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+{"text": "Cytotoxic microspheres have been developed for intra-arterial use in patients with liver metastases. Following injection, the distribution of microspheres reflects the pattern of hepatic arterial blood-flow. Vasoactive agents, such as angiotensin II, by producing vasoconstriction in normal liver, might divert arterial blood toward tumour and thereby enhance the delivery of drug-loaded particles. Using a double isotope technique, the distribution of radiolabelled microspheres to tumour and normal liver tissue was measured before and after angiotensin II infusion in nine patients with multiple liver metastases. The median increase in tumour: normal ratio following angiotensin II infusion was by a factor of 2.8 . This novel approach to regional chemotherapy, using a combination of angiotensin II infusion and cytotoxic microspheres, increases the exposure of tumour to cytotoxic agents and may, therefore, enhance tumour response rates."}
+{"text": "Both biodegradable emboli and pharmacological agents can enhance regional therapy for hepatic targeting. Using a rat model with similar haemodynamic characteristics to human colorectal liver tumour and a radio-labelled marker of similar molecular weight to Adriamycin, we have combined the two approaches to see if the effect was addictive. Following induction of liver tumour in male hooded rats by intrahepatic injection of HSN sarcoma cells, the relative distribution of marker, 99mTc methylene diphosphonate (MDP), was studied in three groups given the following by injection into the hepatic artery. (1) Saline (Control) + MDP; (2) Degradable Starch Microspheres (DSM) + MDP; and (3) Angiotensin II + DSM + MDP. Both Degradable Starch Microspheres alone (P less than 0.001) and Degradable Starch Microspheres + Angiotensin II (P = 0.003) significantly increased the retention of marker in liver and tumour at 1 min following injection, with a 12-fold improvement over controls, but the tumour:liver ratio was unaltered. By 90 min the MDP levels in normal hepatic parenchyma had returned to control values. There was relatively less washout with significant retention in tumour tissue in both DSM (P = 0.03) and combination treated animals (P = 0.001), with a significantly improved (P = 0.001) tumour to liver ratio (5.22:1) in combination treated animal relative to those treated with DSM alone."}
+{"text": "There is increasing interest in the use of microspheres, loaded with chemotherapeutic agents, for regional therapy to hepatic metastases. It is necessary to deliver these particles predominately to tumour rather than to normal liver. This study investigates factors influencing the distribution of regionally injected microspheres. Discreet tumour was induced in rats by subcapsular hepatic inoculations of HSN cells. At 20 days, 12.5 microns, 25 microns or 40 microns diameter, radiolabelled albumin microspheres were administered, in various concentrations, via the gastroduodenal artery. Tumour to normal liver microsphere distribution ratios were determined and median values ranged from 0.1 (0.2 mg ml-1 12.5 microns microspheres) to 1.8 (20 mg ml 40 microns microspheres). Concentrated suspensions (20 mg ml-1) of large microspheres (40 microns) produced the most favourable tumour to normal liver distribution ratios. These results not only have implications for the therapeutic administration of microspheres but also for their use in blood-flow studies."}
+{"text": "Regional chemotherapy for colorectal liver metastases has not demonstrated a convincing survival benefit over systemic chemotherapy. This may be due to poor delivery of chemotherapeutic drugs to hypovascular liver tumour. Since vasoactive agents may influence hepatic blood flow this study investigated the effects of systemic and regional vasoconstrictors on the delivery of a regionally delivered marker in an experimental model of liver tumour. Systemic administration of angiotensin II caused a significant retention of marker in normal liver, but not in tumour compared to controls. Regional delivery of angiotensin II and phenylephrine caused significantly greater retention of marker in tumour than liver with an overall 4-fold increased retention of marker one minute after its injection. Ninety minutes after injection there was still significant retention of marker compared to control animals. Regional delivery of hepatic artery vasoconstrictors increase delivery of marker and may increase delivery of chemotherapeutic drug to liver tumour."}
+{"text": "Regional chemotherapy is commonly used to treat patients with colorectal liver metastases. However, improvement in survival has still not been demonstrated. Cytotoxic loaded albumin microspheres for arterial administration have been described as a means of improving the the therapeutic index, but their distribution depends upon the prevailing pattern of arterial blood-flow at the time of injection. In this study, the ability of the vasoactive drug angiotensin II to target arterially injected microspheres to colorectal liver metastases is assessed in nine patients using scintigraphic planar and tomographic imaging. The median tumour: normal ratio in nine patients with colorectal liver metastases was 3.4:1 before the administration of angiotensin II. The corresponding ratio after administration of angiotensin II was 7.3:1. The median improvement factor was 1.8 (P less than 0.05). The data suggest that worthwhile tumour targeting can be achieved with angiotensin II in patients with colorectal liver metastases."}
+{"text": "Angiotensin II (AT-II) has been used to target regionally-administered cytotoxic microspheres in patients with intrahepatic tumours. The optimisation of vasoconstrictor targeting requires a knowledge of the blood flow changes induced by agents such as AT-II. We therefore assessed duplex/colour Doppler sonography (DCDS) as a means of evaluating the effects of AT-II infusion on hepatic arterial blood flow (HABF) and arterial resistance in patients with intrahepatic tumours. HABF was measured continuously in nine patients using DCDS before, during and after an infusion of AT-II  via a hepatic artery catheter. In seven patients with less than 30% hepatic replacement by tumour, the baseline level of HABF was 331 +/- 85 ml min-1 (mean +/- s.d.), and this was reduced by 75-80% within 30 s of the start of AT-II infusion. HABF recovered rapidly from the end of the infusion, and increased by up to 20% above the baseline for approximately 2 min. In two patients with greater than 50% hepatic replacement, HABF showed no reduction but rose continuously from the start of AT-II infusion, increasing by a factor of 2-2.5 after 3-4 min. Arterial resistance showed reciprocal changes in all cases. We conclude that DCDS is effective in assessing the temporal changes in hepatic arterial blood flow caused by AT-II. In order to optimise tumour targeting, the injection of microspheres loaded with cytotoxic drugs should be completed before the end of the AT-II infusion. The targeting advantage of AT-II in patients with a high percentage hepatic replacement by tumour should be re-assessed."}
+{"text": "Regionally-administered, drug-loaded microspheres have a potential role in the treatment of renal tumours. Vasoactive agents, for example, angiotensin II, may allow selective delivery of microspheres to tumour. The present study defines the regional advantage that may be obtained from angiotensin II by quantifying tumour and normal kidney blood flow using radiolabelled microsphere renal perfusion studies and per-operative laser-doppler flow measurements. Angiotensin II increased microsphere distribution to tumour, relative to normal kidney, by a factor of four. This enhancement was associated with an absolute increase in tumour blood flow."}
+{"text": "The outlook for patients with colorectal liver metastases is poor. Microspheres have been combined with cytotoxics and administered via the hepatic artery in an attempt to improve tumour drug exposure within the liver. However, it has been suggested that arteriovenous connections might occur in association with intrahepatic tumours causing loss of regional advantage, and that the administration of microspheres further exacerbates arteriovenous shunting. In seven patients with colorectal liver metastases, base-line shunting was measured using a tracer quantity of radio-labelled albumin microspheres. The shunted fraction of a 'therapeutic quantity' of microspheres was subsequently measured in the same group of patients using albumin microspheres carrying a different radio-label. Base-line shunt for 0.5 x 10(6) microspheres was found to be 2.2 +/- 1.8% (mean +/- s.d.); the percentage shunt of a therapeutic quantity (40-80 x 10(6)) of microspheres was 3.0 +/- 0.8%. We conclude that arteriovenous shunting in patients with colorectal liver metastases is minimal, and is not significantly increased by the administration of therapeutic quantity of microspheres during regional chemotherapy."}
+{"text": "Incorporation of cytotoxic drugs into microspheres reduces but does not eliminate systemic toxicity. The extent of liver to lung shunt was measured in 26 patients with colorectal liver metastasis. Liver to lung shunting correlated with proportion of liver replacement but did not exceed 4.4% and therefore is unlikely to cause systemic toxicity."}
+{"text": "The effect of degradable starch microspheres (DSM) on the intrahepatic distribution of a low molecular weight marker, 99Tcm-labelled methylene diphosphonate (MDP), was studied in rats with hypovascular HSN liver tumours. MDP was injected regionally, via the hepatic artery, alone or co-administered with DSM, with or without subsequent occlusion of either the hepatic artery or the portal vein. Tumour vascularity was measured with 57Co-labelled microspheres. Co-injection with DSM immediately significantly increased hepatic retention of marker in both tumour (T) (median 22.40 (range 16.82-39.58)% injected dose) and normal liver (N) (9.08 (4.85-12.59) %ID) the greater effect seen in T (P < 0.01). After DSM degradation, very little MDP remained in N (0.61 (0.28-1.40) %ID) but there was significant retention in T (10.01 (6.73-20.28) %ID, P < 0.01). Clamping the hepatic artery had minimal effect on the retention of MDP when administered alone. Regional injection of 16.5 microM 57Co microspheres resulted in a N:T ratio of 2.25:1. Concomitant injection of the 40 microM DSM was 57Co microspheres reversed this ratio to 1:2. The results indicate that DSM selectively enhances the retention of MDP to a hypovascular hepatic tumour, not by causing intra-tumour stasis, but by directing a greater arterial flow to hypovascular areas in the liver."}
+{"text": "Selective internal radiation therapy (SIR therapy) is a technique whereby metastatic liver cancer is irradiated by embolising microspheres containing the radionuclide yttrium-90 into the hepatic arterial circulation. To date this technique has not been used as an adjuvant therapy, but rather to treat established metastases in the liver. This study evaluated the use of two intrahepatic radiation doses delivered on radioactive microspheres for the treatment of small, growing micrometastases. Three groups of five rats were each inoculated with tumour spheroids into the portal vein. The resultant liver micrometastases were treated with either 10 or 20 MBq of yttrium-90 microspheres or a sham dose of non-radioactive microspheres injected into the portal vein 2 days following tumour inoculation. The livers of each animal were examined for the presence of metastases after a further 21 days and liver function tests were performed. At the time of sacrifice there was no obvious normal liver damage in any of the rats treated with microspheres. The livers of the sham-treated animals contained extensive signs of tumour deposition. A mean of 34 tumours were taken from the livers of each of the sham-treated animals, whereas only a single tumour was found in one animal treated with 10 MBq of yttrium and eight small tumours from two animals treated with 20 MBq. Liver function tests demonstrated a significant short-term increase in alkaline phosphatase levels in the radiation-treated animals compared with shams, but there were no other indications of any effects on liver function. These results indicate a potential role for SIR therapy in an adjuvant setting with colorectal cancer."}
+{"text": "A computer-based learning experience was developed using cognitive flexibility theory to overcome the pitfalls often encountered in existing medical education. An earlier study (not published) showed significant pretest-posttest increase in scores, as well as a significant positive correlation between choosing to complete the module individually or in pairs.This experience was presented as part of a second-year course in medical school with randomized assignment for students to complete the program as pairs or individuals.Sixty-six scores of 101 medical students (31 from students working as singles and 35 from 70 working in pairs) were analyzed. Out of 47 possible points, the mean pretest score was 15.1 . The mean posttest score was 22.9 . Posttest scores were statistically significantly higher than pretest scores . Both pairs and singles showed pre-to-post test score gains, but the score gains of pairs and singles were not significantly different.This learning module served as an effective instructional intervention. However, the effect of collaboration, measured by score gains for pairs, was not significantly different from score gains of students completing the assignment individually. Often the traditional teaching model produces learners unable to recall, use, or transfer knowledge and skills in novel situations. Whitehead7A predominant share of the misconceptions (and networks of misconception) that we have identified reflect one or another kind of oversimplification of complex material \u2026. Misconceptions of advanced material result from both interference from earlier, simplified treatments of that material and from a prevailing mode of approaching the learning process in general that fosters simplificational strategies and leaves learners without an appropriate cognitive repertoire for the processing of complexity.Feltovich, Spiro, and CoulsonThus, ill-structuredness  and the goals of advanced knowledge acquisition can, in combination, create a difficult problem for both teachers and students when they use simple strategies to present and learn complex subject matter. In response to this problem of reductive bias, Spiro and his colleagues proposed CFT as a means to create learning environments that provide the complexity and multidimensionality of content required for advanced knowledge acquisition in ill-structured domains.Avoid oversimplifying instruction. The complexity of multiple possibilities in terms of symptoms, causes, and data sources was represented.Emphasize case-based instruction. A variety of cases were chosen to avoid the reductive bias issues inherent in using a small number of prototypic cases.Provide multiple representations of content. In addition to history and laboratory tests, perspectives were available from several colleagues, basic information was found in a textbook, and a collection of \u201csimilar cases\u201d was also provided.Support context-dependent knowledge. All events in these cases represent actual experiences of our advisors and so are well within the clinical context where they are likely to be encountered later by the learner.Emphasize knowledge construction, not transmission. Learners must construct complex schemata including procedural and factual knowledge in order to successfully complete each case.Support complexity. Multiple perspectives on and between cases allowed learners to experience some of the complexity of transfusion medicine.The computer-based module \u201cHandling Transfusion Hazards\u201d was developed using these theoretical principles in a series of case-based instructional simulations for curriculum-specific content in Blood Banking and Transfusion Medicine. The principles of CFT  as reflected in the development of this learning module for medical students included the following:We hypothesized that collaborative learning would further enhance the CFT-based education process. We evaluated this effect by organizing students to complete the module individually or in pairs and comparing test results for these groups. The collaborative learning process was seen as both consistent with these principles and a natural aspect of medical education and practice.The program that was developed as a result was case-based, using a series of cases. Each case had a brief introduction, a report of the case history and physical examination, a range of typical laboratory tests from which to choose, assessment questions, and a range of management options. The practice cases also included a series of opinions and observations by colleagues  and a bank of 24 similar cases that showed how such situations had been handled by other doctors. The test cases were presented in the same format but did not provide the extensive help functions available in the practice cases. In the test cases, students could be evaluated for their ability to assess and manage the clinical problem presented. Two transfusion medicine experts reviewed the test cases for content validity, comprehensiveness, and accuracy. Changes were made to the test cases based on the experts\u2019 feedback. A simulated textbook was available during practice and test cases.\u201cHandling Transfusion Hazards\u201d avoids oversimplification and emphasizes case-based instruction. The textbook, history with physical exam, perspectives from other people, and even the bank of similar cases, all provide multiple representations of content. As each case unfolds, the learners must use the non-compartmentalized information provided in clinical context to construct their own problem-solving approach, a process which supports context-dependent knowledge, emphasizes knowledge construction, and supports complexity.When the students encounter a list of cases to explore, and see the information resources available, they realize (or are reminded of) the complex, ill-structured nature of the transfusion medicine domain. This is the first example of how Research Questions - The main question we sought an answer to was \u201cIs completing the computer program \u2018Handling Transfusion Hazards\u2019 an effective way to learn topics in Transfusion Medicine?\u201d No specific measure of sufficiency was established: any indication of increased performance on our test was of interest to us. A second question was \u201cDoes completing this program in collaboration with another student enhance its effect?\u201d We also wanted to know how much time the students spent on the program and how they felt about the learning experience.Preliminary Study - One year prior to the study described, we observed a class as they passed through the course. At this time, the computer program was piloted to determine if our anticipated measurements and interventions were practical and if medical students using a computer-based learning program would effectively collaborate and use this software. The pilot study participants were second-year medical students. The program used in the pilot study consisted of nine cases, two pretest cases, five practice cases, and two posttest cases. All students participated, and the material was effectively distributed to the students.To determine if program users would effectively collaborate, five volunteer pairs were videotaped using the program. A coding scheme containing two main interaction types (on-task and off-task) and four verbal interaction categories (questions or statements for both interaction types) was developed as an observation checklist. The four interaction categories were further subdivided into two categories: (a) suggestions, opinions, directions, and (b) explanations, evaluations. The coding scheme also contained a provision for categorizing an interaction as either cognitive conflict (argumentative) or co-construction. The categories which the coders applied to each event in the interaction were On-task/Off-task, Question/Statement, Suggestion/Explanation, and Argumentative/Co-constructive.Average intercoder agreement was .95. Although the sample was small, the data suggest that the medical students remained highly task-focused during the program phases observed. Furthermore, the interaction patterns demonstrate that the students concentrated their efforts in giving and receiving explanations, which are positive collaborative behaviors. Finally, the coders failed to document any cases of cognitive conflict, suggesting that these pairs created a co-constructive, collaborative environment. Since this portion of the preliminary study generated sufficient observational evidence that the students worked collaboratively, the investigators felt no need to further document that finding.Scoring \u2013 The module presents several clinical case-based scenariosLab tests: Experienced practitioners assigned the scores as 2 , 1 (possibly appropriate), 0 (irrelevant but harmless) or -1 .Assessment: Each action selected for assessing a case received a score of -l or +1 depending on the validity and appropriateness of the selection.Management: Each management action received a score of \u2212l or +1 depending on the appropriateness of the selection for the case.The three test cases contained a total of 172 lab tests options, 19 assessment issues, and 16 management choices. Appropriate responses could earn 30 lab test points, 9 assessment points, and 8 management points for a total perfect score of 47 points. No score was determined as \u201cadequate\u201d and the scoring was not adjusted to reflect traditional percentage to letter-grade correspondence.Participants - One hundred thirty-two second-year medical students, 70 men and 62 women, participated in this study. The subjects completed \u201cHandling Transfusion Hazards\u201d during the three-week Hematology segment of the Pathophysiology of Disease course. All subjects completed the program, but 31 students were excluded from the final analysis because of missing or inaccurate data. Thus, 101 students were included in final statistical analysis .Design \u2013 The experimental design employed the pretest-posttest control group design espoused by Campbell & Stanley,1 OR O X2 OR O X1 represents the unpaired condition and X2 represents the paired condition. Each subgroup can serve as control for the other and the pre-treatment observation provides a control for the entire population as described in the first paragraph of the discussion. The study's design established the submitted score as the unit of analysis rather than the individual student's score, so students assigned to the collaborative condition tested together rather than separately. Pairs achieved a single score that was analyzed along with scores earned by students completing the program as individuals. This allowed the pretest results to be easily compared to the posttest results, as all subgroups (scores from pairs and scores from singles) were handled identically.where XThe scores on the pretest and posttest were based on the lab tests ordered and how the three test cases were assessed and managed. These cases were conceptually similar to the cases presented during the learning phase of the program.Random Assignment \u2013 For the main study, students were randomly assigned to work individually or in pairs so that roughly 1/3 would work individually and 2/3 of the class would form pairs. This would yield about as many scores from individual students as from pairs.Lecture Hall Intervention \u2013 During a 30-minute lecture on the first day of the hematology section, the students were introduced to the computer module. In this lecture, the transfusion medicine curriculum manager discussed the program content and learning objectives, the logistics of obtaining a computer disk, the requirement to complete the program, and grading criteria. The students were also told that they must complete the program as assigned .Program Structure - As a result of the non-equivalent nature of the four test cases used in the pilot study, one test case was re-designed to fit into the practice cases section. The remaining three test cases  were used as both a pretest and a posttest, thus assuring equivalence. The portion of score differences attributable to testing effects was considered to be insignificant. The case structure now consisted of the three test cases as a pretest, five practice cases, and then the same three test cases as a posttest.Data Collection and Statistical Analysis-The data disks recorded pretest and posttest results (selections and scores), as well as other data such as time information, student explanations and summaries, and program critiques. These data were extracted from the disks, checked for accuracy, and hand entered into the Statistical Package for the Social Sciences (SPSS 7.5 for Windows) data editor. This database served as the master file for the statistical analyses. This study examined the effects of collaboration on knowledge acquired by advanced learners in a cognitive flexibility theory-based computer microworld. The independent variable was method of instruction (single or pairs). The dependent variable was the score achieved on a posttest consisting of three transfusion medicine cases. Analysis of covariance (ANCOVA) was used to test the experimental hypotheses: scores will improve from pretest to posttest and scores from pairs will show greater improvement. Further evaluation using a spreadsheet with appropriate formulas for Cohen's dStudent Comments - Subjects were given the opportunity to provide constructive feedback after finishing the program. These comments were independently coded by two raters into categories such as Time or Instructional Method. Where disagreement occurred, the raters discussed and revised their codes to achieve consensus.Score information, learning time, and program effectiveness - A total of 66 scores were analyzed. These scores came from 101 medical students, 31 from students working as individuals and 35 from the 70 students working in pairs. Out of 47 possible points, the overall mean pretest score was 15.1 and the overall mean posttest score 22.9. The total posttest scores were statistically significantly higher than pretest scores (p<.0001 by the ANCOVA), showing an average gain of 7.8 points. Both pairs and singles showed statistically significant (p<.0001) and very large  pre-post test score gains . By calculating effect size from the F statistic and the sample sizeAnalysis of Comments - Table Cognitive flexibility theory (CFT) has been advanced as a means to overcome the problem of inert knowledge formation by advanced learners in complex and ill-structured knowledge domainsEffect size measures reported in the previous section indicate that both statistically and practically significant amounts of learning took place. The statistically insignificant and negligible effect size estimates regarding collaboration, despite the encouraging observational data from the preliminary study, indicates that confounding factors may have suppressed the hypothesized effect. Ensuring a strictly random assignment to the paired condition, for instance, may have interfered with an essential component of forming successful collaborative groups such as unrestricted choice of partners. In any event, score gains for pairs did not appear to differ significantly from those of singles, and students engaged in this program as singles or pairs performed equally well in test cases.Recommendations for Future Research - This study raises several areas for further research. A qualitative approach might yield more useful insight into the dynamics of knowledge acquisition with such case-based instructional simulations. A larger question must be answered regarding the integration of cognitive flexibility theory or any other active, student-centered learning environment into the medical education arena. The students studied were accustomed to memorizing and reproducing information and may not have been prepared to deeply analyze and synthesize the information. The learning program might be better accepted in the third year where clinical evaluation, including interpretation of laboratory tests, and managing cases becomes the predominant approach to learning. This study points to the need to address the question of what conditions are necessary to successfully implement an active, student-centered learning environment."}
+{"text": "Prior to the publication of the book reviewed here, the only books dealing specifically with fossil spiders have been primarily taxonomic monographs, such as those on Dominican and Baltic amber inclusions by , 2004, oThe book begins with an introduction to spiders from a palaeontological perspective, putting their geological longevity into context of the other known fossil and extant arachnid orders, including comparative data for numbers of described extant and fossil species for all orders. The relative diversity of spider sub-orders today is compared with that of the past. The authors provide reasoned support for the importance of fossil spiders in addressing large-scale palaeobiological questions on a global scale, and over long periods of time, including how information on past changes in biogeographical distributions can be correlated with changes in palaeoclimatological factors and the potential of this for predicting the consequences of current climate change. Information is also provided on how the fossil spiders are dated.PageBreakThe fossil record of spiders is discussed with regard to non-amber fossils , including the different modes of preservation with photographic examples (often of holotypes) throughout. The amber spider fossil record follows a similar format with a table of 42 important localities. There is also a section on fossilized spider silk. An important aspect of interpreting fossil assemblages is an understanding of bias in the fossil record and this is covered for both amber and non-amber deposits.PageBreak It includes techniques for preparing both amber and non-amber fossils, including how to obtain the best photographs, and also the application of new imaging techniques which allow non-destructive digital dissection of the fossils, such as x-ray computed and synchrotron technology. The images included here are quite exceptional. This is followed by a discussion of the problems associated with identifying and naming fossil spiders, particularly in a neontological context, including critical examples of techniques recently proposed to assist in this process.Next follows a highly informative chapter on techniques for the preparation and study of fossil spiders, which will be of great use for new students of palaeoarachnology.There is a chapter on the strictly fossil spider families, which lists all known species and their deposit of origin.The taxonomic status of each family is discussed and it is noted that many are in need of revision. Again, this section includes good photographs including of holotypes.Approximately half way through, the focus of the book switches from understanding and interpreting the fossil spiders to the application of the palaeontological data. Much use is made of the spider evolutionary tree, which was created by superimposing the cladogram of Araneae over the geological time scale and calibrating it with described fossils. This tree is up-to-date and even includes the recently erected family Penestomidae and the recent synonymy of Microphocommatidae with Anapidae. There is a handy table referencing the oldest fossil of each of the 73 extant spider families known in the fossil record, used to create the evolutionary tree. Topics in this section include originations and radiations , mass extinctions, and predator-prey co-radiations, including updated analyses (based on new data) of previously published work by the authors.The last chapter concerns how fossil spiders can contribute to our understanding of past and present biogeography. The authors present some rather remarkable examples of disjunct distributions between the fossil and extant spider faunas. A table of spider family distributions over time, including recent, Cenozoic and Mesozoic distributions presents the available data in a user-friendly fashion. Importantly, the authors note that it is just as important to look at which families do not appear in the fossil record and provide a table of data for these also, and discuss possible reasons for their exclusion. The book ends with a comprehensive reference section of some 265 entries and an index to family citations. There is a handy reference figure of the geological timescale on page 6.In conclusion, this is an absolutely unique book in the spider literature to date. It is authoritatively written by two of the leading researchers in this field and provides broad coverage of their combined 50 years experience and expert knowledge. The book is hard back and presented on high quality glossy paper. It is richly illustrated thoughout by numerous high quality photographs of fossil spiders. In conclusion, this book deserves a place on the shelves of the libraries of all professional and amateur arachnologists alike, in addition to those of invertebrate palaeontologists.http://www.siriscientificpress.co.ukThe book can be ordered via the author: by emailing siri.press@live.co.uk. Price \u00a332.00 plus post and packing. For further details, including ordering online, etc. visit"}
+{"text": "Acrocephalus arundinaceus), a passerine bird breeding in Eurasia and wintering in sub-Saharan Africa. We were specifically interested in seasonal strategies in routes and schedules of migration. We found that the great reed warblers  migrated from the Swedish breeding site in early August. After spending up to three weeks at scattered stopover sites in central to south-eastern Europe, they resumed migration and crossed the Mediterranean Sea and Sahara Desert without lengthy stopovers. They then spread out over a large overwintering area and each bird utilised two (or even three) main wintering sites that were spatially separated by a distinct mid-winter movement. Spring migration initiation date differed widely between individuals (1-27 April). Several males took a more westerly route over the Sahara in spring than in autumn, and in general there were fewer long-distance travels and more frequent shorter stopovers, including one in northern Africa, in spring. The shorter stopovers made spring migration on average faster than autumn migration. There was a strong correlation between the spring departure dates from wintering sites and the arrival dates at the breeding ground. All males had a high migration speed in spring despite large variation in departure dates, indicating a time-minimization strategy to achieve an early arrival at the breeding site; the latter being decisive for high reproductive success in great reed warblers. Our results have important implications for the understanding of long-distance migrants\u2019 ability to predict conditions at distant breeding sites and adapt to rapid environmental change.Recent technological advancements now allow us to obtain geographical position data for a wide range of animal movements. Here we used light-level geolocators to study the annual migration cycle in great reed warblers ( Many species of birds, turtles and sea mammals conduct astonishing long-distance journeys between their breeding and wintering grounds . These iOenante oenante), red-backed shrike (Lanius collurio) and common swift (Apus apus) , , , . In thisIn 2008 and 2009, male great reed warblers that were known to be successful breeders from at least the preceding season were fitted with a geolocator . We used geolocator model Mk10S in 2008 and Mk12-SAD in 2009 (British Antarctic Survey (BAS), Cambridge, UK; www.birdtracker.co.uk). The light sensor was fitted on an 8 mm stalk pointing up and backwards (30\u00b0 angle) in order to minimise shading effects caused by the bird\u2019s plumage. The geolocators measured light intensity every minute, but stored only the maximum value for each 10 minute interval. The geolocator was mounted on the bird\u2019s back using leg-loop harnesses . The lenBASTrak (BAS) for downloading data from retrieved geolocators and to correct for clock drift . After decompressing the raw data, TransEdit2 (BAS) was used to visualise light data. A light threshold value of 2 was chosen at which the program assigned transitions between light and dark phases (sunrises and sunsets). To establish the sun azimuth angle corresponding to this threshold value, we used three different methods. First, we used LocatorAid (BAS) to calculate the sun angle for the periods when the birds were at a known position (i.e. breeding site) for the year when the geolocator got attached (from date of affixing the geolocator in May to 5 July), and one year later when the birds returned to the study site . Secondly, we used the Hill\u2013Ekstrom calibration procedure, which is based on the observation that the latitude error of the determined fixes increases with increasing mismatch between light threshold value and inferred sun angle. The latitude error is of opposing magnitude on either side of an equinox, so it is possible to evaluate the sun angle by assessing the effect of different sun angles on the latitude curve and choosing the one that minimises these distortions . T. TBASTraIn order to determine periods when equinoxes had negative effects on the accuracy of the data, each bird\u2019s longitude and latitude positions were plotted on a graph and visually checked for erratic patterns during times of equinoxes (between 18 and 42 days of data around the equinoxes were excluded). Before plotting the tracks on a map, the original positions were smoothed twice by applying a running average for each fix point and its two adjacent fix points on either side of the focal fix point (thus covering a 36 hour period) . n = 10; estimates for 6 individuals prior to departure and after arrival and for 4 individuals only before departure; ArcMap (ArcView 9.3 (ESRI)) \u2013 namely 9 positions for individual 1V-87 that were larger than 3 times the SD of the distance error for all breeding site positions of all birds (i.e. > 3 \u00d7 884.4 km). When these extreme positions were excluded the estimation of the accuracy resulted in a mean error distance for all individuals of 134.2 km . The estimation of the accuracy for the calculated positions on the breeding grounds resulted in a mean error distance for all individuals of 205.7 km .Active migratory movements were differentiated from stopovers by their clearly directional pattern compared to the clustered pattern of stopovers. Only a cluster of at least 3 fixes (36 hours), which then was laying within a 210 km radius, i.e. roughly twice the median error distance for precision estimates . These between-year return rates were similar to non-logger attached males . Due to technical problems with some of the geolocators, complete data over the full annual cycle were available for 6 males , autumn On autumn migration, the great reed warblers followed a southerly directed route to their first stopover site that showed a wide distribution with stopovers located between north-eastern Germany and north-western Greece . Four ouWintering sites were located throughout the western part of the species\u2019 sub-Saharan wintering range, from West Africa into Central Africa, from Senegal in the west to Chad in the north, and the Congo basin in the east . Note, however, that there is a tendency for a significant relationship with a correlation coefficient that is relatively high and a statistical power that is rather low due to low sample size.The departure date from the breeding grounds ranged between 27 July and 14 August with a mean date of 2 August, whereas the time period from the breeding site to the last stopover site in Europe varied substantially between individuals . The meaS = 0.94, n = 6, p = 0.03; S = 0.09, n = 6, p = 0.84).The timing of the mid-winter movements was unsynchronized with departure dates from the first to the second wintering site from 21 November to 2 February . On averS = 0.08, n = 6, p = 0.88).The speed of migration of individuals for which we had data over the whole route (n = 6) was on average 62% faster in spring (mean 220 km/day) than in autumn , whi, whi40])The departure of the birds from the breeding site in southern Central Sweden occurred surprisingly early, already in early August. Feeding conditions at the breeding grounds are favourable at least for another 2-3 weeks in August, which indicates that deteriorated conditions at the breeding site were not the underlying cause for the early timing of autumn migration in male great reed warblers. After leaving the breeding grounds, most individuals chose stopover sites in south-eastern Central Europe, which fits the distribution of earlier ringing recoveries of birds from our study population . When leOur data clearly show that individual great reed warblers utilize two, or in one case three, main wintering sites, and the presence of a distinct mid-winter movement. The occurrence of a mid-winter movement is in line with De Roo and Deheegher\u2019s  observatA completely unexpected result \u2013 although an older source had noted an increase of great reed warblers in Tunisia during spring  \u2013 was thThe speed of spring migration in the great reed warblers was relatively constant over long distances and in total faster than during autumn. A similar pattern has also been seen in other bird species ,11, 46]46]. ThatS = 0.94), and this has also been found in red-backed shrikes, another trans-African long-distance migrant . F. FS = 0. grounds ). To conclude, our findings are consistent with the hypothesis that ecological barriers strongly influence migratory strategies; in the case of the great reed warbler, crossing the Mediterranean Sea and the Sahara Desert was clearly associated with increased travel speed and brief stopover periods. We further conclude (i) that there exist prominent differences between autumn and spring migration with respect to stopover sites, speed and timing; (ii) that individuals utilise multiple wintering sites rather than single sites or even have a nomadic life-style; and (iii) that birds from a small breeding area in Sweden exhibit a wide wintering range throughout western sub-Saharan Africa. The departure dates from the wintering site differed between individuals (1-27 April), which may potentially be explained by differences in local environmental conditions, and was strongly correlated to arrival to the breeding grounds. The consistent high spring migration speed indicates a time-minimization strategy for the migratory journey and also highlights the importance of the timing of departure. In the future, repeated tracks of individual great reed warblers might offer fascinating insights how constrained or flexible long-distance migrating passerines are in their routes and timing of migration. Figure S1Inferred migration route and timing of male great reed warblers determined by geolocators. Hexagons indicate breeding site (black), stop-over sites during autumn (orange) and spring (green) as well as wintering sites (blue). Lines with arrows indicate autumn migration routes (orange), winter movements (blue) and spring migration (green) whereby some parts are covered with slower migration speed (filled line indicate speed of <500 km/d) than other (double lines indicate speed of >500 km/d). Dashed lines represent great circle routes and do not necessarily represent the actual route taken by the bird due to analytical reasons . Dates inferred from latitudinal and longitudinal data are given for departure and arrival at each site (in black); dates inferred from longitudinal movements only are given in grey. 1) The black bar in C (7V-50) indicates a stop-over where the actual position could not be determined. 2) In H (7V-72), only longitudinal data is given for the last position and the actual position is located anywhere along the grey line.(PDF)Click here for additional data file.Table S1Sun angles for different stages during the year for each individual. Breeding site before autumn migration (Sweden (departure)), autumn migration, wintering site after autumn migration until change to next wintering site (autumn equinox), second wintering site before spring migration (spring equinox), spring migration, and breeding site after spring migration ) are given.(XLSX)Click here for additional data file.Table S2Accuracy of geolocator position data at the breeding site based on distances between measured and known geographical positions.(XLSX)Click here for additional data file.Table S3Geolocator data over the annual cycle for individual great reed warblers.(XLSX)Click here for additional data file."}
+{"text": "The tracking of small avian migrants has only recently become possible by the use of small light-level geolocators, allowing the reconstruction of whole migration routes, as well as timing and speed of migration and identification of wintering areas. Such information is crucial for evaluating theories about migration strategies and pinpointing critical areas for migrants of potential conservation value. Here we report data about migration in the common swift, a highly aerial and long-distance migrating species for which only limited information based on ringing recoveries about migration routes and wintering areas is available. Six individuals were successfully tracked throughout a complete migration cycle from Sweden to Africa and back. The autumn migration followed a similar route in all individuals, with an initial southward movement through Europe followed by a more southwest-bound course through Western Sahara to Sub-Saharan stopovers, before a south-eastward approach to the final wintering areas in the Congo basin. After approximately six months at wintering sites, which shifted in three of the individuals, spring migration commenced in late April towards a restricted stopover area in West Africa in all but one individual that migrated directly towards north from the wintering area. The first part of spring migration involved a crossing of the Gulf of Guinea in those individuals that visited West Africa. Spring migration was generally wind assisted within Africa, while through Europe variable or head winds were encountered. The average detour at about 50% could be explained by the existence of key feeding sites and wind patterns. The common swift adopts a mixed fly-and-forage strategy, facilitated by its favourable aerodynamic design allowing for efficient use of fuel. This strategy allowed swifts to reach average migration speeds well above 300 km/day in spring, which is higher than possible for similar sized passerines. This study demonstrates that new technology may drastically change our views about migration routes and strategies in small birds, as well as showing the unexpected use of very limited geographical areas during migration that may have important consequences for conservation strategies for migrants. Limosa lapponica baueriApus apus  from two breeding sites in Sweden. The swift is a highly aerial species, only leaving its aerial habitat during the breeding season and during occasional roost events in trees Long-distance migration by birds is typically carried out as cycles of fuelling at stopovers followed by flight towards the next suitable stopover The autumn mean initial migration direction was 182\u00b0 for the tracked birds, with one bird taking a more easterly initial direction . Four ofAll six birds spent the winter in the same general area in Central Africa between latitudes 0.97\u00b0N\u20133.20\u00b0S and longitudes 10.42\u00b0E\u201319.38\u00b0E , during Spring migration routes were similar to the autumn routes, while five of the birds visited a rather restricted area in SW West Africa (Liberia), where they stopped over for on average 7 days , before continuing towards NNE across Sahara . One birThe autumn migration route was on average 53% longer than the direct route between the breeding site and the wintering area, while it was slightly more direct during spring migration with a 43% detour. The difference between autumn and spring detours was however not significant .The duration of the entire autumn migration was on average 69 days (range 30\u201399 days), divided into 30 days of travelling and 39 days of stopover . The large variation was due to that three individuals spent lengthy stopovers of 56\u201382 days in West Africa . CorrespOverall migration speed is the rate of travel when including time for actual movement and time for refuelling at stopovers 1,58\u200a=\u200a20.3, P<0.0001; season: F1,48\u200a=\u200a9.5, P\u200a=\u200a0.0034).The overall migration speed was significantly higher in spring  in West Africa. The first leg of spring migration towards Liberia was more to the south than the corresponding leg during autumn migration, when the birds visited the Savannah zone The extensive detours of on average 53% and 43% during autumn and spring migration respectively, suggest there is some ecological advantage of migrating via West Africa instead of along a direct N-S axis. Detours can occur for several reasons, such as when the direct route involves the crossing of an ecological barrier  where fuelling is not possible. A detour to avoid crossing the barrier, or one involving a shorter barrier crossing, may be favourable if the longer detour allows migration with smaller fuel reserves than needed for a direct flight across the barrier 1,106\u200a=\u200a15.2, P\u200a=\u200a0.0002, wind assistance F1,105\u200a=\u200a32.3, P<0.0001; latitude \u00d7 wind assistance F1,105\u200a=\u200a11.7, P\u200a=\u200a0.0009).The quadratic relationship between travel rate (including days of travel) and latitude indicates that relatively more time was devoted to directed flights, and less to en-route foraging, about latitudes 25\u201330\u00b0N. This pattern may also arise if wind assistance varies in relation to latitude. In fact, analysing travel rate in relation to latitude and wind assistance showed that both variables significantly contributed to the variation in travel rate, except for wind data at the lowest altitude  spent on average 64% of the time at stopovers, while the corresponding numbers for spring migration was 24% The migration paradigm for passerines involves alternate cycles of stopovers for fuelling and flight b is the relative benefit from en-route foraging as reduced effective flight power consumption, c is the cost as reduced travel speed due to foraging, and p is the power ratio  and Pdep is rate of energy accumulation at stopover b is relatively large, c is small or p is large, or a combination of these factors satisfying the inequality. A high b and low c are likely satisfied by the swift, since it can forage in flight during migration with a small reduction in travel speed, while the power ratio is likely to be low. Swifts have an efficient aerodynamic design that will give a relatively low power required to fly A comparison with flight speed of swifts can inform about the migration strategy. The flight speed of swifts on spring migration in southern Sweden measured by tracking radar was 10.6 m/s Because the swift can combine foraging and migration and since it can sample food abundance continually during migration, it will probably not experience the search/settling time and energy costs of avian migrants that depend on terrestrial stopovers Based on our migration tracks of swifts the average annual time allocation could be estimated for breeding, migration and wintering as 19%, 27% and 54%, respectively. Autumn migration took longer time than spring migration, which may be explained by differing strategy between the seasons (see above). More than half the year is spent on the wintering grounds, presumably without coming to the ground except during rare occasions Using gelocators we have been able to reveal the details of the migrations of six Swedish swifts. Despite a small sample size, we have learned more about migration routes, wintering areas, timing of migration, travel rates and migration strategies of this species than from a century of bird ringing. For example, not a single ring recovery is known from any European ringing schemes from the Liberia region, which appears to be a major stopover area in spring. Taken together, the swift has many analogies with seabirds. Just as seabirds, swifts live in an environment where food may be found virtually everywhere. Furthermore, swifts have a mixed fly-and-forage strategy, making stopovers in areas where feeding conditions are likely to be outstanding, such as Liberia in spring. This is very similar to seabirds that probably combine migration and foraging to a large extent, but also make stopovers in areas with high food abundance We attached eight archival Mk10 geolocators from the British Antarctic Survey (BAS) to common swifts in two breeding colonies in southern  and central Sweden  in May, July and August 2009. In 2010 six of the geolocators were recovered . This is comparable to published data on annual survival rates in common swifts at about 80% Light-level data were linearly corrected for clock drift using the program BASTrak Overall, we obtained two positions per day, and both midnight and noon locations were used in our analyses. We distinguished between movements and stationary periods  by inspecting subsequent positions. Due to the inaccuracy of positioning data, stopovers shorter than two days could not be distinguished from slow movements. For further analysis and plotting we calculated 3-day mean positions (i.e. means for 6 subsequent position estimates). Total migration distance is the sum of the length of segments based on 3-day means, in which stationary periods are excluded. The direct distance is the Great Circle Route between breeding and wintering site.The period over which to calculate mean positions affect the estimated migration distance and derived properties such as detour and migration speed. We consider 3-day means for the positional data as a reasonable compromise between using shorter periods that will inflate migration distances due to noise in the data, and using longer periods that will underestimate the true migration distance due to the omission of real movements away from the straight line between successive positions. To illustrate the effect of period on the estimated migration distances we calculated mean positions for 1-day, 2-day and 5-day periods in addition to the 3-day means used for the analyses for autumn and spring migration, respectively. As expected, 1-day and 2-day means resulted in increased estimated migration distance compared with 3-day means by 37% and 8.6%, respectively, while 5-day means resulted in reduced migration distance by 5.4% compared with 3-day means . The corresponding numbers for estimated migration distances for spring were increase by 36% and 15% (1-day and 2-day means), and a decrease by 7.8% (5-day means), when compared with 3-day means. The effects on derived properties were very similar.Positions derived from light-level geolocators are marred with errors of magnitudes estimated at 143\u00b162 km  and 186\u00b1114 km (mean \u00b1 SD) for latitude position http://www.cdc.noaa.gov). For every 12 h-segment, wind data were extracted for the start, mid- and endpoint, in which the midpoint was given twice as much weight during averaging. We subsequently calculated, for every segment, the tailwind component of the average wind vector, which is the amount of wind blowing parallel to the general migration direction . For the spring migration, two general migration directions were defined, the direction from the wintering area to the stopover area in Liberia, and the direction from Liberia to the breeding site. For individual \u20187964\u2019, which did not make a detour via Liberia, a single general migration direction was used (direction from wintering area to breeding site). The amount of tailwind experienced by the swifts was averaged per individual and per travel leg . Negative tailwind values represent headwinds. These calculations were repeated for different pressure levels , which correspond to different altitudes .For the wind analysis, location data were smoothed twice cf. , giving Figure S1Maps showing migration routes, breeding sites and wintering areas for the individual common swifts tracked using light-level geolocators.(PDF)Click here for additional data file.Table S1Key numbers of migration and wintering for individual common swifts tracked by light-level geolocators.(DOCX)Click here for additional data file."}
+{"text": "Progne subis from the Amazon basin to two breeding sites in eastern North America. Spring 2012 was the warmest on record in eastern North America, but contrary to predictions, this did not result in earlier departure, faster migration, or earlier arrival at breeding areas compared with earlier years. Temperatures and rainfall in the Amazon basin at the time of departure were not higher in 2012, and conditions along migration routes did not give consistent signals of a warmer spring at the breeding site. Once in North America, individuals likely had limited opportunity to speed up their migration because this final portion of the journey was already very rapid . Migration timing over the entire journey was best predicted by breeding latitude and sex and was not sensitive to ecological cues  at departure from South American overwintering sites or en route, in contrast to recent studies of other songbirds. Our results provide the first direct evidence for a mismatch between higher spring temperatures at breeding sites and departure schedules of individual songbirds, and suggest phenotypic responses to short-term climatic warming may be limited for some species. Further direct-tracking data with greater geographic and temporal scope is needed to test for individual plasticity in response to temperature and rainfall along migratory routes for this, and other, species.The decline of long distance migratory songbirds has been linked to an increasing mismatch between spring arrival date and timing of food availability caused by climate change. It is unclear to what extent individuals can adjust migration timing or en route rate in response to annual variation in temperature at breeding sites. We tracked the ca. 7300 km spring migration of 52 purple martins  Many animals have shown long-term advancement in spring phenology in response to climate change Progne subis, a declining We examined migratory schedules of purple martin This study was conducted in accordance with the recommendations of the Ornithological Council 'Guidelines to the Use of Wild Birds in Research' and was approved by the York University Animal Care Committee ).Purple martins were fitted with geolocators  during the nesting period  at two eastern breeding locations  during the main departure period from Brazil (March 15 to April 15). We also compared spring warmth sum  and rate (ANOVA) between 2012 and earlier years, for each breeding population. Next, we used linear mixed-effects models fit by REML to assess the influence of temperature and rainfall on migration timing and rate at the core wintering and stopover locations. We examined timing at four locations  and rate in three migration zones . We fit a separate timing and rate model for each location and zone. For arrival date at stopover sites and migration rate in each zone, we used temperature and rainfall amounts at the preceding site as factors, reasoning that timing and rate would be most influenced by conditions prior to arrival (i.e. at the previous site). The full models included fixed factors of population (PA or VA), sex, rainfall and temperature with a random effect of year. We dropped individual explanatory variables one by one, then used Akaike\u2019s Information Criterion weights to select the best-fit model. To assess the significance of each variable, we removed them one by one from the full model, then compared each reduced model to the full model using a likelihood ratio test. All analyses were conducted using R t\u200a=\u200a\u22123.42, df\u200a=\u200a49.38, P\u200a=\u200a0.001, VA: t\u200a=\u200a\u22123.09, df\u200a=\u200a54.51, P\u200a=\u200a0.003) and spring warmth sum was 46% (PA) and 25% (VA) higher than previous years. However, in 2012 birds departed significantly later, not earlier, from wintering sites  and there was no difference between years in the timing of crossing the Gulf of Mexico (23.4\u00b0N) or arrival at breeding sites  during passage through South America , Central America  and North America . Migration rate was not significantly faster in 2012 versus prior years . Migration duration from the Yucatan Peninsula to the breeding colony was only 4\u20135 days on average .Rate of spring migration varied significantly . On average, males left 5 days earlier than females and individuals breeding at the more southerly Virginia colony departed 14 days earlier than birds breeding in Pennsylvania. We had similar results for migration timing en route at Panama, Yucatan and breeding arrival where best-models included only sex and breeding location. Both factors were significant in the models except for sex at the Yucatan . None of the factors in our models of migration rate during each of the three stages of migration  were significant, illustrating that birds did not differ their rate in response to temperature or rainfall en route and that rate did not differ by breeding location or sex.Based on our model results, neither rainfall amount or temperature were significant factors predicting departure date from South American overwintering sites in the Amazon Basin. Our best-fit models of migration departure date included only sex and breeding location (PA or VA) and both were significant in the model  and total sample size for spring migration (excludes tags that failed prior to spring migration). Most (75%) geolocators were 1.1 g with a <10 mm stalk  and were mounted using a leg-loop backpack harness (DOCX)Click here for additional data file."}
+{"text": "Alpha-lipoic acid (a-LA) is an antioxidant shown to ameliorate age-associated impairments of brain and cardiovascular function. Human milk is known to have high antioxidant capacity; however, the role of antioxidants in the developing brain is largely uncharacterized. This exploratory study aimed to examine the dose\u2013response effects of a-LA on piglet growth and neurodevelopment.Beginning at 2\u2009days of age, 31 male pigs received 1 of 3 diets: control (CONT) (0\u2009mg a-LA/100\u2009g), low a-LA (LOW) (120\u2009mg a-LA/100\u2009g), or high a-LA (HIGH) (240\u2009mg a-LA/100\u2009g). From 14 to 28\u2009days of age, pigs were subjected to spatial T-maze assessment, and macrostructural and microstructural neuroimaging procedures were performed at 31\u2009days of age.P\u2009=\u20090.01) fractional anisotropy (FA) in the internal capsule of HIGH-fed pigs compared with both the CONT (P\u2009<\u20090.01)- and LOW (P\u2009=\u20090.03)-fed pigs, which were not different from one another. Analysis of axial diffusivity (AD) within the internal capsule revealed a main effect of diet (P\u2009<\u20090.01) in which HIGH-fed piglets exhibited smaller (P\u2009<\u20090.01) rates of diffusion compared with CONT piglets, but HIGH-fed piglets were not different (P\u2009=\u20090.12) than LOW-fed piglets. Tract-based spatial statistics, a comparison of FA values along white matter tracts, revealed 1,650 voxels where CONT piglets exhibited higher (P\u2009<\u20090.05) values compared with HIGH-fed piglets.No differences due to diet were observed for bodyweight gain or intestinal weight and length. Spatial T-maze assessment did not reveal learning differences due to diet in proportion of correct choices or latency to choice measures. Diffusion tensor imaging revealed decreased (The lack of differences in intestinal and bodyweight measures among piglets indicate a-LA supplementation does not impact overall growth, regardless of concentration. Additionally, no observed differences between CONT- and LOW-fed piglets in behavior and neuroimaging measures indicate a low concentration of a-LA does not affect normal brain development. Supplementation of a-LA at a high concentration appeared to alter white matter maturation in the internal capsule, which may indicate delayed neurodevelopment in these piglets. Alpha-lipoic acid (a-LA), also known as thioctic acid, is an endogenously derived antioxidant found in animal and human cells alike, where it is a cofactor in mitochondrial dehydrogenase complexes. Although production of this dithiol occurs within the body, a-LA is known to be readily absorbed from the diet and is active in both aqueous and lipid-rich tissue. Lipoic acid is referred to as the \u201cuniversal antioxidant\u201d due to its multitude of functions including metal chelation, free radical quenching, antioxidant regeneration, and regulation of gene and protein expression . MoreoveTo date, much of research on a-LA supplementation has focused on its impact in the aging brain. Iron deposition that occurs in disease states such as Parkinson\u2019s and Alzheimer\u2019s increases occurrence of free radicals and peroxidation of polyunsaturated fatty acids . DihydroThe brain is one of the most highly metabolic organs in the body. It contains a high concentration of polyunsaturated fatty acids and accumulates redox-active metals, thereby rendering it susceptible to oxidative stress . ThroughUse of MRI techniques in the neonatal piglet has previously established the pig as a clinically relevant translatable model for human infants in the context of nutrition and neurodevelopment \u201317. To tAll animal care and experimental procedures were in compliance with National Research Council Guide for the Care and Use of Laboratory Animal Care and Use Committee and approved by the University of Illinois Urbana-Champaign Institutional Animal Care and Use Committee.ad libitum access to water, as well as a rubber ball and towel for enrichment - or 29 (replicates 2 and 3)-day trial period. The trial was completed in 3 replicates (12\u201314 piglets per replicate), with piglets selected from 12 total litters to control for genetics and initial body weight. Piglets were housed individually in stainless steel cages with clear Plexiglass facades and side walls bearing small openings to allow for proper ventilation. Individual piglets had richment .Clostridium perfingens C. perfingens antitoxin C\u2009+\u2009D per manufacturer\u2019s recommendations . Piglets exhibiting sickness behavior, defined as diarrhea for more than 48\u2009h, were placed on an electrolyte solution and administered a single dose of sulfamethoxazole and trimethoprim oral suspension . Piglets that failed to thrive were euthanized prior to study end.Ambient room temperature was maintained between 27 and 29\u00b0C and heat lamps provided supplemental heat within the cage. A 12-h light/dark cycle was maintained with light from 0600 to 1800 hours. Prior to placement in the artificial rearing system, piglets were administered 5\u2009ml of All researchers involved with conducting the study and acquiring and analyzing the study results remained blinded to identity of dietary treatments until final statistical analyses had been completed. Piglets were provided either a control (CONT) diet or the control diet supplemented with a low a-LA concentration (LOW) or high a-LA concentration (HIGH) for the duration of the feeding trial. Concentrations of a-LA  in test diets were formulated as follows: CONT (0\u2009mg a-LA/100\u2009g milk replacer powder), LOW (120\u2009mg a-LA/100\u2009g milk replacer powder), and HIGH (240\u2009mg a-LA/100\u2009g milk replacer powder). A prebiotic blend of polydextrose (PDX) and galactooligosaccharides (GOS) was provided in all dietary treatments as follows: . All dietary treatments were supplemented with docosahexaenoic acid (DHA)  and arachidonic acid (ARA) . Experimental piglet diets were manufactured to human quality standards  with adjustments in the nutritional profile made to meet or exceed nutrient requirements for the neonatal pig  were as follows: CONT (0\u2009mg a-LA/l), LOW (240\u2009mg a-LA/l), and HIGH (480\u2009mg a-LA/l). On the first day in the artificial rearing system, piglets received small volumes of milk formulas to provide an adjustment period prior to the standard feeding regimen. Piglets were fed at 285, 305, 310\u2009ml of reconstituted diet per kilogram of BW starting on 3, 6, and 12\u2009days of age, respectively. Meal allotment was determined by recording daily body weight of individual piglets. Each diet was reconstituted fresh at each feeding and provided up to five times a day, approximately every 4\u2009h, between 0700 and 2200 hours. Piglets were fasted prior to spatial T-maze assessment to incentivize the milk reward offered in the behavioral task. On days that cognitive assessment occurred (between 17 and 28\u2009days of age) piglets were provided four meals instead of five, while maintaining the aforementioned daily volume per kilogram body weight feeding rate.Spatial working memory was assessed using a validated behavioral task in a specially designed T-maze for the young pig , 20. Begvia intramuscular injection of Telazol . Once sedated, piglets were transferred to the MRI scanner and maintained on 2% isoflurane/98% oxygen  for the entirety of the 60-min scan. Piglet vital signs were monitored throughout the scan using an MRI-compatible pulse oximeter, and upon completion of the scan, piglets were maintained under anesthesia and immediately euthanized for tissue collection. Detailed methods for manual brain segmentation, volumetric assessment, voxel-based morphometry, and diffusion tensor imaging were previously described (At 29 (replicate 1) and 31 (replicates 2 and 3) days of age, piglets were subjected to magnetic resonance imaging (MRI) procedures. Scanning procedures were implemented on a Siemens MAGNETOM Trio 3\u2009T Imager using a 32-channel head coil. Upon arrival to the Biomedical Imaging Center located in the Beckman Institute for Advanced Science and Technology, piglets were anesthetized escribed , 16.http://pigmri.illinois.edu/) was used in place of human brain templates  assessment of fractional anisotropy (FA) data. FA images, previously generated from diffusion data, were manually extracted, and all FA data from individual subjects were aligned using the FSL non-linear registration tool FNIRT. Upon alignment, the study-specific mean FA image was created, and a mean FA skeleton representing the center of all common tracts was established. A threshold of 0.2 was determined to be sensitive for mean FA tract delineation. Once the study-specific mean FA skeleton was created, each subjects\u2019 aligned FA data were projected onto the mean FA skeleton and the resulting voxel-wise cross-subject data were used for statistical analyses , 22. Foremplates . For TBSvia intracardiac administration of sodium pentobarbital . Following euthanasia, brain and small intestinal tissues were collected. The entire small intestine  was removed and immediately measured for total length. The small intestine was further dissected into three segments: duodenum , jejunum (middle 75%), and ileum . All sections were flushed with saline prior to being weighed, as described previously (Immediately following magnetic resonance imaging procedures (described above) and while remaining under anesthesia, piglets were euthanized eviously . Data weP\u2009<\u20090.05 with trends accepted at 0.05\u2009<\u2009P\u2009<\u20090.10.An analysis of variance (ANOVA) was conducted using the MIXED procedure of SAS 9.4  and was applied to differentiate the effects of the CONT, LOW, and HIGH diets provided to young pigs. For outcomes collected at a single time-point , data were analyzed by a one-way ANOVA. Any data collected from the same animal on more than one occasion  were analyzed as a two-way, repeated-measures ANOVA. Both statistical models included replicate as a random effects and the level of significance was set at P\u2009=\u20090.229) or the main effect of diet (0.463) for daily bodyweight throughout the feeding trial. However, a main effect of day (P\u2009<\u20090.001) was evident  and as such were euthanized prior to study end. Thus, only 31 piglets are included in the subsequent analyses.Overall, there was no observed interactive effect  or total absolute (P\u2009=\u20090.783) or relative (P\u2009=\u20090.216) intestinal weights (data not shown). Moreover, absolute weight of each small intestinal segment did not differ by diet , and neither did relative intestinal segment weights  (data not shown).No main effect of diet was observed for small intestinal length . Additionally, one piglet (CONT diet) had 1\u2009day of acquisition and 1\u2009day of reversal removed and another piglet (HIGH diet) had 1\u2009day of acquisition removed, all due to non-compliance on those days.Using piglet participation criteria set forth previously , four piP\u2009=\u20090.207) or main effect of diet (P\u2009=\u20090.580); only a main effect of day (P\u2009=\u20090.019) was observed  or main effect of diet (P\u2009=\u20090.963); only a main effect of day (P\u2009=\u20090.001) was observed. Analysis of proportion of correct choices during the reversal phase also indicated no interactive effect (P\u2009=\u20090.637) or main effect of diet (P\u2009=\u20090.645); only a main effect of day (P\u2009=\u20090.001) was observed. Finally, latency to choice during the reversal phase resulted in no observed interactive effect (P\u2009=\u20090.587), main effect of diet (P\u2009=\u20090.105), or main effect of day (P\u2009=\u20090.824).Analysis of proportion of correct choices during the acquisition phase indicated no interactive effect  P\u2009=\u20090.07 or maiP\u2009=\u20090.25; data not shown). Analysis of absolute volumes of individual brain regions also resulted in no volumetric differences due to diet . Absolute volumes of individual brain regions were analyzed relative to total brain volume within subject, and relative proportion of total brain volume was analyzed for each region. Dietary treatment tended (P\u2009=\u20090.087) to affect cerebellar relative volume, in which piglets provided the HIGH diet had a proportionally larger cerebellum (P\u2009=\u20090.037) compared with LOW-fed piglets. No differences due to diet were observed for any relative total volume of individual brain regions (P\u2009>\u20090.139).No differences in absolute total brain volume were observed between dietary treatments (P\u2009<\u20090.05) in the internal capsule axial diffusivity (AD) and FA measures  of dietary treatment was observed for internal capsule AD, with piglets provided the HIGH diet exhibiting smaller (P\u2009<\u20090.01) AD values compared with CONT piglets, but they were not different (P\u2009=\u20090.124) than LOW-fed piglets. Low-fed piglets tended to have smaller (P\u2009=\u20090.064) internal capsule AD values when compared with CONT piglets. A main effect of diet (P\u2009=\u20090.012) was also observed in internal capsule FA measures. Piglets provided the HIGH diet exhibited lower FA values compared with CONT (P\u2009<\u20090.01)- and LOW (P\u2009=\u20090.029)-fed piglets. No difference between CONT- and LOW-fed piglets was observed in internal capsule FA values.Diffusion tensor imaging revealed a main effect of diet  for AD, radial diffusivity (RD), and mean diffusivity (MD) measures of the thalamus (Table P\u2009=\u20090.031)- and LOW (P\u2009=\u20090.054)-fed piglets. Moreover, piglets provided the HIGH diet exhibited higher RD values compared with CONT (P\u2009=\u20090.066)- and LOW (P\u2009=\u20090.049)-fed piglets. MD values within the thalamus were also noted, with piglets fed the HIGH diet exhibiting higher MD values compared with CONT (P\u2009=\u20090.035)- and LOW (P\u2009=\u20090.038)-fed piglets. Finally, the main effect of diet tended to be significant for FA values in the cerebellum (P\u2009=\u20090.067). Piglets provided the HIGH diet exhibited lower FA values compared with CONT (P\u2009=\u20090.036)- and LOW (P\u2009=\u20090.052)-fed piglets, but no difference in cerebellar FA values was observed between CONT- and LOW-fed piglets.The main effect of diet tended to be significant P\u2009<\u20090.0 for AD,P\u2009<\u20090.05) FA values compared with HIGH-fed piglets (Figure Tract-based spatial statistical analysis revealed 1,650 voxels within the predetermined white matter tracts in which CONT piglets exhibited higher (s Figure . There wPiglets in this study were supplemented with varying levels of a-LA from 2 to 31\u2009days of age to determine the impact of a-LA supplementation on overall body growth and brain development. Most antioxidant research to date focuses on the effects of supplementation in the context of brain aging, with very little focusing on the needs and efficiency of antioxidants throughout neurodevelopment. It is known that infants, notably preterm infants and those having experienced intrauterine growth restriction, are at an increased risk for oxidative stress due to immature antioxidant systems and elevated metabolic rates, polyunsaturated fatty acid concentrations, and non-protein bound iron concentrations . As suchIn this study, learning and memory were assessed using a spatial T-maze behavioral task. Over the course of the 8-day acquisition period and the 6-day reversal period, there was no observed effect of diet on piglet performance in proportion of correct choices. Moreover, piglet latency to choice also did not differ between dietary treatment groups throughout the acquisition or reversal phases. A main effect of day was observed for proportion of correct choices during acquisition and reversal, indicating learning did in fact occur. These behavioral observations appear to be consistent with previous studies where a-LA supplementation improved memory in aged mice, but not in young mice . It shouDiffusion tensor measures revealed differences due to diet in AD and FA within the internal capsule. For both the FA and AD measures, an apparent dose\u2013response was evident with CONT piglets consistently exhibiting the highest values, HIGH piglets exhibiting the lowest values, and LOW piglets being intermediate to the other two treatment groups. Interestingly, analysis of AD and FA in the internal capsule revealed LOW piglets were not different than CONT piglets, suggesting that the LOW dose of a-LA supports normal internal capsule development in the piglet. AD is most commonly described as a measurement of water movement along an axon tract. As development occurs, fiber coherence and myelination increase, thus increasing AD measures. Additionally, FA values tend to increase in brain regions as myelination occurs . A recenThalamic and cerebellar diffusion tensor measures resulted in trending significance that was consistent with observations made in the internal capsule. Cerebellar FA values revealed numerically lower measures in HIGH piglets compared with CONT and LOW piglets. Moreover, RD and MD tend to decrease as white matter matures during development, thus the thalamic RD and MD measures resulted in values indicative of delayed development in HIGH piglets. While these measures are only trending, they are important to highlight because imaging piglets at a later time point may have revealed significant differences between dietary treatments consistent with observations in the internal capsule. Recent work suggests that the internal capsule is the earliest myelinating brain region with other regions starting to myelinate at approximately 6\u2009months of age in the human infant . One weeTract-based spatial statistics comparing CONT- and HIGH-fed piglets revealed differences along white matter tracts that are consistent with diffusion tensor measures described above. Analysis of FA values along the predominant white matter tracts resulted in 1,650 voxels in which CONT piglets exhibited higher measures compared with HIGH-fed piglets. Moreover, visual inspection of these voxels revealed the largest differences occurring in the internal capsule and to a lesser extent the corpus callosum. Together with DTI and TBSS analyses, it is evident that supplementation of high concentrations of a-LA specifically altered development within the internal capsule. Given that the internal capsule is known to be myelinating at this time and these measures indicate HIGH piglets might be experiencing delayed maturation, it is possible that this is due to alterations in myelination status within this region. In the early postnatal period, the brain is rapidly accreting iron due to its pivotal action as a co-factor in myelinating events . a-LA isin vitro findings, further extending the observation of altered brain development due to excess antioxidants in a translational piglet model.To the best of our knowledge, no studies have been conducted to directly assess the effects of oral a-LA supplementation on brain development. Antioxidant research in the context of neurodevelopment appears to be limited to cell culture and suggests that excess supplementation of antioxidants negatively impacts development. Castagn\u00e9 and colleagues proposed that neuronal cells might have an optimal redox status that operates within a tight range of antioxidant capacity . They suThis study is one of the first to assess the impact of a-LA supplementation on the neonatal piglet brain. Supplementation of a-LA at any level had no impact on daily bodyweight, suggesting no detrimental impacts to overall growth or health of the animals. Additionally, no differences in spatial T-maze behavioral assessments were observed, suggesting observed structural differences in the brain may not have functional implications, at least for the cognitive functions required to perform this specific task. Results obtained from DTI and TBSS analyses suggest supplementation of HIGH may have delayed brain development. It is important to note the lack of difference between CONT and LOW piglet neurodevelopmental measures, thereby suggesting a low concentration of a-LA supports normal brain development. Because of the clear dose\u2013response observed in MRI outcomes, future research should be performed at doses closer to the LOW concentration to determine if a-LA supplementation can confer benefits to neurodevelopment. From this study, we conclude that a diet containing a low concentration of a-LA has little impact, whereas a high concentration of a-LA may have delayed structural brain development.RD, RW, and BB were involved in project conceptualization. AM and RD were involved in daily project activities. AM and RD were involved in data collection. AM and RD were involved in data analysis. All authors were involved in data interpretation and manuscript preparation.RD received grant funding and has consulted for Mead Johnson Nutrition. RW and BB are employees of Mead Johnson Nutrition."}
+{"text": "Depression has been shown to be associated with elevated leptin levels, low-grade inflammation and insulin resistance. These derangements are often measured in mixed gender cohorts despite the different body compositions and hormonal environments of men and women and gender-specific prevalence and responses to depression.A cross-sectional analysis was carried out on a cohort of 639 participants from the ADDITION-Leicester dataset to assess differences in markers of diabetes risk, cardiovascular risk and inflammation in depressed and non-depressed individuals. Depressive symptoms were determined using the WHO (Five) well-being index. Multivariate linear and logistic regression analyses were adjusted for age, sex, ethnicity, body mass index, smoking, social deprivation and activity levels for continuous and binary variables respectively. Further analysis included stratifying the data by gender as well as assessing the interaction between depression and gender by including an interaction term in the model.Women with depressive symptoms had a 5.3% larger waist circumference (p = 0.003), 28.7% higher HOMA IR levels (p = 0.026), 6.6% higher log-leptin levels (p = 0.01) and 22.37% higher TNF-\u03b1 levels (p = 0.015) compared with women without. Conversely, depressive symptoms in men were associated with 7.8% lower body fat % (p = 0.015) but 48.7% higher CRP levels (p = 0.031) compared to men without. However, interaction analysis failed to show a significant difference between men and women.Depressive symptoms are associated with metabolic derangements. Whilst women tended to show elevations in biomarkers related to an increased risk of type 2 diabetes , men showed a marked increase in the cardiovascular disease risk biomarker CRP. However, perhaps due to the cohort size, interaction analysis did not show a significant gender difference. Depression is associated with a significantly increased risk of developing type 2 diabetes (T2DM) and cardiovascular disease (CVD)\u20133. A metThe causal pathways linking depression with metabolic dysregulation have not been fully elucidated, however elevations in insulin resistance (IR), low grade inflammation and leptin have been repeatedly reported , 10\u201313. Most studies analysing depression and metabolic dysfunction have not considered effect modification by gender. This is despite the marked differences in body mass, body composition and hormonal milieu of each gender which in turn affects basal levels of CVD markers , an Anglo-Danish-Dutch randomised control trial assessing of the cost effectiveness of population screening and intensive multi-factorial intervention for Type 2 diabetes. Details of this cohort have been published elsewhere . The AddOf the 987 participants with biomarker data, 307 were excluded because they had missing WHO (Five) data. Additionally, a further 41 participants were excluded for: missing ethnicity data (n = 1), social deprivation scores (n = 7), physical activity scores (n = 31) and/or smoking status data (n = 3). Therefore 639 participants were included in the current analysis. An analysis of the n = 346 participants that were not included  questionnaire, n = 307) showed that they were: older, less likely to be White European, less physically active, more likely to have screen detected T2DM and IGR and showed a higher index of multiple deprivation; as such, non-inclusion of these participants may have introduced some sampling bias .White Europeans between the ages of 40\u201375 years and Black and Minority Ethnic (BME) participants (predominantly South Asian) between the ages of 25\u201375 years were included in the study. A lower age cut-off for BME participants was chosen due to the reported higher risk of T2DM and CVD. People with the following pre-existing conditions are excluded , T2DM, terminal illnesses with a likely prognosis of less than 12 months, psychiatric illness likely to hinder informed consent, pregnancy and lactation.Baseline demographic data captured at screening included: age, sex, ethnicity, BMI, smoking, index of social deprivation and activity levels. Ethnicity status was self-assigned using UK population census categories. Weight (to the nearest 0.1kg) and body fat % (to the nearest 0.1kg) was measured using Tanita 611 scales . Height was measured to the nearest 0.1cm using a stadiometer. Waist circumference was measured by trained staff using a non-stretching measuring tape over the tops of the iliac crests. Smoking was self-reported. Postcode was used to calculate the Index of Multiple Deprivation (IMD), which is a deprivation score for small areas in England based on a combination of domains encompassing economic, social and housing factors, and enabled ranking of areas according to their specific level of deprivation . Physicag for 10 minutes to produce plasma, this was then frozen at \u221280\u00b0C for subsequent measurement of research biomarkers. Quantitative analysis of plasma for human CRP, ApoA1 and ApoB was carried out on the ABX Pentra clinical chemistry analyser and latex-enhanced immunoturbidimetric assay . TNF-\u03b1 and IL-6 were analysed using the Quantikine high sensitivity ELISA for human TNF-alpha /TNFSF1A and human IL-6 kit, . Leptin and resistin was assayed using the Mediagnost human enzyme-immunoassay ELISA kits . Human adiponectin, insulin and 8-Iso prostaglandin F2 (8-Iso-PG F2) were assayed fluormetrically using the AutoDELFIA 1235 automatic immunoassay systems . All samples were analysed in duplicate and with all duplicate samples having a CV% of \u226420%. All research biomarker assessments were carried out by University of Leicester Scientists in conjunction with Unilever personnel at the Unilever Corporate Research Laboratory, Bedford UK.Prior to screening, participants were instructed to fast for 12 hours before the study visit. Venous blood was collected in EDTA tubes, centrifuged at 1500 a priori on the basis of previously reported associations with depression, diabetes or cardiovascular disease. The covariates included were: age, sex, ethnicity, BMI, IMD, smoking status and physical activity. Adjusted means were calculated from linear regression models for groups with and without depressive symptoms, whilst odds ratios were calculated from logistic regression models for the group without depressive symptoms. Models were fitted for the whole cohort and subsequently stratified by gender. Additionally, the interaction between depression and gender was assessed by including an interaction term in the model. Additionally, the analysis was repeated substituting BMI with waist circumference. Data was analysed using Stata V12.1. No adjustments were made for multiple testing. P values of \u22640.05 were considered statistically significant.Demographic variables were presented as mean (SD) or n (%) for continuous and categorical variables respectively. Differences between the groups with and without depressive symptoms were estimated using chi-squared tests for categorical variables and t-tests for continuous variables. Linear regression models were fitted for continuous response variables and logistic regression models were fitted for binary response variables. All models included a binary depression case status variable, as well as additional known confounding variables. These covariates were selected The descriptive statistics of the whole cohort are summarised in 2 = 0.188. Despite the depression-linked elevation in IR in women, HOMA IR levels were higher in men compared with women (p = 0.005)HOMA IR was not significantly higher in the group with depressive symptoms when analysing the entire mixed gender cohort . However2 = 0.368. Leptin was significantly higher in women vs. men (p = <0.001).Levels of leptin were examined as continuous, log-transformed values and were higher with depressive symptoms .When analysing the entire mixed gender cohort, TNF-\u03b1 was not significantly higher in the group with depressive symptoms , howeverCRP was also higher with depressive symptoms when assessing the entire cohort  and these findings agree with previous studies , 10, 13.2 = 0.188 and 0.368), suggesting that other pathways, such as depression-linked excitation of the HPA axis, may also play a significant role in inducing metabolic derangement.Systematic reviews have found a significant relationship between adiposity and depression , 42; witde novo depression/onset depression-like symptoms [The above discussion suggests that depression leads to increases IR and leptin, however, due the cross-sectional design of this study the directionality of the association can not be proven. Some studies have shown that high baseline levels (or administration) of leptin and TNF-\u03b1 lead to increased rates of symptoms , 50. TheThe finding that HOMA IR was 28.7% higher with depressive symptoms suggests an increased risk of developing T2DM. Additionally, a prospective study by Facchini et al (2001) reported that IR was an independent predictor of cardiovascular disease, stroke, hypertension and cancer . The elePrevious reports have shown that CRP is significantly elevated with depression, particularly in men and the results of this study agree with these findings , 57. AgaIn line with women, men also showed a trend towards higher IR, leptin and TNF-\u03b1 with depressive symptoms but within this cohort this did not reach significance . This faCRP is recognised as an accurate predictor of future cardiovascular events with, 0-1mg/L predicting<6% risk of a cardiovascular event in the next 10 years (low risk), 1-3mg/L predicting a 6\u201320% risk (average risk) and >3-10mg predicting >20% risk (high risk) . The malAs already outlined, due to the cross-sectional design of the present study it is possible that increases in inflammation and markers of T2DM and CVD precede the onset of symptoms of depression. This data potentially suggest that, for example, depression in women may respond to therapies aimed to reduce omental fat (diet and exercise) and TNF blockade medications . ConversThe main strengths of this study were that the data was adjusted for a wide range of potential confounders. Additionally, this study assessed a broad range of clinical and research biomarkers associated with T2DM, CVD and inflammation. However, we feel that the due to the relatively low number of participants included in this study (n = 639) we were unable to prove a significant difference between depressive symptoms and metabolic status in men and women. Additionally, this is a secondary analysis of a study designed to test a different primary hypothesis and therefore some measurement bias and residual confounding is likely. The temporal relationship between depressive symptoms and metabolic disturbances could not be established due to the cross-sectional design of the study. History of depression (depression duration and severity) and antidepressant/psychiatric medications were not taken into account, additionally, clinical diagnosis of depression was not confirmed in accordance with the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). One furThis study supports the theory that symptoms of depression are associated with metabolic disturbances. Whilst women with depressive symptoms had significantly higher risk factors associated with diabetes , depression in men was associated with higher levels of CRP which potentially suggests an increased risk of CVD. However, within the present cohort whilst each gender showed distinct trends, no significant gender difference was found. It is possible that further work assessing larger cohorts may reveal a significant gender difference between depressive symptoms and metabolic status.S1 Tablea P-values test for a difference between participants included and excluded from the main analysis, and were estimated using chi-squared tests for categorical variables and t-tests for continuous variables.All values are n (%) or mean (sd). (DOC)Click here for additional data file.S2 TableaAdjusted for: age, ethnicity, waist circumference, Index of Multiple Deprivation, International Physical Activity Questionnaire score and smoking status.(DOC)Click here for additional data file."}
+{"text": "The major drawback for the more widespread use of paclitaxel and its precious precursor, 10-deacetylbaccatin III (10-DAB III), is that they require large-scale extraction from different parts of yew trees (Taxus species), cell cultures, taxane-producing endophytic fungi, and Corylus species. In our previous work, a novel online two-dimensional heart-cut liquid chromatography process using hydrophilic interaction/ reversed-phase chromatography was used to introduce a semi-preparative treatment for the separation of polar (10-deacetylbaccatin III) and non-polar (paclitaxel) taxanes from Taxus baccata L. In this work, a combination of the absorbent (Diaion\u00ae\u00a0HP-20) and a silica based solid phase extraction is utilized as a new, efficient, and cost effective method for large-scale production of taxanes. This process avoids the technical problem of two-dimensional preparative liquid chromatography. The first stage of the process involves discarding co-extractive polar compounds including chlorophylls and pigments using a non-polar synthetic hydrophobic absorbent, Diaion\u00ae\u00a0HP-20. Extract was then loaded on to a silica based hydrophilic interaction solid phase extraction (silica 40-60 micron). Taxanes was eluted using a mixture of water and methanol at the optimized ratio of 70:30. Finally, the fraction containing taxanes was applied to semi-preparative reversed phase HPLC. The results revealed that using this procedure, paclitaxel and 10-DAB III could be obtained at 8 and 3 times more, respectively than by the traditional method of extraction. There is no other naturally occurring defense agent against cancer that has a stronger effect than paclitaxel, commonly known under the brand name of Taxol Tests determined that taxotere has excellent activity better than paclitaxel in some assays and were therefore developed as a parallel drug to paclitaxel , first introduced by Alpert, is an efficient technique used for the separation of polar and basic compounds because of its complementary selectivity against RP-HPLC , meaningBased on a previous study on production and purification of important taxanes including paclitaxel and 10-DAB III -24, SPE In this work, an offline combination is introduced using an absorbent for preliminary purification of taxanes. This is followed by a hydrophilic interaction solid phase extraction (SPE) based on a silica stationary phase. Consequently, a taxane containing fraction was eluted by an aqueous-organic mobile phase. Subsequently, this fraction was applied on a semi-preparative reversed-phase HPLC. Plant materials Taxus baccata L. gathered in June 2011 from the Botanical Garden of the University of Tehran, Iran. These Taxus needles were air-dried at room temperature. Stems were cut from plants, and mature needles were stripped manually from the stem, powdered and kept at -20 \u00b0C pending the extraction process. The investigation was performed on needles of \u00ae\u00a0HP-20 column followed by hydrophilic interaction SPE for obtaining polar and non-polar taxanes (paclitaxel and 10-DAB III).Chemicals n-hexane, dichloromethane were provided from Merck Co. . HPLC-grade acetonitrile was obtained from Caledon Co. . Ultrapure water was obtained from a Milli-Q water system. Diaion\u00ae\u00a0HP-20 was purchased from Sigma-Aldrich\u00ae (3050 Spruce Street saint louis MO 63103 USA) with Product number of 13608 SupelcoTM. Celite 521 (from Acros Organic with CAS number: 61790-53-2) was received as a gift from the Pasteur Institute of Iran.Paclitaxel standard (98%) was purchased from Sigma Co. . The chemicals methanol, ethanol, Liquid-liquid extraction (LLE) n-hexane (2 \u00d7 10 mL). The hexane extract that contained lipids, waxes and pigments was discarded and the aqueous layer was extracted with dichloromethane (5 \u00d7 10 mL). During this stage the polar compounds such as carbohydrates remained in the aqueous phase and taxoids were extracted to the organic phase. Dichloromethane extracts were combined, evaporated under reduced pressure and the residue was dissolved in 15 mL acetonitrile.LLE was carried out according to Glowniak and Mroczek (18). An aliquot of 10 mL of methanol extract was mixed with 10 mL water and extracted with Instrumentation -1 was applied. The maximum absorption wavelength was 227 nm, and 20 \u00b5L of the sample was injected.Analytical separation was performed on an Agilent 1200 Series HPLC  fitted with variable-wavelength UV-Vis absorbance detector. Determination was performed using a C18 analytical column  and an isocratic elution with a mixture of acetonitrile and water at the ratio of 45/55 for paclitaxel and 30/70 for 10-DAB III. The flow rate of 1 mL minSemi-preparative purification was performed on a semi-preparative Knauer equipment including a WellChrom preparative pump K-1800, ultraviolet (UV) detector K-2501, a B\u00fcchi fraction collector B-684 , and YMC-Pack ODS-A . Separation of taxanes was carried out using acetonitril: water at the ratio of 45/55 for paclitaxel and 30/70 for 10-DAB III. Levels of flow rate, maximum wavelength and injection volume were 8 mL/min, 227 nm and 8 mL, respectively.TM LCQ TM DECA instrument, comprising an ion trap. An ionization device was used for sample analyses . The Xcalibur 2.0 SR2 software (copyright Thermo Electron Corporation 1998\u20132006) was used.Mass spectra were acquired by a Finnigan Silica based SPEIn this work, 10 g of the underivatized silica (40-60 micron particle size) was packed in to a 12 mL polypropylene column (13 \u00d7 2.5 cm). After loading the sample, column was washed with 500 mL deionized water and was subsequently washed with 500 mL of solvent mixtures from different water/methanol ratios. Absorbent treatment\u00ae\u00a0HP-20, it was soaked and swelled in 100% (v/v) methanol for 12 h prior to use in order to omit impurities and the solvent was removed through washing with an adequate amount of distilled water. The washed wet resin was then weighed and used. After removing the major pigments and chlorophylls by the use of n-hexane, the methanolic extract was dried (G\u0142owniak and Mroczek 1999) and suspended in 100 mL water. Resulting aqueous suspensions were treated by an absorbent in both column and dispersive modes . In the dispersive mode, absorbent was added directly to the extract. After 10 min stirring, absorbents were collected and then washed using 300 mL methanol. After evaporation of the solvent, extract was loaded on to a hydrophilic interaction column. The same procedure was applied in cases of column mode except that the resulting aqueous extract was applied to a cartridge packed with the absorbent. After loading of the sample, the column was washed with methanol. Fraction containing taxanes was dried using rotary evaporator under reduced pressure and dissolved in 15 mL acetonitrile.Absorbents in these tests, in both cases were used either as a dispersed agent or as a column. In the case of Diaione.g. 10-DAB III) and nonpolar (e.g. paclitaxel) taxanes . Then the column was eluted using different percentages of methanol. There are some reports about the application of Diaionproducts , 27. In \u00ae\u00a0HP-20 both as a dispersed agent and a column.Using methanol (100%) as eluent leads to elution of all taxanes (including 10-DAB III and paclitaxel). Although using absorbent as a dispersed agent showed more efficiency for cleaning up of a plant matrix, recovery percentage was not satisfactory. et al. This column was eluted using an aqueous-organic solvent including a water/methanol mixture. Percentages of methonal were optimized and the amount of packing for washing the most important polar and nonpolar taxanes such as paclitaxel and 10-DAB III. The fraction was analyzed using reversed-phase chromatography including C18 column. At the next stage fraction containing taxanes was loaded on to a homemade column, where a hydrophilic interaction solid phase extraction including a silica column was utilized according to the method cited in Hajnos \u00ae\u00a0HP-20 column followed by hydrophilic interaction SPE for obtaining polar and non-polar taxanes (paclitaxel and 10-DAB III). Final purification was done using semi-preparative reversed-phase liquid chromatography. + ion of paclitaxel and the observed mass at m/z of 554 corresponds to the [M+H]+ ion of 10-DAB III. To confirm separated compounds, liquid chromatography-mass spectrometry . Comparison of mass spectrometry data and previously reported data verified the structures of these separated compounds. In summary and as a conclusion, a combination of non-polar absorbents, hydrophilic interaction liquid chromatography solid phase extraction and reversed-phase chromatography demonstrates the ability for simultaneous separation and isolation of polar and non-polar taxanes. Also, as the solvent used in hydrophilic interaction liquid chromatography is compatible with reversed-phase chromatography, on line two-dimensional combinations could be used as a fast separation technique for isolation of natural products"}
+{"text": "Amentotaxus argotaenia (Taxaceae) to investigate population genetic variation and the effects of environmental heterogeneity on genetic structure.New microsatellite markers were developed for the vulnerable conifer species A. argotaenia through a Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, of which 23 were polymorphic. These markers yielded 1\u201313 alleles and 1.0\u20137.9 effective alleles per locus; levels of observed and expected heterozygosity varied from 0.000\u20131.000 and 0.000\u20130.873, respectively. In total, 18 of the markers were transferable to the related species A. yunnanensis.A total of 27 microsatellite loci were developed from A. argotaenia.These polymorphic markers are a valuable genetic resource for investigating population genetic variation and the potential for local adaptation in  Amentotaxus argotaenia (Hance) Pilg. (Taxaceae) is a dioecious evergreen conifer species that grows to 7 m in height. It has the widest distribution among all Amentotaxus Pilg. species and is found in southern and central China, northern Vietnam, and Laos  and showed that 10 of those markers could also be amplified in A. argotaenia. However, when we used these SSR loci to examine population genetic variation in A. argotaenia, we found their amplification efficiency to be low. We developed a new set of polymorphic SSR markers for A. argotaenia with high amplification efficiency.Ho et\u00a0al.  developeA. argotaenia from four natural populations in China  protocol for separating microsatellite\u2010containing DNA fragments from genomic DNA de novo , genomic DNA was ligated to an MseI adapter nucleotide pair (5\u2032\u2010TACTCAGGACTCAT\u20103\u2032 and 5\u2032\u2010GACGATGAGTCCTGAG\u20103\u2032) using T4 DNA ligase. The 10\u2010fold diluted digestion\u2010ligation mixture was subsequently amplified using the adapter\u2010specific MseI\u2010N primers (5\u2032\u2010GATGAGTCCTGAGTAAN\u20103\u2032) with the following PCR conditions: 24 cycles at 94\u00b0C for 30 s, 53\u00b0C for 60 s, and 72\u00b0C for 60 s. The linker\u2010adapted fragments were enriched by hybridization to single\u2010stranded 5\u2032\u2010biotinylated microsatellite (AC)15 probes based on the reaction conditions of Deng et\u00a0al.  at 16\u00b0C for 16 h and transformed into E. coli DH5\u03b1 competent cells by transient thermal stimulation . Recombinant positive clones were selected by blue\u2013white screening according to Cui and Su , and SSRs were selected using SSRHunter software version 1.3 with parameters set to more than four repetitions for di\u2010, tri\u2010, and tetranucleotide repeats , 2 \u03bcL of 10\u00d7 PCR buffer (containing Mg2+), 1.6 \u03bcL of dNTPs (2.5 mM each), 0.5 \u03bcL of each forward and reverse primer (10 \u03bcM), and 1.25 units of Taq DNA polymerase (TaKaRa Biotechnology Co.). The PCR conditions were: initial denaturation at 94\u00b0C for 5 min; followed by 35 cycles at 94\u00b0C for 50\u00a0s, the optimal annealing temperature for each SSR  along with the GeneScan 500 LIZ Internal Size Standard (Applied Biosystems). DNA fragment analysis and genotyping were performed using GeneMarker version 1.65 .Specific SSR primers were designed using Primer Premier version 5.0  using the following parameters: primer size of 17\u201325 bp, product size of 90\u2013400 bp, GC content of 40\u201360%, primer melting temperature of 47\u201360\u00b0C, and no complementarity between primer pairs. They were used to assess all 56 A total of 160 positive clones were sequenced, 92 of which contained SSR loci. After discarding the short flanking regions, we selected 27 primer pairs that generated clear and reproducible bands. In total, 23 of these exhibited polymorphism and four were monomorphic (Table\u00a0GenAlEx version 6.41 (Peakall and Smouse, A. argotaenia, number of alleles and number of effective alleles per locus ranged from 1\u201313 and 1.0\u20137.9, respectively (Table\u00a0A. formosana (Ho et\u00a0al., A. argotaenia was relatively high. We also evaluated the 27 SSR markers in A. yunnanensis and found that 18 of them were polymorphic (Table\u00a0Within populations of ly Table\u00a0. Levels ly Table\u00a0. Among tly Table\u00a0. Three lly Table\u00a0. In compA. argotaenia, 18 of which were successfully amplified in the congener A. yunnanensis. These SSR markers are a valuable genetic resource and may be used to evaluate overall population genetic structure, to quantify genetic variation, and to assess the potential for adaptation in A. argotaenia.We developed 23 new polymorphic SSR markers for"}
+{"text": "Helicobacter pylori (H. pylori) is the major risk factor for the development of gastric cancer. Our laboratory has reported that the Sonic Hedgehog (Shh) signaling pathway is an early response to infection that is fundamental to the initiation of H. pylori-induced gastritis. H. pylori also induces programmed death ligand 1 (PD-L1) expression on gastric epithelial cells, yet the mechanism is unknown. We hypothesize that H. pylori-induced PD-L1 expression within the gastric epithelium is mediated by the Shh signaling pathway during infection. To identify the role of Shh signaling as a mediator of H. pylori-induced PD-L1 expression, human gastric organoids generated from either induced pluripotent stem cells (HGOs) or tissue (huFGOs) were microinjected with bacteria and treated with Hedgehog/Gli inhibitor GANT61. Gastric epithelial monolayers generated from the huFGOs were also infected with H. pylori and treated with GANT61 to study the role of Hedgehog signaling as a mediator of induced PD-1 expression. A patient-derived organoid/autologous immune cell co-culture system infected with H. pylori and treated with PD-1 inhibitor (PD-1Inh) was developed to study the protective mechanism of PD-L1 in response to bacterial infection. H. pylori significantly increased PD-L1 expression in organoid cultures 48 hours post-infection when compared to uninfected controls. The mechanism was cytotoxic associated gene A (CagA) dependent. This response was blocked by pretreatment with GANT61. Anti-PD-L1 treatment of H. pylori infected huFGOs, co-cultured with autologous patient cytotoxic T lymphocytes and dendritic cells, induced organoid death. H. pylori-induced PD-L1 expression is mediated by the Shh signaling pathway within the gastric epithelium. Cells infected with H. pylori that express PD-L1 may be protected from the immune response, creating premalignant lesions progressing to gastric cancer. th most common cancer worldwide and the 3rd most common cause of cancer-related death. Helicobacter pylori (H. pylori) infection is the major risk factor for the development of gastric cancer. Our laboratory has reported that the Sonic Hedgehog (Shh) signaling pathway is an early response of the gastric epithelium to infection and fundamental to the initiation of H. pylori-induced gastritis. H. pylori also induces programmed death ligand 1 (PD-L1) expression on gastric epithelial cells, yet the mechanism is unknown. PD-L1 is a protective ligand that is known to suppress the immune system by shutting down T cell effector function. We hypothesized that H. pylori-induced PD-L1 expression within the gastric epithelium is mediated by the Shh signaling pathway during infection. Moreover, we showed that metaplastic cells may survive chronic inflammation by expressing the immunosuppressive ligand PD-L1 for the persistence of infection and progression of disease to cancer.Gastric cancer is the 5 Helicobacter pylori (H. pylori) infects nearly 50% of the world's population and is the number one risk factor for gastric cancer [H. pylori infection treated with antibiotics is cleared, once a patient has progressed to a metaplastic phenotype, elimination of the bacteria does not reduce the risk of developing gastric cancer [H. pylori induces pathogenesis by injecting one key virulence factor cytotoxic associated gene A (CagA) into the gastric epithelial cells [H. pylori infection [H. pylori induces the secretion of Shh from the acid-secreting parietal cells [H. pylori-induced gastritis [H. pylori infection programmed death ligand 1 (PD-L1) expression on the gastric epithelium is drastically increased [c cancer . Albeit c cancer . H. pyloal cells . Importaal cells , 5. Shh nfection . Upon inal cells . Followial cells . These mal cells , 6. Overastritis , 5. It hncreased . The expncreased , 9. Thusncreased , 11.H. pylori infection combined with the atrophy of the acid secreting parietal cells leads to the development of spasmolytic polypeptide/Trefoil Factor (TFF) 2-expressing metaplasia (SPEM) [H. pylori-induced PD-L1 expression is mediated by Shh signaling as an early epithelial response to infection and a mechanism by which the bacteria evades the immune response. We also demonstrate here that SPEM cells may survive chronic inflammation by expressing the immunosuppressive ligand PD-L1 for the persistence of infection and progression of disease to cancer.a (SPEM) , 13. SPEa (SPEM) , 15. In a (SPEM) , 9. HereH. pylori induces PD-L1 expression in the stomach, we first collected gastric biopsies from uninfected normal patients , and infected patients that exhibited metaplasia . Compared to the normal control patients , there was an increase in PD-L1 expression in response to H. pylori infection . PD-L1 expression within the infected stomach co-localized with SPEM glands that co-expressed Trefoil factor 2 (TFF2) and CD44v9 [Fig 1D and 1E).To determine whether d CD44v9 , 17 withH. pylori infection on the gastric epithelium was then investigated using gastric organoids derived from human induced pluripotent stem cells (HGOs) . PSC-derived HGOs are truly na\u00efve gastric tissue that has never been exposed to any commensal or pathogenic bacteria. In addition, HGOs can be generated into regionally specific gastric organoids that have either fundic or antral epithelium thus allowing us to investigate the unique effects of the two different epithelia. Fundic/corpus (FHGOs) and antral (AHGOs) gastric organoids were infected with H. pylori for 72 hours. Histological evaluation revealed that compared to control  FHGOs, there was the development of a dysplastic epithelium in response to H. pylori infection . Treatment of infected FHGOs with Hedgehog signaling inhibitor GANT61, resulted in the inhibition of the development of dysplasia . FHGOs infected with a mutant G27 H. pylori strain bearing a CagA deletion (\u0394CagA) did not exhibit that same morphological changes in the epithelium as that observed with the wild type G27 strain  despite colonization of that both bacterial strains within the organoids . While H. pylori infection also induced dysplasia in AHGOs, in contrast to FHGOs, GANT61 treatment did not inhibit this response . Fig 2A\u20132D are representative images of the grading scale used to score the histology of infected HGOs. To identify whether the morphological changes observed in the HGOs in response to H. pylori infection were metaplastic changes, we immunostained sections prepared from organoids for gastric cancer stem cell and SPEM marker CD44v9 . While CD44v9 was not expressed in either the control or \u0394CagA infected FHGOs , there was a robust induction of this marker in FHGOs infected with H. pylori  and this correlated with a significant increase in epithelial cell proliferation . The proliferative response was abrogated with GANT61 treatment of FHGOs . A similar response was observed in AHGOs infected with H. pylori, although GANT61 did not block the proliferation as seen in the FHGOs .The effect of +,K+-ATPase positive parietal cells within the epithelium of FHGOs . Importantly, in response to histamine, Acridine Orange accumulated in cell vesicles as indicated by the increase in the shift in red fluorescence and increase in the ratio of F458 (red)/F488 (green) . AHGOs treated with histamine did not exhibit accumulation of Acridine Orange as documented by a lack in an increase in F458/F488 ratio . FHGOs infected with H. pylori for 24 hours also exhibited areas of acidic accumulation within the epithelium .Acridine Orange is a dye known to show green fluorescence (F488) at a neutral pH and a shift in the fluorescent spectrum to red (F458) when it accumulates in the acidic organelles, as the secretory canaliculus of parietal cells . ImmunohH. pylori, that was not observed in infected AHGOs that were devoid of parietal cells . The response was CagA dependent . Collectively, these data demonstrate that Shh expression is induced in acid-secreting FHGOs by H. pylori infection.Studies have demonstrated that Sonic Hedgehog (Shh) is found within the gastric parietal cells and processed from a 45kDa to a 19kDa bioactive protein via a mechanism that is acid- and protease-dependent . SupportGriffonia Simplicifolia II (GSII) revealed increased PD-L1 expression within metaplastic glands of infected organoids . Treatment of infected FHGOs with GANT61 inhibited the PD-L1 expression that was triggered in response to H. pylori , when compared to control  and GANT61 (minus H. pylori)  treated groups. Organoids infected with the G27 H. pylori strain that expressed a deletion of CagA (\u0394CagA) did not differ from the controls with regards to PD-L1 expression . In contrast to FHGOs, H. pylori infection did not induce PD-L1 expression as assessed by and western blot .Immunofluorescence staining and western blot analysis of FHGOs for the expression of PD-L1 and co-expression of SPEM markers TFF2 and Fig 6A), when compared to AHGOs . Hedgehog signals are regulated based on the positive feedback loop via GLI1 and negative feedback loop via patched 1 (PTCH1), patched 2 (PTCH2), and hedgehog interacting protein (HHIP) [Fig 6C). A response that was not observed in the AHGOs . Collectively, these data demonstrate that H. pylori-induced PD-L1 is localized to the fundic/corpus epithelium, and this response is mediated by Hedgehog signaling.Consistent with changes in protein expression, quantitative RT-PCR data showed a significant increase in PD-L1, Shh and SPEM markers clusterin (CLU) and Human Epididymis Protein 4 (HE4) gene expression specifically within the FHGO epithelium (n (HHIP) . We alsoH. pylori induces PD-L1 within the human gastric corpus epithelium, we developed a 2D/monolayer culture of H. pylori infection using human-derived gastric organoids. We first established fundic organoids derived from human stomachs (huFGO). After 4 days of culture, huFGOs were transferred into 2D dense planar cultures of polarized epithelial cells according to a modification to a published protocol  [Ulex Europeus I (UEAI) to demonstrate apical expression of this pit cell marker in the polarized gastric cultures  and parietal cell specific H+,K+-ATPase . The monolayers expressed all major mature gastric cell lineages .To identify the mechanism by which  . Forty eFig 8A, 8B and 8G). A similar response was observed in monolayers infected with H. pylori . Acid secretion was also induced in human-derived gastric organoids (huFGOs) in response to histamine . The accumulation of Acridine Orange was also observed within resident parietal cells of huFGOs in response to a 24 hour H. pylori infection . Stimulation of acid secretion in response to H. pylori infection, correlated with increased Shh expression within H+,K+-ATPase positive parietal cells . The induction of Shh was CagA dependent .As discussed, Acridine Orange is a dye that accumulates in the acidic organelles such as the secretory canaliculus of parietal cells leading to a fluorescence shift from green to red . As obseH. pylori with or without pretreatment with Hedgehog signaling Gli inhibitor GANT 61 . We observed an increase in PD-L1 membrane-specific expression following H. pylori infection  that was blocked with GANT 61 treatment . Quantitative RT-PCR confirmed the significant induction of PD-L1 and Shh expression in response to H. pylori infection, and this response was mediated by canonical Hedgehog signaling . This response was blocked by a second Hedgehog inhibitor vismodegib (VIS), and appeared to by CagA dependent . Thus, our studies further demonstrate a role of Hedgehog signaling as a mediator of H. pylori-induced PD-L1 expression during early infection.Human gastric-derived monolayer cultures were infected with Fig 10 demonstrates that the increase in PD-L1 expression in response to H. pylori infection was localized to GSII/TFF2 co-expressing cells within the monolayers . These data are consistent with the presence of SPEM markers as confirmed by the increase in CLU and HE4 in response to infection . Interestingly, treatment with GANT61 or VIS not only inhibited PD-L1 expression but also the increase in SPEM markers CLU and HE4 , suggesting a role of Hedgehog signaling in the emergence of metaplasia.H. pylori infection, monolayers were infected with the bacteria for a period of 0 to 24 hours, harvested and analyzed by flow cytometry using markers specific for parietal , mucous neck (GSII) and chief (PgA) cells . Compared to the unstained controls , at baseline (0 hours) there was no expression of Shh localized within parietal cells . However, over a period of a 24 hour infection Shh expression was induced within parietal cells . H. pylori infection also induced increased GSII/PgA co-expressing cells . As the mucous neck cells (GSII-expressing) migrate toward the base of the gastric gland, these cells differentiate into the zymogen/chief cells (PgA-expressing) [H. pylori infection . These data suggest that PD-L1 may be induced specifically within SPEM glands in response to H. pylori infection.To identify the cellular origin of Shh and PD-L1 expression in response to ressing) , 25. In H. pylori infection, we developed an organoid/immune cell co-culture system . We obtained autologous patient blood from which dendritic cells were cultured and FACS sorted . From the blood, CTLs were also isolated and cultured together with the patient's own gastric organoids . After the organoid/immune cell co-culture was infected for 72 hours, CTLs were extracted from the culture by a CD8 positive selection kit. These T cells were analyzed for activation and proliferation by flow cytometry and CFSE uptake . Within the co-culture, H. pylori significantly induced CTLs to express PD-1, IL-2 and IFN\u03b3 . While H. pylori infection resulted in a decrease in CTL proliferation, treatment with PD-1Inh induced high CTL proliferation . Unselected cells were then immunostained for CD11c (myeloid-derived dendritic cells) and epithelial marker EpCAM. These cells were then FACs sorted and the EpCAM positive cells were collected and analyzed for PD-L1 expression and cell viability . HuFGOs infected with H.pylori had a significantly increased population of EpCam positive cells that expressed PD-L1 when compared to control uninfected huFGOs . These data suggest that while bacterial infection results in decreased CTL proliferation, inhibition of PD-L1/PD-1 interactions induced proliferation of CTLs within the co-culture in the presence of H. pylori infection.PD-L1 interacts with programmed death 1 (PD1) on the surface of cytotoxic T lymphocytes (CTLs) rendering them unable to induce apoptosis , 26. PD-Fig 12). Compared to the organoid/immune cell co-cultures in the control  or PD-1Inh alone  treatment, H. pylori infected organoids exhibited a significant increase in cell death with a concomitant increase in PD-L1+ve expressing epithelial cells . However, a further significant increase in epithelial cell death was observed in co-cultures treated with PD-1I that was reflected by a decrease in PD-L1+ve expressing epithelial cells . PD-1Inh alone without immune cells present had no effect on PD-L1 or organoid viability . H. pylori alone, in the absence of immune cell in culture, continued to have a significant increase in epithelial cell death, however not to the extent as that observed in combination with immune cells and PD-1Inh . Importantly, H. pylori infection significantly induced PD-L1 expression in the absence of immune cells from the culture . The co-cultures confirm that H. pylori induces the expression of PD-L1 within the gastric epithelium. In addition, the decreased CTL effector function in response to bacterial infection is inhibited by the PD-1Inh leading to PD-L1 expressing epithelial cell death.To investigate the interaction between the infected gastric epithelium and the host's immune response, infected organoids were co-cultured with the patient's DCs and CTLs in the presence and absence of a PD-1Inh and epithelial cell death was measured . The involvement of CagA in H. pylori-induced PD-L1 expression is significant because CagA is associated with an increased risk of developing gastric cancer [H. pylori allow for the persistence of infected gastric epithelial cells.In the current study we show that PD-L1 is induced following initial nfection , 27. Thec cancer , 28. OurH. pylori-induced PD-L1 expression. Consistent with our findings, we have shown that H. pylori infection induces an increase in Shh secretion and signaling via a CagA dependent pathway [H. pylori infection. Shh signaling in the epithelium of infected antral differentiated organoids was not observed. An explanation for this observation is data demonstrating that Shh is secreted from the acid-secreting parietal cells within the fundic region of the stomach [H. pylori, a response that was ablated with GANT 61 and vismodegib treatment. Parietal cells in this region secrete Shh which subsequently induces the secretion of acid, normal epithelial cell function and regeneration [Shh signaling mediates  pathway . We furt stomach , 30. Indneration , 31\u201333. neration .H. pylori infected patients, PD-L1 expression co-localized with proteins that classically mark SPEM cells including TFF2 and CD44v9 [H. pylori infection is evident from our studies. The use of human PSC-derived antral and fundic gastric organoids has allowed us to identify how these unique regions of the human stomach differentially respond to H. pylori infection.In biopsies collected from d CD44v9 , 17. Difd CD44v9 \u201337. DiffH. pylori highly expressed PD-L1 and suppressed CTL proliferation. CTLs are the main pro-apoptotic cell within the gastric cancer microenvironment [H. pylori infected organoids were co-cultured with CTLs and treated with a PD-1 inhibitor (PD-1Inh) there was an increase in proliferating CTLs and a decrease in live PD-L1 expressing gastric epithelial cells. Therefore, this suggests that PD-L1 expression was protective to the infected cells. These results are significant because once a patient progresses to a metaplastic state, the eradication of H. pylori does not decrease the risk of developing gastric cancer [To identify whether PD-L1 expression protects the epithelium from chronic inflammation, we developed an organoid/autologous immune cell co-culture system. Organoids infected with ironment . When H.c cancer .H. pylori and treatment with immune checkpoint inhibitors. From this study, we proposed that PD-L1, that is induced by parietal cell-derived Shh, may be protective to SPEM cells in the presence of bacterial infection . When the interaction between PD-1 and PD-L1 is inhibited, activated CTLs may target the SPEM glands . These models could be used to devise a therapy for patients that have progressed to a metaplastic state and would therefore not benefit from eradication of H. pylori. In addition, this co-culture system could possibly be used to discover new therapies for gastric cancer.PD-L1 expression lasts through to the development of gastric cancer. Up to 69% of all gastric cancers express PD-L1 . Here weHuman gastric tissue and blood was collected during sleeve gastrectomies were specifically collected for this study with the approval of the Institutional Review Board . All subjects provided informed written consent. A parent or guardian of any minor participant provided informed consent on their behalf.2  in 2mL EEM. On d2, 1 mL fresh EEM was added to wells. On days 3 and 5, 1 mL hESC media  was added to the existing media in each well. Starting on d5, wells underwent a complete daily media change with 2.5 mL hESC media. Putative iPSC colonies were then manually excised and replated in feeder free culture conditions consisting of matrigel (BD BioSciences) and mTeSR1 (Stem Cell Technologies). Lines exhibiting robust proliferation and maintenance of stereotypical human pluripotent stem cell morphology were then expanded and cryopreserved at ~ passage 10 [For generation of iPSC263_10 whole blood from a healthy blood donor was obtained from the CCHMC Cell Processing Core, Division of Experimental Hematology and Cancer Biology. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll centrifugation in SepMate tubes (Stem Cell Technologies). PBMCs were then frozen in cryopreservation media (90% FCS + 10% DMSO) until iPSC generation. PBMCs were thawed and 1-5x10e6 cells were primed for iPSC generation by culture in erythroid expansion media for 8 days . During priming, 1mL of fresh EEM was added to existing media every 2 days. At the completion of priming (d0), 1x10e6 cells were transduced for 3 h with recombinant VSV-G pseudotyped polycistronic lentiviral particles co-expressing reprogramming factors Oct4, Klf4, Sox2, cMyc and dTomato  in the presence of 8ug/mL polybrene. Transduced cells were then plated on 0.1% gelatin-coated dishes containing 2 x 10e4 irradiated MEFs/cmssage 10 , 41. DonTM following a published protocol [2+ and Mg2+ supplemented with 1% Penicillin/Streptomycin. Epithelial tissue was further digested in DMEM/F12  containing collagenase A  and bovine serum albumin (2 mg/ mL) for 15\u201330 min to liberate glands from tissue. The reaction was stopped using DMEM/F12  supplemented with Kanamycin (50 mg/ml) and Amphotericin B (0.25 mg/ml)/Gentamicin (10 mg/ml), and the glands were filtered through sterile gauze and allowed to settle on ice for 10 mins. Glands were washed with PBS supplemented with Kanamycin (50 mg/ml) and Amphotericin B (0.25 mg/ml)/Gentamicin (10 mg/ml) and suspended in MatrigelTM. Organoids were plated at a density of 50 \u03bcL/well and cultured in 3D human gastric organoid media , 100 ng/mL Noggin, 20% R-Spondin Conditioned Media, 50% Wnt Conditioned Media, 200 ng/mL FGF10, 1 nM Gastrin, 10uM Y-27632, Kanamycin (50 mg/ml) and Amphotericin B (0.25 mg/ml)/Gentamicin (10 mg/ml)). Following 6\u20137 days 3D organoids grew from glands. Following this time 3D organoids were infected with H. pylori or transferred to 2D organoid monolayers.Human stomach was digested to glands and embedded into Matrigelprotocol , 42. BriTM using cold PBS. Organoids were suspended in 2D media containing  and plated onto MatrigelTM coated plates. Briefly, MatrigelTM was diluted tenfold into cell culture grade water and allowed to coat 2 well chamber slides or 12 well plates at 37\u00b0C for 1 hour. Excess water was removed from the plate and MatrigelTM coating was allowed to dry for 1 hour at room temperature.Human-derived gastric epithelial monolayers were prepared according to a modified published protocol . OrganoiH. pylori strain G27 [cagA::cat) [Brucella broth containing approximately 2X105 bacteria using a Nanoject II (Drummond) microinjector. Gastric epithelial monolayers cultured for 4 days were infected with 50 \u03bcL of DMEM/F12  containing 5-8million bacteria.rain G27 , 44 and gA::cat) , were grgA::cat) . HGOs cuTM tubes (Stemcell) and LymphoprepTM (Stemcell) were used to separate out red blood cells and platelets according to manufacturer\u2019s protocol. Briefly, 50 mL SepmateTM tubes were filled at the bottom with 15 mL of LymphoprepTM. Whole blood was diluted with phosphate-buffered saline containing 2% fetal bovine serum. Diluted whole blood was added to the tube containing LymphoprepTM. The tubes were centrifuged at 1200 g for 10 minutes. Following this supernatant was poured into a separate tube. The supernatant was diluted with phosphate-buffered saline containing 2% fetal bovine serum. The supernatant was centrifuged at 300 g for 8 minutes. The supernatant was discarded, and the pellet was re-suspended in phosphate-buffered saline containing 2% fetal bovine serum. The pellet was centrifuged at 120 g for 10 minutes. The resulting peripheral blood mononuclear cells were cultured in dendritic cell media or put through the negative selection EasySepTM Human CD8+ T cell Enrichment Kit (Stemcell).Whole blood was collected from young sleeve gastrectomy patients (15\u201321 years old). The Sepmate2 . On day 6 mature dendritic cells were shorted by fluorescence-activated cell sorting (FACs) for the expression of HLA-DR (Biolegend). On day 7 FACs sorted mature dendritic cells were co-cultured with control or H. pylori huFGOs.PBMCs were matured into dendritic cells using a published protocol . PBMCs aTM Human CD8+ T cell Enrichment according to manufacturer\u2019s protocol. Briefly, PBMCs were suspended in EasySepTM buffer (Cell Separation Buffer) (Stemcell) in a 14mL round bottom centrifuge tube (Corning). 50 \u03bcL/mL of Enrichment Cocktail was added to PBMCs and allowed to incubate at room temperature for 10 minutes. Magnetic particles were mixed by vortexing for 30 seconds. 150 \u03bcL/mL of magnetic particles were added to the PBMCs and allowed to incubate for 5 minutes at room temperature. The PBMC cocktail was topped up to 5 mL using EasySepTM Buffer. The PBMC cocktail was added to \u201cThe Big Easy\u201d magnet (Stemcell) and allowed to incubate at room temperature for 5 minutes. The CD8+ T cells are the cells that have no bound magnets. These were poured into a fresh 15 mL conical and centrifuged at 1200 rpm for 5 minutes and plated in T cell media containing RPMI 1640 (Invitrogen), 10% fetal calf serum, \u03b2-mercaptoethanol (50 \u03bcM), 1% Pennecillin/Streptomycin, 1% Insulin-tellurium-selenium (Thermofisher), IL-2 (30 U/mL) (Thermofisher) and IL-7 (0.5 ng/mL) (Thermofisher) [CD8+ T Cells were extracted from PBMCs isolated from whole blood using the EasySepofisher) .TM with cold DMEM/F12 and centrifuging the organoid suspension at 400 g for 5 minutes. CD8+ T cells were harvested and centrifuged at 300 g for 5 minutes. CTLs were suspended in a 5 \u03bcM Carboxyfluorescein succinimidyl ester (CFSE) for 20 minutes at 37\u00b0C. Following this cells were washed with DPBS and centrifuged at 300 g for 5 minutes. CTLs were then incubated in huFGO full media for 10 minutes at 37\u00b0C. CTLs were then centrifuged at 300 g for 5 minutes. Mature dendritic cells were centrifuged at 300 g for 5 minutes. HuFGOs, CD8+ T cells and dendritic cells were suspended in MatrigelTM and plated in 4 well plate. HuFGOs were injected with 200 nL of Brucella broth containing approximately 2*105 bacteria using a Nanoject II (Drummond) microinjector. One well of uninfected huFGOs co-cultured with CD8+ T Cells and dendritic cells and one well of H. pylori infected huFGOs co-cultured with CD8+ T Cells and dendritic cells were treated with Nivolumab , a PD-1 inhibitor. Cells were co-cultured for 5 days.HuFGOs were harvested from MatrigelGriffonia simplicifolia (GSII) , anti-rat 594, anti-rabbit 647 and counter stained with . huFGOs co-cultured with immune cells were incubated overnight at 4\u00b0C with primary antibodies specific for CD8a , CD11c  and E-cadherin . Organoids were then treated with secondary antibodies anti-mouse 594, anti-rabbit 488 or anti-goat 647 and counter stained with  for 1 hour at room temperature. Organoids were visualized using the Zeiss LSM710.Tissue slides were hydrated with ethanol, xylenes and water. Slides were then blocked with 20% donkey serum at room temperature for one hour and incubated with primary antibodies for PD-L1 , TFF2  or CD44v9 , PD-L1  or GSII  overnight at 4\u00b0C. Slides were washed in 0.01% triton x-100 in PBS and treated with a secondary for donkey anti-rat 488, anti-rabbit 647 or donkey anti-rat 488, donkey anti-rabbit 555 and GSII 647 as well . Media was removed from HGOs, 2D organoid monolayers or huFGO co-cultures with immune cells and 3.7% formaldehyde was added to the organoids for 15 minutes at room temperature. The cultures were washed with PBS and then permeabilized with 0.5% Triton X-100 in DPBS for 20 minutes at room temperature. Blocking was done with 2% normal donkey serum for 1 hour at room temperature. Monolayer cultures were then incubated overnight at 4\u00b0C with primary antibodies specific for H+/K+ ATPase  and E-cadherin . HGOs and monolayers were incubated overnight at 4\u00b0C with PD-L1  and TFF2  or HK  and Sonic Hedgehog . Monolayers were incubated for 1 hour at room temperature with secondary antibodies donkey anti-mouse 594, UEAI , donkey anti-goat 647 or donkey anti-mouse 555 and donkey anti-goat 647 and counter stained with . Monolayers and HGOs were incubated for 1 hour at room temperature with Helicobacter pylori and CD44v9)  or 20% goat serum (H+/K+ ATPase). Slides were then incubated with a 1:2000 dilution of PCNA , 1:1000 dilution of H+/K+ ATPase  or 1:1000 dilution of CD44v9  overnight at 4\u00b0C. Helicobacter pylori  stained slides were incubated with the pre-diluted antibody for 28 minutes at 37\u00b0C. Slides were then biotinylated with an IgG secondary antibody for either rabbit, mouse or rat for 30 minutes at room temperature. Finally slides were incubated with ABC reagent  for 30 minutes at room temperature. The color of each set of slides was then developed with 3,3\u2019-diaminobenzidine (DAB) from the DAB Substrate Kit . The slides were counterstained with hematoxylin , dehydrated and mounted with Permount.Organoids were fixed in 4% paraformaldehyde for 15 minutes. They were then embedded in paraffin and cut into 5 \u03bcM sections. Slides were then deparaffinized and antigen retrieval was done by heating slides for 10 minutes at 100\u00b0C in 0.01 M sodium citrate buffer . Endogenous peroxide activity was then blocked by incubating slides with 0.3% hydrogen peroxide in methanol for 20 minutes. Slides were then incubated with 20% horse serum  containing 5\u20138 million bacteria. Histamine was added to the medium of 3D huFGOs, iPSC-derived HGO or 2D gastric epithelial monolayers at a concentration of 6.67 mM (Sigma Aldrich). Images were analyzed using the Zeiss LSM710 Microscope and background corrected 550-620/620-700 nm ratio values were converted to fold change corresponding to pH change using Prisim Graph Pad software.Dye was added to the culture and monitored on the Zeiss LSM710 microscope. Organoids and monolayers were harvested in cold DMEM/F12 and lysed in M-PER Mammalian Protein Extraction Reagent (Thermofisher) supplemented with protease inhibitors (Roche) according to the manufacturer\u2019s protocol. Cell lysates were suspended in 40 \u03bcL of Laemmli Loading Buffer containing \u03b2-mercaptoethanol (BioRad). Samples containing 20 \u03bcg of protein were then loaded onto 1 4\u201320% Tris-Glycine Gradient Gels (Invitrogen) and run at 120 V for 1.5 hours before transferring the protein onto nitrocellulose membranes  at 105 V for 1.5 hours at 4\u00b0C. Membranes were blocked for 1 hour at 23\u00b0C using KPL Detector Block Solution . Next membranes were incubated overnight at 4\u00b0C with a 1:1000 dilution of anti-PD-L1  a 1:1000 dilution of anti-Shh  or 1:2000 dilution of anti-GAPDH . The membranes were washed 3 times for 5 minutes each. Following this, the membranes were incubated with a 1:1000 dilution anti-mouse, 1:1000 dilution anti-goat or anti-rabbit Alexa Fluor 680 (Invitrogen). The blots were then imaged using a scanning densitometer along with analysis software (Odyssey Infrared Imaging Software System).+/K+ ATPase ATP4B (Hs01026288_m1), Pepsinogen C (Hs00160052_m1) Pepsinogen A (Hs05416800_g1) and Mist 1 (Hs00703572_s1). PCR amplifications were done with a pre-validated 20X TaqMan Expression Assay primers and a 2X TaqMan Universal Master Mix (Applied Biosystems) and a cDNA template in a total volume of 20 \u03bcL. Amplifications were performed in duplicate wells in a StepOne Real-Time PCR System (Applied Biosystems). Fold change was calculated at (Ct-Ct high) = n target, 2ntarget/2nGAPDEH = fold change where Ct = threshold cycle.Total RNA was isolated from tissue, glands, 3D organoids, HGOs and 2D organoid monolayers using TRIzol (Life Technologies) according to manufacturer\u2019s protocol. A High Capacity cDNA Reverse Transcription Kit synthesized cDNA from 100 ng of RNA following protocol provided by Applied Biosystems. Real-time PCR assays were utilized for the following genes in HGOs and 2D organoid monolayers: GAPDH (Hs02786624_g1), PD-L1 (Hs01125296_m1), SHH (Hs00179843_m1), TFF2 (Hs00193719_m1), Clustrin (Hs00156548_m1), and HE4 (Hs00899484_m1). Cell lineage markers were determined in tissue, glands, hFGOs, 2D monolayers fundic and antral HGOs by RT-PCR for Mucin 5AC (Hs01365616_m1), Mucin 6 (Hs01674026_g1), HH. pylori. The cultures were treated with accutase for 10 minutes at 37o C. The organoids were then passed through a 27 1/8-gauge syringe in order to dissociate the organoids into single cells. CTLs were extracted using the EasySep Human CD8 Positive Selection Kit II . Breifly, cells were incubated with 100 \u03bcL/mL of sample selection cocktail and incubated at room temperature for 3 minutes. 50 \u03bcL/mL of sample RapidSpheres were added to the sample and incubated at room temperature for 3 minutes. Samples were topped up to 5 mL with EasySep Buffer  and incubated at room temperature for 3 minutes. Cells that did not bind to the magnetic beads were collected in a 50 mL conical. Samples were wash two more times with 5 mL of Easy Sep Buffer . Cells that were adherent to magnetic beads were CTLs and were collected and centrifuged at 300 g for 5 minutes. Cells that did not bind to the magnetic beads were DCs and epithelial cells. These cells were also centrifuged at 300 g for 5 minutes. Fluoresence Assisted Cell Sorting was using to collect epithelial cells from the epithelial cell/DC mixture. This cell mixture was stained with EpCam (Biolegend) and CD11c (Biolegend). EpCam positive cells were collected during sorted and CD11c cells were disgarded. EpCam positive cells were suspended in 100 \u03bcL of a 1:1000 dilution of the zombie red cocktail (BioLegend). 1 \u03bcL of a 1:1000 dilution of the calcein violet cocktail (BioLegend) was added to this cell suspension. The cells were incubated in this cocktail for 20 minutes at room temperature. The cells were then washed with 1 mL of 5% BSA at 300 g for 5 minutes. The cells were then suspended in 100 \u03bcL of 5% BSA, treated with 1 \u03bcL of anti-PD-L1(BioLegend) and incubated for 15 minutes at room temperature. The cell suspension was then incubated at room temperature for 15 minutes with 100 \u03bcL of Reagent A (Thermofisher). The cells were then washed with 1 mL of 5% bovine serum albumin at 300 g for 5 minutes. CTLs that were extracted from culture were suspended in 100 \u03bcL of 5% BSA, treated with 1 \u03bcL of anti-CD8 (BioLegend) and anti-PD-1 (BioLegend) and incubated for 15 minutes at room temperature. The cells were then incubated at room temperature for 15 minutes with 100 \u03bcL of Reagent A (Thermofisher). The cells were then washed with 1 mL of 5% bovine serum albumin at 300 g for 5 minutes. The cells were suspended in 100 \u03bcL of Reagent B (Thermofisher) and 1 \u03bcL of anti-IL2 and 1 \u03bcL of anti-IFN-\u03b3 were added to the cells. This cocktail was incubated at room temperature for 20 minutes. The cells were washed with 1 mL of 5% bovine serum albumin at 300 g for 5 minutes. All cells were then suspended in 500 \u03bcL of 5% bovine serum albumin. Samples were run on the CANTO 3 and analyzed by FlowJo data analysis.The media was removed from huFGO co-cultured with immune cells and infected or uninfected with t-test using commercially available software . A P value <0.05 was considered significant.The significance of the results was tested by one-way ANOVA, two-way ANOVA or student\u2019s"}
+{"text": "To support AIDS service organisations and other community-based organisations\u2019 use of research evidence to inform HIV-related programmes, services and policies, the Ontario HIV Treatment Network (OHTN) developed a Rapid Response Service. The final product of the rapid response process at the OHTN, which is more streamlined than that of traditional systematic reviews, consists of a detailed report answering questions regarding an HIV-specific issue and how the findings apply within the local context. In 2016, the OHTN conducted an evaluation to assess the effectiveness of its Rapid Response Service. This article reports on the development of this service as well as the results of the evaluation.n\u2009=\u2009102) were analysed using univariate analyses. Frequency distributions were determined for the following variables for each rapid response: populations observed, topics covered, requestor affiliations and number of downloads from the OHTN\u2019s website. Requestors of rapid responses were also interviewed regarding perceived helpfulness and utility of the service and final products, and suggestions for changes to the service. Six-month follow-up interviews were conducted to determine how affiliated organisations used the evidence from the rapid response they requested.All rapid responses published between January 1, 2009, and September 30, 2016, by the OHTN  contains supplementary material, which is available to authorized users. The use of research evidence to inform policy and practice has been widely accepted among public health professionals \u20136. HowevTypically, rapid reviews are completed within 1\u201312\u2009months and range from annotated bibliographies, reference lists and abstract summaries to rapid systematic reviews and full health technology assessments , 10. TheInitiatives designed to promote the use of research evidence among health system and policy decision-makers has steadily increased , 20\u201322; In the context of HIV-related public health initiatives in Canada, community actions and programmes have contributed substantially since the beginning of the epidemic . CBOs haGrowing from this research, the Ontario HIV Treatment Network (OHTN) \u2013 a non-profit collaborative network aiming to improve the health and lives of people living with and at risk of HIV by using data and evidence to drive change \u2013 began to address these challenges by developing a Rapid Response Service in 2009. The OHTN\u2019s Rapid Response Service works primarily with CBOs, both HIV-focused (i.e. AIDS Service Organisations (ASOs)) and non-HIV-focused, that lack the capacity and resources to acquire and assess research evidence. It also provides services to policy-makers , healthcare providers and academic researchers. With respect to assessing the impact of health research, the literature has identified rapid response programmes as a key mechanism to facilitate what is known as \u2018user-pull\u2019 or decision-makers\u2019 efforts to identify research, particularly where a crucial gap in knowledge exists , 28, 29.The rapid response process at the OHTN was developed with a commitment to respond to questions from ASOs and other CBOs by systematically and transparently identifying and synthesising relevant research evidence in days or weeks . The prThe final product consists of three to five key messages, an outline of the issue and its importance, a detailed summary of findings, and potential factors related to how the findings apply within the local context. All of this information is presented in plain language and in a format that is compliant with legislation in Ontario, Canada, for ensuring accessibility for people living with disabilities . All completed rapid responses \u2013 from 2009 to the present \u2013 are housed on a publicly accessible page on the OHTN website , which wIn 2016, the OHTN conducted an evaluation to assess the effectiveness of its Rapid Response Service. The evaluation consisted of analysing previously published rapid responses and conducting qualitative interviews with requestors to explore how the rapid responses were used by community organisations and other stakeholders to inform services, programmes or policies, create or improve them, or secure new funding .The goal of this article is to share the development process of the OHTN\u2019s Rapid Response Service, report on the evaluation of the programme and outline potential next steps for the Rapid Response Service to address issues raised through this evaluation by community organisations as well as implications.Wilson et al.\u2019s  indicaton\u2009=\u2009102) were analysed by the dedicated Knowledge Synthesis and Rapid Response Service staff members using univariate analyses. Each rapid response was categorised within the following variables: \u2018populations observed\u2019, \u2018topics covered\u2019, \u2018requestor affiliations\u2019 and \u2018number of downloads from the OHTN website\u2019. Frequency distributions of rapid responses were determined for each respective variable. These variables were chosen to reflect assessment of programme organisation, as described by Wilson et al.  helped us write the background of our paper\u201d [Being provided with a list of references was described as most useful by 13% of requestors (r paper\u201d .The What We Found section provided the information we needed to give consideration to the programming we wanted to develop\u201d [Two requestors (9%) felt that the \u2018what we found\u2019 section of the rapid responses provided a strong background for future research, or offered the appropriate foundation from which programmes and services could be refined: \u201cdevelop\u201d .this section helped create a baseline of information \u2026 a quick take was needed on where the research was. We needed a starting point and that was what the rapid response was for\u201d [\u2018The issue and why it\u2019s important\u2019 section was identified by one participant (4%) as creating a starting point from which to begin their own research, who stated that \u201cwas for\u201d .n\u2009=\u200915) said that they did not find any aspect of the rapid responses unhelpful. Thirteen percent of requestors (n\u2009=\u20093) who did find limitations within the rapid responses stated that the evidence itself was the least useful aspect of the rapid responses. Some of these requestors explained that the evidence was insufficient, either because it did not reveal anything new or because it was not necessarily relevant to their specific contexts. A lack of detail in the information provided was described by 13% (n\u2009=\u20093) of requestors, but most conceded that there was a paucity of Canadian research evidence on their respective areas of interest. For example, one participant indicated that \u201cmost of the data cited was American \u2026 there was a shortage of Canadian data\u201d [Almost two-thirds of requestors , when asked if there was anything the Rapid Response Service could do differently, responded with \u2018No\u2019. Those who did feel that there was a need for improvement, suggested that the Rapid Response Service should provide recommendations , decrease the time necessary to produce the rapid response , place more focus specifically on the requestor\u2019s question , provide a data extraction table with all references , expand the \u2018key take-home messages\u2019 section , and allow the reviews to answer multiple questions  [Many requestors  .n\u2009=\u200918) responded. These requestors were ASO staff (n\u2009=\u20098), researchers from universities or other institutions (n\u2009=\u20093), staff from non-HIV-focused CBOs (n\u2009=\u20092), policy-makers (n\u2009=\u20092), lawyers (n\u2009=\u20092) and healthcare providers (n\u2009=\u20091). Of the six requestors who did not respond, affiliations were as follows: CBO staff (n\u2009=\u20091), OHTN staff (n\u2009=\u20091), healthcare provider (n\u2009=\u20092), policy-maker (n\u2009=\u20091) and ASO staff (n\u2009=\u20091) [We contacted each of 24 requestors who completed the initial qualitative survey by email for 6-month follow-up telephone interviews and 75% ( (n\u2009=\u20091) .n\u2009=\u200914), some thought the communication between OHTN staff and requestors could be improved. Some respondents felt they were unaware of the \u2018commitment\u2019 that the rapid response process entailed with regards to developing a research question and would have liked to have been more prepared for the process as a whole. Others expressed a desire to be more involved in the writing of the rapid response or to have had more discussions with OHTN staff during the writing process. Some felt that their organisations lacked internal research capacity and required further resources or communication with the OHTN for community members to conduct their own searches in the future. For example, two participants shared the following feedback:It may be helpful to have some resources on how community members and others without the expertise can do their own \u2018quick\u2019 research, some capacity building\u201d [\u201cuilding\u201d .It might be nice for the OHTN to have a conversation once the rapid response is submitted with the requestor for capacity building purposes. A dialogue might help the translation of the rapid response  be as true to the evidence as possible\u201d [\u201cossible\u201d .Of the requestors who offered suggestions about improving the Rapid Response Service , feedback of the key features of the programme, and qualitative interviews to provide insight for whether and how the products were used. As few programmes that support the use of research evidence within the community context have been established , we haveHowever, some limitations to this evaluation do exist. Firstly, the process entailed contacting previous and current users of the programme. In certain instances, this proved difficult, since some contacts had left their organisation. Interviews were therefore only conducted with people who had requested rapid responses between 2014 and 2016. Insights from requestors who received rapid responses before this time would have been valuable for this evaluation to compare comments as the Service evolved from 2009 to 2016. There is also the possibility of recall bias ; due to With the increasing demand for rapid reviews by public health decision-makers, there is a growing need for evaluation of interventions designed to promote access to these products, and investigation of whether and how they are being used , 16\u201319. The information gleaned from our analyses of rapid responses and requestor interviews provides many insights into how the Rapid Response Service can be improved to better inform the service, programme, and policy efforts of CBOs, government agencies and other stakeholders. While the service is being accessed by CBOs, there is a need to extend the reach of the Rapid Response Service. Profiles of rapid responses show that roughly 39% of them focused on the general HIV-positive population and not on any one specific population. These numbers suggest that organisations serving certain populations may not be aware of the OHTN\u2019s Rapid Response Service . FurtherThe Rapid Response Service could also focus on actively promoting awareness of the service by sharing published rapid responses more widely. Facilitating \u2018user-pull\u2019 may be improved by prompting partners for research where gaps in literature are identified and building their capacity to access and apply research evidence within their local contexts .The fact that many requestors applied reference lists and key take-home messages to programmes, services and policies, but wanted further recommendations to be provided, demonstrates that requestors are willing to engage with research, but are not always sure how to do this. An explicit decision was made by the OHTN to not include recommendations in the Rapid Response Service, and this decision is similar to those of other rapid evidence services (e.g. ). While The desire for greater transparency in the research process is evident in responses to the \u2018Least useful aspect\u2019 and \u2018What the OHTN could do differently\u2019 survey sections. Requestors said they would like rapid responses to describe the search strategies used and data extraction tables; one requestor even suggested that the rapid response should include a chronology of the process from the initial research question to the end product . TranspaIndeed, increasing communication with requestors during the rapid response process for capacity-building purposes may be another area to improve. Reviews of rapid review programmes have suggested that close relationships and personal contact facilitate the use of knowledge products and the use of research evidence , 51. ReqThis speaks to a growing discussion in healthcare emphasising collaborative approaches  to knowMetrics categorising the uses for rapid reviews among CBOs would be important for other evaluations of rapid response services designed for these organisations. Moore et al.\u2019s study among policy-makers demonstrated that not only did policy-makers use rapid reviews for instrumental, conceptual and symbolic purposes, but also that these products could have multiple types of uses at once, with benefits beyond those for which they were requested . Moore eIndeed, much focus is being directed by researchers towards whether and how rapid review products are being used to make public health decisions; however, there also remains the enduring need to investigate whether their use has helped to make better decisions. While it is believed that the use of research evidence to inform decision-making  is the most appropriate and easily assessed measure of research impact in public health , and it Organisations that have used the OHTN\u2019s Rapid Response Service describe it as a valuable service useful for the development of programmes and policies. The service must ultimately fit into a larger knowledge translation initiative in order to reach its full potential. Increasing capacity-building efforts, and working more collaboratively with CBOs throughout the rapid response process, may maximise its utility. Future research efforts should also be focused on exploring what facilitates the use of rapid response products as well as the types of activities that they are being used for. In the meantime, outreach to additional CBOs and other organisations should be extended to foster further collaboration. Describing the process and findings of this evaluation provides lessons to be learned for the development of similar programmes that aim to promote the use of research evidence among CBOs and other stakeholders.Additional file 1:Rapid response evaluation requestor interview questionnaire. Interview questionnaire document given by email or telephone to requestors who requested a rapid response between 2014 and 2015. (DOCX 14 kb)Additional file 2:Rapid response evaluation 6-month follow-up interview guide. In-depth 6-month follow-up interview document emailed to requestors who completed an initial interview in 2016. (DOCX 12 kb)Additional file 3:Characteristics of individual rapid responses published by the Ontario HIV Treatment Network between 2009 and 2016. Table of the characteristics of individual rapid responses, including title, year published, population of interest, topics covered and number of downloads as of June 30, 2018. (DOCX 32 kb)"}
+{"text": "P\u00a0<\u00a00.05). The top 10 lncRNAs with the most significant differences were LINC01977, RP11\u2010363E7.4, RP3\u2010483K16.4, RP11\u2010547D24.1, RUNDC3A\u2010AS1, AC093609.1, CTD\u20102008L17.2, HAGLROS, UNC5B\u2010AS1, and LINC01354. In addition, CTD\u20102008L17.2, HAGLROS, AC093609.1, UNC5B\u2010AS1, and RUNDC3A\u2010AS1 were shown to play vital roles in determining the histological cancer type. Furthermore, RP11\u2010547D24.1 and UNC5B\u2010AS1 could distinguish patients with different stages of PTC. The lncRNA RP11\u2010547D24.1 was validated by loss\u2010of\u2010function assays, revealing that downregulation of this lncRNA regulates thyroid tumor cell proliferation and apoptosis, invasion, and migration. This study demonstrates the potential for using lncRNAs to interpret the pathogenesis and development of PTC.Long noncoding RNAs (lncRNAs) are known to be key regulators of numerous biological processes, and substantial evidence supports that abnormal lncRNA expression plays a significant role in tumorigenesis and tumor progression. However, the mechanism by which lncRNAs function in thyroid carcinoma are still unclear. To investigate the role of lncRNAs in the tumorigenesis of papillary thyroid carcinoma (PTC), we analyzed lncRNA data in The Cancer Genome Atlas RNA\u2010Seq database. A comparison of lncRNAs in cancerous thyroid tissues and normal tissues revealed hundreds of differentially expressed lncRNAs. Of 7589 lncRNAs identified in 561 thyroid cancer cases , the expression levels of 144 were found to be aberrant (|log2 fold change| >2 and adjusted  Furthermore, high\u2010throughput RNA sequencing and microarray analyses of different types of cancerous tissues have revealed that thousands of lncRNAs are expressed aberrantly.To date, only a few lncRNAs in thyroid tumors have been identified. To discover more TC\u2010associated lncRNAs, we used The Cancer Genome Atlas (TCGA) RNA sequencing database of TC tissues and adjacent normal tissues to perform genome\u2010wide analyses and differentially profile lncRNA expression in TC.We found the expression of RP11\u2010547D24.1 to be extremely upregulated in TC tissues compared with that in paratumorous (PT) tissues. Thus, we chose to use RP11\u2010547D24.1 in additional cell experiments. RP11\u2010547D24.1 is known to be located on human chromosome 1p36.33 and to have a 2318\u00a0bp transcript . However, there have been no reports on the expression and biological function of the lncRNA RP11\u2010547D24.1 in PTC.22.1The high\u2010throughput RNA sequence data from confirmed PTC cases were downloaded from TCGA on 6 August 2017. These data were acquired on an Illumina HiSeq RNA\u2010Seq platform and included 502 PTC tissues and 58 adjacent noncancerous thyroid tissues. Currently, studies related to TCGA are widely acknowledged since TCGA is a community resource project.P\u00a0<\u00a00.05 and absolute Log >2). Some lncRNAs were excluded from our analysis because their expression level fold changes were less than 1 in more than 10% of the samples. In addition, the expression level of each lncRNA was log 2 transformed for further analysis.PTC includes 60\u00a0483 mRNAs that cover 7589 lncRNAs, as described by RNA\u2010Seq data from the NCBI and Ensembl databases. We next assessed the differential expression of these lncRNAs with the R language package DESeq (adjusted 2.2https://cancer.sanger.ac.uk/census) using the key word \u201cthyroid\u201d, and genes associated with the cancer pathway were also downloaded from the KEGG database . Pearson correlation analysis  was carried out to analyze the link between RP11\u2010547D24.1 and related mRNAs. For these lncRNAs, uni\u2010 and multivariate Cox analyses were also needed. The Kaplan\u2013Meier method was utilized to reveal the prognostic significance of the lncRNAs, and the log\u2010rank test was conducted to analyze survival time.To value the diagnostic effectiveness of the lncRNAs in PTC, we constructed receiver operating characteristic (ROC) curves and subjected the top 10 lncRNAs of the area under the ROC curve (AUC) to further analysis. Student's t test was used to analyze the top 10 lncRNAs differentially expressed between PTC and PT tissues. To further study the potential proteins related to RP11\u2010547D24.1, we downloaded genes from the CGC database  supplemented with 10% fetal bovine serum , 10\u00a0units/mL penicillin and 10\u00a0mg/mL streptomycin at 37\u00b0C in a humidified 5% CO2 atmosphere. TPC\u20101 and K1 cells were transfected with RP11\u2010547D24.1 siRNA  using Lipofectamine 3000 transfection reagent  in accordance with the manufacturer's protocol. All cell lines were plated in 6\u2010well plates before transfection. RP11\u2010547D24.1 cells were silenced by treatment with 10\u00a0\u03bcL of siRNA and 4\u00a0\u03bcL of Lipofectamine 3000 for 48\u00a0hours. The RP11\u2010547D24.1 siRNA sequences used in our research targeted the following sequences: forward 5\u2032\u2010GAGUUAAAUAGAUAUCCAAdTdT\u20103\u2032 and reverse 5\u2032\u2010UUGGAUAUCUAUUUAACU CdTdT\u20103\u2032.2.4We used lysis buffer to extract protein, which was isolated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein was then transferred to a polyvinylidene fluoride membrane, blocked with a primary anti\u2010active \u03b2\u2010catenin antibody  overnight at 4\u00b0C and incubated with an anti\u2010mouse horseradish peroxidase\u2010conjugated secondary antibody  after being washed with Tris\u2010buffered saline at 37\u00b0C for 1\u00a0hour. Protein quantification was performed using an enhanced chemiluminescence reagent . GAPDH was used as a loading control.2.5g for 10\u00a0minutes and washed with PBS 2 times. The cells were stained with propidium iodide  containing 0.1\u00a0mg/mL RNase A and 0.6% NP\u201040  in the dark for 30\u00a0minutes at room temperature, collected by centrifugation at 167.7\u00d7g for 10\u00a0minutes at 4\u00b0C, and washed with PBS. For the apoptosis assay, the cells were stained with FITC\u2010Annexin V and PI from the Annexin V\u2010FITC/PI kit . Flow cytometry was performed using a FACSCalibur flow cytometer . FlowJo 10.0.4 software  was used to analyze the data. All procedures were performed 3 times.The cells were washed with PBS and then fixed with Hank's buffered salt solution  containing ice\u2010cold 80% ethanol for 30\u00a0minutes. The cells were then collected by centrifugation at 167.7\u00d72.66\u00a0cells/well) were treated with the indicated reagents, and wounds were created using a 1\u00a0000\u00a0\u00b5L plastic pipette tip. The cells were then photographed every 12\u00a0hours from 0\u00a0hour to 36\u00a0hours. Five random fields of view were chosen, and the images were captured under microscopic magnification (\u00d720). Experiments were carried out independently in triplicate.TPC\u20101 and K1 cells  with coated Matrigel . The transfected cells were treated with trypsin/EDTA solution, washed once with serum\u2010free RPMI 1640 medium for centrifugation. A total of 1\u00a0\u00d7\u00a0105 cells were re\u2010suspended in 0.2\u00a0mL serum\u2010free RPMI 1640 medium and were seeded into the upper chamber. RPMI 1640 medium containing 10% FBS (0.5\u00a0mL) was added to the lower chamber as a chemoattractant. After 24\u00a0hours incubation at 37\u00b0C in a 5% CO2 incubator, cells on the top surface of upper chamber were removed by wiping with a cotton swab. Cells that invaded to the bottom surface of the filter were fixed with 4% paraformaldehyde for 10\u00a0minutes, stained in 0.1% crystal violet for 30\u00a0minutes, then washed in PBS and photographed by microscope. The values for invasion were obtained by counting 5 random fields per membrane. These experiments were performed 3 times in triplicate.2.8Fifty PTC tissues and paired adjacent noncancerous thyroid tissue specimens were obtained from the Department of Head & Neck Surgery, Fudan University Shanghai Cancer Center. The paracancerous tissues were one cm from the edge of tumor, and there were no obvious cancer cells, as evaluated by an experienced pathologist. All tissue samples were snap\u2010frozen in liquid nitrogen immediately after thyroidectomy, and were transferred to the freezer at \u221280\u00b0C before use. All of the tissue specimens were obtained for this study with patient informed consent, and the use of the human specimens was approved by the Institutional Ethics Committee of Fudan University Shanghai Cancer Center and all procedures performed in our study were consistent with the ethical standards of our institutional research committee. Total RNA was extracted from PTC tissue and normal thyroid tissue with TRIzol reagent , and the quality and concentration of RNA were assessed with a SmartSpec Plus spectrophotometer . RNA purity was evaluated by the A260/A280 ratio. One microgram of total RNA was reverse transcribed using the All\u2010in\u2010One RNA RT\u2010quantitative real\u2010time PCR (qPCR) Detection Kit . qPCR was performed using a standard protocol from the SYBR Green PCR kit  on Applied Biosystems 7300 real\u2010time PCR system . \u03b2\u2010actin were used as references for mRNAs.2.9P values\u00a0<\u00a00.05 were considered significant.All data are representative of each assay repeated independently at least 3 times. Quantitative data are presented as the mean\u00a0\u00b1\u00a0SD. We analyzed the data using STATA . Two\u2010tailed Student's t test was used to analyze the data between 2 groups. Categorical variables are expressed as frequency and percentage values. The chi\u2010square test or Fisher's exact test was used to describe the differences. 33.1DESeq R was used to assess the expression level of each lncRNA. In total, 143 lncRNAs with aberrant expression  value was used for drawing the relative expression of ten selected genes in Figure P\u00a0=\u00a00.037, Table P\u00a0=\u00a00.012), RP11\u2010547D24.1 (P\u00a0=\u00a00.000), and UNC5B\u2010AS1 (P\u00a0=\u00a00.031) could be used to distinguish PTC patients in early stages from those in advanced stages. CTD\u20102008L17.2 (P\u00a0=\u00a00.007), RP11\u2010547D24.1 (P\u00a0=\u00a00.018), RUNDC3A\u2010AS1 (P\u00a0=\u00a00.029) and RP3\u2010483K16.4 (P\u00a0=\u00a00.012) were associated with lymph node metastasis. AC093609.1 (P\u00a0=\u00a00.040), LINC01354 (P\u00a0=\u00a00.013), UNC5B\u2010AS1 (P\u00a0=\u00a00.010), and LINC01977 (P\u00a0=\u00a00.028) had links to distant metastasis.The top 10 aberrantly expressed lncRNAs Table , LINC0193.3P\u00a0<\u00a00.05 was used as the cut\u2010off criterion. mRNAs were classified into 3 functional groups: molecular function, biological process, or cellular component. Significant results of the gene ontology (GO) enrichment and KEGG pathway analyses of mRNAs and lncRNAs in PTC are shown in Figure The genes coexpressed with the 10 lncRNAs were identified by weighted correlation network analysis (WGCNA), revealing 617 genes that are potentially coexpressed with these 10 lncRNAs in PTC. Among these genes, 37 had a relationship with AC0936091, and 255 had coexpression relationships with CTD\u20102008L172 and with the other key lncRNAs . Biological annotation of the mRNAs identified from an integrated analysis of microarray data, especially for the lncRNA RP11\u2010547D24.1 in PTC, was performed using the DAVID online analysis tool; RP11\u2010547D24.1\u2010coexpressed genes were most enriched in the Rap1 signaling pathway, focal adhesion pathway and Ras signaling pathway. The most enriched GO terms for mRNAs coexpressed with RP11\u2010547D24.1 were angiogenesis, positive regulation of angiogenesis and vasculogenesis. Additionally, the most enriched GO terms were related to all of the top10 lncRNAs. Positive regulation of transcription by the RNA polymerase II promoter was the most enriched GO term.3.4https://cancer.sanger.ac.uk/census and selected the genes with the key word \u201cthyroid\u201d. Furthermore, the genes associated with the cancer pathway were downloaded from the KEGG database (https://www.kegg.jp/kegg/). Then, the relationships of the 24 and 17 genes pulled from the CGC and KEGG databases, respectively, with the top 10 lncRNAs were calculated, and a heatmap was drawn using an illustrator . As shown in the figures, consistent results were found in PTC, revealing that the RP11\u2010547D24.1 level was markedly higher in PTC tissues than in PT tissues and positively associated with PAX8 (P\u00a0=\u00a00.000), PPARG (P\u00a0=\u00a00.001), FLT4 (P\u00a0=\u00a00.000), FZD4 (P\u00a0=\u00a00.000), NOTCH4 (P\u00a0=\u00a00.000) and FGFR2 (P\u00a0=\u00a00.03) , FGFR2 (P\u00a0=\u00a00.000), FZD4 (P\u00a0=\u00a00.000) and NOTCH4 (P\u00a0=\u00a00.000)  could affect the development of the kidney, eye, thyroid gland, central nervous system, and organs derived from the M\u00fcllerian duct.5This study identified new mechanisms underlying TPC tumorigenesis. We found that the highly expressed lncRNA RP11\u2010547D24.1 could promote the development of malignant thyroid nodules from benign nodules by altering the proliferation of thyroid cells, which is potentially attributed to its ability to alter thyroid cell cycle progression. Targeted drugs for RP11\u2010547D24.1 can provide an important theoretical basis for clinical reversal of the malignant PTC phenotype.The authors have no conflict of interest to declare."}
+{"text": "Among them, arugosin N (4) and 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenzoxepin-11(6H)-one  were obtained as the tautomeric mixtures. The structures of isolated compounds were determined by detailed spectroscopic analysis. In addition, the absolute configurations of these three pairs of new enantiomers were determined by quantum chemical ECD calculations.Three pairs of new isopentenyl dibenzo[ Up to now, about nine isopentenyl dibenzooxepinones had been reported from fungi in Nature, including arugosins A\u2013D from Aspergillus rugulosus oxepin-11(6H)-one  from Penicilium sp. oxepinone enantiomers ((+)-(5S)-arugosin K (1a), (\u2212)-(5R)-arugosin K (1b), (+)-(5S)-arugosin L (2a), (\u2212)-(5R)-arugosin L (2b), (+)-(5S)-arugosin M (3a), (\u2212)-(5R)-arugosin M (3b)), a new isopentenyl dibenzooxepinone, arugosin N (4), and two known compounds 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenzoxepin-11(6H)-one (5) and chrysophanol (6) (4) and 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenzoxepin-11(6H)-one (5) were a pair of tautomers, and they were isolated as mixtures. Detail of the isolation and structural elucidations of compounds 1\u20136 are presented herein.ompounds ,9. In ouompounds ,13,14,15ompounds ,13,14,15anol (6) . The abs1 was obtained as a yellow solid. The quasi-molecular ion at m/z 355.1550 [M + H]+ obtained by HRESIMS indicated the molecular formula of 1 was C21H22O5 with 11 degrees of unsaturation. The 13C-NMR spectrum combined with the DEPT-135 spectrum showed 21 signals (2 quaternary carbons + by HRESIMS indicated the molecular formula of 2 was C22H24O5 with 11 degrees of unsaturation. Except for the loss of an oxygenated methyl at \u03b4C 56.9 (5-OCH3) and the appearance of an additional oxygenated methylene at \u03b4C 65.3 (C-1\u2032\u2032) and methyl at \u03b4C 14.8 (C-2\u2032\u2032), the NMR data of 2   , the plaablished .1 and 2 coexist in the same strain, 2 was also a racemic mixture, and it displayed two peaks in a Phenomenex Lux Cellulose-2 chiral column (2a (tR = 22.6 min) and (\u2212)-2b (tR = 26.6 min) were isolated from 2 by a Phenomenex Lux Cellulose-2 chiral column using CH3OH/H2O  as eluent at a flow rate of 0.7 mL/min. The ECD spectra and 2a ) and 2b ) showed their enantiomeric relationship. Since the ECD data of 2a were similar to that of 1a  as shown in 1H\u20131H COSY experiment and molecular formula, the HMBC correlations  and (\u2212)-3b (tR = 22.1 min) were isolated from 3 by a Phenomenex Lux Cellulose-2 chiral column using CH3OH/H2O  as eluent at a flow rate of 0.7 mL/min. The ECD spectra and 3a ) and 3b ) showed their enantiomeric relationship. The absolute configurations of 3a and 3b were determined by quantum chemical ECD calculations at the APFD/6-311++g level. The predicted ECD curves of (5S)-3 and (5R)-3 were in good accordance with the experimental ECD for 3a and 3b, respectively , the 1H NMR and 13C-NMR data of 4   , the pla5 was fourteen atomic mass units less than 1. Except for the loss of an oxygenated methyl at \u03b4C 56.9 (5-OCH3), the 1H-NMR and 13C-NMR data of 5  -dibenzoxepin-11(6H)-one +; ESI-MS (negative): m/z 253 [M \u2212 H]\u2212 indicated the molecular weight was 254. It was identified as chrysophanol by comparing its data  with literature values oxepin-11(6H)-one (5). Then, arugosin K (1) and arugosin L (2) may originate from 5 by methylation and ethylation, respectively. Arugosin M (3) may originate from 4 by methylation.Based on the structural features of compounds proposed . Chrysopdization ,18, ringdization ,18, redudization ,18, and dization , which mMethanol (MeOH) was purchased from Yuwang Industrial Co. Ltd. . Cyclohexane, ethyl acetate (EtOAc), and petroleum ether (PE) were purchased from Tianjin Fuyu Fine Chemical Co. Ltd. .3: \u03b4H 7.26/\u03b4C 77.0) as internal standard. The analytical HPLC was performed on a Dionex HPLC system equipped with an Ultimate 3000 pump, an Ultimate 3000 diode array detector (DAD), an Ultimate 3000 Column Compartment, an Ultimate 3000 autosampler  using a Gemini C18 column  . The semi-preparative HPLC was performed on a Shimadzu LC-6-AD Liquid Chromatography with an SPD-20A Detector using a Phenomenex Gemini C18 column (Phenomenex Inc.) . The chiral HPLC was performed on a Shimadzu LC-6-AD Liquid Chromatography with an SPD-20A Detector using a Phenomenex Lux Cellulose-2 chiral column .Optical rotations were measured on a P1020 digital polarimeter . UV data were recorded using a Jasco V-550 UV/Vis spectrometer. IR data were recorded on a Jasco FT/IR-480 plus spectrometer. ECD spectrum was recorded on a Jasco J-810 spectrophotometer using MeOH as the solvent. The ESI-MS spectra were recorded on a Finnigan LCQ Advantage MAX mass spectrometer . The HR-ESI-MS spectra were obtained on a Micromass Q-TOF mass spectrometer . The NMR spectra were measured with Bruker AV-400 and Bruker AV-600 spectrometers  using the solvent signals  and the 5.8S rRNA gene sequences (ITS1-5.8S-ITS2) of the strain have been deposited at GenBank as KX011167. The fungus was cultured on slants of potato dextrose agar at 25 \u00b0C for 5 days. Agar plugs were used to inoculate four Erlenmeyer flasks (250 mL), each containing 100 mL of potato dextrose broth. Four flasks of the inoculated media were incubated at 25 \u00b0C on a rotary shaker at 200 rpm for 5 days to prepare the seed culture. Fermentation was carried out in 20 Erlenmeyer flasks (500 mL), each containing 70 g of rice. Distilled H2O (105 mL) was added to each flask, and the rice was soaked overnight before autoclaving at 120 \u00b0C for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the seed culture containing mycelia and incubated at room temperature for 50 days.The strain AHK07-3 was isolated from the soil, collected at the wetland of Ahongkou, Sinkiang Province, China. The strain was identified as v/v aqueous MeOH (400 mL). The methanolic solution was further extracted with cyclohexane (400 mL) to afford a cyclohexane fraction C (4.6 g). Fraction C was subjected to silica gel (200\u2013300 mesh) column chromatography initially eluting with cyclohexane (500 mL) and then with MeOH (500 mL) to afford subfractions CC and CM. Subfraction CC (3.3 g) was further separated by open silica gel column chromatography eluting with cyclohexane\u2013EtOAc (100:0 (150 mL), 99:1 (150 mL), 98:2 (150 mL), 97:3 (150 mL), 96:4 (150 mL), 95:5 (150 mL), 94:6 (150 mL), 93:7 (150 mL), 92:8 (150 mL), 91:9 (150 mL), 90:10 (150 mL), 50:50 (150 mL), and 0:100 (150 mL), v/v) to yield four sub-subfractions (CC1 to CC4). Sub-subfraction CC2 (2.8 g) was subjected to open silica gel column chromatography eluting with PE (petroleum ether)\u2013EtOAc (100:0 (100 mL), 99:1 (100 mL), 98:2 (100 mL), 97:3 (100 mL), 96:4 (100 mL), 95:5 (100 mL), 94:6 (100 mL), 93:7 (100 mL), 92:8 (100 mL), 91:9 (100 mL), 90:10 (100 mL), 50:50 (100 mL) and 0:100 (100 mL), v/v) to yield four sub-subfractions (CC2a to CC2d). Sub-subfraction CC2b (1.1 g) was isolated by semi-preparative HPLC purification using MeOH\u2013H2O  at a flow rate of 3 mL/min to yield 1 (617.4 mg), 2 (2.6 mg), and 3 (24.7 mg). Sub-subfraction CC2c (0.6 g) was purified by semi-preparative HPLC purification using MeOH\u2013H2O  at a flow rate of 3 mL/min to yield 6 (1.1 mg). Sub-subfraction CC4 (220.1 mg) was isolated by semi-preparative HPLC purification using MeOH\u2013H2O  at a flow rate of 3 mL/min to yield the mixtures of 4 and 5 (12.5 mg). Enantioseparation of 1\u20133 was carried out on a Phenomenex Lux Cellulose-2 chiral column using CH3OH/H2O  as mobile phase.The culture was extracted with EtOAc (3 \u00d7 5000 mL). Removal of EtOAc under reduced pressure yielded a crude extract (19.7 g) that was dissolved in 90% (\u00b1)-Arugosin K (1): yellow solid; UV (MeOH) \u03bbmax (log \u03b5) 205 (3.75), 221 (3.63), 304 (3.35), 359 (3.29) nm; IR (KBr) \u03bdmax 3438, 2920, 1708, 1619, 1590, 1421, 1206, 1052 cm\u22121; ESI-MS (positive): m/z 355 [M + H]+, ESI-MS (negative): m/z 353 [M \u2212 H]\u2212; HRESIMS (positive) m/z 355.1550 [M + H]+ ; 1H- and 13C-NMR data see (+)-S-Arugosin K (1a): c 0.5, CHCl3); ECD  \u03bbmax (\u0394\u03b5) 208 (+12.50), 294 (\u22125.71), 334 (+3.76), 382 (\u22121.77) nm.(\u2212)-R-Arugosin K (1b): c 0.5, CHCl3); ECD  \u03bbmax (\u0394\u03b5) 208 (\u221211.38), 295 (+6.96), 334 (\u22123.88), 382 (+2.82) nm.(\u00b1)-Arugosin L (2): yellow solid; UV (MeOH) \u03bbmax (log \u03b5) 206 (3.70), 222 (3.57), 304 (3.30), 359 (3.23) nm; IR (KBr) \u03bdmax 3440, 2916, 1624, 1590, 1422, 1206, 1057 cm\u22121; ESI-MS (positive): m/z 369 [M + H]+; ESI-MS (negative): m/z 367 [M \u2212 H]\u2212; HRESIMS (positive) m/z 369.1695 [M + H] + ; 1H- and 13C-NMR data see (+)-S-Arugosin L (2a): c 0.5, CHCl3); ECD  \u03bbmax (\u0394\u03b5) 208 (+13.90), 295 (\u22126.07), 335 (+5.07), 382 (\u22121.91) nm.(\u2212)-R-Arugosin L (2b): c 0.5, CHCl3); ECD  \u03bbmax (\u0394\u03b5) 208 (\u221213.79), 295 (+7.98), 335 (\u22123.44), 382 (+3.40) nm.(\u00b1)-Arugosin M (3): yellow solid; UV (MeOH) \u03bbmax (log \u03b5) 206 (3.85), 221 (3.73), 299 (3.42), 357 (3.36) nm; IR (KBr) \u03bdmax 3441, 2913, 1620, 1464, 1202, 1050 cm\u22121; ESI-MS (positive): m/z 355 [M + H]+, ESI-MS (negative): m/z 353 [M \u2212 H]\u2212; HRESIMS (positive) m/z 355.1541 [M + H]+ ; 1H- and 13C-NMR data see (+)-S-Arugosin M (3a): c 0.5, CHCl3); ECD  \u03bbmax (\u0394\u03b5) 209 (+14.24), 290 (\u22126.29), 334 (+6.67), 381 (\u22123.30) nm.(\u2212)-R-Arugosin M (3b): c 0.5, CHCl3); ECD  \u03bbmax (\u0394\u03b5) 209 (\u221212.84), 292 (+7.26), 334 (\u22125.93), 381 (+4.27) nm.Arugosin N (4): 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenzoxepin-11(6H)-one (5): yellow solid; UV (MeOH) \u03bbmax (log \u03b5) 206 (3.75), 222 (3.65), 286 (3.29), 356 (3.16) nm; IR (KBr) \u03bdmax 3443, 2917, 1628, 1589, 1420, 1353, 1173, 1052 cm\u22121; ESI-MS (positive): m/z 341 [M + H]+, m/z 363 [M + Na]+; ESI-MS (negative): m/z 339 [M \u2212 H]\u2212; HRESIMS (positive) m/z 363.1213 [M + Na]+ ; 1H- and 13C-NMR data see S)-1, (5R)-1, (5S)-3, and (5R)-3 were converted into SMILES codes, respectively. Conformer databases were generated in CONFLEX version 7.0  using the MMFF94s force-field, with an energy window for acceptable conformers (ewindow) of 5 kcal\u00b7mol\u22121 above the ground state, a maximum number of conformations per molecule (maxconfs) of 100, and an RMSD cutoff (rmsd) of 0.5 \u00c5. Then each conformer of the acceptable conformers was optimized with HF/6-31G(d) method in Gaussian09 oxepinones with intact 6-7-6 tricyclic systems have been reported from the genera of Aspergillus, Emericella, Pestalotiopsis, Graphiopsis, and Penicilium. Except the absolute configuration of cephalanone D that had been verified by X-ray crystallography oxepinones established by ECD experiments. Furthermore, 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenzoxepin-11(6H)-one (5) is a known compound, but no NMR data was previously reported. The assignments of NMR data of 5 are thus provided for the first time.Up to now, about nine isopentenyl dibenzo["}
+{"text": "Malalignment of the femoral stem in total hip arthroplasty (THA) can detrimentally affect outcome. Poor preparation of the femur intraoperatively is an important cause of stem malalignment.The objective was to compare coronal alignment of three different stems using three different broaches.Retrospective study of three groups of 60 patients following primary THA via direct anterior approach, by the same surgeon, between January 2015 and January 2016. Each group had a similar designed stem . Groups were matched for age, body mass index, gender, side, neck shaft angle and indications. The significant difference between groups was the broach shape. Broaches for the Corail and Meije stems had a prominent shoulder laterally, while the broach of the Targos stem had a rounded less prominent shape laterally. Coronal alignment was determined radiologically at 2 months.The mean varus was significantly lower for the Targos stems (1.1\u00b0 +/-0.8) compared to the Corail (2.3\u00b0 +/-1.5) and Meije stems (1.9\u00b0 +/-1.2) (p<0.0001). There were significantly less Targos stems with varus greater than 3\u00b0  compared to the Corail  and Meije stems  (p<0.001).A femoral broach with a prominent lateral shoulder when performing a THA via direct anterior approach will increase the risk of varus femoral stem alignment compared to a less laterally prominent broach. Femoral alignment is considered important for both survivorship and outcome in total hip arthroplasty (THA). Multiple studies have demonstrated varus stems are associated with poor clinical outcomes and increased complications e.g. aseptic loosening, secondary subsidence , 2 and tSeveral factors can influence stem positioning, in particular the anatomical femoral bone shape , minimalThe surgeons involved in this study use DAA for all primary THA. The exception is when significant femoral shortening or correction is required as part of the femoral preparation . The posterior approach is used in this situation.The objective of this study was to compare the preoperative plan to the definitive coronal alignment of three different stems implanted relative to different broach geometry.This is a retrospective study comparing three groups of patients following primary THA via DAA. The inclusion criteria for the three groups were first 60 patients operated for primary THA via DAA by the same surgeon between January 2015 and January 2016 using a Corail stem (Depuy), Targos stem (Lepine) or a Meije stem (Tornier). The surgeon was experienced in THA via DAA and is his routine approach for primary THA.Exclusion criteria were previous surgery or trauma on the operated hip, femoral congenital deformities and femoral neck fractures.The demographic characteristics are summarized in the Intraoperative complications were recorded. At two-month routine follow up standard AP and lateral X-rays was taken and reviewed.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.The Advisory Committee on Research Information Processing in the Field of Health (CCTIRS) approved this study in Paris under number 2018-AO1653-5. For this type of study formal consent is not required (retrospective and anonymous study). All data were fully anonymized before their access. The ethics committee did not require an informed consent.The three cementless stems  are of similar design. Each had a nonporous fully hydroxyapatite coating on a forged titanium alloy stem . The desA double offset broach handle was used in all cases  to avoidThe Corail and Meije broaches had a prominent shoulder laterally while the Targos stem broach laterally had a rounded shape . All broThe aim was to prepare the femur appropriately to place all stems according to preoperative planning aligned with the coronal femoral axis.All measurements were recorded by the same observer on an anteroposterior pelvic view radiograph at 2 months. Inter-observer reliability was assessed for 10 patients in each group. Radiological complications were recorded. Coronal alignment of the stem was determined by measuring the angle formed between the long axis of the prosthesis and the long axis of the femur. Positioning was recorded as positive for varus and negative for valgus. Coronal alignment greater  than 3\u00b0 was considered a varus placed stem.The width of stems in the proximal and distal part of the diaphyseal segment of each stem  were measured and compared. The Targos stem was designed with a larger distal portion as a method to regulate stem insertion distally and prevent malalignment.The continuous variables were averaged and reported with standard deviation and extremes. They were compared using Student t-test or Wilcoxon nonparametric test. The categorical variables were compared using a Fisher exact test. A p value <0.05 was considered statistically significant in each analysis. The intra-observer and inter-observer reliability of these measurements were evaluated by an intraclass correlation coefficient. The statistical analyses were performed with XLstat (2015 Addinsoft).There were significantly less Targos stems placed in a varus position  compared to both the Meije  (p<0.001) and Corail stems  (p<0.001). The number of Meije stems in a varus position was significantly less than the Corail stems (p<0.05). All results are detailed in At 2 months no patient had a major complication requiring hospital admission or surgical revision. No patient had radiological subsidence of the stem. The mean varus of stems was 2.3\u00b0 +/-1.5 Corail, 1.9\u00b0 +/-1.2, Meije and 1.1\u00b0 +/-0.8 Targos groups . The meaNo patient had a valgus position greater than -3\u00b0. Only five patients had a valgus coronal alignment between -0.4\u00b0 and -1.4\u00b0 without significant difference between the three groups .The mean ratio between the proximal part and the distal part of the diaphyseal segment of stems was different between the three stems, though not significantly .Radiological measurement of the stem alignment was reliable, with both a strong intraclass correlation (ICC = 0.86) and interclass correlation (ICC = 0.81).The most important finding of this study is that there was a significantly higher rate of stems of a similar design placed in varus greater  than 3\u00b0 when the femoral broach had a prominent lateral shoulder via DAA. This is the first study to compare varus positioning of different femoral stems relative to broach shape for DAA. This is important factor to be aware of when preparing the femur using a broach with a prominent lateral shoulder and may help in preventing varus alignment relative to preoperative planned alignment from occurring.Murphy et al. studied the femoral alignment of 200 uncemented THA with the Corail hip system. They reported that varus positioning could sometimes be related to femoral morphology, particularly coxa vara deformity, and was not necessarily a technical failure . HoweverThe group with a less prominent lateral shoulder of the broach (Targos) resulted in only one stem in varus greater than 3\u00b0 and a low mean varus relative to the preoperative plan and femoral axis. This less prominent broach allows work on the femoral shaft smoothly in a medial to lateral movement. This is because during early femoral preparation there is decreased initial contact with either/or the proximal lateral femoral shaft, lateral edge of greater trochanter and superolateral femoral neck cortical bone. Any or all of these three bone contact points can displace preparation and stem placement into varus. This is particularly important to be aware of in difficult cases when the initial broach may be placed in varus and not corrected with each successive increase in size until the definitive size is reached. The two other groups had a significantly greater number of stems in varus greater than 3\u00b0, with Corail greatest at 40%.Further comparison between the broaches of the Targos (one stem (1.7%) in varus) to the Corail (24 stems (40%) in varus) was an increased aggressiveness or sharpness of the Targos. This would result in removal of bone with the Targos broach rather than impaction which occurred with the Corail during femoral preparation. A broach that impacts bone is forced away from harder cortical bone towards softer metaphyseal bone. If with initial broaching the hardest contact is prominent lateral cortical bone of either the greater trochanter or superior femoral neck, as described above, the broach could be forced into varus. If the first broach is in varus and not recognized then the malalignment will be propagated as broach size is increased sequentially.The Targos stem compared to the literature had a very low varus rate (1.7%) where varus rates have been reported up to 25% , 18, 19.Several complications have been described with a stem in varus, particularly thigh pain, femoral loosening, stem undersizing with postoperative subsidence , 11, 20.This study had several limitations. It was retrospective and patients were not randomized in each group. Each group were matched with the choice of the implant by availability independent of femoral anatomy. Three similar but not identical designs of implants with their own specific instruments were assessed rather than only one design. The offset of the broach handles is slightly different between the three implants, but this difference is too small to explain the varus rate. The stem design is also a little different between the three . However, there was no significant valgus positioning for the three stems, and the difference of the ratio of the proximal and distal diaphyseal part of each was not significant. Thus, we believe that the larger diaphyseal stem of the Targos is not the sole explanation for the different varus rate. It is a strength that this single surgeon study removes surgeon experience. The DAA could be a risk factor of varus positioning with inappropriate technique. The surgeon was experienced for this approach and the double offset broach handle is standard use in our department . Our resFemoral alignment is an important parameter during THA implantation. Multiple factors can adversely affect the alignment, in particular the design of the instruments to prepare the femur. Comparing three stems and their instrumentation, this study showed that there were less stems in varus when a more aggressive broach that had a less prominent lateral shoulder was used via DAA. Femoral stem positioning is influenced by the design of the instruments used to prepare the femur. To be aware of this risk and anticipate will decrease the chance of a femoral stem in varus alignment using the DAA.S1 TableAll anonymized data of this study are reported on a table in supporting information.(XLSX)Click here for additional data file."}
+{"text": "Bladder cancer (BC) patients with advanced disease have poor outcomes. The use of patient\u2010reported outcomes (PROs) could lead to improvements in symptom management and hence quality of life (QoL). The aim of this study is to report correlations between selected PROs and QoL and thus to present symptoms that influence QoL. Identification of these symptoms during treatment can lead to earlier symptom management and thus secure improvements in QoL.BC patients in chemo\u2010 or immunotherapy for locally advanced or metastatic disease reported weekly PROs for the duration of their treatment. The PROs included EORTC QLQ\u2010C30 and QLQ\u2010BLM30 and 45 selected PRO\u2010CTCAE items. Spearman's correlation analysis was performed for all PRO\u2010CTCAE items and QLQ\u2010C30 global QoL and subdomains.rs\u00a0=\u00a0\u22120.603, P\u00a0<\u00a0.0001), concentration  and cognitive function , discouraged (F) and emotional function , fatigue (S) and role function  and sad (F) and emotional function . The weakest correlations were found for the PRO\u2010CTCAE items urinary frequency, incontinence and urge, all with variations in the direction and significance of the correlations.In this study, 78 BC patients reported 724 questionnaires. Spearman's analysis showed significant correlations between almost all PRO\u2010CTCAE items and the expected domain of QoL. The PRO\u2010CTCAE items with the strongest correlations with QoL were anxiety  and emotional function (This study delivers information on which PROs may influence QoL for patients in clinical trials or daily clinic. Psychological issues have a strong impact on QoL and should be dealt with during treatment to secure the best possible QoL for BC patients. For bladder cancer patients, psychological items have a strong impact on the quality of life while urinary items have a weak impact on the quality of life. Toxicity reporting from clinical trials and supportive care in daily practice may be incomplete and not reflective of the full picture of symptomatology for bladder cancer patients. In perspective, we are interested in directing the supportive care in the right direction for this patient group thereby securing focus on timely symptom management and the impact of such on QoL and clinical outcomes. At the same time, we hope that this information on pivotal symptomatic items for the bladder cancer population could be helpful when designing clinical trials regarding which items are relevant to this population to reflect the total toxicity burden during treatment and its effect on QoL.22.1Bladder cancer (stages T2\u2010)Initiating treatment with chemotherapy  or immunotherapy (pembrolizumab) as the standard neoadjuvant or metastatic treatment.Able to read Danish.No serious cognitive impairment as evaluated by the treating team of physician and/or nurse.Data for this study were obtained from two prospective clinical studies (study 1 and 2) in patients with locally advanced or metastatic bladder cancer receiving chemo\u2010 or immunotherapy at two university hospitals (H1 and H2), Copenhagen, Denmark. The purposes of these two studies were to collect PROs longitudinally, report clinical outcomes (study 1&2) and evaluate electronic PRO completion (study 2), data on these issues will be reported elsewhere. Study 1 was conducted at H1 with both paper and electronic completion of questionnaires whereas study 2 was conducted at H1 and H2 with only electronic capture of PROs. Criteria for inclusion in both studies were as follows:2.2In both studies, the patients completed the same questionnaires weekly by paper or electronically through the Ambuflex software system in a web\u2010browser.For patients completing questionnaires by Web they were prompted to do so weekly by receiving a notification in e\u2010Boks, a national communication system between authorities, companies, and citizens ensuring delivery of official communication regardless of geographical home address ensuring secure data delivery.The specific scores from the EORTC and HADS questionnaires are hence the aim of this study reported elsewhere.2.3The PRO\u2010CTCAE Item library consists of 78 symptom items explored by 128 questions as many symptoms are explored by attributes on frequency (F), severity (S), interference with daily activities (I) and/or presence (P).2.4s, in either direction from 0) of 0\u20100.19 were regarded as very weak, 0.2\u20100.39 as weak, 0.40\u20100.59 as moderate, 0.6\u20100.79 as strong and 0.8\u20101 as very strong correlation. A PRO\u2010CTCAE symptom was included in the above rs categories if one or more correlation coefficient between a PRO\u2010CTCAE item and the expected domain was found within the given intervals. Descriptive analysis was used to describe the symptom burden from PRO\u2010CTCAE scores. Linear regression analysis was performed to estimate the relation between summarized PRO\u2010CTCAE scores and QoL and its subdomains.To test the association between two instruments as a way of testing for construct validity when developing new measurement tools correlation analyses were performed. These analyses verify that the tool in question can distinguish between groups of patients with differences in the symptom under study. The analyses can also identify shifts in symptom severity within the same patient when using a well\u2010known tool as the anchor, in this case, the EORTC QLQ\u2010C30. Scatter plots were used to check for monotonicity, as a determination of strength and direction of the correlation in question, between the given PRO\u2010CTCAE item and calculated global QoL score, including all subdomains: physical function, role function, emotional function, cognitive function, and social function. The spearman's test was then performed to test for correlations between the PRO\u2010CTCAE item and QoL and its subdomains. Correlation analyses were performed for the total population and for the locally advanced and metastatic population separately. Absolute values of rho  did not fulfill inclusion criteria due to either poor performance status inhibiting treatment initiation , enrollment into a clinical trial , initiating radiotherapy instead of chemo\u2010/immunotherapy , synchronic metastatic cancer of another site  or lack of access to electronic communication with authorities through e\u2010Boks\u2122 . A further three patients (2%) were missed at treatment initiation and three (2%) declined treatment altogether. Participation was declined by 10/122 patients (8%) leaving 79 patients who completed written informed consent, one of whom did not initiate questionnaire completion due to a cerebral stroke on the day of completing informed consent. The clinical data of the participating patients are listed in Table\u00a0A total of 711 questionnaires were completed by the participating 78 patients. The median number of completed questionnaires was 9 (range 0\u201020). The overall completion rate was 76%. Twenty patients completed questionnaires on paper and 58 by web\u2010browser in Ambuflex. Analysis of missing data revealed an overall completion of 93% and missing data in 7% of the 724 questionnaires, all with 158 items equaling 8007 missing item responses out of a total of 114.392 item responses. After imputing zeros in the PRO\u2010CTCAE items following a zero in the frequency item, the missing data analysis showed 6% missing data. The transformation of values 5 and 6 to \u2018missing\u2019 for the item \u2018decreased libido\u2019 was not included as missing data in this analysis as they were completed by the patients.rs\u00a0=\u00a0\u22120.603, P\u00a0<\u00a0.0001), concentration (S), and cognitive function , discouraged (F) and emotional function , fatigue (S) and role function , memory (S) and cognitive function , and sad (F) and emotional function , all with the expected direction of correlation with QoL, see Table\u00a0rs\u00a0>\u00a00.8). The PRO\u2010CTCAE items concerning abdominal pain, dizziness, memory, muscle pain, nausea, pain, and shortness of breath all reached moderate correlations of Spearman's rho 0.4\u20100.59. The items with the lowest rank\u2010order correlations with the expected QoL domain were the PRO\u2010CTCAE items concerning pain and swelling at the injection site (P) , urinary frequency (F) , urinary incontinence (F) , and urinary urgency (F) , all with very weak correlations, nonsignificant and with converging directions of the correlations. For the corresponding interference with daily activities items (I) of the urinary items, the correlations were very weak or weak but significant within the expected domain of QoL. No significant differences were found between the patients with locally advanced disease vs. metastatic disease although patients with locally advanced disease generally experienced higher correlations between a larger amount of symptoms (more symptoms with weak rs) and QoL than the metastatic population (more symptoms with very weak rs).When analyzing the relationship between all single PRO\u2010CTCAE items and QLQ\u2010C30 quality of life including subdomains (see Table P\u00a0<\u00a0.0001 for all analyses, see Table\u00a0Figure\u00a04This study identifies imperative symptoms for bladder cancer patients during chemo\u2010 or immunotherapy most likely to influence QoL. Interestingly the symptoms with the highest correlations are predominantly of a psychological nature whereas urinary symptoms known to be frequent for this population and over time anticipated to influence QoL the most, do not show positive or significant correlations with QoL. We find that our data may inform caregivers to focus on supportive care in these areas during treatment in order to best assist bladder cancer patients to achieve a better QoL. Likewise, this study may inform future investigators of imperative symptoms to report from a clinical trial for a comprehensive collection of the full symptom burden of enrolled patients for more transparent toxicity reporting in clinical trials.Our findings of the correlations between the PRO\u2010CTCAE and the EORTC QLQ\u2010C30 are similar to that of Dueck et al in the validation process of the PRO\u2010CTCAE instrument in 2015.The symptoms with moderate or strong correlations are all general symptoms likely to reflect symptomology of a broad group of cancer patients and not specific to bladder cancer patients or to the stage of disease. With the added attention toward active management of PROs during treatment, this finding speaks for a general, nondisease specific, model of PROs during treatment if the aim is to reflect QoL. This approach has also been shown to be beneficial for patients in previous trials testing the impact of PROs.We found significant associations between summarized PRO\u2010CTCAE scores and QoL, including subdomains, in our linear regression model. When performing the same analysis without the inclusion of the psychological items found to have strong correlations from Table\u00a0The strengths of this study include the prospective collection of data in a broad cohort of bladder cancer patients receiving active oncological treatment. This study is, to the best of our knowledge, the first to perform these analyses in a population of patients receiving chemo\u2010 or immunotherapy for advanced disease. We believe that the data are representative and that the results are applicable for a wide spectrum of bladder cancer patients. Also, with the disease\u2010specific PRO\u2010CTCAE item selection performed prior to our analysis and described in detail in a separate publication we believe that we in this study capture PROs relevant for the bladder cancer patients without adding substantial burden of questionnaire completion for the patients while reporting.Some limitations do however need to be addressed. First, although correlation analysis from 724 questionnaires have been presented, only 78 patients report in this study thereby including a single patient's reports over time multiple times in the analysis. Patients responding to treatment and thus reporting for a longer period will therefore weigh more in this analysis potentially skewing the data and underestimating symptom burden in Figure\u00a0With the increasing focus on PROs as part of clinical trials investigators should be aware of the pitfalls of item selection when planning trials and especially of the items imperative for the population at question. Bladder cancer patients with advanced disease have a poor prognosis, partly due to a burden of comorbidity troubling treatment adherence.5The Danish National Data Protection Agency approved the conduction of studies included in this manuscript. The studies described in the manuscript were carried out prospectively during treatment for all patients completing written informed consent. In Denmark, the Danish Health Authority waivers the need for ethical approval for scientific studies assembling questionnaire data only. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institution and the national research committee, the Danish Health Authority, and also with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Supplementary MaterialClick here for additional data file."}
+{"text": "Our study aimed to assess the safety and protective effect of maternal influenza vaccination on pregnancy and birth outcomes.The study population comprised 1253 healthy nulliparous pregnant women in South Australia between 2015 and 2018. Participants were followed prospectively, with vaccination status , pregnancy, and birth outcome data collected by midwives. Adjusted relative risks (aRRs) and adjusted hazard ratios (aHRs) were estimated accounting for time-varying vaccine exposure and temporal nature of each outcome.Maternal influenza vaccination  reduced the risk for pre-delivery hospitalisation with influenza like illness . Maternal influenza vaccination was not associated with spontaneous abortion , chorioamnionitis , gestational hypertension , pre-eclampsia , gestational diabetes  nor preterm birth . No associations between antenatal influenza vaccination and congenital anomalies, admission to the neonatal care unit, low Apgar scores, and mechanical ventilation were observed. Results were not materially changed after adjustment for pertussis vaccination. We observed a protective effect of maternal influenza vaccination on low birth weight  and a marginal protective effect on small for gestational age births  during periods of high influenza activity.These results support the safety of maternal influenza vaccination and suggest a protective effect in reducing the rates of low birthweight and small for gestational age births.There was no funding for this study. We searched PubMed for English language studies published until March 31, 2020, with no start date restriction, with the terms \u201cinfluenza\u201d, \u201cinfluenza vaccine\u201d, \u201cmaternal influenza vaccination\u201d, maternal influenza immunisation\u201d, \u201chumans\u201d and \u201cpregnancy\u201d. The World Health Organization (WHO) considers pregnant women as a priority group for seasonal inactivated influenza vaccination due to their vulnerability to influenza infection and its resulting morbidities. Previous studies have shown that inactivated influenza vaccine during pregnancy is safe and provides passive antibodies to the infant, as well as clinical protection for both mother and infant < 6 months of age against influenza infections and influenza-related hospitalisations. Despite the recommendation of maternal influenza vaccination from immunisation advisory groups internationally including WHO, it has not been implemented in most low-resource countries, and even in high income countries where it is incorporated into standard antenatal care, vaccination uptake is often suboptimal. While the body of literature regarding the safety of influenza vaccination during pregnancy is mounting, there are relatively few prospectively designed or clinical trials that include pregnant women. In high income countries where maternal influenza vaccination is recommended, prospectively designed studies with advanced statistical approaches are likely to be the only way to comprehensively assess the safety of influenza immunisation during pregnancy and its important potential protective effects in reducing low birth weight, small for gestational age birth and preterm birth, for which evidence is conflicting.This prospective cohort of healthy pregnant women, with confirmed vaccination status and accurate pregnancy and infant outcome data used robust nuanced time-to-event analyses. The study showed that influenza vaccination during pregnancy is not associated with adverse pregnancy, foetal or birth outcomes. This study also presents evidence that inactivated influenza vaccination decreases the risk of pre-delivery hospitalisation with maternal influenza-like illness by 39% and reduces the risk of low birthweight and small for gestational age births during periods of high influenza activity.reducing low birth weight and small for gestational age births during periods of widespread influenza activity. These findings need to be replicated in other countries as it is plausible that the impact of maternal influenza vaccine on these birth outcomes may vary with the underlying local influenza epidemiology and demographic characteristics. Our findings could be pivotal for countries weighing the additional benefits of implementing maternal influenza immunisation programs. This may be particularly important for low income countries where the rates of low birthweight and small for gestational age births are very high, and known to be strong risk factors for neonatal and early childhood morbidity and in which health systems have poor capacity to mitigate short and long-term effects.These findings provide further reassurance to women and health care providers about the safety of inactivated influenza vaccination during pregnancy. Importantly, our results provide evidence in support of maternal influenza vaccination Alt-text: Unlabelled box1Pregnant women are vulnerable to serious complications from influenza including preterm labour, pneumonia, hospitalisation and death, particularly during seasonal and pandemic influenza outbreaks ,2. NewboA major challenge for achieving high uptake of influenza vaccination during pregnancy relates to relatively limited published evidence of vaccine safety for pregnant women and their foetus Most observational research into vaccine safety and efficacy during pregnancy has been retrospective, due to the relatively cheaper cost, fewer ethical concerns, and difficulty in recruiting pregnant women to randomized controlled trials (RCTs). Whilst providing timely reporting, this approach has limitations. In most retrospective studies, authors have been unable to establish if a pregnancy complication preceded vaccination nor account for the time-dependant nature of exposure to vaccination during pregnancy. In countries where maternal influenza vaccination is recommended, prospectively designed studies are likely to be the only way to accurately determine the true risk or potential benefits of maternal vaccination beyond prevention of influenza for pregnant women and their infants. Our study aimed to prospectively assess maternal and birth outcomes following inactivated influenza vaccination during pregnancy, while also taking into account the most comprehensive set of potential confounding variables considered to date.22.1+\u00a00 and 16+0 weeks\u2019 gestation were enroled. Women were excluded if they were considered already at high risk of pregnancy complications at screening (i.e. experienced three or more previous miscarriages or with pre-existing hypertension or diabetes). Participants were followed prospectively, with vaccination, pregnancy, and birth outcome data collected by research midwives. Written informed consent was obtained from all participants included in the STOP study. The original STOP study protocol was approved by the Human Research Ethics Committee of the Women's and Children's Hospital Adelaide Australia (HREC/14/WCHN/90), registered at Australian New Zealand Clinical Trials Registry, ACTRN12614000985684.The current study draws on data collected as part of a prospective cohort study (STOP), which aims to develop screening tests to identify adverse pregnancy outcomes. Healthy nulliparous women were recruited in pregnancy at two major maternity hospitals, the Lyell McEwin Hospital, the tertiary hospital serving the low socio-economic community in Adelaide's Northern suburbs and the Women's and Children's Hospital, the primary tertiary maternity hospital for complex care, accounting for around 50% of the 16,000 annual births in metropolitan Adelaide, South Australia. Between March 2015 and December 2017, nulliparous women with a singleton pregnancy attending their first antenatal clinic between 9\u00a02.2The exposure of interest was trivalent inactivated influenza vaccination during pregnancy, defined as a vaccine received between the first day (date) of the last menstrual period and the end of pregnancy. A research midwife interviewed and collected maternal vaccination status of the women during their first study visit at 9\u201316 weeks\u2019 gestation and during their second study visit interview at 32\u201336 weeks\u2019 gestation. Vaccination date and gestation of administration were recorded. Following delivery, a research midwife interviewed the participants and verified final vaccination status by reviewing medical case notes and Pregnancy-Hand-Held-Record to confirm the reported vaccination status. Pregnancy-Hand-Held-Records are the main medical record of pregnancy care in South Australia and are reviewed and updated at antenatal appointments.2.3Pregnancy outcomes assessed were pre-delivery admission due to influenza-like illness, spontaneous abortion after inclusion in the STOP study, gestational diabetes, gestational hypertension, pre-eclampsia, severe pre-eclampsia, chorioamnionitis, premature rupture of membranes, spontaneous preterm birth, preterm birth and stillbirth. Birth outcomes included congenital anomalies, small for gestational age (SGA), low birthweight (< 2500\u00a0g) (LBW), low birthweight at term (\u2265 37 weeks\u2019 gestation), Apgar scores at 1 and 5\u00a0min, neonatal care unit admissions, respiratory distress and mechanical ventilation.Pregnancy and birth complications were diagnosed using the Brighton Collaboration consensus list of terms, and international guidelines. Gestational hypertension was defined as  hypertension [systolic BP (SBP) \u2265 140\u00a0mmHg or diastolic BP (DBP) \u2265 90\u00a0mmHg] after 20 weeks of gestation in previously normotensive women. Pre-eclampsia was defined as gestational hypertension with proteinuria (24\u00a0h urinary protein \u2265 300\u00a0mg or spot urine protein: creatinine ratio \u2265 30\u00a0mg/mmol creatinine or urine dipstick protein \u2265 2+) or any multi-organ complication of pre-eclampsia. Severe pre-eclampsia was defined as pre-eclampsia with one or more of the following clinical features: BP of \u2265 160/110\u00a0mmHg or hypertension requiring intravenous therapy with an antihypertensive agent or magnesium sulphate after 20 weeks of gestation. Preterm birth was defined as any birth before 37 and after 20 completed weeks of gestation. SGA was defined as neonates with a birthweight below the <10th percentile customized for maternal factors such as maternal height, booking weight, ethnicity and gestational age at delivery. The estimated date of delivery was calculated from a certain last menstrual period (LMP) date and was only adjusted if either (1) a scan performed at < 16 weeks of gestation found a difference of \u22657 days between the scan gestation and that calculated by the LMP or (2) on 20-week scan a difference of \u2265 10 days was found between the scan gestation and that calculated from the LMP. If the LMP date was uncertain, then scan dates were used to calculate the estimated date of delivery.2.4During the first study visit at 9\u201316 weeks\u2019 gestation, information was obtained regarding baseline socio-demographic, lifestyle and clinical characteristics such as age, ethnicity, level of education, household income, employment, exercise, smoking, supplement use, intake of alcohol and recreational drugs, medical and obstetric history, and complications during the current pregnancy. Participating women also completed the Perceived Stress Scale (PSS-10), to assess perceived stress levels in the past month, the short form of the Spielberger State\u2013Trait Anxiety Inventory (STAI), assessing current anxiety symptoms, and the Edinburgh Postnatal Depression Scale (EPDS), assessing depressive symptoms during pregnancy.2.5Demographic, lifestyle and clinical characteristics of participants were summarized descriptively, by influenza vaccination exposure during pregnancy. Continuous variables were summarized as mean with standard deviation (SD) or median with interquartile range (IQR), as appropriate, while counts and percentages were used to summarize categorical variables. To investigate if there was an association between influenza vaccination status and each of the outcome variables, we initially conducted independent samples t-tests or Mann-Whitney U tests, as appropriate, for continuous variables and chi-square tests of association for binary and categorical variables.+6 weeks of gestation. Cox proportional-hazards models with gestational age in weeks as the underlying time metric were used to derive hazard ratios (HRs) that compared the hazard rates for time-sensitive outcomes such as spontaneous abortion or preterm birth between vaccinated and unvaccinated women. Vaccination status was treated as a time-varying exposure in these models, in that each vaccinated woman's pregnancy was decomposed into an unvaccinated exposure period and a vaccinated exposure period. In sensitivity analyses, we estimated HRs and adjusted HRs of time-dependant pregnancy or birth outcomes by trimester of influenza vaccination during pregnancy. To assess the impact of the intensity of influenza activity on the association between maternal influenza vaccination and key birth outcomes, we also stratified analyses by the level of influenza activity at time of delivery using the South Australian Influenza Surveillance Report The timing for vaccination exposures and time at risk windows were calculated for each time sensitive pregnancy and birth outcome accounting for the temporal nature of each outcome of interest. For example, women were at risk for preterm birth from 20 weeks until 36We used log-binomial models to estimate risk ratios (RR) and adjusted risk ratios (aRR) comparing risk of late onset or early postpartum adverse pregnancy outcomes and adverse birth outcomes including congenital anomalies, low Apgar score, admission to neonatal unit, respiratory distress syndrome and mechanical ventilation in infants of vaccinated and unvaccinated mothers. Finally, we used a multivariable linear regression model to predict the difference in mean gestational age at delivery and mean birthweight by vaccination status. For all multivariable (i.e. adjusted) models, annual household income, level of education, ethnicity, maternal health risk factors ), use of micronutrient supplements, asthma and current psychological states were amongst the variables selected as potential confounders based on evidence in the literature Role of Funding Source: Not applicable3n\u00a0=\u00a010); all 28 were excluded from our final analyses. So as not to confound any observed associations, we excluded 83 women who had influenza vaccination prior to pregnancy. Our final cohort consisted of 1253 women  were vaccinated in first trimester, 20\u20222% (n\u00a0=\u00a0122) in second trimester, and 55\u20227% (n\u00a0=\u00a0336) in third trimester. Both influenza and pertussis vaccinations occurred in 555 of 1253 (44\u20222%) pregnancies. Unvaccinated women were more likely to be younger, Aboriginal and/or Torres Strait Islander, in lowest household income group, smoke cigarettes, use illicit drugs, physically inactive, have lower educational attainment and less likely to take micronutrient supplements pre-conception or during pregnancy, and give birth during Autumn compared with vaccinated pregnant women ; of the vaccinated women, 24\u20220%  had spontaneous abortions < 20 weeks\u2019 gestation, seven had terminations (0\u20225%), six had stillbirths (0\u20224%), and 1201 (95\u20228%) delivered a live infant . The mean gestational age at delivery was 39\u20222 weeks (SD 2\u20220 weeks). Overall, 95 of 1253 (7\u20225%) women were admitted to hospital due to influenza like illness during pregnancy; mostly in the third (93 of 95) trimesters of pregnancy. The time-dependant Cox proportional hazards regression model shows that women vaccinated at any time during pregnancy had a significant lower risk of pre-delivery hospitalisation with influenza like illness compared to unvaccinated women  . After aThere was no association with spontaneous abortion for women who were vaccinated for influenza prior to 20 weeks\u2019 gestation  . Our Coxn\u00a0=\u00a03) and unvaccinated women (n\u00a0=\u00a03). Our time-dependant analysis showed no association between influenza vaccination through to 37 weeks\u2019 gestation and preterm birth , preterm premature rupture of the membranes , and spontaneous preterm birth   . Materna3.2Maternal influenza vaccination was protective against delivering LBW term infants in our Cox proportional hazard regression analyses  . This efOur study found no increased risk for SGA delivery after influenza vaccination during pregnancy  . Materna4In robust nuanced analyses that account for timing of maternal influenza vaccination and the time risk of adverse pregnancy outcomes, we show maternal influenza vaccination is safe in a prospective cohort of healthy pregnant women, with confirmed vaccination history and accurate, pregnancy and infant outcome data. There was no evidence of associations between influenza vaccination administered at any time in pregnancy and adverse pregnancy or foetal outcomes including spontaneous abortion, congenital anomalies, shortened gestation, gestational diabetes, chorioamnionitis or gestational hypertensive disorders, consistent with the literature In contrast to our findings, a recent Bayesian meta-analysis of 28 cohort studies showed maternal influenza vaccination protects against preterm birth Consistent with previous studies, , 27 we dOur study has a number of strengths and some potential limitations. The major strength is the prospective cohort design that recruited a large number of nulliparous women with singleton pregnancies at low risk for obstetric complications at two major maternity hospitals, reducing potential confounding by indication. Such bias could have occurred if women with known comorbidities and/or high-risk factors were more likely to receive the influenza vaccine during pregnancy and have a higher baseline risk of adverse pregnancy outcomes than healthy women leading to an underestimation of vaccine safety. The opposite effect  due to a \u2018healthy vaccinee bias\u2019 could also have occurred. Vaccinated women in our study were more likely to engage in healthy lifestyles i.e. pregnancy micronutrient supplementation, exercise regularly and were less likely to smoke or use illicit drugs in pregnancy than unvaccinated women. The analysis framework used herein adjusted for putative risk factors, including psychosocial factors, to mitigate the impact of any \u2018healthy vaccinee bias\u2019 on our findings.Our use of Cox proportional-hazards models accounting for time-varying vaccine exposure within pregnancy, minimized the introduction of immortal time bias in our data Evidence from previous influenza pandemics, and seasonal influenza demonstrates that pregnant women and their infants are at high risk of severe influenza-related complications , 2. Our The datasets generated and/or analysed during the current study are available upon reasonable request to Prof. Claire Roberts (claire.roberts@adelaide.edu.au) and subject to regulatory approvals.HSM has been an investigator on clinical trials funded by pharmaceutical companies including Pfizer, GSK Sanofi-Pasteur, Novartis. Her institution receives funding for Investigator led research. HSM receives no personal payments from Industry. The remaining authors have no conflicts of interest to declare."}
+{"text": "Welche Bedeutung hat eine Lockerung der Kontaktbeschr\u00e4nkungen f\u00fcr die Erholung der deutschen Wirtschaft, und welche Schlussfolgerungen f\u00fcr das angemessene Niveau der Kontaktbeschr\u00e4nkungen sind in den kommenden Monaten zu ziehen? Um diese Fragen zu beantworten, wird abgesch\u00e4tzt, welche Bedeutung die Ma\u00dfnahmen zur Kontaktbeschr\u00e4nkung, die von Bund und L\u00e4ndern seit Mitte M\u00e4rz 2020 auf den Weg gebracht worden sind, f\u00fcr den derzeit zu beobachtenden Einbruch der Wirtschaftsaktivit\u00e4t in Deutschland tats\u00e4chlich haben. Zudem werden die verschiedenen plausiblen Szenarien zur Infektionsverbreitung und -eind\u00e4mmung unter verschiedenen Optionen zur Lockerung der Kontaktbeschr\u00e4nkungen dargestellt."}
+{"text": "Gastroesophageal reflux disease (GERD) is a common comorbidity in chronic obstructive pulmonary disease (COPD) and has been associated with increased risk of acute exacerbations, hospitalization, emergency room visits, costs, and quality-of-life impairment. However, it remains unclear whether GERD contributes to the progression of COPD as measured by lung function or computed tomography.To determine the impact of GERD on longitudinal changes in lung function and radiographic lung disease in the COPDGene cohort.1) and forced vital capacity (FVC)] and quantitative computed tomography (QCT) metrics of airway disease and emphysema using multivariable regression models. These associations were further evaluated in the setting of GERD treatment with proton-pump inhibitors (PPI) and/or histamine-receptor 2 blockers (H2 blockers).We evaluated 5728 participants in the COPDGene cohort who completed Phase I (baseline) and Phase II (5-year follow-up) visits. GERD status was based on participant-reported physician diagnoses. We evaluated associations between GERD and annualized changes in lung function [forced expired volume in 1\u2009s , \u2212\u20095.43 to 0.37) or FVC , but the odds of rapid FEV1 decline of \u226540\u2009mL/year was higher in those with GERD (adjusted odds ratio (OR) 1.20; 95%CI, 1.07 to 1.35). Participants with GERD had increased progression of QCT-measured air trapping , but not other QCT metrics such as airway wall area/thickness or emphysema. Among those with GERD, use of PPI and/or H2 blockers was associated with faster decline in FEV1  and FVC .GERD was reported by 2101 (36.7%) participants at either Phase I and/or Phase II. GERD was not associated with significant differences in slopes of FEV1 decline and QCT-measured air trapping, but not by slopes of lung function. The magnitude of the differences was clinically small, but given the high prevalence of GERD, further investigation is warranted to understand the potential disease-modifying role of GERD in COPD pathogenesis and progression.GERD was associated with faster COPD disease progression as measured by rapid FEVNCT00608764. Gastroesophageal reflux disease (GERD) is a common comorbidity in chronic obstructive pulmonary disease (COPD). The prevalence of self-reported GERD in those with COPD is reported to be between 17%  while stCross-sectional studies that evaluated the relationship between GERD and lung function have revealed conflicting results \u2013 some studies observed worse airflow obstruction , 18, 19 ClinicalTrials.gov Registration # NCT00608764) is an ongoing multicenter, longitudinal study designed to investigate the genetic and epidemiologic characteristics of smoking-related lung disease. A complete description of the protocol has been published previously [COPDGene  \u22650.7 and an FEV1\u00a0<\u200980% predicted in current or former smokers without COPD, is not currently included in the GOLD guidelines, but was used previously [1/FVC \u22650.7 and an FEV1\u00a0>\u200980% predicted in former or current smokers [1 and FVC changes per year were calculated by dividing the differences between Phase I and Phase II by the number of years between visits.Our primary outcome variable was rate of lung function decline, as assessed by post-bronchodilator spirometry. Participants underwent spirometry  before and after administration of 180\u2009\u03bcg of albuterol  at Phase I and Phase II. Percent predicted and lower limit of normal (LLN) values were obtained using National Health and Nutrition Examination III reference equations for spirometry . COPD seeviously . Preserv smokers , 24. Rathttp://www.vidadiagnostics.com) software and Thirona (https://thirona.eu/) software. QCT outcomes included airway wall thickness (AWT)-Pi10 , airway wall area , air trapping  [Our secondary outcomes were QCT-based measures of lung disease. Participants included in this analysis had high-resolution CT scans at full inspiration at Phase I and Phase II to assess emphysema and airway disease. Quantitative imaging analysis were performed using VIDA  , emphysetory CT) . Rates o1% predicted at Phase I; and Model 3 included covariates in Model 2 and whether or not the patient had \u22651 acute exacerbation of COPD between Phase I and Phase II.Demographic, clinical, and lung health characteristics were compared between those with and without GERD using descriptive statistics. GERD was assessed identically at the Phase I and Phase II visits, and in our primary analysis, we categorized those with GERD as reporting GERD at either visit and compared outcomes to those with no GERD at either visit. Associations between GERD and longitudinal changes in spirometry and QCT chest measurements were assessed using linear regression models with sandwich standard errors. We present the outcome data using three models: Model 1 is unadjusted; Model 2 covariates included age, sex, race, whether the patient smoked between Phase I and Phase II, body mass index (BMI), clinical center, and FEV1 decline (defined as FEV1 decline of \u226540\u2009mL/year) for those with vs. without GERD after adjustment for the same covariates. Lastly, we explored associations between GERD treatment (PPI and/or H2 blocker at Phase I and/or Phase II) and changes in lung function using linear regression models.Secondary analyses compared changes in spirometry between  those win\u00a0=\u20094031) and without (n\u00a0=\u20091697) available QCT measurements at both visits are provided in the Supplemental Table\u00a0Data were available for 5728/10,720 (53.4%) participants who were former or current smokers and completed both Phase I  and Phase II  study visits , \u2212\u20096.56 to \u2212\u20090.73) and FVC , after adjustment for age, sex, race, smoking, BMI, clinic center, and FEV1% predicted , \u2018persistent GERD\u2019 (Phase I\u2009=\u2009\u2018yes\u2019 and Phase II\u2009=\u2009\u2018yes\u2019), and \u2018resolved GERD\u2019 (Phase I\u2009=\u2009\u2018yes\u2019 and Phase II\u2009=\u2009\u2018no\u2019) (Table\u00a01 decline (n\u00a0=\u20092572) was significantly increased for those reporting \u2018any GERD\u2019 , \u2018persistent GERD\u2019 , and \u2018incident GERD\u2019 ; the only participants that did not have an increased odds of rapid decline was the \u2018resolved GERD\u2019  group  was associated with faster decline in FEV\u2019) Table\u00a0. The odd2 blockers by 260 (6.5%), of whom most (81%) reported GERD at either visit, though 19% did not report GERD at either visit. Among those with GERD, treatment with PPI and/or H2 blocker at either Phase I and/or Phase II was associated with faster decline in lung function  and FVC  were faster in those receiving PPI and/or H2 blocker compared to those who are not receiving either medication. Among those without GERD, PPI and/or H2 blocker treatment was not associated with lung function decline, though these estimates had less precision than in those with GERD due to the smaller sample.Among our 5728 study participants, pharmacologic treatment with PPIs was reported by 990 (24%) and H2 blockers compared to those who were not taking medications , then stratified GERD into \u2018persistent\u2019, \u2018incident\u2019, and \u2018resolved.\u2019 Rapid decline in FEV1 is a strong predictor of mortality and COPD-related hospitalization [1 decline, using a multivariate logistic regression model controlling for age, sex, race, smoking status, BMI, FEV1% predicted at baseline, and acute exacerbations. Participants with \u2018resolved GERD\u2019 do not appear to have increased odds of rapid FEV1 decline raising the question about the potential role of GERD treatment in slowing lung function decline, which will need to be addressed in future clinical trials.We evaluated loss of lung function both as continuous variables (mL/year) and as a categorical variable , while Rogha et al. [p\u00a0=\u20090.005) supporting our findings. In contrast, several other studies found no association between lung function and GERD, possibly due to relatively small sample sizes [Smaller cross-sectional studies that have evaluated the relationship between lung function severity and GERD have shown mixed results. Mokhlesi et al.  found thle sizes , 33\u201335. In addition to adding a temporal dimension in the assessment of the impact of GERD in lung function, we also evaluated whether GERD contributes to the progression of small airway disease and emphysema over time using QCT. Small airway obstruction and emphysematous lung destruction reflect abnormalities in lung function . Airway 2 blocker among participants with GERD was associated with an accelerated decline in FEV1 and FVC. Using the same COPDGene cohort, Martinez et al. showed that the use of PPI was associated with improved SGRQ total score, but also increased exacerbations highlighting the possibility of confounding-by-indication [2 blocker use in this cohort is also likely due to confounding-by-indication. Xiong et al. suggested that treatment of GERD with PPI in patients with COPD is associated with delayed deterioration of FEV1 after 1-year follow-up [The association between GERD and lung health bring into question the role of anti-reflux treatment in management of COPD. We observed that pharmacologic treatment with PPI and/or Hdication . The sigollow-up . Howeverollow-up , 44\u201346. ollow-up . We did Our study has limitations. GERD was based on self-report of a physician diagnosis, not on validated reflux questionnaires, pH monitoring, or esophageal manometry. Therefore, GERD misclassification might have affected our results. Because this is an observational study, we cannot establish causal inferences.1 decline and QCT-measured air trapping, but not by slopes of lung function. The magnitude of the differences was clinically small, but given the high prevalence of GERD, further investigation is warranted to understand the potential disease-modifying role of GERD in COPD pathogenesis and progression.GERD was associated with faster COPD disease progression as measured by rapid FEVAdditional file: Supplemental Figure 1S. Participant flow diagram. Supplemental Table 1S. Cohort characteristics at Phase I by availability of quantitative CT (QCT) measurements at both visits. Supplemental Table 2S. Multivariable linear regression models of the association between treatment with proton pump inhibitor (PPI) and/or H2 blocker (n = 960) and slopes of quantitative CT (QCT) measures of lung disease among those with gastroesophageal reflux disease (GERD)."}
+{"text": "Staphylococcus aureus causing community-acquired infections as the pathogen develops resistance towards multiple antibiotics. The recent emergence of community strains of S. aureus harbouring methicillin-resistant (MRSA), vancomycin-intermediate (VISA) and vancomycin-resistant (VRSA) genes associated with increased virulence is challenging. Despite the few significant developments in antibiotic research, successful MRSA therapeutic options are still needed to reduce the use of scanty and expensive second-line treatments. This paper provides an overview of findings from various studies on antibacterial secondary metabolites from basidiomycetes, with a special focus on antistaphylococcal activity.Fungi are a rich source of secondary metabolites with several pharmacological activities such as antifungal, antioxidant, antibacterial and anticancer to name a few. Due to the large number of diverse structured chemical compounds they produce, fungi from the phyla Ascomycota, Basidiomycota and Muccoromycota have been intensively studied for isolation of bioactive compounds. Basidiomycetes-derived secondary metabolites are known as a promising source of antibacterial compounds with activity against Gram-positive bacteria. The continued emergence of antimicrobial resistance (AMR) poses a major challenge to patient health as it leads to higher morbidity and mortality, higher hospital-stay duration and substantial economic burden in global healthcare sector. One of the key culprits for AMR crisis is  Staphylococcus aureus with the emergence of multidrug-resistant strains such as methicilin-resistant (MRSA), vancomycin-intermediate (VISA) and vancomycin-resistant (VRSA) S. aureus , C. clelandii, C. [D. kula], C. memoria-annae, C. persplendidus, C. sinapicolor and C. vinosipes demonstrated IC50 values of \u22640.09 mg/mL against S. aureus  and C. persplendidus, and eleven unknown Cortinarius spp. were reported to exhibit anti-Pseudomonas activity with IC50 \u2264 0.09 mg/mL [Bioactive compounds isolated from fruiting bodies of 09 mg/mL .Pleurotus eous. The petroleum ether extracts of P. eous possessed several fatty acids compounds such as stearic acid (28), heptadecanoic acid (29) and tartronic acid (30) that attributed to high growth inhibitory activity against E. coli (DIZ = 11 \u00b1 0 mm) than K. pneumoniae. The antimicrobial activity of this fungus is related to the presence of fatty acids which traditionally could be used for pain, fever and inflammatory disorders [Potential antibacterial compounds have been isolated from organic extracts of the fungus isorders .The present review focuses on antistaphyloccocal activity of basidiomycetes globally and their isolated secondary metabolites. An exhaustive literature search was performed and only those basidiomycete extracts (and isolated compounds) with positive antistaphyloccoccal activity were included. For further extensive scientific studies, it will certainly prove useful to investigate the metabolites derived from this fungal group.Staphylococcus strains. Increasing reports in recent years have also shown the potential ability of some natural extracts in potentiating the activity of standard antibiotics [The emergence of several staphylococcal resistance strains linked to nosocomial infections requires a new antimicrobial solution. Literature reports indicated that extracts and/or compounds from various basidiomycete species exhibited potential antibacterial activity against ibiotics ,90. FurtThe numerous methodologies used for the assessment of antimicrobial activity of basidiomycete extracts or isolated compounds made it difficult to compare between the published data. The factors involved like type of solvent, conditions such as temperature and time, and the structure of compounds that causes variation in the extraction of bioactive compounds has to be optimised to attain a greater overall output and efficiency of the target compounds. The geographic location, different fungal cultivation method and types of growth media could possibly affect the content and amount of active compounds in the extracts . Thus, tThe available literature studies are mostly focused on screening of antibacterial properties of basidiomycete extracts. The prospect of discovering new secondary metabolites from them is highly valued. The lack of data on mechanism of action of the antimicrobial compounds from Basidiomycete fungus does not support their use as a sole antibiotic. Further studies are needed before these compounds are made available for human use. Identifying individual compounds that exhibit antibacterial activity and explaining their mode of action is inevitable in drug discovery. This provides an avenue for the creation of pharmaceuticals that are effective against selected microorganisms resistant to traditional therapy in tandem with the growing multidrug resistance crisis in recent decades."}
+{"text": "Objective: To determine the safety and tolerability of escalating doses of three cannabis oil formulations, containing predominantly CBD, THC, or CBD and THC (1.5:1) vs. placebo in dogs.Design: Randomized, placebo-controlled, blinded, parallel study.Animals: Twenty healthy Beagle dogs .Methods: Dogs were randomly assigned to one of five treatment groups : CBD-predominant oil, THC-predominant oil, CBD/THC-predominant oil (1.5:1), sunflower oil placebo, medium-chain triglyceride oil placebo. Up to 10 escalating doses of the oils were planned for administration via oral gavage, with at least 3 days separating doses. Clinical observations, physical examinations, complete blood counts, clinical chemistry, and plasma cannabinoids were used to assess safety, tolerability, and the occurrence of adverse events (AEs). AEs were rated as mild, moderate, or severe/medically significant.Results: Dose escalation of the CBD-predominant oil formulation was shown to be as safe as placebo and safer than dose escalation of oils containing THC (CBD/THC oil or THC oil). The placebo oils were delivered up to 10 escalating volumes, the CBD oil up to the tenth dose , the THC oil up to the tenth dose , and the CBD/THC oil up to the fifth dose . AEs were reported in all dogs across the five groups and the majority (94.9%) were mild. Moderate AEs  and severe/medically significant AEs  manifested as constitutional  or neurological (ataxia) symptoms and mainly occurred across the two groups receiving oils containing THC (CBD/THC oil or THC oil).Conclusions and clinical significance: Overall, dogs tolerated dose escalation of the CBD oil well, experiencing only mild AEs. The favorable safety profile of 10 escalating doses of a CBD oil containing 18.3\u2013640.5 mg CBD per dose (~2\u201362 mg/kg) provides comparative evidence that, at our investigated doses, a CBD-predominant oil formulation was safer and more tolerated in dogs than oil formulations containing higher concentrations of THC. As a result of changing cannabis regulatory frameworks and social perceptions globally, there is a renewed interest in the potential therapeutic properties of cannabinoids across multiple stakeholders, including the veterinary community. Currently, there are no authorized veterinary drugs containing cannabinoids in the United States (U.S.) or Canada and state or federal laws legalizing the use of medical cannabis in either jurisdiction do not apply to uses in animals , 2. NotwWhile there are existing reviews on the safety and toxicology of cannabinoids, namely CBD \u20138 and THWhile minimal, there are published data on the safety and efficacy of cannabinoids in companion animals. Dogs have been used in the course of developing the drug safety profile of Cesamet\u2122  and Sativex\u00ae , both approved drugs in the U.S. and/or Canada for chemotherapy-induced nausea and vomiting (Cesamet\u2122), or spasticity or pain in multiple sclerosis or advanced cancer (Sativex\u00ae) , 15. PubThe primary objective of this study was to determine the safety of three cannabis oil formulations, predominant in CBD, THC, or CBD and THC (1.5:1) in dogs. Since a hallmark dosing strategy for cannabis initiation is to \u201cstart low and go slow\u201d so as to avoid AEs associated with THC , a slow The study was randomized, placebo-controlled, and blinded. Twenty-four healthy Beagle dogs were acclimated to study conditions for 24 days prior to treatment allocation. Twenty healthy adult purpose-bred Beagle dogs  were randomized to one of five treatment groups with four dogs per group : (i) CBD-predominant oil; (ii) THC-predominant oil; (iii) CBD/THC-predominant oil (1.5:1); (iv) medium-chain triglyceride (MCT) oil placebo; or (v) sunflower (SF) oil placebo.Up to 10 escalating doses of the oils were planned for administration , with atTwo different placebo oils were used since the cannabinoid oils included either SF or MCT oil as solvents. Moreover, the amounts delivered across the two placebo oils differed to match the volumes administered with the cannabinoid oils. The dosing schedule of the placebo oils was such that two placebo doses were administered prior to the start of cannabinoid oil dosing to ascertain tolerability to increasing volumes.Treatments were administered to fasted dogs by oral gavage. Each cannabinoid or placebo oil administration was followed by a 10 mL water flush to ensure full uptake. A small wet meatball was given immediately after dosing to disrupt negative association with repeated oral gavage. If an animal was observed vomiting within 30 min of test or placebo administration, the dose was re-administered.Randomization was stratified by sex and conducted using a random number generator in Microsoft EXCEL\u00ae 2016 . Cannabinoid and placebo oil formulations were also randomly assigned a code name using a random number generator in Microsoft EXCEL\u00ae 2016. Once the dogs were assigned to a group (Groups 1 through 5), the study coordinator randomly allocated each group to a coded product by drawing lots. All technicians collecting data and administering the investigational products were blinded to treatment allocation. All bottles containing the cannabinoid or placebo oil formulations were over-labeled with opaque white labels. Information about treatment groups and their respective treatment conditions were securely kept in the VivoCore Inc. archive room for the duration of the study.The cannabinoid oils  and placebo oils (SF or MCT oils) were acquired from Tweed Inc.  and stored between 19.6 and 21.9\u00b0C. The cannabis plants used to prepare the cannabinoid oils were grown indoors under tightly controlled environmental conditions. Within one lot, all plants were genetically identical. Upon harvest, plant material was trimmed, dried, and extracted. Extraction was performed by super-critical carbon dioxide, and the extracted resin was diluted with a food-grade oil (SF or MCT oil) to the target concentration: 18.3 mg/mL CBD in the CBD-predominant oil, 24.9 mg/mL THC in the THC-predominant oil, and 7.0 mg/mL CBD + 4.8 mg/mL THC in the CBD/THC-predominant oil.An independent laboratory  analyzed the composition of the cannabinoid oil formulations using validated methods. Levels of phytocannabinoids and terpenes in the oil formulations are outlined in The purpose-bred animals were acquired from a colony at VivoCore Inc. . The inclusion criteria for the study were good general health as determined by the veterinarian; stable weight over a 10-day period preceding study start and a weight of 9\u201315 kg; and, under 8 years of age. Exclusion criteria were pregnant or lactating dogs; use of medications or supplements during the course of the study; receipt of cannabinoid or cancer-related therapies in 2 months preceding study start; receipt of any test substance within a month prior to study start; existing or a history of cancer, blood- or immunology-related disease or other chronic morbidity .ad libitum in stainless steel bowls. Upon study completion, animals were returned to their colony at VivoCore Inc.The dogs were individually housed in stainless steel metabolic cages  and those in the same treatment group were exercised together. Environmental controls for the animal housing area were electronically set to maintain a temperature of 18.7\u201326.2\u00b0C and a 12-h light/dark cycle. Animals were fed a standard commercial dry canine diet in stainless steel bowls (Purina ProPlan Savor Adult\u2014Chicken and Rice Formula) once daily, 7\u201310 h after dosing. Food was left for 1 h and the food quantity offered (1.25\u20132.75 cups) was based on body weight. Water was available Throughout the study period, food consumption and 24-h activity  were measured daily and animal health observations occurred twice daily. Body weights were collected throughout the acclimation period, once during the study period, and once upon study completion. Following administration of the cannabinoid or placebo oils, animals were monitored up to 9 h post-dose for body temperature , respiration rate (by observation), and heart rate (stethoscope). Observations of the animals were also conducted every 1\u20133 h post-dosing through the first 9 h and then at 12 and 24 h. Subjects were observed for any signs that would not be expected in normal dogs and for the occurrence of AEs. Experienced veterinary technicians and/or a licensed veterinarian conducted the clinical observations and physical assessments.AEs were rated for severity as (i) mild\u2014activities of daily living (ADL) not impacted and no intervention indicated; (ii) moderate\u2014ADL moderately limited (non-invasive intervention may be indicated); or (iii) severe/medically significant\u2014ADL significantly limited . If one 2EDTA tube. These blood collections occurred during acclimation (baseline), mid-study , and 7 days following the final dose of the cannabinoid or placebo oils. Additionally, they occurred 24 h following the final dose of the cannabinoid oils. With the occurrence of a severe AE, blood was drawn immediately, and 24 h and 7 days thereafter.For analysis of Complete Blood Count (CBC) and clinical chemistry, 4 mL of blood was drawn by direct venipuncture from a cephalic or jugular vein; 2 mL was placed into an evacuated serum separator tube (SST) and another 2 mL into an evacuated K2EDTA tube. These blood collections occurred before and after the ninth dose of the CBD oil and THC oil . These blood collections also occurred 7 days following the final dose of the cannabinoid or placebo oils.For analysis of CBD, THC, and their metabolites (7-COOH-CBD and 11-OH-THC), 2 mL of blood was drawn and placed into an evacuated K2EDTA tubes were centrifuged at 2,800\u20133,000 revolutions per minute (rpm) for 10 min at 4\u00b0C. The SSTs and K2EDTA tubes were stored at 2 to 8\u00b0C and on the same day as blood collection were transported to Antech Diagnostics  for analysis  or further processing. For the K2EDTA tubes intended for analysis of CBD, THC, and their metabolites, plasma was separated into two equal aliquots and stored at \u221280\u00b0C until shipment on dry ice to the bioanalytical laboratory for analysis .Blood in the evacuated SSTs was allowed to clot for a minimum of 30 min but no more than 1 h following collection, then was centrifuged at 1,525\u20131,992 relative centrifugal force (rcf) at 20\u00b0C for 10 min. K3, CBD-d3, 11-OH-THC-d3, Toronto Research Chemicals); paclitaxel was used as the internal standard for 7-COOH-CBD. All four compounds were analyzed in one run.CBD, THC, 7-COOH-CBD, and 11-OH-THC were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) . The analytes CBD, THC and 11-OH-THC were purchased as analytical reference solutions from Sigma-Aldrich; 7-COOH-CBD was purchased from Toronto Research Chemicals. The analytes were chromatographically separated on a Phenomenex Kinetex Phenyl-Hexyl column  using gradient elution (mobile phase A = 0.1% formic acid in water and mobile phase B = 0.1% formic acid in acetonitrile) at a flow rate of 0.4 mL/min. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode with a turbo ion spray interface. Internal standards were deuterated analogs of THC, CBD, and 11-OH-THC (THC-d2EDTA as anticoagulant). Ten calibration standards over the range of 0.25\u20132,000 ng/mL  or 0.5\u20132,000 ng/mL  were used and included a blank sample  and a zero sample . Plasma standards and samples were extracted by the addition of a solution of 50/50 methanol/acetonitrile containing the internal standards to precipitate the proteins. A sample batch consisted of the following: 3 replicates of a system suitability standard , calibration standards in ascending order including a blank sample , a zero sample , and 10 non-zero standards, the assay samples, followed by the 3 replicates of the system suitability sample. Calibration standards bracketed an analysis batch of >40 samples.Calibration standards were prepared in blank pooled dog plasma  that at least 75% of the non-zero calibration standards be included in the calibration curve with all back-calculated concentrations within \u00b120% deviation from nominal concentrations , (2) the correlation coefficient (r) of the calibration curve must be \u22650.99, and (3) the area ratio variation between the pre- and post-run injections of the system suitability samples is within \u00b125%.www.graphpad.com.Measures of central tendency (mean), variability , and all figures were generated by GraphPad Prism version 8.1.2 for Windows, GraphPad Software, San Diego, California, USA, n = 1), recurring ear infections (n = 1), and inadequate maintenance of body weight (n = 2). Therefore, 20 dogs were randomized to treatment groups (with 4 dogs per group). The mean (SD) body weights of each group at baseline were as follows: MCT oil: 12.2 kg (1.1 kg); CBD oil: 10.3 kg (0.4 kg); THC oil: 12.3 kg (1.0 kg); SF oil: 11.9 kg (1.5 kg); CBD/THC oil: 11.3 kg (0.9 kg).Four of 24 dogs evaluated for inclusion into the study were excluded from study enrollment following the acclimation period due to recurring loose stool , 8.1% (CBD oil), 27.9% (THC oil), and 44.7% (CBD/THC oil), with no changes observed between these periods with SF oil (data not shown). Dogs were not exercised/socialized on dosing days and physical activity (measured by Actiwatch-64\u00ae) was thus reduced across all groups on dosing days as compared to non-dosing days by 12.9% (MCT oil), 19.2% (CBD oil), 19.8% (THC oil), 24.4% (SF oil), and 40.7% (CBD/THC oil) (data not shown). Despite these changes, body weights remained stable throughout the study period across the five groups.The two placebo oils were tested up to 10 escalating volumes while the CBD oil, THC oil, and CBD/THC oil were tested up to the tenth, tenth, and fifth dose, respectively; titration to maximum doses of 640.5 mg CBD ~62 mg/kg), 597.6 mg THC (~49 mg/kg), and 140.8/96.6 mg CBD/THC (~12 mg/kg CBD + 8 mg/kg THC), respectively, was thus achieved , 2. For  mg/kg, 5One of four dogs in the THC oil group experienced severe ataxia at the 7th dose and was discontinued from further dosing. Two of four dogs in the CBD/THC oil group experienced severe ataxia and/or lethargy at the fourth or fifth doses (one dog at the fourth dose and a second dog at the fifth dose) and thus further dosing ceased for all dogs in this group. No dogs were discontinued from the CBD oil or placebo oil groups as a result of AEs.n = 505), 104 AEs occurred in the placebo groups across 10 escalating volumes (77 AEs with MCT oil and 27 AEs with SF oil), and 401 AEs occurred across the three cannabinoid groups: 80 AEs with CBD oil (10 doses), 206 AEs with THC oil (10 doses), and 115 AEs with CBD/THC oil (five doses)  . The SF e doses) .Across the three groups receiving cannabinoid oils, the fewest AEs were reported in the CBD oil group (across 10 doses) as compared to the THC oil group (across 10 doses) and the CBD/THC oil group (across five doses) . CompareThe majority of AEs in each of the five groups, and collectively across all five groups, were mild. Mild AEs accounted for 479 of the 505 total study AEs (94.9%) . Mild AEThere were no moderate AEs in the CBD oil group at any of the doses tested. Moderate AEs accounted for 22 of the 505 total study AEs (4.4%) and occurred in 40% of the subjects (8 of 20 dogs) across three groups: MCT oil (two dogs at the tenth dose), THC oil (two dogs at the third or seventh doses), or CBD/THC oil (four dogs across the five doses tested) . ModeratThere were no severe/medically significant AEs in the CBD oil group at any of the doses tested. Severe AEs accounted for 4 of the 505 total study AEs (0.8%)  and occuFor the cannabinoid oil groups, blood was collected at baseline, and 24 h and 7 days after the final dose. Overall, hematological parameters were generally normal for the dogs across these groups at 24 h and 7 days after the final dose, with a few exceptions as outlined below.With respect to changes in clinical chemistry parameters suggestive of altered liver function, while possibly not clinically pathologic, we interpreted a notable change to be: (i) at least a 2-fold increase from baseline to post final dose timepoints (24 h or 7 days) in total bilirubin or plasma levels of liver enzymes ; and (ii) the post final dose level (24 h or 7 days) in the above parameters reached or exceeded the upper limit of normal. Applying these criteria, one dog in the CBD oil group and one dog in the CBD/THC oil group experienced 2.9-fold or 3.6-fold increases in ALP from baseline to 24 h following the last administered dose, which was the tenth dose (CBD oil) or the fifth dose (CBD/THC oil), respectively . MoreoveWith respect to the remaining CBC and clinical chemistry parameters, blood collected 24 h following the final dose of the cannabinoid oils showed only a few abnormalities as based on laboratory reference ranges. These abnormalities occurred in 1 or 2 dogs in the cannabinoid oil groups and consisted of low creatine phosphokinase (1 dog in CBD oil group); low amylase, high sodium, or high pancreatic sensitive lipase (2 dogs in THC oil group); high pancreatic sensitive lipase (1 dog in the CBD/THC oil group) (data not shown). All abnormalities resolved 7 days following the final dose.n = 4) or THC oil (n = 3), plasma levels of CBD, THC, and their metabolites  were measured 1, 2, 4, 6, and 24 h post-dose; levels of these parameters were also measured pre-dose  led to steady inclines in plasma CBD, 7-COOH-CBD and THC over the 6-h post-dose period . Levels Following intake of the ninth dose of the THC oil , mean (SEM) plasma THC and 11-OH-THC reached maximum levels of 69.8 \u00b1 28.8 ng/mL  and 5.9 \u00b1 2.7 ng/mL , respectively, with steady declines observed thereafter over the 6-h post-dose period. Mean plasma THC at 6 h post-dose (21.1 \u00b1 5.1 ng/mL) was similar to its level 24 h post-dose (18.2 \u00b1 7.6 ng/mL). Mean plasma 11-OH-THC at 6 h post-dose (3.7 \u00b1 1.8 ng/mL) was similar to its level 24 h post-dose (4.5 \u00b1 1.7 ng/mL).n = 4), CBD was detected in all four dogs (3.6\u201331.7 ng/mL) while levels of 7-COOH-CBD were detected in half the dogs (1.4\u20131.8 ng/mL) (data not shown). Seven days following intake of the last (tenth) dose of the THC oil , THC was detected in all three dogs (2.8\u20138.2 ng/mL), while 11-OH-THC was not detected in any dogs (data not shown). Seven days following the intake of the last (fifth) dose of the CBD/THC oil , CBD and THC were detected in all three dogs , while 7-COOH-CBD was detected in one of three dogs (1.4 ng/mL) and 11-OH-THC was not detected in any of the three dogs (data not shown).Across the cannabinoid oil groups, cannabinoids and their metabolites were measured 7 days following the last administered dose. Seven days following intake of the last (tenth) dose of the CBD oil  led to only mild AEs and no moderate or severe AEs. Importantly, the number and type of AEs that occurred in the CBD oil group were comparable to the corresponding placebo group (MCT oil). In both groups, the majority of AEs were gastrointestinal, which could have been due to discomfort with oral gavage, oil volume, and/or the MCT oil carrier.The safety and tolerability of CBD, as determined primarily in rodents and humans, has been extensively reviewed \u20138. In huPrevious studies have reported increases in liver enzymes, specifically ALP, in dogs receiving CBD orally at doses ranging from 2 mg/kg (twice daily) to 10 mg/kg (twice daily) for 4, 6, or 12 weeks , 20, 21 Only in the CBD/THC oil group were severe AEs experienced by more than one dog causing cessation of dosing after the fifth dose. Researchers have acknowledged that CBD can have interactions with THC  and thatOur data show a clear distinction in AEs associated with a CBD-predominant oil vs. oils containing higher levels of THC in that constitutional  and neurological  AEs most commonly occurred in dogs receiving the THC oil or the CBD/THC oil. Suppression of locomotor activity (hypolocomotion), catalepsy, and hypothermia have been observed across species  and associated with exposure to cannabis, THC, or THC and CBD in combination, but not CBD alone , 42\u201345. Plasma levels of CBD, THC, and their metabolites were highly variable between dogs in the same treatment group receiving either the ninth dose of CBD oil or THC oil. Others have also observed high variability in cannabinoid  blood concentrations across dogs in single dose pharmacokinetic studies , 19 and max and higher Cmax achieved for THC in the fasted condition. The approximate cumulative CBD dose administration from the first to ninth dose was 2122.9 mg. Detected plasma levels of CBD may also be reflective of CBD accumulation in plasma with dose escalation over time.Contrary to earlier assertions that CBD has low bioavailability after oral administration to animals, including dogs , 50, ourmax levels for plasma THC of 1.25 h in fasted dogs orally dosed with Bedrocan\u00ae (1.5 mg THC/kg). It is reported in the literature that following oral ingestion of cannabis, maximum plasma THC levels are reached within 1\u20132 h (Fasted dogs receiving the ninth dose of the THC oil (cumulative THC from first to ninth dose = 1653.5 mg) achieved maximum THC plasma levels at 1-h post-dose. Related findings are those reported by Lebkowska-Wieruszewska et al.  who calcin 1\u20132 h . Decreasin 1\u20132 h . Indeed,n = 4) or THC oil , at levels ranging from 3.6 to 31.7 ng/mL CBD (CBD oil) and 2.8 to 4.6 ng/mL THC (THC oil). This finding is relevant to future studies which apply a crossover design and include a washout period. That quantifiable levels of CBD were observed one week following dose exposure is also interesting given that CBD has been reported to have a relatively rapid elimination in dogs [CBD has been shown to be bio-transformed in dogs via hydroxylation, carboxylation, and conjugation  and a final formulation (extraction procedures used), our findings are most relevant to our investigated oil-based formulations and may not be applicable to the safety of other marketed formulations consisting of a different profile of cannabinoids and other cannabis constituents  and delivered in a different matrix . Our study was also a preliminary safety study and, as such, a small number of animals were used, which is a limitation. Additional larger studies that investigate the safety of longer-term cannabinoid dosing in dogs are needed.in vivo metabolism of CBD vs. THC when delivered at higher dose levels were generated, which showed that CBD is absorbed more slowly than THC.Overall, our study provides novel data that separates the relative safety and tolerability of dose escalation of oil formulations predominant in plant-derived CBD, THC, or CBD and THC in combination (1.5:1) in dogs. Of the three cannabinoid oil formulations tested, dose escalation of the CBD-predominant oil formulation was the most tolerated by dogs up to a maximum dose of 640.5 mg CBD (~62 mg CBD/kg) and only mild AEs were experienced. Novel data on the Research on the potential health benefits of CBD in dogs is beginning to emerge. Existing studies show its potential as a single therapy for pain reduction in osteoarthritic dogs  or as anphil.shaer@canopygrowth.com).The datasets generated for this study are not publicly available to allow for commercialization of research findings. Reasonable requests to access the datasets should be directed to Phil Shaer  and in accordance with the Principles of the Animals for Research Act  and guidDV and JK were responsible for conception of the study, data analysis, and provided intellectual input on the manuscript. LP was responsible for data analysis and interpretation and writing of the manuscript.DV, JK, and LP are employed by Canopy Animal Health, which is a division of Canopy Growth Corporation. Staff at VivoCore Inc., and not the authors, were responsible for study conduct and data collection."}
+{"text": "Periophthalmus chrysospilos, the Gold\u2010spotted mudskipper, within the Mekong Delta are facing extirpation risks due to indiscriminate harvesting for the growing aquarium and food\u2010fish trade. This study provides some of the first information on reproductive ecology\u2014such as spawning type and season, length at first maturity, and batch fecundity\u2014of this species, to be used in their management. The sex ratio of wild populations, based on 1031 individuals  is 1:1. The gonadosomatic index (GSI) values are exhibit a non\u2010normal distribution and changed with gender, season, and site. A combination of GSIs and the monthly appearance of mature gonads suggest that this species reproduces throughout the year, with peak from July to October. This species exhibits sexual and spatial variation in size at first maturity (mL) as mL is 6.2\u20138.6\u00a0cm in males and 6.4\u20137.3\u00a0cm in females. The batch fecundity  exhibits non\u2010normal distribution and varies with site, with the highest values at Dam Doi, Ca Mau  and the lowest at Tran De, Soc Trang (6654 \u00b1 851 SE). In addition, batch fecundity is directly proportional to body size due to high determination relationships between batch fecundity and fish size . Information derived on the reproductive biology of this species can inform its conservation, sustainable exploitation, and ex situ propagation.Populations of  mL) as mL is 6.2\u20138.6\u00a0cm in males and 6.4\u20137.3\u00a0cm in females. The batch fecundity  exhibits non\u2010normal distribution and varies with site, with the highest values at Dam Doi, Ca Mau  and the lowest at Tran De, Soc Trang (6654 \u00b1 851 SE).A combination of GSIs and the monthly appearance of mature gonads suggest that this species reproduces throughout the year, with peak from July to October. This species exhibits sexual and spatial variation in size at first maturity ( The pH Fishes were caught by hand over the final three days of each month. This species is easily distinguishable from sympatric congeners by the extent of fusion of the pelvic fin and the pattern of the first dorsal fin (Murdy and Jaafar ). Tricai2.2In the laboratory, sex of specimens was determined from the urogenital papillae Figure . In maleG/W)  of males and females at each site was calculated from the formula: P = 1/(1 + exp[\u2212r \u00d7 (TL\u2013mL)])   Zar, .F = (n \u00d7 G)/g   between four sites. One\u2010way ANOVA with Tukey's post hoc was used to test the change in GSI for sites and months when the GSI variances were equal, but one\u2010way ANOVA with Tamhane's T2 was analyzed if the GSI variances were not. In contrast, Kruskal\u2013Wallis test was performed to verify if GSI varied with site and months when GSI variable did not display normal distribution. This test was also applied to confirm the variation of F among four sites if F was not a normal distribution. The logarithmic regression was applied when testing relationships of fish size (TL and W) and F  were sampled over 1\u00a0year at four sampling sites  were thin, smooth, pale white, and measured 1.16 \u00b1 0.02\u00a0mm in diameter Figure . At this3.4p < .01) and changed by gender , season  and site . Higher GSI values were recorded in the wet season compared to the dry season for both sexes at all four sites  and GSI values of males and females showed monthly fluctuations . Notably, mean values of GSI in females were the highest in August 2020 at TV (3.19 \u00b1 0.45 SE); in January 2021 at ST (4.53 \u00b1 0.41 SE); in December 2020 at BL (4.60 \u00b1 0.43 SE); and in September 2020 at CM (4.08 \u00b1 0.56 SE). The higher GSI values  in females were observed during the wet season. Similar patterns were observed in males, where GSI values were highest in the wet season from July to October. For example, the highest mean of male GSI was found in July 2020 at TV (0.27 \u00b1 0.03 SE); in October 2020 at ST (0.29 \u00b1 0.04 SE); in August 2020 at BL (0.27 \u00b1 0.03 SE); and in October 2020 at CM (0.23 \u00b1 0.05 SE).At each site, the GSI values of females were significantly higher than those of males  of Ps. chrysospilos differed between the four sites; this value fluctuated for both male and female individuals from 6.2 to 8.6\u00a0cm and 6.4 to 7.3\u00a0cm, respectively  and varied between sites, fluctuating between values 2614 eggs/female and 23,465 eggs/female . The highest average fecundity values were from individuals recovered from BL  and CM , while the lowest values were from those caught from TV (7516 \u00b1 418 SE) and ST (6654 \u00b1 851 SE). The F values are positively correlated with total length and weight due to the high 2r\u00a0value of the relationships between F and fish size  boddarti  septemradiatus , secondary spermatocytes (SC2), and spermatozoa (SZ) in stage IV of mature testes; as well as oogonia (O), primary oocytes (PO), primary vitellogenic oocytes (PVO), and secondary vitellogenic oocytes (SVO) in stage IV of mature ovaries indicate that  Miller, . This moF of Ps. chrysospilos  changed significantly between the four sites in this study\u2014the highest fecundity was recovered at CM  while the lowest was at TV (F = 4927\u20139941). In gobioid fishes, F values can also vary greatly from one species to another. For instance, one of the lowest F was reported from Eviota lacrimae with only 100 eggs, while one of the highest was reported from Awaous guamensis with approximately 500,000 eggs . Gobioid species sympatric with the gold\u2010spotted mudskipper within the MD also had a positive correlation between fertility and fish body sizes such as Butis butis  tends to be longer. The length of maturity for Ps. chrysospilos is similar to Ps. barbarus .Males and female mudskippers mature at different lengths ; Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Software ; Supervision ; Validation ; Visualization ; Writing \u2013 original draft ; Writing \u2013 review & editing . Ton Huu Duc Nguyen: Data curation ; Formal analysis ; Investigation ; Methodology ; Resources ; Software ; Writing \u2013 original draft ; Writing \u2013 review & editing . Tran Thi Huyen Lam: Formal analysis ; Investigation ; Resources ; Writing \u2013 original draft ; Writing \u2013 review & editing . Tien Thi Kieu Nguyen: Conceptualization ; Investigation ; Writing \u2013 original draft . Ngon Trong Truong: Investigation ; Writing \u2013 review & editing (supporting). Zeehan Jaafar: Conceptualization ; Investigation ; Methodology ; Validation ; Visualization ; Writing \u2013 original draft ; Writing \u2013 review & editing ."}
+{"text": "The association between nonalcoholic fatty liver (NAFL) or liver fibrosis and diabetic peripheral neuropathy (DPN) has not been well studied. We aimed to investigate the association of NAFL or liver fibrosis indices and DPN in individuals with type 2 diabetes. In this observational study, we included 264 individuals with type 2 diabetes, and calculated non-alcoholic fatty liver disease (NAFLD) liver fat score, NAFLD fibrosis score, and Fibrosis-4 (FIB-4) index to evaluate the status of NAFLD or liver fibrosis. DPN was diagnosed when the Michigan Neuropathy Screening Instrument\u2014Physical Examination score was\u2009\u2265\u20092.5. The NAFLD fibrosis score and FIB-4 index were significantly higher in individuals with DPN than in those without DPN. Logistic analyses showed that the NAFLD fibrosis score and FIB-4 index were associated with DPN after adjustment for covariates . In the subgroup analysis, this association was only significant in the group with a high NAFLD liver fat score (>\u2009\u2212\u20090.640). Serum levels of fetuin-A, a hepatokine, were decreased in individuals with abnormal vibration perception or 10-g monofilament tests compared with their counterparts. The present study suggests that liver fibrosis might be associated with DPN in individuals with type 2 diabetes. It encompasses a spectrum of diseases, extending from nonalcoholic fatty liver (NAFL) through nonalcoholic steatohepatitis (NASH), advanced fibrosis to cirrhosis, and end-stage liver disease. The natural history of NAFL is variable, with the majority of individuals having benign disease with steatosis without inflammation2. However, 40% of individuals with NAFL develop advanced fibrosis, which can result in cirrhosis3. The prevalence of NAFLD in individuals with type 2 diabetes is higher than that in the general population, ranging from 40 to 70%2. In addition, individuals with type 2 diabetes showed an increased risk of developing NASH, advanced fibrosis, and cirrhosis7. NAFLD in individuals with type 2 diabetes is associated with an increased risk of developing cardiovascular disease (CVD)9 and an increased risk of microvascular complications, such as nephropathy and retinopathy12.Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease worldwide, affecting approximately 25% of the global population13. Risk factors for DPN include hypertension, smoking, hyperglycaemia, diabetes duration, age, dyslipidaemia, and obesity, which are also known risk factors for CVD14. Considering that NAFLD, CVD, and DPN share risk factors and that NAFLD is associated with an increased risk of other microvascular complications, it is reasonable to hypothesize that individuals with diabetes and NAFL or liver fibrosis would also have a high risk for DPN.Diabetic peripheral neuropathy (DPN) is the most common form of diabetic neuropathy and affects approximately 50% of individuals with diabetes15.In the present study, we examined the association of NAFL or liver fibrosis with DPN using noninvasive methods for evaluating NAFLD and liver fibrosis. In addition, we tested the association between DPN and fetuin-A, a hepatokine that is known to be elevated in individuals with NAFLD1c (HbA1c), and aspartate aminotransferase (AST) levels were significantly higher in individuals with DPN than in those without DPN (Table p\u2009=\u20090.493). However, both the NAFLD fibrosis score and Fibrosis-4 (FIB-4) index were significantly higher in individuals with DPN than in those without DPN.The present study included the data of 264 individuals with type 2 diabetes but without chronic liver diseases; 38.2% had DPN. Body mass index (BMI), fasting plasma glucose (FPG), haemoglobin AWe stratified individuals by NAFLD liver fat score (\u2264\u2009\u2212\u20090.640 or\u2009>\u2009\u2212\u20090.640) and the presence of DPN. Compared with individuals with a low NAFLD liver fat score (\u2264\u2009\u2212\u20090.640), individuals with a high NAFLD liver fat score (>\u2009\u2212\u20090.640) were more obese and had higher blood pressure (BP), triglyceride levels, AST, alanine aminotransferase (ALT), and homeostatic model assessment-insulin resistance (HOMA-IR) Table . HoweverLogistic regression analyses showed that the NAFLD liver fat score was not associated with DPN. However, the NAFLD fibrosis score and FIB-4 index were significantly associated with DPN: adjusted odds ratio (aOR) 1.474 , and aOR 1.961 , respectively (Table p\u2009=\u20090.956). Serum fetuin-A levels were significantly lower in individuals with abnormal vibration perception  and in those with an abnormal 10-g monofilament test  compared with their counterparts. The area under the receiver operating characteristic curve (AUROC) of the fetuin-A levels for the absence of abnormal vibration perception was 0.671  and for the absence of abnormal 10-g monofilament test was 0.736  , serum fetuin-A levels were 613.5\u2009\u00b1\u2009181.0\u00a0\u00b5g/ml in individuals without DPN and 611.3\u2009\u00b1\u2009182.7\u00a0\u00b5g/ml in those with DPN  method and a vibration perception threshold (VPT) assessment for the diagnosis of DPN. They showed a positive association between NAFLD and DPN in Italian individuals with type 1 diabetes . Lv et al.17 used ultrasonography for the diagnosis of NAFLD, and diagnosed DPN based on physical examination. They showed a negative association between NAFLD and DPN in hospitalised Chinese individuals with type 2 diabetes . Kim et al.18 used ultrasonography for the diagnosis of NAFLD, and used a nerve conduction study, a current perception threshold test, and physical examination for the diagnosis of DPN. They showed no association between NAFLD and DPN in Korean individuals with type 2 diabetes , which was consistent with our results. Potential explanations for these differences are the different characteristics of participants in each study and different diagnostic criteria for DPN. Otherwise, considering that the majority of individuals with NAFLD in previous studies were estimated to have NAFL, which is an early stage of NAFLD, other medical conditions or more severe stages of NAFLD might be more important contributors to DPN than NAFL per se.Previous studies have shown conflicting results regarding the association between NAFLD and DPN. Mantovani et al.19. Our study is consistent with previous observations, and we further found that the association between DPN and liver fibrosis indices was only significant in individuals with a high NAFLD liver fat score (>\u2009\u2212\u20090.640). This can be explained by increased vulnerability of the liver to injuries such as oxidative stress or cytokines, as reflected by higher BMI, AST, ALT, and HOMA-IR levels in individuals with a high NAFLD liver fat score (>\u2009\u2212\u20090.640) compared with those with low NAFLD liver fat score (\u2264\u2009\u2212\u20090.640).A previous cohort study reported that an elevated lower-limb vibration perception threshold was associated with markers of liver fibrosis, such as the NAFLD fibrosis score, and with liver stiffness measurement in individuals with type 2 diabetes20. During the development of NAFLD, hypercaloric diets can induce intestinal dysbiosis and excess fat storage in adipose tissue, skeletal muscle, and liver, which result in inflammation and insulin resistance. Insulin resistance results in hyperglycaemia and hyperinsulinaemia. During the progression of NAFLD, glucolipotoxicity increases reactive oxygen species (ROS) generation and endoplasmic reticulum stress, resulting in cell death. Together, these dead cells combined with infiltrated inflammatory cells in the liver, free fatty acids, intestine-derived lipopolysaccharides, and transforming growth factor (TGF)-\u03b2 from Kupffer cells activate hepatic stellate cells (HSCs). Activated HSCs increase the extracellular matrix, leading to liver fibrosis. Among these insults related to the progression of NAFLD, hyperglycaemia, insulin resistance, oxidative stress, and inflammation are also involved in the pathogenesis of DPN21.The \u2018multiple hit hypothesis\u2019 suggests that multiple insults might be generated in individuals with type 2 diabetes due to altered inter-organ crosstalk between the intestine, adipose tissue, skeletal muscle, liver, and pancreas, and that these insults would synergistically result in the development and progression of NAFLD23. Interestingly, patients with NASH exhibited higher hepatic and serum glyceraldehyde-derived AGEs levels than those with simple steatosis or healthy controls24. In addition, glyceraldehyde-derived AGEs increase ROS generation and upregulate fibrogenic genes such as \u03b1-smooth muscle actin, TGF-\u03b21, and collagen type I\u03b12 in human hepatic stellate cell line in vitro25. These results suggest that glyceraldehyde-derived AGEs may contribute to the pathogenesis of NASH. Considering the potential role of AGEs and RAGEs in the pathogenesis of both DPN and NAFLD, our finding that the NAFLD fibrosis score and FIB-4 index were associated with DPN appears reasonable.Advanced glycation end products (AGEs) are implicated in the pathogenesis of DPN. The formation of AGEs increases under chronic hyperglycaemia in diabetes. Interaction of AGEs with their receptors (RAGEs) activates intracellular signalling pathways and increases oxidative stress and inflammation, ultimately resulting in neuronal injuries27, and we evaluated an association between fetuin-A and DPN. Serum fetuin-A levels were negatively associated with abnormal vibration perception and abnormal 10-g monofilament tests. Considering a previous study that showed TGF-\u03b21 signalling suppression by fetuin-A28, and a previous study that showed high TGF-\u03b21 levels in individuals with DPN29, our results seem to suggest a possibility of link between fetuin-A and DPN. Although, fetuin-A cannot be used as a diagnostic tool for DPN, this link suggests the possibility of loss of protection sensation.The progression of NAFLD alters the secretion of hepatokines such as fetuin-A, fetuin-B, and dipeptidyl peptidase-4This study has several limitations. First, it cannot establish a causal relationship because of its cross-sectional nature. Second, liver biopsy, the gold standard method for the diagnosis of NAFLD and liver fibrosis, was not performed. Third, neurophysiological studies were not used for the diagnosis of DPN. Despite these limitations, this study provides valuable insight implying that the progression of NAFL to liver fibrosis might affect the development of DPN and suggests the possible role of fetuin-A in specific feature of DPN, a loss of protection sensation.In conclusion, liver fibrosis might be associated with DPN in individuals with type 2 diabetes and suspected NAFLD. Notably, this association was independent of previously known risk factors. The present study suggests the need for special attention to DPN in individuals with type 2 diabetes and NAFLD, especially those with a high NAFLD fibrosis score or FIB-4 index. Future studies to investigate the molecular mechanism of the association between liver fibrosis and DPN are necessary.\u22121 [1.73\u00a0m]\u22122), pregnancy, and severe diabetic foot ulcers or previous amputation. This is a subset study analysing data from individuals who were enrolled during the initial 3-year period (January 2017 to January 2020). We recruited 300 individuals with type 2 diabetes from Seoul National University Bundang Hospital (SNUBH), a tertiary academic hospital. In the present study, the following individuals were excluded: (1) individuals (n\u2009=\u200919) with cirrhosis of any etiology or chronic liver disease due to excessive alcohol consumption  or viral hepatitis based on a medical history and medications; (2) individuals (n\u2009=\u200915) with incomplete data needed to calculate the NAFLD liver fat score, NAFLD fibrosis score, or FIB-4 index; and (3) individuals (n\u2009=\u20092) aged under 35\u00a0years due to poor performance of NAFLD fibrosis score and FIB-4 index for a diagnosis of liver fibrosis in those aged\u2009\u2264\u200935 years30. The remaining 264 individuals with type 2 diabetes were included in the final analysis , ex-smoker (\u2265\u2009100 cigarettes in lifetime and currently a nonsmoker), and current smoker (\u2265\u2009100 cigarettes in lifetime and currently a smoker). Positive exercise was defined as exercising for\u2009>\u2009150\u00a0min/week. Blood samples were collected after an overnight fast. FPG levels were measured by the hexokinase method, and HbA1c levels were measured by high-performance liquid chromatography . Serum insulin levels were measured by immunoradiometric assay . Total cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, and LDL cholesterol were measured by enzymatic colorimetric assay. Liver function tests, including AST and ALT, and renal function tests were measured by the protocol of the central laboratory of SNUBH. HOMA-IR was calculated using the following formula32: HOMA-IR\u2009=\u2009(fasting insulin [\u03bcIU/ml]\u2009\u00d7\u2009FPG [mg/dl]/405). Among individuals with a high NAFLD liver fat score (>\u2009-0.640), serum fetuin-A levels of 41 individuals with DPN and 41 individuals without DPN were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits .Anthropometric indices and neurologic tests were measured by a well-trained research nurse. BMI was calculated as weight (kg) divided by the square of the height (m). Waist circumference was measured at the midpoint between the margin of the lowest rib and the iliac crest. Systolic BP and diastolic BP were measured by an electronic blood pressure metre after 10\u00a0min of rest in a sitting position. Alcohol consumption was assessed by two questions from the Alcohol Use Disorders Identification Test-Consumption: (1) the usual frequency of drinking, (2) the typical quantity of drinking33. The MNSI-PE is scored for abnormalities of foot appearance such as deformities, dry skin, calluses, infections and fissures , ulceration , vibration perception at great toe , and ankle reflexes . The total possible score is 8 points for both feet. DPN was diagnosed when the MNSI-PE score was\u2009\u2265\u20092.5, based on prior studies35. A 10-g monofilament test was considered abnormal when an individual had a sensation of fewer than seven points on one of the two feet36. Abnormal appearance was defined as the presence of any abnormality except ulceration, as ulceration was defined separately. Ankle reflexes were tested using a tendon hammer at the Achilles tendon. Abnormal vibration perception was defined as the absence of vibration perception on either side of the great toe using a 128-Hz tuning fork. A trained nurse performed all neurologic examinations.DPN was assessed using the MNSI, which includes two separate assessments: a 15-item self-administered questionnaire (MNSI-Q) and a lower extremity physical examination (MNSI-PE)37. The NAFLD fibrosis score was calculated according to the following formula: \u2212\u20091.675\u2009+\u20090.037\u2009\u00d7\u2009age (years)\u2009+\u20090.094\u2009\u00d7\u2009BMI (kg/m2)\u2009+\u20091.13\u2009\u00d7\u2009impaired fasting glucose or diabetes \u2009+\u20090.99\u2009\u00d7\u2009AST/ALT\u2009\u2212\u20090.013\u2009\u00d7\u2009platelets (109/l)\u2009\u2212\u20090.66\u2009\u00d7\u2009albumin (g/dl). A NAFLD fibrosis score\u2009>\u20090.676 was used to identify liver fibrosis38. The FIB-4 index was calculated according to the following formula: (age [years]\u2009\u00d7\u2009AST [U/l])/(platelets [109/l]\u2009\u00d7\u2009ALT1/2 [U/l]). A FIB-4 index\u2009\u2265\u20091.3 was used to identify liver fibrosis39. However, for individuals aged\u2009\u2265\u200965\u00a0years, a FIB-4 index\u2009\u2265\u20092.0 was used to identify liver fibrosis as previously reported30.The NAFLD liver fat score was calculated according to the following formula: \u2212\u20092.89\u2009+\u20091.18\u2009\u00d7\u2009metabolic syndrome \u2009+\u20090.45\u2009\u00d7\u2009type 2 diabetes \u2009+\u20090.15\u2009\u00d7\u2009fasting serum insulin (IU/l)\u2009+\u20090.04\u2009\u00d7\u2009AST (U/l)\u2009\u2212\u20090.94\u2009\u00d7\u2009AST/ALT. A NAFLD liver fat score\u2009>\u2009\u2212\u20090.640 was used to identify suspected NAFLD according to a previous report that a score\u2009>\u2009\u2212\u20090.640 detected NAFLD with a sensitivity of 86% and specificity of 71%t tests. Categorical variables were compared using \u03c72 tests. The associations between the presence of DPN and NAFLD liver fat score, NAFLD fibrosis score, and FIB-4 index were analysed using logistic regression models. Multivariable logistic regression analysis was performed including known risk factors for DPN. The prediction performance of liver fibrosis indices and serum fetuin-A levels for DPN and for the absence of abnormal vibration perception or absence of abnormal 10-g monofilament test was assessed by analysing receiver operating characteristic (ROC) curves, and the AUROC was calculated. Based on various cut-off values, we calculated the sensitivity, specificity, positive predictive value, and negative predictive value. In all cases, p\u2009<\u20090.05 was considered statistically significant. Statistical analyses were performed using IBM SPSS version 25.0 . Figures were drawn using GraphPad Prism software .Data were expressed as the mean\u2009\u00b1\u2009standard deviation (SD) or number (%). Variables with a nonnormal distribution were log-transformed prior to analysis. Comparisons of continuous variables between individuals with and without DPN were performed using Student\u2019s unpaired Supplementary Table S1."}
+{"text": "Motoric cognitive risk syndrome (MCR) is a recently defined concept combining objective slow gait and subjective cognitive complaints, in the absence of dementia or significant functional impairment. MCR is associated with an increased risk for dementia but its prognostic value for other mental and physical health conditions is less studied. MCR is quick, inexpensive and easy to measure, making it a potentially useful clinical tool. This review aims to be the first to synthesise all mental and physical health outcomes associated with MCR since the term was coined in 2013. Results from multiple databases  were screened independently and risk of bias was assessed. [Results are currently preliminary but definitive findings will be available in time for the conference] In total, 1057 references were screened, resulting in 34 studies being included, of which 14 will be meta-analysed. Eleven longitudinal studies examined MCR in relation to incident cognitive impairment or dementia conversion. Other prospective studies found that MCR predicted higher risk of mortality (n=2), falls (n=3), post-fall fractures (n=1), gait dysfunction (n=1), and cardiovascular risk factors and diseases (n=1). The results from the 23 cross-sectional studies reporting associations with MCR highlight areas for further study to better understand the biological mechanisms of MCR. By synthesising the latest evidence, this review reinforces the value of MCR for predicting incident dementia, but also adds weight to its value in relation to other important age-related health outcomes."}
+{"text": "In this study, we focused on the influencing factors of renal anaemia in patients with IgA nephropathy and constructed a nomogram model. We divided 462 patients with IgA nephropathy diagnosed by renal biopsy into anaemic and non-anaemic groups. Then, the influencing factors of renal anaemia in patients with IgA nephropathy were analysed by least absolute shrinkage and selection operator (LASSO) regression and multivariable logistic regression, and a nomogram model for predicting renal anaemia was established. Eventually, nine variables were obtained, which are easy to apply clinically. The areas under the receiver operating characteristic (ROC) curve and precision-recall (PR) curve reached 0.835 and 0.676, respectively, and the C-index reached 0.848. The calibration plot showed that the model had good discrimination, accuracy, and diagnostic efficacy. In addition, the C-index of the model following internal validation reached 0.823. Decision curve analysis suggested that the model had a certain degree of clinical significance. This new nomogram model of renal anaemia combines the basic information, laboratory findings, and renal biopsy results of patients with IgA nephropathy, providing important guidance for predicting and clinically intervening in renal anaemia. Among patients with more advanced disease and those on dialysis, approximately 90% have anaemia [IgA nephropathy is currently the most common glomerular disease worldwide, with a high incidence in the Asia-Pacific region , and is The pathogenesis of renal anaemia is complex; the primary mechanism is the reduced production of erythropoietin (EPO) by the paratubular apparatus. In addition, excessive expression of hepcidin, a persistent microinflammatory state, and uraemic toxins have an effect on anaemia ,6,7,8. ATherefore, the early diagnosis of and intervention for renal anaemia is necessary, but the individualized prediction of IgA nephropathy complicated by renal anaemia has been rarely reported and is an urgent problem to be solved. The aim of this study was to establish the first nomogram model for the personalized prediction of the risk of IgA nephropathy complicated by renal anaemia to guide clinical screening of high-risk groups and develop more targeted clinical decisions.22.1A total of 658 patients with IgA nephropathy diagnosed by renal biopsy were enrolled from January 2015 to January 2016. The method of inclusion and exclusion was based on a previous study . Finally2.2According to WHO recommendations, anaemia can be diagnosed in males aged \u226515 years with haemoglobin <130\u2009g/L or in adult non-pregnant females with haemoglobin <120\u2009g/L in regions at sea level [2; CKD stage 2: eGFR: 60\u201389\u2009mL/min/1.73\u2009m2; CKD stage 3: eGFR: 30\u201359\u2009mL/min/1.73\u2009m2; CKD stage 4: eGFR: 15\u201329\u2009mL/min/1.73\u2009m2; and CKD stage 5: eGFR < 15\u2009mL/min/1.73\u2009m2) [To evaluate renal function, we referred to the National Kidney Foundation Kidney Disease Outcomes Quality Initiative clinical practice guidelines for classifying subjects into different stages of CKD based on eGFR , which w1.73\u2009m2) . In thisThe 2017 updated Oxford pathological evaluation criteria  were use2.3https://www.R-project.org). All clinically collected data  were enumeration data and are expressed as frequency (%). Least absolute shrinkage and selection operator (LASSO) regression was performed by the \u201cglmnet\u201d package in R software, the \u201crms\u201d package was used to draw the nomogram and calibration curve, the \u201cpROC\u201d package was used to illustrate the receiver operating characteristic (ROC) curve, the coords function was used to return the values of the variables used in the calculation of the ROC curve, the \u201cmodEvA\u201d package was used for plotting the precision-recall (PR) curve, and the \u201crmda\u201d package was used to draw the decision curve analysis (DCA) curve. LASSO regression is a compression estimation regression method. Its greatest advantage lies in the fact that by performing penalized regression on all variable coefficients, the coefficient of the most relatively insignificant independent variable becomes zero, so that it is excluded from the modelling, improving the modelling stability, and solving the problem of having highly correlated variables in the traditional model [P-value were calculated . A nomogram prediction model was developed based on the results of logistic regression analysis [P < 0.05 was considered statistically significant. As there were a few missing values for some variables in the data, we assumed that the data were missing at random. In R software, we performed multiple interpolations for the missing values by chained equations [The study design and statistical analysis of this study were carried out in strict accordance with the TRIPOD statement for prediction models . All datal model . Therefoanalysis , and allanalysis . A calibanalysis . The DCAanalysis . The nomanalysis , and thequations .33.1A total of 462 patients with IgA nephropathy were divided into an anaemia group (132 cases) and a non-anaemia group (330 cases). 3.2A total of 17 potential risk factors for renal anaemia were included in this study. The 17 variables were reduced by the LASSO regression reducing dimension algorithm, and representative risk factors for renal anaemia were selected. The lambda parameter value with the smallest 10-fold cross-validation error was used as the optimal value for the model, and the number of variables with a non-zero regression coefficient was also counted . The res3.3The results of logistic regression analysis using age, sex, DBP, ALB, CHOL, TG, CKD stage, M, and T are shown in 3.4A model containing the above independent predictors was developed and presented as a nomogram . To appl3.5The DCA of the renal anaemia nomogram is shown in 4With the change in the medical model from evidence-based medicine to precision medicine, the latter has rapidly become the focus of attention of the global medical community. The era of big data provides unlimited possibilities for the realization of individualized medicine, in which treatment plans can be tailored according to the individual characteristics of each patient. Clinical prediction models are increasingly widely used in clinical diagnosis, treatment decisions, and patient prognosis management through comprehensive statistical analyses of various clinical data and have become increasingly important as the value of clinical risk prediction and benefit assessment has increased . The nomWe obtained nine variables that are easy to apply clinically and used them to develop and validate a new tool to predict the risk of IgA nephropathy complicated by renal anaemia. Risk factors extracted from among demographic characteristics, laboratory findings, and pathological findings were included in the nomogram for the individualized prediction of disease occurrence. Internal validation of the cohort data showed that the model had good discrimination and calibration ability, especially via its high C-index, indicating that the model can be widely and accurately used in a large number of clinical samples. These nine variables were age, sex, DBP, ALB, CHOL, TG, CKD stage, M, and T, which were associated with IgA nephropathy complicated by renal anaemia. The nomogram suggests that age \u226560 years (score = 43), female sex (score = 31), DBP \u226469\u2009mm\u2009Hg (score = 36), ALB \u226430\u2009g/L (score = 43), serum CHOL <5.72\u2009mmol/L (score = 23), serum TGs <1.7\u2009mmol/L (score = 23), CKD stage 3 or higher (score = 30 for CKD stage 3 and score = 100 for CKD stages 4\u20135), mesangial hypercellularity , and tubular atrophy/interstitial fibrosis (score = 5 for T1 and score = 38 for T2) may be key factors in determining renal anaemia in patients with IgA nephropathy.In this study, we found that age \u226560 years was an independent predictor of renal anaemia in patients with IgA nephropathy, similar to the study by Melissa E. Stauffer, in which the incidence of anaemia tended to increase with increasing age in elderly CKD patients . The reaIn the nomogram model, the other two factors accounting for the higher weight score and that were independent predictors of concurrent renal anaemia in patients with IgA nephropathy were patients with CKD stages 3\u20135 and pathological findings of more severe tubular atrophy/interstitial fibrosis (T2). We demonstrated that the prevalence of anaemia increases with CKD stage, similar to previous studies . We alsoIn addition, we analysed ROC and PR curves to validate the nomogram model and evaluated the clinical usability and benefits of the prediction tool through DCA, which suggested that this model can be applied to larger clinical samples. However, this study has some limitations. First, our study is a single-centre study, and the model may require further external validation through a multicentre sample study. Second, the risk factors did not include all potential factors affecting renal anaemia, and thus the model may be somewhat biased.In conclusion, we constructed a nomogram model for predicting the risk of renal anaemia in patients with IgA nephropathy. The model achieved a reasonable accuracy, discrimination, and predictive ability, indicating its potential usefulness for the clinical screening of high-risk patients and the development of more targeted intervention strategies."}
+{"text": "This study evaluated the epidemiological implications of arbovirus infections and coronavirus disease (COVID-19) co-occurrences in Esp\u00edrito Santo, Brazil.  This ecological study of dengue, chikungunya, zika, and COVID-19 was performed from January 1 to July 31, 2020.  Esp\u00edrito Santo registered 44,614, 8,092, 3,138, and 91,483 cases of dengue, chikungunya, zika, and COVID-19, respectively . In the 27 and four municipalities with a high incidence of dengue and chikungunya, respectively, the incidence of COVID-19 was 647.0-3,721.7 and 1,787.2-3,403.0 cases per 100,000 inhabitants, respectively.  Esp\u00edrito Santo experienced an overlap of epidemics, especially in urban areas. Aedes aegypti, such as dengue, chikungunya, and zika, are significant public health concerns in Brazil and other countries with the simultaneous occurrence of these diseases. The coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has imposed additional challenges in territories with overlapping epidemics, increasing the demand for healthcare services-,,,Viral infections transmitted by -,,,Dengue, chikungunya, zika, and COVID-19 may present with similar clinical manifestations and laboratory features in the early stages of the diseases, such as an acute undifferentiated febrile illnessesIn Esp\u00edrito Santo State, Brazil, an area with co-circulation of dengue, chikungunya, and zika viruses, the first case of COVID-19 was reported in February 2020 during the seasonal period of arboviral transmissions. The local government adopted many actions during the COVID-19 pandemic such as suspension of activities in educational, commercial, financial, and alimentary sectors. The present study evaluated the concurrent occurrence of these arbovirus infections and COVID-19 in the state and the possible implications of this epidemiological scenario.An ecological study was performed based on the number of probable cases of dengue, chikungunya, and zika and confirmed cases of COVID-19 reported to the Health Department of the Esp\u00edrito Santo State from January 1, 2020 to July 31, 2020 and accessed on September 1, 2020.2 land area. The state\u2019s population is estimated to be approximately 4,018,650 individuals. Its climate is tropical humid, with an annual precipitation of >1,400 mm and an average temperature of 23\u00b0CThe Esp\u00edrito Santo State is located in the southeast region of Brazil and has 78 municipalities in a 46.089,390 kmhttps://mosquito.saude.es.gov.br/planilhasegraficos) along with those of COVID-19 (https://coronavirus.es.gov.br/painel-covid-19-es). Data on the municipalities\u2019 population were obtained from the same source as that for the arbovirus data.Publicly available official data on the reports of dengue, chikungunya, and zika were accessed . The cumulative incidence of arbovirus infections was presented in three levels-low , medium , and high , according to the Health Department of Esp\u00edrito Santo State criteria. The cumulative incidence of dengue was presented similarly according to the Brazilian Ministry of Health criteriaDescriptive analysis of simple frequency was conducted considering the weekly reports between the epidemiological weeks 1 to 31 . The incidences of dengue, chikungunya, zika, and COVID-19 per 100,000 inhabitants were calculated for all municipalities for the entire study period. Maps were produced using QGIS 3.14.15 and shapefile (https://geobases.es.gov.br/downloads). The analyses were performed using Microsoft ExcelFrom January 1, 2020 to July 31, 2020, the Esp\u00edrito Santo State Surveillance Department registered a high number of cases and incidences of dengue, chikungunya, and zika . The incFrom February 29, 2020 to July 31, 2020, the state registered 91,483 confirmed cases of COVID-19. Laboratory confirmation was obtained in 92.1% cases, while the clinicoepidemiological criteria was used to confirm other cases . The inc(Four municipalities experienced a high incidence of dengue and chikungunya, with an incidence of >300 cases per 100,000 inhabitants for both diseases. In these municipalities, the incidence of COVID-19 was 1,787.2-3,403.0 cases per 100,000 inhabitants. In the 27 municipalities with a high incidence of dengue, the incidence of COVID-19 was 647.0-3,721.7 cases per 100,000 inhabitants, while in the four municipalities with a high occurrence of chikungunya, the incidence of COVID-19 was 1,787.2-3,403 cases per 100,000 inhabitants. In the four municipalities with an incidence of zika of >100 cases per 100,000 inhabitants, the incidence of COVID-19 was 1,787.2-2,761.7 cases per 100,000 inhabitants .A. aegypti-,,,Esp\u00edrito Santo State experienced an overlap of epidemics with the introduction of SARS-CoV-2 infection by travelers, followed by its local and community transmission during the peak of dengue, chikungunya, and zika seasonal transmissions. Urban areas were more affected than rural areas, especially in the metropolitan region and municipalities that are regional hubs of economic development. Areas with high human population density favored the transmission of COVID-19 and the arbovirus infections reported in this study, since urban environment is propitious for breeding sites for Health systems dealing with concomitant infectious disease epidemics tend to experience challenges in their different service areas, including laboratory, primary healthcare, hospital, and epidemiological surveillance systems. The high incidence of COVID-19 among health professionals in Esp\u00edrito Santo State, which may affect the capacity of the health system, emphasizes the importance of this challenge. In this state, <30% of dengue, chikungunya, and zika cases were confirmed by laboratory tests. Although laboratory confirmation for arbovirus transmission in endemic countries has a surveillance purpose,Primary healthcare, the entry-level to the health system for patients, is essential in epidemic situations. Professionals at this level must be well trained to manage co-occurring diseases with similar manifestationsMany infectious disease reports also affect epidemiological surveillance, increasing the efforts required to investigate and follow-up the reported casesThis study presents some limitations inherent in ecological studies using secondary data, mainly because COVID-19 continues to affect the state\u2019s health systems. Therefore, the number of reported cases may increase in the months after the study period due to the epidemiological service updates. This study evidences the impact of simultaneous epidemics that went beyond the socio-economic losses and was responsible for numerous deaths in Esp\u00edrito Santo State, with many municipalities affected by at least two of concurrent epidemics, compromising the health system response. Additional studies should be performed to assess the effects of the COVID-19 pandemic in the state on the epidemiological profiles of non-communicable diseases, such as cardiovascular disease, cancer, chronic respiratory disease and diabetes, and communicable diseases, including dengue, chikungunya, and zika."}
+{"text": "Throughout the COVID-19 pandemic, wearing facemasks has become mandatory in many parts of the world. While wearing a mask has a vital role in protecting people against the spread of infection, it has not been free of complications. We are reporting two unique cases of oromandibular dyskinesia induced by wearing a surgical mask.A 58-year-old right-handed man was brought to our attention because of abnormal movements in the oromandibular area (Case 1). His wife reported that the abnormal movements started 3 months earlier, when he started wearing a surgical mask due to the COVID-19 pandemic. The patient denied any inner urge to perform the movements or abnormal sensation over this area; he was unaware of the movements and was not disturbed by them as the movements did not interfere with his daily activities such as speaking and eating. He reported a 1-year history of hand tremor present at rest, for which he had tried primidone, clonazepam, and propranolol without benefit but had not received anti-Parkinson medications. He had no history of dopamine receptor blocking drugs usage, tics, or obsessive compulsive disorder. He had a normal dentition and no history of oral or facial sensory complaints.1). However, wearing a surgical mask elicited abnormal movements in the form of oromandibular dyskinesia which was further enhanced by distraction (video 2). When he took away the mask, the abnormal movements completely disappeared. We asked him to wear a different kind of mask (an N95 mask) and we observed an almost complete resolution of the dyskinesia (video 3). His neurologic examination was otherwise entirely normal, including normal cranial nerves, and normal motor and sensory examination.On neurologic examination, he had normal mental state and cognition (MoCA score was 30 out of 30). He had mild hypokinesia and tremor of the left limbs present at rest. There might have been very occasional and inconsistent low-amplitude side-to-side jaw movements in the absence of wearing the mask; distracting/activating tasks did not bring out any abnormal movements, nor did voluntarily activating facial muscles, talking or protruding the tongue .Removing the surgical mask resulted in complete disappearance of the abnormal movements  induced by wearing a surgical mask.The COVID-19 pandemic has deeply impacted the lives of the world\u2019s population; one of them being the need to use respiratory protective devices including facemasks in public spaces. Wearing masks has been associated with a number of complaints, including fatigue, headache, impaired concentration, and drowsiness.3 Although wearing a\u00a0facemask is not a skilled movement, it does result in some tension and contraction of facial muscles.Whether these movements should be considered similar to a task-specific dystonia is uncertain. Task-specific dystonia is defined as a focal isolated dystonia that presents as loss of motor control occurring almost exclusively during a skilled movement or specific action.4 Patients with oromandibular dystonia usually use sensory tricks such as chewing gum or holding something in their mouth in order to improve their symptoms.5 Wearing a mask might hypothetically serve as a sensory trick for patients with oromandibular dystonia and result in the alleviation of their symptoms. However, in these cases, we see the opposite effect of the facemask as the inducer of the abnormal movement. In Case 1, this seemed to be selective for a softer facemask than a firmer more molded N95 mask, possibly due to the greater sensory input from more contact with the skin of the face in the former.Sensory tricks, also called geste antagoniste, represent the temporary alleviation of dystonia by a specific maneuver; sometimes even thinking or preparing to perform the specific maneuver results in amelioration of the dystonia, pointing to a disturbance of sensory motor integration in the pathophysiology of dystonia.6 In the orolingual region, tardive orobuccolingual dyskinesia and edentulous dyskinesia are important examples of this phenomenon.7 The ameliorative effect of wearing dentures in both of these disorders8 is an interesting contrast to the exacerbating effect of wearing the surgical mask in our patients.One point against calling this movement disorder dystonia is the lack of awareness of its presence in the first patient. This anosognosia-like feature is not at all characteristic of dystonia but is more typical of some choreic disorders such as levodopa-induced dyskinesia and chorea in Huntington\u2019s disease.The first patient had some additional Parkinsonism and evidence of diffuse small vessel disease on brain MRI. Whether his abnormal movements on wearing a mask could be attributed to these findings is unclear. However, the second patient had a completely normal neurologic examination without the mask on, and a normal brain MRI which stand against this possibility.To conclude, we report two patients with oromandibular dyskinesia exclusively induced by wearing a surgical mask. To our knowledge, this is the first report of abnormal movement induced by respiratory protective devices."}
+{"text": "S-nitrosation is a redox-based post-translational modification that mediates nitric oxide (NO) regulation of various aspects of plant growth, development and stress responses. Despite its importance, studies exploring protein signaling pathways that are regulated by S-nitrosation during somatic embryogenesis have not been performed. In the present study, endogenous cysteine S-nitrosation site and S-nitrosated proteins were identified by iodo-TMT labeling during somatic embryogenesis in Brazilian pine, an endangered native conifer of South America. In addition, endogenous \u2013S-nitrosothiol (SNO) levels and S-nitrosoglutathione reductase (GSNOR) activity were determined in cell lines with contrasting embryogenic potential. Overall, we identified an array of proteins associated with a large variety of biological processes and molecular functions with some of them already described as important for somatic embryogenesis . In total, our S-nitrosoproteome analyses identified 18 endogenously S-nitrosated proteins and 50 in vitro S-nitrosated proteins (after GSNO treatment) during cell culture proliferation and embryo development. Furthermore, SNO levels and GSNOR activity were increased during embryo formation. These findings expand our understanding of the Brazilian pine proteome and shed novel insights into the potential use of pharmacological manipulation of NO levels by using NO inhibitors and donors during somatic embryogenesis.Cysteine  SE is aormation . In geneormation . In coniormation . Howeverormation .S-nitrosation of cysteine residues. Among them, protein S-nitrosation, the reversible covalent attachment of a NO molecule to the thiol side chain of cysteine, can be considered the most relevant due to its effects on protein structure, activity and subcellular localization (S-nitrosation reactions (Nitric oxide (NO) is a free radical reactive gas acting as a biological mediator in many physiological processes in plants  . S-nitrolization . GSNO leeactions .S-nitrosation may impact biological processes related to growth and development, phytohormone signaling, immune responses, abiotic stress responses and photosynthesis  events controlling plant developmental processes  Kuntze], an endangered native conifer of South America. Furthermore, we evaluated the endogenous content of \u2013SNO and the activity of GSNOR in cell lines with different embryogenic potential. The stable bond between cysteine (Cys) and iodo-TMT reagent allowed us to sequence the labeled peptides using mass spectrometry, providing both the protein identification and the unequivocal position S-nitrosated Cys residues. Using this strategy, we identified proteins associated with a large variety of functional categories such as oxidation-reduction process, defense response, cellular process, carbohydrate metabolic process and translation-related process. In addition, three proteins already described as important for somatic embryogenesis  were detected as S-nitrosated providing additional evidence for the importance of nitrosative signaling during SE.In the present work, we used a protocol adapted from the Biotin-Switch , replaciA. angustifolia were collected from three open-pollinated trees grown in the Parque Estadual de Campos do Jord\u00e3o  , Campos do Jord\u00e3o, S\u00e3o Paulo, Brazil. Seeds were mixed to form a pool and then kept at room temperature (up to 48 h) before processing.Immature seeds of Embryogenic culture induction was carried out according to To determine the ability to develop somatic embryos, embryogenic cell lines were screened according to g for 10 min at 4\u00b0C. The supernatant was incubated at 37\u00b0C with 1 mM NADH and 5 mM GSNO . GSNOR activity was determined by monitoring the consumption of NADH at 340 nm. Protein concentration was measured by the Bradford assay with BSA as the standard . Samples were then incubated for 60 min at \u221220\u00b0C and centrifuged again at 16,000 \u00d7 g for 20 min at 4\u00b0C. Acetone was discarded, and the pellets were resuspended in the same extraction buffer. An aliquot of 400 \u03bcL of extract or standard was mixed with 1.5 \u03bcL of a 5 mM diaminorhodamine-4M (DAR-4M) solution in DMSO and then divided into two aliquots of 200 \u03bcL. One of these aliquots remained in absolute darkness at room temperature for 5 min, while the other one was transferred into a well in a 0.2 mL PCR plate and placed face-down in a transilluminator , where it remained for 5 min exposed to UV light (302\u2013312 nm provided by 8 W lamps). Afterward, the volume of both aliquots was adjusted to 1 mL with 50 mM phosphate buffer (pH 7.2), and fluorescence was measured in a PerkinElmer LS-55 spectrofluorometer with excitation and emission recorded at 560 and 575 nm, respectively. The differences between fluorescence in UV-treated and -untreated aliquots were used to estimate the concentration of nitrosylated proteins in the extracts by comparing them to a standard curve of GSNO. Protein concentration was measured by the Bradford assay with BSA as the standard .iodo-TMT specific labeling, enrichment and detection of S-nitrosated proteins using Western Blot analysis and detection of TMT labeled peptides using LC-MS/MS were performed following the manufacturer\u2019s protocols , and protein concentration was determined by BCA assay (Pierce BCA Protein Assay Kit) following the manufacturer\u2019s manual. Protein concentration was adjusted at 1 mg/mL in 1 mL of HENS buffer. For in vitro S-nitrosation, proteins were incubated with 500 \u03bcM GSNO  for 30 min at room temperature in the dark. The proteins were incubated with 20 mM methyl methanethiosulfonate (MMTS) at 50\u00b0C for 30 min in the dark with frequent vortexing for blocking free cysteine thiols. Residual MMTS was removed by precipitation for 1 h with three volumes of \u221220\u00b0C acetone. Proteins were resuspended in 1 ml of HENS buffer. Labeling with iodo-TMT was achieved by adding 10 \u03bcL of iodo-TMT Reagent  dissolved in methanol and 1 M sodium ascorbate for 1 h at 37\u00b0C in the dark. The labeling reaction was quenched with 0.5 M dithiothreitol at 37\u00b0C for 15 min. Then, proteins were alkylated with 0.5 M iodoacetamide in 1 mL of HENS buffer at 37\u00b0C for 1 h.For proteomics analyses, PEMs were collected after 14 days of cultivation in MSG proliferation medium, and after 2 and 4 months of somatic embryo maturation. Proteins were extracted from embryogenic cell lines  frozen in liquid nitrogen, and ground to a fine powder in a pre-chilled mortar. Aliquots of 4 g of powder were further ground in 4 ml of HENS buffer . Cell debris was removed by centrifugation   and digested overnight at 37\u00b0C. The peptides were acidified with 10% (v/v) trifluoroacetic acid (TFA) to final concentration of 0.5% and desalted with 50 mg Waters Sep-Pak tC18 columns and eluted with 70% (v/v) acetonitrile (ACN) and 0.1% (v/v) TFA. For peptide enrichment, the labeled peptides were dried by vacuum centrifugation and resuspended in 120 \u03bcL TBS buffer . The peptide samples were incubated with 200 \u03bcL anti-TMT resin for 2 h at room temperature with end-over-end rocking. After the supernatant was removed, the resin was washed four times with 200 \u03bcL TBS followed by three washing steps with 100 \u03bcL HPLC-grade water. Peptides were eluted twice with 400 \u03bcL TMT elution buffer , and pooled eluates were dried by vacuum centrifugation.Alkylated samples were precipitated with acetone for 60 min, centrifuged by 10,000 \u00d7 A. angustifolia transcriptome database  were used for bioinformatics analyses. Proteins were reported with at least one matching peptide since, in many cases, only one site in a protein is S-nitrosated  and analyzed using an EASY-nLC system  coupled to LTQ-Orbitrap Velos mass spectrometer . The peptides were loaded onto a C18 PicoFrit column  and separated with a gradient from 100% mobile phase A (water with 0.1% FA) to 45% phase B (95% Acetonitrile with 0.1% FA) during 100 min, 45\u201395% in 5 min and 15 min at 95% phase B at a constant flow rate of 300 nL/min. The LTQ-Orbitrap Velos was operated in positive ion mode with data-dependent acquisition in positive ion mode. The three most abundant peptide ions found in full scan were selected for MS/MS and dynamically excluded for a duration of 30 s. The acquisition method is a top 3 \u00d7 2, which consists of three HCD events followed by 3 CID events on the same ions selected for the first 3 HCD events. The first scan is a FTMS full scan with resolution of 15000 FWHM and 380\u20131800 m/z mass range. The three most intense ion found in the full scan were selected and fragmented using HCD with a normalized collision energy of 50, default charge state 3, isolation width 3 m/z and activation time 30 ms. The fragment ions obtained were acquired in the Orbitrap analyzer with a resolution of 7500 FWHM. The same three most intense ions were subsequently fragmented using CID with a normalized collision energy of 35, isolation width 3 m/z and activation time 30 ms. All raw data were accessed in the Xcalibur software  and Proteome Discoverer software version 1.4.0.288  was used to search the HCD MS/MS spectra against a concatenated database consisting of the 48,246 EST sequences in the database . The seatrosated .Proteins labeled with the iodo-TMT reagent were resolved on 12% SDS-PAGE gels and transferred onto nitrocellulose membrane using a semi-dry apparatus , at 100 V for 90 min at 4\u00b0C. The membrane was stained with Ponceau S to check equal protein loading. Immunoblots were blocked in 5% non-fat dry milk in TBST buffer for 1 h and were probed with anti-TMT antibody  at 1:1000 dilution for 1 h. For chemiluminescence detection, blots were probed with anti-mouse IgG-HRP conjugate (Pierce) at 1:20,000 dilution and detection was made as suggested by the manufacturer using SuperSignal West Pico Chemiluminescent Substrate .1 with default settings. Search for conserved domains was carried out using SMART  with sequence identity 77.47% was used as template for modeling 3D structure of A. angustifolia Chitinase IV. Structure was generated using the Swiss-model  terms were assigned using PANNZER annotation program 1 with deng SMART 2, PROSIT PROSITE 3, and InInterPro 4. Proteitor tool 5. The crss-model  and USCFss-model  was appl-SNO 1.0 6 and iSNO-AAPair 7. Cysteib server 8.t-test was used to determine statistically significant differences (P < 0.05) in the mean values between pairwise comparisons (cell lines Y1 \u00d7 B2 during proliferation and cell lines Y1 \u00d7 B2 during maturation). Analyses were performed using \u201cR\u201d .For physiological analyses, PEMs were collected after 14 days of cultivation in MSG proliferation medium, and after 2 and 4 months of somatic embryo maturation. For determination of endogenous -SNO levels four independent biological replicates (obtained from four Petri dishes) were used . For GSNOR activity three independent biological replicates (obtained from three Petri dishes) were used . Student\u2019s After 4 months of cultivation, two cell lines were selected according to their potential to differentiate somatic embryos. PEMs of cell line Y1  demonstrP < 0.05) in cell line Y1 than that observed for cell line B2 (P < 0.05) in cell line B2 compared to Y1  for the Y1 cell line compared to the B2 cell line (P < 0.05) than the cell line responsive to the development of somatic embryos (Y1)  . After 2yos (Y1) . Howeverin vivo and in vitro S-nitrosation) were identified as containing iodo-TMT-labeled cysteine residues  and 13 peptides were observed in both in vitro and in vivo samples (S-nitrosated cysteine residue and 3 peptides (4.23%) with two S-nitrosated cysteine residues. A Chitinase IV (UniProt ID Q5NTA4) identified in cell lines Y1 and B2 after 2 months of maturation showed the highest number of S-nitrosated peptides   .S-nitrosation sites, enabling the identification of 28.57% of Cys residues labeled with iodo-TMT reagent (S-nitrosation in vivo) against 20.68% of those predicted by ISO AAPAIR  presented the best results for prediction of O AAPAIR . In the residues .S-nitrosated proteins (S-nitrosated proteins in cell lines Y1 and B2 was detected only after cell lysate treatment with 500 \u03bcM GSNO (in vitro S-nitrosation)  in the Y1 cell line, and 10 S-nitrosated cysteine residues (corresponding to 10 proteins) in the B2 cell line  . Using tell line , 2. Of tell line . In the in vitro S-nitrosated proteins, we carried out Gene Ontology (GO) analyses to functionally classify S-nitrosated proteins into their biological process, molecular function, and cell component , transport process (6.25%), carbohydrate metabolic process (6.25%), cell wall organization or biogenesis (6.25%), protein catabolic process (6.25%), adenosine triphosphate (ATP) biosynthetic process (6.25%), and S-adenosylmethionine cycle (6.25%). For B2 cell line, 20% of the identified proteins were associated with protein folding, 20% with S-adenosylmethionine cycle/biosynthetic process and 20% with oxidation-reduction process. Finally, translation-related process, amino acid metabolism, carbohydrate metabolic process, and proteolysis were annotated, each category containing approximately 10% of total identified proteins. Among the cellular component classification, in vitro S-nitrosated proteins were predicted to localize to the cytosol, ribosome and apoplast in both Y1 and B2 cell lines. For the Y1 cell line in vitro S-nitrosated proteins were also localized to the nucleus, mitochondria, vacuole and integral component of membrane, whereas for the B2 cell line, proteins were associated with the plasmodesma and chloroplast. In the molecular function classification, catalytic activity and binding were the two main categories annotated for cell lines Y1 and B2.In order to elucidate the regulatory roles of identified omponent . In cellS-nitrosated proteins (in vivo S-nitrosation) during somatic embryo formation. As a control, tests were also carried out treating the cell lysate obtained after 2 and 4 months of maturation with GSNO (in vitro S-nitrosation). In total, we identified 14 endogenously S-nitrosated cysteine residues (corresponding to 10 proteins) in cell line Y1 after 2 and 4 months of maturation (in vitro S-nitrosated cysteine residues (corresponding to 43 proteins) (in vitro S-nitrosated cysteine residues (corresponding to 19 proteins) after 2 and 4 months of maturation  . For celroteins) . Finallyturation .114VSTWTC120EYDTLPGVPQDEGK was detected with S-nitrosated Cys residues in positions Cys114 and Cys120 on both Y1 and B2 cell lines, with Cys120 being identified as a hydrophobic ligand binding site. Moreover, in cell line B2, a S-nitrosated Cys residue (Cys82) was also detected in a position before a tyrosine residue (Y) was also identified as a hydrophobic ligand-binding site. For Class IV chitinase, two S-nitrosated peptides AINSMEC272NGGNPSEVSSR and KYPC191VSGK were detected in both Y1 and B2 cell lines, being Cys272 localized one position after a glutamic acid residue (E) identified as a sugar-binding site. Furthermore, in the B2 cell line, a S-nitrosated Cys residue (Cys170) was detected occupying a position before a tyrosine residue (Y) identified as a sugar-binding site.Six proteins, namely 40S ribosomal protein S11-beta, ribosomal protein 1, endo-beta-N-acetylglucosaminidase, mitochondrial benzaldehyde dehydrogenase, putative intracellular pathogenesis-related protein and Class IV chitinase, were identified as endogenously S-nitrosated in both Y1 and B2 cell lines after callus cultivation in the presence of ABA and osmotic agents . Among tS-nitrosated proteins were also carried out for samples obtained in vivo (only for proteins detected after 2 months of embryo maturation as after 4 months it was not possible to detect in vivo S-nitrosated proteins)  analyses to functionally classify roteins)  and afteroteins) . ConsideS-nitrosation sites to evaluate the possible effects of S-nitrosation (Cryptomeria japonica (PDB code: 5H7T). The in silico structural analysis demonstrated that C170 and C272 NGGNPSEVSSR, KYPC(4)VSGK, EIAAFFANAAHETGGFC(17)YTEER and TAVWFWMVNSNC(12)HSAITSGK] were shown in Taking into account the importance associated with Class IV chitinase activity during somatic embryogenesis in Angiosperms and Gymnosperms  and for rosation . To perfand C272  are situS-nitrosated proteins during PEMs proliferation. Moreover, GSNOR activity was significantly higher in cell line Y1 in comparison to B2 during PEMs proliferation  (in vivo and in vitro) using two different Brazilian pine cell lines. Using this strategy, we observed that some of the S-nitrosated-Cys residues detected in the in vivo analysis were also observed for the in vitro fraction  of each category differed markedly during PEMs proliferation and somatic embryo maturation. Considering that endogenously S-nitrosated proteins were not detected during PEMs proliferation, cell lysate treatment with GSNO was used to identify potential targets for S-nitrosation. It is likely that immediately after the transfer of the PEMs from the proliferation to the maturation medium, the embryogenic cells may be exposed to nitrosative/oxidative stress conditions. According to S-nitrosation during the first days of somatic embryo maturation due to limited source material, we considered that the detection of S-nitrosated proteins in vitro is relevant for the identification of potential signaling pathways affected by S-nitrosation during the initial differentiation of PEMs into early somatic embryos. Below, we discuss selected GO functional categories  to gain a better insight into the potential biological significance of S-nitrosated proteins identified during Brazilian pine somatic embryogenesis.Normally, works reporting protein O donor) . In our fraction . Similarr plants , S-nitro vacuole . With a in vitro S-nitrosated proteins during PEMs proliferation of cell line Y1  was S-nitrosated after treatment with GSNO in both cell lines, although at different cysteine residues , 2. FBA  sativus . Beyond  sativus . During  sativus . In assomulation . In Cyclpersicum  and Cyat Sternb.  pyruvate Sternb. , 2021. CDuring somatic embryo maturation in cell lines Y1 and B2, we detected three endogenously S-nitrosated glycosidases, namely endo-\u03b2-N-acetylglucosaminidase  , 4. To tin vitro S-nitrosated  is a component of the tricarboxylic acid cycle and catalyzes the interconversion of malate and oxaloacetate coupled to NAD reduction  were identified during PEMs proliferation as trosated , 2. Malaeduction . In leavtabolism . NO dono pattern . L-ascor pattern . Xanthin pattern . In addi pattern . To the S-nitrosated proteins associated with the oxidation-reduction process were the primary biological process observed in the blocked cell line B2 (35.7% of detected proteins) (S-nitrosation in plants. In Brazilian pine dehydroascorbate reductase (DHAR) is more abundant in cell lines responsive to maturation promoters (ABA and osmotic agents) in comparison to blocked cell lines (24PFSQR) was S-nitrosated after 2 months of maturation in B2, and after 4 months of maturation in Y1 , (at Cys128 in cell line Y1 and at Cys345 in cell line B2)  . In controteins) . Accordill lines , and in on in Y1 , 4. Afteline B2) , 4. BALDline B2) . BA was  process , suggestin vitro S-nitrosated during PEMs proliferation only in the B2 cell line  was detected as ell line . MAT andell line . MAT  were identified by iodo-TMT labeling qualitative proteomics during PEMs proliferation and somatic embryo development. Notwithstanding the contrasting embryogenic potential in cell lines Y1 and B2, an overall increase in the content of endogenous SNO and GSNOR activity was observed during cell line cultivation in the medium supplemented with ABA and osmotic agents. In vitro and in vivo S-nitrosated proteins were associated with a large variety of biological processes, with some of them already described as important for somatic embryo formation . To the best of our knowledge, this is the first time that protein S-nitrosation is demonstrated during somatic embryogenesis. By identifying new and previously described plant S-nitrosated proteins as differently present at distinct contexts of somatic embryogenesis, our data facilitates future studies focusing on elucidating how the activity and function of these proteins is affected by this PTM during somatic embryo differentiation.To summarize, endogenous SNO levels and GSNOR activity were determined during somatic embryogenesis in Brazilian pine. Furthermore, cysteine PXD033177.The data presented in the study are deposited in the ProteomeXchange Consortium repository, accession number: AB, GC, RZ, RS, and MR performed the experiments. RS, BM, LF, EF, and AW analyzed the data. AW conceived and coordinated the research. RS, LF, EF, and AW wrote the manuscript. All authors contributed to the article and approved the submitted version.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": "Lipoid congenital adrenal hyperplasia (LCAH) is a rare and severe disorder that is caused by mutations in the steroidogenic acute regulatory protein (StAR). Non-classic LCAH is defined as late-onset glucocorticoid deficiency and even complete male external genitalia in 46,XY individuals. However, to date, few cases of non-classic LCAH have been reported.It was attempted to describe the clinical characteristics of a male child with complete male external genitalia in terms of age of onset, adrenal function, and biochemical indicators. Previously reported cases were also reviewed to investigate the relationship of age of onset with enzymatic activity in non-classic LCAH.STAR gene were identified via genetic testing. The literature review resulted in identification of 47 patients with non-classic LCAH from 36 families. The mutational analysis showed that c.562C>T mutation was prevalent in patients with non-classic LCAH, accounting for 37.2% of the total mutant alleles, which could reflect the founder effect on the non-classic LCAH population. In total, 28 46,XY patients were reported, including 22 (78.5%) cases with complete male external genitalia and six (21.5%) cases with different degrees of hypospadias.The patient with complete male external genitalia was diagnosed with non-classic LCAH, in which the reason for his referral to a local hospital at the of age 1.25 years was progressive skin hyperpigmentation, and plasma adrenocorticotropic hormone (ACTH) level was elevated to higher than 1,250 pg/ml. The compound heterozygous mutations c.772C>T/c.562C>T in The clinical phenotypes of non-classic LCAH are highly variable. Routine physical examination, laboratory measurement, genetic testing, and, importantly, enzymatic activity assay may facilitate the early diagnosis of non-classic LCAH. The age of primary adrenal insufficiency (PAI) onset may not be a diagnostic basis for non-classic LCAH, and enzymatic activity assay determination may be more effective. STAR gene located on chromosome 8p11.2 and consists of seven exons. It plays an indispensable role in the delivery of cholesterol from the outer to the inner mitochondrial membrane in the initial steps of steroid synthesis  is a rare and severe disorder that is caused by mutations in the steroidogenic acute regulatory protein (StAR) . StAR isof onset . Non-claIn the present study, the patient has undergone extensive physical and laboratory examination for the evaluation of primary adrenal sufficiency. Collection of his clinical features and laboratory data was attempted when he was initially admitted to a hospital for adrenal insufficiency. Routine examination included testing of liver and kidney functions and measurement of the levels of serum insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-1-binding protein 3 (IGFBP3). Furthermore, assessment of renin-angiotensin-aldosterone system (RAAS) function and gonadal function, measurement of plasma adrenocorticotropic hormone (ACTH) and cortisol levels, and ACTH stimulation test were performed. Bone age was assessed using the Greulich\u2013Pyle (G-P) and Tanner\u2013Whitehouse 3 (TW3) methods. Clinical data on the first and follow-up visits were collected, including birth history, history of growth and development, physical examination, laboratory data, and radiological data.STAR gene sequence from GenBank. The STAR gene was amplified using polymerase chain reaction (PCR). The enzymatic activity of four mutants of the STAR gene was herein investigated, including some mutations that had been functionally analyzed previously or merely reported. Non-steroidogenic COS-7 cells  were cultured in Dulbecco\u2019s modified Eagle\u2019s medium  supplemented with 12% fetal bovine serum and 1% antibiotics (50 units/ml penicillin and 50 \u03bcg/ml streptomycin) at 37\u00b0C in an incubator (5% CO2 with humidity of 95%). Cells were seeded into 12-well plates and transfected using Lipofectamine 2000 reagent  with 1 \u03bcg of empty plasmid, wild-type STAR, or mutant STAR constructs together with 1 \u03bcg of F2 plasmid, which were kindly provided by Walter L. Miller, UCSF, San Francisco, USA . Primers were designed according to the sco, USA , at a co\u201cCongenital lipoid adrenal hyperplasia\u201d was searched in the PubMed and Wanfang databases to retrieve English and Chinese published articles. A total of 106 articles were published until September 2021, in which the diagnosis of non-classic LCAH was confirmed by genotyping, and participants\u2019 clinical characteristics were described.All data were analyzed using Microsoft Excel software.3 and the right testis was 17 * 11 * 10 mm3, and the adrenal ultrasound displayed normal data without enlargement.The Chinese male child was born at a gestational age of 37 weeks with complete male external genitalia . His birth weight was 3.34\u00a0kg, and his birth length was 50\u00a0cm. He had no history of adrenal insufficiency, and the only reason for his referral to a local hospital at the age of 1.25 years was progressive skin hyperpigmentation. Laboratory examination revealed plasma ACTH of more than 1,250 pg/ml , cortisol 8\u00a0a.m. was 1.36 \u03bcg/dl , cortisol 4 p.m. was 0.97 \u03bcg/dl , and testosterone level was <0.1 ng/ml, and he was therefore diagnosed with primary adrenal insufficiency (PAI). Hydrocortisone was given as treatment for PAI. At the age of 43 months, he was referred to our department for the elevated ACTH level and a micropenis. His height and weight were 111 cm [+2.9 standard deviation (SD)] and 24 kg ( +3 SD), respectively; his blood pressure was 86/56 mmHg. Examination showed generalized hyperpigmentation of the skin. His stretched penile length was 1.5\u00a0cm, testicular volume was 3\u00a0ml, and Tanner stage of PH1 was recorded. Laboratory examination revealed that the levels of sodium and potassium were 135  and 4.22  mmol/L, and no hyperkalemia and hyponatremia were detected. Moreover, the following laboratory data were collected: plasma ACTH level of 1,744  pg/ml, basal 17-hydroxyprogesterone (17-OHP) level of 0.07  ng/ml, cortisol level of 0.2  \u03bcg/dl, plasma RAAS activity of 9.99  ng/ml/h, aldosterone (Ald) level of 0.1  pg/ml, angiotensin II level of 134.22  pg/ml, and testosterone level of 0.01  ng/ml. The ACTH stimulation test showed no changes in the levels of cortisol and 17-OHP Table\u00a01.STAR mutation for c.772C>T (p.Q258X) and c.562C>T (p.R188C). Pedigrees of his family with STAR gene mutations and Sanger sequencing confirmation of compound heterozygous mutations analyzed by the direct DNA sequencing are shown in The results of exome sequencing revealed compound heterozygous STAR gene indicating the location of mutations (including enzymatic activity) identified in patients with non-classic LCAH. These mutations included 14 missense mutations without nonsense, frameshift, and splice-site mutations. Moreover, c.562C>T,STAR-R188C was detected as the most common mutation of STAR gene in non-classic LCAH patients, accounting for 37.2%, including 14 homozygous mutations and one compound heterozygous mutation. Enzymatic activity assay showed that c.562C>T,STAR-R188C mutation had reserved 13.6%  . Gene sequencing is the most appropriate diagnostic method to differentiate these diseases. Ishii et\u00a0al.  and external genital development that is consistent with the chromosomal karyotype . The litThe clinical phenotypes of non-classic LCAH are highly variable. Routine physical examination, laboratory measurement, genetic testing, and, importantly, enzymatic activity assay may facilitate the early diagnosis of non-classic LCAH. Further study is required to verify the findings of the present case report.The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Ruijin Hospital Ethics Committee, Shanghai JiaoTong University School of Medicine. Written informed consent to participate in this study was provided by the participants\u2019 legal guardian/next of kin.WL, TZ, and LZ as co-first authors wrote and translated this article. XW, JW, LY, YX, ZD, WW, and SS collected all the clinical information of these patients. RH, GN, CL, and XM are the corresponding authors who edited and reviewed this manuscript and approved the version to be published.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": "Time spent on being with others  and being alone (solitude) in day to day life might reflect older adults' agentic regulatory strategies to balance the needs to belong and to conserve energy. Motivated from a joint lifespan psychological and social relationship theoretical perspective, this study examined how time spent on social interactions and solitude alternatively unfolds within individuals in daily life, relating to individual differences in trait\u2010level well\u2010being and fatigue. Over 21\u2009days, a total of 11,172 valid records of social interactions were collected from 118 older adults (aged 65\u201394\u2009years) in a smartphone\u2010based event\u2010contingent ambulatory assessment study in Switzerland. On average, a social interaction episode lasted 39\u2009min and a solitude episode lasted 5.03\u2009hr. Multilevel models showed that, at the within\u2010person level, a longer\u2010than\u2010usual social interaction preceded and was followed by a longer\u2010than\u2010usual solitude episode. Moderator analyses showed that older adults with higher trait life satisfaction and lower trait fatigue spent even more time in social interactions after longer solitude episodes, amplifying the solitude\u2010then\u2010interaction association. Our findings suggest that whereas social interaction is a means to improve well\u2010being, solitude is also an integral part in older adults' daily life supporting energy recovery. Individuals' lifestyle is, at least in part, formed by personal choices of time and energy investment on different daily activities and is closely related to health and well\u2010being in older age  and time spent alone (i.e. solitude) alternatively unfolding in daily life might reflect older adults' agentic regulatory processes to meet and balance the sense of belonging and the sense of energy. Further evidence is needed to form the basis for the development of guidance for lifestyle interventions for older adults. We draw from both social relationship and lifespan psychological theories and research to inform the current study.Several psychological theories have proposed that to belong and to be affiliated are fundamental psychological needs of human beings. For example, the belongingness hypothesis proposes that the need to belong is a pervasive drive that motivates human beings to form at least a minimum quantity and quality of interpersonal relationships and that failure to satisfy this fundamental need has a detrimental impact on well\u2010being  subsequently influences the other interpersonal activity duration . Some studies have described time spent on lifestyle activities, including social and solitary activities, by the so\u2010called \u2018yesterday interview\u2019  prior solitude episode duration and subsequent social interaction duration; and (2) prior social interaction duration and subsequent solitude episode duration. As shown in Figure\u00a0 prior soSecond, we aimed to examine whether and how the within\u2010person processes differed between older adults with different trait\u2010level well\u2010being and energy. We expected older adults with more personal resources (indicated by high trait\u2010level well\u2010being and low trait\u2010level fatigue) would strategically spend more time in social interactions (for their need to belong/well\u2010being) and less time in solitude episodes (for energy conservation). As shown in Figure\u00a0This study aimed to examine alternation patterns of social interactions and solitude in healthy and community\u2010dwelling older adults. To do so, we use data from a larger project on digitalization and social lives of older adults  intraindividual effects and cross\u2010level moderation effects with a power of \u03b2\u00a0=\u00a0.80. Participants were recruited via advertisements in local and national newspapers and through a database of participants hosted at the University of Zurich. Participants had to meet the following criteria: Using digital devices to communicate, having sufficient hearing and vision, being fluent in German, and being 65\u2009years or older. Participants were compensated with 150 Swiss Francs for their participation.A total of 120 older adults from German\u2010speaking regions of Switzerland participated in the study. Based on prior research on daily social interactions  out of 21 possible days. Some participants completed interaction reports beyond the 21\u2010day period. Number of social interactions reported per day decreased over the study period. We excluded data from two participants: One participant did not complete the take\u2010home questionnaire and the other one misunderstood the instructions on recording social interactions.After providing informed consent, participants took part in a baseline session where they received detailed instructions on the study protocol and a take\u2010home questionnaire to collect their demographic information and psychological variables, including positive and negative affect, life satisfaction and fatigue. After the baseline session, participants were given an iPhone 4S to complete a 21\u2010day event\u2010contingent observation period. The questionnaires were administered with the app \u2018iDialogPad\u2019 (G Mutz). Participants were asked to record any spoken interactions  that lasted longer than 5\u2009min and any text\u2010based conversations . The five min cut\u2010off was implemented on the basis of earlier research on meaningful social interactions in daily life to reduce participant burden during the data collection period  by answering the following question: \u2018How long did the conversation last?\u2019 (German \u2018Wie lange hat das Gespr\u00e4ch gedauert?\u2019). Conversations with the same person that happened intermittently within the same context, not necessarily engaging in a lengthy discussion but just exchanging a few words now and then, were counted as one social interaction. For example, watching a movie together over 2\u2009hr was counted as one social interaction, because they may have chatted from time to time during the movie. We removed 265 (0.02%) interactions that were reported as shorter than 5\u2009min as they did not meet the inclusion criteria. Different from spoken interactions, participants did not report the duration of text\u2010based conversations . Intermittent text\u2010based conversations that extend over hours often include other ongoing activities. We counted each text\u2010based conversation  as 5\u2009min. Changing the duration to 10\u00a0min did not impact our findings.SD\u00a0=\u00a010.47\u2009hr, range\u00a0=\u00a00.43\u2013159.83\u2009hr [6.7\u2009days]).Based on information on social interaction onset and duration, we calculated the time in solitude when no social interactions were reported. Some social interactions overlapped in time and the solitude episodes in between were smaller than zero. For example, a text message could be sent during a telephone call. A total of 719 records (0.06%) of these negative solitude episodes were recoded as zero, indicating that there was no interval between two overlapping social interactions. A follow\u2010up analysis excluding these records led to identical findings. Additionally, sleeping time was counted in the solitude episodes. In our study, there were 2403 (22%) solitude episodes that lasted overnight from a previous day to the next day(s). The average duration of these episodes was 16.94\u2009hr  to 5 (extremely), participants indicated their answer to the question \u2018how often have you felt this feeling during the last year\u2019. Scores were averaged for each affective factor. Higher scores indicated higher affect levels in each domain. Cronbach's alpha was 0.80 for trait positive affect and 0.84 for trait negative affect.The German version of The Positive And Negative Affect Schedule , number of physician\u2010diagnosed health conditions during the last 2\u2009years, and marital status . We also controlled for the number of days of the study  and a binary variable indicating weekday (=\u20090) versus weekend (=\u20091).We controlled for covariates that have been found to be closely related to social interactions and well\u2010being  of prior within\u2010person activity duration on the outcome; and ui1 is the deviation of a participants' slope from the sample\u2010level slope.Multilevel models were used to examine the alternation of time spending on the two activities. Alternation is operationalized as the associations between prior activity duration  and subsequent activity duration . According to procedures that are typically used to analyse ambulatory assessment data . We first examined models without any covariates and then added the covariates as main effects into the models. We standardized all moderators with a Likert scale  into z\u2010scores  for ease of interpretation and centred the covariates of age, gender, health conditions and marital status at the sample level. Analyses were conducted in R  as it is and added moderators to the Level 2 Equations (2) and (3), turning them into Equations (4) and (5) as follows:SDage\u00a0=\u00a05\u2009years, rangeage\u00a0=\u00a065\u201394\u2009years; 40% women). Most participants (97%) were retired and 55% of them were married. After secondary school education , the majority of participants (98%) completed further training and about 22% completed university education. About 52% participants lived in cities, 16% in small towns and 33% in villages and small villages. About 18% of participants had an income up to 4\u2032000 Fr., 53% of them had an income between 4\u2032001 to 8\u2032000 Fr., and 22% of them had an income of more than 8\u2032000 Fr per month (5% participants did not report their income).We examined a total of 11,172 valid records of social interactions. The 118 participants had a mean age of 72\u2009years  and a solitude episode lasted 5.03\u2009hr . On a 1\u20135 scale, participants reported an average trait positive affect of 3.82 (SD\u00a0=\u00a00.51) and an average trait negative affect of 1.62 (SD\u00a0=\u00a00.52). They reported an average score of 5.39 (SD\u00a0=\u00a01.12) in trait life satisfaction on a 1\u20137 scale and 29.41 (SD\u00a0=\u00a014.13) in fatigue on a 0\u2013100 scale. Participants had on average 2.30 (SD\u00a0=\u00a01.93) health conditions.On average, a social interaction episode lasted 0.65\u2009hr . That is, when the prior solitude episode was 1\u00a0hr longer than usual, participants subsequently engaged in a 0.01\u2009hr (i.e. 0.6\u00a0min) longer\u2010than\u2010usual social interaction. In reverse, when the prior social interaction was 1\u00a0hr longer than usual, participants subsequently experienced a 0.28\u2009hr (i.e. 16.8\u2010min) longer solitude episode . Please refer to Figure\u00a0Table\u00a001\u00a0=\u00a0\u22121.68, SE\u00a0=\u00a00.62, p\u00a0=\u00a0.008, STE\u00a0=\u00a0\u22120.24) and lower trait life satisfaction  were associated with overall higher solitude duration (i.e. main effects), while trait negative affect and trait fatigue were unrelated to overall solitude duration. None of the trait well\u2010being and fatigue variables moderated the within\u2010person interaction\u2010then\u2010solitude association. As shown in Table\u00a001\u00a0=\u00a0\u22120.12, SE\u00a0=\u00a00.05, p\u00a0=\u00a0.033, STE\u00a0=\u00a0\u22120.20) was related to lower overall social interaction duration (i.e. main effect), while trait positive and negative affect and trait life satisfaction were unrelated to overall social interaction duration. Furthermore, trait life satisfaction  and trait fatigue  moderated the within\u2010person solitude\u2010then\u2010interaction association. That is, compared to participants with a sample\u2010average trait life satisfaction and trait fatigue, after a 1\u00a0hr longer\u2010than\u2010usual solitude episode, older adults with 1 SD higher trait life satisfaction experienced a 0.01\u2010hr (\u03b311) even longer social interaction and older adults with 1 SD higher trait fatigue experienced a 0.01\u2010hr (\u03b311) shorter social interaction. Please refer to Figure\u00a0Results of the second research aim on between\u2010person differences in the within\u2010person associations are shown in Tables\u00a0This study examined alternation of time spent on social interactions and solitude in older adults as a reflection of resource allocation in the social domain in later life . The similar effect sizes suggest that compared to the own typical time in social interaction and in solitude, for each individual, the time change from social interaction to solitude was of similar size to the relative time change from solitude to social interaction. In line with our expectation, duration of solitude and social interactions mutually and positively interacted with each other within individuals. In other words, time spent on one activity is related to time spent on the preceding activity. We interpret that these positive within\u2010person associations reflect an agentic regulatory process involved in how time is spent in old age. As shown in Figure\u00a0At the within\u2010person level, our findings show that a longer\u2010than\u2010usual solitude episode preceded and was followed by a longer\u2010than\u2010usual social interaction. Notably, the effect sizes (STE) for both associations were comparable, although the unstandardized coefficients (duration in minutes) were different . Although trait\u2010level well\u2010being and fatigue might explain time spent on social interactions conditional on prior solitude episodes, they did not seem to have an effect on time spent on solitude conditional on prior social interactions. To understand the results, we offer the following perspectives.First, in line with our conception of a regulatory process consisting of social interactions as a means to well\u2010being and solitude as an opportunity to take a break and recharge, our findings may have suggested that engagement in social interactions differed between older adults with different available resources, but everybody might have returned to solitude of similar duration as a default state after a social interaction. More specifically, the transition from solitude to social interaction might need motivation and energy to engage in  and thus individuals with lower trait levels of well\u2010being and fatigue might have difficulties to engage in social interactions. In contrast, the transition from social interaction to solitude might require less energy and be more under older adults' control (e.g. leaving a conversation) and, thus, there may be less room for between\u2010person differences to be moderated by our target variables. Additionally, the association between solitude duration and energy recovery might not be linear. Thus, there might be a threshold when energy levels are restored and there is no benefit of additional solitary time. Thus, individual differences in trait\u2010level well\u2010being and fatigue do not necessarily need to have an association with time spent on solitude.Second, solitude is a complex phenomenon  as indication that these episodes also included meaningful waking solitude time (besides sleeping). We thus considered the bias that might be introduced by including sleep time to be limited. Nevertheless, we acknowledge that sleeping is crucial for energy restoration and the role that sleep might play in the process of alternating time allocation should be carefully examined by future research. Third, we note that although the effect sizes of the interaction\u2010then\u2010solitude association and the solitude\u2010then\u2010interaction association were similar, the explained variances of the two associations beyond the covariates differed .Tables S1\u2010S3Click here for additional data file."}
+{"text": "P\u2009=\u20090.027), and 6.7% in all pregnant women. Results also demonstrated that gestational diabetes mellitus (GDM) and MetS were more likely to appear in the maternal age \u226535\u2009years group than the maternal age 20\u201334 years group . Risk for preterm delivery and eclampsia were increased with raised MetS (RR 3.434 and RR 1.800); MetS in women aged \u226535 years had the largest area under the curve (AUC) , and its optimal cutoff point was \u226524.998\u2009kg/m2, and the optimal cutoff point for total cholesterol (TC)  predicting MetS was \u22654.955\u2009mmol/l. MetS in pregnancy are associated with the occurrence of preterm delivery and eclampsia, and pre-BMI and TC can predict MetS in the maternal age \u226535 group.We aimed to explore the association of BMI in pre-pregnant women with metabolic syndrome in pregnancy in advanced maternal age. A total of 229 maternal women and 536 maternal women participated in this study. Pregnancy women underwent a 75\u2009g-oral glucose tolerance test and maternal lipid profile test between 24 and 28 weeks. Data about biological and sociodemographic characteristics were recorded for each case. The metabolic equivalent (Met) was 9.6% in the maternal age \u226535 group, 5.4% in the age 20\u201334 group ( Less research about MetS in pregnant women has been investigated \u20133. The nOur research was aimed at discussing the associations between BMI in pre-pregnant and metabolic syndrome during gestation in advanced maternal age and exploring the roles of maternal age and lipid profiles in the metabolic syndrome of pregnancy. In addition, we hope to find more evidence to predict metabolic equivalence in pregnant women and to develop better perinatal care in older mothers.This study enrolled pregnant women during the period from January 2017 to December 2018 in Cangzhou Central Hospital. Women during singleton pregnancy underwent a 75\u2009g oral glucose tolerance test and maternal lipid profile test between 24 and 28 weeks. Women with previous diagnoses of diabetes, hypertension, and dyslipidemia during a singleton pregnancy and women with overt diabetes diagnosed before 20 weeks of pregnancy were excluded.A questionnaire with information about biological and sociodemographic characteristics was used for each case. These data included prepregnancy weight, used for calculating the pregestational BMI, and history of adverse pregnancy. In addition, at enrollment, this information included blood pressure, height, and current weight used to calculate BMI and circumference during pregnancy and to analyze the full lipid profile  blood collection.We excluded participants with pregestational diabetes mellitus (DM), hypertension, psychiatric disorders, chronic maternal diseases , congenital malformations, and multiple pregnancies. Subjects with imperfect data were also excluded.2); (2) high blood sugar; (3) hypertension; (4) lipid metabolism disorder. The maternal factors investigated were pre-BMI, anthropometric measurements, blood pressure (BP), and metabolic profile. GDM was diagnosed meeting one of the following standards: fasting glycemia \u22655.1\u2009mmol/l; one-hour levels \u226510\u2009mmol/l; two-hour levels \u22658.5\u2009mmol/l, respectively, after a 75\u2009g-OGTT. Hypertension was defined as follows: systolic blood pressure \u2265140\u2009mmHg or diastolic blood pressure \u226590\u2009mmHg, or both, after 20 weeks of gestation, but before delivery or miscarriage on at least 2 occasions separated by at least 4 hours. Maternal pregnancy BMI was categorized into underweight (<18.5\u2009kg/m2), normal weight (18.5\u201324.9\u2009kg/m2), overweight or obesity  according to the World Health Organization BMI classification [ATPIII, CDS, and IDF had published the diagnostic criteria for MetS, but there was no confirmed diagnostic gold standard. There are also no clear diagnostic criteria for the gestational metabolic syndrome. MS was diagnosed according to the classification by the Diabetes Branch of the Chinese Medical Association (CMA) . Women wAdverse pregnancy outcomes include maternal and fetal outcomes. Maternal pregnancy outcomes include miscarriage, preeclampsia, oligohydramnios, polyhydramnios, anemia, premature rupture of membranes, placental abruption, and preterm birth, and women with a history of adverse events during pregnancy, including previous miscarriage, fetal death, stillbirth, and fetal malformations. Low birth weight, macrosomia, fetal malformation death or death, fetal distress, and a low Apgar score (Apgar score \u2264 7 at 1 or 5 minutes) were defined as fetal outcomes.Follow-up was performed until the end of pregnancy (delivery or miscarriage). For women with GDM, the CDS-recommended protocol for maternal hyperglycemia control was followed. Diseases that occur during pregnancy are treated under the guidance of a professional doctor.t-test. The Mann\u2013Whitney U test was used when the variable distribution was abnormal, and the \u03c72 test or Fisher's exact test was used to compare categorical variables. Through ROC curve analysis, the optimal cut-off points of maternal lipid profiles in prepregnancy and midpregnancy for predicting the metabolic syndrome in pregnancy were determined. Each optimal cutoff point was based on the maximum Youden Index (sensitivity\u2009+\u2009specificity\u2009\u2212\u20091). AUC was calculated to evaluate the predictive powers. The confidence interval was set as 95% and the significance level was set at P\u2009<\u20090.05.Statistical analyses were done using Statistical Product and Service Solutions (SPSS), version 23.0 . The Kolmogorov\u2013Smirnov test was employed to determine the measurement data normality. Normally distributed continuous variables were analyzed using the P\u2009=\u20090.001; 2  vs. 1 ; 3  vs. 1 , both P\u2009<\u20090.001).Overall, 229 aged \u2265 35 and 536 aged 20-34 maternal women participated in this research . The anaP\u2009<\u20090.001). The prevalence of overweight and obesity were more likely to happen in the maternal age \u226535 group than in the maternal age 20\u201334 group , and the prevalence of underweight was more likely to occur in the maternal age 20\u201334 group than the maternal age \u226535 group . The incidence of normal weight between the two groups has no statistical difference.BMI in the maternal age \u226535 group was higher than those in the 20\u201334 age group, and the difference had statistical significance , and 6.7% in all pregnant women. Prevalence of metabolic syndrome components showed that GDM and MetS were more likely to happen in maternal age \u226535 group than in the maternal age 20\u201330 group .Met frequency was 9.6% in the maternal age \u226535 group, 5.4% in the maternal age 20\u201334 group (P\u2009<\u20090.001), and the birth weight in the maternal age \u226535 group was more than that in the maternal age 20\u201334 group . The prevalence of macrosomia between the two groups has no statistical difference. . We also found that the GWG in the maternal age \u226535 group was less than those in the maternal age 20\u201334 group .We found that women aged \u226535 years exhibited shorter gestational weeks than the younger controls , and the frequency of eclampsia was 13.6% and 1.4% (P\u2009=\u20090.013) in the subgroups with and without the MetS. The frequency of preterm labor was 17.2% and 5.7% (P\u2009=\u20090.030) and the frequency of eclampsia was 27.6% and 2.0% (P\u2009<\u20090.001) in both subgroups with the maternal age 20\u201334 group. Results showed that macrosomia was more likely to happen in the maternal age \u226535 group than in the maternal age 20\u201334 group   .For preterm delivery and eclampsia, the relative risk at study participants are shown in In participants aged \u226535 years, we discovered that pre-BMI  and the TC level  were associated with an increased risk of MetS. In women aged 20\u201334 years, we observed that pre-BMI , TG level , LDL-C level , and age  were related to an increased risk of MetS .2; the optimal cutoff points for TC  predicting MetS were \u22654.955\u2009mmol/l. Pre-BMI predicting MetS had the largest AUC ). The optimal cutoff points were \u22651.075\u2009mmol/L for TG predicting MetS and \u22652.745\u2009mmol/L for LDL-C predicting MetS in women aged 20\u201334 years  and its optimal cutoff point was \u226524.998\u2009kg/m34 years .Older women are at an increased risk of adverse outcomes caused by the decay of reproductive function. There are several definitions to characterize MetS , 7. In 2Analysis of adverse pregnancy outcomes showed that the incidence of GDM in women aged \u226535 years is higher. Considering this, our study suggested that an advanced maternal age accelerated the occurrence rate of adverse pregnancy outcomes , 11. EldIn this chapter, we observed significant differences between the two groups regarding the kinds of adverse pregnancy outcomes. Some scholars also believe that there is insufficient evidence to prove whether an advanced maternal age is an independent direct risk factor for preterm birth and small-for-gestational-age birth \u201315. We fOur current study showed that pre-BMI, blood pressure (BP), and metabolic syndrome may be related to premature delivery and eclampsia. Given this, it is meaningful to have the necessary counseling and education about weight management and blood pressure control during pregnancy before conception . The utiOur study indicates that pre-BMI can predict MetS in pregnancy. Our findings suggested lipid profiles can predict MetS, TC in women with maternal age \u226535 years, and TG and LDL-C in maternal women aged between 20 and 34. Examining lipid profiles in pre-BMI and midtrimester can provide insight into the underlying mechanisms of MetS and contribute to a better understanding of the risk factors underlying each condition. Maternal obesity increases the risk of premature death and cardiovascular diseases. Pregnancy and early postpartum offer opportunities for interventions to identify obesity and reduce its adverse outcomes , 18.MetS in pregnancy are more to occur in the maternal age \u226535 group than in the younger controls. MetS are related to the occurrence of preterm delivery and eclampsia; pre-BMI and TC can predict MetS in the maternal age \u226535 group. Advanced maternal age women should have more attention to weight control before pregnancy."}
+{"text": "Ube3a mouse models to investigate the maturation of medium spiny neurons (MSNs) from dorsomedial striatum. MSNs of mutant mice matured properly till postnatal day 15 (P15) but remained hyperexcitable with fewer excitatory synaptic events at later ages, indicative of stalled striatal maturation in Ube3a mice. Reinstatement of UBE3A expression at P21 fully restored MSN excitability but only partially restored synaptic transmission and the operant conditioning behavioral phenotype. Gene reinstatement at P70 failed to rescue both electrophysiological and behavioral phenotypes. In contrast, deletion of Ube3a after normal brain development did not result in these electrophysiological and behavioral phenotypes. This study emphasizes the role of UBE3A in striatal maturation and the importance of early postnatal reinstatement of UBE3A expression to obtain a full rescue of behavioral phenotypes associated with striatal function in AS.Angelman syndrome (AS) is a severe neurodevelopmental disorder (NDD) caused by loss of functional ubiquitin protein ligase E3A (UBE3A). Previous studies showed that UBE3A plays an important role in the first postnatal weeks of mouse brain development, but its precise role is unknown. Since impaired striatal maturation has been implicated in several mouse models for NDDs, we studied the importance of UBE3A in striatal maturation. We used inducible   Neurodevelopmental disorders (NDDs) are brain diseases caused by heterogenous genetic lesions that often generate overlapping clinical phenotypes, including intellectual disability, autism spectrum disorder (ASD), motor dysfunction, and epilepsy , 2. SuchLoss of neuronal ubiquitin protein ligase E3A (UBE3A) results in AS, a severe NDD characterized by intellectual disability, absence of speech, gross and fine motor deficits, behavioral abnormalities, and often epilepsy . ClinicaImpaired fine and gross motor dysfunction and lack of speech are distinct clinical phenotypes in individuals with AS  and presUbe3a mice, we identified the critical window for rescuing the electrophysiological and the behavioral operant conditioning phenotypes.Here, we used whole-cell patch clamp recordings, to determine how lack of UBE3A affects the maturation of MSNs of the DMS during development. Additionally, we used the lever press task to validate previously identified deficits in operant learning of AS mice. Furthermore, using inducible mStop/p+Ube3a (LSL) and m+/p+Ube3a wild-type (WT) littermates. Upon increasing depolarization current, we observed a significant left shift in the average firing frequency versus injected current (F-I) relationship between MSNs from LSL and WT mice .To identify how absence of UBE3A changes the electrophysiological properties of MSNs, we performed whole-cell current clamp recordings from MSNs in striatal slices from adult (P110\u2013P175)  WT mice , pointin WT mice . Values Next, we asked whether the changes in rheobase reflected changes in active, passive, or both properties of MSNs. To investigate the active properties that are dependent on voltage-gated ion channels, we focused on several parameters, including maximum firing rates, the F-I slope, and the AP firing threshold. We found no differences in any of these parameters between LSL and WT mice, including the maximum firing rate , F-I sloNext, we investigated whether absence of UBE3A affects excitatory postsynaptic currents of MSNs. We voltage-clamped the MSNs from DMS at \u201370 mV, to record spontaneous excitatory postsynaptic currents (sEPSCs) . LSL micTogether, these findings indicate that absence of UBE3A enhances the intrinsic excitability and decreases the frequency of sEPSCs in MSNs from DMS, which from now on we will refer to as electrophysiological phenotypes.In rodents, the striatum matures during the first 4 weeks postnatal \u201345. To iThe F-I relationship between MSNs from LSL and WT was similar at P15 but started to left shift at P21 and became significantly different at this time point . In lineIn contrast to the passive properties, the F-I slope, maximum firing rate and AP threshold remained unchanged between genotypes , A\u2013C, thTo find when changes in synaptic transmission start to appear, we voltage-clamped MSNs at \u201370 mV to record sEPSCs . SimilarCollectively, these results show that at P15, MSNs of both LSL and WT mice have similar electrophysiological phenotypes, and differences between genotypes emerge at P21.F values for each model  = B* + Constant]. In contrast, the rest of the parameters, including the F-I slope, maximum firing frequency, and AP threshold, were not well fit by any of the tested models  curve estimation function . Althougch model  suggested models .F = B* + Constant] fit for LSL versus WT , and sEPSC frequency (B parameter) and lower \u201csteady-state mature\u201d   . Next, wonstant) . For botonstant) . These vonstant)  and the onstant)  showed tonstant) . SimilarUbe3a gene reinstatement. To test this hypothesis, we used our previously validated inducible AS mouse model mStop/p+-CreEsr1*+Ube3a (Ube3a gene. We treated the mice with either tamoxifen (LSL-TAM) or vehicle (LSL-VEH) at P21 or P70 and performed electrophysiological recordings after P120  mice in which the Ube3a gene is efficiently deleted upon TAM injection  does not affect striatal function. Moreover, mice exclusively expressing UBE3A-Iso3 at 70% of total UBE3A levels also show no behavioral deficits  brain development in cortical, striatal, or dopaminergic neurons. Our study indicates that combining these approaches with electrophysiological measurements, as well as behavioral testing , will allow us to further understand how neuronal changes lead to behavioral dysfunction. Such knowledge is critical to identify therapies and to understand when during development these therapies should be administered to get optimal therapeutic benefit.The striatal deficits observed by us and others  in the mUbe3a mouse lines: mStop/p+Ube3a    mouse line was provided by the Philpot laboratory, the Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill . The tm1YelgUbe3a line was maintained in the 129S2 background (129S2/SvPasCrl) by crossing male m+/pStopUbe3a mice with female 129S2 WT mice. The tm1.1BdphUbe3a line was maintained in the C57BL/6J background (Charles River) by crossing male m+/pFloxUbe3a mice with C57BL/6J WT females. The mouse lines do not show audiogenic seizures   and Ube35882092) , 49. Them+/pStopUbe3a mice with WT C57BL/6J males (Charles River) to generate mStop/p+Ube3a (referred to as LSL) and their WT littermate controls, m+/p+Ube3a (referred to as WT), in a 129B6F1 hybrid background. The mice in this group were between 15 and 180 days old, as further detailed in the results. To establish the critical window of the described phenotypes (m+/pStopUbe3a with homozygous B6Tg(CAG-Cre/Esr1*)5Amc/J  . To test if UBE3A is necessary after neurodevelopment (m+/pFloxUbe3a mice with homozygous 129S2Tg(CAG-CRE/Esr1*)5Amc/J (MGI:2182767) male mice to generate mFlox/p+-CreEsr1*+Ube3a, whereupon TAM treatment, UBE3A levels are reliably decreased to the levels expressed by the mStop/p+Ube3a mice . For simplicity, we used an abbreviated nomenclature for the different genotypes and treatments used in this study  , to genely shown , 37, andelopment , we cros/p+ mice , 49 and is study . The micTAM (0.10 mg of TAM/g of body weight) or vehicle (oil) was delivered by intraperitoneal injections for 5 consecutive days as previously described , 48, 49.2PO4, 26 NaHCO3, 10 glucose, 7 MgSO4, and 0.5 CaCl2, as previously described  with infrared illumination and differential interference contrast video microscopy. Patch electrodes (3\u20134 M\u03a9) were backfilled with internal solution that contained the following (in mM): K-gluconate 125, NaCl 10, HEPES 10, EGTA 0.2, MgATP 4.5, NaGTP 0.3, and K-phosphocreatine 10, adjusted for pH to 7.2\u20137.4 using KOH. Whole-cell recordings were obtained from MSNs in the DMS using Multiclamp 700B amplifiers (Axon Instruments). Signals were low-pass\u2013filtered at 4 kHz and digitized at 20 kHz using Digidata 1440A (Molecular Devices) acquisition interfaces, and acquisition and analysis were performed using Clampex (Axon Instruments) or Minianalysis software (Synaptosoft).Electrophysiological experiments were performed on animals between P15 and P180. Coronal striatal slices of 300 \u03bcm were prepared in ice-cold, modified ACSF containing the following (in mM): 125 NaCl, 3 KCl, 1.25 NaHescribed . The recFor current clamp recording, series resistance and pipette capacitance were monitored and cancelled using bridge and capacitance neutralization. To obtain the passive properties and the firing pattern, we recorded voltage responses in current clamp mode from neurons held at around \u201380 mV, while we injected a family of 500 ms square pulses starting from \u2013100 pA with 10 pA increments, delivered at 0.2 Hz.The pipette capacitance was compensated, and series resistance was continuously monitored but was not compensated. Only recordings with a stable series resistance of less than 20 M\u03a9 were used for analysis. sEPSCs were recorded in gap-free protocol in Clampex, for 10 minutes, while the cells were voltage clamped at \u201370 mV.g, at 4\u00b0C for 5 minutes), supernatants were collected, and concentration was measured using the BCA protein assay kit (Thermo Fisher Scientific). Lysate concentrations were adjusted to 1 mg/mL. A total of 20 \u03bcg of each sample was loaded on the gel, and a semidry transfer was performed . The blotted nitrocellulose membrane was probed with antibodies directed against E6AP  and actin . A fluorophore-conjugated secondary goat anti-mouse antibody  was used, and the protein was detected using Odyssey Scanner system (LI-COR Biosciences). Quantification was done using Odyssey 3.0 software (LI-COR Biosciences).For the Western blot analysis, frontal cortex and striatal tissue were dissected from adult mice and immediately frozen in liquid nitrogen. The lysates were prepared by adding lysis buffer  supplemented with protease inhibitor mixture  to the tissue, and homogenization was achieved by sonication. After centrifugation . To analyze the frequency, events were counted over 10 minutes of recording. To obtain the average events, for each cell, at least 100 nonoverlapping events were detected and averaged. The peak amplitude of the average sEPSCs was measured relative to the baseline current.To obtain the best fit line to each parameter measured at different postnatal time points, we used the regression analysis and curve estimation module in SPSS. The data from LSL obtained at different time points were pulled and a series of mathematical functions including linear, logarithmic, inverse, quadratic, cubic, power, compound, S-curve, logistic, growth, and exponential were compared based on their relative goodness of fit. The single dependent variable (measured parameter) was predicted by a single independent variable  . After iP values for each statistical test are reported either in figure legends or in tables, as indicated. P \u2264 0.05 was considered statistically significant. When only 2 groups were compared, we used the 2-tailed, unpaired t test. When more than 2 groups were compared, we used either 1-way RM ANOVA, 2-way/factorial ANOVA, or 2-way RM ANOVA. If a significant effect of the independent factors was observed, we applied post hoc analysis, as indicated.The statistical analysis was performed in IBM SPSS software. The statistical tests performed for data are indicated in figure legends. The exact t test in Specifically, to test the effect of genotype  on the electrophysiological properties, we used the following: 1-way RM ANOVA (firing frequency as repeated measures) in Ube3a gene reinstatement/deletion, we used the following: 2-way RM ANOVA (firing frequency as RM) followed by post hoc Bonferroni\u2019s in To analyze the interaction between treatment and genotypes and of both factors on the electrophysiological phenotypes and the To test the interaction between sex  and genotype  and of both factors on the lever press phenotype, we used the following: 2-way RM ANOVA (days as repeated measures) in All animal experiments were conducted in accordance with the European Commission Council Directive 2010/63/EU . Mice were housed in individual ventilated cages and kept on a  12-hour light/12-hour dark cycle, at 22\u00b0C \u00b1 2\u00b0C and had ad libitum access to food and water. Animal welfare was checked daily by animal caretakers and experimenters throughout the experiments. All cages contained basic bedding and nesting material.DCR designed, performed, and analyzed the electrophysiological experiments and wrote the manuscript. IW performed the Western blots and the behavioral experiments and contributed to the figures. MDV performed and analyzed electrophysiological and behavioral experiment and contributed to the manuscript. JVDB performed the behavioral experiments. YE designed and supervised the research and wrote the manuscript."}
+{"text": "This study aimed to assess the efficacy of dual-tracer [18F-FDG and 68Ga-DOTA-SSAs. Specifically, we analyzed the imaging results using \u03c72 tests for classification variables, paired-sample tests for number of BMs, Wilcoxon's signed rank test for number of lesions, and the Kruskal\u2013Wallis test for standard uptake value (SUV) ratio comparison. The correlation of dual-tracer SUVmax with Ki-67 index was analyzed by Spearman's correlation coefficient.  We retrospectively enrolled 74 GEP-NEN patients with BMs from two centers, who underwent dual-tracer PET/CT from January 2014 to March 2021. We compared and analyzed effectiveness of the dual PET/CT imaging techniques on the BMs, based on 68Ga-DOTA-SSAs was better at detecting BMs for NET G3 (P=0.049 for SUVT/B and P=0.026 for the number of metastatic lesions). In addition, statistical significance was found among osteogenesis group, osteolysis group, and the no-change group . What is more, liver and bone SUVmax and Ki-67 index were positively correlated in 18F-FDG imaging , and negatively correlated in 68Ga-DOTA-SSAs imaging .  The detection efficiencies of dual-tracer PET/CT imaging in patients with different pathologies showed discordant for detecting liver metastases and BMs in group neuroendocrine tumor (NET) G3, 68Ga-DOTA-SSAs was superior to 18F-FDG for detecting BMs in NET G1/G2 (well and moderately differentiated NETs), as well as in NET G3 (poorly differentiated NETs). Relatively good differentiation was observed in the osteogenesis group. In addition, dual-tracer PET/CT imaging results were observably correlated with tumor differentiation. Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are heterogeneous neoplasms that show neuroendocrine differentiation, with peculiar histomorphological and clinical features . NENs ar18F-fluorodeoxyglucose (FDG) and 68Ga somatostatin receptor analogs (SSAs), including 68Ga-DOTA-Tyr3-octreotate (68Ga-DOTA-TATE), 68Ga-DOTA-D-Phe1-Tyr3-octreotide (68Ga-DOTA-TOC), and 68Ga-DOTA-1-Nal3-octreotide (68Ga-DOTA-NOC), which are targeted to somatostatin receptors Ga-FAPI, which indicated high uptake of [68Ga]Ga-FAPI in NET of different origins in several case reports [The current study conducted more investigations than previous studies of BMs in patients with GEP-NENs. For example, Scharf et al.  examinedg et al.  demonstr and BMs . Since tDOTATATE . An emer reports , 41. AltT/B to reduce the impacts caused by different tomographs from two centers, we should not ignore this data distribution problem in the analysis. In addition, this retrospective analysis has an inherent limitation: conclusions need to be confirmed in future randomized controlled studies.The current study also had some limitations. The sample size was small for the NET G3 and NEC groups. Besides, despite using SUV68Ga-DOTA-SSAs was superior to 18F-FDG for detecting BMs in terms of the number and SUVT/B of the detected lesions. We analyzed patients with tumors with different degrees of differentiation separately, including G1, G2, and G3 NET groups. We also observed that patients with osteogenesis showed relatively well-differentiated lesions, which might provide promising evidence for NEN differentiation. In addition, dual-tracer PET/CT imaging showed strong complementary correlations with tumor differentiation. Further randomized controlled trials are needed to explore the detection advantages of dual-tracer imaging in patients GEP-NENs with BMs.In this study, we compared the multidimensional performances of dual-tracer PET/CT imaging in GEP-NEN patients with BMs."}
+{"text": "Women and Clinical Microbiology 2021 is an inaugural Research Topic in Frontiers in Cellular and Infection Microbiology Clinical Microbiology aimed at commemorating the accomplishments of women in science by compiling articles focused on women\u2019s health or research studies conducted by women as a primary or corresponding author. This Research Topic was chosen because less than 30% of researchers worldwide are women, which is likely due to long-standing biases and gender stereotypes that have discouraged women from entering science-related fields. To help promote gender equality and overcome these stereotypes, Frontiers in Cellular and Infection Microbiology introduced this topic to promote research by women scientists studying cellular and infection microbiology. The nine publications, 8 research studies and 1 minireview, presented by women scientists highlight the diversity of research performed across the entire breadth of clinical microbiology, from female reproductive health to diagnostic approaches and beyond.Gao et\u00a0al., Li et\u00a0al., Huang et\u00a0al., and Lu et\u00a0al.) tackled different issues in relation to maternal health during pregnancy. One study by Gao et\u00a0al. compared the Copan Walk Away Specimen Processor (WASP), an automated diagnostic tool, to conventional manual culture methods. The research team used this tool to detect pathogens such as Chlamydia spp., Neisseria gonorrhoeae, Candida albicans, group B Streptococcus (GBS), and Ureaplasmaurealyticum, from swabs collected from the lower reproductive tract of pregnant women. Because the presence of these pathogens can lead to perinatal complications, including death of the fetus, accurate identification of these microbes is critical. The data revealed that conventional methods could distinguish more pathogen types compared to WASP; however, WASP was better at detecting single colonies. The Copan swabs were also tested using fluorescence-based qPCR and immunochromatography to identify Chlamydia trachomatis, but only the qPCR approach worked. Since the Copan WASP has been successfully applied to other specimen types , there is potential for this approach in women\u2019s health. Indeed, the quick turnaround time as well as the use of less resources  makes automated diagnostics a worthy pursuit.Four manuscripts , the vaginal and intestinal microbiota were characterized in three groups of women. These groups included pregnant women with AMA over 35 years of age, pregnant women of non-advanced maternal age (NMA) less than 35 years, and non-pregnant women over the age of 35 (control group). The results showed that the AMA group had significantly increased \u03b1-diversity and decreased \u03b2-diversity as well as enriched Bifidobacterium and decreased Lactobacillus johnsonii in the vaginal microflora compared to NMA group. As L. johnsonii can inhibit the growth of pathogens and reduce the incidence of preterm birth, the authors suggested that decreased levels were associated with an increased risk of complications during pregnancy in women with AMA. A third study by Li et\u00a0al. evaluated how clinical and microbiological characteristics of mixed vaginitis in the third trimester impacted adverse pregnancy outcomes, which is an understudied area of research. Over 1,600 women in late-stage pregnancy were included in this analysis and 66 had mixed vaginitis. Independent risk factors for developing mixed vaginitis included previous vaginitis during pregnancy, positive glucose tolerance test during pregnancy, sex during pregnancy, and history of genital infection before pregnancy. The main adverse outcome for pregnant women with mixed vaginitis, however, was found to be peripartum infection.A second maternal health study, which was conducted by In the fourth manuscript relevant to maternal health, Lu et\u00a0al. examined the role of human milk lactoferrin in the prevention of GBS disease by investigating its antimicrobial and anti-biofilm activity against a diverse set of 25 phenotypically and genetically diverse isolates. Importantly, lactoferrin showed growth inhibition and anti-biofilm activity against 14 and 21 of the clinical GBS isolates, respectively. The maternal colonizing GBS isolates were more susceptible to inhibition than the neonatal invasive isolates. In addition, variation in susceptibility was observed across multilocus sequence types when treated with 750 \u00b5g/mL of lactoferrin, while different capsular types had differences in susceptibility at 250 \u00b5g/mL of lactoferrin. Lactoferrin also had better anti-biofilm activity against isolates with enhanced biofilm production, highlighting its potential for reducing bacterial densities and subsequently limiting transmission to neonates.Boucherabine et\u00a0al.), the development and validation of a rapid predictive risk scoring function , and the analysis of a diagnostic approach . To begin with, Boucherabine et\u00a0al. reported the potential of health care worker (HCW) mobile phones and environmental surfaces to serve as reservoirs for SARS-CoV-2 and multidrug-resistant superbugs. The point prevalence study was conducted in the emergency room, which was divided into a non-COVID-19 patient zone and a zone dedicated to confirmed or suspected COVID-19 patients. Three methicillin-resistant Staphylococcus aureus isolates and one pan-drug resistant carbapenemase-producing Acinetobacter baumannii isolate were recovered from mobile phones. A shotgun metagenomics analysis revealed that a total of 76 and 46 different antibiotic resistance genes were found in the mobile phone and environmental samples, respectively. In addition, one mobile phone from the COVID-19 zone tested positive for SARS-CoV-2, demonstrating that phones serve as fomites and may contribute to the transmission of clinically relevant bacterial and viral pathogens. The authors therefore recommended the introduction of highly efficient phone sanitization methods in hospitals and public areas.Other areas of focus within this topic include molecular surveillance  in intensive care unit (ICU) patients by evaluating clinical risk factors as well as lymphocyte subtyping. IC is highly prevalent in ICU patients and outcomes are poor especially if the initiation of antifungal therapy is delayed. A total of 1054 ICU patients were included of which 69 had IC and were included in the analysis. Multivariate logistic regression was used to identify which clinical risk factors and lymphocyte subtypes significantly correlated with IC. Notably, a CD8+ T-cell count \u2264143 cells/mm3, receipt of high-dose corticosteroids , receipt of carbapenem/tigecycline, APACHE II score \u226515, -\u03b2-D-glucan (BDG) positivity, and emergency gastrointestinal/hepatobiliary (GIT/HPB) surgery were highly predictive for rapid diagnosis of IC in ICU patients. Risk scores were also stratified into three different cohorts based on the former factors. Together, this risk assessment has the potential to be useful to predict IC in ICU patients.The aim of Senok et\u00a0al. evaluated a novel lateral flow assay for the detection of Panton Valentine leukocidin (PVL), a virulence factor that can impact the severity of Staphylococcal infections. S. aureus isolates from 129 patients were screened for PVL by the lateral flow assay and molecular detection of pvl. Excellent correlation was observed between the new PVL assay and molecular testing as 76 isolates were negative by both molecular and antigen testing, while 53 isolates were positive by both molecular and antigen testing. However, the authors noted 4 isolates with weak antigen positivity and as a precaution state a diagnostic sensitivity of 92.5%, a negative predictive value of 95% as well as specificity and positive predictive value of 100% can be expected. This work supports the implementation of an accurate assay for PVL detection from Staphylococcal isolates diagnostically. It will be interesting to see in the future the clinical utility of this assay.In another study, Urrea-Quezada et\u00a0al.) and a minireview  were also included in this Research Topic. The work of Urrea-Quezada et\u00a0al. focused efforts on the gp15 protein as a novel vaccine target for Cryptosporidium, a parasitic cause of gastroenteritis that can be severe in children and the immunocompromised. To identify immunogenic candidate peptides, a library of 5 synthetic peptides (4 against gp15 and 1 against gp40) was created and screened for IgM and IgG responses in serum collected from 39 human cases of cryptosporidiosis and 90 healthy individuals as controls. In comparison to the control group, the authors observed a statistically significant response in two of the synthetic peptides (A133 and A32) designed from gp15. This strong antibody response supports the further investigation of gp15 as a vaccine candidate for cryptosporidiosis. Finally, the mini-review by Moubareck and Halat summarized studies dealing with the link between COVID-19 infections and carbapenemase-producing Enterobacteriaceae and Acinetobacter. Concerning Enterobacteriaceae, their search revealed reports showing increases in colonization or infection with carbapenemase-producing Enterobacteriaceae in COVID-19 versus non-COVID-19 patients. Furthermore, they highlighted reports showing a marked increase in carbapenemase-producing Enterobacteriaceae in regions with recent decreases of such organisms, or a shift in carbapenemase-producing Klebsiella pneumoniae dominant genetic types. Similarly, they highlighted studies reporting an increase in infections caused by carbapenem-resistant A. baumannii in COVID-19 patients, identifying A. baumannii as the most common pathogen isolated from COVID-19 patients, with carbapenem resistance rates above 90%. Together, these studies further demonstrate the need for antibiotic stewardship and widespread surveillance for carbapenemase-producing pathogens in order to contain their spread.A description of initial studies that can aid in the development of a vaccine (In conclusion, this inaugural Research Topic highlighted the impactful research being conducted by women scientists around the world. Not only are they advancing the field of clinical microbiology in terms of women\u2019s health, but in other important areas as well.KP-W, SM, and SV participated in the editorial review process for these manuscripts. KP-W, AC, MK, and SV wrote the first draft the manuscript. All authors contributed to manuscript revisions, read, and approved the submitted version.This work was supported in part by funds and/or facilities provided by the Cleveland Department of Veterans Affairs to KMPW, the Veterans Affairs Merit Review Program Award 1I01BX002872 to KMPW from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development. SM is funded by the MSU Foundation. MK is supported by JSPS KAKENHI Grant Numbers JP22H03300 and JP22K19622.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.The contents do not represent the views of the U.S. Department of Veterans Affairs or the U.S. Government."}
+{"text": "A broad spectrum of lethal kidney diseases involves the irreversible destruction of the tubular structures, leading to renal function loss. Following injury, a spectrum of tissue\u2010resident epithelial stem/progenitor cells are known to be activated and then differentiate into mature renal cells to replace the damaged renal epithelium. Here, however, we reported an alternative way that tissue\u2010resident cells could be activated to secrete multiple factors to promote organ repair. At single\u2010cell resolution, we showed that the resident SOX9+ renal epithelial cells (RECs) could expand in the acutely injured kidney of both mouse and human. Compared to other cells, the SOX9+ RECs overexpressed much more secretion related genes, whose functions were linked to kidney repair pathways. We also obtained long\u2010term, feeder\u2010free cultured SOX9+ RECs from human urine and analysed their secretory profile at both transcriptional and proteomic levels. Engraftment of cultured human SOX9+ RECs or injection of its conditional medium facilitated the regeneration of renal tubular and glomerular epithelium, probably through stimulating endogenous REC self\u2010activation and mediating crosstalk with other renal cells. We also identified S100A9 as one of the key factors in the SOX9+ REC secretome. Altogether, the abilities to extensively propagate SOX9+ RECs in culture whilst concomitantly maintaining their intrinsic secretory capacity suggest their future application in cell\u2010free therapies and regeneration medicine. The secretome of SOX9+ RECs, especially soluble S100A9 protein, could play a critical role in renal tubules regeneration by triggering resident SOX9+ REC proliferation and intercellular communications among adjacent tubular epithelial cells (TECs), immune cells and vascular endothelial cells (VECs). However, controversy remains concerning the identity of SOX9+ cells responsible for mature epithelial repair after kidney injury.in vivo and in vitro.Recently, a body of evidence has bolstered that the paracrine mechanism is also responsible for AKI therapy.In our study, we identified the endogenous SOX9+ RECs could be activated after damage in mouse and human. Single\u2010cell transcriptional analysis revealed the secreted signature of SOX9+ RECs, whose function contributed to renal restoration. Based on a feeder\u2010free culture system, we showed that the long\u2010term, cultured SOX9+ RECs could participate in renal tubular and glomerular epithelium regeneration by secretory function.22.1All animal experiments were carried out in accordance with Chinese National Guidelines GB/T 35892\u201320181, as well as under the guidance of the Institutional Animal Care and Use Committee of Tongji University. To establish a unilateral partial nephrectomy (UPN) mouse model, 8\u201310\u2009weeks C57BL/6 mice were euthanized by intraperitoneal injection of 3.7% chloral hydrate (0.5\u00a0g/kg body weight). The left lateral peritoneum was cut to expose the left kidney, and the renal artery was clamped with hemostatic forceps. About one\u2010third of the left kidney was cut off from the upper pole of the kidney using a surgical blade. After cleaning off the blood, the incision was sealed with FuAiLe Medical glue (FAL), and the hemostatic forceps on the renal artery were removed. The muscle layer was closed with sutures , followed by the closing of the skin. For the unilateral ureteral obstruction (UUO) injury model, the abdomen was opened with a midline incision and the left kidney and upper ureter were exposed. Mice were subjected to surgical cautery of the left ureter 15\u2009mm below the pelvis. Kidney samples were harvested on day 0, day 1, day 10 and day 20 post\u2010surgery for analysis. Sham surgery kidneys in UPN and UUO models were also harvested.For the unilateral ischemia\u2013reperfusion injury (UIRI) model, 6\u20138\u2009weeks NOD SCID mice were anaesthetised using 3.7% chloral hydrate (0.5\u00a0g/kg body weight), and the mice were put prostrate on a heating pad at a temperature of 37\u00b0C for the duration of the surgical procedure. The left renal pedicle was occluded for 30\u201340\u2009min using a microaneurysm clamp, during which time the kidney was moistened by phosphate\u2010buffered saline (PBS) every 3\u00a0min. Then the clamp was removed, and reperfusion was confirmed by observing tissue colour change. The kidney was returned to the abdomen with an intraperitoneal injection of 200\u2009\u03bcl penicillin/streptomycin (P/S) to prevent infection. For sham operation, mice had only incisions in the skin and muscle layer, but the renal pedicles were not clamped.For the Adriamycin (ADR) induced glomeruli injury model, BALB/C mice of 8\u2009weeks were treated with a single dose of ADR (Sangon Biotech), 10.5\u00a0mg/kg, by tail vein injection. All mouse injury experiments were performed on male mice and were randomly allocated to the experimental groups.2.2For mouse REC cloning, 8\u201310\u2009weeks mice bred in a specific pathogen free (SPF) facility were collected to obtain renal cortex, medulla and papilla samples. For human kidney tissue sampling, percutaneous renal needle biopsies were performed to obtain patient tissue with membranous nephropathy (MN) by ultrasound\u2010guided core tissue biopsy needles (18 gauge). All renal specimens were subjected to pathological diagnosis. All the human renal tissues were obtained following clinical standard operating procedure (SOP) under the patient's consent and approved by the Hospital Ethics Committee.2.33 sizes to a viscous and homogeneous appearance. The minced tissue was then digested with dissociation buffer including DMEM/F12 , 2\u00a0mg/ml protease XIV , 0.01% trypsin  and 10\u00a0ng/ml DNase I  in 37\u00b0C incubator 2\u00a0h with gentle agitation. Digested cell suspensions were washed with cold\u2010wash buffer and passed through 70\u2009\u03bcm Nylon mesh  to remove aggregates. Cell pellets were collected by centrifuge of 200g and then seeded onto a feeder layer of lethally irradiated 3T3\u2010J2 cells in modified SCM\u20106F8 medium. Human SOX9+ RECs were generated from urine samples and expanded as previously described.For RECs isolation from kidney tissues, samples were washed with cold wash buffer  and minced by sterile scalpel into 0.2\u20130.5\u2009mmFor better visualization of colony growth, td\u2010Tomato+ RECs derived from mT/mG mice were used. For GFP labelling of cultured SOX9+ RECs, medium containing lentivirus was added to the cell culture together with 10\u00a0\u03bcg/ml polybrene (1:2000)2.4\u2212\u0394\u0394Ct method. The following primer pairs were used:Total RNA was prepared from cultured RECs using a Rneasy Mini kit (QIAGEN) according to the manufacturer's instructions. All RNA was digested with DNase I . One micro gram total RNA and PrimeScript RT Master Mix  was used for reverse transcription in a SimpliAmp Thermal Cycler . RT\u2010qPCR was performed in triplicate using a QuantStudio3 Sequence Detection System and SYBR Premix Ex Taq II . DNA primer pairs were designed to span exons, when possible, to ensure that the product was from mRNA. The following cycling conditions were used: 1 cycle of 95\u00b0C for 30\u2009s, 35 cycles of 95\u00b0C for 5\u00a0s and 60\u00b0C for 34\u2009s. The specificity of the amplified product was evaluated using the melting curve analysis. Internal control glyceraldehyde 3\u2010phosphate dehydrogenase (GAPDH) was used to normalize the result in each reaction, and relative fold change was calculated by the 22.5For cells immunofluorescent (IF) staining, cells were fixed by 3.7% formaldehyde and then permeabilized with 0.2% Triton X\u2010100 for 5\u20138\u00a0min. For cryo\u2010section, tissue samples were fixed in 3.7% formalin overnight at 4\u00b0C, then embedded by the Tissue\u2010Tek O.C.T compound  after PBS washed, frozen sections (5\u2010\u03bcm thick) were made by cryotome . For the paraffin section, the fixed tissue was dehydrated by ethanol gradient processed in an automatic tissue processor and then embedded into the paraffin blocks. Five to seven 5\u20137\u00a0micrometre sections were using a microtome . Fixed and paraffin sections were further blocked with 10% donkey serum. Then followed by incubation with indicated primary antibody overnight at 4\u00b0C, and incubation with secondary antibody for 2\u00a0h. Fluorescent images were visualized and captured with an Olympus IX73 microscope. Quantitative image analyses were performed using the ImageJ software. Haematoxylin and eosin (H&E) staining was performed in standard protocol. The tubular injury score was determined as followed: 0, 0%\u20135%; 1, 5%\u201310%; 2, 11%\u201325%; 3, 26%\u201345%; 4, 46%\u201375% and 5, >76%.Antibodies used for immunofluorescence include:2.6g for 45\u2009min at 4\u00b0C to collect concentrate whose mass was more 10\u2010kD and then overnight freeze\u2010drying. After measuring protein concentration, samples were loaded and separated on 15% precast polyacrylamide gels, and then transferred to PVDF membranes (Roche) at 300\u2009mA for 30\u2009min. Membranes were blocked with 5% no\u2010fat powdered milk, and then incubated with primary antibodies overnight, followed by secondary antibodies. The specific signals were detected by Immobilon Western Chemiluminescent HRP Substrate (Millipore) and Tanon image system.Cells were digested and lysed in RIPA buffer (CST) containing protease inhibitors cocktail (Roche) followed by standard Western blotting procedure.Antibodies used for Western blotting include: S100A9 , \u03b1\u2010tubulin  and HRP\u2010conjugated anti\u2010rabbit IgG (H\u2009+\u2009L) as a secondary antibody .2.7g and the cell pellets were washed twice with cold wash buffer including F12 medium, 5% FBS , 1%P/S , 1% l\u2010glutamine  and resuspended in cold PBS. Details were prepared as described.All steps were performed on ice or at 4\u00b0C. Urine samples were collected from 52 (12\u2009+\u200940) healthy volunteers with two different age ranges. Urine specimens from 40 healthy older volunteers were obtained from the department of nephrology in Tongji Hospital, Tongji University. Midstream of voided urine specimen was collected for each donor in the morning. Once the urine volume was measured, the sample was transferred into a 50\u2009ml conical tube and centrifuged immediately at the speed of 3902.8NormalizeData in Seurat was used to scale and log transform the dataset. Two thousand highly variable genes were identified by using the FindVariableGenes function. ScaleData function regressed out the variants arising from library size and percentage of mitochondrial genes. Principal component analysis (PCA) was performed on the variable genes with RunPCA function. Additionally, uniform manifold approximation and projection (UMAP) was used on the top 50 principal components for visualizing the cells. FindClusters function was used to identify cell clusters with a resolution of 0.2, which produced seven clusters. VlnPlot function showed the markers used to define each cluster. To understand the consistencies between our dataset and other urinary datasets, a previously published kidney single\u2010cell dataset was downloaded and analysed.Single cells were captured and barcoded in 10\u00d7 Chromium Controller (10\u00d7 Genomics). Subsequently, RNA from the barcoded cells was reverse\u2010transcribed, and sequencing libraries were prepared using Chromium Single Cell 3'v3 Reagent Kit (10\u00d7 Genomics) according to the manufacturer's instructions. Sequencing libraries were loaded on an Illumina NovaSeq with 2\u2009\u00d7\u2009150 paired\u2010end kits at Novogene, China. Raw sequencing reads were processed using the Cell Ranger v.3.1.0 pipeline from 10X Genomics. Data were aggregated and normalized to the same sequencing depth, resulting in a combined gene\u2010barcode matrix of all samples. Seurat R package (version 4.0.2) was used for dataset analysis. According to the number of detected genes and the percentage of mitochondrial genes in the urine samples, low\u2010quality cells with mitochondrial gene content >25% and detected genes less than 200 or more than 9000 were removed. This filtering step resulted in 15,760 genes\u2009\u00d7\u20091010 cells sampled from the younger urine species, and 8594 genes\u2009\u00d7\u20091774 cells from the older urine species. Then the function 2.9To explore potential intercellular crosstalk between SOX9+ RECs and other cell types from healthy control (HC) and AKI samples, we implied the ligand\u2010receptor distribution and expression of SOX9+ RECs and other cell types with a standard pipeline implemented in R using CellChat R package,2.10Protein extraction and pre\u2010treatment of enzymolysis and desalination at Novegene, China. Then polypeptides were labelled by Tandem Mass Tags. Subsequently, combining the polypeptides, fractionate, clean up and perform QE HF\u2010X LC\u2013MS/MS analysis. Raw sequencing data were processed and quantitated using Proteome Discoverer Software 2.2. Six serum samples divided into three groups were analysed. The differential expression of each two groups was performed using the DESeq2 R package. Due to poor sample repeatability, some protein data with high Foldchange values were deleted, and then the remaining data were averaged. The protein dataset had been normalized by the housekeeping gene GAPDH to reduce the extraneous variation among samples. PCA analysis was performed using the gmodels R package (version 2.18.1) and visualized by the ggthemes R package (version 4.2.4). Heatmap showed the average expression of three groups. Ward's minimum variance method was used and the result was visualized by using the ggplots R package (version 3.1.1). Protein\u2013protein interaction (PPI) network was constructed to map the differentially expressed genes (DEGs) to the protein data by using Cytoscape (3.9.0).2.11p\u2009\u2264\u20090.05 were considered statistically significant.Results were expressed as means\u2009\u00b1\u2009SD. Matched results were assessed by two\u2010way ANOVA. Comparisons among multiple groups were analysed using Tukey's multiple comparison test. GraphPad Prism (version 7.0a) or R programming was used for data management, quantitative analysis and graph generation. Differences with 33.1in vivo lineage tracing studies have indicated a strong correlation between SOX9+ cell activation and early transcriptional response to murine renal injury.Previous in vivo immunostaining data, the kidney tissue subjected to UPN yielded approximately five\u2010fold more clones, which surged as early as 1 dps  culture system.s Figure\u00a0.Sox9 and Pax2 suggested that the activated RECs shared similar biological characteristics with resident SOX9+ RECs under normal conditions  function analysis showed that the differentially expressed genes of SOX9+ RECs were associated with not only renal development, but also the secretory regulation processes, manifested by genes including F Figure\u00a0. TherefoF Figure\u00a0. While tIn HC group, CellChat detected 61 significant ligand\u2010receptor pairs in the SOX9+ RECs group, which were further categorized into 12 signalling pathways Figure\u00a0. NetworkUnder the damaged condition, the categorized pathways of activated SOX9+ RECs included VEGF, COMPLEMENT, SPP1 and CALCR Figure\u00a0. We dete3.3n\u00a0=\u00a012, median age\u00a0=\u00a024) and older  healthy people. In total, we sequenced 1104 cells from the younger age group and 2386 cells from the older age group. After stringent quality controls, we eventually analysed 1010 and 1774 cells, respectively. Our clustering indicated seven clusters under unsupervised graph\u2010based clustering (Seurat method) of the dataset and visualization by UMAP , podocytes (PODXL+), urothelium cells (PSCA+) and immune cells   Figure\u00a0. CompareSOX9+ cells were found in both younger and older urine samples Figure\u00a0. The perS100A9, MUC1 and NOTCH2. To compare the secretory capability of different groups, we calculated the average expression level of the gene set in each group. We found that SOX9\u2009+\u2009RECs demonstrated significantly higher expression for secretome profiles compared to SOX9\u2010 cell groups (p\u00a0<\u20090.05) in our dataset  enrichment analysis demonstrated that the differentially expressed genes identified urinary SOX9+ RECs enriched in specific processes, including wound healing, gland development, secretory granule lumen and epidermal growth factor receptor (EGFR) signalling pathway Figure\u00a0. Networkt Figure\u00a0 and othet Figure\u00a0. Altoget3.4SOX9, CD24, NOTCH2 and SALL1,LRP2, ATP1A1, AQP1, UMOD and SLC12A3)  Figure\u00a0.To further characterize the cells after continuous culture, SOX9+ RECs were propagated for >8 passages. Quantitative flow cytometry analysis showed a high percentage of SOX9 expression  during the culture process and a low percentage of mature epithelial markers  injection route. Interestingly, we found that the I.P. injected GFP\u2010positive cells distributed at the surface of the unilateral injured kidney on the right side selectively, whereas no cells resided in the contra\u2010laterally healthy kidney Figure\u00a0. Similarg for 15\u2009min to remove any dead cells and cell debris. By S.C. injection of CM, we observed consistent therapeutic effects with less injured KIM1+ tubules, increased expressions of healthy proximal tubule markers (ATP1A1+/AQP1+), activated more endogenous SOX9+ cells and decreased the serum creatinine levels  injection resulted in remarkable glomeruli tuft and glomerular adhesion of the renal capsular membrane, whereas these damages were ameliorated by S.C. injection of REC\u2010derived CM like the previous UIRI study Figure\u00a0. Histolo3.6To identify the secreted proteins involved in renal injury and repair, we applied a proteomic strategy to study the blood serum of SOX9+ REC engrafted mice. We used a multiplexed quantitative proteomics approach following tandem tag\u2010based mass spectrometry, a technology that enables protein identification and quantitation from multiple samples simultaneously.To further systematically assess REC secretome treatment induced changes and identify proteins of interest, we subjected the proteomic datasets to hierarchical clustering based on Ward's minimum variance method,S100A9 overexpression in the sc\u2010RNA\u2010Seq data of urine\u2010derived SOX9+ RECs, which was confirmed here in cultured SOX9+ RECs by RT\u2010qPCR  protein in Cluster III is known as a critical biomarker for monitoring renal toxicity or harm in clinical trials,R Figure\u00a0. S100A9 R Figure\u00a0. Therefo3.7S100A9 is a member of the S100 calcium\u2010binding protein family. S100A9 deficiency displayed a phenotype with enhanced renal damage, revealing that S100A9 could contribute to kidney repair.in vivo, which could be related to the regeneration of the proximal tubular epithelium (ATP1A1+ and AQP1+)   Figures\u00a0 and S9B.o Figure\u00a0. Mice ino Figure\u00a0 and S9D.o Figure\u00a0.4In the current study, we noted that the endogenous SOX9+ RECs could expand in the acutely damaged kidney of both mice and human. At the single\u2010cell resolution, the SOX9+ RECs displayed the dominantly secreted signature, which was closely linked to kidney regeneration pathways. Based on a feeder\u2010cell\u2010independent regenerative cloning (R\u2010Clone) system, we successfully obtained the cultured SOX9+ RECs from human urine, maintaining stable cell characteristics and secretory function. Transplantation of cultured human SOX9+ RECs or administration with its CM ameliorated renal tubular and glomerular epithelium damage. We also confirmed S100A9 as one of the critical factors in the SOX9+ REC secretome by quantitative proteome analysis and functional studies.The isolation and expansion system of epithelial cells from adult tissues are fundamental techniques supporting cell\u2010based regeneration studies. In 1975, Green et al. established the first successful example of adult human epidermal stem cell culture.In recent decades, the mechanism of kidney regeneration in mammalians has been widely studied, and SOX9+ cells are found to be stimulated to participate in this process.Moreover, here we clarified the potential molecular mechanism underlying the SOX9+ REC secretome\u2010mediated kidney repair by quantitative proteomic analysis. The experiments showed up\u2010regulation of several renoprotective factors, including S100A9. In a study by Mark C. Dessing et al., they observed that S100A9 deficiency led to sustained renal pathological state and dysfunction following renal UIRI,In conclusion, our study clarified the secretory function of SOX9+ RECs to regenerate renal epithelium, which elucidated a potential organ/tissue repair mechanism and provided novel translational opportunities for cell\u2010free strategies.Wei Zuo, Yu Ma and Yujia Wang designed the study. Hao Nie, Zixian Zhao, Dewei Zhou, Dandan Li, Xutao Liu and Yujia Wang involved in data collection and analysis. Wei Zuo, Hao Nie and Zixian Zhao drafted the manuscript. All authors read and approved the final manuscript.All authors declare that they have no conflict of interest.Figure S1. Cloning mice RECs from UUO injury model. (A) The percentage of SOX9+ cells at 0 and 1 dps. Data expressed as mean\u2009\u00b1\u2009SD . (B) Representative serial images showed single cell\u2010derived td\u2010Tomato+ REC clones' expansion within 27\u2009h. 1\u20133 indicated the clones with high proliferation capacity. Asterisks indicated the clones with low proliferation capacity. Scale bar, 500\u2009\u03bcm. (C) REC colonies derived from sham and UUO injured kidneys stained with SOX9 and PAX2 antibodies. Scale bar, 50\u2009\u03bcm.Figure S2. Cell lineage analysis by single\u2010cell RNA\u2010sequencing in human subjects. (A) Twelve distinct cell clusters were visualized by UMAP plotting. (B) UMAP plot of cell clusters from different specimens of HC and AKI. The colour of the cells represented group origin. (C) Dot plots showed gene expression patterns of cluster\u2010enriched markers. (D) Enriched GO of SOX9+ RECs between HC and AKI subjects.Figure S3. The signature of urinary SOX9+ cells in the published dataset. (A) Distribution and quantification of SOX9+ cell fractions. (B) Violin plots presented each cluster's marker genes and highlighted the selected marker genes for each cluster. (C) Heatmap exhibition of differentially secreted gene expressions between SOX9+ RECs and SOX9\u2010 cells.Figure S4. Identifying human REC colonies from renal tissue and urine specimen. (A) Human REC colonies isolated from renal tissue (RECs\u2010tissue) of two patients with membranous nephropathy (MN) stained with indicated markers (representative images of n\u00a0=\u00a03 independent experiments). Scale bar, 20\u2009\u03bcm. (B) Long\u2010term cultured RECs from urine stained with SOX9 and mature epithelial markers of ATP1A1 and CDH1 at early and late passage. Scale bar, 100\u2009\u03bcm.Figure S5. Quantitative analysis of tubular necrosis and glomerulus injury. (A) Quantitative scores of tubular necrosis post UIRI and RECs S.C. engraftment. (B) Quantitative scores of tubular necrosis post ADR and REC\u2010derived CM S.C. injection. (C) Quantification of the whole kidneys about FN1 and SYNPO after REC\u2010derived CM administration. Data shown in (A), (B) and (C) were represented as mean\u2009\u00b1\u2009SD .Figure S6. SOX9+ RECs can repair the renal injury by I.P. injection. (A) Representative image of H&E\u2010staining after REC therapy. Sham, no surgery; UIRI, 3T3 cells pellets with 2\u20134^106 after UIRI; UIRI+ RECs, equivalent\u2010number of REC pellets I.P. injection after UIRI. Scale bar, 100\u2009\u03bcm. (B) Quantitative scores of tubular necrosis post UIRI and RECs I.P. engraftment. (C) The merged image of injured NOD\u2010SCID mouse kidney (left) and contralateral healthy kidney (right) 7\u2009days after GFP\u2010RECs I.P. transplantation along the midabdominal line.  Immunostaining (D) and quantification (E) of the whole kidneys after REC I.P. treatment . Scale bar, 200\u2009\u03bcm. (F) Serum creatinine level showed a reduction after REC treatment. Data shown in (B), (E) and (F) were represented as mean\u2009\u00b1\u2009SD .Figure S7. The CM of SOX9+ RECs reverse tubular epithelial cell damage caused by UIRI. (A) Representative H&E and IF post REC derived CM therapy like UIRI study. Sham, no surgery; UIRI, PBS subcutaneous injection after UIRI; UIRI\u2009+\u2009CM, equivalent volume CM subcutaneous injection after UIRI. Scale bar, 100\u2009\u03bcm (HE); Scale bar, 200\u2009\u03bcm (IF). (B) Quantitative scores of tubular necrosis post UIRI and RECs derived CM injection. (C) Quantification of representative markers after CM treatment. (D) Serum creatinine level showed a reduction after CM injection. (E) Isolation of RECs from patients followed by culture in SOX9+ RECs derived CM from two healthy volunteers (1# and 2#) resulted in higher cell viability by CCK\u20108 test. Data shown in (B), (C), (D) and (E) were represented as mean\u2009\u00b1\u2009SD .Figure S8. GO analysis of secreted proteins in each cluster. (A\u2013D) Dot plots of enriched GO terms in Cluster I (A), Cluster II (B), Cluster III (C) and Cluster IV (D). The X\u2010axis showed the fold enrichment of each GO term, whereas the colour denoted the p\u2010value and the size of the dot denoted the number of IDs assigned to each GO term.Figure S9. Recombinant S100A9 protein restores kidney damage by I.V. injection. (A) H&E staining confirmed less kidney injury in the recombinant S100A9 protein treatment group. Scale bar, 50\u2009\u03bcm. (B) Quantitative scores of tubular necrosis post UIRI and recombinant S100A9 protein therapy. (C) Quantification of the immunostained whole kidneys after recombinant S100A9 protein I.V. injection. (D) Serum creatinine level significantly decreased after recombinant S100A9 protein treatment. (E) Isolation of RECs followed by culture in DMEM or DMEM plus 10\u2009\u03bcg/ml recombinant S100A9 protein. Cell viability validated by CCK\u20108 test. Data shown in (B), (C), (D) and (E) were represented as mean\u2009\u00b1\u2009SD .Click here for additional data file."}
+{"text": "Consumers have difficulty understanding alcoholic units and low risk drinking guidelines (LRDG). Labelling may improve comprehension. The aims of this rapid evidence review were to establish the effectiveness of on-bottle labelling for (i) improving comprehension of health risks; (ii) improving comprehension of unit and/or standard drink information and/or LRDG, and (iii) reducing self-reported intentions to drink/actual drinking.Electronic database searches were carried out (January 2008-November 2018 inclusive). Papers were included if they were: published in English; from an Organization for Economic Co-operation and Development country; an experimental/quasi-experimental design. Papers were assessed for quality using the Effective Public Health Practice Project Quality Assessment tool. Ten papers were included. Most studies were moderate quality (n\u2009=\u20097).Five themes emerged: comprehension of health risks; self-reported drinking intentions; comprehension of unit/standard drink information and/or LRDG; outcome expectancies; and label attention. Labelling can improve awareness, particularly of health harms, but is unlikely to change behaviour. Improved comprehension was greatest for labels with unit information and LRDG.Alcohol labelling can be effective in improving people\u2019s comprehension of the health risks involved in drinking alcohol enabling them to make informed consumption decisions, and perhaps thereby provide a route to changing behaviour. Thus, effective alcohol labelling is an intervention that can be added to the broader suite of policy options. That being said, the literature reviewed here suggests that the specific format of the label matters, so careful consideration must be given to the design and placement of labels.The online version contains supplementary material available at 10.1186/s12889-023-16327-x. Globally in 2017, among those aged 15\u201349 years, alcohol was the leading risk-factor for ill-health, disability and death, accounting for 6.5% of total disability adjusted life years (DALYs) [\u201cTo keep health risks from alcohol to a low level, it is safest not to drink more than 14 units/week on a regular basis. If you regularly drink as much as 14 units/week, it is best to spread your drinking evenly over three or more days\u201d [Drinking guidelines vary internationally. UK guidelines are based around the concept of units . There are around 2.5 units in a pint of beer, 2.3 units in a 175ml glass of wine and 1.4 units in a shot of spirits. The low risk drinking guideline (LRDG) is set at the level where 1% of deaths are alcohol-related and are communicated as follows: re days\u201d . Units are days\u201d .Consumers have difficulty understanding units and LRDG. In England, one in four drinkers could report the LRDG, and even fewer reported using the guideline to monitor their own consumption . ConsumeThe aims of this rapid evidence review were to establish the effectiveness of labelling approaches on bottles of alcohol for (i) improving comprehension of the health risks of consuming alcohol; (ii) improving comprehension of unit and/or standard drink (SD) information and/or a LRDG, and (iii) reducing self-reported intentions to drink/actual drinking.The findings of this review were used to inform an experimental study which tested different label designs on an online sample of adult drinkers in England to understand their impact on consumer understanding of LRDGs .A rapid evidence review was used which balances resource and time constraints by streamlining the systematic review approach to synthesise evidence to inform decision makers . An inteElectronic database searching included four databases  and was carried out in November 2018. These databases were those the authors had access to that were relevant to the topic at hand. Papers published between January 2008-November 2018 (inclusive) were included. Database searching was supplemented by hand-searches on Google Scholar and consultations with expert groups listed in Supplementary Material To be eligible for inclusion in this review, papers needed to meet the following criteria:Published between January 2008-November 2018 (inclusive).Published in English.Data collected from Organization for Economic Co-operation and Development countries.Relevant outcome measures.Experimental/quasi-experimental.p-value for between groups differences without the mean score and error estimates. Research funded by the alcohol industry was also excluded in line with previous evidence reviews [Studies were excluded if the populations of focus were: under the legal drinking age; pregnant women; or those with alcohol dependence. While pregnant women are a key group in relation to alcohol health literacy, this review focuses on labelling messages for the general population and its findings will be used to inform an experimental study testing labels on the general population. Furthermore, pregnant women and children are advised to abstain from alcohol entirely and thus require different messaging to the general population. Studies evaluating the effectiveness of media campaigns/advertisements were excluded . Editori reviews .After applying the eligibility criteria, studies were further refined using a sequential process. Study titles and abstracts were scrutinised to determine eligibility. Following this, or when relevance was unclear from title and abstract alone, full texts were examined.A test of inter-rater agreement was conducted by two researchers. Both conducted a title and abstract screen of 100 articles and found an agreement rate of 95%; this process produced a kappa statistic of 0.71, indicating a high level of agreement.Key variables were systematically extracted by one author using the PICO ) framework . MeasureThe Effective Public Health Practice Project (EPHPP) Quality Assessment tool was used to assess methodological quality . This toThe study selection process can be seen in Fig.\u00a0Five studies were from Australia, three from the UK, two from Canada, one from the United States of America, and one which simultaneously recruited participants from Luxembourg and Germany. Most studies were moderate quality (n\u2009=\u20098), two weak, and two strong. Between-subjects designs, where participants were exposed to different experimental groups, were utilised in seven of the studies, with the remaining three utilising within-subjects designs, whereby the same participants were exposed to all conditions. An overview of study characteristics is given in Table\u00a0Five themes emerged and are presented as a narrative synthesis, as the papers were too diverse in measures and outcomes for any other approach. This involved looking for emerging themes inductively within the results and classifying them into groups. These were: comprehension of the health risks of alcohol; self-reported drinking intentions; comprehension of unit/SD information and/or LRDG; outcome expectancies; and label attention.Three between-subjects experiments tested the impact of labels on participants\u2019 abilities to accurately identify units, SDs and the LRDG Study 1 \u201321. In aA second between-subjects experiment aimed to understand the effectiveness of labels with SD information and Canada\u2019s LRDG compared to only ABV% labels . In an oA third between-subjects experiment aimed to understand the effectiveness of labels with SD information compared to ABV% labels . The expThese three studies suggest that not all labels are created equal: labels that help participants generalise to the weekly limit appear to help participants best understand drinking limit information. However, the latter two studies may have been influenced by their choice of dependent variables , 21. BotFive studies explored the impact of labelling approaches on participants\u2019 comprehension of alcohol\u2019s health risks: 4 between-subjects designs and 1 within-subjects design \u201326.An Australian between-subjects design aimed to assess whether exposure to alcohol warning statements relating to specific chronic diseases increases consumers\u2019 beliefs that alcohol is a risk factor for those diseases . ParticiA between-subjects study aimed to investigate the effectiveness of alcohol labels on perceptions of the health risk of alcohol use . ParticiA between-subjects study aimed to investigate the impact of cancer warnings on consumers\u2019 level of agreement that alcohol causes cancer . OverallAn Australian between-subjects study aimed to investigate the acceptability of cancer warning statements for alcoholic beverages . A contrA within-subjects study recruiting students from the USA aimed to explore how message formats  influence risk perceptions about alcohol-attributable cancer . ResultsThese studies show that labels do help educate people about specific health risks, especially when current awareness of that specific disease/health complication is low. Further, two suggests that warning labels of this kind would be well received by consumers , 26.In an online, between-subjects experiment, participants were randomised to view one of eight health warnings that varied in their specificity (e.g. \u201ccancer\u201d vs. \u201cbowel cancer\u201d), framing  and health message  Study 2\u00a0. SpecifiAn Australian between-subjects design aimed to assess whether exposure to warning statements relating to specific chronic diseases influences consumption intentions . ParticiA between-subjects design recruiting participants from Luxemburg and Germany aimed to investigate the effectiveness of tailored pictorial warning labels formulated as questions, e.g. \u201cDo you really want alcohol to help you test your limits?\u201d or statements, e.g. Yes, alcohol helps test your limits\u201d . No signIn a between-participants study, participants viewed one of three warnings: no warning, text-only warning, or a pictorial warning . PictoriThese studies suggest that vivid labels  are best at reducing participants\u2019 intentions to drink. Formulating the labels as questions does not appear to affect self-reported intentions to drink less.A single between-subjects design explored the impact of message framing on alcohol outcome expectancies \u2013 a measure of the perceived expectancies associated with consuming alcohol (can be positive or negative) . Particiand size [A multi-method within-subjects design was used to deliver four labelling conditions: (i) a control, (ii) enhanced colour, (iii) size, and (iv) enhanced colour and size . The firThe aims of this rapid review were to establish the effectiveness of approaches to alcohol labelling for improving comprehension of units, the LRDG, and alcohol-related health risks, and any effect of labelling on self-reported intentions to drink/actual drinking. The review identified 11 studies (ten experiments).A range of labelling approaches were effective at increasing participant comprehension, particularly for approaches that used pictorial warnings  and messThe effect of labels on drinking intentions were mixed, and no studies reported on actual drinking. A single strong study demonstrated motivations to drink less were higher for cancer and negatively framed messages , but theThis review highlighted a lack of awareness of unit  information and LRDG. It suggests that enhanced labels can reduce this knowledge deficit, particularly when both unit and LRDG information is given simultaneously , 20. It A lack of clear evidence precludes firm conclusions relating to message framing and outcome expectancies . FurtherThis review informed an experimental study testing different label designs on an online sample of adult drinkers in England to understand their effect on consumer understanding that has since been published . Given oThis review, and the subsequent experiment, supports more recent work that investigated alcohol labels in Yukon, Canada \u201339. Theyperiodically introduce high-intensity labels to recapture the initial impact. That being said, previous research that focused on the familiarity of the item, rather than familiarity with the warning label, found that familiarity tended to improve compliance with the label [The studies in this review typically measured a single  label exposures at a solitary time-point in contrast to repeated exposures experienced in day-to-day life. It is possible that responses, such as changes in drinking, only develop after repeated exposures to information received on alcohol labels or in tandem with other skills building and behaviour change interventions. Indeed, the research reviewed here suggests that warning labels are typically low intensity \u2013 small and difficult to notice . Alternahe label . This suFinally, the use of a rapid review protocol leaves this work more vulnerable to bias and errors. For instance, the search process in rapid reviews is typically less comprehensive. To try and compensate for this, we consulted expert groups for potentially missing literature (see Supplementary Material Well-implemented alcohol labels play an important role in increasing comprehension of risk and understanding of units and LRDGs, although there is room for improvement in label design. This understanding underpins a fundamental right to knowledge and can enable informed choice. Public support for labelling is high . OpportuBelow is the link to the electronic supplementary material.Supplementary Material 1"}
+{"text": "This article was migrated. The article was marked as recommended.Introduction: The escalating problem of unprofessionalism calls for teaching medicalprofessionalism in a manner that should lead to deeper learning. Early clinicalexposure (ECE) to an Intensive Care Unit (ICU) presents the issues pertaining tomedical professionalism to the students in a more explicit and emotionallychallenging manner. And reflection note writing evokes the critical process ofthought and analysis required for learning. We conducted the present study tosensitize the pre-clinical students towards medical professionalism using thesetwo tools, ECE and Reflection.Methods:Two hundred students of 1st MBBS were given an ObjectiveStructured Clinical Examinations (OSCE). The students were then taken for ECE toan ICU. There, the students observed different ongoing activities and criticalpatients, a doctor discussed some cases with them, and they also interacted withthe relatives of patients admitted in the ICU. Thereafter, students wrote a\u2018reflection\u2019 note describing what did you see? so what? and nowwhat? Students were again given an OSCE, similar to the one given before theECE, for assessing any change in their professional behaviour.Analysis ofreflection notes was done thematically and of OSCE scores using paired t-test(p<0.05).Results: The analysis of reflection notes revealed the budding of differentelements of professionalism among the students. Post-visit OSCE scores alsoshowed significant improvement.Conclusion: Incorporation of reflection note writing along with ECE is helpful inlaying the foundation of medical professionalism among pre-clinicalstudents. Medical field is increasingly becoming plagued with unprofessionalism  and reflection  /IEC/2017-18/6792) and a written informed consent of the students wastaken.The empirical research involved 200 students of 1st MBBS in the college. There were both qualitative andquantitative components: qualitative component included analysis of reflectionnotes, using a post-course design, and quantitative component includedassessment of OSCE results and feedback; OSCE was incorporated as before andafter design.Our study was a mixed methods experimental research. The experiment includedreflection note writing, ECE being a regular part of the curriculum for1An objective structured clinical examination (OSCE) was given to all thestudents. The OSCE included 3 stations where the students had to performdifferent components of clinical examination on subjects. The subjects werehealthy people from among the staff of the college who had volunteered to besubjects for the same. Each student spent 5 minutes at a station. Among othersteps of the clinical examination proper, the students were evaluated for theirprofessional behavior towards the subject and their communication with him/her:1) greeting the subject, 2) asking his/her name, age, occupation, residence,chief complaints 3) explaining the procedure of performing the examination tothe subject, 4) reassuring him/her, 5) taking his/her consent to perform theexamination on him/her, 6) exposing the body part required for examination in agentle and dignified way, 7) being gentle in examination, 8) covering back theexposed part after examination, 9) informing the subject about the completion ofexamination and its result, and 10) thanking the subject for his/hercooperation. The evaluation for each step of OSCE, including those assessingprofessionalism, was done by awarding marks from 0 to 1. One (1) mark wasawarded when the response of the student was satisfactory or correct and zero(0) when it was incorrect. If the response was less than satisfactory, but notincorrect, the student was awarded less than 1 but more than 0 mark.what did you see (what was your observation)? sowhat (what were your feelings and thoughts about it)? andnow what (what do you intend to do about it in future)?For, the purpose of early clinical exposure, the students were taken to anintensive care unit. The students were given a brief introduction as to what anintensive care unit is. They were also told what a reflection/reflection note isand were asked to write and submit the same after the visit. To help them writeit, the students were given handouts carrying clues about writing reflection:Thereafter, the students were taken for visiting an ICU in medicine department ofhospital attached to the medical college. The students were divided into threebatches. Each batch was taken for visit on a separate day. The students werefurther subdivided into groups of 10-12 students. Only one group went inside theICU at a time and the other groups interacted with the relatives of the patientsadmitted in the ICU. Each group spent about 30 minutes inside the ICU under theguidance of a doctor, who discussed with them the ICU set-up, few cases/patientsadmitted there, and also answered their queries. During their interaction withthe relatives of patients, students enquired about the problems they were facingregarding treatment of their patient and their stay in hospital. After thevisit, the students were given time to discuss the visit among themselves andwith the teacher. The students then wrote reflection notes and submitted them.Copies of the reflection notes were kept and the original ones were returned tothem. Then, another OSCE, similar to the one given before the ICU visit, wasgiven to the students and their evaluation was done with respect to the sameaspects of empathy, communication and professional attitude, as before.Thereafter, a feedback on the visit was collected from the students by means ofa validated questionnaire. The questionnaire had 13 items meant to be valued ona five point Likert scale, ranging from strongly agreeing with an item tostrongly disagreeing with it. 7 of these items were related to theidentification of the elements of medical professionalism by the students.The qualitative data of reflection notes was analyzed thematically. All thepoints mentioned by the students were taken into consideration, coded andtabulated by both the authors, separately. The authors then exchanged notes anddiscussed the themes, coding and interpretations for ensuring exhaustive studyof the reflection notes and for cross-checking the results. For analysis of OSCEresults, only the scores assessing professionalism were taken intoconsideration. A paired t- test with p<0.05 as significance level wasused. Feedback was also analyzed quantitatively by calculating percentage ofstudents agreeing with a particular value of an item. Thematic analysis was doneusing QDA Miner Lite 2.0.5 and quantitative analysis using Microsoft ExcelProfessional 2015.The 200 students included 92 females and 108 males .The reflection notes revealed the dynamics of perception and attitude of thestudents as they were remodeled by the clinical exposure and experience. Thereflection notes were scrutinized under three domains: what did you see? sowhat? and now what? Several themes emerged each with its own set of relevantcodes . Analysi: \u201cwhen we entered the ICU and when I saw the patients,I got to know what must be their mental condition: nothing but painful andhelpless. But for this how a doctor takes standing is something that cannever be neglected by me.\u201dExemplar 1- reveals development of empathy for patients, and of a sense ofresponsibility.\u201c.. after all this I realized that to become a doctoris not an easy task, it requires a lot of hard work... in starting, I wastaking studies very lightly, but when I saw patients in ICU, I realized weare the future doctors who would deal with patients\u2019 lives. Andbefore all this we should acquire all knowledge ...\u201dExemplar 2: - reveals realization of importance of hard work for continuous improvement inknowledge and skill, and willingness for striving for excellence.\u201cThe family was in agony and we could see theirimpatience and helplessness. For them we were all doctors. So, thepatient\u2019s wife asked me if he was out of danger. I felt veryhelpless. At the same time, I understood what this white coatsignifies.\u201dExemplar 3: - reveals development of empathy for relatives of patients and of sense ofaccountability.\u201c... and just knowledge is not enough. My bodylanguage, my words, what I say in front of relatives of my patients, whobelieve that he will be well as he has come to me, the way I talk, I dressand my overall behavior with staff also matters. And henceforth I need toinculcate all these things in my behavior and most importantly study hardeverything thoroughly.\u201dExemplar 4: - reveals realization of importance of having good communication skills.\u201c... After coming outside, I saw another battle ofdoctors: one of the relatives was so firm in his belief that he was debatingwith the doctor. But she (doctor) was trying to convince him that they aretrying their best to save the patient. But still he was not able tounderstand.\u201dExemplar 5: - reveals development of empathy for doctors  andrealization of importance of having good communication skills.\u201c... One of the things that I noticed was the waydoctor interacted with the patient and staff. I am glad that I had such apositive experience. I want to be a good doctor, so it is important for meto stay connected with patient...\u201dExemplar 6: - reveals realization of importance of working in association with other healthprofessionals, and that of need of developing good communication skills.\u201cWhat I felt is we should help them at leastemotionally. And if possible financially. As we waste a lot of money onother things which are sometimes not useful for us. Instead of that weshould help them. This is the most valuable work . In rural hospitals, we can serve food fortheir relatives which can help them to a certain extent.\u201dExemplar 7: - reveals a sense of social justice and altruism being developed.\u201c... just stay honest towards the profession and workhard for your patients.\u201dExemplar 8: - reveals inculcation of sense of integrity.\u201cThe doctor-patient relationship is the foundation ofmedical ethics. Patients, the innocent problem holders, come up to doctorsfor all sorts of problems, be it physical, mental or social. They expectdoctors to give solution to every kind of problems. And so, it is our dutyto stand up to their mark.\u201dExemplar 9: - reveals a sense of accountability and integrity.Most of the students strongly agreed with the positive influence of ICU visit onvarious aspects of their medical professional learning (Supplementary file 1).The students either agreed or strongly agreed that seeing critically illpatients aroused their interest in the profession (89.0%), that the agony ofrelatives for their patients taught them to look at patients sympathetically(91.5%) and that they now had better understanding of importance ofcommunication skills (91.5%). The students also agreed or strongly agreed thatthe experience motivated them to learn more (95.0%). Most of them agreed orstrongly agreed that the experience changed their perception of medical field(82.5%), that they became more sensitive towards their profession (84.5 %), andthat they found that their professional attitude has changed after the visit(77.5%). Also, the experience was rated as being quite relevant to pre-clinicalphase (88.5%) by the students and found to be helpful in enhancing academiclearning (95.0%). These findings suggest that the students were indeed able toidentify the different elements of professionalism with the help of the ICUvisit and their reflection on it.The Experiential Learning Theory given by Kolb , states et al., 2009;et al., 2018). In the present study, the students were takenfor a visit of an ICU, the early clinical exposure. The students were then made towrite a note on the visit \u2018reflecting\u2019 on it. This made them revisittheir experience in mind and made them \u2018think and analyze itcritically\u2019. It made them become more aware of the experience and helped themin developing an insight into it. In turn, this made them seek rationalizations fortheir thoughts and feelings. Their critical comprehension then reformed theirattitude and perception of the experience. And different components of theexperience inculcated different elements of medical professionalism among thestudents  The author obtained her postgraduate degree in the year 2013 and has since dedicatedly worked in academics.While teaching undergraduate and post graduate students, she realized that there isimmense need of improvising medical education. This research work is her first stepin this direction.Dr. Alka Rawekar (ORCID iD: https://orcid.org/0000-0002-1372-6332) The author is a professor ofPhysiology and is currently positioned as Dean- Allied Health Sciences, JNMC, DMIMS(DU), Wardha. During her academic career of more than 15 years now, she has severalarticles, both in physiology and medical education, to her credit as she continuesto work in the direction of improvising medical education. This review has been migrated. The reviewer awarded 4 stars out of 5. This is aninteresting paper that promotes early clinical exposure as a method of instillingthe beginnings of proessionalism in Year 1 medical students. It is valuable, but youneed to be careful aboutoverinterpeting you r results.Does the OSCE as outlinedmeasure some aspect of professionalism. there is a significant debate about whetherit is possible to measure professionalism in an OSCE. I may measure some aspect ofthe approach to a patient. this is only a very small part of professionalism.Thereflections are important in meeasuring the impact of the ECE, but it is not clearif it represents a method of teaching prrofessionalism.However sepite my concernsthis is an important paper and you should consider extending it. This review has been migrated. The reviewer awarded 4 stars out of 5. I thank theauthors for their insightful response to the reviewers comments and the way thatthey have restructured their revised paper. I am pleased that the authors haveextensively reviewed the limitations of this paper and have striven not to be toodogmatic in the paper's conclusions. As a result of this and I think because theauthors have shown due diligence in their second version of the paper, I would nowrecommend this paper. I feel that the authors have learned from this exercise- itwas indeed a very impressive piece of work, and I now look forward to readingfurther examples from the authors as they build upon developing professionalism intheir students- not an easy task and one which requires repetitive learninginterventions- but one that remains very important in the curriculum. This review has been migrated. The reviewer awarded 5 stars out of 5. This is aninsightful study in medical education for medical students in low in come countries.it can be replicated every where . i recommend this study strongly. This review has been migrated. The reviewer awarded 3 stars out of 5. An interestingpaper dealing with laying the foundations of medical professionalism amongpre-clinical students, with an emphasis on using reflection. The authors begin byclearly identifying the problem of unprofessionalism, and then use an exposuresession  in an ICU as the stimulus for thereflection. The paper does have a few issues that need to be addressed:\u2022 Theinstitutional context needs to be more clearly identified.\u2022 The OSCE process needsto be laid out in more detail .\u2022 The qualitative data (Exemplar 1 to Exemplar 9) are interesting, andgive a good insight into the students\u2019 mindset. When laying out qualitative data,however, it is preferable to identify the theme first, and then to cite examples. Inaddition, many of the identified themes appear to overlap, and the theme is more ofa general description of each exemplar than an actual theme. Part of theming is theprocess by which separate and succinct themes are identified, and then the data usedto support that.\u2022 I am not entirely clear what Table 1 is. It appears to be thereflective notes and then analysis of a single student, given as an example, but itcould also be information taken from several students. So, I think that, while thedata make interesting reading, the authors need to describe a little more explicitlythe source of the data in Table 1.\u2022 In Table 2, there appear to be errors in thedata. The pre mean score for the males and females are given, but the overall meanscore is higher than both. As a rough calculation, if the male and female scores arecorrect, I would estimate that the overall pre mean score should be around 62.77,not 64.39 as given in the table. If this error is merely an error of transcription,then it can be easily corrected; the authors, though, should double-check to ensurethat their other associated data (e.g. standard deviations) are not affected. \u2022 Thedata in the supplementary file are actually important data for the paper, so I wouldrecommend that the authors include the table in the body of their paper. \u2022 The datain the supplementary file should, however, be presented as raw figures first, andthen percentages in parentheses afterwards, not the other way round.\u2022 Figure 4should rather come at the end of the Discussion (before the limitations), and thenthe Conclusion used to summarise the overall findings of the study.\u2022 There are manyminor language errors in the paper. For version 2 of the paper, I recommended thatthe authors proof-read their manuscript very carefully in order to correct theseerrors.So, I think this is a useful study, but the paper itself does needimprovement, and I hope that these suggestions will lead the authors to submit animproved Version 2. This review has been migrated. The reviewer awarded 2 stars out of 5. I truly enjoyedreading this paper for the ideas it so well expressed, and for the exemplars itprovided of professionalism derived from the students' reflections. Along with otherreviewers, I feel the paper did not provide enough details about the OSCEs to fullyendorse the paper. I think the paper could be published if rewritten to address thiscomponent of the students' evaluation. This review has been migrated. The reviewer awarded 2 stars out of 5. Although thisis an interesting paper to read and does cover an important area, I am not sure thatI could be as positive as the previous reviewer.I do think that this paper is anexercise in what constitutes good research and what we can positively take away fromthe research. I worried with the authors' opening statements regarding the need forteaching- in too many instances teaching doesn't lead to learning and if we are notcareful, it only leads to superficial learning. I would agree with the authorsregarding their thoughts regarding early clinical exposure and reflectiveportfolios, both useful learning strategies, but only if used in the correct way. Ihave no doubt that the students were exposed to a situation- the ICU- that couldlead to deep learning, but again, only if used correctly and our results were basedon longer term effects.The results from the students were to be expected. Althoughwe are not given the structure of the OSCE- was it a one station or multiple stationOSCE and what was the internal structure of the scenario (s)?- the students had beentold what was appropriate and I daresay, this was also reflected by their increasedscores on the questionnaire. We were not told of how the reflective portfolios wereused or scored and what sort of feedback was given, but these are surely importantelements to recognise if the student has really learned. At the moment thisevaluation lies at only Kirkpatrick 1 and 2- to be as dogmatic as the authors' arein their take home messages I feel that there needs to be a longer-term evaluationto at least level 3 and 4.I am sorry but I am afraid I cannot recommend this paperin its present form, but I do wish the authors well in their future research in thisimportant area. This review has been migrated. The reviewer awarded 5 stars out of 5. I'm encouragedby this study. in my own work I have been grappling with teaching reflection tostudents of public health as a way of encouraging public health practiceprofessionalism. Unlike clinical medicine education , I cannot find a practicum sitesimilar to ICU experience for students. I find this article relevant to my work."}
+{"text": "Low HDL-C is associated with an increased risk of sepsis-associated AKI and subsequent decline in eGFR. HDL-C possesses anti-inflammatory, antioxidant, and endothelial repair-promoting properties. The use of Apo A-I mimetic peptides, which are the main structural components of HDL-C, has been shown to improve renal function in animal models of sepsis. However, the diagnostic value of low HDL-C in persistent sepsis-associated AKI remains unclear.This is a retrospective cohort study based on MIMIC IV (V 2.2). The study population consisted of all adult septic patients admitted to the Beth Israel Deaconess Medical Center Intensive Care Unit from 2008 to 2019, with plasma HDL-C measured within 24\u00a0h of ICU admission. The primary endpoint was persistent severe sepsis-associated acute kidney injury (SA-AKI) and the secondary endpoint is kidney replacement therapy (KRT). Logistic regression was used to assess the correlation between HDL-C and persistent severe SA-AKI and KRT, and receiver operating characteristic (ROC) curve analysis was performed to evaluate predictive ability.A total of 604 cases of SA-AKI patients were included in the analysis, among which 88 cases (14.5%) experienced persistent severe SA-AKI. The median (IQR) HDL-C level in the group with persistent severe SA-AKI was lower (33.0 [24.0\u201345.5]) compared to the non-persistent severe SA-AKI group (42.0 [31.0\u201353.0]). However, HDL-C showed poor discriminatory ability with an AUROC [95%CI] of 0.62 [0.56\u20130.69]. Clinical prediction models based on serum creatinine concentration, 24-h creatinine change, APSIIIscore, lactate levels, APTT, and heart rate performed well in predicting persistent severe SA-AKI with an AUROC [95%CI] of 0.876 [0.84\u20130.91]. However, adding HDL-C to this model did not improve predictive performance.The plasma HDL-C measured within 24\u00a0h after admission to the ICU does not provide a good prediction for persistent severe SA-AKI, and it does not improve the clinical predictive ability compared to conventional variables.The online version contains supplementary material available at 10.1186/s40001-023-01513-9. Acute kidney injury (AKI) is a highly prevalent disease worldwide, with sepsis being the most common factor leading to AKI in critically ill patients, accounting for 40% of cases . A recenIn clinical practice, the identification of persistent AKI is of great clinical significance. Firstly, the duration of AKI is closely related to patient prognosis and the risk of end-stage renal failure. Recent evidence has shown that two-thirds of AKI patients recover kidney function within 3\u20137\u00a0days, while those with persistent AKI have significantly lower one-year survival rates . AdditioHigh-density lipoprotein (HDL) possesses anti-inflammatory, antioxidant, and endothelial repair-promoting properties, participating in the regulation of various pathological processes that influence the progression of sepsis associated acute kidney injury (SA-AKI). HDL increases liver clearance of LPS through scavenger receptor class B type 1 (SR-B1) , therebyThe purpose of this study is to determine whether plasma HDL-C measured within 24\u00a0h after admission to the ICU can predict persistent severe acute kidney injury and KRT. The secondary objective is to evaluate the potential use of HDL-C in combination with routine clinical data.This is a retrospective cohort study using the MIMIC-IV (version 2.2) database to investigate different populations. The MIMIC-IV database is a publicly available multi-parameter intensive care unit (ICU) database provided by the Massachusetts Institute of Technology (MIT). It includes critically ill patients admitted to the ICU at Beth Israel Deaconess Medical Center in Boston, Massachusetts, from 2008 to 2019 . Since tThis study selected adult patients from the MIMIC-IV database who were admitted to the ICU once and had HDL-C measurements within 24\u00a0h after admission. Patients who met the criteria for sepsis 3.0 were included in this study, with inclusion criteria being: presence of infection and Sequential Organ Failure Assessment (SOFA) score\u2009\u2265\u20092 . This stExtracted variables from the database using PostgreSQL 14.5 include demographic information, vital signs, medical history, laboratory test results, scoring data, and prognosis data for patients. All comorbidities are diagnosed based on International Classification of Diseases (ICD) codes from the 9th and 10th editions. HDL-C and other laboratory test results are obtained within 24\u00a0h after admission to the ICU. Considering that laboratory data is measured multiple times within a 24-h period, this study extracted the worst value for each day. For missing experimental data that accounts for less than 15% of the total population, multiple imputation methods were employed .t-tests or rank-sum tests. Categorical data were presented as frequency (N) and percentage (%), with group comparisons analyzed using chi-square tests. Variables that were statistically significant on univariate analysis were included in multivariate analysis. Multivariable analysis was performed with a logistic regression model. We considered p\u2009<\u20090.05 to indicate statistical significance. The ability to predict persistent severe AKI as well as KRT was assessed using receiver operating characteristic curve (ROC) analysis. All analyses were performed using R software version 4.62.Normally distributed continuous data were presented as mean\u2009\u00b1\u2009standard deviation (X\u2009\u00b1\u2009s), while non-normally distributed continuous data were presented as median (interquartile range) [Median (IQR)]. Group comparisons were performed using p\u2009=\u20090.043) and cerebrovascular disease . The incidence of coronary atherosclerotic heart disease was higher in patients with persistent severe SA-AKI . Patients with persistent severe SA-AKI had higher scores for disease severity, such as median [IQR] SOFA score on the day of ICU admission  and median [IQR] APS III score .Patients with persistent severe SA-AKI present with more severe kidney injury, as reflected by higher median [IQR] serum creatinine levels on the day of admission to the ICU  and higher median [IQR] blood urea nitrogen levels .This study included 846 patients with sepsis, of which 716 were diagnosed with SA-AKI. After excluding 112 patients who did not have plasma creatinine and urine output measurements within 72\u00a0h after SA-AKI diagnosis, a total of 604 SA-AKI patients were finally included for analysis. Among them, 88 cases (14.5%) experienced persistent severe SA-AKI (stage 3), while 516 cases (85.4%) had non-persistent severe SA-AKI , KRT rate , and usage of vasoactive drugs . The low HDL-C group also had higher SOFA scores , APS III scores  and serum creatinine levels   and blood urea nitrogen     can stimulate the activity of eNOS through SR-B1, and eNOS is involved in regulating the pathological process that affects the progression of SA-AKI. During sepsis, decreased eNOS activity can lead to microcirculatory dysfunction, which may result in local renal ischemia and contribute to kidney damage and the development of SA-AKI . In a smThe HDL-C levels are associated with poor prognosis in sepsis patients. Research has found that during the early stage of sepsis, HDL-C concentration rapidly decreases and whether it recovers or continues to decline affects the survival status of sepsis . In a smHowever, it should be noted that our study only focused on the static value of HDL-C within 24\u00a0h after admission to the ICU. This result suggests that HDL-C within 24\u00a0h of ICU admission is not very effective in predicting persistent severe SA-AKI. However, our study and previous clinical research results suggest a negative correlation between HDL-C and serum creatinine levels as well as severity scores, and low HDL-C is associated with long-term decline in glomerular filtration rate . These rIn summary, we have found that the HDL-C concentration within 24\u00a0h of ICU admission is not a good indicator for distinguishing between persistent and non-persistent severe SA-AKI, and it does not improve clinical prediction.Additional file 1: Table S1. Integrated discrimination improvement (IDI), category-free net reclassifcation improvement (cfNRI) with the addition of HDL-C. Fig. S1. Differences between the high HDL-C group and low HDL-C group. Fig. S2. Correlation between HDL-C group and serum creatinine, blood urea nitrogen and differences in HDL-C among different groups."}
+{"text": "Pterocarya macroptera (a vulnerable relict species in China) under future climate scenarios.Understanding adaptive genetic variation and whether it can keep pace with predicted future climate change is critical in assessing the genetic vulnerability of species and developing conservation management strategies. The lack of information on adaptive genetic variation in relict species carrying abundant genetic resources hinders the assessment of genetic vulnerability. Using a landscape genomics approach, this study aimed to determine how adaptive genetic variation shapes population divergence and to predict the adaptive potential of FST) and genotype\u2013environment association (GEA) methods. We further dissected the effect of geographical/environmental gradients on genetic variation. Finally, we predicted genetic vulnerability and adaptive risk under future climate scenarios.We applied restriction site-associated DNA sequencing (RAD-seq) to obtain 8244 single-nucleotide polymorphisms (SNPs) from 160 individuals across 28 populations. We examined the pattern of genetic diversity and divergence, and then identified outliers by genetic differentiation (P. macroptera: the Qinling-Daba-Tianmu Mountains (QDT), Western Sichuan (WS) and Northwest Yunnan (NWY) lineages, which showed significant signals of isolation by distance (IBD) and isolation by environment (IBE). IBD and IBE explained 3.7\u20135.7 and 8.6\u201312.8 % of the genetic structure, respectively. The identified GEA SNP-related genes were involved in chemical defence and gene regulation and may exhibit higher genetic variation to adapt to the environment. Gradient forest analysis revealed that the genetic variation was mainly shaped by temperature-related variables, indicating its adaptation to local thermal environments. A limited adaptive potential was suggested by the high levels of genetic vulnerability in marginal populations.We identified three genetic lineages within P. macroptera. Marginal populations may be at high risk of extinction, and thus proactive management measures, such as assisted gene flow, are required to ensure the survival of these populations.Environmental gradient mainly shaped the population differentiation of  Climate change potentially alters habitat suitability at the regional scale and results in local extinctions , and hasAdvances in landscape genomics have enabled the elucidation of the molecular genetic basis of the local adaptation of tree species . GenotypTrees are the main components of forest ecosystems . Relict Pterocarya macroptera is a vulnerable Cenozoic relict tree species in China that grows at an altitude of between 1100 and 3500 m  obtained from restriction site-associated DNA sequencing (RAD-seq) data, we formulated the following objectives: (1) to infer population genetic differentiation and genetic diversity; (2) to quantify the contributions of environmental and geographical variables in shaping the spatial distributions of genetic variation; and (3) to assess the vulnerable populations with a mismatch between genotype and environment.In this study, we sequenced 160 individuals of  P. macroptera populations representing its entire geographical range. Samples were dried and kept in silica gel. Genomic DNA was extracted from tissue using a Plant Genomic DNA Kit . A total of 160 individuals were selected for sequencing, with four to eight individuals per population  ligation was performed on 200 ng DNA. Ligated DNA was pooled, purified, and PCR-amplified by an ABI GeneAmp 9700. DNA fragments with sizes between 300 and 500 bp were selected based on AMPure XP bead-based size selection steps. RAD libraries were sequenced on an Illumina HiSeq\u2122 platform using 150-bp paired-end reads at Major Bio Pharm Technology, Shanghai, China.Samples of healthy and mature leaves were collected from 28pulation . SamplesPterocarya stenoptera; The quality of the RAD data was assessed using FastQC . AdapterP. stenoptera gene models using SnpEff v4.3t (https://www.uniprot.org/), we determined the possible molecular function of each gene and the biological process involved.VCFtools v0.1.13 was used to discard loci with missing data present in at least 20 % of individuals and to keep only biallelic SNPs (K) was set to vary between 1 and 10. The optimal number of clusters was determined based on the lowest cross-validation error rate. For each K value, we performed a 10-fold cross-validation. The genetic differentiation among populations was calculated based on all SNPs using principal component analysis (PCA) in the R package adegenet 2.1-5 (FST) among lineages was calculated based on different SNP datasets using VCFtools v0.1.13 (NP), percentage of polymorphic loci (PL), mean observed heterozygosity (HO) and mean expected heterozygosity (HE) using PLINK v1.9 with default settings based on all SNPs and all outlier SNPs (\u03c0) was conducted using pixy v1.2.7 for all loci (including non-polymorphic loci) and all outlier loci following a window length of 10 kb  using ADMIXTURE v1.30 . For ADMet 2.1-5 . Populatier SNPs . The unbof 10 kb . For eacP. macroptera with correlation coefficients |r|\u2005<\u20050.7 and the top three variables identified by the GF analysis were retained. Finally, eight variables  were used for subsequent analysis (P. macroptera (see section Prediction of genetic vulnerability). Given that the correlation between the top three variables identified by GF and the other variables may affect the final prediction of GF, we also used bioclimatic variables with correlation coefficients |r|\u2005<\u20050.7  to build the final GF model.Nineteen bioclimatic variables for the current period (1970\u20132000) at 30 arcsec resolution were downloaded from the WorldClim v.2.1 database based on the geographical coordinates of the sampled populations . Elevatianalysis . The eigFST-based methods identified outlier SNPs as those with levels of differentiation above those of neutral SNPs among populations  (K\u2005=\u20053) captured most background genetic variation based on the results of ADMIXTURE and PCA (see section Genomic divergence and genetic diversity). The SNPs that deviated significantly from the neutral background structure along the principal components  were identified as putatively genetic differentiation loci. OutFLANK employs an improved likelihood approach to estimate the null distribution of population differentiation for neutral loci  in BAYENV. An allele frequency versus environment variable covariance matrix among populations was calculated with 106 iterations in five independent runs. Based on the prior null hypothesis distribution model, we calculated the posterior distribution P-values of the correlation between allele frequency and environmental variables. The Bayes factor (BF) was generated by calculating the correlation between the allele frequency and environmental variable covariance matrices after 105 runs. The BF was calculated five times, and the average BF was treated as the final matrix of the BF. SNPs with BF values > 10 and among the top 5 % with absolute value of Spearman rank correlation coefficients (\u03c1) were considered as significantly environment-associated loci. To reduce the FDR, the associations between genotypes and environmental variables were determined using the LFMM method, which considers the population structure. Genetic data were converted into LFMM format using the R package LEA 2.0.0 . Missing SNP data were imputed based on the inferred genetic structure using the built-in SNMF function in the LEA package. Then, we ran ten independent operations to simulate the correlation between allele frequency and environmental variables for a burn-in period of 5,000 steps followed by 10,000 iterations. We used the lfmm.pvalues function in the LEA package to adjust the P-values. SNPs with adjusted P-values < 0.001 strongly support associations between allele frequencies and environmental variables. To verify the robust detection of outlier loci, we further corrected the P-values at an FDR of 0.05 using the R package fdrtool 1.2.17  putative GEA SNPs detected jointly by BAYENV and LFMM; and (4) putative outlier SNPs identified by the FST and GEA methods. Finally, we used a Venn diagram to evaluate the consistency of outlier SNPs identified across the FST and GEA methods, respectively.Using the results of these four approaches, we divided our dataset into four categories for the subsequent analyses: (1) all SNPs; (2) putative FST/(1\u2005\u2212\u2005FST)) was calculated based on all SNPs using the R package hierfstat 0.5.11 (Isolation by distance (IBD) and isolation by environment (IBE) were inferred using the R package vegan 2.5.6 to investigate the role of geographical and environmental variables in shaping spatial genetic differentiation . The gent 0.5.11 . Simple FST, GEA and all outlier SNPs) were performed to distinguish the independent effects of environment and geography by reciprocally constraining geographical and environmental variables. Significance was assessed using the randomization procedure implemented in the function ANOVA.cca with 999 randomizations.We used RDA to assess the relative contribution of environmental and geographical distances to population genetic differentiation using the R package vegan 2.5-6  to imputP. macroptera. The GF model was tested using 2000 regression trees per SNP. The Euclidian distance between current and future genetic compositions was calculated; this represents the scale of genetic change needed to match environmental change (i.e. genetic offset), with higher values indicating greater vulnerability of the population  and all outlier SNPs (0.25\u20130.58). Based on all SNPs, the genetic differentiation among different lineages was the lowest (0.09\u20130.28). For all SNPs, the genetic diversity indicated no significant difference among the three lineages , respectively . We founP. macroptera ] was highly correlated with geographical distance, suggesting a strong signal of IBD  were located in the Sino-Himalayan Forest subkingdom and one (QDT) in the Sino-Japanese Forest subkingdom. This pattern of population structure is consistent with previous studies in East Asia (P. macroptera populations is higher than that in other closely related wingnut taxa (P. stenoptera: 0.067)  were both involved in the triterpenoid biosynthetic process. As significant chemical defence compounds for the growth and development of plants and for coping with a stressed environment, terpenoids directly act as antimicrobials or signals for resisting herbivores and other natural enemies . The identified GEA genes involved in chemical defence and gene regulation may exhibit high genetic variation to adapt to the environment (temperature and precipitation). The PST018122 and PST019941 genes (detected by the P. macroptera had a high level of genetic vulnerability, suggesting that these populations are potentially at higher risk of in situ extinction under future climate changes. These marginal populations may be less resilient to future climates because genotypes were not sufficiently correlated with predicted climate variables. This result reinforces our understanding that ecologically marginal populations may be separated not only by distance from the core of the species\u2019 distribution but also experience different biotic and abiotic environments (Understanding the genetic basis of adaptation and determining the adaptive ability of species to future climate conditions are crucial in the context of climate change (P. macroptera predicted in this study may be related to the species-specific tolerance to environmental variables and the complex topography of mountains. Overall, we expect that long-term sustained climate change will result in marginal populations with high genetic vulnerability (The genetic vulnerability of P. macroptera.There is a growing interest in evolutionarily informed management strategies that rely on the spatial distribution of genetic diversity and genetic vulnerability (P. macroptera have higher genetic vulnerability. Assisted gene flow from populations with genotypes preadapted to future climate may help those marginal populations mitigate future climate change (P. macroptera (Gradient forest analysis and RONA have been widely used in assessing genetic vulnerability. Our results suggested that marginal populations of e change . Thus, mhttps://academic.oup.com/aob and consist of the following.Supplementary data are available online at FST outlier SNPs in P. macroptera identified by PCADAPT and OutFLANK. Figure S3: unique and shared outlier SNPs for the top 20 % FST SNPs identified by PCADAPT and OutFLANK. Figure S4: Manhattan plot of SNPs called by BAYENV in P. macroptera with eight environment variables. Figure S5: Manhattan plot of SNPs called by LFMM in P. macroptera with eight environmental variables. Figure S6: unique and shared outlier SNPs associated with the top four environmental variables. Figure S7: cumulative importance of genetic variation along environmental gradients in P. macroptera. Figure S8: partial redundancy analysis by condition geography using all SNPs, FST SNPs identified in PCADAPT and OutFLANK, and GEA SNPs identified in BAYENV and LFMM. Figure S9: prediction of genetic offset to future climate change based on five environment variables for all SNPs and GEA SNPs. Table S1: summary of statistical information on sequencing quality for 28 populations of P. macroptera. Table S2: details of population locations, sample size and current environmental variables for 28 populations of P. macroptera. Table S3: predicted environmental variables for the years 2081\u20132100 under two shared socio-economic pathways for P. macroptera. Table S4: genetic information statistics on mapping rate and missing rate of 160 individuals in 28 populations. Table S5: statistical information for 8244 SNPs. Table S6: functional annotation of 8244 SNPs based SnpEff software. Table S7: genetic diversity for 28 populations of P. macroptera. Table S8: unbiased estimation of nucleotide diversity for NWY, WS and QDT lineages of P. macroptera. Table S9: outlier SNPs detected by FST-based methods. Table S10: functional descriptions of genes associated with SNPs identified by FST- and GEA-based methods. Table S11: outlier SNPs detected by GEA-based methods. Table S12: accuracy importance of each environmental variable identified by GF modelling for P. macroptera. Table S13: partial Mantel test in P. macroptera conditioned with environmental and geographical distance. Table S14: partitioning of the variance and accumulated constrained eigenvalues associated with environment based on partial redundancy analysis for all SNPs, FST SNPs, GEA SNPs, and all outlier SNPs. Table S15: summary of risk of non-adaptedness calculated for SSP126 and SSP585 in P. macroptera populations based on future climate predictions for 2081\u20132100.Figure S1: ADMIXTURE bar plots of the proportion of genetic membership for each ancestry. Figure S2: mcad083_suppl_Supplementary_FiguresClick here for additional data file.mcad083_suppl_Supplementary_TablesClick here for additional data file."
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