitum.59 This\u201ccosmology of mutant strains\u201d constitutes a challenge to biopower andmedical triumphalism: since pathogens are always ahead of scientists, the\u201cbold dreams of control and eradication\u201d must be replaced with\u201cmodest schemes of response and relief.\u201d60Foucault-influenced anthropologists have likewise devoted attention to theconcept of EIDs to explain the cosmological underpinnings of Western medicalgovernance, and the challenges these diseases pose to biopolitics. Carlo Caduff hasargued that at \u201cthe heart of the concept of emerging viruses is a particulartemporality\u201d that \u201cnaturalizes the idea of a permanent threat\u201d.This \u201ccosmology of mutant strains,\u201d which emerged in the early 1980s,draws attention to the \u201cever-evolving nature of viruses.\u201d61 To Wald, these stories follow a \u201cformulaicplot\u201d, derived from past narratives of epidemics and nineteenth-centurydetective novels. It commonly starts with a mysterious outbreak in an isolated placein Africa or South America. Traveling globalized networks, this faraway menacebegins threatening American society. A team of doctors is then called to solve themystery in a race against time. The heroes eventually succeed: the outbreak iscontained and the disease eradicated, at least in the US.62 To Wald, these accounts are a tool \u201cformaking the invisible appear\u201d: they reveal obscure biological entities and thehidden realities of globalization.63 Ironically, if AIDS gave a new life to the outbreak narrative,its trajectory does not follow the formulaic plot, because it was never totallycontained.64 One questionsif the ongoing COVID-19 and monkeypox pandemics will have a similar fate.Along with history and anthropology, literary criticism has also providedinsights into the birth of the EID concept and its sustained popularity. Accordingto Priscilla Wald, North American publics have primarily been informed about therisks of EIDs through books, newspapers, and movies.In short, critical studies of the historical, anthropological, andrhetorical aspects of the EID concept have emphasized not only biological factors,but also the social, political, and economic realities that explain the emergence ofthe concept, its diffusion and popularization. Such studies have, however, beenlargely anchored in North American and Western European experiences, paying littleattention on how the concept circulated, was adapted, and reconstructed in placessuch as Latin-America, Africa, and South or East Asia, commonly seen by Westernersas the sources of EIDs.EmergingInfections Diseases journal, in 1995, \u201cinfectious diseasesemerging throughout history have included some of the most feared plagues of thepast.\u201d65 This claimshowcases, firstly, how history was mobilized by a generation of doctors andscientists to legitimize the EID concept.66 Secondly, it suggests to historians the need to track theontological emergence of diseases.As Stephen Morse remarked in his opening paper of the 67 Asdiscussed below, this approach to disease emergence has been applied by historiansin two different directions: firstly, with an emphasis on the spread of diseasesamong human communities with little or no immunity, and secondly, with an emphasison spillover of the disease from animals to humans.Indeed, as remarked by historian Monica Green, every disease, from smallpoxto Ebola, was at one point emerging. Historians interested in the ontologicalemergence of a disease must examine not only the factors that led to the originalspillover event, but also those that transformed them into epidemics and pandemics,and what allowed these to persist. Advances in ancient DNA sequencing and medicine,as well as historical sciences are of use here: they allow historians to pinpointthe deep histories of diseases and to challenge established interpretations basedonly on historical writings.68 According tohis definition, pathocenosis is the ensemble of diseases that existed among apopulation in a given time and space, which tend to stay in equilibrium. However,this equilibrium could be broken by external factors. Two examples are the arrivalof black rats to Europe, which led to the Black Death, and the spread oftuberculosis, which led to the retreat of leprosy, since TB provides cross immunityagainst leprosy's Hansen bacillus.69The concept of pathocenosis, as developed by Grmek in 1969, is an importanttheorical milestone for a reflection on the ontological emergence ofdiseases.Virgin SoilEpidemics, and by William McNeill in his book Plagues andPeoples,70 bothpublished in 1976. Synthesizing historical analyses with the most advancedscientific knowledge at his disposal, Crosby stressed how the low-immunity ofindigenous Americans to infectious diseases, such as smallpox, brought by Europeancolonizers was a major reason for their demographic collapse and for Europeanconquest.71McNeill's work complemented this argument and argued that the biggestdemographic disasters of humanity, such as the Black Death, the fall of the Aztecand Inca Empires, and the nineteenth century cholera waves, were the result ofenvironmental imbalances caused by the arrival of new diseases to places withnon-immunized populations. For McNeill, imperial expansion and commerce were toblame for the rupture of environmental equilibrium because they connected differentenvironments and allowed people, animals, parasites, and micro-organisms tocirculate between them. For instance, the Black Death was directly connected withthe expansion of the Mongol Empire, because it bridged plague reservoirs among wildrodents in inner Asia with black rats in Europe.72 It is worth noting that McNeill was the only historian toparticipate in the 1989 conference that coined the EID concept.73 As he remarked in the introductionto the third edition of Plagues and Peoples in 1998, the spread ofHIV/AIDS around the world confirmed his arguments of how disease emergence andpandemics are connected with processes of globalization.74As remarked by M\u00e9thot, Grmek's insights anticipated some ofthe main arguments developed by Alfred Crosby in his essay Civilizations, which won the 2019 Grand Prix du Romande l'Acad\u00e9mie Fran\u00e7aise. Binet imagines a worldwhere pre-Columbian populations met with Vikings adrift in the Caribbean by the year1000, from whom they pick-up iron, horses, and smallpox, which became, one couldsay, part of the American pathocenosis. Therefore, in this imagined past, nodemographic annihilation occurs with Columbus' arrival. Smallpox does notarrive in the Americas after 1492 because in his story it had arrived five hundredyears earlier. Indigenous Americans managed then to defeat Columbus, seize hiscaravels, and invade and ultimately conquer Western Europe.75Crosby and McNeil's interpretation of the conquest of America have,however, been met with criticism, because they offer a fatalistic conclusion thatindigenous American populations were fated to disappear once their pathocenoticequilibrium was broken and smallpox and other infectious disease emerged in theAmericas, no matter if by violence or by peaceful contact. Their interpretation ishowever still potent in popular culture, as evidenced by Laurent Binet'snovel Contagion. By using modernknowledge on the epidemiology of diseases and archival research, Harrison emphasizedthe major role played by trade in the circulation of pathogens and vectors, such asrats and fleas, and showed how measures to combat them were as much driven bypolitics as they were by the threat posed by the disease itself.76 Myron Echenberg, likewise, hasshown how the sea-traffic of goods, people, and also of rats and fleas between\u201cplague ports\u201d spread plague between 1894 and 1901 from its endemicareas in China to new locations across the globe, including South America,Australia, South Africa and the United States. Echenberg also reflected on why thedisease was better controlled in some of these ports than in others. To him, theports that focused on rat destruction ,were more successful in fighting plague emergence thanthe ports of the BritishEmpire, which relied more on disinfection.77 In short, Harrison and Echenberg convincingly showed howpolitical economy and growing global capitalism were important factors in the spreadof plague. Nonetheless, the price paid in these two accounts was sometimes toovershadow science's own historicity. For instance, albeit rats and fleaswere framed in both books as main villains for the emergence of plague as a globalscourge, these animals were not considered as such before the late nineteenthcentury. Even after that time, vigorous debates took place around the world on therole of rats and fleas in spreading plague and on the feasibility of killingthem.78Among historians, McNeill's argument of how the flow of people andgoods between continents led to the emergence of diseases has more recently beenexamined in Mark Harrison's 79 Jacques P\u00e9pin, a formermedical officer in the rural Democratic Republic of Congo, likewise showed howcolonial medical interventions, \u201crequiring the massive use of re-usablesyringes and needles\u201d helped turn HIV into an epidemic. 80 Most recently, WilliamSchneider's edited volume on the Histories of HIVs has alsodemonstrated the potential for syntheses of history and biology in clarifying theemergence of diseases. He sees the history of HIV as key in understanding the futureof the COVID-19 pandemic.81HIV/AIDS, like COVID-19, has provided a painful lesson in how \u201canimal virusescan transition to become epidemic or pandemic human viruses.\u201d The fact thatgenetic evidence suggests that \u201cnew HIVs emerged multiple times\u2026meansthey cannot be seen as random or chance events\u201d but instead \u201ca humanmade disaster.\u201d 82John Iliffe's synthesis of the biological and social history ofHIV/AIDS in Africa provides another good example of this ontological approach inaction, this time focused on social factors and spillover events that transformedAIDS into a pandemic. Mobilizing up-to-date genetic studies, Iliffe situated theorigins of HIV/AIDS within multiple zoonotic spillovers from simians in the1930s-1950s, which were transformed into a silent epidemic through colonialupheavals, the migration of labor, and the inequalities of capitalism.These ontological approaches have thus synthesized modern science andhistorical methods to pinpoint the appearances of pathogens in periods in whichthese were not yet mentioned in archives. Moreover, modern epidemiology has allowedhistorians to reconstruct the probable causes of their spread and understand whysome places were more affected than others. Simultaneously, archival work hasprovided pointers as to the social, economic, and political factors that transformeddiseases into epidemics and pandemics. Fascinating as such work is, it has oftenneglected the epistemology of disease emergence.83 To examine thebroader historiography on disease emergence as a social and scientific phenomenon,one might focus on plague and on trypanosomiasis, which is a family of diseasesprevalent in sub-Saharan Africa, South Asia, and South America. These two diseasesprovide an excellent illustration of how social and intellectual changes can(re)invent new diseases.Infectious diseases that were emerging, either because they passed fromanimal to human hosts or started spreading within new communities, were oftenperceived differently by different peoples. Perhaps one of the richest ways in whichhistorians have engaged with the idea of emerging diseases has been to understandthe emergence, or appearance, of diseases in the past as an epistemological andsocial process. In other words, how and why a disease was considered a new entityfollowed upon transformations in science or transformations in cultural and socialattitudes. A good introduction to theoretical and methodological issues in thesematters can be found in Ludwik Fleck's studies on the epistemological andcultural roots of the changing nature of syphilis. Also useful is CharlesRosenberg's concept of framing disease, which stresses the scientific andsocial forces behind new categorizations of diseases, along with how the biologicalcharacteristics of each disease served as limiting factors to socialframes.84 Therefore,Cunningham concluded, Yersin and Kitasato transformedplague's identity, because from 1894 onwards, a plague outbreak would bedeclared only after its bacillus was identified in a laboratory.Plague was reframed by microbiology between the late nineteenth and earlytwentieth centuries, in the first years of what was later called the \u201cThirdPlague Pandemic.\u201d Historian Andrew Cunningham examined the transformation ofplague by studying the identification of its bacillus during the Hong Kong outbreakof 1894, which started the pandemic. Cunningham argued that the French-Swissmicrobiologist Alexandre Yersin and the Japanese microbiologist Shibasaburo Kitasato\u201chad taken into their laboratories a disease whose identity was constitutedby symptoms; they had emerged with a disease whose identity was constituted by itscausal agent.\u201d85More recently, numerous historians have further developedCunningham's argument. They have shown, for instance, that reframing plagueas a microbiological entity involved more than just the identification of thebacillus. It involved the production of sera and vaccines against it, the proposingof theories about its spread, and the invention of new sanitary measures to controlthe bacillus and its animal \u201creservoirs.\u201d Moreover, reframing plaguewas more of a global process than assumed by Cunningham. As we have shown inprevious works, the microbiological reframing of plague involved a network of actorsand laboratories in places such as Brazil, India, and South Africa. Scientific,diplomatic, and imperial forces connected them and knowledge about plague circulatedbetween them and was transformed by them.Trypanosoma brucei, a blood parasite transmitted betweenwildlife, livestock, and humans by the tsetse fly. Both diseases, the former inlivestock and the latter in humans, cause progressive, gradual emaciation, alongwith neurological disturbances, followed by death. African pastoralists in numerousareas had long contended with these symptoms in livestock, and often attributed themto a disease caused by the bite of the tsetse fly. The term \u201cnagana\u201ditself is an Anglicization of unakane, an isiZulu word that hasbeen translated as \u201ccontinual pestering action,\u201d referring to theinfuriating bite of the fly.86Despite trypanosomiasis's long history under a plethora of different names,numerous historians have argued that it became a problem of colonial governance inthe late nineteenth to early twentieth century, during a period in which imperialpowers were attempting to develop lands they had violently seized from indigenouspeople.87 Africantrypanosomiasis was first identified bacteriologically in the 1890s, in Zululand,and named \u201ctsetse fly disease or nagana\u201d by Scottish-Australianmicrobiologist David Bruce and his research partner and wife Mary Bruce. Later, itwas studied across Africa by a host of scientists. European colonists feared thatthis disease would become endemic across much of the continent and even spread intoother parts of their empires.88If old diseases were reframed by microbiology, this new science, with thehelp of tropical medicine and indigenous medical experts, was also responsible forconstructing \u201cnew\u201d diseases, such as trypanosomiasis in Africa and inSouth America. African trypanosomiasis is a term that encompasses two diseases\u2013 nagana and sleeping sickness \u2013 caused by three subspecies of89 AsMaryinez Lyon notes, in her seminal study of trypanosomiasis in the Belgian Congo,it was a \u201ccolonial disease\u201d: the political, ecological, and labordisruptions caused by colonial governance created ecological conditions conducive tothe emergence of devastating trypanosomiasis epidemics.90 In response, historians have documented howEuropean states attempted to prevent the disease from spreading and how Africansresponded to such efforts. This involved the attempted wholesale extermination ofwildlife in agricultural areas, trapping and killing tsetse flies in enormousnumbers, slashing and burning vegetation in which the flies nested, the forcedresettlement of Africans, and ultimately, the dusting and soaking of parts of thecontinent in DDT.91 In short,although sleeping sickness symptoms were long known in the continent, the disease\u201cemerged\u201d and was framed as a particular concern to colonial powers inthe twentieth century due to the scramble for Africa. Colonists transformed it intoa persistent series of devastating epidemics, that killed hundreds of thousands ofAfricans. Changes in human and animal relations were critical here. In Zululand,South Africa, for example, colonial game-protection laws coupled with the suspicionof Zulu ideas that large game was pathogenic, produced significant outbreaks ofnagana, and extended its endemic area.92Scholars such as ecologist John Ford have argued that European activitieswere at times responsible for the emergence and persistence oftrypanosomiasis.Trypanosoma cruzi. Following thisidentification, Chagas located the parasite's vector, known asbarbeiro in Brazil, a common blood-sucking insect present inthe hinterland of South America that nests in holes in wooden houses. Chagasidentified, as a symptom of the disease, lesions in the heart, caused by theparasite, as well as related symptoms, such as fatigue and hyperthyroidism. The pathfollowed by Chagas was unusual, not just because he started from the parasite ratherthan from the symptoms, but because he made a threefold discovery. Thus, as has beenargued, the identification of this new disease demonstrated the Braziliancontributions to the field of microbiology and tropical medicine at the turn of thetwentieth century.93 It alsofostered connections between Brazilian scholars and their counterparts in scientificcenters, such as Germany and France. This carved out a place for Brazil in theglobal field of tropical medicine.94The emergence of American trypanosomiasis, or Chagas disease, as a newnosological entity in the early twentieth century has intriguing counterpoints toits African counterpart. In 1909, while conducting work on malaria in Lassance, avillage in the Brazilian state of Minas Gerais, Carlos Chagas, of the InstitutoOswaldo Cruz in Rio de Janeiro, made a breakthrough discovery. In the blood of a fewpeople that he imagined might be infected with malaria, he found a differentparasite, which he christened 95 Indeed,the 1910s-1920s were a period of intense discussion about the problems of Brazilianagriculture, coupled with the commemorations of the first independence centenary in1922. In this context, Chagas disease was used by medical, political, intellectualelites as \u201cthe symbol of a \u2018sick\u2019 and \u2018backwards\u2019country, devastated by endemics that disable the rural population, but its discoverywas also seen \u201cas the herald of [a new] science\u201d.96 Therefore, a strong association wasformed among the Brazilian elites, which framed Chagas disease as \u201ca diseaseof Brazil.\u201d97 Its presencein the territory was understood as an obstacle to the modernization of the country,which could be overcome through vector-destruction, sanitation, andhome-improvement. Thus, the emergence of Chagas disease as a new pathology wasintertwined with nation building and modernization projects in Brazil. Therefore,the history of Chagas disease in Brazil, like sleeping sickness in Africa, shows howdisease emergence can be a matter of the scientific tools available to a society,and a result of political and social forces.98However, as shown by the historian Simone Kropf, \u201cChagas disease wasrepresented as a medico-scientific entity and at the same time as a socialquestion.\u201dIn conclusion, we would like to emphasize some challenges that humanitiesscholars face in studying the concept of emerging infectious diseases. Firstly,there are several gaps in the historiography of the EID concept, such as itscirculation outside the USA and Western Europe, how scientific and politicalcommunities in the rest of the world have utilized it, and the evolution of theconcept. There is considerable scope for future research into EIDs in global andnon-western frameworks, particularly for historians working with multiple archivesacross various regions.Secondly, it is worth reflecting on the utility of using techniques such asphylogenetics in tracing the ontological emergence of diseases in the past. On theone hand, they are vulnerable to charges of presentism and problematic in that theytake modern science as a series of timeless facts that can be projected onto eras inwhich such knowledge never existed. On the other hand, when cognizant of thislimitation, scientific methods, such as genetic studies, can provide valuable toolsfor writing histories of the emergence and persistence of the pathogens themselves.They allow historians to demonstrate the impacts of these nonhumans on multispeciesdemographies and geographies in periods where written records are scarce. Likewise,they can provide counterpoints to the prejudices of colonial archives.99 Moreover, they are commonly blamedfor the emergence of new diseases,100 notably avian influenza,101 HIV,102and more recently, COVID-19. Yet, even in studies that explicitly focus on zoonotictransfers of disease, animal subjects often recede into the background and are castas tools for human experimentation, vectors of infection, or passive victims ofdisease control policies, rather than as active beings that have shaped theemergence of diseases, and the knowledge produced about them. In taking animalsseriously as historical subjects, one rich topic of enquiry would be to reverse thestandard story of emerging infectious diseases and examine EIDs as a problem of wildanimals. In addition to examining how contact with wild animals leads to spilloversof disease into human populations, we might also examine how diseases of humans ordomestic animals spill into wild animal populations. Here, scholars could examinethe attempts of scientists to protect mountain gorillas from human diseases, Africanwild dogs from diseases of domestic dogs, or even zoo and farm animals fromCOVID-19. The relationship between the global movements of animals and diseaseemergence constitutes another potential area of study. Along with the better-knownstories of accidental introductions of rats and insects around the globe, historianscould examine the epidemiological consequences of the trade in horses and cattle orwild animals for zoos, the migration of wild animals beyond international borders,or of sea creatures across the oceans.Thirdly, although animals have not escaped the attention of historians ofEIDs, there is still much more work to be done on animals as historical subjects inanalyses of emerging or re-emergent diseases. Animals have played an important rolein the microbiological reframing of old diseases, such as plague.A final challenge is how to transcend national/local boundaries when aanalyzing the epistemological emergence of diseases. While some work has been donehere regarding the history of plague, it is still lacking in other areas such as thestudy of trypanosomiasis. Despite the richness of the works mentioned above, theyoften regard the framing of trypanosomiasis as new entities as the result ofprocesses sealed within national/imperial borders. However, Chagas disease, nagana,and sleeping sickness emerged almost in parallel, perhaps because both regions wereexposed to similar global political, economic, and scientific processes. Thus,future works could highlight the connections between African and South Americancontexts, along with other places where trypanosomiases were studied, such as Indiaor the Philippines. In short, global histories of disease emergence as anepistemological and social process constitute a promising field that can bedeveloped beyond the cases discussed in this essay.These are just a few suggestions that can be adapted to the study of thenumerous infectious diseases not mentioned here. We have not aimed to be exhaustive,as this might have encompassed much of the history of infectious disease. Moreover,we did not examine the ontological and epistemological emergences of non-infectiousdiseases, nor how these diseases were examined or neglected by the EID concept.Despite these gaps, we hope our essay has provided a guide to works that haveengaged critically with the EID concept, along with studies that, in connection withthe burgeoning interest in this concept, have sought to understand the history ofthe ontological and epistemological emergence of new infectious diseases acrossspace and time."}
+{"text": "The paper discusses one of the key features in the multiaxial fatigue strength evaluation\u2014the procedure in which the stress path is analyzed to provide relevant measures of parameters required by multiaxial criteria. The selection of this procedure affects the complete equivalent stress derived for any multiaxial load combinations. Three major concepts\u2014the minimum circumscribed circle, minimum circumscribed ellipse, and moment of inertia methods\u2014are described. Analytical solutions of their evaluation for multiaxial stress state with components described by harmonic functions are provided. The concepts are validated on available experimental data when included into six different multiaxial fatigue strength criteria. The results show that the moment of inertia results in too conservative results. Differences between both methods of circumscribed entities are much smaller. There are indications however that the minimum circumscribed ellipse solution works better for critical plane criteria and for the criteria based on stress tensor transformation into the Ilyushin deviatoric space. On the other hand, the minimum circumscribed ellipse solution tends to shift integral criteria to the conservative side. Methods of multiaxial fatigue analysis should cope in some kind with one key problem: If the load history of individual stress tensor components is not proportional, the load path created by the end point of the stress vector gets multidimensional\u2014it is not a straight line anymore. Most of practically used or developed fatigue estimation methods focus on detecting individual closed cycles in the load history, and on describing them by the most basic characteristics . If the object of such description\u2014the load path within the detected cycle\u2014gets more complicated than the line is, the decision which geometric features could serve well for such a definition is not simple.There are several types of multiaxial fatigue criteria, and the way the load path is treated differs for some of them . The One of the early attempts to summarize this problem, to describe it, and to propose the optimum solution can be found in the paper by Papadopoulos et al. . In addiOne of the key parts of the analysis presented in  is the dThe calculation speed for the MCC problem is not the decisive issue, which is why some other authors proposed to adopt another strategy. Li et al. in  object tThe principle of the maximum prismatic hull (MPH) was therefore defined . As withAll these concepts are based on the assumption that the minimum circumscribed circle method is wrong in the way the shear stress path signal is treated, because it does not differentiate sufficiently well between proportional loading and non-proportional loading. For non-proportional loading, the discussed modified definitions of the shear stress amplitude tend to result in higher values than the MCC variant would result. Such a claim should be supported by appropriate experimental data, but most of the validations done until now are inconclusive. Papuga et al.  explain Within those sensitivity analyses, all multiaxial fatigue strength computations were performed while using the MCC concept. The number of other concepts invented to replace this method is large, so the analysis in this paper is extended to cover also those methods. There are not many such comparisons based on real experimental data and mostly they have been already cited here. They are often analyzed on relatively small data sets, into which also proportional load cases are included. The computational outputs for the proportional load cases do not differ for any of the stress path description methods, so these attempts to highlight the differences are weakened by the decision to include such cases. This is e.g., the case of Scalet  or MamiyThis paper provides an analytical formulation of the load path trajectory either when projected onto a specific plane or if transformed into the Ilyushin deviatoric space. The formulations extend the study by Papadopoulos et al. , who derThese derivations for the first time prove that such projections are invariably ellipses, which can be analytically described. Thanks to this geometric feature, only three concepts of stress path processing  provide different estimates, while the other mentioned concepts (MPH or convex hull) result in the output identical with MCE. The paper further focuses on validating the output of these concepts, when integrated into six different multiaxial criteria of various types. Data items from the FatLim database of experiments  are primx axial, t tangential and r radial directions and the shear stress in the a amplitude and m mean parts. The use of the harmonic signal is effective thanks to a simpler control of the machine drive, which would get complicated if any sharp peaks are induced in the signal. The complete stress tensor components look then:The paper focuses on the most often practically used multiaxial load configurations\u2014a combination of acting axial, torsion and pressurizing load channels, which induce the stress tensor components:see also . The othP.Various phase shifts The two Euler angles The normal stress on N:The mean value of the normal stress is derived from all time-independent addends of After several mathematical steps, the normal stress amplitude can be written as:l and k are lying on n is the normal line. The unit vectors l a k can be written as:To describe the course of the shear stress The shear stress on If the matrix multiplication is performed, we get to:f, g, p and q are:After some further mathematical manipulation, this record is obtained:Equations  and 14)14) correThe centre of either the MCE or the MCC is therefore computed from these values as:Semi-axes of the ellipse stress path are:The smallest circumscribed circle has its radius equal to the longer semi-axis of the ellipse:The smallest circumscribed ellipse is identical with the stress path shape, and thus the shear stress amplitude can be derived from semi-axes written above in Equation  as theirTo illustrate the output stress path, a quite complex load case TAK10 was chosen, see The shear stress path description by Equations  and 14)14) is alThe perimeter of the elliptic shear stress path is defined:The polar moment of inertia:Typically, the multiaxial criteria based on the load path analysis in the Ilyushin deviatoric space are based on combining the hydrostatic stress with the second invariant of the stress deviator. Hydrostatic stress equals:Its mean value is set from the time-independent terms:The amplitude of hydrostatic stress is obtained when the mean value is subtracted from the total:The stress deviator is derived from the stress tensor and from hydrostatic stress:The second key stress parameter in multiaxial criteria processed in the Ilyushin deviatoric space\u2014the square root of the second invariant of the stress deviator\u2014is obtained from:If the transformed coordinates are used:This effectively means that only 3-parametric space is sufficient to describe the load path given by Equation  in its eThe formulas in Equations \u2013(47) desThe length of both ellipse semi-axes is defined:The minimum circumscribed ball enveloping the elliptic load path in the Equation  into theThe fact that, for the given composition of load channels and harmonic loading  defThe mean value The amplitude of the stress tensor deviator is again defined:The stress path described in Equation  forms thA more practical comparison of the output of individual stress path description methods is thus desirable. Papuga et al. designed in  a speciaThe same procedure was also processed here. Whereas  focused ion, see ):(70)\u03c3eqFIN, see  or 21]))\u03c3eq,a. Ta, b, c or d represent material parameters derived from basic uniaxial load conditions. The parameters a and b are usually obtained from fatigue strengths at fully reversed axial loading and at fully reversed torsion loading. To derive parameters c and d, e.g., repeated axial loading and repeated torsion tests are required in addition to provide the necessary fatigue strengths.In all Equations \u201375), th, th75), Another critical plane concept\u2014the critical plane selected as the plane of maximum shear stress range\u2014was evaluated on the example of the Matake criterion :(72)The request to find the maximum shear stress range first to locate the critical plane can be solved in different ways. It can be defined as the longest shear stress path projection onto a specific direction on the evaluated plane. However, it can also be the The Papuga PIN criterion  :(73)\u03c3eq, method,  or 21]))(73)\u03c3eq, If the chosen stress combination processed in the multiaxial fatigue strength criterion gives the equivalent stress amplitude equal to the given fatigue strength in fully reversed axial loading In the sensitivity study presented here, the acting stress levels at each load combination described by criteria  also shoFor most criteria, the result trends for the MCE and MCC concepts do not differ much for cases with sufficiently big shear stresses  obtained. The change in trends for both of them is therefore very similar. MCE shifts some of the results to more conservative estimates compared with MCC. The analysis of data in the FatLim database shows that this change concerns the cases of extra-ductile materials  for which the load ratio with prevalent axial stress over shear stress was applied. This is complies with the previous observations based on The explanation as to why the cases with prevalent axial stress over shear stress are more affected can be manifested very well on graphs in There are 17 cases in the FatLim database in which the Matake criterion gets to higher If the change related to the switch from the MCC concept to the MCE concept was observable only for some items from those OOP cases studied with the critical plane criteria above, the change by integral methods (PIN and Liu & Zenner) affects most of the evaluated load cases. This observation has a logical reason hidden again in maps presented in a material parameters over the whole range of a parameter causes the One point has not been discussed until now. How it could occur for some stress combination that the integral method can lead to lower In the case of the Crossland criterion, the change when switching from MCC to MCE is very profound. The well-visible non-conservativeness provided when MCC is used is avoided when MCE is applied. For many cases  the Though the output seems quite conclusive, open questions remain. The MCE concept proved itself to be the best choice for the critical plane criteria and also for the Crossland method processed in the Ilyushin deviatoric space. The validation, however, bases this decision only on the stress path forming the ellipse. For such stress path, the identical output will be provided by various MCE approaches (see ), by theThe paper analyzed the problem of the stress path evaluation in the multiaxial fatigue strength criteria. It started with the analysis of the most complex multiaxial non-contact load case, which is the pressurized tube loaded additionally in the axial direction and in torsion. Even if all these load signals are involved with whichever phase shifts between their harmonic functions, but with identical frequencies, the ellipse will be the output stress path either on any evaluated plane or in the Ilyushin deviatoric space . Thanks to that, most methods invented replacing the Minimum Circumscribed Circle (MCC) result for such load cases in the shear stress amplitude identical to the Minimum Circumscribed Ellipse (MCE). Parameters of this ellipse are analytically described in this paper. In addition to those two methods, the concept of Moment of Inertia (MOI) is also analyzed in this paper.The MOI concept results in a too conservative output because it exaggerates the phase shift effect too much.For the tested critical plane criteria (PCRN or by Findley), which define the critical plane by the maximum damage reached, the use of MCE improves the prediction quality in comparison with using MCC.For the Matake critical plane criterion using the maximum shear stress range to select the critical plane, MCE largely improves the quality of estimates compared with MCC. The use of MCE brings the prediction results close to the output of the Findley critical plane method of the maximum damage type.The use of the MCE concept for the Liu and Zenner method and for the Crossland criterion results in making both methods insensitive to the phase shift effect.The validated integral criteria seem to lose some of the prediction quality when using the MCE concept. The use of MCC should be preferred.The Crossland method benefits from the switch from MCC and MCE, as the latter solution mends its too non-conservative output for out-of-phase loading discussed e.g., in . This chThese conclusions from various analyses provided in the paper can be made:"}
+{"text": "Retrospective analysis of the utility of adjuvant radiation (RT) or chemoradiation (CRT) and identify prognostic features for patients with high\u2010risk head and neck salivary gland cancers.From 1/1997 to 12/2017, 108 patients underwent surgery, and RT (n = 50) or CRT (n = 58) for positive lymph node(s), extracapsular extension, perineural invasion, lymphovascular space invasion, positive/close margin, and/or grade 3 disease. Outcomes were estimated with the Kaplan\u2010Meier method. Significant predictors identified through regression analyses were incorporated into multivariable regression (MVA). Toxicities were compared using chi\u2010square.P\u2009<\u2009.01). On MVA, stage 3 or 4 disease predicted worse outcomes including overall survival . Increasing number of RFs predicted worse disease\u2010free survival, distant metastasis\u2010free survival, and overall survival , but not locoregional control (P = .54). So, adjuvant CRT may have provided comparable locoregional control for patients with more adverse features, but the CRT did not translate into improved distant control. There was no difference in acute or late grade 3+ toxicities, or parenteral nutrition , respectively.The median follow\u2010up was 52\u2009months (range: 3\u2010226). The number of risk factors (RFs) between RT and CRT groups were: 0 to 1 (44% vs 7%), 2 to 3 (48% vs 41%), or 4 to 6 (8% vs 52%), respectively (Adjuvant CRT provides adequate locoregional control in patients with more adverse RFs. The absolute number of RFs serves prognostic significance and should be considered in future prospective trials. Treatment is based on retrospective data given the relatively small number of affected patients and paucity of randomized trials to guide treatment decisions. Surgery is considered the mainstay of treatment. However, several high\u2010risk features have been identified such as: perineural involvement (PNI), lymphovascular space invasion (LVSI), positive/close (<2\u2009mm) margin, grade 3 disease, extracapsular extension (ECE), or positive lymph nodes (+LN).Adjuvant radiation has been used with success to improve locoregional control in high\u2010risk disease,While large prospective randomized trials continue to accrue  or report (NCT01488838), there remain limited comparisons between adjuvant chemoradiation (CRT) to radiation therapy (RT) alone in patients with high\u2010risk salivary gland cancer. Moreover, the prognostic value of different high\u2010risk features, or a combination of which remains poorly understood. While we wait for this large prospective evidence, we investigate the utility of adjuvant CRT compared to RT alone and identified prognostic features from our 20\u2010year institutional experience.22.1Patients with histologically confirmed high\u2010risk head and neck salivary gland carcinomas were retrospectively identified through the electronic medical record. All patients signed the institutional review board (IRB17\u20100489) protocol consent. Those identified included those that received definitive surgical resection followed by adjuvant RT alone or CRT. Patients who previously received RT to the head and neck were excluded from data collection. Pre\u2010treatment evaluation included assessing pathologic specimens, and operative findings. The risk factors (RFs) included: +LN, ECE, PNI, LVSI, positive/close margin, and grade 3 disease. These RFs were summed to reach the number of factors (ranging from 0 to 6).Patients had to have a confirmation of localized disease through head and neck computerized tomography (CT) and/or magnetic resonance imaging (MRI) and at least CT chest to rule out metastatic disease. Disease stages were coded according to the AJCC 7th edition.2.22/d\u2009\u00d7\u20095\u2009days), hydroxyurea (500\u2009mg PO twice\u2010daily [BID]), with/without 5\u2009days of paclitaxel (100\u2009mg/m2 on day 1), and either 1.8 to 2 Gy daily (QD) RT or 1.5 Gy BID RT from days 1 to 5. The cycle of CRT is then followed by a 9\u2010day break, after which the next cycle of CRT begins. The regimen used varied over the years as treatment protocols evolved in an effort to improve efficacy. While earlier patients were treated with FH and QD fraction RT, more recent patients received TFH and either QD or BID RT.The adjuvant RT cohort received 2 Gy daily fractions. The adjuvant CRT utilized most frequently comes from a previously reported practice of 4 to 6 alternating \u201ccycles\u201d of weekly TFH or FH with RT.2.3Follow\u2010up data was gathered from the notes in the electronic medical record, which included: radiation oncology, otolaryngology, medical oncology, telephone encounters, and event notes. Head and neck acute and late toxicities were assessed with the use of RTOG toxicity scales. Acute toxicities were defined as those occurring within 90\u2009days of treatment, whereas late toxicities were defined as those occurring after 90\u2009days of treatment. Due to the lack of laboratory reports in early versions of the electronic medical record, hematologic toxicities were not recorded. The use of artificial feeding tubes or total parenteral nutrition (TPN) was documented when the use of these methods for supplemental nutrition occurred as a result of RT or CRT.Locoregional control was defined from the time of surgery until evidence of recurrence within the primary tumor bed, adjacent to the tumor bed, or within the regional lymphatics. Distant metastasis\u2010free survival was defined from the time of surgery until evidence of recurrence outside of the head and neck region. Disease\u2010free survival was defined from the time of surgery until recurrence or death. Overall survival was defined from the time of surgery until death from all causes.2.4The survival outcomes were estimated using the Kaplan\u2010Meier method and compared using the log\u2010rank test. Multivariable Cox proportional hazards model (MVA) was performed for any significant factors on univariate analysis. From this initial model, backward stepwise regression was then performed to remove factors that were not significant, and to find the most parsimonious model that minimized Akaike Information Criteria and Bayesian Information Criterion. RTOG toxicity tables were used to score the maximum acute and late head and neck toxicities and compared using the chi\u2010square test.3From January 1997 to December 2017, 108 patients were treated with surgery, followed by adjuvant RT alone (n = 50) or CRT (n = 58) for high\u2010risk pathologic features. The median follow\u2010up duration was 52\u2009months (range: 3\u2010226). There were a total of 11 different histologies, with the most common being: adenoid cystic carcinoma (n = 30), followed by mucoepidermoid (n = 20), and salivary duct adenocarcinoma (n = 18). The most salivary gland location was the parotid (n = 73). Twenty\u2010two percent were in minor salivary gland locations. The rest of patient characteristics are shown in Table 3.1P = .15) in the adjuvant RT and CRT groups, respectively. In the CRT group, 50% received BID treatments. Both 3D conformal radiation therapies (6.9% vs 16%) and intensity\u2010modulated radiotherapy (IMRT)  techniques were utilized in the RT alone and CRT groups, respectively.The median dose of RT was 66\u2009Gy , followed by FH (17%), then weekly cisplatin (2%), and unknown/other (2%). Only three patients received induction carboplatin/paclitaxel followed by CRT.3.2P = .05, Figure P = .02, Figure P = .02, Figure P\u2009<\u2009.01, Figure P = .57), disease\u2010free survival (P = .37), distant metastasis\u2010free survival (P = .80), or overall survival (P = .42).The unadjusted 5\u2010year locoregional control estimates were . Figure P = .02).On MVA, locoregional control was only associated with stage 3 or 4 disease compared to stage 1 or 2 .In addition to stage, the total number of RFs was associated with disease\u2010free survival , and 95% of patients with ECE (P\u2009<\u2009.01) were treated with adjuvant CRT, thus comparison with the RT alone group was not feasible. Figures P = .16) and ECE (P\u2009<\u2009.01), respectively. The MVA and model outputs are shown in Table Of note, imbalances in therapies existed: 87% of patients with lymph node positivity (3.3P = .98). There was one acute grade 5 toxicity in the RT alone group, which occurred in a patient who succumbed to a ventricular arrhythmia due to the inability to take amiodarone secondary to acute mucositis. Furthermore, there were no differences in RTOG head and neck late grade 3+ toxicities , or rates of G\u2010tube/TPN use . When comparing those that received 3D conformal RT to IMRT, there were no differences in acute toxicities , late toxicities , or G\u2010tube/TPN rates . A tabular form of the toxicities is shown in Table Overall, there were no differences in RTOG head and neck acute grade 3+ toxicities between the adjuvant RT alone group (36.0%) compared to the adjuvant CRT group  in the CRT group compared with the RT alone group Table , this stP\u2009<\u2009.01).In addition to previous reports for nodal stage,The central implication of this mismatch is that the use of CRT may have compensated for high\u2010risk features in the treatment group, resulting in comparable locoregional control. Thus, the use of adjuvant CRT may be considered for patients with four or more high\u2010risk features to provide comparable locoregional control. Importantly, while 5\u2010year locoregional control may be comparable  have been explained.Conceptualization: Benjamin OnderdonkData Curation: Benjamin OnderdonkFormal Analysis: Benjamin OnderdonkInvestigation: Benjamin Onderdonk, Michael GwedeMethodology: Benjamin Onderdonk, Daniel HarafSupervision: Daniel Haraf, Everett VokesVisualization: Benjamin OnderdonkWriting \u2010 Original Draft Preparation: Benjamin OnderdonkWriting \u2010 Review and Editing: Benjamin Onderdonk, Everett Vokes, Daniel Haraf, Elizabeth Blair, Nishant Agrawal, Michael GwedeAll authors have read and approved the final version of the manuscript.Daniel Haraf had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.Figure S1 Locoregional Control by TreatmentKaplan\u2010Meier locoregional control (LRC) estimates by adjuvant radiation (RT) alone compared to adjuvant chemoradiation (CRT).Click here for additional data file.Figure S2 Disease\u2010free Survival by TreatmentKaplan\u2010Meier disease\u2010free survival (DFS) estimates by adjuvant radiation (RT) alone compared to adjuvant chemoradiation (CRT).Click here for additional data file.Figure S3 Distant Metastasis\u2010Free Survival by TreatmentKaplan\u2010Meier distant metastasis\u2010free survival (DMFS) estimates by adjuvant radiation (RT) alone compared to adjuvant chemoradiation (CRT).Click here for additional data file.Figure S4 Overall Survival by TreatmentKaplan\u2010Meier overall survival (OS) estimates by adjuvant radiation (RT) compared to adjuvant chemoradiation (CRT).Click here for additional data file.Figure S5 Locoregional Control by StageKaplan\u2010Meier locoregional control (LRC) estimates by AJCC 7th edition pathologic stage.Click here for additional data file.Figure S6 Overall Survival by Lymph Node PositivityKaplan\u2010Meier overall survival (OS) estimates by lymph node (LN) positivity.Click here for additional data file.Figure S7 Overall Survival by Extracapsular ExtensionKaplan\u2010Meier overall survival (OS) estimates by extracapsular extension (ECE) positivity.Click here for additional data file."}
+{"text": "Bleeding from the proximal suture line after replacement of the aortic root can be very difficult to control. Such bleeding is an important predictor of morbidity and mortality. Here, we detail a reliable method that is simple to perform and effectively prevents surgical bleeding. We adopted a technique of overlapping the pledgeted interrupted U-stitches of the proximal sutures on the prosthetic graft, which can eliminate the bleeding from the proximal suture line. The description of aortic root replacement using a composite graft by Bentall and De BonoAll surgical approaches were conducted through a median sternotomy on cardiopulmonary bypass (CPB) via right atrial drainage and cannulation of the femoral or right axillary artery for arterial return. We instituted moderate hypothermia and infused cold cardioplegic solution from either the antegrade (directly into the coronary ostia) or retrograde (into the coronary sinus) direction, or both.After reaching cardioplegic arrest, the aortic root aneurysm is excised, leaving only the two coronary buttons and approximately 5\u2009mm of aortic wall attached to the aortic annulus. The abnormal aortic valve is also excised and the appropriate sized graft and valve selected.Assistants make the composite graft in the operating room. The composite graft must have a short skirt that is 3 to 4\u2009mm in length for sewing the proximal margin. After sewing the valve to the graft, fibrin-sealant spray is administered to the outside of the suture line of the composite graft.Interrupted 2\u20130 polyester pledgeted U-stitches are then started on the annulus in an everting mattress fashion, with the pledgets lying on the outside of the annulus. Overlapping sutures are never made directly on the annulus to avoid the needle piercing the previous suture. Piercing the previous suture induces shear force on the annulus which may result in dissection of the annulus. All interrupted mattress sutures on the annulus are inserted in this fashion.The composite graft is then brought to the operative field and the annular sutures passed through the sewing skirt of the composite graft. An overlap technique is used on the graft, placing the first needle of the subsequent mattress suture behind the second thread of the previous suture in the graft, overlapping by approximately 1 to 2\u2009mm and using slightly larger bites than usual . Care mWhen all sutures have been passed through the graft, the composite graft is lowered into the annulus and all sutures were tied and divided.The proximal suture line can then be tested for bleeding by filling the left ventricular cavity with blood using the vent catheter and manually squeezing the left ventricle gently into the clamped aortic graft.After confirming a watertight suture line, the left and right coronary buttons can be attached to the graft in that order. The left coronary button is attached to the graft by means of continuous 5\u20130 polypropylene sutures without Teflon felt pledgets. We then test the left coronary button suture line for bleeding by administering antegrade blood cardioplegia through the aortic graft.After confirming hemostasis of the left coronary ostium, we perform the distal anastomosis, followed by suturing of the right coronary button to the aortic graft. The distal anastomosis and the right coronary anastomosis are performed with a running 4\u20130 polypropylene suture and 5\u20130 polypropylene suture, respectively. Before removing the aortic clamp, we spray fibrin sealant onto all suture sites.After rewarming and deairing, the patients are weaned from CPB.From February 2003 to March 2015, we performed the Bentall procedure using this technique on 71 consecutive patients, 13 (18.3%) of whom were emergent cases , and 30 (42.3%) of whom required aortic arch replacement. None of these patients needed surgical reexploration. In-hospital death occurred in only one patient due to acute coronary syndrome.Bleeding at the level of the proximal anastomosis remains a problem with the Bentall procedure, especially when the bleeding originates from the posterior aortic circumference. That portion of the aorta is almost impossible to reach after completion of the procedure. Mobilizing the graft after left main coronary artery anastomosis is a high-risk maneuver.Recently, many modifications of the original technique have been proposed to prevent the problem. The \u201coverlap suture technique\u201d and \u201cimbricated suture technique\u201d have already been described in previous reports.However, our technique differs from these previous descriptions. Our technique has two important distinctions as follows: (1) the composite graft is created with a short skirt 3\u20134\u2009mm) and (2) the sutures overlap on the graft but not on the aortic annulus ,3. Th\u20134\u2009mm andIn conclusion, our surgical technique is simple to perform and provides an effective means to prevent surgical bleeding.Do you feel that adding the 3-mm skirt is important? Why is it not sufficient to pattern your sutures in the same way, but place them into the valve cuff of a conventional mechanical valved conduit?It is important because the valve cuff is so thick that overlapped suture might pierce the previous suture in the valve cuff. On the other hand, graft skirt is thin enough to make it easy to confirm the suture is not piercing the previous suture while overlapping.Do you use the same method for biological valves, as well as mechanical valves?Exactly, we use the same method for biological valves, as well as mechanical valves."}
+{"text": "Human biomonitoring of oxidative stress relies on urinary effect biomarkers such as 8-oxo-7,8-dihydro-2\u2032-deoxyguanosine (8-oxodG), and 8-iso-prostaglandin F2\u03b1 (8-isoprostane); however, their levels reported for similar populations are inconsistent in the scientific literature. One of the reasons is the multitude of analytical methods with varying degrees of selectivity used to quantify these biomarkers. Single-analyte methods are often used, requiring multiple injections that increase both time and cost. We developed a rapid ultra-high-performance liquid chromatography\u2013tandem mass spectrometry (UPLC-MS/MS) method to quantify both urinary biomarkers simultaneously. A reversed-phase column using a gradient consisting of 0.1% acetic acid in water and 0.1% acetic acid in methanol/acetonitrile (70:30) was used for separation. The MS detection was by positive (8-oxodG) and negative (8-isoprostane) ion-mode by multiple reaction monitoring. Very low limit of detection (<20 pg/mL), excellent linearity (R2 > 0.999), accuracy (near 100%), and precision (CV < 10%) both for intra-day and inter-day experiments were achieved, as well as high recovery rates (>91%). Matrix effects were observed but were compensated by using internal standards. Our newly developed method is applicable for biomonitoring studies as well as large epidemiological studies investigating the effect of oxidative damage, as it requires only minimal clean up using solid phase extraction. Oxidative stress is a major contributor to the pathophysiology of a variety of diseases . It repr2\u03b1 (8-isoprostane). 8-oxodG is one of the major compounds resulting from oxidative damage to DNA . F2. F22-iso in vivo . In clinoutcomes .Several analytical methods have been developed to quantify 8-oxodG and 8-isoprostane in different biological matrices, including blood, saliva, urine, and exhaled air condensate (EBC). Urine is the preferred matrix in biological monitoring because its collection involves a simple, non-invasive sampling method. Both biomarkers can be quantified by two principal analytical approaches: liquid (LC) or gas (GC) chromatography coupled with mass spectrometry (MS) or enzyme-linked immunosorbent assay (ELISA) ,19,20. L2Gua), 8-isoprostane, and N-acetyl-S-(tetrahydro-5-hydroxy-2-pentyl-3-furanyl)-L-cysteine (HNE-MA) with solid-phase extraction -8-oxodG (CAS Number 569649-11-2) was used as the internal standard (IS) and bought from Cambridge Isotope Laboratories, Inc. . 8-Isoprostane -9,11,15-trihydroxyprosta-5,13-dien-1-oic acid; \u226595%, CAS Number 27415-26-5) and its deuterated isomer (IS) 8-isoprostane-d4 -9,11,15-trihydroxyprosta-5,13-dien-1-oic-3,3,4,4-d4 acid; CAS Number 211105-40-7), were obtained from Cayman Chemical . LC-MS grade solvents, water, methanol, and acetonitrile, were obtained from Carlo Erba Reagents . LC-MS grade acetic acid was obtained from Honeywell . MilliQ water was produced in the laboratory with a water purification system (MilliQ Advantage) from Merck . Solid phase extraction (SPE) cartridges (Chromabond C18 ec SPE 500 mg 3 mL) were purchased from Macherey-Nagel .Three urine samples collected from healthy, consenting adults were aliquoted in tubes (8 mL) before storing at \u221220 \u00b0C. We chose to focus our study on volunteer hydration to investigate its consequences on matrix effects; thus, we chose urine samples by color and creatinine concentration. Indeed, even if urine is a relatively clean matrix, it contains many compounds that can interfere with the analysis . This isCalibration curves were prepared by spiking \u201cmedium urine\u201d with six different concentrations of 8-oxodG and 8-isoprostane and a constant concentration of IS . Water (400 \u00b5L), IS (100 \u00b5L), and 10% formic acid (100 \u00b5L) were added to form the SPE loading solution. SPE cartridges were first conditioned with methanol (2 mL) and water (2 mL). Urine samples were loaded onto the SPE, washed with water (2 mL) and then 5% methanol (2 mL), dried with air , and eluted with methanol (3 mL). The extract was filtered (0.45 \u00b5m), evaporated under a nitrogen flow with a Pierce Reacti-Therm III evaporator , and reconstituted in the injection solvent (500 \u00b5L 0.1% acetic acid in water). Calibration standards and QC were treated identically to the samples. Analysis of 8-oxodG and 8-isoprostane was performed using a UPLC  coupled with a triple-stage quadrupole mass spectrometer  equipped with a heated electrospray ionization source (ESI) operated in positive ion mode for 8-oxodG and in negative ion mode for 8-isoprostane.2-isoprostane isomers\u2019 separation was not achieved with a mobile phase of 100% methanol [The compounds were separated using a C18 column . The column temperature was maintained at 30 \u00b0C. The mobile phase consisted of: eluent A composed of 0.1% acetic acid in water, and eluent B of 0.1% acetic acid in methanol/acetonitrile . The solvent gradient program was: t = 0 min: 0% B, t = 1.1 min: 55% B, t = 12 min: 65% B, t = 12.5 min: 90% B, t = 14.5 min: 90% B, t = 15.5 min: 0% B, t = 22 min: 0% B, at a flow rate of 250 \u00b5L/min. Using methanol and acetonitrile mixture as the mobile phase (B) was based on previous work from Prasain et al. (2013) reporting that Fmethanol .Multi-reaction monitoring (MRM) transitions and ESI parameters can be found in ClinicalTrials.gov Identifiers: NCT03589989) approved by the Ethics committees of Bern, Geneva, and Lausanne (Project-ID: 2017-02332), Switzerland. The study was conducted in accordance with the ethical principles of the World Medical Association Declaration of Helsinki and the International Committee on Harmonization for Good Clinical Practice and Swiss law. All participants provided a written informed consent and the following information: age, gender, and anthropometric data .For method application, urine samples were collected from an on-going randomized controlled trial on smoking cessation: \u201cEfficacy, Safety, and Toxicology of Electronic Nicotine Delivery Systems as an aid for smoking cessation: the ESTxENDS multicenter randomized controlled trial\u201d  and had donated their first-void urine sample (first morning urine sample). We validated the smoking abstinence by assessing total urinary nicotine equivalent (<2 nmol/mg creatinine). The urine samples were first stored at 4 \u00b0C (for 1 to 7 days), and urine aliquots were then stored at \u221220 \u00b0C until analysis.Fifty-six participants provided first-void urine samples for the quantification of 8-oxodG and 8-isoprostane. Mean age of the participants was 43.5 years old with a BMI mean of 26. Twenty-six participants were women (46%) and 30 participants were men (54%). Participant demographics are described in Urinary concentrations of 8-oxodG and 8-isoprostane were adjusted with creatinine concentration to account for the hydration status of the participants and allow inter-individual comparison. There is an acceptable correlation between creatinine corrected spot-urine and 24 h urine ,31,32,33N-glucoronide, nicotine-N-glucoronide, and trans-3-hydroxycotinine-O-glucoronide). In this study, only four metabolites were included (TNE 4) as it was sufficient for smoking status verification. Nicotine, cotinine, trans-3\u2032-hydroxycotinine, and norcotinine were analyzed at the Unit of Forensic Toxicology and Chemistry, University Center of Legal Medicine  by LC-MS/MS with a routine method based on an application note of Thermo Fisher Scientific . TNE was calculated as TNE = (nicotine/162.23 + cotinine/176.22 + trans-3\u2032-hydroxycotinine/192.22 + norcotinine/162.19)/creatinine, expressed in nmol/mg creatinine).Total nicotine equivalent (TNE) is considered as the gold standard biomarker of daily nicotine intake . In mostMethod validation parameters were calculated based on the peak areas that were integrated by the UPLC-MS/MS software. Description of these parameters can be found in v/v; B) at a flow rate of 250 \u00b5L/min. Retention times were 4.7 min for 8-oxodG and 10.2 min for 8-isoprostane (After the SPE on C18 cartridge (optimization description in prostane . InternaESI mode interface was operated in positive ion mode for the first segment of the run (0.5\u20138 min) and in negative ion mode for the second segment (8.5\u201314 min) to optimize the detection of both analytes. Ion source parameters, as well as m/z transitions for the multiple reaction monitoring, were determined by infusion of aqueous standard of 8-oxodG (5 \u00b5g/mL) and 8-isoprostane (5 \u00b5g/mL). Mass transitions, collision energy, and RF lens are shown in 2-isoprostane isomers in urine (MRM transitions for 8-isoprostane showed the probable presence of other Fin urine . SeparatWe tested a lower concentration of acetic acid in the mobile phase (0.01%), but it did not increase 8-isoprostane signal and decreased 8-oxodG signal. Use of formic acid (0.1%) reduced the signals of both analytes.2 > 0.999. Slopes of the calibration curves were similar for urine and water: 2% \u00b1 7% for 8-oxodG and 3% \u00b1 6% for 8-isoprostane.We determined LODs at 10 pg/mL for 8-oxodG and 20 pg/mL for 8-isoprostane (S/N \u2265 3) and the LOQs at 30 pg/mL for 8-oxodG and 50 pg/mL for 8-isoprostane (S/N \u2265 10 and coefficient of variation <20%) in aqueous solution. In urine, our lowest calibration standard was 0.5 ng/mL for 8-oxodG and 0.1 ng/mL for 8-isoprostane. These concentrations were low enough to quantify these biomarkers in participants\u2019 urine samples and were in accordance with previous published methods. Therefore, calibration curves were constructed with six levels from 0.5 to 20 ng/mL for 8-oxodG and 0.1 to 5 ng/mL for 8-isoprostane in urine. Linear regression with 1/x weighting was performed on analyte/IS peak area ratio versus standard concentrations. Linearity of the working ranges was observed with a regression coefficient of RIntra-day precision and accuracy were determined by analyzing three replicates of two urine samples spiked at three concentrations: 0.5, 1, and 10 ng/mL for 8-oxodG and 0.1, 0.2, and 0.5 ng/mL for 8-isoprostane. Intra-day accuracy ranged from 92% to 114% with a coefficient of variation lower than 5.7% for 8-oxodG, and from 97% to 114% with a coefficient of variation lower than 7% for 8-isoprostane. Injections were performed for three days to determine the inter-day precision and accuracy. The inter-day accuracy for 8-oxodG ranged from 92% to 103% with a coefficient of variation lower than 10%, and from 97% to 114% with a coefficient of variation lower than 8.1% for 8-isoprostane. Accuracy and precision details are shown in During the method development, extraction recoveries were calculated for each concentration used in the calibration curve. We observed stable extraction recoveries. The extraction recovery and the matrix effects for the concentrations corresponding to the highest calibration curve levels, 20 ng/mL for 8-oxodG and 5 ng/mL for 8-isoprostane, are presented. To determine extraction recovery, three replicates in urine spiked before SPE with 8-oxodG and 8-isoprostane and three replicates in urine spiked after SPE with the same solution were analyzed. Extraction recovery was 97% for 8-oxodG and 91% for 8-isoprostane. To determine absolute matrix effects, three replicates in water spiked with 8-oxodG and 8-isoprostane (without SPE) were compared to three replicates in urine spiked after SPE with the same solution. Matrix effects were found to be urine-dependent, and we observed matrix effects up to 20% for 8-oxodG and 70% for 8-isoprostane for \u201cmedium urine\u201d (100% corresponds to no matrix effects). Variation of the analyte to IS ratio was lower than 4% indicating that the observed signal reduction was compensated by using a stable isotopic internal standard.Matrix effects were also observed for \u201clight urine\u201d (67% for 8-oxo-dG and 83% for 8-isoprostane) and \u201cdark urine\u201d (4% for 8-oxodG and 25% for 8-isoprostane), estimated by the IS variation. A simple dilution by a factor of two of the \u201cdark urine\u201d reduced matrix effects to 19% for 8-oxodG and 58% for 8-isoprostane  over a 6-month period. 8-oxodG concentration was 8\u20139% higher after 6 months for both low (0.65 ng/mL) and high QC (3.42 ng/mL). The variation of the concentration over the whole period (65 injections) was less than 5% for both. 8-isoprostane concentration was 15% lower after 6 months for low QC (0.09 ng/mL) and 7% higher for high QC (0.46 ng/mL). The variation of the concentration over the whole period (65 injections) was 13% and 7% for low and high QC, respectively.Stability of the analytes in processed urine at room temperature was also monitored by the QC (low and high). QCs were injected three times in an injection sequence , seven hours apart. The average of the intra-sequence variation of 8-oxodG was 1.42% and 1.34% for low and high QC, respectively. The average of the intra-sequence variation of 8-isoprostane was 8.9% and 6.1% for low and high QC, respectively. There was no tendency for signals to increase or decrease between the 1st and the 3rd injection , meaning that the analytes were stable in processed urine during this period.Oxidative stress biomarkers\u2019 concentrations were determined in 56 morning urine samples obtained from the participants. The two analytes were quantified in all samples. After creatinine correction, the median of 8-oxodG concentration was 4.04 ng/mg creatinine (1st quartile\u20133rd quartile: 3.42\u20135.37 ng/mg creatinine) and the median of 8-isoprostane concentration was 0.23 ng/mg creatinine (1st quartile\u20133rd quartile: 0.14\u20130.28 ng/mg creatinine). Details are shown in We successfully optimized the simultaneous quantification of urinary 8-oxodG and 8-isoprostane by LC-MS/MS and efficiently applied the method to 56 urine samples from participants . The creatinine-adjusted concentrations ranges of 8-oxodG and 8-isoprostane were in agreement with the reference values of the population ,37. ThisMatrix effects are commonly observed in analysis of biological fluids and can obscure the signal in an otherwise selective and sensitive LC-MS method. The matrix effects\u2019 mechanisms are not fully understood, but they involve co-elution of matrix components that induce a loss of response  or an increase of response . As all urine samples have different compositions, Matuzewski et al. (2003) recommended performing method validation in five different sources instead of a single one . We hypothesized that \u201cdark urine\u201d samples  cause greater matrix effects than \u201clight urine\u201d samples . We selected urine samples according to the aspect (color) and urinary creatinine concentration. The latter is dependent on hydration status, as an increased amount of water in urine will lower the creatinine concentration. We demonstrated that matrix effects were proportional to urinary creatinine concentrations. This finding is of primary importance, because even if the matrix effect is compensated by the use of internal standards, the sensitivity of the method is decreased due to signal suppression . Furthea value at pH ~5) [We planned initially to include malondialdehyde (MDA), with 8-oxodG and 8-isoprostane, in the method. Simultaneous analysis of different biomarkers presents many advantages such as saving time and money. It can be challenging if the analytes have different physicochemical properties. This is the case for 8-oxodG, 8-isoprostane, and MDA. 8-oxodG is composed of a purine and is a polar molecule due to the presence of polar functional groups . Moreover, it is uncharged under low and neutral pH and forms anions and dianions at higher pH values  ,40. 8-ist pH ~5) . MDA is t pH ~5) .We used dinitrophenylhydrazine (DNPH) solution (5 mM) in water/acetonitrile/acetic acid (6.5:1:2.5) for the derivatization as described by Martinez and Kannan (2018) . HoweverWe were able to quantify low concentrations of oxidative stress biomarkers (0.5 ng/mL for 8-oxodG and 0.1 ng/mL for 8-isoprostane) in 56 morning urine samples from non-smoking healthy participants. Matrix effects were observed during the analysis of urine samples and their magnitude was directly linked to the urinary creatinine concentration, a measure of hydration level of the individual. This also meant that the sample clean-up was not complete as matrix components induce MS signal suppression effects. Solid-phase extraction with a reversed-phase cartridge was chosen because it retained both analytes well. However, 8-oxodG is more polar than 8-isoprostane and eluted with low percentage of methanol (10%). Therefore, the SPE washing step could not be optimized further to remove more matrix components without losing 8-oxodG. Both 8-oxodG and 8-isoprostane are negatively charged at high pH. Nevertheless, 8-isoprostane was not recovered during the elution step for the two anionic SPE cartridges we tested. Due to the different physicochemical properties of the two analytes, the reversed-phase was a good compromise.2-isoprostane isomers can complicate the 8-isoprostane quantification due to possible co-elution [2-isoprostane isomers but this was not explored further.The various existing F-elution . As we oThe obtained concentration medians for 8-oxodG (4.04 ng/mg creatinine) and 8-isoprostane (0.23 ng/mg creatinine) in our participants were comparable to the values in healthy adults reported in two systematic reviews by Graille et al.  ,37. For Oxidative stress biomarkers and inflammation markers have been analyzed together in several studies. Helmersson et al. (2004) and Tatsch et al. (2015) showed respectively that type 2 diabetes led to chronic inflammation followed by oxidative damage and that patients with higher 8-oxodG concentrations had higher degrees of inflammation and higher insulin resistance ,45. AlteOf these nine studies, only one used LC-MS as an analytical technique and only two studies quantified both analytes with two separate analyses. The method we propose would provide a simultaneous quantification of 8-oxodG and 8-isoprostane. By addressing both analytes in one run, this method saves time and consequently money and can thus be used in larger epidemiological studies. This method can help in gaining a better understanding of the relationship between oxidative stress and inflammation, and to understand the underlying mechanisms as currently these biomarkers are not completely understood. We would also highlight that the measurement variability of our method is lower than the intra-individual variabilities for both 8-oxodG and 8-isoprostane, which renders them excellent in molecular epidemiological studies ,53.From a clinical perspective, 8-isoprostane and 8-oxodG are important oxidative stress biomarkers, which have a diagnostic and prognostic value and correlate with disease degree. Therefore, screening for 8-isoprostane and 8-oxodG in a fast and cost-effective way could help to identify people at risk and monitor the potential effect of interventions.Our concurrent analysis of urinary 8-oxodG and 8-isoprostane method is rapid, stable, and robust. We recommend using a stable isotopic internal standard to compensate for matrix effects. The matrix effect was related to creatinine content; consequently, we suggest diluting \u201cdark urine\u201d (high creatinine concentration) to reduce ion suppression effects and increase the loading volume of \u201clight urine\u201d (low creatinine concentration) to allow quantification. We successfully analyzed 56 urine samples from healthy non-smoking participants and were able to quantify background levels of oxidative stress biomarkers. Our method is suitable for large epidemiological or biomonitoring studies."}
+{"text": "Neuromorphic vision sensors have been extremely beneficial in developing energy-efficient intelligent systems for robotics and privacy-preserving security applications. There is a dire need for devices to mimic the retina\u2019s photoreceptors that encode the light illumination into a sequence of spikes to develop such sensors. Herein, we develop a hybrid perovskite-based flexible photoreceptor whose capacitance changes proportionally to the light intensity mimicking the retina\u2019s rod cells, paving the way for developing an efficient artificial retina network. The proposed device constitutes a hybrid nanocomposite of perovskites (methyl-ammonium lead bromide) and the ferroelectric terpolymer (polyvinylidene fluoride trifluoroethylene-chlorofluoroethylene). A metal-insulator-metal type capacitor with the prepared composite exhibits the unique and photosensitive capacitive behavior at various light intensities in the visible light spectrum. The proposed photoreceptor mimics the spectral sensitivity curve of human photopic vision. The hybrid nanocomposite is stable in ambient air for 129 weeks, with no observable degradation of the composite due to the encapsulation of hybrid perovskites in the hydrophobic polymer. The functionality of the proposed photoreceptor to recognize handwritten digits (MNIST) dataset using an unsupervised trained spiking neural network with 72.05% recognition accuracy is demonstrated. This demonstration proves the potential of the proposed sensor for neuromorphic vision applications. The illustration depicts capacitive photoreceptor (CPR) mimicking the rod cell and its functionality simulation. CPRs connected to the sensing circuit followed by a spiking neural network mimics the retina functionality. A change in paradigm from sensing to perception aided by machine learning and deep neural networks; revolutionizing perceptive intelligence such as computer vision and voice processing. Our human brain receives most of the information (80%) through the eyesight5. Various hierarchical perceptive processes take place in the eye to form the vision in the brain. This includes photoreceptors\u2019 photon reception and encoding the illumination information into varying spike frequencies of the cells through ganglion cells of the retina6. The photoreceptor cells, namely rod cells, and cone cells absorb the light in the retina. The rod cells\u2019 density in the retina is much higher than the cone cells, and they are responsible for light sensing in low light conditions7. Whereas the cone cells are responsible for light sensing in bright conditions. The typical schematic of the rod cell is shown in Fig. 8. Hence, artificial retina networks are faster and smarter than the conventional image processing devices10.The biomimetic microelectronic devices are indispensable for human-inspired robotics and neuromorphic computing applications4 demonstrated a hemispherical eye developed using silicon (Si) nano-membrane in the origami approach, and they have employed Si photodetectors as photoreceptors. Recently, Gu et al.3 demonstrated a perovskite-based hemispherical biomimetic eye for robotics and visual prosthesis applications. They have fabricated a perovskite nanowire array as photoreceptors and demonstrated image sensing using the eye. However, each pixel in these biomimetic eyes requires biasing that leads to high static power consumption since they are photodetectors and closer to the conventional image sensors. One of the popular biological vision cameras, called dynamic vision sensing (DVS) cameras, also employs photodiodes as photoreceptors11. The capacitors are usually used to mimic the cell membrane in CMOS-based electrical neurons9. Moreover, the capacitive neural networks, rather than the resistive/conductance-based approach14, featured better emulation of neural functionalities and low static power consumption15. Thus, there is a need for a tunable photoreceptor to develop artificial retina networks for efficient, perceptive intelligent applications.To construct the artificial retina network, it requires a tunable artificial neuron with functionalities of photoreceptors cells and ganglion cells. In other words, the membrane potential in photoreceptor neurons is affected by the light signals in addition to the electrical signals of other neurons. The traditional integrate-and-fire neuron model for data processing doesn\u2019t possess the functionality of photoreceptors. There are few inspiring works that perform image sensing mimicking the human eye; Zhang et al.17. These thin-films exhibited varying dielectric properties upon light illumination but not under visible wavelength. Such a phenomenon has been useful in developing photosensitive and photostrictive actuators18. There are also reports on visible light photo capacitors for the charge storage using dye-sensitized semiconducting nanoparticles19, phosphors20, photosensitive conjugated polymers21, and through a hybrid plasmonic effect of Ag nanowires22. However, hybrid perovskites24 attracted considerable attention because of their exceptional optoelectronic properties such as excellent light absorption, long carrier lifetime, low trap density, giant optical anisotropy25, and high carrier mobility28. Thanks to these properties, higher photocurrents, and higher quantum efficiencies were observed in perovskite-based optoelectronic devices, namely; solar cells32, photodetectors35, photo-transistors36, and devices for various applications like photo-sensing37, lasing39 and emission40. The hybrid perovskites are sensitive to moisture and oxygen, resulting in degradation of the device performance, which is the major hurdle for the commercialization of perovskite devices34. On the other hand, polyvinylidene fluoride (PVDF) based ferroelectric polymers have an electroactive phase and are widely used as a dielectric medium in various energy storage applications41, fractional-order capacitors43, optoelectronic devices44, and plastic solar cells45. Especially, a terpolymer of PVDF such as PVDF-TrFE-CFE exhibits a high dielectric constant , and low dielectric losses46, which can offer high capacitance and high dynamic range in the tunability of capacitors. Moreover, it is a relaxor ferroelectric polymer, high charging and discharging efficiencies are achievable for capacitive energy storage applications48. The choice of flexible substrates and the design of interconnects on flexible substrates for wearable electronics is crucial. One of the promising and commercially available substrates is Kapton (polyimide), and the design of interconnects on this substrate is also well known. Hence, Kapton substrate is the appropriate choice to explore flexible sensors and circuit architectures49. Moreover, it provides an opportunity to develop bio-compatible systems and implement telecommunication protocols50 between flexible sensors and flexible peripheral electronics.Furthermore, there are studies on the capacitors using lead zirconate titanate (PZT) thin films that are sensitive to UV illumination9. To fabricate CPRs with excellent light tunable properties, we require materials that are photosensitive and materials that have tendencies to tune the dielectric properties. In order to obtain the combination of exceptional optoelectronic and ferroelectric properties, we prepared a hybrid composite of methylammonium lead bromide perovskite (MAPbBr3) and the terpolymer polyvinylidene fluoride trifluoroethylene-chlorofluoroethylene (PVDF-TrFE-CFE). We demonstrated the fabrication and characterization of flexible CPR, which has a frequency-dependent capacitance within the range of 1\u2212100\u2009kHz. The hybrid perovskites as fillers in the ferroelectric polymer attribute to modulate the dielectric properties proportional to the intensity and wavelength of the incident light. The capacitive change with respect to the wavelength of the incident light mimics the spectral sensitivity curve of human photopic vision with the maximum response in the greenish-yellow regime. The photoresponse of these CPRs is reproducible with negligible hysteresis. Furthermore, the fabricated device is resistive to humidity and oxygen due to the encapsulation of the hybrid perovskites in the hydrophobic ferroelectric terpolymer (FP). To the best of our knowledge, we report the longest stability measurement of hybrid perovskites (~129 weeks) owing to their PVDF-TrFE-CFE encapsulation. The proposed device is modeled with an RC network and integrated with a novel low power spike oscillator to generate the spike train with a firing rate proportional to the incident light intensity and wavelength (color). Then, the functionality of the proposed CPR and sensing circuit is demonstrated through simulation to recognize the handwritten digits (MNIST) dataset using an unsupervised trained spiking neural network.Herein, we demonstrate the light intensity capacitive photoreceptor (CPR) that mimics the retina\u2019s rod cells. The capacitance of CPRs is dependent on visible light illumination and can lead to the development of the artificial retina by integrating with peripheral electronics3 nanocrystals embedded in the FP  was sandwiched between metallic electrodes Fig. , where t FP Fig. . The X-r54. The broad absorption of PFNC thin-film until 563\u2009nm in the visible regime is attributed to the MAPbBr3 nanocrystals in the composite. The PFNC composite exhibited strong photoluminescence (PL) due to the presence of MAPbBr3 nanocrystals in the ferroelectric polymer, as shown in PL spectra , green (~ \u03bbpeak\u2009=\u2009520\u2009nm), greenish-yellow (~ \u03bbpeak\u2009=\u2009560\u2009nm), yellowish-orange (~ \u03bbpeak\u2009=\u2009590\u2009nm), and red (~ \u03bbpeak\u2009=\u2009630\u2009nm). The beam profile and the intensities of these LEDs were homogenized using a custom-built setup, as shown in Fig. C\u03b1), which was estimated from the impedance and phase angle of the device . The C\u03b1 of the CPRs increased with the increasing light intensity, and frequency-independent behavior at each light condition is further evident in the capacitive response shown in Fig. The CPR with PFNC is very sensitive to light due to the presence of MAPbBr2 greenish-yellow light is overlapped with the response of devices illuminated to 247 \u03bcW/cm2 violet light, as depicted in Fig. 2, the change in C\u03b1 is negligible, where the absorbance of the composite is almost negligible. The observed electrical response is in-line with the discussed UV\u2212VIS and PL spectra of the PFNC composite. The capacitive response was measured under other LEDs, and Nyquist impedance plots are shown in Figs. 9, the capacitance of the CPR is found to be variable with light intensities. The trend in variation of Z and C\u03b1 indicates that there is an increase in the conductivity of the dielectric medium56 due to the photo-generated charge carriers  spectroscopy. The phase transitions, which are the indications of degradation, result in the peak broadening or shift59. It is evident that even after 100 weeks of aging, there is neither significant linewidth broadening nor peak PL wavelength shift in the PL spectra . This indicates that the composite performance is very stable, and there is no degradation of MAPbBr3 nanocrystals as they are encapsulated in the PVDF-TrFE-CFE polymer. Since PVDF polymers are more hydrophobic60, PFNC is more resistant to oxygen and moisture absorption. The absorbance spectra are also undisturbed, as seen in Fig. 62, the solar cell efficiency has been increased in perovskite solar cells with PVDF polymer as an additional layer due to the reduction in electron\u2212hole recombination. Hence, the PFNC composite can lead to exploring a wide variety of efficient and stable optoelectronic devices.Halide perovskites are highly sensitive to polar molecules such as water and oxygen, owing to their ionic nature. This results in the phase transition of the perovskites, which eventually leads to poor optical performancetra Fig.  of the dC\u03b1 at 10\u2009kHz for a long time, and the negligible deviation from the baseline was observed , B1 is the earliest batch, and B3 is the recent batch of fabricated devices. These devices\u2019 response was tested under homogenized conditions at various intervals to understand devices\u2019 air stability after the storage. Interestingly, the devices (batch-B1) are stable even after 129 weeks of storage in robust ambient conditions , and the response of all devices that were tested after several weeks is comparable to as-deposited devices Fig. . This sh65. The generation of non-equilibrium charge carriers upon light illumination is affecting the dielectric properties. To further understand the effect of photo-generated charge carriers, the CPR was characterized by varying applied AC voltage magnitude (Vac) under the dark and the greenish-yellow light conditions. As shown in the C\u03b1 response  to recognize handwritten digits. The pixels of the handwritten digits were encoded proportional to the light intensity, then converted to a spike train, as discussed in the previous subsection. This SNN is a single-layer network with 100 output neurons employing a winner-take-all (WTA) mechanism followed by a statistical output classifier. The network was trained with simplified spike-timing-dependent plasticity, STDP, with a leaky integrate-and-fire neuron model3NH3PbBr3) and ferroelectric polymer (PVDF-TrFE-CFE). The purity and morphology of the perovskite nanocrystals are undisturbed in the nanocomposite and exhibited excellent light absorption properties. The characterized CPR\u2019s capacitance is tunable and reproducible with the light intensity and frequency independent in the 1\u2212100\u2009kHz regime at each light intensity. The mechanism of the CPR was studied through optoelectric characterizations. The stable and non-degradable performance of metal halide perovskite nanocrystals-based devices is observed for 129-weeks. The highly sensitive CPR mimics the rod cells of the retina. The developed CPR is interfaced with simple circuitry and a simple neural network to demonstrate its functionality as a biomimetic retina. The demonstrated system has massive potential in developing artificial retina networks and biomimetic eyes for perceptive intelligence applications. In addition, such CPRs find their unique place in developing photosensitive actuators for robotic applications, and these can also be explored as optoelectronic devices for optical communications.We demonstrated the facile fabrication of tunable and flexible capacitive photoreceptors using a hybrid nanocomposite of metal halide perovskite (CH3NH3PbBr3 (MAPbBr3) solution39 was prepared from the commercial methyl ammonium bromide (CH3NH3Br) and lead bromide (PbBr2), the mixture was added to the N, N Dimethylformamide (DMF) solvent and ultra-sonicated for 24\u2009h. In the second step, 100\u2009mg of commercial ferroelectric terpolymer (FP) PVDF-TrFE-CFE was stirred constantly in 1\u2009ml of DMF solvent for 24\u2009h. The final PFNC solution was prepared by mixing 1\u2009ml of the FP solution with the 1\u2009ml of MAPbBr3 solution. The mixture was stirred constantly for 24\u2009h to obtain a homogenous PFNC solution. We have employed an aluminum-coated Kapton Polyimide sheet that serves as the flexible substrates for flexible CPR. 300 \u03bcL of PFNC solution was drop-casted on the square-shaped (2 \u00d7 2 cm2) Al coated polyimide sheet pasted on the carrier substrate. The substrate was heated under vacuum for 3\u2009h at 90\u2009\u00b0C for solvent evaporation. 120\u2009nm thick transparent indium tin oxide (ITO) was RF sputtered on the PFNC thin-film using a shadow mask for the top electrode (3\u2009mm circular form). It was observed that by adding the MAPbBr3 solution as filler in the FP solution, the phase angle could be tuned to frequency-independent as reported for semiconducting fillers in the FP solution55. The final weight percentage of FP and MAPbBr3 solutions of PFNC was optimized to get a frequency-independent phase angle under the dark condition was achieved. A similar process can also be followed on a rigid metal-coated Silicon substrate, and flexibility can be achieved through the soft-backside etch of the silicon substrate75. The PVDF-CrFE-TFE powder was purchased from Piezotech. DMF  solvent, PbBr2 (99%), CH3NH3Br (98%) powders were purchased from Sigma-Aldrich.The solution of the nanocomposite was prepared in two steps. At first, 0.5\u2009M CHA FEI- Quanta 600 FEG scanning electron microscope operated at 10\u2009kV was used to capture the cross-sectional image of the perovskite ferroelectric nanocomposite (PFNC). The CPR sample was dipped in liquid nitrogen to have a sharp cross-section cut for imaging. The sample was coated with a layer of 3\u2009nm of iridium. The transmission electron microscope imaging was performed using FEI-Tecnai Spirit Biotwin. The TEM sample was prepared by drop-casting the PFNC solution on the copper grid. The UV\u2013visible absorbance of the PFNC was measured using a Perkin Elmer\u2019s UV\u2212VIS spectrophotometer lambda 950, which was equipped with an integrated sphere accessory to measure reflectance/absorbance of thin-films. The absorbance was measured in the range of 200\u2013850\u2009nm at a scanning speed of 1\u2009nm/s. The X-ray diffraction studies on both PVDF and PFNC were performed using the XRD Bruker D2 Phaser instrument. Samples for study were exposed to the X-ray source for 4\u2009min. The XRD measurements were studied on PFNC/Au/Si and FP/Au/Si samples. Photoluminescence analysis was performed with the WITec Apyron spectrometer, and samples are excited with laser (\u03bb\u2009=\u2009473\u2009nm).The electrical characterization was performed using an Agilent 4980A LCR meter. In all the experiments, unless otherwise mentioned, the AC voltage bias is 0.1\u2009V. The LEDs were biased using a voltage source. A LabVIEW data acquisition software was designed to acquire the data used in all the experiments.L2 norm of the relative error of the real and the imaginary parts of the admittance over the frequency range. The normalized root mean square error of the extracted model is less than 3.37% in the worst case.Finding a circuit model for the fabricated CPR is essential to be incorporated in the retina simulators and to design suitable interface circuits. In order to characterize our device, a shunt capacitor, and a shunt conductance in addition to parallel RC branches, inspired from Fig. A Flexible Capacitive Photoreceptor for the Biomimetic Retina"}
+{"text": "P > 0.050). The scores on the BMQ-S of the unremitted group were significantly lower than those of the remitted group (P < 0.001). The HAMD scores were significantly reduced in both groups after the eight-week treatment (P < 0.001). There was no significant difference in the BMQ-S scores before and after the treatment (P > 0.050). The medication beliefs of the unremitted inpatients after the treatment were still lower than those of the remitted inpatients (P < 0.001). Logistic-regression analysis showed that low BMQ-S scores at the baseline were an independent risk factor for antidepressant efficacy. Beliefs about medication at baseline may be correlated with the therapeutic efficacy in inpatients with first-diagnosed depression under supervised therapeutic compliance.The aim of the present study was to explore the effect of baseline beliefs about medication on therapeutic outcomes of antidepressants in inpatients with first-diagnosed depression under supervised therapeutic compliance. Ninety-seven inpatients with first-diagnosed depression were included to collect their baseline demographic data to evaluate the Hamilton depression rating scale (HAMD) scores and the beliefs about medicine questionnaire-specific (BMQ-S) scores at baseline and the end of the eight-week treatment. Additionally, we explored the relationship between inpatients\u2019 medication beliefs and therapeutic effect of antidepressants. The inpatients were divided into remitted depression and unremitted depression groups according to outcomes at the end of the eight-week treatment. There was no significant difference in the baseline HAMD between the two groups ( Depression is one of the most common, harmful, and burdensome mental illnesses. Approximately 5\u201312% of males and 9\u201326% of females will experience at least one depressive episode in their lifetime, and approximately 50% of depressed patients will experience a second depressive episode . PatientPoor medication adherence is one of the factors influencing the progression to treat-resistant depression \u201313. CompStandard drug treatment for patients with first-diagnosed depression is the main therapeutic measure for reducing the recurrence rate of depression and preventing progression to treatment-resistant depression . EffectsPatients with first-diagnosed depression were individually recruited by experienced psychiatrists from outpatient and inpatient department of Psychology, Suzhou Psychiatric Hospital in Jiangsu Province from January 2018 to December 2019. The inclusion criteria for patients were as follows: (1) met the diagnostic criteria for depression listed in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) published by the American Psychiatric Association; (2) scores > 17 on the 17-item Hamilton depression rating scale (HAMD-17); (3) ages from 18 to 60 years old; (4) patients were not treated for antidepressants before hospitalization and regularly taking medication under the supervision of the nurse for eight weeks. (5) signed the informed consent. The exclusion criteria of this study were as follows for the candidate patients: (1) depression caused by other diseases, such as bipolar depression; (2) depression accompanied by severe organic brain disorder; (3) severe physical illness; (4) other psychiatric disorders; (5) high risk for suicide (\u2265 3 suicide score on HAMD-17); or (6) pregnancy or lactation. The 17-item HAMD scores and beliefs about medicine questionnaire-specific (BMQ-S) scores at the baseline and the end of the eight-week treatment were examined and divided into the remitted group (Remitted) and Non-remitted group(Non-Remitted) groups according to the therapeutic outcomes. This study included a total of 97 patients with first-diagnosed depression, including 39 males and 58 females, with their ages ranging from 18\u201360 years old (mean age: 39.00 \u00b1 12.29 years). The study was approved by the Ethics Committees of the Suzhou Psychiatric Hospital, The Affiliated Guangji Hospital of Soochow University, and all participants signed an informed consent form. All study procedures were in accordance with the Declaration of Helsinki.Self-rated survey questionnaires were used to collect the basic clinical data of the participants, such as age, sex, disease course, years of education, marital status, economic condition and previous medication. Married or cohabiting for more than 6 months means in marital status, otherwise unmarried. Economic status poverty refers to the family per capita annual income of less than 3,000 yuan (RMB), or higher income. Previous medication history was divided into: 0, no medication; 1, sleeping pills; 2, other drugs ; 3, 1 and 2.The HAMD-17 was used to evaluate the severity of depression. Most of the items in the scale were evaluated using a five-point scale (0\u20134 scores), and some individual items were evaluated using a three-point scale (0\u20132 scores). The total score was the sum of all items in the scale. Patients were divided into two groups based on the total score of the HAMD-17, including the severe-depression group  and the mild-to-moderate depression group .The BMQ-S, established by Horne in 1999, is mainly used to assess the beliefs about medication  and const-tests or analyses of variance and are presented as mean \u00b1 standard deviation P < 0.05 was considered as a statistically significant difference.SPSS24.0 software  was used for statistical analysis in this study. The normally distributed data were analyzed by All participants gave written informed consent and ethical approval was obtained from the Ethics Committee of Suzhou Guangji Hospital (2017-036).The dataset used and analysed during the current study are available from the corresponding author on reasonable request.P > 0.05). The patients with non-remitted had significantly lower BMQ-S scores than those of the patients with remitted , the main effect of time , and mutual effect between groups and time . Pairwise comparisons showed that there was no significant difference in HAMD scores between groups before the treatment  but that there was a significant difference in HAMD scores between groups after the treatment . A significant difference in the HAMD score was found within each group before and after antidepressant treatment ; however, there were no significant differences in the main effect of time  or in the mutual effect between groups and time . Pairwise comparisons showed significant differences in BMQ-S scores between groups before and after antidepressant treatment . No significant difference in BMQ-S scores was found within each group before or after antidepressant treatment  were considered as dependent variables, and the age, sex, disease course, years of education, marital status, economic conditions, previous medication, HAMD scores and BMQ-S scores at baseline were considered as independent variables. The variable screening was performed by the Enter method. Multivariable logistic regression analysis showed that the BMQ-S scores at baseline were independently and negatively correlated with the treatment outcomes (OR: 0.386, In patients with first-diagnosed depression, the initial beliefs about medication were the only contributing factor to the short-term therapeutic efficacy of antidepressants. This result indicated that non-remitted patients had stronger concern beliefs than necessity beliefs prior to treatment. Antidepressants alleviated the depressive symptoms of the patients but did not change the patients\u2019 beliefs about medication.Depressed patients with stronger medication beliefs at baseline were more likely to obtain symptomatic relief after antidepressant treatment but non-remitted patients with weaker medication beliefs at baseline less likely to be relieved, suggesting that the therapeutic outcomes of depressed patients were highly correlated with their initial beliefs about medication. Depressed patients with suicidal attempt have weaker baseline necessity beliefs, and suicidal patients reporting hopelessness during follow-up have relatively strong concern beliefs . Weak neOur study showed that the severity of depression in patients was improved after antidepressant treatment, but patients\u2019 beliefs about medication were not changed following treatment. Previous studies have rarely reported the effects of antidepressant treatment on patients\u2019 medication beliefs, and the factors affecting beliefs about medication are poorly understood. Les\u00e9n et al.  have shoThe present study showed that beliefs about medication were the only influencing factor affecting the short-term therapeutic outcomes in patients with first-diagnosed depression. No similar reports were published in previous depression-related studies. Zayed et al.  have shoThe present study has certain limitations. Firstly, this is a retrospective in hospital investigation. Selection bias may result from outpatients\u2019 intention to be hospitalized. The results need to be interpreted with caution. Secondly, this study didn\u2019t analyze the correlation between beliefs of necessity or concern and the treatment outcome, and therefore could not determine the specific role of each factor on the prognosis. Previous papers have examined the effects of the two factors on compliance and prognosis respectively, and this study has examined the integrated impact of patients\u2019 medication belief on treatment outcome. Therefore, the results of this study have important complementary value to previous studies. Thirdly, the study didn\u2019t perform hierarchical analysis of the patient\u2019s initial antidepressants, and differences in the effects of variant antidepressants may affect the prognosis of patients. Fourthly, the mechanism by which medication beliefs influence short-term prognosis of depression independently of compliance remains to be clarified. These unmet clinical needs need to be addressed in future large-scale clinical studies. Nevertheless, the medication regimen of this study follows the treatment guideline, the 8-week hospitalization period not only ensures the patient\u2019s medication compliance, but also provides a reliable medical record for judging the effect. The clear results of this study deepen the understanding of the influence of medication belief on the prognosis of patients with depression.This study confirms the significant effect of medication belief on the outcome of antidepressant treatment. For the first time, this study raises the possibility that the effect of medication belief on the antidepressant outcome may have additional mechanism independent of therapeutic compliance. The results of this study warrant further studies on excavating the potential value of the scale for developing new strategies to treat depression."}
+{"text": "Pollinators are essential to produce fruits in apple production. Bumble bees are among the most effective pollinators in orchards during the blooming season, yet they are often threatened by the high levels of pesticide use in apple production. Hedgerows and flower strips are infrequently sprayed by pesticides and are thus potentially good shelter for bumble bees. This study evaluated the influence of landscaping in the form of hedgerows and flower strips on the abundance and number of bumble bee species found in apple orchards. The number of bumble bee species found in orchards with hedgerows or flower strips was higher than in orchards without such landscape enhancements. Similarly, three species were more abundant in orchards with landscaping than orchards without those enhancements. Our work provides additional evidence that landscaping in the form of hedgerows and/or flower strips improves bumble bee presence in apple orchards and should therefore be considered as a means to enhance and ensure pollination within farms.Bombus ternarius was lower in orchards with high levels of pesticide use. Apples had fewer seeds when collected in orchards with landscape enhancements and were heavier in orchards that used more pesticides. Our work provides additional evidence that landscape enhancements improve bumble bee presence in apple orchards and should therefore be considered as a means to enhance pollination within farms.Bumble bees are among the most effective pollinators in orchards during the blooming period, yet they are often threatened by the high levels of pesticide use in apple production. This study aimed to evaluate the influence of landscape enhancements  on bumble bee queens in apple orchards. Bumble bee queens from 12 orchards in southern Qu\u00e9bec (Canada) were marked, released, and recaptured in the springs and falls of 2017 to 2019. Half of the 12 orchards had landscape enhancements. Apples were harvested in 2018 and 2019 to compare their quality  in sites with and without landscape enhancements. Species richness, as well as the occurrence of three species out of eight, was higher in orchards with landscape enhancements than in orchards without such structures. The occurrence of  Apples are one of the most widely eaten fruits on the planet . The adaOne particularity of apple production is the cross-pollination required for fruit set. Not only must pollen grains come from another flower but they also usually need to come from another apple variety . In factBombus are often the most efficient pollinators for apples, in terms of flower visitation rate, flower constancy, and pollen deposition ). A greater number of species occurred at orchards with enhancements than at those without enhancements . Species richness was higher at orchards with low levels of pesticide use compared to orchards with higher levels of pesticide use .Species richness was greater in spring than in fall . Apple weight was higher in orchards with high pesticide use than in orchards with lower levels of pesticide use  but did not vary with the presence of landscape enhancements . In contrast, apple diameter did not vary either with landscape enhancements  or with the intensity of pesticide use . The sugar level in apples followed the same pattern . Finally, the number of seeds was lower at sites with landscape enhancements than at sites without enhancements  but did not vary with the intensity of pesticide use . Markov chain Monte Carlo diagnostics indicated that the generalized linear mixed models on apple characteristics converged  and that chains were sufficiently long (MCMC error < 0.5% of the posterior standard deviation). Residual diagnostics and posterior predictive checks suggested model fit  of bumble bee queens, namely, ructures B. These The increased presence of bumble bees in orchards that we documented can potentially be financially rewarding for apple growers. Indeed, Quebec apple producers generally pay large sums of money to rent hives of honey bees, a species not native this region, to ensure the pollination of apple trees . OrchardAs expected, landscape enhancements had a positive effect on bumble bee queen richness. This result is consistent with that of other studies showing a positive effect of either flower strips or hedgerows on bee species richness ,40,68. FContrary to our hypothesis, we found no effect of landscape enhancements on apple characteristics, except for the number of apple seeds which was lower at sites with landscape enhancements. However, these results are not unlike those found in the literature, despite not explicitly investigating apple yield. Flower strips generally increased the abundance and richness of pollinators, but this increase did not consistently lead to increased yield . In factB. ternarius . This is surprising given that pesticides are used to increase yield by, among other things, enhancing individual apple quality. Although our sampling effort may have been too small  to detect an effect, other studies in cucumber and watermelon cropping systems also failed to find a relationship between crop yield and pesticide use ,88. In fB. ternarius site occupancy, it was only positively associated with apple weight. To our knowledge, this study is one of the first to assess the influence of landscape-related effects on bumble bee site occupancy and species richness in agroecosystems while controlling for imperfect species detection . Our"}
+{"text": "A series of 10 analogs were synthesized of which compound 6 showed an improved potency/selectivity (EC50 0.2 \u00b1 0.1 \u00b5M) against MNV; good activity was also observed against the HuNoV GI replicon (EC50 1.2 \u00b1 0.6 \u00b5M). Time-of-drug-addition studies revealed that analog 6 acts at a time point that coincides with the onset of viral RNA replication. After six months of selective pressure, two compound 6res variants were independently selected, both harboring one mutation in VPg and three mutations in the RdRp. After reverse engineering S131T and Y154F as single mutations into the MNV backbone, we did not find a markedly compound 6res phenotype. In this study, we present a class of novel norovirus inhibitors with a high barrier to resistance and in vitro antiviral activity.Human noroviruses (HuNoVs) are the most common cause of viral gastroenteritis resulting in ~219,000 deaths annually and a societal cost of ~USD60 billion. There are no antivirals or vaccines available to treat and/or prevent HuNoV. In this study, we performed a large-scale phenotypical antiviral screening using the mouse norovirus (MNV), which included ~1000 drug-like small molecules from the Drug Design and Synthesis Centre . Compound 3-sulfonyl)-5-chloroindole-N-(phenylmethanol-4-yl)-2.carboxamide (compound  Caliciviridae family which causes gastroenteritis resulting in vomiting and watery non-bloody diarrhea. HuNoV infections occur worldwide and are an important health issue that affects all age groups. After the introduction of two rotavirus vaccines, HuNoV is becoming the most common cause of viral gastroenteritis, resulting annually in 200,000 deaths [Human norovirus (HuNoV) is a (+)ssRNA virus, that belongs to the 0 deaths . Infecti1) as an interesting hit compound. Based on the initial results, a test set of 10 analogues, 2\u201311, was synthesized to further explore this class of norovirus inhibitors; among them, analog 6 showed an improved potency and selectivity against MNV and was studied in more detail.In the search of new anti-norovirus molecules, we performed a large-scale antiviral drug screening, which included ~1000 drug-like small molecules from the Drug Design and Synthesis Centre . Due to the lack of a robust and high-throughput cell culture system for HuNoV, the screen was performed using mouse norovirus (MNV). From the initial screening, we identified 3sulfonyl) 5 chloroindole-N (pheylmethanol 4 yl)-2-carboxamide in tetrahydrofuran at 25 \u00b0C for 4 h tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and triethylamine in N,N-dimethylformamide at 40 \u00b0C for 18 h under argon stream  with EC50 values ranging from 0.16 \u00b5M (compound 6) to 35.95 \u00b5M (compound 11), and five compounds (2\u20135 and 8) showed EC50\u2032s >100 \u00b5M. In this assay, the cytotoxic concentrations  of compounds 1, 2 and 6 were >100 \u00b5M, and those of compounds 3\u20135 and 7\u201311 were in the 3.48 (compound 4)\u201388.69 (compound 3) \u00b5M range.Compounds 1 with the homologue N-4-(hydroxymethyl)benzyl (compound 6) improved the inhibitory activity against both MNV and HuNoV replicon, meanwhile the CC50 value was >100 \u00b5M. The full N-4-(hydroxymethyl)benzyl end tail played a key role for the inhibitory activity. In fact, if a compound is deprived of the 4-hydroxymethyl group (compound 2) [3) [4 and 11) the activity was remarkably less compared to compounds 1 and 6 . Replacement of the sulfonyl bridging group by a methylene, carbonyl, or sulfur group provided less active or inactive compounds against both strains (compare compound 6 with compounds 7\u20139). Compounds 1 and 6 with the 3,5-dimethylsulfonyl group at position 3 of the indole were superior to the corresponding 3-phenylsulfonyl derivatives 5 and 10. Compound 6 was the most potent inhibitor of both MNV and the HuNoV replicon within the series, with EC50 values of 0.16 \u00b5M and 1.24 \u00b5M, respectively, and CC50 >100 \u00b5M. Thus, compound 6 was selected for more in-depth in vitro studies (Replacement of the N-4-(hydroxymethyl)phenyl group at the indole-2-carboxamide nitrogen of compound pound 2) , of the  studies .1) were synthesized and tested for anti-norovirus activity. Out of these, compound 6 had improved antiviral activity against MNV (EC50 (CPE) of 0.16 \u00b1 0.06 \u00b5M, EC50  of 0.21 \u00b1 0.03 \u00b5M), with a 50% toxic concentration (CC50) >12.50 \u00b5M sulfonyl)-5-chloroindole-N-(phenylmethanol-4-yl)-2.carboxamide  experiment .To assess at which moment compound periment . First, 6 acts at the same time point in the viral replication cycle as polymerase inhibitors, we could assume that the compound inhibits the active site of the MNV polymerase by directly competing with their natural substrate, as a nucleoside analog would. However, no direct competition between the norovirus polymerase and the natural substrate was observed when the compound was present.Given that compound 6 we cultured the MNV under sub-optimal compound concentrations. After six months, three independent resistant variants were selected after 30 passages. Of those, two of these variants  play a role in the compound observed . As cont6, againstmultiple norovirus genotypes.In the search for an antiviral against HuNoV infections, we performed a large-scale antiviral drug screen, which included ~1000 drug-like small molecules from the Drug Design and Synthesis Centre  for their potential anti-norovirus activity. We here report on the anti-norovirus activity of the 3-sulfonyl)-5-chloroindole-N-(phenylmethanol-4-yl)-2.carboxamide analog, compound 50 values around 0.5 \u00b5M and 2 \u00b5M, respectively. The analog, compound 6, had an improved antiviral activity as it inhibited MNV and the HuNoV GI.1 replicon with EC50 values around 0.2 \u00b5M and 1.7 \u00b5M, respectively. In addition, compound 6 had a better antiviral effect against MNV and the HuNoV GI.1 replicon than 2CMC, which is currently used as the benchmark antiviral compound in norovirus research [The early hit compound 1 sulfonyl)-5-chloroindole-N-(phenylmethanol-4-yl)-2.carboxamide), inhibited the replication of MNV and the HuNoV GI.1 replicon in vitro with ECresearch ,14.6 was achieved by studying the effect of adding this compound at different time points during infection. We determined that compound 6 acts at a post-entry step, most likely acting at the time viral genome replication starts. In fact, the presence of compound 6 is required at the same time points as that of the nucleoside analog 2CMC [6 was also an RdRp targeting compound. However, a polymerase assay showed that the compound could not directly inhibit the active site of the enzyme. Still, compound 6 rendered the catalytic site of the MNV RdRp less efficient, thus it could act as an allosteric inhibitor of the RdRp.A first insight into the mechanism of action of compound log 2CMC . Hence, 6 would easily arise (thus the class of compounds would have a low barrier to resistance) or if many passages (months) and multiple mutations would be required to confer resistance to the compound (high barrier to resistance), we passaged the virus in the presence of compound 6 up to 30 consecutive times [50 against compound 6 (6\u20138 fold). After deep sequencing, we found these two mutants had the same exact four mutations, one mutation in the VPg (F69L) and three mutations in the RdRp . By reverse engineering S131T and Y154F as single mutations into the MNV backbone, we did not find a markedly compound 6res phenotype but the S131T did confer a small 2-fold shift in EC50 value. Insertion of the other single mutations or combination of mutations is ongoing. Since there is a possibility that the mutant viruses would replicate without inducing a cytopathic effect, which is the used indicator of viral replication, we also measured the levels of viral RNA in samples by RT-qPCR after three passages in BV-2 cells. Despite repeated passage, we found no signs of viral RNA replication. This might also suggest that these amino acid changes make the virus unviable and that these amino acids play a critical role in the viral replication.Resistance development is a major obstacle in antiviral therapy, and almost all active antiviral agents have shown to select for resistance mutations. To assess whether resistant variants to compound ve times ,17,18,19ve times . Out of 6. However, in the case of noroviruses there are two equally active versions of its polymerase (Pol), the mature polymerase and a protease-polymerase precursor protein (ProPol), the latter thus contains both protease and polymerase activities [We can speculate that the mutations acquired in the RdRp play an important role in the resistance against compound tivities . The nuctivities . It coultivities . To undeThe high barrier to resistance is a crucial property of an antiviral drug, preferably multiple rather than single mutations are required, and resistant variants have a seriously compromised fitness. As selection for resistant virus showed to be so difficult, we need to take into consideration that this compound might impede an interaction with a cellular factor. As viruses require cellular factors to translate their transcripts, targeting these offers another strategy to develop broad antiviral drugs.2. To obtain each virus stock, once full CPE was observed, cells underwent two freeze-thaw cycles and the virus was harvested from the supernatants after centrifugation  and stored at \u221280 \u00b0C. The viral titer was determined by endpoint titration.Murine norovirus  was propagated as described earlier . HG23 ceThe compound library was synthetized and supplied by Prof. Romano Silvestri  and dissolved in dimethyl sulfoxide .4 cells/well) were infected with MNV.CW1 in the presence of a dilution series of compounds. Antiviral activity and cytotoxicity were determined using a colometric assay using 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). The 50% effective concentration (EC50) was defined as the compound concentration that protected 50% of the cells from CPE. The cell viability % was calculated as (ODtreated/ODCC) \u00d7 10 and the 50% cytotoxic concentration (CC50) was defined as the compound concentration that reduces the number of viable cells by 50%.Norovirus: The experiment was performed as described earlier ,23. In s2 cells/well) were seeded into the wells of a 96-well plate without G418 (Geneticin Selective Antibiotic). After 24 h of incubation, serial dilutions of compounds were added. Cells were further incubated for 72 h, then cells were collected for RNA load quantification by RT-qPCR. To determine relative levels of Norwalk virus replicon RNA, \u03b2-actin was used as a normalizer and ratios were calculated as previously described [50 values were defined as the compound concentration that resulted in 50% reduction of the relative HuNoV GI.1 replicon RNA levels.HuNoV GI.1 replicon: The experiment was performed as described earlier with minor modifications . In shorescribed . The EC5The supernatants of treated or untreated infected cells were harvested. The viral RNA was isolated from supernatant using the NucleoSpin RNA Virus Kit (Macherey\u2013Nagel), according to the manufacturer\u2019s protocol and was quantified by RT-qPCR. A one-step RT-qPCR was performed in a reaction mixture of 20 \u03bcL containing 10 \u03bcL reaction mix from the iTaq Universal Probes One-Step Kit (Bio-Rad), 0.5 \u03bcL reverse transcriptase, 4 \u03bcL template RNA, primers, and probe .5 cells/mL) were infected with MNV (MOI of 1). After 1 h at 4 \u00b0C, cells were washed with cold (4 \u00b0C) DMEM to remove unbound viruses. Cells were incubated at 37 \u00b0C to start the infection. To study the replication kinetics of MNV supernatant and cells were harvested, from untreated cultures, every 2 h until 24 h post-infection (pi) and viral RNA was quantified by RT-qPCR. In parallel, another set of infected cultures were treated with compound. The compound was added at 2 h intervals and cultures were further incubated until 24 h pi, supernatant and cells were collected separately for determination of the viral RNA by RT-qPCR. The isolation of the extracellular MNV RNA from cell culture supernatant was done by the NucleoSpin RNA Virus Kit (Macherey\u2013Nagel), and the intracellular RNA was extracted from cells by the RNeasy kit (Qiagen).RAW 264.7 cells  template  and 100 \u00b5M ATP, was followed for 1 h at 30 \u00b0C on a TECAN Infinite 200 PRO microplate reader, measuring the growth of PicoGreen fluorescence (Ex/Em = 485/530 nm) after the addition of 3.4 \u00b5M MNV RdRp and of increasing amounts of compound 6 (from 0 to 100 \u00b5M), in a reaction mixture containing 20 mM Tris/HCl (pH 7.5), 25 mM NaCl, 0,3 mM MnCl2, 5 mM MgCl2, 1 mM DTT, 2 U RiboLock Ribonuclease inhibitor (Life technologies). The final fluorescence values were calculated as the average of four independent experiments. Measurement of the activity  vs. compound 6 concentration was used to estimate the IC50 value of the compound with the program GraFit5 (Erithacus software).Compound 4 cells/well) were seeded into the wells of 96-well plate and were infected with MNV.CW1 in the presence of a dilution series of compounds. When CPE was visible in the virus control (no compound), all the wells were scored for visible CPE. Virus was harvested from the wells in which CPE was observed under the highest compound pressure. The resistant virus was then diluted to 1:5 and used to infect a new 96-well plate under the same conditions to continue. After 25 passages (~6 months), the viral RNA was isolated using the NucleoSpin RNA Virus Kit (Macherey\u2013Nagel). MNVres viruses were analyzed by deep sequencing by the group of Prof. Matthijnssens as described previously [RAW 264.7 cells  according to the manufacturer\u2019s protocol. Red fluorescent protein (RFP) was used as a positive control for transfection. Transfected cells were incubated for 48 h, cells underwent two freeze thaw cycles and the virus was harvested from the supernatants after centrifugation  and stored at \u221280 \u00b0C. Virus stocks were passaged for three times on BV-2 cells and followed by two consecutive passages on RAW 264.7 cells. Viral titers were determined by serial dilutions on RAW 264.7 cells seeded in 96-well plates.The MNV.CW3 mutant viral stocks were obtained by transfecting the mutated plasmid pMNV CW3  in Huh 7 Lunet cells expressing the MNV receptor CD300lf  . Huh-7 C50 was used for each virus in the antiviral assays. RAW 264.7 cells (1 \u00d7 104 cells/well) were seeded in the presence of a dilution series of compounds and infected. Antiviral activity was determined using the MTS method. The 50% effective concentration (EC50) was defined as the compound concentration that protected 50% of the cells from CPE.The assessment of the antiviral activity of compounds on resistant viruses was performed similarly as the regular antiviral assay on MNV. First, the titer of the resistant viruses was determined by endpoint titration. A concentration of 30\u00d7 TCID\u22121. Proton nuclear magnetic resonance (1H NMR) spectra were recorded with a Varian Mercury (300 MHz) or a Bruker Avance (400 MHz) spectrometer in the indicated solvent, and the corresponding fid files were processed by MestreLab Research SL MestreReNova 6.2.1-769 software. Chemical shifts are expressed in \u03b4 units (ppm) from tetramethylsilane. The purity of tested compounds was checked by high pressure liquid chromatography (HPLC). The purity of the tested compounds was found to be >95%. The Thermo Fisher Scientific Inc. Dionex UltiMate 3000 HPLC system consisted of an SR-3000 solvent rack, an LPG-3400SD quaternary analytical pump, a TCC-3000SD column compartment, a DAD-3000 diode array detector, and an analytical manual injection valve with a 20 \u03bcL loop. Samples were dissolved in acetonitrile (1 mg/mL). HPLC analysis was performed by using a Thermo Fisher Scientific Inc. Acclaim 120 C18 column , at 25 \u00b1 1 \u00b0C with an appropriate solvent gradient (acetonitrile/water), flow rate of 1.0 mL/min and signal detector at 206, 230, 254, and 365 nm. Chromatographic data were acquired and processed by Thermo Fisher Scientific Inc. Chromeleon 6.80 SR15 Build 4656 software.All reagents and solvents were handled according to the material safety data sheet of the supplier and were used as purchased without further purification. Organic solutions were dried over anhydrous sodium sulfate. Evaporation of solvents was carried out on a B\u00fcchi Rotavapor R-210 equipped with a B\u00fcchi V-850 vacuum controller and a B\u00fcchi V-700 vacuum pump. Column chromatography was performed on columns packed with silica gel from Merck (70\u2013230 mesh). Silica gel thin layer chromatography (TLC) cards from Merck  were used for TLC. Developed plates were visualized with a Spectroline ENF 260C/FE UV apparatus. Melting points (mp) were determined on a Stuart Scientific SMP1 apparatus and are uncorrected. Infrared (IR) spectra were recorded on a PerkinElmer Spectrum 100 FT-IR spectrophotometer equipped with a universal attenuated total reflectance accessory. IR data were acquired and processed by PerkinElmer Spectrum 10.03.00.0069 software. Band position and absorption ranges are given in cmTo a solution of the protect derivative (0.11 mmol) in tetrahydrofuran (20 mL/mmol) tetrabutylammonium fluoride  was added and the resulting mixture was stirred at 25 \u00b0C for 3 h. The solvent was removed from the reaction mixture and the resulting product was purified by column chromatography .N,N-dimethylformamide (20 mL/mmol) was stirred at 40 \u00b0C for 18 h under an argon stream. After cooling, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried, and filtered. Evaporation of the solvent gave a residue that was purified by column chromatography .A mixture of acid (0.30 mmol), triethylamine (0.90 mmol), amine (0.90 mmol), and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (0.30 mmol) in tert-butyldimethylsilyl chloride (0.89 mmol) and imidazole (0.97 mmol) was added. The reaction mixture was stirred at 25 \u00b0C for 1 h, then diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried and filtered. Evaporation of the solvent gave a residue that was purified by column chromatography .To a solution of 4-(hydroxymethyl)aniline (0.89 mmol) in chloroform (10 mL/mmol) N-(4-(hydroxymethyl)phenyl)-1H-indole-2-carboxamide (compound 1). It was synthesized according to general procedure A, starting from 19. Yield 40%, mp 220\u2013221 \u00b0C (from ethanol) as a white powder. 1H NMR : \u03b4 \u03b4 2.29 , 4.49 , 5.18 , 7.25\u20137.26 , 7.34-7.36 , 7.55 , 7.64 , 7.68 , 7.92\u20137.93 , 10.85 , 13.21 ppm . IR: \u03bd 1632 and 2984 cm\u22121.5-Chloro-3-sulfonyl)-N-Benzyl-5-chloro-3-sulfonyl)-1H-indole-2-carboxamide (compound 2). It was synthesized as previously reported [N-(2-hydroxyethyl)-1H-indole-2-carboxamide (compound 3). It was synthesized as previously reported [5-Chloro-3-sulfonyl)-reported .N-(4-hydroxybenzyl)-1H-indole-2-carboxamide (compound 4). It was synthesized according to general procedure B, starting from compound 18 and 4-hydroxybenzylamine. Yield 60%, mp 236\u2013237 \u00b0C (from ethanol), as a cream colour powder. 1H NMR : \u03b4 2.26 , 4.46 , 6.73-6.75 , 7.22-7.24 , 7.33 , 7.51\u20137.53 , 7.55-7.56 , 7.94 , 9.32 , 9.34 , 13.03 ppm . IR: \u03bd 1684 and 3105 cm\u22121.5-Chloro-3-sulfonyl)-N-(4-(hydroxymethyl)phenyl)-3-(phenylsulfonyl)-1H-indole-2-carboxamide (compound 5). It was synthesized according to general procedure A, starting from compound 20. Yield 12%, mp 152\u2013155 \u00b0C (from ethanol), as a pale white powder. 1H NMR : \u03b4 4.47 , 5.17 , 7.30\u20137.34 , 7.52\u20137.60 , 7.67\u20137.70 , 7.92\u20137.93 , 8.05-8.08 , 10.87 , 13.23 ppm . IR: \u03bd 1523 and 3291 cm\u22121.5-Chloro-N-(4-(hydroxymethyl)benzyl)-1H-indole-2-carboxamide (compound 6). It was synthesized according to general procedure B, starting from compound 18 and (4-(aminomethyl)phenyl)methanol. Yield 18%, mp 124\u2013126 \u00b0C (from ethanol), as a white powder. 1H NMR : \u03b4 2.28 , 4.50 , 4.57 , 5.17 , 7.25\u20137.26 , 7.31 , 7.32\u20137.35 , 7.40 , 7.54 , 7.59\u20137.60 , 7.94 , 9.42 , 13.06 ppm . IR: \u03bd 1658 and 2984 cm\u22121.5-Chloro-3-sulfonyl)-N-(4-(hydroxymethyl)benzyl)-1H-indole-2-carboxamide (compound 7). It was synthesized according to general procedure B, starting from 16 and (4-(aminomethyl)phenyl)methanol. Yield 22%, mp 171\u2013174 \u00b0C (from ethanol), as a white powder. 1H NMR : \u03b4 2.16 , 4.34 , 4.49 , 5.16 , 6.75\u20137.76 , 6.83 , 7.18 , 7.25\u20137.30 , 7.41 , 7.55 , 8.57 , 11.54 ppm . IR: \u03bd 1613 and 2917 cm\u22121.5-Chloro-3--N-(4-(hydroxymethyl)benzyl)-1H-indole-2-carboxamide (compound 8). It was synthesized according to general procedure B, starting from compound 15 and (4-(aminomethyl)phenyl)methanol. Yield 15%, mp 271\u2013274 \u00b0C (from ethanol), as a pale white powder. 1H NMR : \u03b4 2.30 , 4.24 , 4.47 , 5.14 , 7.15 , 7.24 , 7.27\u20137.32 , 7.55 , 9.56 , 12.80 ppm . IR: \u03bd 1646 and 2922 cm\u22121.5-Chloro-3--N-(4-(hydroxymethyl)benzyl)-1H-indole-2-carboxamide (compound 9). It was synthesized according to general procedure B, starting from compound 12 and (4-(aminomethyl)phenyl)methanol. Yield 15%, mp 212\u2013215 \u00b0C (from ethanol), as a pale yellow powder. 1H NMR : \u03b4 2.11 , 4.42 , 4.52 , 5.13 , 6.65-6.66 , 6.78-6.79 , 7.15\u20137.16 , 7.28 , 7.45 , 7.53 , 8.74 , 12.51 ppm . IR: \u03bd 1636 and 3219 cm\u22121.5-Chloro-3-thio)-N-(4-(hydroxymethyl)benzyl)-3-(phenylsulfonyl)-1H-indole-2-carboxamide (compound 10). It was synthesized according to general procedure B, starting from compound 13 and (4-(aminomethyl)phenyl)methanol. Yield 24%, mp 182\u2013185 \u00b0C (from ethanol), as a white powder. 1H NMR : \u03b4 4.47 , 4.53 , 5.17 , 7.27\u20137.38 , 7.49, 7.59 , 7.91\u20137.98 , 9.41 , 13.07 ppm . IR: \u03bd 1523 and 3214 cm\u22121.5-Chloro-N-(4-hydroxyphenethyl)-1H-indole-2-carboxamide (compound 11). It was synthesized according to general procedure B, starting from compound 14 and 4-hydroxyphenethylamine. Yield 56%, mp 231\u2013234 \u00b0C (from ethanol), as a white powder. 1H NMR : \u03b4 2.29 , 2.76 , 3.49 , 6.67 , 7.08 , 7.24\u20137.31 , 7.50 , 7.58\u20137.59 , 7.90\u20137.91 , 8.99 , 9.19 , 13.01 ppm . IR: \u03bd 1620 and 2934 cm\u22121.5-Chloro-3-sulfonyl)-N-(4-(((Tert-butyldimethylsilyl)oxy)methyl)phenyl)-5-chloro-3-sulfonyl)-1H-indole-2-carboxamide (compound 19). It was synthesized according to general procedure B, starting from compound 18 and 4-(((tert-butyldimethylsilyl)oxy)methyl)aniline. Yield 13% as an oil. 1H NMR : \u03b4 0.08 , 0.90 , 2.28  4.70 , 7.24\u20137.25 , 7.33\u20137.35 , 7.55 , 7.63\u20137.64 , 7.69 , 7.92 , 10.85 , 13.25 ppm . IR: \u03bd 1648 and 2929 cm\u22121.Tert-butyldimethylsilyl)oxy)methyl)-N-((5-chloro-3-(phenylsulfonyl)-1H-indol-2-yl)methyl)aniline (compound 20). It was synthesized according to general procedure B, starting from compound 17 and 4-(((tert-butyldimethylsilyl)oxy)methyl)aniline. The crude product was used without further purification.4-(((Tert-butyldimethylsilyl)oxy)methyl)aniline (compound 21). It was synthesized according to general procedure B, Yield 67% as an oil. 1H NMR : \u03b4 0.00 , 0.86 , 4.48 , 4.94 , 6.50 , 6.93 ppm . IR: \u03bd 3529 and 4018 cm\u22121.4-(((In conclusion, we here present the discovery, synthesis and activity of a novel class of anti-norovirus compounds. Overall, these findings confirm once again that targeting the HuNoV proteins is an attractive target for the development of norovirus antivirals. However, more research efforts are necessary in the search for effective HuNoV antiviral agents useful for the treatment of HuNoV infections."}
+{"text": "Bisphenol A is a compound commonly found in products meant for daily use. It was one of the first compounds to be identified as an endocrine disruptor that was capable of disrupting the endocrine system and producing very similar effects to those of metabolic syndrome. It has recently gained popularity in the scientific arena as a risk factor for obesity and diabetes due to its ability to imitate natural oestrogens and bind to their receptors. The aim was to study the possible relationship between the Bisphenol A endocrine disruptor with diabetes and obesity. The analysis of the articles allows us to conclude that Bisphenol A is an additional risk factor to consider in the development of diabetes and obesity, since it is capable of stimulating the hypertrophy of adipocytes and altering the endocrine system by mimicking the effects of the oestrogen molecule, since epidemiological studies carried out have suggested that the same disruptions seen in experimental studies on animals can be found in humans; however, despite many countries having developed policies to limit exposure to this disruptor in their populations, there is a lack of international agreement. Understanding its relationship with obesity and diabetes will help to raise awareness in the population and adopt public health campaigns to prevent exposure\u2014especially among young people\u2014to these substances. Obesity is a chronic metabolic disease affecting people of all ages and is one of the 21st century\u2019s major public health problems, which the WHO considers to be an epidemic that affects both adults and the child-youth population  i signals and insulin release. In addition, there is an activation of the transcription of the insulin gene via ER\u03b1\u2014ERK1/2 that activates the transcription factor NeuroD1  of E2 (17-\u03b2-oestradiol) or BPA induced an increase in plasma insulin. They also saw that longer exposures induced an increase in the insulin content in pancreatic \u00df cells in response to a stimulus of their oestrogen receptors. They began to see these effects at doses as low as 10 \u03bcg/kg and from day two. After several days of treatment with E2 or BPA, the mice developed chronic hyperinsulinaemia [Gestational insulin resistance is a natural phenomenon that appears physiologically during pregnancy to more easily direct nutrients present in blood to the growing foetus ,76; howelinaemia .A similar effect can be observed after taking hormonal contraceptives with oestrogens, which is why they have been associated with changes in carbohydrate metabolism and increased insulin resistance . Some stSeveral studies have analysed the risk of exposure to low doses of BPA on the metabolism in animals, finding hyperinsulinaemia, lower body temperatures, and lower physical activity in exposed mice compared to the control group . When exExposure during pregnancy in women causes glucose intolerance and high levels of insulin, triglycerides, and leptin in plasma compared to the control group, which seems to indicate that exposure to BPA during pregnancy promotes glucose intolerance . The lonOther studies showed that exposure to BPA can cause increased adipogenesis in human cells. Their results indicated that it is not just BPA, but also its main metabolite that is capable of stimulating adipocyte differentiation ,87. ExpoThe lack of consensus on the safety of BPA combined with the fact that more and more articles have indicated that exposure to BPA is directly related to an increased risk of causing endocrine system disruptions drove us to carry out this literature review to better understand the mechanisms by which endocrine disruptors, specifically BPA, disrupt the molecular pathways of the endocrine system.The literature search was carried out on PubMed and Web Of Science. The search strategy was carried out by combining the MeSH terms \u2018bisphenol A\u2019, \u2018BPA\u2019, \u2018endocrine disruptors\u2019, \u2018obesity\u2019, \u2018insulin resistance\u2019, and \u2018glucose intolerance\u2019, combined with each other using Boolean operators. Articles from within the last 10 years were selected, giving a total of 17 to review. All the articles included the Bisphenol A endocrine disruptor and its relationship with insulin resistance or glucose intolerance. They have been divided into two groups for review and analysis. On the one hand, those dealing with experimental research studies in animals  and, on There is more and more evidence to point to endocrine disruptors being an additional risk factor to take into account. Understanding the relationship between them and obesity or diabetes would help ensure that appropriate measures are taken to raise awareness among the population and to prevent their negative effects on health.In recent decades, endocrine disruptors have been subject to much research and debate, and the amount of information about them today is overwhelming. There are many articles dealing with endocrine disruptors and the trend is for publications on them to increase given that it is a fairly recent topic with many unresolved issues. Conversely, as indicated above, phenols are widely-used compounds in modern society in developed countries and are increasingly being used in developing countries. This means that science cannot ignore whether one of the many properties of this compound is to disrupt the endocrine system. The numerous studies published in recent years\u2014both experimental and epidemiological\u2014have contributed to our understanding of some of the properties and to better understand how they affect people.Some studies have shown how adipocytes can hypertrophy when exposed to certain concentrations of BPA as occurs in obesity, as well as presenting a higher prevalence of obesity, abdominal obesity, and insulin resistance ,96,102. The result of this review suggests that bisphenol A is capable of acting as an endocrine disruptor through the modifications produced by this chemical in glucose and insulin homoeostasis ,92, specThe tissues most susceptible to BPA are related to embryonic development  as well as postnatal development under maternal influence (such as breast milk and colostrum). It is, therefore, logical that experimental studies in mice showed transgenerational effects in offspring, even though they have not been exposed to BPA. Increased insulin secretion stands out among these effects, which is the body\u2019s physiological response to carbohydrate intolerance, which occurs in metabolic syndrome and type 2 diabetes.In addition to observing that offspring had increased insulin secretion compared to the control groups, other arguments supporting the role of BPA as an endocrine disruptor are that the levels of leptin, triglycerides, and glycerol also increased . These fOne of the current unanswered questions about BPA is at what concentrations its effects occur and whether there is a limit beyond which its ability as an endocrine disruptor decreases. Alonso-Magdalena et al. and Wei et al. obtained similar conclusions when it was observed that the disruptions mainly occurred at low doses of BPA and not at higher doses. This suggests that there may be a critical narrow concentration range for the action of BPA and that exposures above or below that range would be less harmful to the body ,95.Several authors have analysed what happens in prolonged foetal exposure to BPA during the neonatal period and have evaluated the metabolic disruptions that exposure to environmentally equivalent concentrations normally received by humans in mice and their offspring could have and concluded that females exposed to BPA during childhood showed signs of obesity and metabolic disturbances such as increased triglyceride levels, hyperinsulinaemia, and insulin resistance ,93,95,99The disruptions produced by BPA in glucose metabolism and insulin homoeostasis can also occur physiologically in humans at the time of gestation, in which a physiological or pathological insulin resistance occurs, as in type 2 diabetes. Nutrition plays a key role in this second case, which is why the effects of prenatal BPA exposure on adipose tissue and metabolism in goats were analysed, introducing a novel factor of analysing whether a high-calorie, high-fat diet would exacerbate the disruptions caused by BPA. The combination of both factors was not seen to increase endocrine disruptions; nevertheless, they did find that the disruptions produced by both were similar: insulin resistance and adipocyte hypertrophy . The resIn the second part of the results of this literature review, we analysed articles that studied the levels of BPA in humans ,102,103.The results obtained from these studies add to the conclusions already drawn from experimental studies in animals. In pre-adolescent females exposed to BPA, the baseline levels of oestradiol and androstenedione were significantly higher than in those who were not exposed, with the elevated levels of these hormones and greater insulin resistance remaining one year later . This suAlthough concentrations of BPA are more difficult to measure in blood due to its short half-life, many participants have also presented with disrupted basal blood glucose levels and insulin resistance . We alsoThe main limitation of this work is that most of the articles analysed are animal studies and not human studies due to the logical ethical difficulties of carrying out this type of study in humans. Another limitation is that the disruptions in the endocrine system produced by bisphenol A have been studied without considering possible interactions with other external factors.As indicated above, there is not a general consensus of all countries in the establishment of a limit as a safe dose, so as not disrupt the glucose-stimulated insulin response in humans ,64,103. In conclusion, endocrine disruptors may be an additional risk factor to consider in the development of obesity as they are capable of stimulating adipocyte hypertrophy and this appears to confirm a positive association between the levels of BPA in the body and obesity. The results of the experimental studies mostly point to BPA having the ability to disrupt the endocrine system by mimicking the effects of the oestrogen molecule, which is why more experimental and epidemiological research will be necessary to establish the scale of the effects caused by BPA in large populations and its association with insulin resistance and diabetes. Epidemiological studies carried out on humans suggest that the same disruptions seen in experimental studies on animals may be found; however, despite many countries having developed policies to limit exposure to BPA in their populations, there is a lack of international agreement. Understanding the relationship between EDs and obesity will help to raise awareness in the population and adopt public health campaigns to prevent exposure\u2014especially among young people\u2014to these substances."}
+{"text": "Advances in mass-spectrometry have generated increasingly large-scale proteomics datasets containing tens of thousands of phosphorylation sites (phosphosites) that require prioritization. We develop a bioinformatics tool called HotPho and systematically discover 3D co-clustering of phosphosites and cancer mutations on protein structures. HotPho identifies 474 such hybrid clusters containing 1255 co-clustering phosphosites, including RET p.S904/Y928, the conserved HRAS/KRAS p.Y96, and IDH1 p.Y139/IDH2 p.Y179 that are adjacent to recurrent mutations on protein structures not found by linear proximity approaches. Hybrid clusters, enriched in histone and kinase domains, frequently include expression-associated mutations experimentally shown as activating and conferring genetic dependency. Approximately 300 co-clustering phosphosites are verified in patient samples of 5 cancer types or previously implicated in cancer, including CTNNB1 p.S29/Y30, EGFR p.S720, MAPK1 p.S142, and PTPN12 p.S275. In summary, systematic 3D clustering analysis highlights nearly 3,000 likely functional mutations and over 1000 cancer phosphosites for downstream investigation and evaluation of potential clinical relevance. Dysregulated phosphorylation is well-known in cancers, but it has largely been studied in isolation from mutations. Here the authors introduce HotPho, a tool that can discover spatial interactions between phosphosites and mutations, which are associated with activating mutation and genetic dependencies in cancer. Recent advances in mass-spectrometry have generated increasingly large-scale proteomics datasets in multiple cancer types4, each containing tens of thousands of phosphosites that urgently require prioritization. Missense somatic mutations and phosphorylations, independently or through mutual interactions, can affect the physicochemical properties of the residue side chains and modulate protein functions or stability in oncogenic pathways. Thus far, mutation and phosphorylation have been largely studied in isolation by genomics and proteomics approaches. Integrated methodologies are required to reveal their interactions and prioritize both types of events with functional significance.Dysregulated phosphorylation of oncogenic proteins alters pathway activity and contributes to tumor phenotypes7, yet these studies did not consider the 3-dimensional structures of proteins. We and others previously demonstrated that mutations in cancer genes form 3-dimensional (3D) spatial clusters\u2014defined by high local concentrations of mutations on protein structures\u2014enriched for functional missense mutations10. We hypothesize that co-clustering mutations and phosphosites in spatial hotspots will also enrich for functional events of both categories. Systematic analyses of mutations from sequencing data and phosphosites from global proteomics data will enable us to investigate beyond currently-interrogated phosphosites with available targeting antibodies and reveal functionalities of phosphosites.Previous works highlighted the potential functionality of mutations that are linearly adjacent to phosphosites in cancer driver genesHere, we report on the development and application of a bioinformatics tool called HotPho to systematically discover spatial interactions of mutations and phosphosites. We find 474 significant hybrid clusters (defined as clusters containing both co-clustering phosphosites and mutations) that prioritize 1255 phosphosites and 2938 mutations on protein structures from large-scale proteomics and genomics data. Many co-clustering mutations are associated with high functional scores, expression changes, and known recurrent/activating events that expose genetic dependency; whereas many co-clustering phosphosites are found in kinase domains and verified in primary tumor samples. We specifically prioritize phosphosites co-clustering with activating mutations of BRAF, EGFR, and PIK3CA. Collectively, our approach of 3D spatial clustering on protein structures systematically highlights likely functional mutations and phosphosites for downstream investigation.8, HotPho enables investigation of proximal and structural information of phosphosites with their neighboring mutations and domains, both on a single protein structure or co-crystallized binding partners in a protein complex .Extending beyond the originating framework of an earlier mutation-clustering tool we developed, namely HotSpot3Dlex Fig.\u00a0. Briefly4 (\u201cMethods\u201d). We also included 791,489 missense mutations from 9062 samples across 33 cancer types from a filtered set of Multi-Center Mutation Calling in Multiple Cancers project (MC3) mutation calls from the TCGA PanCanAtlas11, taken in account their recurrence in the MC3 cohort. Both mutations and phosphosites are mapped by HotPho and analyzed based on 5950 processed human proteins from UniProt12 having at least one PDB structure.We demonstrated the capability of HotPho for identifying co-clustering cancer mutations and phosphosites using data comprising 225,151 unique phosphosites from PTMcosmos compiled from multiple databases and CPTAC cancer proteomic cohortsTo assess whether the co-clustering between aforementioned sets of mutations and phosphosites is non-random, we analyzed the clusters against a set of permutated data as follows: the original mutation backbone was maintained while phosphosites were randomly populated 100 times, keeping the corresponding ratios of residue types of phosphosites constant (\u201cMethods\u201d). We found a higher fraction of hybrid clusters in the original HotPho output (8.1%) at the top 5% of the cluster closeness score compared to the null distribution from the permutations Fig.\u00a0.13 versus other genes. While hybrid clusters involving driver genes showed a higher density at the higher-score mode, driver gene status did not guarantee high scores  may permit false-positives if the simulated phosphosites only contain negatives, we observed many of the clusters containing activating or recurrent mutations with cluster closeness scores close to the threshold . Notably, the highest counts of hybrid clusters were found for genes known for recurrent mutations, including TP53 (10 hybrid clusters), PIK3CA (8), CTNNB1 (6), EGFR (6), and other genes involved in cancers, such as HIST1H2BC (6) and PLG (5) Fig.\u00a0. These cAmong the 1,255 co-clustered phosphosites, 291 sites directly overlap and 356 sites are proximal (within 2 amino acid residues linearly) to their co-clustered mutations (Supplementary Data\u00a014 and the NCI-Nature Pathway Interaction Database15 using Enrichr16 (Supplementary Data\u00a0P\u2009<\u20091E\u221212), which is reaffirmed by the Focal Adhesion-PI3K\u2013Akt\u2013mTOR-signaling pathway being one of the top enriched WikiPathways (adjusted P\u2009=\u20098.8E\u221216). These findings suggest the possible involvement of hybrid clusters and co-clustering phosphosites in oncogenic signaling pathways.We then examined whether proteins containing hybrid clusters are enriched in specific biological pathways curated by WikiPathways17. Mapping residues to PFAM domains, we identified 26 PFAM domains significantly enriched for mutations and phosphosites in hybrid clusters when comparing to the background of all mapped mutations and phosphosites . We found 29 hybrid clusters containing 90 of these activating mutations in 17 genes, suggesting the functional relevance of the 54 co-clustering phosphosites , as well as p.R132C implicated in several cancer types . In its homolog protein IDH2, p.Y179 co-clustered with p.R140Q affecting 6.5% of LAML , supporting the view that co-clustered mutations should be prioritized.We evaluated whether HotPho can effectively prioritize functional mutations in hybrid clusters by comparing with functional scores predicted by VESTs25 Fig.\u00a0. Within 27. Using the TCGA Reverse Phase Protein Arrays (RPPA) dataset for each of the 33 cancer types, we conducted a differential expression analysis to search for protein/phosphoprotein markers expressed at different levels in carriers of clustered mutations (\u201cMethods\u201d), identifying 24 significant  gene-cancer associations  are highlighted herein: we validated that TP53 co-clustering mutations associated with higher protein expression in all three cancer types. In colorectal cancer, KRAS mutations in cluster 9458.0 affecting p.G12, p.G13, p.V14, and p.Q61 are associated with higher KRAS expression, whereas HNF4A mutations in cluster 7977.0 are associated with low HNF4A expression. Other notable findings include that ESR1 mutations in cluster 1357.1 are associated with high ESR1 in ovarian cancer, whereas AKT1 mutations in cluster 756.0  each comprised of 78\u2013126 samples  directly overlapping phosphosites, (2) proximal to phosphosites, and (3) co-clustering with phosphosites, we noticed that considering co-clustering mutations contribute significantly to the fraction of potentially functional mutations in many cancer genes including EGFR, KRAS, and PIK3CA  of the co-clustering mutations were determined as activating compared to only 30.2% (138/457) of the other mutations determined as activating , with 33/35 co-clustering mutations being validated in Ba/F3 and 21/22 in MCF10A; its binding partner PIK3R1 also shows suggestive enrichment in Ba/F3 . Significant enrichment of activating mutations was also observed for EGFR  and BRAF  co-clustering mutations in Ba/F3. In MCF10A, we also noted suggestive associations for BRAF, where 7/18 co-clustering mutations are activating (P\u2009=\u20090.068), and ESR1, where 7/18 co-clustering mutations are activating (P\u2009=\u20090.068). The enrichment of activating mutations in hybrid clusters suggests structural adjacency to phosphosites implies functional significance in oncogenes.We then examined whether the co-clustered mutations show significant enrichment of activating mutations compared to the other mutations on a gene-by-gene basis Fig.\u00a0. Co-clusP\u2009=\u20090.91) or MCF (P\u2009=\u20090.40) cell line  or MCF10A (P\u2009=\u20090.023) cell lines. But, when adding the phosphosite co-clustering status to the regression model, the mutation functionality was no longer associated with recurrence (P\u2009>\u20090.21), but strongly associated with the co-clustering status in both BAF3 (P\u2009=\u20091.4e\u221210) and MCF10A (P\u2009=\u20091.5e\u22124) cell lines . Strikingly, cancer cell lines with co-clustered mutations showed significantly higher dependency (or more vulnerability upon genetic knockout) than those with missenses in 14 lineages , most notably lung, colorectal, skin, pancreas, and gastric cancer cells  projectTo verify co-clustering phosphosites, we sought evidence of these events being observed in the CPTAC proteomic cohorts of prospective primary tumor samples of 123 breast invasive carcinoma (BRCA), 83 ovarian carcinoma (OV), 97 colorectal adenocarcinoma (CRC), 103 uterine corpus endometrial carcinoma (UCEC), and 41 clear cell renal cell carcinoma (CCRCC). Of the 1255 co-clustered phosphosites, 259 were detected in at least one of the 5 cohorts Fig.\u00a0. Some ph31; CTCF p.T374 that, along with a few nearby residues, were shown to be phosphorylated during mitosis and to decrease its DNA-binding activity32; BRAF p.T599 and p.S602 that are conserved from C. elegans to mammals and required for activation of the B-Raf kinase34, and RB1 (Rb) p.S567 that is uniquely phosphorylated by MAPK11 (p38), triggering Rb-Hdm2 interaction and apoptosis35. These findings further validate the functionality of selected co-clustering phosphosites HotPho identified and suggest other sites in hybrid clusters may be prioritized for downstream investigations.Finally, we conducted a systematic literature review of co-clustering phosphosites regulated or implicated in cancer (\u201cMethods\u201d), finding 25 unique phosphosites across 18 proteins that were experimentally linked to cancer  discovered in these datasets highlight the urgent need for enhanced annotation and prioritization using approaches like HotPho. There are still significant limitations to identifying functional hybrid clusters, as prioritization necessarily relies on known mutations or functional domains. Thus, while we also discovered phosphosite-only clusters, we cannot yet effectively determine their significance until we enhance our understanding of functional phosphosites.37.Our investigation supports the functional relevance of co-clustering phosphosites and mutations. For example, we found that these phosphosites and mutations are enriched in functional domains of kinases and histones, that co-clustering mutations tend to be functionally active and confer genetic dependency. Among the 1255 co-clustering phosphosites, only 25 were previously known to be associated with cancer  version 2018.01, PhosphoSitePlus (snapshot on the date 2018\u201302\u201314), and CPTAC2 MS phosphoproteome data. A PTM site was included if it satisfied either of the following: (1) included in UniProtKB and was reported in at least one publication or by sequence similarity. (2) included in PhosphoSitePlus and was reported in at least one publication or validated internally by Cell Signaling Technology. (3) included in CPTAC2 experiments and was detected in at least one of the samples. To match phosphosites between multiple databases, we used transvar40 to map amino acid residues on different protein isoforms to their unique genomic positions.We gathered 225,151 human phosphorylation sites from PTMcosmos, following a procedure similar to our recently published study11 mutation annotation file (MAF) (syn7824274). These mutations were further filtered based on flagged artifacts, hypermutators, and pathology to a driver discovery dataset of 9062 samples with 791,489 missense mutations, as described in the recent PanCanAtlas somatic driver paper25.We used somatic mutations from the TCGA cohort as provided by the publicly-available MC318, OncoKB19, KinDriver20, and ClinVar41. We subsequently required an activating mutation to be seen in at least 2 of these sources, collecting a total of 367 activating mutations.We curated experimentally validated mutations identified as neutral or activating from multiple databases and papers, including the Cancer Biomarkers database within the Cancer Genome InterpreterWe used the GRCh37 assembly and Ensembl release 75 to preprocess residue pair data for all human proteins in RCSB PDB as of 22 May 2017, which includes PDB structures of 6002 genes.Some chains or structures from PDB were filtered out due to the following types of artifacts in the data file annotations, (1) chains with inconsistent PDB to UniProt coordinate ranges from DBREF given any alterations from SEQADV length changes, (2) chains where SEQADV describes REMARK 999, which indicates absent residues explained by free text, and (3) structures in which site mismatches were identified  even after converting between the PDB and UniProt coordinates designated by the DBREF line.HotPho reads a site input file where each phosphosite must contain the HUGO symbol, Ensembl transcript accession ID (ENST), its residue position within the given transcript, and feature description. The sites are then combined and run through the HotSpot3D search step to produce pairwise data between mutations and phosphosites, comprising mutation\u2013mutation pairs, mutation-site pairs, and site-site pairs. Even though HotPho calculates offsets in residue numbers in PDB structures and transcripts, some offsets provided by structure uploaders resulted in the erroneous mapping of residues. In the resulting pairwise files with phosphosites (.musite and.sites files), we, therefore, filtered out the sites where the mapped residue on the PDB structure differs from those documented in our original input phosphosite file, ultimately retaining 785,867 mutation\u2013mutation pairs, 376,614 mutation-site pairs , and 1,010,011 site-site pairs .8 and enables co-clusterings of mutations and phosphosites on protein structures , where V is a subset of the input mutations and phosphosites and E is the set of proximal pairs from V. Considering vi, vj\u2009\u2208\u2009V for i, j \u2208 {1,2,\u2026,N} where N is the number of vertices in V, the clusters are built up using the Floyd\u2013Warshall shortest-paths algorithm, initiated by the distance matrix of the edges, to obtain the geodesics, gi,j between each vi and vj. For each vi\u2009\u2208\u2009V where i\u2009\u2260\u2009j, the cluster centrality, c(vi), is then calculated as:HotPho extends beyond the originating HotSpot3D algorithmres Fig.\u00a0. Brieflyc(vi), and the cluster closeness score (Cc) is calculated as:For each of the cluster, the centroid is identified as the vertex showing the highest 8. Here in our hybrid cluster analysis, we not only show the top 5% clusters show sensitivity in distinguishing cancer driver genes vs. other genes, but also in observed vs. randomly simulated clusters.A high Cc score indicates a dense 3D cluster enriched in recurrent mutations and phosphosites on the protein structure. In the pan-cancer study of mutation-only clusters, clusters with known cancer proteins showed significantly higher Cc score than those without cancer-related proteins, and a threshold at top 5% showed a notably significant difference between cancer- and non-cancer-related proteins13 versus other genes using a ROC curve analysis  scores (ie. more closely packed clusters) we chose the top 100 genes having high CC clusters. Next, for each of these genes, we found the number of phosphosites in the original dataset, which are covered by at least one structure. After that, we generated a permutated phosphosites-dataset by randomly populating the sites at possible covered phosphosite residue locations keeping the original residue ratios the same. HotPho clustering was performed for 100 such simulated phosphosites-datasets and the maintained TCGA MC3 mutation call backbone given the non-random distribution and occurrence count of mutation calls. Finally, the clusters from the original HotPho run and the simulated runs were compared focusing on the number of clusters and their Cc score distribution. We further evaluated the sensitivity, specificity, and ROC curves using different threshold of the Cc score  versus co-clustered status (co-cluster with mutation or not). Although this test is exact, we followed the general rule-of-thumb for table testing of only evaluating those cases where there were at least 5 mutations and phosphosites in the domain. Resulting P-values were corrected to FDR values using the standard Benjamini-Hochberg procedure.We conducted a domain enrichment analysis of co-cluster phosphosites in PFAM domains (Pfam 31.0 released March 2017)27, TCGA level-3 normalized RPPA expression data of the tumor samples were downloaded from Firehose (2016/1/28 archive). The protein/phosphoprotein expression percentile of individual proteins in each cancer cohort was calculated using the empirical cumulative distribution function (ecdf), as implemented in R. Where there are at least 3 carriers within each cancer type, we then applied the linear model to evaluate the protein/phosphoprotein expression percentile between carriers of co-clustered mutations and all other cancer cases. The age at initial diagnosis, gender, and ethnicity are included as covariates to account for potential confounding effects. The resulting P values were adjusted to FDR using the standard Benjamini-Hochberg procedure for tests across all cancer types.Similar to our previous analyses of a different set of mutations43 and prospective collection . For each hybrid cluster, protein levels were compared between samples with and without cluster mutations (Wilcoxon rank-sum test).We analyzed the effects of clustered mutations using samples from the CPTAC2 retrospectivep-values represent the alternative hypothesis that the dependency score distribution of the cell lines with co-clustered mutations is located left  of that of without co-clustered mutations, and they are multiple-testing adjusted using the BH method for FDR.Within each lineage, we carried out a Wilcoxon rank-sum test between the cell lines with co-clustered missense mutations versus those with either (1) other missense mutations, or (2) other non-synonymous mutations. The 25. The abstracts of all publications associated with a phosphosite were then retrieved from Europe PMC using their Digital Object Identifier (DOI) or PubMed identifier (PMID). We determined a paper to be cancer-related if its abstract contained the keyword \u201ctumor\u201d and/or \u201ccancer\u201d. We then closely examined whether the exact co-clustering phosphosites identified by HotPho showed any alterations on cancer-related phenotypes in these publications. Additionally, we included all disease-associated sites in PhosphoSitePlus (snapshot on the date 2018\u201302\u201314) that were connected to any type of cancer.First, we confined our search space to 71 cancer genes with hybrid clusters by limiting our search space to 299 cancer driver genesFurther information on research design is available in the\u00a0Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1-12Reporting Summary"}
+{"text": "Several types of PMO NP (bis(triethoxysilyl)ethane (BTEE) as matrix, co-condensed with trialkoxysilylated aminopyridine (py) and trialkoxysilylated bipyridine (Etbipy and iPrbipy)) were synthesized by means of the sol-gel procedure, then characterized with different techniques . A systematic evaluation of CO2 adsorption was carried out at 298 K and 273 K, at low pressure. (3) Results: The best values of CO2 adsorption were obtained with 6% bipyridine: 1.045 mmol\u00b7g\u22121 at 298 K and 2.26 mmol\u00b7g\u22121 at 273 K. (4) Conclusions: The synthetized BTEE/aminopyridine or bipyridine PMO NPs showed significant results and could be promising for carbon capture and storage (CCS) application.(1) Background: Due to human activities, greenhouse gas (GHG) concentrations in the atmosphere are constantly rising, causing the greenhouse effect. Among GHGs, carbon dioxide (CO Indeed, CO2 emissions between 1974 and 2004 were greater than those during the 600,000 previous years ethylenediamine [2 capture and valorization is growing significantly [2 has been less described than the use of bulk materials.To overcome the problems of conventional liquid amines, new studies have gradually focused on the synthesis of solid adsorbents modified by amines. Recently, porous silica and periodic mesoporous organosilicas (PMOs) have become popular solid adsorbents for CO capture ,23. An ig amines , amines rocedure . The improcedure . Howeverediamine ,28. Furtficantly . The use2 capture. We believed nanoparticles could be very efficient due to their very high developed specific surface area. Indeed, a recent publication reported the high potential of bis(triethoxysilyl)ethane-based nanocubes for CO2 sequestration [2 photoreduction. However, to our knowledge, pyridine and bypyridine-based PMO nanoparticles have not yet been investigated for CO2 capture. Therefore, PMO NPs were prepared through the sol-gel procedure with either 1-(6-aminopyridin-2-yl)-3-(3-(triethoxysilyl)propyl)urea (py), 5,5\u2032-bis(triisopropoxysilyl)-2,2\u2032-bipyridine (iPrbipy) or 5,5\u2032-bis(triethoxysilyl)-2,2\u2032-bipyridine (Etbipy) and 1,2 bis-triethoxysilylethane (BTEE) as the PMO NPs matrix. The synthetized PMO NPs were observed by TEM, chemical functionalization was confirmed by FTIR molecular spectroscopy, and the measurement of CO2 adsorption on the PMO NPs was performed in pure CO2 gas at 273 K and 298 K.In the course of our work concerning the synthesis and applications of periodic mesoporous organosilica nanoparticles (PMO NPs) , we werestration . The prestration ,35,36,37Three BTEE/aminopyridine PMO NPs were synthetized with 1,2-bis(triethoxysilyl)ethane (BTEE)  and 1-, 1600 cm\u22121 (amide II band), and 1690 cm\u22121 (amide I band). The presence of these bands proved the success of the co-condensation between the BTEE and the aminopyridine precursors. Moreover, an increase in the nitrogen content was confirmed by elementary analysis  having the highest aminopyridine content (15%) compared to the 100% BTEE (0.81 mmol\u00b7g\u22121). This shows the potential of adding aminopyridine to BTEE for CO2 capture. However, different results were observed at 273 K, where the CO2 adsorption for the 100% BTEE was higher than with others PMOs, showing in this case the potential of PMO 1 for CO2 capture. CO2 adsorption increases with decreasing temperature. Indeed, adsorption being an exothermic process, increasing the temperature of the system decreases the adsorption capacity [2 pressure, where the highest interaction sites can be found. The shape of the sorption isotherms is almost linear up to 266 hPa, which confirms the rather poor affinity of these materials for carbon dioxide, regardless of temperature. In these cases, similar Henry\u2019s constants can be derived (~1.5\u00b710\u22123 mmol\u00b7g\u22121\u00b7Pa\u22121 at 298 K).COnd 273 K , Table 2capacity . In termThree BTEE/iPrbipyridine PMO NPs were synthetized with BTEE and 5,5\u2032-bis(triisopropoxysilyl)-2,2\u2032-bipyridine (iPrbipy)  by incre\u22121 appeared. This band can be attributed to the C=N bonds from the two aromatic cycles. An increase in the intensity of this band with the bipyridine molar fraction was observed, due to the four-fold increase in nitrogen content . The derivation of the pore size distribution using the desorption branches of the sorption isotherms gave two populations of pores, the most important located at ~2.5 nm and a second one located at 3.7 nm. The latter is reminiscent of cavitation effects when desorption occurs in small mesopores connected to large cavities, which is precisely the case with hollow materials.The three iPrbiPyPMO materials were mesoporous, as indicated by a clear uptake at intermediate relative pressures followed by saturation . However2 capture, the bipyridine-based PMOs behaved differently compared to the aminopyridine-based PMOs . A straightforward explanation is that the greatest specific surface area is found with this material, compared to the other PMOs of the series .In terms of COsed PMOs , Table 4e series . In termThree BTEE/Etbipyridine PMO NPs were synthetized with BTEE and 5,5\u2032-bis(triethoxysilyl)-2,2\u2032-bipyridine (Etbipy) , by incr\u22121 increased with the Etbipyridine molar fraction, a feature of the co-condensation between the BTEE and the Etbipyridine precursors. This band did not appear for the aminopyridine precursor, probably due to the difference in chemical structure with the presence of two aromatic cycles with nitrogen for the iPrbipyridine and the Etbipyridine, against only one aromatic cycle for the aminopyridine precursor. An increase in the percentage of nitrogen with the increase in the molar fraction of Etbipyridine was observed. However, these percentages were lower than with the other precursors  for a large Etbipyridine molar fraction (15%), and for a low iPrbipyridine molar fraction (6%).Despite the similarity of the chemical structures between the iPrbipyridine and the Etbipyridine precursors, the CO2 capture and the field has been comprehensively reviewed [2 amount obtained for the best materials is around 1 mmol/g which corresponds to 45 mg/g. Using APTES-grafted MCM-41, Mello et al. obtained 33 mg/g at 20 \u00b0C [2 could be adsorbed at very low pressure (0.05 atm) [Our results compare well with some of the studies which use amine-modified porous silicas for COreviewed ,41. Noteat 20 \u00b0C  with APTat 20 \u00b0C . However.05 atm) .2 amounts have been captured (135 mg/g).Under more favourable conditions : 1.04 mmol\u00b7g\u22121;EtbipyPMO 15 (85% BTEE/15% Etbipyridine): 0.95 mmol\u00b7g\u22121.pyPMO 15 (85% BTEE/15% aminopyridine): 0.92 mmol\u00b7gFinally, according to the different types of synthesized PMO NPs, the highest CO\u22121), higher CO2 adsorption capacity was observed with 29% (iPrbipyPMO 6 NPs), 17% (EtbipyPMO 15 NPs) and 14% (pyPMO 15 NPs), which showed the strong impact of the polycondensation between the BTEE and the precursor containing aminopyridine groups, amines having an important affinity with the CO2 molecule. On the one hand, the best CO2 adsorption values were obtained with the bipyridine and the Etbipyridine precursors, proving the importance of the presence of two aromatic cycles with nitrogen for CO2 capture. On the other hand, CO2 adsorption was favored with a large aminopyridine (or Etbipyridine) molar fraction (15%), and with a low iPrbipyridine molar fraction (6%). In terms of CO2/PMO affinity, these three materials exhibited different features, as can be seen in Compared to the 100% BTEE  which suggests two sorption regimes. At very low coverage, sorption is likely to mostly happen on the iPrbipyridine moieties, while at higher coverage, sorption proceeds on the whole surface of the material. To investigate these observations, we performed two sorption cycles using the procedure described in the experimental section , and potassium bromide, were purchased from Sigma-Aldrich . 5,5\u2032-bis(triisopropoxysilyl)-2,2\u2032-bipyridine was purchased from TCI. 5,5\u2032-bis(triethoxysilyl)-2,2\u2032-bipyridine was purchased from Sikemia. Absolute ethanol was purchased from Fisher Chemicals. Cetyltrimethylammonium bromide (CTAB), sodium hydroxide, 3-(triethoxysilyl)propylisocyanate, 1,2-bis(triethoxysilyl)ethane (BTEE), 2,6-diaminopyridine, ammonium nitrate , 437 \u00b5L of NaOH solution (2 mol\u00b7L\u22121), and 333 \u00b5L of 1,2-bis(triethoxysilyl)ethane  were dissolved in 60 mL of deionized water. The mixture was stirred for 2 h at 80 \u00b0C/750 rpm. The reaction mixture was cooled to room temperature and centrifuged for 20 min at 20,000 rpm. The CTAB surfactant was removed by 45 min sonication at 40 \u00b0C, with a 30 mL solution of ammonium nitrate (NH4NO3) in EtOH (6 g\u00b7L\u22121), followed by one water wash and two EtOH washes (30 mL each). BTEENPs were dried in a vacuum oven at 70 \u00b0C.For COor BTEE,  (9 \u00d7 10\u2212\u22123 mol) and 0.472 g of NaOH solution (1.18 \u00d7 10\u22122 mol) were dissolved in 31.8 mL of deionized water. The total number of moles for the two precursors (BTEE/aminopyridine or iPrbipyridine or Etbipyridine) was 5.18 \u00d7 10\u22123, the molar fraction being different. For example, with iPrPMO 6 (94% ethane/6% bipyridine), a solution of 1.8 mL of BTEE (4.86 \u00d7 10\u22123 mol) and 0.181 g of 5,5\u2032-bis(triisopropoxysilyl)-2,2\u2032-bipyridine (3.20 \u00d7 10\u22124 mol) was added and the mixture was stirred for 24 h at room temperature, at 750 rpm. The reaction was stirred for 2 h at 95 \u00b0C and 750 rpm. The reaction mixture was cooled to room temperature and centrifuged for 20 min at 20,000 rpm. Surfactant was removed by by 45 min sonication at 40 \u00b0C, with a 30 mL solution of ammonium nitrate (NH4NO3) in EtOH (6 g\u00b7L\u22121), followed by one diethyl ether wash and two EtOH washes (30 mL each). Finally, the NPs were dried in a vacuum oven at 70 \u00b0C. This process was mainly used with the iPrbipyridine and the Etbipyridine precursors  is a non-destructive analysis technique for measuring the hydrodynamic diameter of particles suspended in a liquid. Analysis of the scattering of light from the laser of the device by particles was performed with the Cordouan Technologies DL 135.\u22121). Absorptions in this zone correspond to the movement  of organic functional groups and can be used to deduce the chemical bonds and the structural details of the material. Analyses were performed using the Spectrum Two FT-IR Spectrometer .Fourier transform infrared spectroscopy (FTIR) is a non-destructive analysis technique based on the absorption of electromagnetic radiation between 2.5 and 25 \u00b5m (wavelength between 400 and 4000 cmTransmission electron microscopy (TEM) is an analytical technique which enables observation of the local pore arrangement of a material by means of the interactions that occur when an electron beam accelerated by a high voltage travels through the material. For TEM characterization, the nanoparticles of the material were dispersed in EtOH and carefully deposited on a copper grid with porous carbon films. The JEOL 1400 Plus (120 kV) microscope was used to record the image size, the shapes and the pores of the synthetized nanoparticles.Prior to the sorption measurements, the nanoparticles were evacuated under a secondary vacuum at 80 \u00b0C for 8 h. Because our materials are hybrid materials, the organic moieties in the materials cannot withstand high activation temperatures. We optimized the activation temperature, and 80 \u00b0C was found to be acceptable. The nitrogen sorption isotherms showed that the mesoporosity is accessible to nitrogen, which supports our choice.2 as cross sectional area for nitrogen. T-plot analysis of the sorption isotherms revealed that the prepared materials were not microporous, which allowed the use of the BET model for deriving the specific surface areas. The pore size distributions were derived using the BJH (Barrett-Joyner-Helenda) approach on the desorption branches of the sorption isotherms, starting from p/p\u00b0 = 0.95 downwards. The pore geometry was assumed to be cylindrical.The sorption isotherms were obtained at 77 K using a Micromeritics TriStar device. The specific surface area of the various materials was derived using the BET (Brunauer-Emmet-Teller) method, taking 0.162 nm13C-NMR analysis was carried out on 50 mg of NPs using a VARIAN 300 MHz (Wild Bore) solid spectrometer (3.2 mm MAS probe) device.Nuclear magnetic resonance (NMR) spectroscopy is an analytical technique for determining the structure of an organic molecule, exploiting the magnetic properties of certain atomic nuclei, such as hydrogen, carbon, or silicon. Solid Elemental analysis is a qualitative and quantitative technique for revealing, by means of combustion or pyrolysis, the elemental composition of an organic compound and therefore the mass percentage of elements such as carbon, hydrogen, nitrogen, sulfur and oxygen. Elemental analysis was carried out using an Elementar Vario Micro Cube device with a sample of 15 mg.2 adsorption isotherms were measured using a Micromeritics 3Flex device. Two sorption temperatures were investigated, namely 273 K and 298 K, in order to provide the isosteric heat of adsorption. Prior to the sorption measurements, the materials underwent a degassing stage at 80 \u00b0C for 8 h under a secondary vacuum.The CO2 capture application. A significant impact on the polycondensation between the BTEE and the aminopyridine or bipyridine  precursors was observed, compared to the synthesis of PMO 1 NPs with 100% BTEE. Specifically, an increase in the CO2 adsorption of 29% , 17% , and 14%  was observed compared to PMO 1 NPs (0.81 mmol\u00b7g\u22121). The addition of aminopyridine or bipyridine groups improved the affinity of the PMO with CO2. In conclusion, PMO NPs represent an interesting tool for CO2 capture applications. Furthermore, the prepared PMO NPs could be useful for carbonate synthesis from epoxides [2 to formic acid or methanol [Various syntheses of PMO NPs were successfully achieved for the COepoxides  or hydromethanol ."}
+{"text": "JAMA Psychiatry reveals that drug overdose mortality rates have increased sharply in the USA since the pandemic started, in 2020. Using data from the Centers for Disease Control and Prevention (Wide-ranging Data for Epidemiologic Research) and the National Center for Health Statistics, they calculated drug overdose death rates by race and ethnicity for 1999\u20132020 in the USA.The COVID pandemic has highlighted widespread existing health inequalities in the UK and elsewhere. A research letter just published in They found that drug overdose mortality rates had been rising since 2015 most rapidly among \u2018Black, Hispanic and Latino\u2019 communities. In 2020 the largest percentage increase (48.8%) was in Black individuals compared with White individuals (26.3%). They also report that in American Indian and Alaskan natives, who between 1999 and 2017 had the same overdose mortality as White individuals, the overdose mortality rates climbed in 2019 (at 28.9 per 100\u00a0000 compared with 25 for the White population) and became the highest (41.4 per 100\u00a0000) in 2020; that was 30.8% higher than the rate increase in White individuals.The authors discuss the role of illicit drug use and toxic polysubstance availability of synthetic opioids, benzodiazepines and highly purified methamphetamines as contributing to the worsening overdose crisis in the USA. They observe that the high and unpredictable potency of illicit drug supply may be disproportionately harming racial and ethnic minority groups with deep-seated inequalities in social conditions  playing a major part.They conclude that overdose mortality has increased since the COVID-19 pandemic and they believe that their records from 2020, which were provisional, may be underestimating the mortality rates. They observe that overdose mortality is \u2018increasingly becoming a racial justice issue in the US\u2019 and ask for the social/health inequalities to be addressed.FriedmanJR, HansenH. Evaluation of increases in drug overdose mortality rates in the US by race and ethnicity before and during the COVID-19 pandemic. JAMA Psychiatry [Epub ahead of print] 2 Mar 2022. Available from: 10.1001/jamapsychiatry.2022.000.Despite our richer experience and wide-ranging vocabulary accumulated over the years, as we grow old, we often find ourselves unable to find the right word at the right time. Frustrating as it is, our brain refuses to play ball. What is the problem?Investigators from the Max Plank Institute for Human Cognitive and Brain Sciences and Leipzig University investigated the underlying brain mechanisms involved in this. They compared a group of younger people aged 20\u201335 and older people aged 60\u201370 years. The participants were asked to name words in different categories  while undergoing magnetic resonance imaging (MRI) scanning. Although both age groups were able to find words, the older people were slower compared with the younger ones. This difference in performance, the researchers found, was down to functional brain network connectivity. More specifically, the younger people's brain activity showed more intensive exchange between the network where factual knowledge is stored (semantic memory) and the network responsible for general functions such as attention and memory (executive network) compared with the older group. In the latter, there was more intensive activity within the executive network, indicating more difficulty in these subjects, whereas the connectivity between the two networks was less effective.Don't despair, as there are some things you can do to help yourself \u2013 exercise, sleep well and stay away from bugs that may get up your nose. For more info on these, please read the following three summary reports.MartinS, SaurD, HartwigsenG.Age-dependent contribution of domain-general networks to semantic cognition. Cereb Cortex2022; 32(4): 870\u201390.34464442As we age and our memory starts failing, we grasp at every piece of advice on how to stop or at least slow this down. Exercise figures high on the list, and Pandora has repeatedly reported on research demonstrating the merits of exercise on both body and mind. A lot has been said about aerobic exercise being one of the most promising approaches for enhancing cognitive function in mature age. Different agencies and apps offer advice on numbers of steps or miles to walk per day, some of us opting for the minimum requirements and others enthusiastically exceeding these. But does aerobic exercise improve memory?A recently published systematic review and meta-analysis tried to clarify matters. They selected 36 randomised control trials with data from 2750 participants without dementia to determine whether aerobic exercise influences episodic memory  in late adulthood (mean age 70 years) and what characteristics of the subjects and interventions may influence the effects. They found that aerobic exercise did indeed improve episodic memory in those with a mean age between 55 and 68 (young-old) but not those between 69 and 85 (old-old). However, where women were overrepresented (65\u2013100%) in the older group, more benefit was noted from aerobic exercise.If you want to know how much aerobic exercise you should do, based on their data, they advised that sessions lasting 15\u201390\u00a0min three times a week for 18\u201339 weeks, to achieve a total of >3900\u00a0min, were effective. Make sure you start early, and don't wait until you get into the old-old age, particularly if you are a man!AghjayanSL, BourniasT, KangC, ZhouX, StillmanCM, DonofrySD, Aerobic exercise improves episodic memory in late adulthood: a systematic review and meta-analysis. Commun Med2022; 2: 15.There is a lot we still don't understand about the physiology of the need to sleep, but we do know that one important function of sleep is to clear our brain from waste generated through our daytime activity. We also know that the cerebrospinal fluid, driven by cardiovascular, respiratory and vasomotor brain pulsations, flows along perivascular spaces and washes away our brain waste material.A recent study from Norway investigated how these pulsations may change during sleep. In a group of 15 healthy volunteers , they recorded simultaneously fast functional MRI, magnetic resonance encephalography (MPEG) and electroencephalography (EEG) during sleep and while awake. They quantified sleep-related changes in the signal frequency and also the strength of the brain pulsations.They found that in non-rapid eye movement sleep, signal frequency was reduced whereas brain pulsation was increased compared with wakefulness. EEG slow oscillation power (delta waves) was increased in regions of the brain that showed sleep-related changes in MPEG pulsation. The increase in respiratory pulsation was greatest in the areas of the brain most used during the day, i.e. the visual cortex, auditory cortex and sensorimotor cortex, which needed more cleaning up during sleep.They concluded that their results support the view that sleep promotes fluid transport in the brain via an increase in brain pulsation and waste clearance. They aim to investigate these functions in brain disorders associated with sleep disturbances such as Alzheimer's dementia and others.HelakariH, KorhonenV, HolstSC, PiispalaJ, KallioM, V\u00e4yrynenT,  Human NREM sleep promotes brain-wide vasomotor and respiratory pulsations. J Neurosci [Epub ahead of print] 7 Feb 2022. Available from: 10.1523/JNEUROSCI.0934-21.2022.Chlamydia pneumoniae, a pathogen that can take residence in the nose. The researchers isolated live organisms from tissues and using immunohistochemical methods showed that this pathogen can, via the olfactory and trigeminal nerves, reach the olfactory bulb and brain in mice within 72\u00a0h. Importantly they also showed that this resulted in dysregulation of key pathways known to be involved in Alzheimer's disease at 7 and 28 days following inoculation. They also detected amyloid beta accumulations close to the C. pneumoniae inclusions, at least in the olfactory system, and in vitro showed the pathogen to infect central nervous system glia.As Alzheimer's dementia is increasing in prevalence, intensive research into its causes is searching in all possible directions. A recent study discovered an unusual or at least not previously thought of culprit, C. pneumoniae could invade the brain via the olfactory and trigeminal nerves and survive in glia, causing amyloid beta deposition.They concluded that ChackoA, DelbazA, WalkdenH, BasuS, ArmitageCW, EindorfT, Chlamydia pneumoniae can infect the central nervous system via the olfactory and trigeminal nerves and contributes to Alzheimer's disease risk. Sci Rep2022; 12: 2759.35177758In the quest to understand anxiety, the body responses to fear have been widely investigated. However, practical and ethical considerations limit the scope of such investigations in terms of the threat used in experiments and the physical measures of the body response. A recent study from the Division of Humanities and Social Sciences at the California Institute of Technology aimed to examine scare responses in a more comprehensive way, incorporating a social component with the psychological and using a hitherto not considered type of threat experience.They recruited 156 individuals, whom they divided into small groups, and exposed them to a 30\u00a0min haunted house experience. The house had 17 rooms, and the subjects went through them with a continuous exposure to threatening experiences such as mimicking suffocation threats, a speeding car and a shower of shots from a firing squad. During the experience in the haunted house, they wore monitoring wristbands to measure their electrodermal activity, sweat-induced changes in the skin's electrical characteristics, including skin conductance level and skin conductance response. Prior to entering the haunted house, the participants rated their expected fear on a scale from 1 to 10; they also rated their actual fear after the visit.The researchers examined four factors from these data: (a) group composition; (b) threat imminence; (c) intrapersonal factors of fear; and (d) the participant's sensitivity to threats. Interestingly, they found positive associations between friends and tonic arousal, i.e. being around friends as opposed to strangers increased the arousal of being scared. There was also a positive association between unexpected threats and subjective fear. Those who felt the most frightened in the haunted house had more rapid changes to their body experiences. Individuals who had strong responses to the first room of the haunted house showed increased responses as they went through the other rooms.A helpful take away message is, if you don't enjoy being scared, make sure that the next time you happen to visit a haunted house, you don't go with a friend!TashjianSM, FedrigoV, MolapourT, MobbsD, CamererCF. Physiological responses to a haunted-house threat experience: distinct tonic and phasic effects. Psychol Sci2022; 33(2): 236\u201348.35001710Do you wonder what you'll experience in those last moments of life? \u2018Near death\u2019 experience has been described by those surviving in various ways, including being outside their bodies, floating in space above and watching themselves being resuscitated. But what the dying brain experiences in the moments prior to final death is not known. Research in rats showed that the dying mammalian brain paradoxically generates neural correlates of heightened conscious processing.An idea of what this is like in humans was unexpectedly observed by doctors at the University of Tartu, Estonia, as they were monitoring an 87-year-old patient, who was having epileptic seizures, with continuous EEG. The patient unfortunately suffered a heart attack and died while still being recorded with EEG. In order to investigate what happened in the 30\u00a0s before and after the heart stopping, they examined the EEG recording and measured 900\u00a0s of brain activity during that time. They saw changes in neural oscillations (brain waves), specifically in gamma but also in theta, delta, alpha and beta oscillations, which are involved in high cognitive functions such as dreaming, memory retrieval and conscious perception, like those associated with memory flashbacks. After cardiac arrest, all oscillations were decreased but higher gamma power was observed compared with that in the interictal interval prior to the cardiac arrest. There was modulation of left hemisphere gamma activity by alpha and theta rhythms across all windows, even after cessation of cerebral blood flow.This is the first evidence from the dying human brain in a real life, non-experimental, clinical setting that demonstrates that the human brain may possess the capability to generate coordinated activity during the dying period. The authors comment \u2018these findings challenge our understanding of when exactly life ends and generate important questions such as those related to the timing of organ donation\u2019. This raises serious ethical questions, and more research is needed to establish more clearly when exactly life ends.VicenteR, RizzutoM, SaricaC, YamamotoK, SadrM, KhajuriaT, Enhanced interplay of neuronal coherence and coupling in the dying human brain. Front Aging Neurosci2022; 14: 813531.35273490"}
+{"text": "Head and neck cancer is the sixth most frequent cancer all over the world, with the majority of subtypes of head and neck squamous cell carcinoma (HNSCC). Cellular senescence-associated genes have been confirmed to play a critical role in cancer and have the potential to be prognostic biomarkers for cancer. Clinical information of HNSCC samples and expression data were acquired from public databases. Expression profiles of genes related to cellular senescence were used to identify molecular subtypes by consensus clustering. To screen differentially expressed genes (DEGs) between different subtypes, differential analysis was performed. We used the univariate Cox regression to identify prognostic DEGs and performed least absolute shrinkage and selection operator (LASSO) to optimize and construct a prognostic model. CIBERSORT, ESTIMATE, and TIDE tools were applied to estimate immune characteristics. Four molecular subtypes were established based on cellular senescence-associated genes. Differential prognosis was observed among different subtypes with C4 having the longest overall survival and C1 having the worst prognosis. C4 subtype also showed the highest immune infiltration. We screened a total of eight cellular senescence prognosis-related genes and established a cellular senescence-related signature score (CSRS.Score) that could stratify samples into high-CSRS.Score and low-CSRS.Score groups. The high-CSRS.Score group had worse prognosis, lower immune infiltration, and lower response to immunotherapy. We further improved the prognostic model and survival prediction by combining CSRS.Score with clinicopathological features using a decision tree model, which had high predictive accuracy and survival prediction. This study demonstrated an important role of cellular senescence in HNSCC. The identified eight cellular senescence-associated genes have the potential to provide ideas for adjuvant treatment and personalized treatment of HNSCC patients. Head and neck cancer (HNC) is the sixth most frequently diagnosed cancer type that causes 500,000 affected individuals per year worldwide . Head anSenescence is a nearly unavoidable feature in all creatures, which is marked by a descending function of multiple cells and tissues. In spite of that degeneration is the most common age-related phenotype, aging allows to generate gain-of-function changes that lead to abnormal cell proliferation . MoreoveSenescence-associated genes also have the great potential to predict cancer prognosis. Althubiti et al. identified 10 plasma membrane-associated proteins expressed in senescent cells to be prognostic biomarkers especially in breast cancer . CoppolaTherefore, in this study, we used cellular senescence-associated genes to identify molecular subtypes. Differential pathways and immune features were observed among different subtypes. Differential expressed genes (DEGs) were screened between different subtypes, and least absolute shrinkage and selection operator (LASSO) regression analysis was used to develop a cellular senescence-related signature scoring (CSRS) system. The CSRS system could define CSRS.Score for each HNSCC sample and classify them into high-CSRS.Score and low-CSRS.Score groups. Importantly, CSRS.Score had the potential to guide immunotherapy and chemotherapy for HNSCC patients. A decision tree and a nomogram based on CSRS.Score were constructed to more accurately predict prognosis than CSRS.Score only.From The Cancer Genome Atlas (TCGA) database (named as TCGA cohort), we downloaded RNA-seq data of HNSCC samples and removed samples that did not have survival time, clinical follow-up information, or status of patients' survival. Ensembl ID was transformed into gene symbol. The median value of gene expression was selected for the genes with multiple gene symbols. GSE65858 and GSE41613 cohorts including gene expression profiles of HNSCC samples were obtained from Gene Expression Omnibus (GEO) database and were used as validation cohorts. We downloaded the annotation information of the corresponding microarray platform and mapped the probes to genes based on the annotation information to remove the probes that match one probe to multiple genes. If certain number probes matched to one gene, the median value was taken as the expression value of that gene. Finally, 499, 253, and 97 samples were remained in TCGA, GSE65858, and GSE41613 cohorts, respectively.https://genomics.senescence.info/cells/), we obtained 279 cellular senescence-associated genes.From CellAge database (We next constructed a consistency matrix by ConsensusClusterPlus to cluster HNSCC samples . The expP < 0.05). LASSO regression using the glmnet R package [\u03a3\u2009\u03b2i \u00d7 Expi, where Expi indicates the gene expression level of prognostic genes and \u03b2i is Cox regression coefficients of the corresponding genes. CSRS.Score was normalized using z-score, and the threshold \u201c0\u201d was determined to classify samples into low-risk and high-risk groups. The Kaplan-Meier (KM) survival analysis was conducted to assess the overall survival of different molecular subtypes. Significant differences were determined using the log-rank test.We identified differentially expressed cellular senescence genes between subtypes using limma R package  and sele package  and step package  were perCIBERSORT algorithm was employed to estimate the proportion of 22 immune cell types . ESTIMATWe used the TIDE algorithm to validate predicted treatment responsiveness. The TIDE algorithm is a computational method for predicting immune checkpoint blockade responsiveness using gene expression profiles . TIDE caGSEA allows to calculate the enrichment score of a gene set for annotating biological function . We usedP < 0.05 was considered as significant.R software (v4.1) was applied to conduct all statistical analysis. The Kruskal-Wallis test was performed in testing the significance among four subtypes. Between high- and low-risk groups, the Wilcoxon test was performed to test the significance. Log-rank test was conducted in the Cox regression analysis and survival analysis. ANOVA was conducted in comparing different groups containing multiple subgroups. P < 0.01). Then, based on the expression data of 28 prognosis-related cellular senescence-associated genes, we clustered 499 HNSCC samples into four clusters (molecular subtypes) through the determination of the CDF and the CDF delta area  among tNext, we analyzed whether differential enriched pathways exist in the different molecular subtypes by GSEA. 37 pathways were identified to be significantly enriched in the C1 subtype in the TCGA cohort. The enriched pathways mainly include cancer-related pathways, such as small cell lung cancer, ECM receptor interaction, and Wnt signaling pathway Figure . The resP < 0.05, P < 0.0001, We used immune cell signatures to assess immune cell infiltration in different subtypes to evaluate their immune characteristics. CIBERSORT revealed that 16 of 22 immune cells had a significant difference among four subtypes, such as regulatory T cells, CD8 T cells, resting memory CD4 T cells, and M0 macrophages  < 0.05 and |log2(fold\u2009change)| > 1. 232 DEGs were identified by the above intersection. And 77 prognostic genes were confirmed by the univariate Cox regression analysis including 31 \u201crisk\u201d and 46 \u201cprotective\u201d genes . Next, LP < 0.0001), and higher CSRS.Score had worse overall survival in the training cohort  for each sample according to the eight-gene model. We classified the samples as the high-risk group if the score was greater than 0 and as the low-risk group if the score was less than 0. The distribution of CSRS.Score for samples in the training set (TCGA cohort) is shown in g cohort . We furtg cohort . For theP < 0.001, To further explore the immune characteristics between two risk groups, we analyzed the estimated proportion of 22 immune cells in high- and low-risk groups in the TCGA cohort . Some imWe further explored the immune response of two risk groups to immunotherapy. The expression of most immune checkpoints was differential between high- and low-risk groups . TIDE prWe constructed a decision tree based on clinical information and CSRS.Score, and only stage, gender, and CSRS.Score were remained in the decision tree . Four suA number of evidences prove that senescence-dependent changes are associated with proinflammatory properties and are involved in the chronic inflammatory microenvironment. Increased levels of inflammation and immune cell infiltration with senescence-dependent changes may lead to tumor formation and malignancy, and their function in HNC is unknown . ExploriSenescence and tumor microenvironment are closely related in tumor progression and invasion. There is an obvious difference of immunity between elder patients and younger patients, where younger patients have more abundant T cells in tumor tissue than elder patients. Senescence leads to a declining immune system which is referred to as immune senescence . It is sBased on the DEGs among different subtypes, we confirmed a total of eight key cellular senescence prognosis-related genes including CDKN2A, PYGL, KRT8, AREG, MAGEA4, DES, EPHX3, and SPINK6. CDKN2A has been shown to mediate the antitumor effects in HNSCC through cell cycle arrest . Low CDKAdditionally, the correlation of molecular subtypes with gene mutations was analyzed, and a significant correlation between the two was detected. The common TP53 and CDKN2A were mutated at a high frequency in the four subtypes. We further evaluated the degree of clinical response of conventional chemotherapeutic drugs paclitaxel, docetaxel, cisplatin, and 5-fluorouracil to CSRS.Score subgroups, and the results showed that high-CSRS.Score to paclitaxel, docetaxel, cisplatin, and 5-fluorouracil was more sensitive. The results suggest that different subgroups have different degrees corresponding to different chemotherapeutic drugs, and perhaps our screened aging-related genes can be used as biomarkers of clinical drug treatment response.Although the prognostic risk model based senescence-associated genes was illustrated to have robust performance in several independent cohorts, this study did not validate the model in the wet experiments. More clinical HNSCC samples should be included to support the reliability and accuracy of the eight prognostic biomarkers in the future.In this study, we developed a prognostic risk model with cellular senescence-associated genes that has great potential as a biomarker for HNSCC patients and provides insights into individualized immunotherapy for head and neck cancer patients."}
+{"text": "Lactobacillus (>90%); the CM community in samples from both pre- and postmenopausal pre-treatment cancer patients was heterogeneous, with a low proportion of Lactobacillus in younger cases. On the genus level, 27 and 11 taxa differentiated healthy controls from cancer patients in pre- and postmenopausal age groups, while 31 and 2 genera differentiated pre- and post-radiation samples and pre-radiation and the follow-up samples, respectively. Microbiome diversity was significantly higher in pre-treatment patients than in healthy controls. The results reveal significant alterations in the CM of cervical cancer patients relative to that in healthy controls; these changes were more striking after CRT. However, further research is needed to determine whether alteration of the CM offers new therapeutic options.The cervical microbiome (CM) is a complex ecosystem that can change in response to gynecological cancers. We aimed to evaluate changes in the CM of patients who underwent chemoradiation (CRT) therapy for locally advanced cervical cancer. Before and after CRT, cervical swab samples were collected from 16 patients with squamous cell carcinoma of the cervix, and 30 healthy women. All samples were subjected to 16s rRNA-Seq analysis. In healthy premenopausal women the CM comprised mostly  Lactobacillus species are the most abundant. The lactobacilli abundance is age-related and depends on the estrogen levels, contributing to production of organic acids, primarily lactate, from glucagon deposited in the mature vaginal epithelium. In turn, acidifying of the vaginal ecosystem provides a protective barrier against viral and bacterial pathogens -related cervical intraepithelial neoplasia and cancer risk  and chemoradiation therapy (CRT) for gynecologic cancers may alter the composition of the cervical microbiome (CM) . A previHere, we used 16S rRNA gene amplicon sequencing to evaluate changes in the CM of patients who underwent CRT for locally advanced cervical cancer. While the lactobacilli abundance dominated CM of premenopausal healthy women, both pre- and postmenopausal cancer patients showed higher CM diversity than those found in both groups of healthy controls. The cancer-related composition of CM was further altered by CRT.2 body surface area) once a week for 5\u20136 weeks. The patients were followed between 2.5 and 4 years, and two patients relapsed for more than 2.5 years after the end of treatment. Patient characteristics are shown in Sixteen patients  with squamous cell carcinoma of the cervix who were indicated for primary RT were recruited. Of them, 6 patients (age 25\u201354) were classified as premenopausal or perimenopausal and 10 others (age 54-62) as postmenopausal, according to STRAW guidelines . Before Cervical swab samples were collected aseptically from each patient using 4N6FLOQSwabs\u2122  1 day before starting EBRT, and then again on the day on which the last fraction of brachytherapy was given. In addition, swab samples were taken from nine and seven patients 3 and 6 months later, respectively. Exclusion criteria included other gynecologic cancers , pregnancy, and treatment with antibiotics or antifungals for at least 2 months before enrollment.The control group comprised 30 healthy women  who were recruited under the human papillomavirus-based cervical cancer screening program. They were HPV negative and had normal Pap smears; none of them was on hormone replacement therapy.All enrolled cervical cancer patients and controls were Polish Caucasians. Each swab sample was transferred to a clean collection tube and stored at \u221280\u00b0C within 1 hour of collection.Microbial genomic DNA was extracted from swab samples using a PureLink\u2122 Microbiome DNA Purification Kit, according to the manufacturer\u2019s instructions. The concentration of bacterial DNA was measured using a Nanodrop ND-1000 spectrophotometer . DNA was stored at -20\u00b0C until further analysis. Bacterial 16S rRNA libraries were prepared using an Ion 16S\u2122 Metagenomics Kit  and an Ion Plus Fragment Library Kit, as previously described \u201328. NextLactobacillus species identification was based on reclassification to Greengenes 13.8.9 database  from pre-treatment cancer samples , p-value in Wilcoxon\u2019s test=2.082e-05. Of 814 taxa (115 found in more than 0.01% of reads), 471 (72) were detected in both groups, while 145 , respectively. From each of the 30 healthy controls, one sample was obtained. While all healthy women were HPV negative, the HPV status of cancer patients was not analyzed. Bacterial DNA extracted and purified from the swab samples was used for PCR amplification of bacterial 16S rRNA gene hyper-variable regions. Prepared libraries were sequenced using the PGM platform. For all but one sample with only 5083 reads, sequencing generated 37104-676457  reads; all of them passed quality control and 94\u2013100%  of sequences were classified using SILVA database version 138 as a reference, and then assigned to Of 46 women included in this study, 21  were premenopausal women and 25  postmenopausal women. After excluding one patient with the low number of reads in the pre-treatment sample and one post-treatment sample with <75% of classified sequences, similarities between the CM community structures of the healthy control and pre-treatment cervical cancer groups, representing either premenopausal or postmenopausal cases and controls, were evaluated using principal coordinate analysis (PCoA) of Bray\u2013Curtis distances. Twelve of the 15 CM samples from premenopausal healthy women and 7 CM samples from postmenopausal healthy women clustered together  and from 0.04% to 45.27%  in premenopausal controls and cases , while in postmenopausal groups Lactobacillus abundance ranged from 0.022% to 99.67%  and from 0.005% to 96.87%  in controls and cases, respectively  , L. iners (100%) and L. unclassified (6-100%), respectively, and 7 postmenopausal healthy women were dominated by L. iners (2 women), L. unclassified (3 women) and L. crispatus (2 women). One woman had also noticeable levels (46%) of L. delbrueckii. All 4 CM of cancer patients with noticeable lactobacilli levels were dominated by L. iners .Eight (62%), 1 (8%), and 4 (30%) of 13 premenopausal healthy women with noticeable lactobacilli levels were dominated by We next analyzed proportion of aerobic and anaerobic bacteria among the studied groups of patients and controls. While significantly lower  and higher  percentage of anaerobic and aerobic bacteria, respectively, was found in premenopausal compared to postmenopausal healthy women, no similar differences were observed between pre- and postmenopausal cancer patients  comprised six genera, one of which were present in all samples, and a core CM of postmenopausal controls comprised 11 genera, 4 of which were present in all samples Figure\u00a04Porphyromonas, Fusobacterium, Peptococcus, Peptoniphilus, Moryella, Stomatobaculum, and Gemella differentiated both groups and the first four are also highly abundant, with more than 10000 reads each  and Gardnerella . The number of differential taxa dropped to 18 and 9 in post-radiation samples in pre- and postmenopausal women respectively , was significantly higher in pre-treatment patients than in healthy controls , samples collected immediately after the treatment  and 3 and/or 6 months later  did not differentiate pre-radiation and post-radiation samples. Of 669 taxa (148 found in more than 0.01% of reads) found in 40 samples of cancer patients, 376  were detStreptococcus, Prevotella, Fusobacterium, Porphyromonas, and Finegoldia . In contrast, an increased percentage of facultatively anaerobic bacteria was found in post-treatment patient . No significant difference was found for anaerobic and facultatively anaerobic bacteria at follow-up samples. No similar findings were not observed in the aerobic bacteria abundances  of 5 samples collected from premenopausal cancer patients before treatment, and in 7 of 16 postmenopausal healthy women and 3 of 10 postmenopausal cancer patients. CMs of 10 and 3 of 20 healthy women with the noticeable lactobacilli levels were dominated by L. crispatus and L. iners, respectively, while all 4 CMs of cancer patients with the noticeable lactobacilli levels were dominated by L. iners.Here, we firstly compared the CM of healthy women with those of cervical cancer patients. The CM was dominated by Lactobacillus-dominated CM, vaginal anaerobic metabolism of glycogen to lactic acid acidifies the vagina to a pH of <4.5. The resulting acidic environment defends the vaginal mucosa and cervical epithelium against cervicovaginal dysbiosis and vaginally transmitted infections. A vaginal microbiota dominated by Lactobacillus is considered to be a marker of a healthy vagina and vice-versa , Eggerthella, and several under-represented members of Streptococcus, Atopobium, Megasphaera, Leptotrichia, and Staphylococcus  dominated by several sub-genera, and h CIN 2+ , lactobah CIN 2+ ; insteadh CIN 2+ . In BV, cies BVAB, Eggerthlococcus , 44\u201347.  cancers \u201319.A study examining the association between the CM and high-grade cervical intraepithelial neoplasia (CIN 2+) in women infected with high-risk HPV revealed that the alpha and beta diversity of the vaginal microbiota were not associated with either CIN severity or oxidative DNA damage . In addiTherefore, our findings are only partly consistent with those reported in other studies , 43, 48.Streptococcus, Prevotella, Fusobacterium, Porphyromonas, and Finegoldia were the most abundant, differentiated pre- and post-radiation samples, and only 2 differentiated pre-radiation and follow-up samples. However, neither microbiome diversity nor microbiome richness differentiated pre-treatment samples from post-treatment samples, either at the end of the therapy or 3 and 6 months later.CRT and RT are curative or palliative therapies for gynecologic cancer that may disturb the composition of the vaginal microbiome . We showGammaproteobacteria, Gemmatimonadetes, Gemmatimonadales, Pseudomonadales, Gemmatimonadaceae, Rikenellaceae, Acinetobacter, Desulfovibrio, Prevotella 9, Rikenellaceae RC9 gut group, and Turicibacter increased with time.A previous pilot study  showed aAlthough there are some inconsistencies between our results and those mentioned above , 21, theLactobacillus, at least in premenopausal patients. Our data provide additional evidences on differences in microbiome composition between pre- and postmenopausal healthy women as well as between pre- and post-treatment cancer samples, which may relate to CRT.While previously published studies evaluated mostly the impact of chemoradiation treatment on the cervicovaginal microbiome , 51, 54,However, further research is needed to determine whether alteration of the cervicovaginal microbiome may offer new therapeutic options leading to a promising strategy. If so, identification of cervicovaginal dysbiosis, related to gynaecological cancers, can be considered an interesting field of investigation.https://www.ebi.ac.uk/ena, PRJEB43410.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by Bioethics Committee at the Maria Sklodowska-Curie National Research Institute of Oncology (69/2017 dated 06.07.2017). The patients/participants provided their written informed consent to participate in this study.Conceptualization, JO and MB; methodology, JO; software, MK; formal analysis, MK and JO, investigation, NZ-L and AP; resources, MB, BL, RK, and AN; data curation, MK; writing\u2014original draft preparation, JO; writing\u2014review and editing, MK and NZ-L; visualization, MK; supervision, JO; project administration, NZ-L; funding acquisition, JO. All authors have read and agreed to the published version of the manuscript.This work was financed by the National Science Center [2017/27/B/NZ5/01504].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "The applications of genetic, and later genomic, metagenomics and transcriptomic techniques to biofilm studies, begun by work on Escherichia coli surface adhesion factors [The first published observations that microorganisms associate to form microbial communities structured as biofilms in natural environments date back to the first half of the last century . However factors , of the  factors .As mentioned above, biofilm growth represents a major concern in microbial infections, since it can lead to tolerance to antimicrobial treatments. This issue is directly connected to the growing emergence of antibiotic resistance as well as to the lack of novel antimicrobial agents. These are issues that, if not addressed, might lead to an estimated staggering amount of 10 million deaths/year caused by pan-antibiotic resistant pathogens by 2050 . Lack ofMicrobial Biofilms: From Molecular Mechanisms and Structure to Antimicrobial Therapy, includes research articles tackling crucial issues in the field of microbial biofilms. One paper published in this Special Issue points to the relevance of the pyrimidine biosynthetic gene pyrD as suitable target for anti-virulence and anti-biofilm agents in adherent invasive Escherichia coli (AIEC), an intracellular pathogen implicated in the onset of Crohn\u2019s disease [Pseudomonas aeruginosa isolates from cystic fibrosis patients, using a machine learning algorithm aimed at the understanding of those molecules that are more active against biofilm formation [Lactiplantibacillus plantarum CCFM8724 appears promising in this respect, being able to impair the formation of a Streptococcus mutans-Candida albicans mixed-species biofilm, with supernatants of L. plantarum cultures strongly affecting the expression of C. albicans adhesion and virulence genes [Staphylococcus aureus clinical isolates from prosthetic joint-associated infections showed the absence of mutations affecting expression of the icaABCD operon, encoding PIA/PNAG biosynthetic genes. Single aminoacid changes in the IcaR regulatory protein resulting in increased PIA/PNAG production led to decreased virulence in a Galleria mellonella model, suggesting that biofilm formation might be detrimental to S. aureus virulence [S. epidermidis clinical isolates, with no apparent role of PIA/PNAG in this process [This Special Issue,  disease . Anotherormation . Anotherce genes . Other wirulence . Surpris process . Thus, tAltogether, the papers featured in this Special Issue cover some of the main topics in biofilm studies, mainly focusing on biofilm control. We believe these studies represent an important contribution to our knowledge in this field."}
+{"text": "Lactobacillus acidophilus La-14 (La-14) and Lacticaseibacillus rhamnosus HN001 (HN001) colonise a healthy human vagina. Furthermore, potential effects on vaginal microbiota and immune markers were explored. Fifty women devoid of vaginal complaints  were randomised into a 2-week intervention with either La-14 and HN001 as the verum product or a comparable placebo. Vaginal swab samples were collected at baseline, after one and two weeks of intervention, and after a one-week follow-up, for assessing colonisation of the supplemented lactobacilli, vaginal microbiota, and six specific immune markers. Colonisation of L. acidophilus and L. rhamnosus was not observed above the assay detection limit . Vaginal microbiotas remained stable and predominated by lactobacilli throughout the intervention, and vaginal pH remained optimal (at least 90% of participants in both groups had pH 4.0 or 4.5 throughout the study). Immune markers elafin and human \u03b2-defensin 3 (HBD-3) were significantly decreased in the verum group  but did not correlate with any microbiota changes. Adverse events raised no safety concerns, and no undesired changes in the vaginal microbiota or immune markers were detected.The primary objective of this randomised, placebo-controlled, triple-blind study was to assess whether orally consumed  Lactobacillus crispatus dominated), CST II (Lactobacillus gasseri dominated), CST III (Lactobacillus iners dominated), CST IV , and CST V (Lactobacillus jensenii dominated). In addition, hormonal changes during the female lifespan and the menstrual cycle, lifestyle, hygiene practices, and ethnicity can affect the composition and stability of the vaginal microbiota [L. crispatus, L. gasseri, L. jensenii, and L. iners have been associated with a healthy vaginal microbiota [L. iners also being linked to transitional and dysbiotic stages [Prevotella spp., G. vaginalis, A. vaginae, and Mycoplasma hominis, are found in a higher abundance when lactobacilli levels are depleted among asymptomatic women and have been associated with bacterial vaginosis (BV) risk [The adult pre-menopausal vaginal microbiota is typically low in diversity and dominated by one or two lactobacilli species or by a mixture of non-lactobacilli genera that form distinct community state types (CSTs), which can be reliably assigned for sample sets originating from various populations ,2,3. Thecrobiota ,5,6. Laccrobiota , with L.c stages ,8. Non-lBV) risk ,9,10.The vaginal immune system is part of the broader female reproductive tract mucosal immune system that is regulated differently during each phase of the menstrual cycle by the sex hormones . FurtherLactobacillus spp. in a dysbiotic vaginal microbiota has been reported even without detectable colonisation in oral, intestinal, or vaginal microbiota samples [Probiotic lactobacilli are an attractive and safe approach to support vaginal health as an adjunct or alternative to conventional antibiotic BV therapies ,21. Prob samples .Lactobacillus acidophilus La-14 (La-14) and Lacticaseibacillus rhamnosus HN001 (HN001). La-14 and HN001 included in the Respecta\u00ae complex  of La-14 and HN001 and 50 mg of bovine lactoferrin per capsule with dosing varying from 1\u20132 daily capsules) have shown beneficial effects for vaginal health in randomised placebo-controlled clinical trials. These include elevating vaginal L. acidophilus and L. rhamnosus levels, reducing Nugent score, which reflects an increase in vaginal lactobacilli, and alleviating BV- and vulvovaginal candidiasis (VVC) associated symptoms [\u00ae complex is a glycoprotein able to both inhibit and enhance the growth of Lactobacillaceae and Bifidobacterium strains in vitro [\u00ae complex (La-14 and HN001) have been reported to have an alike or enhanced efficacy compared to the combination of La-14, HN001, and lactoferrin in attenuating G. vaginalis-induced BV in mice, in inhibiting G. vaginalis growth, and in adherence to human HeLa cells in vitro [L. acidophilus and L. rhamnosus have long been shown to be safe and suitable for human consumption and have been present in human food for decades [In the vaginal tract, probiotics are considered to provide antimicrobial activity by reducing vaginal pH via lactic acid production, producing bacteriocins, and modulating local and systemic immune responses. Furthermore, vaginal probiotics are expected to adhere to vaginal epithelial cells and thereby exclude potential pathogens. However, information is lacking on the effects of probiotic strains on indigenous vaginal microbiota composition and potential immunomodulatory effects in a healthy vaginal tract. The probiotic strains selected for this investigation were symptoms ,30,34. Tin vitro . Vaginalin vitro , and orain vitro . Howeverin vitro , suggestin vitro . La-14, in vitro . Moreovein vitro ,41 and tin vitro ,30. Both decades ,43.The objectives of this clinical trial were to assess whether a two-week oral administration of La-14 and HN001 in a vegan capsule devoid of lactoferrin results in vaginal colonisation of the supplemented species and to characterise the potential effects on vaginal pH, microbiota composition, and immune markers. The target population included healthy premenopausal females devoid of vaginal symptoms, comparable to the study by De Alberti and colleagues , with adThis was a randomised, double-blind, placebo-controlled, parallel group, single center clinical trial performed at an independent clinical research site, CPS Research , under the supervision of Gordon Crawford, MD, as the principal investigator. The trial included a screening phase, a two-week intervention, and a one-week follow-up with five visits: V1 , V2 , V3 , V4 , and V5  . The triAfter pre-screening, participants attended a screening visit (V1) 3 to 42 days prior to randomisation visit (V2) to allow for visits V2 to V5 to be held in between menses for participants with a natural menstrual cycle. During the screening visit, written informed consent was obtained from all participants before any study-specific procedures or assessments were performed. Screening procedures included assessment of eligibility per set criteria , vital sDuring the study, participants were asked to continue their normal routines and lifestyle , to refrain from sexual intercourse, and use of intravaginal products  for 24 h preceding visits, and only to use a provided neutral detergent wash for intimate hygiene.On V2, participants were randomly allocated to receive the investigational product (IP) and instructed to consume one capsule daily after breakfast from Day 1 to the day of V4 . IP compliance was set per protocol at \u226580% and assessed based on the number of returned capsules.\u00ae Feminine pH Test Strips, NutraBlast, Pompano Beach, FL, USA) was measured. Between visits, participants collected information on AEs, CMs, IP compliance, sexual behaviour, contraceptive method used, and menstrual bleeding days on a paper diary. In case of menstrual bleeding on Day 20\u201322, the follow-up visit (V5) was postponed to immediately after the bleeding had ended. Pregnancy test was repeated on V5 as a safety precaution. All AEs were assessed for causality and severity by the investigator.On visits V2 to V5 a gynecological examination was performed, and vital signs were measured. Adverse events (AEs) and updates of contraceptive method, sexual activity, and concomitant medications (CMs) were recorded. For outcome assessments, three vaginal swabs were collected for microbiota and immune marker analyses, and the vaginal pH  and HN001 (ATCC SD5675) in a 4:1 ratio, potato maltodextrin as a carrier, and stearate and silicon dioxide as flow agents. The placebo capsules were identical in shape, texture, and taste to the verum capsules and contained potato maltodextrin adjusted to equal the weight of the verum capsule and the same amounts of stearate and silicon dioxide as added to the verum capsule. Both verum and placebo IP were manufactured and packaged using identical containers and labels by Danisco USA Inc. .Participants were supplemented either with verum or placebo IP for two weeks. One verum capsule  contained 10L. acidophilus and/or L. rhamnosus at species level was assessed as the primary outcome and vaginal pH was followed as the secondary outcome. Exploratory outcomes included strain-specific vaginal colonisation (La-14 and HN001) and the assessment of vaginal microbiota composition and specific immune markers. AEs were collected as a safety outcome.Vaginal colonisation of Vaginal swabs were collected as dry swabs with the Copan FLOQswab Collection Kit  from the sidewalls in the upper third area of the vagina (approximately 5 cm past the introitus) by gently rotating the swab for 10 to 20 s. Before sampling, any excessive secretion or discharge was wiped out and skin contact was avoided. Samples were immediately frozen and stored at \u221280 \u00b0C and all shipments for analysis were conducted on dry ice (temperature range \u221290 to \u221220 \u00b0C).Vaginal swabs\u2019 flocked heads were clipped directly into a Lysis Matrix E bead beating tube . The tubes were beaten using a Precellys 24 Homogenizer , twice for 60 s at 4500 RPM. The bead beating tubes were then spun at 14,000\u00d7 g for 15 min, then 500 \u00b5L of the resulting lysate was carried forward with a FastDNA Spin Kit for Soil  following the manufacturer\u2019s instructions. The purified DNA was quantified using Qubit HS dsDNA kit on Qubit 3.0 Fluorometer .L. acidophilus [L. rhamnosus [Faecalibacterium prausnitzii DSM 17677 for La-14 and L. acidophilus DGCC 8698 or Lacticaseibacillus paracasei DGCC 4981 for HN001) were included on each 96-well plate. Quantification of PCR amplification was performed using the QuantStudio Design and Analysis Software v1.5.1 (Thermo Fisher Scientific). The limit of detection (LOD) values were based on \u00bd the number of genomes calculated to be within the 100 fg standard applied. For the SYBR assays, the dissociation curves were analysed to control for non-specific amplification. to control for non-specific amplification.The vaginal swab DNA samples were analysed as a primary endpoint with absolute quantitative PCR (qPCR) assays for dophilus  and L. rhamnosus , and as hamnosus  and HN00L. acidophilus assay previously successfully applied for detection of vaginal colonisation [L. crispatus DSM 20584, L. jensenii DGCC 11796, L. gasseri ATCC 33323, and L. acidophilus strains La-14 ATCC SD5212, DGCC 8698, ATCC 4356, and DGCC 12900.As a post hoc analysis, an alternative nisation ,30,47 waThe microbiota populations from vaginal samples were analysed by MiSeq 16S amplicon sequencing of the V4 region  and QIIME2 (v. 2021.2) as previously described ,49,50 anImmune marker levels  were determined by ELISA from the eluants of the vaginal swabs collected from the participants at different time points using the Spectramax 250 ELISA analyzer . Briefly, the vaginal swabs were reconstituted in ice-cold phosphate buffered saline (PBS) by vortexing and finally the eluant supernatants were aliquoted in Eppendorf tubes for subsequent ELISA analysis. HBD-1 and HBD-3 levels were measured using Human DEFB1 (Beta-defensin 1) and Human DEFB3 (Beta-defensin 3) ELISA Kits , respectively, and HBD-2 by Human DEFb2/DEFB2 ELISA kit (Elabscience). SLPI and elafin levels were analysed with the human ELISA kits . IgA was determined using Invitrogen Human IgA ELISA kit. All kits were used according to the manufacturer\u2019s instructions and analysed in duplicate on the Spectramax 250 ELISA analyzer using the software SOFTmax PRO v4.3.1 LS. The lower limit of quantification (LLOQ) was defined as the lowest point on the standard curve for each individual analyte.t-test assuming unequal variances and applying a two-sided significance level of 5%. For L. acidophilus the mean difference between the verum and placebo arms was assumed to be 60, with standard deviations of 30 (placebo) and 70 (verum) [L. rhamnosus, the mean difference between the verum and placebo arms was assumed to be 30, with standard deviations of 20 (placebo) and 33 (verum) using 104 genome containing particles/100 ng DNA in qPCR results evaluation as the unit for both endpoints for the power calculation [The sample size was calculated using nQuery software (version 8.5.2) with 90% power and a two-sample  (verum) . For L. culation , althougculation  to accouculation .With the above assumptions, 40 evaluable randomised participants were needed regarding both primary endpoints. Assuming a 20% attrition rate, 50 participants (25/group) were to be randomised to the study.Study participants were allocated to one of two intervention groups in equal proportions using randomly permuted blocks through a computer-generated process. All verum and placebo products were labelled with a randomisation number accordingly.All volunteers, study site personnel, clinical CRO personnel, the monitor, and Sponsor laboratory personnel involved in the study conduct were kept blinded during the entire study period.p-values for the null hypotheses. The secondary endpoint  was analysed utilising a mixed effects cumulative logit model, including the same explanatory variables as the models for primary endpoint. A categorised change in vaginal pH (decrease/no changes/increase) was used as the response. The model was constructed to model the probability of lower ordered categories of vaginal pH.For the primary endpoints, a repeated measures analysis of variance (RM-ANOVA)-model was to be used with fixed effects for supplementation group, repeating factor week  and the interaction between supplementation group and week. The participant was to be used as a random effect in the models. The contrasts between the verum group and the placebo group at the end of the 2-week intervention period was to be estimated from the model with 95% confidence intervals together with two-sided In addition, sensitivity analyses with RM-ANCOVA model and responder analyses, as described in De Alberti et al.,  were plap < 0.05 or p < 0.1 after false discovery rate (FDR) correction by the Benjamini\u2013Hochberg method. Spearman correlation analyses were conducted using the R packages \u2018hmisc\u2019 and \u2018gplots\u2019 for individual visits. P-values were adjusted for multiple comparisons by Benjamini\u2013Hochberg FDR as noted.For the vaginal sequence data, differential taxa were tested for the main effect of group (including model adjustment for visit and random effect of participant or CST at baseline) using the R package \u2018ANCOM2\u2019 . Taxa weDifferences in CST distribution between intervention groups at baseline and for change in CST by timepoint  were checked with Fisher\u2019s exact test. CST distribution and ASV correlations were also assessed for demographic variables including Nugent score, age, body mass index (BMI), contraceptive method , and pregnancies (prior pregnancies/no prior pregnancies) with the Chi-Square test and Spearman correlation, respectively. In addition, the percentages of participants with over 1% increase or decrease from baseline in the prevalence of an ASV were tabulated and tested with Fisher\u2019s exact test as an effort to detect effects related to probiotic consumption.ps. Change from baseline used in the RM-ANCOVA model was calculated using logarithm transformed values. If model assumptions were not met, square root transformation was used when calculating the change from baseline. Model assumptions were confirmed testing residual normality using Shapiro\u2013Wilk test. If normality assumptions were not met with either of the data transformation, nonparametric Wilcoxon rank sum test was applied by visit to compare group differences.For each immune marker, the differences between verum and placebo in change from baseline were analysed with a RM-ANCOVA-model. The models included fixed effects for supplementation group, repeating factor visit and the interaction between supplementation group and week. Baseline value was included in the model as a covariate. Participant was used as the random effect in the models. The contrasts between verum and placebo at different visits were estimated from the model including 95% confidence intervals (CIs) together with two-sided Ps were adjusted for multiple comparisons by Benjamini\u2013Hochberg FDR. Additionally, immune marker concentrations were compared with Wilcoxon test between participants grouped according to CST at baseline (regardless of intervention group).For statistical analysis of microbiota and immune markers, the groups were analysed together. The R package \u2018rmcorr\u2019 was used to correlate immune markers  and ASVs present in at least 40% of the participants using repeated measures and A total of 50 participants were randomised and 47 participants completed the study. Of the participants, 1 was discontinued due to randomisation error and did not receive any IP, leaving 49 participants in the ITT and safety analyses . The twoDNA extraction was successful from all samples, but the efficiency varied considerably, having a range of 206 to 32,400 ng/swab  for all 188 samples.L. acidophilus above the assay\u2019s LOD 5.29 Log10 genomes per swab in either group at any of the sampling timepoints  efficiently, but with two peaks repetitively present in the melt curve analysis (a main peak and a shoulder-like second peak). With L. acidophilus as standard, 82 \u00b0C (dissociation temperature of the main peak) was the dissociation temperature for standard-based quantification of samples in the qPCR analysis. On the other hand, vaginal swab samples (137/141 of the tested samples) and commensal vaginal species L. crispatus, L. gasseri, and L. jensenii all amplified with a uniform amplicon dissociating at 78 \u00b0C with a single peak, although PCR-efficiency was poorer. As the amplicon melting point for L. acidophilus was 4 \u00b0C higher than that of the samples, we were unable to quantify the samples with L. acidophilus as standard and had to reject the uniform amplification seen in the samples as background. With L. crispatus as standard, quantification of samples was possible and results could be calculated as the number of participants presenting an over 2-fold increase from baseline, as previously conducted by De Alberti and colleagues [The nisation ,30,38 amVaginal pH remained stable over the intervention, being 4.0 or 4.5 for at least 95% of the participants during the intervention phase  and no sL. crispatus/acidophilus, L. iners, and L. jensenii being the three most common ASVs within the sequence data (L. crispatus dominated), II (L. gasseri dominated), and V (L. jensenii dominated) were most prevalent (p > 0.1), nor did any taxa differ when comparing between the groups at any visit including the baseline . Similarly, the baseline CST distribution did not vary significantly between groups (Fisher\u2019s exact test p 0.7547).A total of 176 samples (90 verum and 86 placebo samples) had at least 9000 reads and were included in the analysis. For both groups and all timepoints, Lactobacillaceae was predominant, with nce data . Communirevalent . There wNo difference was seen in within-sample species diversity (\u03b1-diversity) between groups or timepoints. For between-sample dissimilarity (\u03b2-diversity), the percent of variation that could be attributed to the study factors was mainly due to individual (77%) and, to a lesser extent, to CST grouping (10%), intervention (3%), BMI (3%), and Nugent score at screening , whereas visit (time) did not have a significant influence on the observed diversity .L. rhamnosus also detected in qPCR, had ASVs assigned to L. rhamnosus. G. vaginalis and predominant Lactobacillus during the intervention. The number of participants with over or under 1% change in ASV abundance did not differ significantly between groups or timepoints (Fisher\u2019s exact test).Abundances of families, genera, and ASVs were comparable between verum and placebo and remained stable over time. One participant from the placebo group, with Prevotella, Finegoldia, Campylobacter ureolyticus, and Streptococcus anginosus correlated positively, whereas ASVs within Lactobacillaceae did not show correlation. Of the demographic variables, BMI correlated positively with Gardnerella vaginalis and Campylobacterium ureolyticus .With all samples combined  the relative abundance of ASVs assigned to For the immune marker analysis , log-trap = 0.022) and HBD-3 (p = 0.028) over all visits .The RM-ANCOVA analysis showed statistically significant differences between the study groups for elafin (l visits . For elap = 0.008).HBD-3 also showed an overall statistically significant decrease in verum compared to placebo with an increasing numerical trend in the difference between the groups adjusted for baseline. On the follow-up visit (V5), the difference between the groups was statistically significant . Statistically significant (p < 0.05) correlations were found for HBD-1, HBD-3, and IgA, but not for HBD-2, elafin, and SLPI, as detailed in Correlations were assessed for immune markers and 16 ASVs that were present in at least 40% of the participants , a trend of higher SLPI and lower HBD-3 was seen in CST I compared to CST III (Wilcoxon test 10 CFU/capsule) before initiating the clinical phase and after the last participant had completed the study. There were no differences in vital signs, menstruation, or vaginal health between groups during the study. After randomisation, only one participant reported reddening of vulva and/or vagina (verum group). Otherwise, all the participants had normal smell of vaginal secretion, normal vaginal appearance, no menstrual bleeding, and no reddening of the vulva and/or vagina.Only four participants had IP compliance < 80% (two in verum and two in placebo). IP stability was confirmed to be above target potency  and placebo (17 events in 10 participants) groups. A total of 11 AEs were classified as potentially related to the IP (for three participants in the placebo group and four participants in the verum group). The most common organ class affected by AEs was the gastrointestinal system . These were equally divided between verum (7) and placebo (7). There were no severe AEs or serious adverse events and no events requiring withdrawal from the study.Gardnerella, Atopobium, and Prevotella, whereas lactobacilli-predominance is associated with a healthy-like vaginal microbiota [L. iners, and the microbial diversity (\u03b1-diversity) is restored even within 8 days, with no significant differences between relative abundances of bacterial taxa remaining after 15 days.A BV-associated microbiota resembles that of CST IV, with pronounced abundance of members from the genera crobiota ,9,10. Bocrobiota ,10. The crobiota ; when BVThese depleted vaginal lactobacilli levels, associated with BV and increased disease risk ,59, can L. acidophilus and L. rhamnosus levels in a comparable study setting with calculations based either on all DNA extracted from the vaginal swab (per swab) or on mass of DNA (per 100 ng of DNA) [L. rhamnosus and 3.3 and 3.9 Log10 genomes per ng of DNA for L. acidophilus, respectively [Lacticaseibacillus paracasei LPC-S01, the vaginal colonisation on strain level was detected at approximately 4\u20136 logs below the level of total bacteria present in a vaginal sample, ranging up to approximately 5 Log10 cells/swab [L. rhamnosus PB01 and L. gasseri EB01) applying shotgun metagenomics [Vaginal colonisation of La-14 or HN001 on species or strain level was not detected with the applied methods allowing detection of 5.29 and 5.11 Log10 genomes per swab, respectively . Previou of DNA) ,30, neit of DNA) , the basectively . For oralls/swab . Moreovegenomics . From thgenomics . RegardlL. rhamnosus assay we applied the same primers as were used in prior studies [L. acidophilus, we applied a different assay [L. acidophilus standard (DGCC 8698 and La-14 both tested as the standard strain through extensive reaction condition optimisation steps) suggesting suboptimal performance of the earlier applied assay, i.e., that there were either two different amplicons being generated during the qPCR, or that the amplicon had a secondary DNA structure leading to a step-wise dissociation. The assay did, however, amplify DNA extracted from the clinical study vaginal swab samples and from pure cultures of L. crispatus, L. gasseri, and L. jensenii, resulting in a uniform single-peak dissociation curve. Due to a difference in the melting point temperatures of these two PCR products, quantification of the samples with L. acidophilus as the standard was not possible with SYBR chemistry. However, when we changed the standard to L. crispatus (likely L. jensenii or L. gasseri would have performed similarly as a standard), we were able to quantify the clinical samples  or predominated by one Lactobacillus species  [G. vaginalis and C. ureolyticus, in alignment with earlier findings by Allen and colleagues [We had recruited participants devoid of vaginal complaints  into an intervention with lactobacilli being orally supplemented as the test ingredient expecting the vaginal pH and microbiota to remain relatively stable. Both outcomes were followed to assess the safety of the verum IP and potential intervention-related fluctuations in a set-up not assessing treatment or prevention. The vaginal pH remained stable and at a healthy level throughout the study, being \u2264 4.5 for almost all participants in both groups at all study visits, although no statistically significant changes within a healthy pH range were detected ; Table 7nificant . The pos and IV) . BMI, whlleagues . Thus, rL. acidophilus, this would not have been possible as the 16S rDNA V4 region does not differentiate between L. acidophilus and L. crispatus, but since the V4 region distinguishes species relevant for vaginal microbial disturbances , it was selected.The noted wide range in DNA extraction efficiency was likely due to varying amounts of host DNA carryover during DNA extraction and may have challenged detection of intervention-emergent changes among less abundant ASVs. DNA extracted from vaginal swab samples may contain over 95% of host DNA if host DNA is not removed during the extraction process . As an eL. acidophilus and L. rhamnosus among women with no vaginal complaints during a 2-week intervention with La-14 and HN001 [6 or 2 \u00d7 108 CFU for three days, L. crispatus CTV-05 colonisation has been shown to be significantly more likely during weekly follow-up visits among women initially devoid of commensal L. crispatus within their vaginal microbiota and not practicing unprotected vaginal intercourse [L. acidophilus, being a gut-oriented lactobacillus, adapts well to simulated vaginal fluid, it would unlikely be able to successfully compete with abundant endogenous vaginal lactobacilli such as L. crispatus, L. gasseri, and L. jensenii [We had aimed to repeat the results published by De Alberti and colleagues, reporting elevation of nd HN001 ; thus, wnd HN001 . By chanercourse . Additioercourse , suggestjensenii . Alignedjensenii . The stujensenii . A furthjensenii , and mayL. rhamnosus Lcr35 [Lactobacillus spp. in healthy vaginal microbiota has an important role in competitive exclusion of pathogenic bacteria, competition for nutrients, production of antimicrobial substances, and on the immune system [L. jensenii, as well as HBD-2 and DNA levels of L. crispatus, in cervicovaginal lavage samples from healthy women. In our study, we found significant correlation of HBD-3 with L. jensenii and of IgA with L. crispatus in 40% of participants irrespective of intervention/group  within the population that we could not detect with 16S sequencing (The current study also provides insights on the effects of probiotics on the mucosal immune system modulation in a healthy vaginal tract. The participants in the verum group showed statistically significant decrease in elafin and HBD-3, with a similar trend in SLPI, when compared with the placebo . Interesus Lcr35 . Accordie system ,71. In te system  showed aon/group . In anot CST III . The othe system . Hence, quencing . Indeed,Taken together, the high and constant lactobacilli predominance observed in the vaginal swab samples of the current study likely left less of a niche for non-commensal lactobacilli strains to colonise the vagina and increased the technical challenges in detecting any potential colonisation, if such existed. DNA extracted from vaginal swab samples are bound to have high but varying amounts of host DNA carryover and, if from healthy volunteers, commensal microbiota likely consisting predominantly of lactobacilli. This underlies the need for stringent optimisation procedures and interpretation of results when PCR-based analyses are applied to detect low levels of supplemented lactobacilli. The stability of the healthy-like vaginal microbiota and pH throughout the study highlights the safety of using La-14 and HN001 as over-the-counter supplements without the risk of jeopardising well-balanced vaginal microbiota. Vaginal immune markers, elafin, HBD-3, and SLPI, should be evaluated further in future probiotic intervention studies elucidating the effects of the supplementation on vaginal health. No safety concerns were raised regarding vital signs, menstruation, vaginal health, or AEs.10 CFU in a 4:1 ratio for two weeks had no aberrant effects on stable and healthy commensal vaginal microbiota and immunological homeostasis.Colonisation of La-14 or HN001 was not observed above the detection limit 5.29 and 5.11 Log10 genomes per vaginal swab, respectively. Oral supplementation of La-14 and HN001 at 10"}
+{"text": "The occurrence and development of tumors are closely related to histone deacetylases (HDACs). However, their relationship with the overall biology and prognosis of glioma is still unknown. In the present study, we developed and validated a prognostic model for glioma based on HDAC genes. Glioma patients can be divided into two subclasses based on eleven HDAC genes, and patients from the two subclasses had markedly different survival outcomes. Then, using six HDAC genes , we established a prognostic model for glioma patients, and this prognostic model was validated in an independent cohort. Furthermore, the calculated risk score from six HDACA genes expression was found to be an independent prognostic factor that could predict the five-year overall survival of glioma patients well. High-risk patients have changes in multiple complex functions and molecular signaling pathways, and the gene alterations of high- and low-risk patients were significantly different. We also found that the different survival outcomes of high- and low-risk patients could be related to the differences in immune filtration levels and the tumor microenvironment. Subsequently, we identified several small molecular compounds that could be favorable for glioma patient treatment. Finally, the expression levels of HDAC genes from the prognostic model were validated in glioma and nontumor tissue samples. Our results revealed the clinical utility and potential molecular mechanisms of HDAC genes in glioma. A model based on six HDAC genes can predict the overall survival of glioma patients well, and these genes are potential therapeutic targets. Glioma is deemed to be the most aggressive tumor in the central nervous system. The annual incidence of glioma has been reported to be approximately 30\u201380/1 million worldwide and increasing by 1\u20132% annually; the 5-year survival rate is only 10\u201320% [Although various cancer therapies have been applied over the past decades, the prognosis of glioma patients remains dismal. After including the isocitrate dehydrogenase (IDH1/2) mutation and whether the 1p19q code is missing, the WHO classification of central nervous system tumors has become more refined . HoweverThe underlying cause of malignant tumors is disordered gene expression systems, including oncogenes, tumor suppressor genes and genes related to DNA repair . With thThe occurrence and development of tumors are closely related to histone deacetylases (HDACs) . StudiesHDACs come in four classes: class I , class II , class III (SIRT1-SIRT7), and class IV (HDAC11) . A previhttps://portal.gdc.cancer.gov/) and Chinese Glioma Genome Atlas  were utilized. Gene mutation data were also obtained from the TCGA database. Drug response data were acquired from the Genomics of Drug Sensitivity in Cancer (GDSC) database (https://www.cancerrxgene.org/downloads). The immune filtration data were from TCIA (https://tcia.at/home). The information on the histone deacetylase genes  was from the Molecular Signatures Database (MSigDB).The mRNA expression data of glioma patients from The Cancer Genome Atlas  and t-distributed stochastic neighbor embedding (tSNE) to further validate the clustering analysis.1 \u00d7 gene1 expression + coef2 \u00d7 gene2 expression + coefn \u00d7 genen expression. Using the established prognostic model, we performed validation in the CGGA dataset. Glioma patients were separated into high- and low-risk groups according to the median risk score. Kaplan\u2013Meier survival curves were used to compare the difference in OS between the high- and low-risk groups. Receiver operating characteristic curves (ROCs) were used to evaluate the predictive ability at 1 year, 2 years, and 3 years of patient OS in TCGA and CGGA. PCA was used to identify the risk type.Using 11 HDAC genes, we developed an overall survival (OS) prediction model in the TCGA dataset. Least absolute shrinkage and selection operator (LASSO) regression was used to select the number entering the model, and then a multivariate Cox regression was performed to obtain the regression coefficient of each HDAC gene. We calculated the risk score of each sample using the following formula: risk score = coefTo investigate the association between the risk score and prognosis in glioma patients, we first performed a stratified analysis of different clinical parameters. Then, we compared the difference in HDAC genes between the two clustering groups, and the risk score distributions were also observed for different clinical parameters. To validate the independence of the risk score, we performed univariate and multivariate Cox regression analyses by adjusting the clinical parameters . We evaluated the diagnostic ability of the risk score and other parameters using ROC curves. We built a nomogram to evaluate the clinical application of the HDAC gene prognostic model, and the nomogram-predicted probabilities of 1-year OS, 3-year OS and 5-year OS were used to assess the model fitting ability.To explore the function and pathway enrichment of different high- and low-risk groups, we performed GO functional enrichment and KEGG pathway analysis using the \u201cclusterProfiler\u201d package. Using the masked copy number segmentation data, we investigated the gene mutation frequency of different risk groups using the \u201cmaftool\u201d package .P < 0.05 were considered significantly correlated.We assessed the difference in 16 immune cell-related infiltrating scores and 13 immune-related pathways between the high- and low-risk groups. Using the TCGA dataset, we calculated the immune and stromal estimate scores of the high- and low-risk groups using the R \u201cestimate\u201d package. To explore the correlation between small molecular drugs and the identified prognostic signature genes, Pearson correlation coefficients were calculated. |R|>0.25 and To validate the expression of six HDAC genes, including the prognostic model , we detected the expression of these HDAC genes in glioma and nontumor tissues. mRNA expression data were collected from 23 samples from epilepsy patients and 157 tumor samples (GEO dataset). The tissue collection was approved by the NCI IRB committee, and informed consent was obtained from all subjects [P < 0.05 was considered significant.Differentially expressed gene analysis was performed using the \u201climma\u201d package. Differences for category variables were performed using the chi-square test. Comparisons of OS curves were achieved using the log-rank test. One-way ANOVA was used to compare the differences in HDAC gene expression among nontumor and different grades of glioma. SNK methods were used for multiple comparisons. All statistical analyses were performed using R software 4.0.1, and https://portal.gdc.cancer.gov/, and CGGA data can be available from the Chinese Glioma Genome Atlas (http://www.cgga.org.cn/). Some data have been provided in the The TCGA data can be obtained from the The ethnic approval is granted because these data were from public database.A flow chart was constructed to comprehensively describe our study . For 11 P < 0.001, We first developed a prognostic model of OS in the TCGA training dataset. Univariate Cox regression indicated that high expression levels of HDAC1, HDAC3, HDAC7 and HDAC9 were associated with poor OS of glioma, and elevated expression levels of HDAC4, HDAC5, HDAC6 and HDAC11 were associated with a favorable prognosis of glioma . HDAC2, P < 0.001, Patients from the CGGA dataset were used to validate the calculated risk score, and they were also separated into high- and low-risk groups according to the median calculated using the formula established in the TCGA training set. Similarly, survival analysis suggested that the high-risk group had a worse OS than the low-risk group . No significant differences between the high- and low-risk groups were observed for the radiotherapy ratio and sex ratio , HDAC3 (P < 0.001), HDAC7 (P < 0.001) and HDAC9 were highly expressed in the high-risk group, while HDAC4 (P < 0.001) and HDAC5 (P < 0.001) were expressed at low levels in the high-risk group (P < 0.001). Then, we compared the risk score differences among the different clinical parameters. Our results indicated that patients >41 years old, advanced WHO stage and higher grade had higher risk scores . We finally identified 2598 differentially expressed genes, including 1723 upregulated genes and 875 downregulated genes, in the high-risk groups , we established a prognostic model for glioma patients, and this prognostic model was validated in an independent cohort population. Furthermore, the calculated risk score from the expression of the six HDACA genes was found to be an independent prognostic factor, able to accurately predict the five-year overall survival of glioma patients. High-risk patients can be attributed to changes in multiple complex functions and molecular signaling pathways, and the gene alterations between high- and low-risk patients were significantly different. We also found that different survival outcomes of high- and low-risk patients could be involved in the differences in immune filtration levels and the tumor microenvironment. Subsequently, we identified several small molecular compounds that could be favorable for glioma patient treatment. Finally, we validated the expression levels of HDAC genes from the prognostic model using glioma and nontumor tissue samples. Our study provides new and simple molecular subtypes and prognostic prediction methods and adds to our understanding of the biology and molecular mechanisms of glioma.We included six HDAC genes in the established prognostic model. HDAC1 and HDAC3 are Class I HDACs. Previous studies have indicated that HDAC1 is overexpressed in diverse human malignancies, such as prostate cancer, breast cancer, liver cancer, and lung cancer \u201321. HDACHDAC3 has become a focus of recent research, and many scholars worldwide have found that it plays a role in carcinogenesis in human tumors. After the expression of HDAC3 is reduced by inhibitors, the growth and invasive abilities of human glioma cells are significantly weakened, which provides a new target for cancer treatment .HDAC4, HDAC5, HDAC7, and HDAC9 are Class II HDACs . HDAC4 iHDAC7 plays an oncogene role in glioma. It was reported that ZNF326 could activate HDAC7 transcription by binding to a specific promoter region via its transcriptional activation domain and zinc-finger structures in glioma cells . FurtherHDAC9, like most class II HDACs, has a conserved histone deacetylase domain, catalyzes the removal of acetyl moieties from the N-terminal tail of histones, and possesses a long regulatory N-terminal domain that interacts with tissue-specific transcription factors and corepressors. The amino-terminal domain contains highly conserved serine residues that are subjected to phosphorylation . Signal-With the emerging and rapid development of disciplines such as structural biomechanics and computer-aided drug design, the development of new HDAC inhibitors with antitumor activity targeting HDACs is bound to have a very broad application space and developmental prospects.We noticed that a recent study also evaluated the role of HDCA genes in glioma . There ain vitro and in vivo.The present study has several limitations. One is that the established model needs to be validated in other cohorts. The other limitation is that we did not explore the specific molecular mechanism of this model in glioma, and some results need to be verified in vivo and in vitro will improve our understanding of the molecular mechanisms of glioma.In summary, our results reveal the clinical utility and potential molecular mechanisms of HDAC genes in glioma. A model based on six HDAC genes  can predict the overall survival of glioma patients well and these genes are potential therapeutic targets. Future research should validate this model in a large cohort, and experiments Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4"}
+{"text": "Mild cognitive impairment (MCI) is an intermediate stage between normal ageing and dementia. The early identification of MCI is important for timely intervention. The visual cognitive assessment test (VCAT) is a brief language-neutral screening tool for detecting MCI/mild dementia. This meta-analysis evaluated the diagnostic efficacy of the VCAT for MCI/mild dementia.Medline, Embase, Google Scholar, and Cochrane Library were searched from their inception until August 2023 to identify studies using VCAT to diagnose MCI/mild dementia. The primary outcome was to assess the diagnostic accuracy of the VCAT for detecting MCI/mild dementia through area under the receiver operating characteristic curve (AU-ROC) analysis. The secondary outcome was to explore the correlation between VCAT scores and MCI/mild dementia presence by comparing scores among patients with and without MCI/mild dementia. Pooled sensitivity, specificity, and area under the curve (AUC) were calculated.p\u2009<\u20090.00001).Five studies with 1,446 older adults (mean age 64\u201368.3\u2009years) were included. The percentage of participants with MCI/mild dementia versus controls ranged from 16.5% to 87% across studies. All studies were conducted in Asian populations, mostly Chinese, in Singapore and Malaysia. The pooled sensitivity was 80% [95% confidence interval (CI) 68%\u201388%] and the specificity was 75% (95% CI 68%\u201380%). The AU-ROCC was 0.77 (95% CI 0.73\u20130.81). Patients with MCI/mild dementia had lower VCAT scores than the controls (mean difference \u22126.85 points, VCAT demonstrated acceptable diagnostic accuracy in distinguishing MCI/mild dementia in cognitively normal older adults. As a language-neutral and culturally unbiased tool, the VCAT shows promise in detecting MCI/mild dementia. Further studies in non-Asian populations are required.https://www.crd.york.ac.uk/prospero/, identifier: CRD42023453453. A coCognitive screening tools, including the Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA), are commonly used , 11. Alt2.2.1.This review was designed and reported in accordance with PRISMA guidelines. Details of the protocol registration are available in PROSPERO (Number: CRD42023453453).2.2.Articles were identified through a search of databases, including MEDLINE, Google Scholar, EMBASE, and the Cochrane Library, spanning from their inception to August 11, 2023. The search was performed employing the following keywords: (\u201ccognitive impairment\u201d or \u201ccognitive decline\u201d or \u201cimpaired cognition\u201d or \u201ccognitive dysfunction\u201d or \u201ccognitive disorder\u201d or \u201ccognitive disability\u201d or \u201ccognitive deficit\u201d) and \u201cvisual cognitive assessment test.\u201d Moreover, to augment the inclusiveness of the literature exploration, controlled vocabulary search terms, including MEDLINE (MeSH) and EMBASE (Emtree), were used. A manual review of the reference lists from the acquired studies was performed to identify additional studies that might be eligible for inclusion. There were no restrictions based on the language in which the studies were published. The search strategy used to scan the MEDLINE database is presented in 2.3.The criteria for article inclusion were as follows: first, studies that employed the VCAT for diagnosing MCI/mild dementia irrespective of ethnicity were considered. Second, studies that provided data on the sensitivity, specificity, and number of patients afflicted with MCI/mild dementia were considered. A diverse range of study designs  were deemed eligible for inclusion in this meta-analysis.Studies falling into any of the following four categories were excluded from the analysis: (1) non-peer-reviewed reports; (2) abstracts, letters, review articles, and case series; (3) studies devoid of information pertaining to MCI/mild dementia or VCAT; and (4) reports for which full-text versions could not be accessed.2.4.Two researchers independently collected the following details: study characteristics , demographics of the enrolled patients, criteria for the definition of MCI/mild dementia, ethnicity, language, education level, sensitivity and specificity values, area under the curve (AUC) value, and the time required for VCAT. Any disagreements were resolved by a third investigator. Efforts were made to contact the authors of the studies to acquire missing or incomplete data.2.5.The main objective was to evaluate the diagnostic precision of the VCAT in diagnosing MCI/mild dementia by calculating the area under the receiver operating characteristic curve (AU-ROC). The criteria for defining MCI/mild dementia were derived from those used in individual studies. The secondary objective focused on the relationship between VCAT scores and the presence of MCI/mild dementia by calculating the score differences between patients with and without MCI/mild dementia.2.6.To evaluate the quality and potential bias of studies assessing the accuracy of diagnostic tests, the Quality Assessment for Diagnostic Accuracy Studies-2 (QUADAS-2) tool was used . The QUA2.7.In this meta-analysis, ROC curve analysis was employed to determine the area under the curve (AUC), offering a comprehensive gauge of diagnostic accuracy. Furthermore, Fagan\u2019s nomogram was applied to estimate the post-test probability of the disease by integrating the pre-test probability with the test likelihood ratios . These r3.3.1.n\u2009=\u200912) and title- and abstract-based exclusion (n\u2009=\u200915). After a thorough full-text reading and assessment of the 10 reports, we decided to exclude 5 of them from our review for various reasons. One study was excluded because it did not provide the necessary outcomes relevant to our review. Two reports were conference abstracts and did not provide comprehensive data for our analysis. Another report was a review, and as our aim was to include the original research, we decided not to incorporate it. Finally, one of the reports was not a peer-reviewed article, and given the importance of peer review in ensuring the quality and validity of research, we deemed it inappropriate for inclusion in our review. Finally, five studies published between 2016 and 2023 were determined eligible for inclusion  or without (n\u2009=\u2009589) MCI/mild dementia. Patients with MCI/mild dementia had lower VCAT values than those without MCI/mild dementia    12, 15,,n\u2009=\u2009857 2\u2009=\u200996%) \u201322.3.6.p\u2009=\u20090.65) (The combined sensitivity was 80% (68%\u201388%), whereas the pooled specificity was 75% (68%\u201380%) . The com\u2009=\u20090.65) . These r4.The increased use of brief cognitive assessments in primary care could improve diagnosis rates and provide early and appropriate treatment to patients. Considering the increasing attention toward cognitive impairment screening tools, to the best of our knowledge, this is the first meta-analysis designed to assess the diagnostic efficacy of the VCAT in identifying cognitive impairment, specifically including both MCI and mild dementia. Based on a meta-analysis of 1,446 patients from studies conducted in Asian countries , patients with MCI/mild dementia had lower VCAT scores than those without MCI/mild dementia, with a mean difference of \u22126.85. VCAT showed a combined sensitivity and specificity of 80% and 75%, respectively, with an overall AU-ROC of 0.77.In the current meta-analysis, VCAT showed a combined sensitivity and specificity of 80% and 75%, respectively, with an overall AU-ROC of 0.77. This finding indicates an acceptable diagnostic performance of the VCAT in distinguishing MCI/mild dementia from the healthy control group. In a previous systematic review, the AU-ROC for the MoCA and MMSE was 0.883 and 0.78, respectively . While tTraditional cognitive tests, including the MMSE and MoCA, have demonstrated limitations when applied to diverse populations, primarily owing to sociodemographic factors and linguistic dependency-related biases \u201328. In cThe emphasis of the VCAT on evaluating episodic memory and executive function, with half of the test specifically geared toward assessing episodic memory, makes it a potential screening tool for early AD detection and monitoring. A previous study indicated that deficits in episodic memory can signal the future development of Alzheimer\u2019s dementia in otherwise healthy adults . Furtherr\u2009=\u20090.819) between the MoCA and VCAT, further highlighting the robustness of the VCAT as a cognitive assessment tool.In the studies included in the current meta-analysis, Low et al.  examinedSeveral studies have highlighted the feasibility of integrating the VCAT into clinical settings, reporting its ease of completion as a significant advantage , 37. TheThe use of a visual-based format in the VCAT not only minimizes potential variables that could influence the results but also facilitates digital implementation . This clThe notable heterogeneity in our meta-analysis can be partially attributed to the study by Soo et al. , which rThe current meta-analysis has several limitations. First, all included studies were conducted in Asian populations, primarily Chinese participants in Singapore and Malaysia. This limits the generalizability of the findings to non-Asian and diverse populations. Further studies on other ethnicities and cultural groups are required. Second, only five studies were included in the meta-analysis. A greater number of high-quality studies would allow for a more robust quantitative synthesis and subgroup analyses to explore potential sources of heterogeneity. Third, the criteria used for defining MCI/mild dementia varied somewhat across studies, which could have introduced inconsistencies. Standardized diagnostic criteria improve the comparability. Fourth, data on key demographic variables, including gender, education, and language, have been inconsistently reported across studies. The impact of these factors on the VCAT accuracy could not be fully evaluated. Fifth, no data have been reported regarding the reliability, learning effects, or responsiveness of the VCAT to repeat administration. This further demonstrates its utility in monitoring cognitive changes over time. Finally, given the significant heterogeneity observed in the pooled sensitivity (94.23%), a systematic review might be better suited than a meta-analysis to present the existing literature on the use of VCAT in diagnosing cognitive decline. While pooling data in a meta-analysis can amplify the risk of misinterpretation owing to such pronounced heterogeneity, our findings underscore the limitations of the included studies and highlight potential directions for future research.5.This meta-analysis of five studies involving 1,446 older adults indicated that the VCAT, as a nonverbal and visual-based cognitive screening tool, has moderate diagnostic accuracy in distinguishing individuals with MCI/mild dementia from cognitively normal controls. The pooled sensitivity and specificity were 80% and 75%, respectively, with an AU-ROC of 0.77. Further rigorous studies are required to confirm its applicability and validity across different populations and settings.The original contributions presented in the study are included in the article/J-HH: Data curation, Investigation, Writing \u2013 original draft, Writing \u2013 review & editing. C-CL: Investigation, Writing \u2013 original draft, Writing \u2013 review & editing. I-WC: Investigation, Visualization, Writing \u2013 original draft, Writing \u2013 review & editing. J-YW: Methodology, Validation, Writing \u2013 original draft, Writing \u2013 review & editing. P-YH: Data curation, Resources, Writing \u2013 original draft, Writing \u2013 review & editing. T-HL: Data curation, Methodology, Writing \u2013 original draft, Writing \u2013 review & editing. K-CH: Conceptualization, Supervision, Visualization, Writing \u2013 original draft, Writing \u2013 review & editing."}
+{"text": "We propose an approach for prediction of\u00a0G4 formation in cells that incorporates epigenetic and chromatin accessibility data. The novel approach termed epiG4NN efficiently predicts cell-specific G4 formation in live cells based on a local epigenomic snapshot. Our results confirm the close relationship between H3K4me3 histone methylation, chromatin accessibility and G4 structure formation. Trained on A549 cell data, epiG4NN was then able to predict G4 formation in HEK293T and K562 cell lines. We observe the dependency of model performance with different epigenetic features on the underlying experimental condition of G4 detection. We expect that this approach will contribute to the systematic understanding of correlations between structural and epigenomic feature landscape.G-quadruplexes (G4s) are secondary structures abundant in DNA that may play regulatory roles in cells. Despite the ubiquity of the putative G-quadruplex-forming sequences (PQS) in the human genome, only a small fraction forms G4 structures in cells. Folded G4, histone methylation and chromatin accessibility are all parts of the complex  VEGF . G4 structures may be implicated in important biological processes, including replication, transcription , telomers. G4 strVEGF , bcl-2  methods that use structure-specific antibodies to detect G4 formed in cellular cultures  and chromatin accessibility (ATAC-seq) were selected for training and evaluation. We aim to predict weaker G4s formed in cells along with stable G4s. We base\u00a0our predictions on a broad set of putative quadruplex-forming\u00a0sequences (PQS) found in the human genome according to the latest motif definitions  and infer the PQS formation probability in cells. The developed approach is designed to transfer the learned features to predict G4 in unseen types of cells based on the underlying sequence and local epigenetic snapshot.python re package in the hg19/GRCh37 human genome assembly, retrieved from the UCSC Genome Browser (http://genome.ucsc.edu/). We broadened the existing definitions of G4 and used the following regular expressions: [G3+L1-12]3+G3+\u2014canonical G4 pattern with extended loop length; [GN0-1GN0-1GL1-3]3+GN0-1GN0-1G\u2014bulged G4 pattern with possible G-run breaks; [G1-2N1-2]7+G1-2\u2014irregular G4 pattern, where L is any of {A, T, C, G} and N is any of {A, T, C}. A total number of guanines greater than or equal 12 was required to allow three-layered G4s. The search was performed on both strands. We filtered the redundant and nested G4s by only considering distinct sequences separated by at least one nucleotide. The overlapping sequences were not merged. The first encountered motif from the 5\u2032 end of the overlapping group is considered. A total of 2\u00a0105\u00a0837 PQS were found for the three types . We filtered out regions with no full coverage of any of the features to ensure continuous data availability for every PQS. All epigenetic data were normalized to a range of values . We then created input arrays of 1000-nt epigenetic profiles for each PQS with bedtools intersect command-line tool. Genomic tracks were visualized with pyGenomeTracks tool  as a baseline for epiG4NN. Model inputs are arrays of shape  for G4NN, sequence-based model, and arrays of shape  for epiG4NN. The first four rows of the input are the one-hot-encoded sequences, and the last row is the normalized epigenetic feature. Output of each residual unit is added to the penultimate layer through a 1D convolution with hidden state of size 32, therefore implementing skip, or residual, connections between units for better convergence and avoidance of vanishing/exploding gradients problem weight, resulting in weights 0.35 and 35.69 for negative and positive classes. We evaluated the models on withheld test samples using accuracy and area under the receiver operating characteristic curve (AUROC) to determine class separability. AUROC x-axis depicts false positive rate (FPR), and y-axis depicts true positive rate (TPR), defined as FPR\u00a0=\u00a0FP/(FP\u00a0+\u00a0TN) and TPR\u00a0=\u00a0TP/(TP\u00a0+\u00a0FN). However, these metrics are not optimal for imbalanced classification problems, therefore we additionally evaluate areas under the precision\u2013recall curve (AUPRC) to determine precision and recall of positive G4 samples. Precision (P) and recall (R) are defined as follows: P\u00a0=\u00a0TP/(TP\u00a0+\u00a0FP), R\u00a0=\u00a0TP/(TP\u00a0+\u00a0FN), where TP is true positives, FP is false positives, and FN is false negatives. Training and evaluation were performed using TensorFlow (v2.4.0) using python keras API. Respective python scripts can be found at https://github.com/anyakors/epiG4NN. For the promoter-enhancer bias assessment, gene promoter data for hg19 were downloaded from the UCSC table browser, and cell line-specific enhancer data were downloaded from the Enhancer Atlas v 2.0 database http://www.enhanceratlas.org/downloadv2.php . We trained our models on A549 G4 data from a RHAU-derived G4-binding protein\u00a0G4P-ChIP-seq experiment  on A549 cell data  and the epigenetic mark of active enhancers, H3K27ac  for G4 formation. On the other hand,\u00a0epiG4NN-H3K4me1,\u00a0epiG4NN-H3K9me3 and epiG4NN-H3K36me3\u00a0gave only moderate improvements  compared to sequence-based G4NN . Combining two best predictive features only resulted in a marginal performance improvement , 38% of K562 (CUT&Tag) and 82% of HeLa G4 peaks are common with A549. To assess how well our model performs on different cell types with distinct underlying epigenetic landscapes, we additionally carried out model evaluation on HEK293T (G4P ChIP-seq) and K562 (CUT&Tag) cell lines. As epiG4NN was trained on G4 peaks from A549 cells obtained with G4P ChIP-seq, we only used data from in situ G4 detection experiments to exclude possible technical variability. We selected the three best models as tested on A549 unseen samples  together with the baseline sequence-only model G4NN and measured their performance on new cell lines with AUROC and AUPRC  and K562 (CUT&Tag) (AUPRC\u00a0=\u00a00.838) cell data, followed by epiG4NN-ATAC for K562 (CUT&Tag) and epiG4NN-H3K27ac for HEK293T. epiG4NN-ATAC, however, performed slightly worse than sequence alone for HEK293T.Populations of G4s in cellular context are shared between cell lines or formed uniquely in some cells. We compared the pre-processed G4 peaks obtained from HEK293T, HaCaT, HeLa cells using G4P ChIP-seq , K562 ceex vivo BG4 ChIP-seq method. Given the same epigenetic profile and the same sequence motifs as for the K562 (CUT&Tag) data, with a different set of G4 peaks to predict, epiG4NN-H3K4me3 and epiG4NN-H3K27ac models were only able to perform marginally better than the G4NN baseline in terms of AUROC and AUPRC; epiG4NN-ATAC resulted in a slightly better evaluation   and K56ChIP-seq ), followChIP-seq  and CUT&ChIP-seq  showed tChIP-seq ) that ims report , while Hs report ,38. Addil DeepG4  on our dl DeepG4 , demonstlqad071_Supplemental_FileClick here for additional data file."}
+{"text": "H) gene usage, these antibodies can be divided in two groups. One group is encoded by the inherently autoreactive VH4-34 gene segment, derives from anti-DNase1L3 germline-encoded precursors, and gains cross-reactivity to dsDNA \u2013 and some additionally to cardiolipin \u2013 following somatic hypermutation. The second group, originally defined as nephritogenic anti-dsDNA antibodies, is encoded by diverse VH gene segments. Although affinity maturation results in dual reactivity to DNase1L3 and dsDNA, their binding efficiencies favor DNase1L3 as the primary antigen. Clinical, transcriptional and monoclonal antibody data support that cross-reactive anti-DNase1L3/dsDNA antibodies are more pathogenic than single reactive anti-dsDNA antibodies. These findings point to DNase1L3 as the primary target of a subset of antibodies classified as anti-dsDNA, shedding light on the origin and pathogenic heterogeneity of antibodies reactive to dsDNA in SLE.Anti-dsDNA antibodies are pathogenically heterogeneous, implying distinct origins and antigenic properties. Unexpectedly, during the clinical and molecular characterization of autoantibodies to the endonuclease DNase1L3 in patients with systemic lupus erythematosus (SLE), we identified a subset of neutralizing anti-DNase1L3 antibodies previously catalogued as anti-dsDNA. Based on their variable heavy-chain (V Antibodies directed against DNA in systemic lupus erythematosus are functionally diverse. This study demonstrates that DNAse1L3 is the primary target of a subset of autoantibodies previously considered specific for double-stranded DNA. According to the clonal selection theory of antibody production, the different autoantigen reactivities in SLE serum should be explained by the simultaneous presence of different autoantibodies, each of them with a unique specificity2. Yet, growing evidence\u2014including the analysis of patient-derived monoclonal antibodies\u2014has shown that the autoantibody landscape in SLE is populated by pathogenic autoantibodies reacting with multiple antigens15. Identifying the antigenic cross-reactivity of these autoantibodies has been of interest to investigators trying to elucidate both the potential triggering and the target antigens in SLE. Anti-dsDNA antibodies are of particular interest in this regard. Although the presence of antibodies to dsDNA is a unifying feature among patients with SLE, not all anti-dsDNA antibodies are pathogenic16. These findings have suggested that anti-dsDNA antibodies comprise a heterogeneous pool of autoantibodies with distinct origins, physicochemical and antigenic properties18, and that fine specificities, in addition to dsDNA binding govern their pathogenic effect in SLE3. For instance, a subset of anti-dsDNA antibodies that bind the N-methyl-D-aspartate receptor can drive neuronal death and neuropsychiatric lupus11, and cross-reactivity with intrinsic renal antigens, such as \u03b1-actinin, has been proposed as a mechanism by which a subset of anti-dsDNA antibodies can mediate nephritis19. The basis of the heterogeneity and cross-reactivity of anti-dsDNA antibodies, however, remains unknown.Systemic lupus erythematosus (SLE) is a complex multisystem disease characterized by the production of antibodies to a diverse number of autoantigens leading to immune-mediated tissue damage21. The enzyme is primarily secreted by myeloid cells (i.e. macrophages and dendritic cells)24, and together with DNase1, is responsible for the DNase activity in circulation25. Different to DNase1, however, DNase1L3 is more efficient in the inter-nucleosomal cleavage of nuclear DNA, suggesting that its major role is the digestion of chromatin from apoptotic and necrotic cells and therefore, in regulating the load of immunogenic DNA27. This notion is supported by the finding that null mutations and hypomorphic variants of DNase1L3 are linked to familial and sporadic SLE, respectively29. In addition, lupus-prone MRL and NZB/W F1 mice are deficient in DNase1L3, and the sole deficiency of this enzyme leads to lupus-like disease in mice30. Mechanistically, DNase1L3 decreases the availability of antigenic cell-free DNA by fragmenting DNA, reducing its exposure on apoptotic cell microparticles31. In the absence of DNase1L3 activity, extracellular self DNA drives TLR-dependent type-I interferon (IFN-I) production and extrafollicular differentiation of antibody-forming cells, driving anti-dsDNA antibodies and SLE32. Interestingly, the recent finding that patients with SLE have autoantibodies to DNase1L3 highlights that the DNase1L3 pathway is also pathogenically targeted in sporadic SLE21.DNase1L3 is a member of the DNase1 family of DNA endonucleases, which was recently found to be the target of autoantibodies in SLEIn this work, we combined clinical and blood transcriptional data, together with an extensive analysis of patient-derived monoclonal antibodies, to understand the origin and immunopathology related to anti-DNase1L3 antibodies in SLE. We found that antibodies to DNase1L3 and dsDNA are associated with both clinical and transcriptional features of SLE disease activity. However, it was intriguing that this association was only significant in patients positive for both autoantibodies compared to patients single positive for either antibody. Through the analysis of SLE serum and patient-derived monoclonal antibodies, we found that a subset of anti-DNase1L3 antibodies is also reactive with dsDNA, providing a rational explanation that SLE disease activity is associated with a single autoantibody with dual reactivity to DNase1L3 and dsDNA. Indeed, these autoantibodies are highly mutated, originate from both autoreactive and non-autoreactive precursors, and have the dual ability of neutralizing DNase1L3 activity and bind to dsDNA with high efficiency, likely amplifying their pathogenic potential compared to monospecific autoantibodies. Based on the binding efficiency of mutated and germline reverted monoclonal antibodies to DNase1L3 and dsDNA, the data support that DNase1L3 is the primary target of these antibodies and dsDNA is the cross-reactive antigen. Collectively, our studies underscore DNase1L3 as the protein antigen and functional target of a subset of pathogenic antibodies catalogued as anti-dsDNA in SLE.33. Demographic, clinical and laboratory features of the SLE cohort are summarized in Supplementary Table\u00a0P\u2009<\u20090.001)  of healthy controls were positive for anti-DNase1L3 antibodies (P\u2009<\u20090.0001). Antibodies to DNase1L3 were significantly associated with anemia, livedo, proteinuria, low complement levels, use of cytotoxic treatment, and a broad range of autoantibodies, including anti-dsDNA, anti-cardiolipin, lupus anticoagulant, anti-\u03b22-glycoprotein I (B2GPI), anti-Ro52 antibodies  vs. 31.2% (15/48), P\u2009<\u20090.0001] were more common with anti-DNase1L3 antibodies , 1.8 0\u201312) vs. 3.4 (0\u201312), 02] Fig.\u00a0, which w02] Fig.\u00a0. Specifiies Fig.\u00a0. Further vs. 3.4 34. To define whether antibodies to DNase1L3 are associated with distinct transcriptional fingerprints in SLE, we used gene expression data from blood collected in parallel with the samples used to measure anti-DNase1L3 antibodies. We identified 584 differentially expressed transcripts (DETs) between anti-DNase1L3 negative and positive SLE patients  , 1319 (1187\u20131472) vs. 1229 (1104\u20131330), respectively, P\u2009=\u20090.025]  Fig.\u00a0. Moreove25] Fig.\u00a0, support34, we conducted single-sample Gene Set Enrichment Analysis (ssGSEA)37 of the blood transcription modules described by Chaussabel et al.38. Interestingly, modules known to be enriched in SLE34 were associated with anti-DNase1L3 positivity  was the only component significantly associated with anti-DNase1L3 positivity 45. To assess whether serum reactivity to DNase1L3 is linked to antibodies containing the VH4-34 encoded heavy chain, antibodies in serum were depleted using the anti-idiotypic antibody 9G4, a monoclonal antibody that specifically targets VH4-34 encoded antibodies46. Interestingly, depletion of antibodies bearing the 9G4 idiotype decreased the reactivity to DNase1L3 by 0% to 80% in anti-DNase1L3 positive SLE sera  B cells and ASCs7  from SLE patients experiencing flaresene Fig.\u00a0. Based oene Fig.\u00a0. The enr47, DNase1L3 activity was determined by absolute quantitation of inter-nucleosomally fragmented genomic DNA using ligation-mediated qPCR (LM-qPCR)49. First, we validated this assay by using increasing amounts of enzyme to digest native chromatin in intact nuclei  n\u201d) Fig.\u00a0. Among tn\u201d) Fig.\u00a0. Antibodn\u201d) Fig.\u00a0.Fig. 5Ha50 . Strikingly, while binding to DNase1L3 was either unchanged (i.e. clone 627A11GL), slightly decreased (i.e. clone 75G12/A11GL) or even enhanced (i.e. cloned 88F7GL) after antibody reversion to germline, the reactivity of these antibodies to dsDNA or cardiolipin was importantly decreased or completely lost  and corresponding light chain (IgL) variable gene sequences to their germline form, and binding to DNase1L3, dsDNA, and cardiolipin were assessed by EC50 Fig.\u00a0. Importaost Fig.\u00a0. TogetheH4-34+ B cells or is a common feature among anti-dsDNA antibodies, we additionally analyzed four SLE-derived non-VH4-34 IgG monoclonal antibodies originally defined as anti-dsDNA from which both the IgH and IgL variable gene sequences are publicly available 51. Their V(D)J usage, CDR3 sequence, and number of SHM are summarized in Fig.\u00a052. In contrast, monoclonal 32.B9 is not nephritogenic in mice52. Antibody 33.H11 has not been tested in vivo. Interestingly, however, this antibody and the nephritogenic monoclonal RH-14 are public clonotypes . As previously described, we confirmed that the four monoclonal antibodies bind dsDNA with high and similar efficiency  and antibodies to DNase1L3 were only determined in a subset of these patients 21, limiting their capacity to find other clinical and serological associations described in our study. Interestingly, the study by Hartl et al. neither found an association between anti-DNase1L3 and anti-C1q antibodies21, which are highly prevalent in patients with renal involvement. Considering that antibodies to dsDNA and C1q are present in up to 80% and 100% of patients with renal disease, respectively56, and that 72% of the patients had renal involvement, it is surprising that Hartl et al. found no associations of these antibodies with anti-DNase1L3 antibodies, unless a significant number of patients with renal disease were seronegative for antibodies to dsDNA and C1q. Alternatively, Hartl et al. only examined the correlation between autoantibody levels, which was not significant, rather than the association with antibody positivity, as we did in our study. For the correlation analysis, Hartl et al. only included small subsets of patients in which the autoantibodies were detected using a homemade bead-based antigen array [i.e. anti-dsDNA in 33/120 (28%) and anti-C1q in 25/120 (21%) patients]. Aside from the small sample size, finding a significant correlation between antibody levels is unlikely because cross-reactive anti-DNase1L3/dsDNA antibodies only correspond to a subset of antibodies detected by the independent anti-DNase1L3 and anti-dsDNA assays.While we found a striking association between anti-DNase1L3 and anti-dsDNA antibodies, which is explained in part by the presence of double reactive antibodies to DNase1L3 and dsDNA, it is puzzling that a recent study by Hartl et al.H4-34, which encodes for a significant number of autoantibodies in active SLE. VH4-34+ B cells, bearing the idiotype 9G4, are inherently autoreactive cells largely excluded from the germinal centers and underrepresented in the memory compartment in healthy individuals57. In patients with SLE, however, 9G4-B cells progress through this checkpoint and successfully participate in germinal center reactions, generating increased levels of IgG memory and plasma cells58. 9G4 antibodies represent 10\u201345% of the total serum IgG in patients with active disease58, which account for the vast majority of anti-B cell CD45 antibodies and a significant fraction of anti-dsDNA in SLE.The analysis of SLE-derived monoclonal antibodies identified two subsets of anti-DNase1L3/dsDNA antibodies according to their origin. A subset uses the variable heavy-chain gene segment V6, which is responsible for their differential binding to cardiolipin. Monoclonal 88F7 is particularly interesting, because it binds DNase1L3 more efficiently in its germline form, suggesting that affinity maturation of this antibody may not have been directly driven by DNase1L3. Since DNase1L3 interacts with dsDNA, it is possible that dsDNA and DNase1L3 are both involved in the process of affinity maturation of this subset of anti-DNase1L3 antibodies, facilitating the production of dual-reactive autoantibodies in which the affinity against either antigen may be stochastically determined.Within a large set of 9G4 SLE-derived monoclonal antibodies, we identified 4 antibodies reactive to DNase1L3, which also bind dsDNA and some to cardiolipin. Further analysis of these antibodies showed that while binding to dsDNA or cardiolipin was decreased in their germline form, binding to DNase1L3 was minimally affected or even enhanced, suggesting that these antibodies originated from autoreactive B cell precursors with preferential reactivity against this endonuclease. The finding that SHM increased the binding of antibody clones 75G12, 75A11, and 627A11 to dsDNA and of clones 75G12 and 627A11 to cardiolipin implicates SHM as the driver of cross-reactivity in these autoantibodies. This notion is further supported by the finding that antibodies 75G12 and 75A11 are clonally related with a single amino acid difference in HCDR2H gene segments. Binding efficiency of this set of antibodies is higher to DNase1L3 than dsDNA, suggesting that DNase1L3 is the primary target and dsDNA is the cross-reactive antigen. Nevertheless, like the VH4-34-derived subset, we cannot exclude the possibility that both DNase1L3 and dsDNA might be involved in the affinity maturation of this set of autoantibodies. In contrast to the VH4-34-derived subset, however, binding of these antibodies to dsDNA and DNase1L3 is entirely dependent on SHM and none of the antibodies showed reactivity to cardiolipin. Together, these findings imply that anti-DNase1L3/dsDNA antibodies are heterogenous regarding their origin, reactivities and can be generated from both autoreactive and non-autoreactive B cell precursors. Moreover, the data demonstrate that these antibodies can be incorrectly categorized as being monospecific for either DNase1L3 or dsDNA\u2014 and some to cardiolipin\u2014when tested by a single assay.The second subset of anti-DNase1L3/dsDNA antibodies, originally defined as nephritogenic antibodies to dsDNA, are encoded by diverse VIndependently of their origin, the presence of autoantibodies with binding capacity to both DNase1L3 and dsDNA has important implications for SLE pathogenesis. By targeting distinct autoimmune pathways in parallel, these antibodies may have the ability to amplify their pathogenic potential. For instance, these antibodies can increase the load of extracellular dsDNA by blocking DNase1L3 activity, while binding to dsDNA can generate immune complexes promoting IFN-I production and cause direct damage by depositing in tissues, such as the kidney. In this regard, it is noteworthy that SLE patients double positive for anti-DNase1L3 and anti-dsDNA antibodies showed the most striking association with disease activity and the IFN and myeloid signatures when compared with single positive patients for either antibody specificity, or negative for both autoantibodies. Thus, the coexistence of serum reactivities to dsDNA and DNase1L3 seems to identify a subset of pathogenic antibodies linked to higher disease activity in SLE. In the context of the analysis of monoclonal antibodies, the most rational explanation is that these pathogenic antibodies correspond to the subset with dual reactivity to DNase1L3 and dsDNA.The finding that some monoclonal antibodies also cross-react with cardiolipin is intriguing, particularly because these antibodies target cardiolipin bound to B2GPI and therefore have the potential to be pathogenic. Although this study did not focus on the analysis of anti-phospholipid syndrome, it is worth noting that anti-DNase1L3 antibody positivity was significantly associated with livedo, lupus anticoagulant, antibodies to cardiolipin and B2GPI, and marginally with arterial thrombosis. Thus, it is possible that triple reactive antibodies\u2014targeting DNase1L3, dsDNA and cardiolipin\u2014may contribute to the pool of anti-phospholipid antibodies in SLE.The study of SLE serum and monoclonal antibodies also demonstrated that not every antibody catalogued as anti-dsDNA or anti-DNase1L3 has dual reactivity to these antigens. There are a significant number of SLE sera that are only positive for anti-dsDNA or anti-DNase1L3 antibodies, not all anti-DNase1L3 antibodies in serum were blocked by dsDNA, and we identified one anti-dsDNA monoclonal antibody (32.B9) with no reactivity to DNase1L3. Monoclonal 32.B9 is particularly interesting because binding to dsDNA is germline encoded, reactivity to dsDNA is enhanced after SHM, and despite having the same binding efficiency to dsDNA as the dual reactive antibodies 33.C9 and RH-14, it is not nephritogenic in mice. Moreover, in contrast to dual reactive antibodies that block DNase1L3 activity, we were surprised that monoclonal 32.B9 enhanced chromatin degradation by DNase1L3, highlighting the incredible heterogeneity in the function of antibodies catalogued as anti-dsDNA in SLE. Whether antibody 32.B9 and others with similar activity may instead be protective for SLE by promoting DNase1L3-mediated chromatin degradation is a hypothesis that will need further exploration. Moreover, these findings underscore that studying the activity of anti-DNase1L3 antibodies in serum or using bulk IgG from SLE patients should be avoided because it will be affected by the presence of different autoantibodies targeting components in the substrate, such as anti-dsDNA antibodies.59 and the identity of the antigen that elicits the production of antibodies to dsDNA remains unclear. Indeed, the previous finding that a peptide surrogate for dsDNA can induce anti-dsDNA antibodies and nephritis in mice supports the notion that a protein antigen can trigger the induction of antibodies reacting with dsDNA60. The discovery of anti-DNase1L3/dsDNA antibodies renews interest in better understanding the significance of cross-reactivity among autoantibodies in SLE, which may shed light on the origin and heterogeneity among the wide range of autoantibodies found in this autoimmune disease.Antibody 32.B9 was also useful to demonstrate that binding to DNase1L3 is not a general feature of anti-dsDNA antibodies. Instead, we propose that anti-DNase1L3/dsDNA antibodies correspond to a distinct subset of autoantibodies in which the primary substrate is DNase1L3. This notion is particularly appealing, as mammalian dsDNA is poorly immunogenic61 cohort were studied to define the prevalence and clinical significance of antibodies to DNase1L3 in SLE. SPARE is a prospective observational cohort that has been extensively described previously61. Briefly, adult patients (age 18 to 75 years-old) who met the definition of SLE per the revised American College of Rheumatology classification criteria were eligible into the study62. Sex and gender of each participant was collected based on self-report. At baseline, the patient\u2019s medical history was reviewed, and information on current medications was recorded. Patients were followed-up over a 2-year period. Patients were treated according to standard clinical practice. Disease activity was assessed using the Safety of Estrogens in Lupus Erythematosus: National Assessment (SELENA) version of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)63 and physician global assessment (PGA)64. C3, C4, anti-dsDNA (Crithidia), complete blood cell count and urinalysis were performed at every visit. Study participants also underwent whole blood gene expression analysis at baseline using the Affymetrix GeneChip HT HG-U133+61. Eighty-seven monoclonal antibodies previously generated from SLE patients experiencing flares were used to screen for anti-DNase1L3 antibodies7. All samples were obtained with written informed consent from the participants. The study protocol was approved the Institutional Review Boards at the Johns Hopkins University School of Medicine and Emory University School of Medicine.The objective of this study was to investigate the origin and immunopathology related to anti-DNase1L3 antibodies in patients with SLE. To evaluate this, sera from 62 healthy controls and 158 SLE patients from the \u201cStudy of biological Pathways, Disease Activity and Response markers in patients with Systemic Lupus Erythematosus\u201d (SPARE)[35S]methionine-labeled DNase1L3 by TNT T7 Quick Coupled Transcription/Translation (Promega). pET-28a-DNase1L3 was used to generate recombinant active DNase1L3 using the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs). The control plasmid encoding dihydrofolate reductase (DHFR) is included in the PURExpress kit.cDNA encoding human mature DNase1L3 (amino acids 21-305) was synthesized using RNA from human PBMCs and cloned into pcDNA3.1 and pET-28a(+). In pET-28a, the 5\u2019 site end in mature DNase1L3 was cloned at the NcoI site in the vector. Thus, the T7 promoter is followed by a ribosome binding site and the DNase1L3 start codon. The protein encoded by pET-28a-DNase1L3 is not tagged. pcDNA3.1-DNase1L3 was used to generate Radiolabeled DNase1L3 was immunoprecipitated with 2\u2009\u03bcl of serum, 10\u2009\u00b5L of cell supernatants from monoclonal antibody producing cells, or with purified monoclonal antibodies in 300\u2009\u00b5L of NP-40 buffer  for 1\u2009hr at 4\u2009\u00b0C. In some experiments, anti-DNase1L3 binding was performed in the presence of 1\u2009mg/mL Salmon Sperm DNA (Thermo Fisher Scientific). Protein A beads were added and incubated for additional 30\u2009min at 4\u2009\u00b0C. After three washes with vortexing in NP-40 lysis buffer, beads were boiled in SDS sample buffer. Samples were separated by gel electrophoresis, and immunoprecipitated proteins were visualized by radiography. Densitometry was performed on all films and values were normalized to a high-titer anti-DNaseL13 serum. Antibody positivity was defined using a cutoff of two standard deviations above the mean anti-DNase1L3 antibody level in healthy sera.7. Single peripheral blood plasmablasts (CD3-CD14-CD19\u2009+\u2009CD20-CD27\u2009+\u2009CD38+) from a healthy female donor were sorted into 96-well PCR plates. IgH and IgL (\u03ba or \u03bb) variable regions were cloned into expression vectors containing human Ig\u03b31, Ig\u03ba, or Ig\u03bb constant regions as previously described65. One monoclonal antibody (C4) was highly expressed and was selected for further characterization. Analysis of the antigen specificity of C4 was performed using the HuProt human proteome microarray (CDI). C4 exhibited broad, non-specific reactivity to 0.25% of the >21,000 proteins on the array, but had no reactivity to DNAse1L3. This antibody was used a monoclonal control. The IgH and IgL variable gene sequences of monoclonal antibodies 32.B9, 33.H11, 33.C9, and RH-1451 were synthetized using the Custom gene synthesis service from Integrated DNA Technologies (IDT), and cloned into expression vectors containing human Ig\u03b31, Ig\u03ba or Ig\u03bb constant regions .The cloning of monoclonal antibodies from single B cells and antibody-secreting cells (ASCs) from SLE patients experiencing flares was previously describedThe V(D)J germline sequences with the lowest number of mismatch nucleotides compared to mutated sequences were obtained using IgBLAST, synthetized using the Custom gene synthesis service from IDT, and cloned into expression vectors containing human Ig\u03b31, Ig\u03ba or Ig\u03bb constant regions.Monoclonal antibodies were produced using 293T cells in high glucose DMEM and 10% ultra-low IgG fetal bovine serum (Gibco), Expi293 cells and ExpiCHO cells (Thermo Fisher Scientific) by co-transfecting plasmids encoding IgH and IgL according to the manufacturer instructions. Supernatants were collected at day 5 after transfection and the antibodies purified using Protein A beads (Pierce). We found no differences among monoclonal antibodies produced by either cell type, although ExpiCHO cells are certainly the most efficient to produce larger amounts of antibodies.49 with some modifications. Briefly, the DHApo1 and DHApo2 oligonucleotides  were annealed by mixing 50\u2009\u00b5L (100 pmol/\u03bcl) of each in 250\u2009\u00b5l of 250\u2009mM Tris (pH 7.7), heating the mixture to 90\u2009\u00b0C for 5\u2009min, incubating at 55\u2009\u00b0C for 15\u2009min, and allowing the mixture to cool to RT. The linker mixture was frozen and thawed on ice before use. Ligation reactions (20\u2009\u00b5L) were performed overnight at 16\u2009\u00b0C using Quick T4 ligase (New England Biolabs), 100\u2009ng DNA and 1\u2009\u00b5L linker. qPCR was performed using the DHApo1 primer and iTaq Universal SYBR Green Supermix (Bio Rad) as follow: 95\u2009\u00b0C for 4\u2009min (1 cycle), 72\u2009\u00b0C for 4\u2009min (1 cycle), followed by 40 cycles of denaturation at 94\u2009\u00b0C for 1\u2009min and annealing/extension at 72\u2009\u00b0C for 3\u2009min.DNA degradation was measured by quantitation of inter-nucleosomally fragmented DNA by LM-qPCR as previously described2, 2\u2009mM CaCl2, pH 7.0) containing 5% bovine serum albumin (BSA) solution (Sigma). PURExpress DHFR was used as negative control. After 30\u2009min at 37\u2009\u00b0C, reactions were stopped by adding 20\u2009\u00b5L of proteinase K buffer , incubation at 50\u2009\u00b0C for 20\u2009min and heat inactivation at 95\u2009\u00b0C for 5\u2009min. DNA was purified by isopropanol precipitation and resolved in a 1.5% agarose gel. In addition, DNA degradation was measured by quantitation of inter-nucleosomally fragmented DNA by LM-qPCR. The amount of inter-nucleosomally fragmented DNA was calculated by the DNase1L3 and control DHFR were generated using the PURExpress In Vitro Protein Synthesis Kit (NEB). DNase1L3 activity was titrated by co-incubating increasing amounts of PURExpress DNase1L3  with 10,000 purified nuclei in 20\u2009\u00b5L of DNase reaction buffer  primarily to manufacture suggestions. The idiotypic rat anti-human 9G4 mAb was saturated within the agarose and cross-linked utilizing a multi-flow through load. 500\u2009\u00b5L of patient sera was utilized and bound overnight at 4\u2009\u00b0C prior to flow through. The elution protocol and column wash resulted in roughly a 1:5 dilution of the sera volume following flow through. Total IgG and 9G4 specific ELISAs were performed on the sera pre- and post-depletion. The total IgG loss ranged from 10 to 27% following the 9G4 column depletion, however the 9G4 IgG loss was >99% in all samples.50 values were calculated using a 4 parameter logistic regression (4PL) model66. The average EC50 was determined from two independent experiments.Anti-DNase1L3 monoclonal antibodies and their germline variants were titrated in duplicate against dsDNA and cardiolipin bound to B2GPI using QUANTA Lite\u00ae dsDNA  and QUANTA lite\u00ae ACA IgG III  ELISA assays, respectively. For antibody binding to DNase1L3, we developed an in-house magnetic bead-based immunoassay. Mature DNase1L3 (amino acids 21-305) containing a N-terminal FLAG-tag sequence (FLAG-DNase1L3) was generated by TNT T7 Quick Coupled Transcription/Translation (Promega) and purified using anti-DYKDDDDK Magnetic Agarose beads (Pierce\u2122). FLAG-IRF3 generated by TNT T7 Quick Coupled Transcription/Translation was used as FLAG-tag protein control. FLAG-IRF3 was chosen because the plasmid was already available in the lab. After extensive washes with NP40-buffer, beads containing FLAG-tagged proteins were blocked for one hour with Protein-Free (TBS) Blocking Buffer (Pierce\u2122) and further incubated in duplicate with decreasing concentrations of monoclonal antibodies for one hour using 96-well plates. A 96-well plate magnet was used to keep the beads in the wells during manual washes.\u00a0After three washes with NP-40 buffer, anti-DNase1L3 antibody binding to FLAG-DNase1L3 or FLAG-IRF3 beads was detected using a horseradish peroxidase (HRP)-conjugated goat anti-human IgG secondary antibody  diluted 1:10,000 in Protein-Free (TBS) Blocking Buffer (Pierce\u2122). SureBlue TMB peroxidase substrate (KPL) was added after washing the beads with NP-40 buffer to visualize antibody binding and an equal volume of 1\u2009M hydrochloric acid was added to stop the colorimetric reaction, before determining the absorbance at 450\u2009nm. Individual values were corrected for background by subtracting the reactivity to FLAG-IRF3 beads. Relative EC33. CEL files were subjected to RMA background correction, and quantile normalization, using the Oligo package34. To select only expressed genes in whole blood, we filtered out transcripts that had a raw signal <100 in <10% of samples with the genefilter R package. All calculations and analyses were performed using R (ver 4.0.2) and Bioconductor (ver 3.13)67. Differentially expressed transcripts (DETs) were analyzed using the R package limma68. Functional gene set enrichment was carried out with the R interface gprofiler2 for the server g:Profiler69.Gene expression analysis from the SPARE cohort was previously described68. The Blood gene expression modules from Chaussabel et al.38 were obtained from the R package \u201ctmod\u201d for Bioconductor70. Module activity at the individual level was calculated by ssGSEA37. Differentially regulated modules according to anti-DNase1L3 status were analyzed with a linear model approach using the R package limma.DETs were analyzed using the R package limmaT test and ANOVA test as indicated. The Mann-Whitney\u2019s U test and Kruskal\u2013Wallis test was used for group-wise comparisons of non-normally distributed variables Fisher\u2019s exact test and \u03c72 tests were used for univariate analysis on SPARE cohort variables, as appropriate. Exact2x2 package in R version 3.5.1 was used for binary variables to obtain p-value, OR, and 95% CI. Multivariate analyses were carried out using multivariate logistic regression or multivariate linear regression as indicated. Unsupervised hierarchical clustering with complete linkage was performed by computing a correlation-based distance between genes (Pearson\u2019s method) and the Canberra metric for the distance between subjects. Heatmap visualization was done using the Complex heatmap R package71. To improve visualization, dendrograms were reordered using the modular leaf ordering methods from the dendsort R package72. Statistical significance was set at p\u2009<\u20090.05. Since 94% of study the study population were female (Supplementary Table\u00a0Comparisons of continuous variables between groups were done using Student\u2019s Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileReporting Summary"}
+{"text": "Arabidopsis thaliana. However, the molecular mechanism by which SMXL6,7,8 negatively regulate drought tolerance and ABA response remains largely unexplored. In the present study, the interacting protein and downstream target genes of SMXL6,7,8 were investigated. Our results showed that the substrate receptor for the CUL4-based E3 ligase DDB1-BINDING WD-REPEAT DOMAIN (DWD) HYPERSENSITIVE TO ABA DEFICIENT 1 (ABA1) (DWA1) physically interacted with SMXL6,7,8. The degradation of SMXL6,7,8 proteins were partially dependent on DWA1. Disruption of SMXL6,7,8 resulted in increased drought tolerance and could restore the drought-sensitive phenotype of the dwa1 mutant. In addition, SMXL6,7,8 could directly bind to the promoter of SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASE 2.3 (SnRK2.3) to repress its transcription. The mutations in SnRK2.2/2.3 significantly suppressed the hypersensitivity of smxl6/7/8 to ABA-mediated inhibition of seed germination. Conclusively, SMXL6,7,8 interact with DWA1 to negatively regulate drought tolerance and target ABA-response genes. These data provide insights into drought tolerance and ABA response in Arabidopsis via the SMXL6,7,8-mediated SL signaling pathway.SUPPRESSOR OF MAX2-LIKE 6, 7, and 8  function as repressors and transcription factors of the strigolactone (SL) signaling pathway, playing an important role in the development and stress tolerance in  SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASES (SnRK2s) and allowing SnRK2s to activate downstream ABA-RESPONSIVE PROMOTER ELEMENTS (AREB)/ABA-BINDING FACTORS (ABFs) in ABA-responsive genes [Drought is a potent abiotic stressor that hampers plant performance and induces yield loss by depressing their physio-biochemical and molecular functions . Abscisive genes ,5.Arabidopsis, SnRK2.2, SnRK2.3, and SnRK2.6 are functionally redundant and are critical for the subsequent response to abiotic stress in the ABA signaling pathway [snrk2.2/2.3/2.6 triple mutant exhibits more severe phenotypes in ABA signaling and water stress response compared with the wild type [TaSnRK2.3 significantly enhances drought tolerance in common wheat [SnRK2s are a family of plant-specific protein kinases that positively regulate ABA signaling . Among t pathway ,8. ABA a pathway . The snrild type . Overexpon wheat . These son wheat .Arabidopsis to recruit the repressors D53 in rice or SMXL6,7,8 in Arabidopsis, resulting in their degradation by the 26S proteasome [Strigolactones (SLs) are a class of carotenoid-derived plant hormones that positively regulate seed germination ; leaf seoteasome ,24.rac-GR24 enhances the drought tolerance of wild-type plants [max3 and max4 mutants, the SL-signaling max2 mutant, and the SL receptor d14 mutant are more susceptible to drought than the wild type in Arabidopsis [SsMAX2 of Sapium sebiferum, MdMAX2, and MdD14 of Malus domestica (apple) in Arabidopsis improve drought and salt stress tolerance [Arabidopsis [Arabidopsis; and BRASSINAZOLE RESISTANT 1 (OsBZR1) in rice to repress the expression of TEOSINTE BRANCHED 1 (TaTB1), BRANCHED1 (BRC1), and FINE CULM 1 (FC1) and promote tillering/branching [D53 expression in rice [SMXL6,7,8 to inhibit their transcription, suggesting that SMXL6,7,8 have a dual function in SL signaling [SLs positively regulate drought tolerance ,25,26. Ee plants . SL-defibidopsis ,25,26. Oolerance ,28,29. Tbidopsis ,31,32. Sbidopsis ,23. It hranching ,34. More in rice . SMXL6,7ignaling ,36.smxl6/7/8 triple mutant, and drought-stress-inducible ABA-response genes were also up-regulated in smxl6/7/8, indicating that SMXL6,7,8 may participate in ABA-mediated drought response through transcriptional regulation of ABA response genes [ABA and SLs are two carotenoid-derived phytohormones that positively regulate drought tolerance in plants. ABA hypersensitivity and stronger drought tolerance were observed in the se genes ,32. Chrose genes . These dSnRK2.3 to repress its transcription and then its response to ABA-mediated seed germination. Accordingly, this work further reveals a possible molecular mechanism by which SMXL6,7,8 regulate drought tolerance and ABA response in plants.In this study, we screened a protein (DWA1) interacting with SMXL6,7,8 that promoted the degradation of SMXL6,7,8 in a 26S proteasome-dependent pathway. Furthermore, SMXL6,7,8 directly bound to the promoter of Arabidopsis thaliana ecotype Columbia (Col-0) was used. The T-DNA insertion mutants smxl6 (SAIL_1285_H05), smxl7 (SALK_082032), smxl8 (SALK_126406), dwa1-1 (SALK_051022), and dwa1-2 (SALK_021789) were obtained from the Nottingham Arabidopsis Stock Centre ). These seeds were used in previous studies [snrk2.2/2.3 mutant in the Col-0 background were originally generated by Hiroaki et al. (2007) [smxl6/7/8, smxl6/7/8/dwa1-1, smxl6/7/8/dwa1-2, smxl6/7/8/snrk2.3, and smxl6/7/8/snrk2.2/2.3, and the single mutant snrk2.3 were generated via genetic crossing in this study, and homozygous lines were identified and used for genetic analyses. The genotyping primers are described in 2 for 5 min, then rinsed five times in sterilized water). Seven-day-old seedlings were transplanted into the soil in a standard growth condition with a 16-hour-light/8-hour-dark photoperiod and 60% relative humidity at 22 \u00b0C.The wild-type xl6 SAIL_85_H05, s-2 SALK_0789 were  studies . Seeds osnrk2.3, snrk2.2/2.3, smxl6/7/8, smxl6/7/8snrk2.3, and smxl6/7/8/snrk2.2/2.3 were germinated and grown on \u00bd MS medium with or without 0.5 \u03bcM and 1 \u03bcM ABA , respectively. The percentage of germinated seeds was recorded every day, and the phenotypes were photographed at 6 days after sowing.All the seeds of Col-0, Drought stress was induced with modification . In brieEcoRI-BamHI sites of the pGBKT7 and pGADT7 vectors, respectively. The primers and cloning sites used for plasmid construction are given in https://www.takarabio.com/ (accessed on 5 November 2018)). Reagents used in yeast experiments were from Clontech and Solarbio. The cDNA library was from Clontech . The Y2H library was screened via the yeast-mating method. Briefly, pGBKT7-SMXL6, pGBKT7-SMXL7, and pGBKT7-SMXL8 constructs were transformed into Y2H Gold as bait. The library strains were combined with bait strains after autoactivation and toxicity tests. Subsequently, the candidate interacting proteins of SMXL6, SMXL7, and SMXL8 were screened using yeast two-hybrid libraries, and then all positive clones were sequenced.To generate constructs fused to the GAL4 DNA-binding domain (BD) and activation domain (AD), the full-length coding sequences were cloned into the For the Y2H assays, bait and prey constructs were co-transformed into Y2H Gold and spotted onto plates containing double-dropout (DDO) medium without leucine and tryptophan (SD/-Leu/-Trp) or quadruple-dropout (QDO) medium without leucine, tryptophan, adenine, and histidine (SD/-Ade/-His/-Leu/-Trp) with or without X-\u03b1-Gal /Aureobasidin A  and incubated at 30 \u00b0C for 3 days.pGADT7-SMXL6,7,8 and the LacZ reporters driven by various promoter fragments. Their binding was observed according to X-Gal  chromogenic reaction on medium (SD/-Leu/-Ura) agar plates after 3 days at 30 \u00b0C.For the Y1H assays, the yeast strain EGY48 was used for the co-transformation of plasmids for SMXL6, 7, and 8 were cloned into pGEX-6P-1, and DWA1 was cloned into pET-30a to obtain the GST-SMXL6, GST-SMXL7, GST-SMXL8, and His-DWA1 recombinant proteins, respectively. The plasmids were grown in E. coli. After ultrasonication, GST-SMXL6, GST-SMXL7, GST-SMXL8, and GST supernatant as bait were incubated with Beaver Beads\u2122 GSH (BEAVER) in PBS , 1% Triton X-100, 1 mM PMSF, and 1% BSA for 4 h at 4 \u00b0C. After the Beaver Beads\u2122 GSH were washed 3 times for 5 min with buffer , the extract from cells producing His-DWA1 was added as prey and incubated for a further 1 h. The beads were washed ten times with wash buffer . The input and proteins retained on the beads were separated on 12% and 8% SDS-PAGE gels and then analyzed via immunoblotting with anti-His monoclonal antibody  or anti-GST monoclonal antibody .A GST pull-down assay was conducted with modification . The codSMXL6,7,8 and DWA1 were cloned next to the N-terminal part of YFP (nYFP) in the pEarleygate202-YN vector and the C-terminal part of YFP (cYFP) in the pEarleygate201-YC vector. Different constructs (including empty vectors) were transformed into the Agrobacterium strain GV3101 and then infiltrated into Nicotiana benthamiana leaves for transient expression. Fluorescence was observed and photographed in leaf epidermal cells 2 days after infiltration using a Confocal laser scanning microscope .The coding regions of dwa1-1 mutants growing on \u00bd MS were extracted in a buffer . Purified GST-SMXL6, GST-SMXL7, and GST-SMXL8 fusions (1 \u03bcg) were mixed with cell extracts (10 \u03bcg) with 10 mM ATP and incubated at 30 \u00b0C for 1 h with or without 50 mM MG132 . Equal amounts of samples were separated by 8% SDS-PAGE gels and analyzed via immunoblotting with an anti-GST antibody.Cell-free assays were conducted with modification . Briefly35S::SMXL6,7,8-GFP  construct, the full-length coding sequences of SMXL6,7,8 were individually cloned into the KpnI-SalI sites of the pCAMBIA1300-GFP vector via the in-fusion method (Clontech). The primers are shown in OE-SMXL6, OE-SMXL7, and OE-SMXL8 transgenic plants, the corresponding constructs were introduced into the Agrobacterium strain GV3101 and then into the wild type via the floral dip method [OE-SMXL6-4, OE-SMXL7-1, and OE-SMXL8-2 were introduced into dwa1-1 to obtain OE-SMXL6/dwa1-1, OE-SMXL7/dwa1-1, and OE-SMXL8/dwa1-1 lines via genetic crossing, respectively.To generate the p method . HomozygOE-SMXL6/7/8 and OE-SMXL6/7/8/dwa1 transgenic lines, these seeds were sown in the same \u00bd MS medium plate and subjected to a 16-hour-light/8-hour-dark photoperiod and 60% relative humidity at 22 \u00b0C in a greenhouse. Fluorescence observation was performed after 5 days. The seedlings of two transgenic lines, such as OE-SMXL6 and OE-SMXL6/dwa1, were placed under the same slide for observing using the layer scanning method with a confocal laser scanning microscope . Then, multiple-layer fluorescent images were superimposed into a single image using a stacking technique in Photoshop. Finally, the overall fluorescence in the multiple roots of the two lines were counted.For the fluorescence observation of the w/v) TB, and softly shaken for 3 h. The excessive TB was washed with water, and then the leaves were photographed.Toluidine blue (TB) staining was conducted as described previously with modification . The fifOE-SMXL6, OE-SMXL7, and OE-SMXL8 lines were grown on \u00bd MS for 7 days, and their seedlings were harvested and extracted in a buffer . The samples with equal amounts were separated by 8% SDS-PAGE gels and analyzed via immunoblotting with an anti-GFP antibody . The Anti-Actin antibody  was used as a normalization control.The seeds of Col-0, smxl6/7/8, OE-SMXL6, OE-SMXL7, and OE-SMXL8 seedlings were prepared using a MiniBEST Plant RNA Extraction Kit  according to the users\u2019 manual. RNA samples were reverse-transcribed using a PrimeScriptTM II 1st Strand cDNA Synthesis Kit (Takara). The qRT-PCR experiments were finished using gene-specific primers  was used as an internal control. A Dual-Luciferase Reporter Assay Kit  was used to detect firefly luciferase (LUC) activity and the Renilla luciferase gene (REN) using a GloMax\u00ae 20/20 Luminometer (Promega). The relative luciferase activity was expressed as Firefly Luc/Renilla Luc.The dual-luciferase reporter assay was performed as described previously . pGreenIEMSAs were performed as described previously with minor modifications . The seqt-test.Three independent experiments were performed, and results were expressed as the mean \u00b1 the standard deviation (SD). Data were analyzed by using GraphPad Prism 7 software. Statistical analyses were performed in SPSS Statistics . We used a one-way analysis of variance (ANOVA) with Duncan\u2019s multiple range tests and a Student\u2019s Arabidopsis Y2H cDNA library was used as the prey to identify the candidate interactors of SMXL6,7,8. The full-length SMXL6,7,8 sequences were individually fused to the GAL4 DNA binding domain of the bait vector . We screened out the interacting clone DWA1, which encodes a substrate receptor for CUL4 E3 ligases. To confirm the interaction between SMXL6,7,8 and DWA1 in yeast, we introduced the full-length DWA1 into the GAL4 activation domain of the prey vector (AD-DWA1). The BD-SMXL6,7,8 and AD-DWA1 plasmids were co-transformed into yeast. The Y2H assays showed that SMXL6,7,8 interacted with DWA1 in yeast  with smxl6/7/8 triple mutant plants. The dwa1-1 and dwa1-2 T-DNA insertions disrupted the production of full-length transcripts  data revealed 729 genes targeted by SMXL6-HA , and theor SMXL6 .SnRK2.3 using a dual-luciferase reporter assay in Arabidopsis mesophyll protoplasts. As expected, the overexpression of individual SMXL6, SMXL7, and SMXL8 inhibited the activity of LUC driven by the SnRK2.3 promoter compared with the expression of GFP alone (SnRK2.3 promoter. The results of multiple rounds of promoter truncation showed that a 101 bp fragment (fragment p2.3-3-1-2) upstream of the start codon of the SnRK2.3 gene was sufficient for SMXL6,7,8 to bind to SnRK2.3  and PIP2;2 for degradation [Arabidopsis [smxl6/7/8 and smxl6/7/8/dwa1 exhibited the drought tolerance phenotype after 16 days of drought stress treatment compared with the wild type, and when the drought treatment time was extended, the drought tolerance of smxl6/7/8/dwa1 was lower than that of smxl6/7/8 after 18 days of drought stress treatment (Arabidopsis.Ubiquitin-conjugating enzymes and E3 ligases can negatively or positively participate in drought stress response. The endoplasmic reticulum-associated degradation (ERAD) component UBIQUITIN-CONJUGATING ENZYME 32 (UBC32) positively regulates drought tolerance in radation . The E3 bidopsis . In thisreatment . Togethedwa1 showed no difference in drought tolerance compared with the wild type [dwa1 mutant showed less tolerance after 16 days of drought stress treatment in this study (dwa1 mutant in response to drought stress may be the difference in drought treatment time, and 16 days of drought treatment time was critical in our working condition and shorter than in Lee\u2019s [SMXL6,7,8 reduced drought tolerance compared with the wild type because of the negative regulatory function of SMXL6,7,8 (dwa1 may contribute to its drought-sensitive phenotype (A previous study reported that ild type . In contis study . This phin Lee\u2019s . We alsoMXL6,7,8 . Thus, ihenotype .Arabidopsis [smxl6/7/8 after drought treatment showed that the drought treatment induced up-regulation of ABA-responsive genes in smxl6/7/8, indicating that SMXL6,7,8 may negatively regulate drought tolerance through an ABA-dependent manner [smxl6/7/8 triple mutant [ABA-induced genes are also involved in regulating drought stress tolerance ,52,53,54t manner . The up-e mutant .SnRK2.3 and SnRK2.6 (SnRK2.3 and SnRK2.6 promoters compared with the expression of GFP alone (SnRK2.3 and SnRK2.6 are also transcriptionally regulated by SMXL6,7,8. The overexpression of SnRK2.3 and SnRK2.6 enhance drought tolerance [snrk2.2/2.3/2.6 mutant significantly reduce the drought tolerance of Arabidopsis, while the overexpression of TaSnRK2.3 in common wheat enhance the drought tolerance [SnRK2.3 and SnRK2.6 were up-regulated in the smxl6/7/8 triple mutant (snrk2.3 and snrk2.2/2.3 restored the ABA-sensitive phenotype of smxl6/7/8 (smxl6/7/8 required functional SnRK2.2/2.3 in seed germination. In addition, we will investigate whether SnRK2.6 is involved in the regulation of ABA response and drought tolerance downstream of SMXL6,7,8 in the future. It is well documented that the ATAACAA motif is essential for SMXL6 to directly bind to the promoter of SMXL7 and repress its transcription [SnRK2.3 and SnRK2.6 (In this study, SMXL6,7,8 could directly bind to the promoters of  SnRK2.6 , which pFP alone . This suolerance ,11. Meanolerance . It has olerance ,57. The olerance . The rele mutant . Geneticmxl6/7/8 , indicatcription . However SnRK2.6 , which nSnRK2.3 and repress its transcription, leading to an ABA response. Our study will prompt us to decipher whether homologous proteins of SMXL6,7,8 in rice, maize, and other crops can also play similar roles in response to drought stress tolerance and ABA, which will enhance a better understanding of the molecular mechanism of SL signaling in response to the drought stress tolerance of crops.Overall, a schematic diagram of how SMXL6,7,8 regulate drought tolerance was proposed . On the"
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