diff --git "a/deduped/dedup_0799.jsonl" "b/deduped/dedup_0799.jsonl"
new file mode 100644--- /dev/null
+++ "b/deduped/dedup_0799.jsonl"
@@ -0,0 +1,44 @@
+{"text": "Cluster randomization design is increasingly used for the evaluation of health-care, screeening or educational interventions. At the planning stage, sample size calculations usually consider an average cluster size without taking into account any potential imbalance in cluster size. However, there may exist high discrepancies in cluster sizes.We performed simulations to study the impact of an imbalance in cluster size on power. We determined by simulations to which extent four methods proposed to adapt the sample size calculations to a pre-specified imbalance in cluster size could lead to adequately powered trials.We showed that an imbalance in cluster size can be of high influence on the power in the case of severe imbalance, particularly if the number of clusters is low and/or the intraclass correlation coefficient is high. In the case of a severe imbalance, our simulations confirmed that the minimum variance weights correction of the variation inflaction factor (VIF) used in the sample size calculations has the best properties.\u03b3) of clusters actually recruit a proportion (\u03c4) of subjects to be included (\u03b3 \u2264 \u03c4)\".Publication of cluster sizes is important to assess the real power of the trial which was conducted and to help designing future trials. We derived an adaptation of the VIF from the minimum variance weights correction to be used in case the imbalance can be a priori formulated such as \"a proportion ( This clustering effect is used during the planning of cluster randomized trials as an inflation factor to increase the sample size required by an individual randomization trial. However, such an approach does not take into account variations in cluster size, which might differ greatly. Indeed, as illustrated by Kerry et al With a Pareto imbalance, the equation can be written as:withwhich leads to Distribution-based correction (denoted d) [noted d) So we have:withthat is to say:One then recognizes the results obtained using the cluster size weights correction.Characteristics of the Pareto-like imbalanceg refers to the number of clusters within each arm and Minimum variance weighs VIFSo we obtain:mi - mj| can be written as:Given the characteristics of the Pareto-like imbalance presented in appendix B, considering that clusters are ordered hierarchically by increasing size, the matrix of the difference |\u03b3g,\u03b3g) (and 0\u03b3)g,(1-\u03b3)g) ((1-are squared matrices of size \u03b3g and (1-\u03b3)g respectively and 1\u03b3g,(1-\u03b3)g) (and 1\u03b3)g,\u03b3g) ((1-are matices of size \u03b3g \u00d7 (1-\u03b3)g and (1-\u03b3)g \u00d7 \u03b3g respectively, containing only 1 s.Where 0Thus:The pre-publication history for this paper can be accessed here:"}
+{"text": "Power for assessing interactions during data analysis is often poor in epidemiologic studies. This is because epidemiologic studies are frequently powered primarily to assess main effects only. In light of this, some investigators raise the Type I error rate, thereby increasing power, when testing interactions. However, this is a poor analysis strategy if the study is chronically under-powered  or already adequately powered (e.g. in a very large study). To demonstrate this point, this study quantified the gain in power for testing interactions when the Type I error rate is raised, for a variety of study sizes and types of interaction.Power was computed for the Wald test for interaction, the likelihood ratio test for interaction, and the Breslow-Day test for heterogeneity of the odds ratio. Ten types of interaction, ranging from sub-additive through to super-multiplicative, were investigated in the simple scenario of two binary risk factors. Case-control studies of various sizes were investigated .The strategy of raising the Type I error rate from 5% to 20% resulted in a useful power gain  in only 7 of the 27 interaction type/study size scenarios studied (26%). In the other 20 scenarios, power was either already adequate , or else so low that it was still weak (below 70%) even after raising the Type I error rate to 20% .Relaxing the Type I error rate did not usefully improve the power for tests of interaction in many of the scenarios studied. In many studies, the small power gains obtained by raising the Type I error will be more than offset by the disadvantage of increased \"false positives\". I recommend investigators should not routinely raise the Type I error rate when assessing tests of interaction. Quantification of effect-measure modification  is an important aspect of epidemiologic research . During However, power for assessing interaction during data analysis is often poor in epidemiologic studies, which are frequently designed primarily for the assessment of main effects only (the term \"main effect\" refers to any variable not involved in an interaction). Researchers who are reluctant to \"miss\" an important interaction due to low power can elect to use a higher Type I error rate when assessing interactions. A error rate of 20%, rather than the traditional 5%, has been suggested . A higheThere are two main drawbacks to the strategy of increasing the power of the interaction test by raising the Type I error rate that are explored in this paper. The first problem occurs if power is at an extremely low level when the Type I error rate is 5% , the power gain obtained from increasing the Type I error rate may not be large enough to boost power to an acceptable level. In this situation, the power might be slightly improved, but will still be very low, at the higher Type I error rate. In this paper, I refer to this chronically under-powered situation as the \"low ground\" scenario.On the other hand, the second problem can occur if the study power is already high enough to detect an interaction of substantive importance. In this situation, there is no real need to boost power, and the effect of raising the Type I error rate is merely to dilute the pool of identified interactions by including a higher proportion of interactions that are of little substantive interest. I refer to this already-adequately-powered situation as the \"high ground\" scenario.There is \"middle ground\" between the \"low ground\" and \"high ground\" scenarios. In the \"middle ground\" scenario, it makes sense to raise the Type I error rate when assessing interactions, because this will usefully boost power from a sub-standard level to a useful level.The purpose of this study is to quantify the size of the \"middle ground\". In other words, how often does raising the Type I error rate for interaction tests result in a useful gain in power? If epidemiologic studies are frequently in the \"middle ground\", there may be a case for universally recommending that the Type I error rate routinely be raised when assessing interactions. On the other hand, if few epidemiologic studies fall into the \"middle ground\", then recommendations suggesting that Type I error rate be raised  are ill-Assume an epidemiologist has conducted a study that assessed multiple exposures and is analyzing the data using a series of logistic regression models, some of which contained interactions. S/he is reviewing computer output that reports measures of effect (such as odds ratios) along with their confidence intervals and p-values. Under a \"test-based paradigm\", s/he will identify a main effect with a p-value above 5% as less predictive of the outcome than a main effect with a p-value below 5%. However, some epidemiologists, attuned to the fact that power for interactions is typically much lower than power for main effects, might elect to raise the Type I error rate to 20% when assessing interactions . They woapriori biological knowledge, in addition to considering p-values, when determining strength of association or assessing modification [Of course, an extensive literature advises epidemiologists to consider measures of effect, confidence intervals, stratum-specific measures and fication ,5-8. Furfication ,9,10. HoEpidemiologists frequently report p-values from tests of interaction, however, my anecdotal impression is that most epidemiologists do not raise the Type I error rate in the manner described above when testing for interactions. To quantify the frequency of the practice of using tests of interaction with a relaxed Type I error rate, I reviewed of all papers published between November 2004 and October 2005 in the American Journal of Epidemiology that included the word \"interaction\" in the title, abstract, or text of the paper. A total of 94 substantive papers were identified that presented some form of quantitative assessment of effect-measure modification. Of these, six papers used tests of interaction with a raised Type I error rate -16. ThreThe outcome of interest in this study was the gain in power obtained by raising the Type I error rate (see Appendix for definition of Type I and Type II error). Power was quantified at four Type I error rates: 5%, 10%, 15%, and 20%. The gain in power due to raising the Type I error rate was studied for ten different hypothetical types of interaction, ranging from sub-additive through to super-multiplicative, across three study sizes. Three commonly used tests of interaction were examined: the Wald test, the likelihood ratio test, and the Breslow-Day test. These tests are described in detail in the Appendix.In the interests of simplicity, the study was focused on case-control studies of two binary exposures. The two binary exposures are referred to as exposure A and exposure B. The standard regression analysis for this data involves fitting a logistic model:where D is coded to 0 for controls and 1 for cases, A and B are binary variables with the non-exposed coded to 0 and the exposed coded to 1, and AB is the product-term interaction obtained by multiplying A by B.Power for tests of interactions in case-control studies has previously been examined in a study that focused on comparing additive and multiplicative models . HoweverThis study examined ten different types of interaction, ranging from joint effects that were less than additive through to greater than multiplicative. These ten scenarios, described in Figure Each interaction is characterized by the expected joint effect under assumptions of perfect additivity, or multiplicativity, of effects. For example, for the sub-additive interaction (S1), the joint effect of double exposure has an odds ratio of 6, which is less than the joint effect expected under perfect additivity (3 + 6 - 1 = 8). For the first super-multiplicative interaction (T1), the joint effect of double exposure is 12, which is greater than expected under perfect multiplicativity (3 \u00d7 3 = 9). The exposures in the M1 interaction are perfectly multiplicative (3 \u00d7 2 = 6) and perfectly additive in the A1, A2, and A3 interactions. Intermediates I1 and I2 are greater than additive but less than multiplicative.I examined studies of three different sizes: 75 cases and 150 controls , 300 cases and 600 controls (large), and 1200 cases and 2400 controls (very large). The case:control ratio was fixed at 1:2.The exposure prevalences examined was also fixed. The exposure prevalence in the non-cases was set at 40% for A and 40% for B, with 40% of the non-cases being exposed to both A and B (doubly exposed), 20% being exposed to A but not to B, 20% being exposed to B but not to A, and 20% unexposed to both A and B (doubly unexposed). The doubly unexposed was the reference category in all analyses. These exposure prevalences ensured adequate numbers of cases and controls in all strata .This study used two methods for studying power: the asymptotic power function and simulations. The asymptotic power function was used to examine the power of the Wald test for the interaction in the logistic model (model 1). The term \"asymptotic power\" refers to the power results obtained from a formula derived under the assumption the study size is large (see Appendix).In addition, simulations were used to confirm the asymptotic results, investigate the coverage of the 95% confidence interval, and extend the results to the likelihood ratio test for interaction and the Breslow-Day test for heterogeneity of the odds ratio. Simulation is a computer-intensive method for empirically studying the properties of a statistic by mimicking the process of conducting a large number of studies. Ten hypothetical populations were created \u2013 one for each of the ten types of interaction studied Figure . One thoFor the purposes of this study, a useful gain in power was deemed to have occurred if: 1) power increased by at least 10% when the Type I error rate was raised from 5% to 20%, and, 2) power reached at least 70% or higher at a Type I error rate was raised of 20%. For the power gain to be less than 10%, power must be high (above 85%) at the 5% Type I error rate. Obviously this criteria is somewhat subjective and is intended as a guide to the interpretation of the results. It should not be seen as a definitive statement about the utility of raising the Type I error rate in a particular study.\u03b23 in the underlying population is zero. Because of sampling variability, however, some M1 samples will return a positive test for a non-zero \u03b23. These are false positive tests for interaction. The power function collapses to the Type I error rate  when data from scenario M1 is analyzed using model 1.The perfectly multiplicative (M1) scenario was excluded from consideration when classifying the power gains as useful or not useful. In scenario M1, the value of Asymptotic power results for the Wald test of interaction are shown in Table bolded.Table italicized scenarios in Table The The unformatted  scenarios in Table Results of the simulations were almost identical to the asymptotic results for all study sizes. The simulation results confirmed that power for the Wald test for interaction, the likelihood ratio test for interaction and the Breslow-Day test of heterogeneity of the odds ratio, are essentially identical.\u03b23  is very low for several types of interaction, even in studies as large as 300 cases and 600 controls.Second, these results call into question the wisdom of addressing the problem of low power for interaction tests by raising the Type I error rate. Power was so low in many of the scenarios studied (44%) that raising the Type I error rate failed to boost power to an acceptable level (defined as 70% in this study). In another 30% of scenarios, power was already above 90% at the 5% Type I error rate, so there was little benefit from raising the Type I error rate. In only about 1/4 of scenarios studied was there a useful gain in power due to raising the Type I error rate. Based on these data, I recommend investigators do not routinely raise the Type I error rate when assessing tests of interaction.apriori hypotheses and biological knowledge), these results may be of limited importance, since these investigators do not use the interaction p-value as the sole basis for determining whether modification exists.The implications of this study for epidemiologic practice depend in part on how individual investigators use tests of interaction. For investigators that see interaction tests as just one portion of an array of information to be utilized in the assessment of modification .Investigators need to be aware that power for testing interactions is probably low in many epidemiologic studies. However, the results of this study suggest that routinely raising the Type I error rate for interaction tests is not an effective solution to the problem of the low power for tests of interaction. I recommend that investigators should not routinely raise the Type I error rate when assessing tests of interaction.The author(s) declare that they have no competing interests.The author conceived this study, conducted all analyses, and wrote the paper. He has read and approved the final manuscript.The Type I error rate, or alpha, is the probability the study finds that an interaction between two exposures exists, when, in truth, there is no such interaction present in the population. This discrepancy arises because of sampling variability, i.e., by chance, the sample is a poor proxy for the population. The Type II error rate, or beta, is the probability that the study fails to detect an interaction between two exposures that, in truth, is present in the population. Power is 1 minus the Type II error rate. Raising the Type I error rate has the effect of decreasing the Type II error rate and thus increases the power of an interaction test.The Wald test statistic for the test of interaction (see model 1) is:The Wald test for interaction follows this power function :F is the cumulative distribution function of the standard normal variate , \u03b1 is the two-sided Type I error rate, and \u03b23 is defined per model 1.where The Wald test statistic follows an approximate chi-square distribution under large sample conditions and the assumption of perfect multiplicativity of joint effects (i.e. assuming the null hypothesis). It has 1 degree of freedom in the situation of two binary exposures.\u03b23 by comparing the log-likelihood for model (1) to the log-likelihood for the same model without the interaction term:The likelihood ratio test can also be used to test The likelihood ratio test for interaction is:Like the Wald test, this statistic is approximately chi-square distributed under large sample conditions and has 1 degree of freedom for two binary exposures.In contrast to these two model-based statistics , the Breslow-Day test for heterogeneity of the odds ratio is based on stratified analysis. The test statistic is:nh11 is the count for the doubly unexposed cell of the hth level of the stratification variable, ORMH is the Mantel-Haenszel odds ratio estimate, and h = 2 if both exposures are binary. Like the other two tests, it has an approximate chi-square distribution under large sample conditions.where"}
+{"text": "The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. These receptors are also ligand-dependent transcription factors responsible for the regulation of cellular events that range from glucose and lipid homeostases to cell differentiation and apoptosis. The importance of these receptors in lipid homeostasis and energy balance is well established. In addition to these metabolic and anti-inflammatory properties, emerging evidence indicates that PPARs can function as either tumor suppressors or accelerators, suggesting that these receptors are potential candidates as drug targets for cancer prevention and treatment. However, conflicting results have emerged regarding the role of PPARs on colon carcinogenesis. Therefore, further investigation is warranted prior to considering modulation of PPARs as an efficacious therapy for colorectal cancer chemoprevention and treatment. Understanding the biology of intestinal epithelial cells may reveal the molecularpathogenesis of a number of digestive diseases. One such disease, colorectal cancer (CRC), leads to significantcancer-related morbidity and mortality in most industrialized countries. Initiation and progression of CRC are a complex processthat results from the loss of the normal regulatory pathways that govern a balance betweenepithelial cell proliferation and death. Forexample, alterations in multiple pathways such as Wnt/APC, COX-2, andRas are known to play major roles in CRC progression. The standard treatment for advancedmalignancies has improved greatly over the past decade but is still notsatisfactory. Therefore, significanteffort has been exerted to identify novel drug targets for both the preventionand treatment of this disease. One groupof compounds found to decrease the risk of colorectal cancer includesnonsteroidal anti-inflammatory drugs (NSAIDs), which target the cyclooxygenaseenzymes (COX-1 and COX-2). However,prolonged use of high doses of these inhibitors (except for aspirin) isassociated with unacceptable cardiovascular side effects . Thus, i\u03b1 (NR1C1), PPAR\u03b4/\u03b2 (NR1C2), and PPAR\u03b3 (NR1C3). Each PPAR isotype displays a tissue-selectiveexpression pattern. PPAR\u03b1 and PPAR\u03b3 are predominantly present in the liverand adipose tissue, respectively, while PPAR\u03b4 expresses in diverse tissues [Dietary fat intake is an environmental factor that is associated with some humandiseases such as diabetes, obesity, and dyslipidemias. Some nuclear hormone receptors play a central role in regulatingnutrient metabolism and energy homeostasis. These nuclearreceptors are activated by natural ligands, including fatty acids andcholesterol metabolites. Among thesereceptors, special attention has been focused on the members of the peroxisomeproliferator-activated receptors (PPARs) family, which were initially identified as mediatorsof the peroxisome proliferators in the early 1990s . PPARsp tissues . In comm tissues , 7, 8. E\u03b3 synthetic agonists, rosiglitazone andpioglitazone, are antidiabetic agents which suppress insulin resistance inadipose tissue. The antiatherosclerotic and hypolipidemic agents including fenofibrate and gemfibrozil are PPAR\u03b1 synthetic agonists that induce hepaticlipid uptake and catabolism. Genetic andpharmacological studies have also revealed important roles of PPAR\u03b4 in regulating lipid metabolism and energyhomeostasis. Genetic studies indicate that overexpression of constitutively active PPAR\u03b4 in mouseadipose tissue reduced hyperlipidemia, steatosis, and obesity induced by eithergenetics or a high-fat diet. In contrast, PPAR\u03b4 null mice treated in similar fashion exhibited an obesephenotype [\u03b4 selective-agonist (GW501516) attenuatedweight gain and insulin resistance in mice fed with high-fat diets [\u03b4 agonists diminished metabolicderangements and obesity through increasing lipid combustion in skeletal muscle[\u03b4 agonists are potential drugs for use in the treatment of dyslipidemias, obesity, and insulin resistance. Therefore,the PPAR\u03b4 agonist (GW501516) is currently in phase III clinical trials to evaluate its use for treatment of patients withhyperlipidemias and obesity. However, recent studies showingthat some agonists of PPARs promote carcinogenesis in animal models have raised concerns aboutusing these agonists for the treatment of metabolic diseases. For example, long-term administrationof a PPAR\u03b1 agonist induces the development ofhepatocarcinomas in mice but not in PPAR\u03b1 null animals, conclusivelydemonstrating that PPAR\u03b1 mediates these effects in promotingliver cancer [\u03b4 agonist (GW501516) acceleratesintestinal polyp growth in ApcMin/+ mice [\u03b4 antagonists as chemopreventive agents.It is well established that modulation of PPAR activity maintains cellular and whole-body glucose and lipid homeostases. Hence, great efforts have been made todevelop drugs targeting these receptors. For example, PPARhenotype . Pharmacat diets  and incrat diets . Furthel muscle. These rr cancer . Furthern/+ mice , 15. The\u03b3 serves as a tumor suppressor. Contradictory evidences suggest that PPAR\u03b4 can act as either a tumor suppressor or tumor promoter. A few evidences support arole of PPAR\u03b1 in CRC.Significant effort has been concentrated on deducing the role of PPARs in CRC and other cancers. A large body of evidence indicates that PPAR\u03b1 in hepatocarcinomas are clear, less is known about the roleof PPAR\u03b1 in human tumors. Generally, activation of PPAR\u03b1 by exogenous agonistscauses inhibition of tumor cell growth in cell lines derived from CRC,melanoma, and glialbrain tumors [\u03b1 expression is elevated in humancancers.Although the tumor-promoting effects of PPARn tumors \u201318. Ther\u03b3 in regulating cellular differentiationprompted a great effort to investigate the function of PPAR\u03b3 in cancer field. While PPAR\u03b3 is elevated in CRC [\u03b3 mutation in CRC from humans, animals, and cultured cellsproduced controversial results. One study showed that 8% of primary human colorectal tumors had a loss of function mutation in one allele ofthe PPAR\u03b3 gene [\u03b3 gene is associated with increasedrisk of CRC [\u03b3 gene has not been detected in human colon tumor samples andCRC cell lines, suggesting that PPAR\u03b3 mutations in human CRC is a rare event [The prominent role of PPARd in CRC , suggestAR\u03b3 gene . Recent k of CRC , 22. There event .\u03b3 results in growth arrest of coloncarcinoma cells through induction of cell-cycle arrest or/and apoptosis. Several potential downstream targets of PPAR\u03b3 for mediating antitumor effects of PPAR\u03b3 have been identified in various cancer cell types. Activation of PPAR\u03b3 negatively regulates cell cycle progression by modulating a number of cell cycleregulators: (1) inhibiting E2F activity in transformed adipogenic cells [\u03b3 has also been reported to inhibit tumor cell growth byupregulation of the transcriptional repressor TSC22 in colon cancer cells [\u03b3 agonists induce apoptosis by inductionof PTEN expression in pancreatic, breast, and colon cancer cells [\u03baB and Bcl-2 expression in colon cancercells [\u03b3 exhibits antiangiogenic effects byinhibiting VEGF expression in tumor cells and VEGF receptors in endothelialcells [\u03b3 agonists suppress tumor cell invasion in colon and breastcancer cells by downregulation of matrix metalloproteinase-7 (MMP-7) andinduction of MMP inhibitors [\u03b3 to suppress tumor growth is also through inhibiting APC/\u03b2-catenin andCOX-2/PGE2 signaling pathways, which are pivotally involved in coloncarcinogenesis [In vitro studies show that activation of PPARic cells , (2) Rb ic cells , 26, (3)ic cells \u201329, and ic cells . Activater cells  and GADDer cells . PPAR\u03b3 aer cells  and inhiercells . Moreovealcells , 36. It hibitors , 38. In ogenesis \u201342.\u03b3 in colorectal cancer progression iscontroversial because there are conflicting results in mouse models of coloncancer. Although PPAR\u03b3 agonists inhibit colorectal carcinogenesis inxenograft models and in the azoxymethane (AOM)-induced colon cancer model [Min/+ mouse. It has been reported that administrationof PPAR\u03b3 agonists significantly increases the numberof colon adenomas in the ApcMin/+ mice [Min/+ and Apc\u03941309) with the PPAR\u03b3 agonist pioglitazone resulted in reduction in the number ofboth small and large intestinal polyps in a dose-dependent manner [\u03b3 is sufficient to increase tumor numberin AOM-treated mice and that intestinal-specific PPAR\u03b3 knockout promotes tumor growth in ApcMin/+ mice [\u03b3 serves as tumor suppressor incolorectal cancer. One possible explanation for the differences in phenotype caused by pharmaceutical versus geneticmanipulation of PPAR\u03b3 in mouse models may be due to the PPAR\u03b3-independent effect of the agonistdrugs, drug doses used, and animal models employed. This controversial extends beyond CRC. For example, data are conflicting from different animal models of breastcancer as well. PPAR\u03b3 agonist suppresses NMU-induced mammarycarcinomas [\u03b3 accelerates mammary gland tumor developmentin MMTV-PyV transgenic mice [However, the role of PPARer model , 44, then/+ mice \u201347 and en/+ mice . Howevert manner , 50. Then/+ mice , 51. Thercinomas . Howevenic mice .\u03b4 has been shown to play an important role inembryo implantation [\u03b4 in colorectal carcinogenesis is more controversial than that of PPAR\u03b3. The first evidence linking the PPAR\u03b4 to carcinogenesis actually emerged fromstudies on gastrointestinal cancer. PPAR\u03b4 is elevated in most human colorectal cancersand in tumors arising in the ApcMin/+ mice, and AOM-treated rats [\u03b4 proteins are accumulated only in human CRC cells with highlymalignant morphology [\u03b4 is correlated with antitumor effects ofdietary fish oil/pectin in rats treated with radiation and AOM [\u03b4 was identified as a directtranscriptional target of APC/\u03b2-catenin/Tcf pathway and as a repressiontarget of NSAIDs [\u03b4 gene [\u03b4 expression and activity are also induced by oncogenicK-ras [2 directly transactivatesPPAR\u03b4 [2 indirectly induces PPAR\u03b4 activation in CRC, hepatocellular carcinoma,and cholangiocarcinoma cells [\u03b4 is a focal point of cross-talk betweenthese signaling pathways.PPARantation , atherogantation , regulatantation , and skiantation , 58. Theed rats , 60. Imprphology . Downreg and AOM . PPAR\u03b4 wf NSAIDs , 63. Ac [\u03b4 gene . PPAR\u03b4 eicK-ras . In addima cells \u201368. Thes\u03b4 alleles in human HCT-116 colon carcinoma cellsdecreased tumorigenicity, suggesting that activation of PPAR\u03b4 promotestumor growth [\u03b4 has been reported to have bothtumor-promoting and tumor-inhibiting effects based on conflicting data obtainedfrom mouse models of colon cancer. Forexample, activation of PPAR\u03b4 by a selective synthetic PPAR\u03b4 agonist (GW501516) or a PPAR\u03b4 endogenous activator (PGE2)accelerates intestinal adenoma growth in ApcMin/+ mice by promoting tumor cell survival [\u03b4 attenuates both small and largeintestinal adenoma growth, and PPAR\u03b4 is required for the tumor-promotingeffects of PPAR\u03b4 ligand (GW501516) and PGE2 in ApcMin/+ mice [\u03b4 in ApcMin/+ micesignificantly reduced growth of tumors larger than a diameter of 2 mm, eventhough PPAR\u03b4 deficiency did not affect overall tumor incidence [\u03b4 serves as tumor accelerator, recent conflictingreports show that PPAR\u03b4 deficiency enhancespolyp growth in ApcMin/+ and AOM-treated mice inthe absence of exogenous PPAR\u03b4 stimulation [\u03b4 ligand (GW0742) inhibits colon carcinogenesis in AOM-treated mice but promotessmall intestinal polyp growth in ApcMin/+ mice [In a murine xenograft cancer model, the disruption of both PPARr growth . Howeversurvival , 66. A snist GW5016 or a Pncidence . In contmulation , 72.Mor/+ mice .Min/+ mice, animal breeding, or possibly to differences in the specific targeting strategy employed to delete PPAR\u03b4. For example, the average number of polyps in 13-week old ApcMin/+ mice on a C57BL/6 genetic background is about 50, while the polyp number in ApcMin/+ mice on a mixed-genetic-background (C57BL/6 \u00d7 129/SV) is about 120. Our results also show that the breeding strategyaffects the number and size of polyps in mice even on the same geneticbackground. Mice generatedby breeding female PPAR\u03b4\u2212/\u2212/ApcMin/+ with male PPAR\u03b4\u2212/\u2212/Apc+/+ exhibit increased adenoma number with a larger average size than those obtained by breeding female PPAR\u03b4\u2212/\u2212/Apc+/+ with male PPAR\u03b4\u2212/\u2212/ApcMin/+. Finally, the PPAR\u03b4 null mice we studied were obtained fromBeatrice Desvergne in Switzerland. These mice were generated by deleting exons 4 and 5 encodingthe DNA binding domain [\u03b4 knockout mice by inserting a neomycin resistance cassette intothe last exon (exon 8) [\u03b4 by the Peters group might have led to a hypomorphic allele, whichretains some aporeceptor function, thus making it difficult to correctlyinterpret their results. Indeed,conflicting results in the context of embryonic lethality have also beenobserved from these two PPAR\u03b4 mutant mouse strains [\u03b4 in colorectal tumorigenesis, it isimportant to investigate the role of PPAR\u03b4 in animal models that are dependent onactivation of other oncogenes or disruption of other tumor suppressors toverify our conclusions that activation of PPAR\u03b4 is proneoplastic.One explanation for these disparate results may be due to differences in thegenetic background of Apcg domain , while P(exon 8) . It has  strains , 75. To\u03b4 serves as a tumor accelerator. A selective PPAR\u03b4 agonist (GW501516) has been shown to stimulate proliferationof human breast, prostate, and hepatocellular carcinoma cells [\u03b4 activation reduced ovarian tumor growth[\u03b4 knockout mice exhibited significantimpaired angiogenesis and tumor growth after these mice were injected s.c. withmouse Lewis lung carcinoma and melanoma cells [\u03b4 agonist (GW501516) accelerated tumor formation, while a PPAR\u03b3 agonist (GW7845) delayed tumor growth [\u03b4 in cancer biologyremains unclear.Studies in other types ofcancer also support the hypothesis that PPARma cells , 76, 77.r growth. PPAR\u03b4 kma cells . In a mr growth . Takent\u03b3 and PPAR\u03b4 in CRC, the role of these receptorsremains highly controversial in this disease. Emerging evidence demonstrates that cooperative interactions betweenWnt, COX-2, and PPARs signaling pathways can initiate cellular transformationand promote progression of colorectal cancer. These studies provide support for evaluating the efficacy of PPAR\u03b4 antagonists for cancer preventionand/or treatment. We propose a potentialworking model as a useful starting point for future studies (see Despite extensive research on both PPARies see ."}
+{"text": "Psychotropic drug prescribing is problematic and knowledge of factors affecting the initiation and maintenance of such prescribing is incomplete. Such knowledge could provide a basis for the design of interventions to change prescribing patterns for psychotropics. The aim of this study was to explore the views of general practitioners (GPs), GP interns, and heads of primary care units on factors affecting the prescribing of psychotropic drugs in primary care.We performed four focus group discussions in Gothenburg, Sweden, with a total of 21 participants . The focus group discussions were transcribed verbatim and analyzed using manifest content analysis.eeking care for symptoms, reflects the participants\u2019 understanding of why patients approach primary care and comprised categories such as knowledge, attitudes, and society and the media. The second theme, Lacking a framework, resources, and treatment alternatives, which reflects the conditions for the physician-patient interaction, comprised categories such as economy and resources, technology, and organizational aspects. The third theme, Restricting or maintaining prescriptions, with the subthemes Individual factors and External influences, reflects the physicians\u2019 internal decision making and comprised categories such as emotions, knowledge, and pharmaceutical industry.Three different themes emerged from the focus group discussions. The first theme SThe results of the present study indicate that a variety of factors may affect the prescribing of psychotropic medications in primary care. Many factors were related to characteristics of the patient, the physician or their interaction, rather than the patients\u2019 medical needs per se. The results may be useful for interventions to improve psychotropic prescribing in primary care. The use of psychotropics is increasing both internationally and in SFor these reasons, there may be a need for interventions aimed at limiting prescriptions of psychotropics to patients who do not clearly benefit from these drugs. Arguably, as polypharmacy may be a problem in itself, such interventions should target the prescribing of psychotropics in general, rather than that of specific drug groups. Consequently, knowledge is needed about factors that can influence the prescribing of this drug group as a whole.Previous qualitative studies, however, have only investigated physician decision making concerning specific psychotropic drug groups, such as hypnotics, antidepFocus group interviews are appropriate to use when the goal is to explore people\u2019s knowledge and experiences. Data coThe focus group discussions were performed in 2011 at two different health care units in Gothenburg . Apart from the participants and the moderator, one additional researcher took part as an assistant moderator, taking field notes. We chose to alternate between moderators in the focus groups because we believed that our different backgrounds could have an impact on the discussions. The researcher with the most experience of focus groups  was moderator twice and the other two researchers acted as moderators once . The study protocol was approved by the Regional Ethical Review Board in Gothenburg, Sweden.Each group discussion started with a short, structured introduction by the moderator, and participant information was again distributed to each person together with a consent form to be signed before the discussion started. To facilitate the discussion, we used an interview guide Table\u00a013], in, in13], The focus group discussions were transcribed verbatim and all transcripts were read by all researchers. Names of participants were replaced by codes . NVivo 9 software was used for data management. We used manifest content analysis, according to Graneheim and Lundman, which ieeking care for symptoms; Lacking a framework, resources, and treatment alternatives; and Restricting or maintaining prescriptions. The latter had two subthemes: Individual factors and External influences. The themes and categories  are presented in Figure\u00a0The focus groups and participants are described in Table\u00a0This theme contains categories describing factors of importance for the decision of the patient to seek care for psychiatric symptoms. According the participants, seeking care was not synonymous, for all patients, with wanting medication.Demography and social factors were considered important for care seeking. Unemployment, divorce, and social problems were brought up as factors of importance. Age was also discussed and the participants meant that youth today cannot handle disappointments and they have high expectations on quick solutions.\u201c\u2026 young people who can\u2019t feel bad for 1 day, just have to get well the same day.\u201d society and the media in terms of how mental diseases are less stigmatized today. The participants perceived that patients do not consider psychiatric symptoms to be as disgraceful as before, and they are more prone to try a psychotropic medication. They also mentioned that the media are contributing to the idea of antidepressants as \u201cthe happy pill\u201d and that there is increased medicalization in society.The participants discussed the importance of \u201c\u2026 the main difference that distinguish psychotropics, and psychiatric diseases as well, is that psychiatric diseases have traditionally somehow, for both health care and patients, been kind of shameful. And that is disappearing to some extent.\u201d friends and relatives, but also staff at nursing homes who may demand treatment for a patient who is considered problematic. The impact of knowledge (the well-informed patient) and attitudes of patients  were also discussed.Other influences mentioned were positive or negative experiences of medication of \u201cSo sometimes I meet people who are negative from the start and they are pretty difficult to convince. And sometimes they finally give in and try for a week. And then they go, All right, it didn\u2019t work. Even though I\u2019ve explained that it may take 3 weeks.\u201d The categories in this theme deal with the conditions of the physician patient consultation. Many of the factors mentioned were considered barriers to rational prescribing.haracteristics of mental illness included difficulties of psychiatric compared with somatic diseases, e.g., diagnosing, where the participants meant there may be a risk of imprecise diagnosing. Further, participants perceived problems in evaluating treatment. The problem of keeping psychiatric treatment evidence-based was also highlighted.The category c\u201c\u2026psychiatry is not a science like others, not as exact. And it leaves room for many interpretations.\u201d \u201cIt depends a lot on the individual, so it\u2019s hard to comply with general guidelines. Each time you have to manage by trial and error, I would say. And that applies to the drug, the selection, and the dosage as well.\u201d patient physician process comprised the importance of communication skills, e.g. the ability of the physician to ask the right questions in order to make the right diagnosis. Further, the participants discussed patient factors, such as the patients\u2019 pre-understanding, or fear of adverse effects, as well as their expectations and desires concerning treatment.The \u201cThere are lots of people who expect that they have the right to feel well all their lives and that health care should sort it out for them.\u201d economy and resources, the costs of medication and engaging locum physicians in primary care were mentioned. Further, the lack of time was discussed both with regard to the patient physician consultation and concerning patient follow-up.Concerning technology. According to the participants, technical issues may increase the issue of renewing prescriptions without proper evaluation of the treatment.Another category was \u201cIt\u2019s easy to press the button, to click accept.\u201d Authors\u2019 comment)(In the multi-dose drug dispensing system in Sweden (ApoDos), all drugs prescribed to an individual can be renewed simultaneously by performing \u201cone click\u201d on the computer. Ordinary prescriptions, on the other hand, need to be renewed one by one. organizational aspects were considered important for the prescribing of psychotropics; namely, level of care and differences between care units. Differences between care units concerned primarily different prescribing traditions at different units. The problem of indeterminate boundaries between psychiatry and primary care, the transfer to primary care of patients previously treated within psychiatric care implicating the inheritance of prescriptions from psychiatry, and the limited possibilities for follow-up in primary care were some of the subcategories of level of care.Two \u201cBecause then you think, if they end up in the right place perhaps it\u2019ll be the right medication and you\u2019re more in control of things. And that\u2019s what it\u2019s all about. As for me, I feel that a lot of this is an organiza-, well, perhaps that\u2019s stretching it a bit, but to some extent anyway, that this is an organizational issue to a large extent, that there are lots of prescriptions.\u201d treatment alternatives from two perspectives, namely, effects/side-effects and availability. Based on the perceived effect of treatment, the choice between pharmacotherapy and psychotherapy is influenced by disease severity, as well as patient\u2019s background and age. Further, the participants suggested that limited access to psychotherapy can increase pharmacotherapy treatment.The participants also discussed \u201cNo psychoanalyst and no God to offer. We just have that security blanket, the pill, you know\u2026\u201d Individual factors and External influences.The last theme describes how the prescribing behavior of physicians is influenced by emotions comprised aspects such as the wish to be, or not to be, updated on new psychotropic drugs, not having the energy to say no to a patient, or the unease about changing drug treatment initiated by colleagues, e.g., specialists in psychiatry:The category \u201cIt\u2019s much harder to withdraw a drug. And if someone else has started it, I feel, Hang on, I\u2019m stepping on this colleague\u2019s toes.\u201d Knowledge, or, rather, lack of knowledge, was another category described to influence the prescribing of psychotropics. The participants mentioned that primary care physicians do not have enough knowledge about new or specialized psychotropics to prescribe them or to evaluate their effect.\u201c\u2026we\u2019ve got problems getting help when we feel we\u2019re not quite, don\u2019t have the right competence\u2026//\u2026So I gather we all feel that our knowledge is not sufficient from time to time, for the prescribing we\u2019re doing.\u201d xperience that a physician\u2019s past prescribing behavior may affect his or her future patient clientele. Participants mentioned that low prescribing of benzodiazepines makes those patients seek care elsewhere. Further, the experience of physicians makes them prescribe a small range of psychotropics, i.e., they have their own personal prescribing repertoire.Participants related the emedical education was also discussed among the participants and many of the participating physicians had gone to medical school at the University of Gothenburg. They said it was their experience that in that academic environment, medication, rather than non-pharmacological treatment, was advocated.The impact of \u201c\u2026 those of us who studied in Gothenburg have had it thoroughly hammered in that it\u2019s the drugs that matter.\u201d Variability in prescribing between individuals was mentioned, with several examples of high prescribers given, that is, physicians that the participants had met in practice. Further, psychiatrists as a group were described as prescribing psychotropics to a higher extent and in higher doses compared to GPs. Participants also felt that the prescribing of psychotropics is often arbitrary.\u201cWe just tinker about with the levels of different chemicals. And then we hope and keep our fingers crossed for it to work, and sometimes it\u2019s for the worse.\u201d pharmaceutical industry, colleagues, and the health care authorities.External factors of importance are the health care authorities mainly concerned regional prescribing guidelines and the list of recommended drugs prepared by the drug and therapeutics committees. These publications were mostly regarded as trustworthy by the participants while some colleagues and, in particular, the drug industry were seen as less dependable.The category In this focus group study, we explored the views of GPs, GP interns, and heads of primary care units on factors affecting the prescribing of psychotropic drugs in general in primary care. Interestingly, many factors emerged other than the medical needs of the patient; the results indicate that prescribing of psychotropics may be initiated or maintained due to patient or peer pressure, or due to the lack of time or of treatment alternatives. Further, the importance of the characteristics of mental illness was more obvious in our study than in previous research in this area-11. PsycEmotions were involved in both restricting, and initiating and maintaining prescriptions of psychotropic drugs. The experienced pressure from patients or from colleagues was described in terms of emotions such as not having the energy to argue with the patient, or the fear of stepping on a colleague\u2019s toes. Indeed, previous research has shown that little effort is used to form a second opinion on a patient\u2019s problem when psychotropic drug treatment has been initiated by another doctor. The dilThe problem of indeterminate boundaries between primary care and psychiatry was another recurrent topic. In Sweden, the level of care depends on the type of psychiatric disease as well as the severity of the disease. Thus, psychosis and severe depression are predominantly treated in psychiatry, whereas mild depression and anxiety are often treated in primary care. Clearly, many patients will fall in-between, and it may be difficult to assign these to the appropriate level of care. The participants described referral blocking of patients to psychiatry, on the one hand, and the transfer to primary care of patients previously treated within psychiatric care, on the other. These boundaries create frustration and additional work for the GPs. The takeover of previous prescriptions from specialists has been highlighted previously, but it is not always described as a problem. For insTechnology has made it possible to renew a number of prescriptions by using a single mouse click, and doing so may be one way of reducing the perceived high workload of GPs. Indeed, this possibility may increase the overall prescribing of psychotropics, as reported in a recent Swedish study. FurtherWe believe that the mix of participants is a strength of the study. The physicians\u2019 workplaces were spread across the city, covering both the city center and the suburbs, thus representing care for different socio-economic groups. Furthermore, the heads of units contributed with a top-down perspective. However, we had no participants from rural areas. In addition, the fact that we recruited by personal contacts may have introduced a source of bias. Thus, there may be some aspects on prescribing of psychotropics that we may not have captured.In this study, we chose to elicit a discussion about psychotropic prescribing in the widest sense. An alternative strategy would have been to focus on certain drug groups (e.g. anxiolytics), or on specific aspects of prescribing (e.g. initiation or maintenance). Our reasons for this choice was, first, the lack of research on psychotropics as a group, and, second, our belief that a more universal approach will be more useful in the design of interventions aimed at increasing the quality of prescribing.We chose to use three different moderators and we cannot rule out that this choice may have had an impact on the results. In addition, one of the researchers had the dual role of a GP intern and a researcher. However, the three moderators followed the same interview guide and were all careful not to steer the discussion, e.g., by not responding in an encouraging way to any one of the comments of the participants. We therThis study illustrates the complexity of the process of psychotropic prescribing. Numerous factors are important in this process, many of which are unrelated to the patient\u2019s medical needs. The results may be useful when targeting interventions aimed at improved prescribing of psychotropics in primary care.The authors report no competing interests.TMH, SAS and SMW were all involved in the design, data collection and analysis of data. TMH drafted the manuscript, which was revised by SAS and SMW. All authors have given final approval of the version submitted.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2296/14/115/prepub"}
+{"text": "Trypanosoma cruzi the agent of Chagas disease is a monophyletic but heterogeneous group conformed by several Discrete Typing Units (DTUs) named TcI to TcVI characterized by genetic markers. The trans-sialidase (TS) is a virulence factor involved in cell invasion and pathogenesis that is differentially expressed in aggressive and less virulent parasite stocks. Genes encoding TS-related proteins are included in a large family divided in several groups but only one of them contains TS genes. Two closely related genes differing in a T/C transition encode the enzymatically active TS (aTS) and a lectin-like TS (iTS). We quantified the aTS/iTS genes from TcII and TcVI aggressive and TcI low virulent strains and found variable aTS number (1\u201332) per haploid genome. In spite of being low TS enzyme-expressers, TcI strains carry 28\u201332 aTS gene copies. The intriguing absence of iTS genes in TcI strains together with the presence of aTS/iTS in TcII and TcVI strains (virulent) were observed. Moreover, after sequencing aTS/iTS from 38 isolates collected along the Americas encompassing all DTUs, the persistent absence of the iTS gene in TcI, TcIII and TcIV was found. In addition, the sequence clustering together with T/C transition analysis correlated to DTUs of T. cruzi. The consistence of TS results with both evolutionary genome models proposed for T. cruzi, namely the \u201cTwo Hybridization\u201d and the \u201cThree Ancestor\u201d was discussed and reviewed to fit present findings. Parasite stocks to attempt genetic KO or to assay the involvement of iTS in parasite biology and virulence are finally available. Trypanosoma cruzi. With an estimated 8\u201310 million people already infected, and about 40,000 new cases/year, Chagas disease represents a major public health, social and economic problem in Latin America where about 100 million people are at risk Chagas disease is a chronic disabling disease caused by the protozoon T. cruzi constitutes a monophyletic but genetically heterogeneous group. Based on various genetic markers and evolutive and population genetics interpretations of data, T. cruzi populations have been classified into six Discrete Typing Units (DTU) namely TcI to TcVI. Recently a seventh group sampled in bats and named TcBat has been added T. cruzi, being involved in host cell adhesion/invasion processes and escape from the complement, the parasite is unable to synthesize this sugar de novo. To circumvent this gap, the parasite expresses the trans-sialidase (TS), that transfers \u03b1-linked sialyl residues among glycoproteins or glycolipids. Circulating TS activity alters the sialylation pattern of the cellular glycoconjugates leading to hematological and immunological abnormalities associated to the disease Although sialic acid is crucial for the life cycle of vs. iTS deduced amino acid sequences shows variations in 20 residues, although the inactivation is entirely due to the single crucial Tyr342His replacement as a consequence of a T/C transition Genes encoding TS are included in a large family composed of at least 1439 members aTS in all analyzed stocks and the striking absence of iTS genes in TcI, TcIII and TcIV DTUs. The consistence of the TS results with current T. cruzi evolutionary genome models was reviewed to fit findings. Parasite stocks to attempt genetic KO or to assay the involvement of iTS in parasite biology and virulence are now available.Previous efforts to associate parasite genetic classification and biological features have allowed us to determine the expression/shed of aTS as a marker of pathogenicity that segregates strains belonging to different lineages The study was carried out in a panel of 38 parasite isolates encompassing all DTUs  obtained from various ecological origins  spanning all the endemic area from Argentina to the USA.DNA from Ac, Hc, K-98, SN, Br, CMA, ChVal, HE, HT, RA, Q501/3, Tulahuen, ML, Alf, FAL and Cvd parasite strains was obtained from peripheral blood trypomastigotes. DNA from Silvio X10, Tu18, M5631, Can III, CL Brener, CID, H1, QUE, CBBcl2, ESMcl3Z2, IVVcl4, MAS1cl1, MVBcl8, X109/2, 3.1, 92122102R, STC10R, STC16Rcl1, MNcl2, SC43cl1, CA15, P63cl1 strains was obtained from epimastigotes. The Blood and Cell Culture DNA Purification Kit (Qiagen) or conventional phenol-chloroform DNA extraction methods were used.T. cruzi DNA samples were genotyped using polymerase chain reaction (PCR) strategies following Burgos et al T. cruzi stocks  were also characterized by MLEE All For PCR characterization, five reactions targeted to the intergenic region of spliced leader genes (SL-IR), 24s\u03b1 rDNA and the A10 fragment were carried out on each DNA sample to determine the parasite DTU. The PCR amplicon size for each DTU was: Tc I: SL-IRac (150 bp), SL-IR-I (475 bp), and 24s\u03b1 HnPCR (140 bp); TcII: SL-IRac (157 bp), SL-IR-II (425 bp), 24s\u03b1 HnPCR (140 bp) and A10-PCR (690 bp); TcIII: SL-IRac (200 bp), 24s\u03b1 HnPCR (125 bp), and A10-PCR (630 bp); TcIV: SL-IRac (200 bp) and 24s\u03b1 HnPCR (140/145 bp); TcV: SL-IRac (157 bp), SL-IR-II (425 bp), 24s\u03b1 HnPCR (125 or 125+140 bp), and A10-PCR (630 bp); TcVI: SL-IRac (157 bp), SL-IR-II (425 bp), 24s\u03b1 HnPCR (140 bp), and A10-PCR (630 bp).5'-TGGGCAAGTATCCATTGGTGATG-3' and 5'-TGATCTCATGCAAACAGTACAGCTT-3'. In the same reaction, a pair of fluorescent-labeled probes specific for the two possible sequences at the mutation position (TACAGCT-3\u20325\u2032-AATTCCGCC coupled to FAM to detect aTS and CACAGCT-3\u20325\u2032-AAAATTCCGCC coupled to VIC which binds to iTS) allowed the quantification of both genes. The analysis was normalized by quantification of the T. cruzi pyruvate dehydrogenase (PVDH)-encoding gene present as a single copy per haploid genome 5'-CGGCGTACCAGCCTGAGAT-3' and 5'-ACCTGAAGGCCCGGAATG-3') and a labeled probe (5'-TACCGTCGTGGCGACT-3') that hybridizes the pvdh gene.To assess the number of genes per haploid genome coding for aTS and iTS in each parasite isolate, a real-time PCR-based strategy was applied. These assays were performed at the facilities of Eurofins Medigenomix GmbH (Germany) on an ABI 7900 HT Sequence Detection System (Applied Biosystems) using the universal mix of ABI TaqMan reagents (Applied Biosystems). Briefly, the region containing the mutation was amplified with the following flanking oligonucleotides: aTS or iTS genes were used as control and during test standardization. In the data analysis, the intensity of each signal was a definite value , which is inversely related to the amount of complementary DNA. The proportion of both genes was calculated for each T. cruzi isolate as the average of two independent determinations.Plasmids containing the 5\u2032-GGAGGCTGTCGGCACGCTCTC-3\u2032 and TS-5, 5\u2032-GCTTCACTGCCGTGACCATCG-3\u2032; 3\u2032 primers: TS-31, 5\u2032-TCACGCAGCGGTACGCATCCT-3\u2032 and TS-3, 5\u2032-CAGCGGGACCACAACCACGCT-3\u2032), so that all the TS sequences annotated in the GenBank were targeted in at least one of the four PCR reactions to be performed. Amplification was carried out with 0.4 \u00b5M of each primer, 5 U of Pfu polymerase enzyme (Promega), 2.5 mM of dNTPs, and 100 ng of genomic T. cruzi DNA as template in 50 \u00b5l final reaction volume. The PCR cycle consisted of 30 rounds of 94\u00b0C for 45 s, 63\u00b0C for 45 s, and 72\u00b0C for 45 s, with a first step of 2 min at 94\u00b0C and one last step of 5 min at 72\u00b0C. PCR products were analyzed by electrophoresis in 2% agarose gel. Amplicons were purified and both strands sequenced with the primers used for amplification. Chromatograms were visually examined to determine the presence of C and/or T in the first position of the codon 342. Firstly TS-51/TS-31 primer set was used and those genomes rendering only the Tyr342 codon were then subjected to the other three PCR reactions. The IUPAC nomenclature for the genetic code was used to define single nucleotide polymorphism (SNP) positions with mixed base identification set to 15% of the highest peak. Sequences were deposited in the GenBank with the accession numbers KC286514, KC286515, KC286516, KC286517, KC286518, KC286519, KC286520, KC286521, KC286522, KC286523, KC286524, KC286525, KC286526, KC286527, KC286528, KC286529, KC286530, KC286531, KC286532, KC286533, KC286534, KC286535, KC286536, KC286537, KC286538, KC286539, KC286540, KC286541, KC286542, KC286543, KC286544, KC286545, KC286546, KC286547, KC286548, KC286549, KC294586 and KC294587.Two upstream and two downstream primers to the region containing the T/C transition of TS-encoding genes were designed  or His codon (complementary to the sequence of iTS genes), respectively. No cross-recognition between the aTS and iTS probes under test conditions was found, as assayed with plasmids harboring the corresponding gene. No Ct could be determined with the iTS-complementary probe for low-virulence TcI strains, indicating no detection of iTS genes carrying the T/C transition. As shown in T. cruzi isolates harbors 28 to 32 copies of aTS-coding genes. On the other hand, data obtained from the aggressive strains RA, Cvd, CL Brener and the clone Q501/3  and Br (TcII) indicated that both the aTS and iTS genes were present with high variability in the gene copy number (aTS/iTS rate was high for TcII (3/1) and quite balanced in TcVI (2/1 and 1/1).To analyze the distribution of TS-encoding genes in the genome of parasites analyzed by Risso et al y number . In thesiTS genes in TcI parasite genome. To further test the differential distribution of aTS and iTS among T. cruzi stocks belonging to different DTUs, the T/C SNP was directly searched by sequencing PCR-amplified gene fragments comprising the surrounding region. To assess the representation of the Tyr342His mutation among these different parasite stocks, two primers were designed upstream and two other downstream the target codon. All TS genes deposited at GenBank were covered given that all these known sequences were targeted at least once in the PCR strategy designed. aTS and iTS genes  indicating the absence of genes coding for iTS in agreement with results shown in The findings presented above strongly suggest the intriguing absence of T. cruzi stocks belonging to the six DTUs. Besides the nucleotide position corresponding to the T/C transition . On the other hand, variable aTS and iTS copy number  were found in TcII and TcVI. These DTUs showed an aTS/iTS ratio that ranged from 1 to 3  in different geographical areas (from the USA to Argentina). We found that aTS genes were present in all 38 parasite populations, emphasizing the central role of this enzyme in parasite biology. It is worth noting that iTS was observed exclusively in stocks from DTUs TcII, TcV and TcVI but intriguingly absent in all TcI, TcIII and TcIV stocks analyzed. The absence of cumulated mutations or stop codons in iTS sequences, together with the fact that we have always found the same T/C transition that encodes the Tyr342His amino acid replacement as the enzyme inactivation mechanism, indicate that the same iTS genes, conserved among all the TcII, TcV and TcVI parasite populations, are probably expressed. The Trp312 and Tyr 119 codons that are crucial in creating the two-aromatic residue-stacking site for the galactosyl portion of the substrate in vitro assays have demonstrated the co-stimulatory properties of iTS proteins on the immune system iTS genes supports that iTS plays an evolutionary selectable role, instead of representing just a collection of pseudogenes. Therefore, an involvement in parasite attachment/invasion to host cells can be postulated because iTS acts as a lectin, able to bind not only small oligosaccharides but also sialylated glycoproteins The m 1 to 3 . These ciTS gene might provide with a nice opportunity to test the actual relevance of iTS in parasite biology and pathogenesis.Interestingly, our findings also reveal the existence of parasites with highly reduced TS genes content that provide models to develop genomic KO, a largely expected tool to extend the study of the biological relevance of TS whose generation has been hampered by the high gene copy numbers always reported for TS. Moreover, the ongoing transfection assays with the T. cruzi evolution and clustering. They remember that the partition of T. cruzi in six principal DTUs could be explained by two alternative models for their origin: the \u2018Two Hybridization\u2019 model giving rise to TcIII and then to TcV and TcVI through hybridization of two ancestors (TcI and TcII) iTS may have originated from aTS genes through a single mutation event, the common ancestor of TcI and TcII should not have had iTS. After iTS consolidation in TcII, its delivery during subsequent hybridization events could explain its presence in TcV and TcVI parasites  of virulent genes may be a key to better understand the mechanism of virulence and its relationship with T. cruzi evolution.Finding an association between clinical manifestations and parasite genotype is a difficult task. The multiclonal nature of most natural infections and the histotropic behavior of different parasites lead to partial characterizations when bloodstream and/or other infected tissue samples are analyzed Figure S1Consensus sequence of TS gene internal region. Sequence alignment of T. cruzi stocks encompassing the 6 DTUs (TcI to TcVI). (.): conserved sites; (\u25be): SNPs that identify a group of parasites (inter-DTU polymorphism). In those positions, depicted nucleotide for each DTU was present in all sequences obtained from all parasites of each DTU (named as IUPAC code); (*): other polymorphic positions not shared by all stocks within a DTU (intra-DTU polymorphisms); TGG: Trp312 codon conserved in all stocks from all DTUs; Box: Tyr342His codon where Thymidine (encoding Tyr) and Cytosine (encoding His) are present in all stocks belonging to TcII, TcV and TcVI whereas only Thymidine was found in TcI, TcIII and TcIV genomes. No other mutations were found in this codon.(TIF)Click here for additional data file.Figure S2UPGMA tree based on TS genes sequence alignment (with ambiguous states) not including hybrid DTUs. To avoid deviations induced by the hybrid nature of T. cruzi TcV and TcVI DTUs, UPGMA tree was built excluding these DTUs.(TIF)Click here for additional data file."}
+{"text": "Hereditary spastic paraplegias (HSP), a group of genetically heterogeneous neurological disorders with more than 56 documented loci (SPG1-56), are described either as uncomplicated (or pure), or complicated where in addition to spasticity and weakness of lower extremeties, additional neurological symptoms are present, including dementia, loss of vision, epilepsy, mental retardation and ichthyosis. We identified a large consanguineous family of Indian descent with four affected members with childhood onset HSP (SPG54), presenting with upper and lower limb spasticity, mental retardation and agenesis of the corpus callosum.Illumina human cytoSNP-12 DNA Analysis BeadChip Kit. Exome sequencing identified a homozygous stop gain mutation (pR287X) in the phospholipase A1 gene DDHD2, in the affected individuals, resulting in a premature stop codon and a severely truncated protein lacking the SAM and DDHD domains crucial for phosphoinositide binding and phospholipase activity.A common region of homozygosity on chromosome 8 spanning seven megabases (Mb) was identified in the affected individuals using the This mutation adds to the knowledge of HSP, suggests a possible founder effect for the pR287X mutation, and adds to the list of genes involved in lipid metabolism with a role in HSP and other neurodegenerative disorders.The online version of this article (doi:10.1186/s13104-015-1227-4) contains supplementary material, which is available to authorized users. Hereditary spastic paraplegias (HSP), are a heterogeneous group of rare inherited neurological disorders . The priHSP is classified clinically as \u201cuncomplicated\u201d (pure or non-syndromic) if symptoms are limited to progressive spastic weakness in the legs, although often accompanied by urinary urgency and subtle dorsal column impairment, or \u201ccomplicated\u201d if associated with additional symptoms involving neurological or systemic abnormalities including dementia, ataxia, mental retardation, neuropathy, distal wasting, loss of vision, epilepsy or ichthyosis.The prevalence of HSP is estimated at 3\u201310 cases per 100,000 populations in Europe , and theTo date the more than 50 distinct genetic loci associated with distinct subtypes of HSP have been mapped , are assWe have identified a large extended consanguineous family of Indian origin with four affected children, two in each branch of the family, sharing the same clinical phenotype of HSP (SPG54). They are all females who have difficulty in walking, and have motor delay, developmental delay, lower limb spasticity, and mental retardation. Brain MRI shows hypoplastic corpus callosum with possible agenesis of the splenium.QIAamp\u00ae mini DNA extraction kit . The DNA was quantified by Nanodrop-2000  spectrophotometer.The subjects who participated in the study are members of a Saudi Arabia based Indian family Figure\u00a0. Prior tIllumina iScan system with HumanCytoSNP-12v12.1 kit following the manufacturer\u2019s protocol (http://www.support.illumina.com/array/protocols.ilmn). Loss of heterozygosity (LOH) regions were detected by analyzing the SNPs for copy number variation with GenomeStudio software.Genomewide homozygosity mapping in four affected and six unaffected individuals was performed using Agilent Technologies). This was followed by sequencing on a HiSeq2000 (Illumina) with 100\u00a0bp paired end reads. Sequence reads were aligned to the reference genome (hg19 build) using Novoalign (Novocraft Technologies Sdn Bhd). Duplicate reads, resulting from PCR clonality or optical duplicates, and reads mapping to multiple locations were excluded from downstream analysis. Depth and breadth of sequence coverage were calculated with custom scripts and the BedTools package.To identify the causative mutation, we undertook whole exome sequence analysis in a trio samples set , using the SureSelect human All Exon kit . Ensembl genome browser (http://www.asia.ensembl.org) was used for obtaining genomic sequence and coding exons information for these genes. Primer sequences were designed for each exon using Primer3 software [http://www.ncbi.nlm.nih.gov/blast). Sequence variants identified were analyzed using BioEdit software (www.mbio.ncsu.edu/bioedit.html).Potential variants identified by whole exome sequencing were validated by Sanger sequencing of all family members. Genomic DNA was PCR amplified and screened by DNA cycle sequencing using Big Dye Terminator v3.1 Cycle Sequencing Kit, together with an ABI 3130 Genetic Analyzer  from a consanguineous Indian family with HSP with one previous miscarriage due to unknown causes in the first trimester  homozygous region at chromosome 8p11.23-8p12, including more than 55 genes, was found in all four affected individuals.We genotyped ten available samples from the family using the DDHD2 gene in the affected individual that was heterozygous in both parents. This mutation at position 38103270, causing a C to T substitution at c.859 (c.C859T) introduced a stop codon at amino acid position 287 (p.R287X). Initially, this variant was not found in our in-house database of over 1,000 exome sequences largely of Caucasian origin or in exome variant server (http://www.evs.gs.washington.edu/EVS/). In order to confirm this mutation and to examine its segregation within the family, Sanger sequencing was performed for all ten family members. This confirmed a homozygous transition mutation (C\u00a0>\u00a0T) at cDNA base position 859 in exon 8 of DDHD2 gene in all four affected individuals. The obligate carriers were heterozygous for this mutation. To further confirm that the mutation was not a common polymorphism, a panel of 192 chromosomes of ethnically matched controls were sequenced. However the variant was not identified within this panel.We undertook whole exome sequencing of both parents and one affected daughter. We searched the chromosome 8p region of homozygosity for non-synonymous variants or stop gain mutations. Based on the assumption that the candidate mutation is autosomal recessive, led us to concentrate on a homozygous stop gain (stop codon) mutation in the DDHD2 (SPG54). SPG54 is characterized by psychomotor delay, cognitive impairment, progressive spasticity, early onset (before the age of 2\u00a0years), thin corpus callosum, periventricular white matter abnormalities, foot contractures, dysarthria, dysphagia, strabismus and/or optic hypoplasia. Our results confirm previously studies reporting DDHD2 mutations in SPG54, extend our clinical knowledge of this condition, give further insights into genotype-phenotype contributions, suggest a founder effect for the p.R287X mutation, and adds to growing list of lipid metabolism genes playing a role in neurodegenerative disorders.We report a large extended consanguineous family of Indian descent, with affected members of this family carrying a homozygous recessive mutation in 1 (iPLA1) family of proteins,  that are involved in organelle biogenesis and membrane trafficking between the endoplasmic reticulum and golgi body [1 family proteins encode a phospholipase that hydrolyze an acyl group and fatty acids at the sn-1ester bonds of phospholipids, and contain a DDHD domain, a WWE domain, a GxSxG lipase motif, and a sterile alpha motif (SAM). SAM and DDHD both bind phatidylinositol 4-phosphate (PI(4)P) which is important in membrane trafficking [DDHD2 (DDHD-domain-containing 2), belongs to an intracellular phospholipase Algi body , 8. iPLAfficking , 8.DDHD2 have been previously reported in 2 Iranian, one Dutch Filipino, one Omani, one Indian, one Canadian, and one Italian family. A wide range of mutations have been reported so far, mostly affecting the SAM and DDHD domains of DDHD2  in the open reading frame of ogenesis .The identification of pR287X mutation in two Iranian families, including one family with two affected siblings carrying this mutation, suggested a founder effect , 11. HowDDHD2 are not known, mutations in genes involved in common intracellular signaling pathways involving HSP, Parkinson\u2019s disease, amyotrophic lateral sclerosis and Alzheimer\u2019s disease have recently been reported [DDHD2 joins a growing list of such genes contributing to HSP and other neurodegenerative disorders [Although the precise pathogenic mechanisms involving reported , 14 and reported . The rolisorders , 9, 15."}
+{"text": "We show that butyrate reduces the frequency of peptide-specific CD8+ T cells and, together with propionate, inhibit the activity of those cells. On the contrary, acetate does not affect them. Importantly, butyrate and propionate inhibit the production of IL-12 and IL-23 in the DCs and exogenous IL-12 fully restores the activation of the MART-1-specific CD8+ T cells, whereas IL-23 has no effect. In conclusion, these results point to a pivotal role of butyrate and propionate in modulating CD8+ T cell activation via the inhibition of IL-12 secretion from DCs. These findings reveal a novel mechanism whereby bacterial fermentation products may modulate CD8+ T cell function with possible implications in anti-cancer immunotherapy.Short chain fatty acids (SCFAs), such as acetate, butyrate and propionate, are products of microbial macronutrients fermentation that distribute systemically and are believed to modulate host immune responses. Recent data have indicated that certain SCFAs, such as butyrate and propionate, directly modulate human dendritic cell (DC) function. Given the role of DCs in initiating and shaping the adaptive immune response, we now explore how SCFAs affect the activation of antigen-specific CD8 Through fermentation of dietary poly- and oligosaccharides resistant to digestion in the small intestine, the distal gut microbiota can metabolize complex carbohydrates to produce small organic acids, the majority of which are comprised of the short chain fatty acids (SCFAs) acetate, propionate, and butyrate4. Acetate, propionate and butyrate are found at molar ratios of 60:20:20 in the intestinal tract, reaching peak concentrations in the gut lumen (50\u2013100\u2009mM). Due to their structure they are easily absorbed into the portal circulation, through which they reach the bloodstream at much lower concentrations than found in the gut lumen9. SCFAs affect cells both by the interaction with their specific G protein-coupled receptors (GPR)-41, GPR43 and GPR109A and independently of these receptors13. Butyrate and, to a lesser degree, propionate inhibit histone-deacetylases (HDAC) thereby affecting host gene expression14 and inducing autophagy15. Several evidences highlight the importance and broad role of SCFAs from regulating energy homeostasis16 to modification of immune cell function and responses18. We have shown that human monocyte-derived dendritic cells (DC) express GPR41 and GPR109A, and that SCFA treatment affect gene expression in lipopolysaccharide (LPS)-activated DCs19. In peripheral tissues, DCs are found in an immature stage specialized in detecting and capturing antigens through expression of innate pattern recognition receptors. When activated by pathogen-associated molecular patterns, immature DCs undergo phenotypic and qualitative changes to become mature DCs and migrate from the periphery into the draining lymph nodes where they present pathogen-derived peptides to CD4+ and CD8+ T cells22. The environment in which the DCs are activated greatly shapes their ability to activate and differentiate T cells20. Cytotoxic CD8+ T cells (CTLs) are pivotal for the killing and clearance of virus-infected cells and cancer cells. The generation of antigen-specific memory and effector CTLs from na\u00efve CD8+ T cells relies on antigen presentation by DCs25 in combination with surface expression of co-stimulatory molecules like CD80, CD83, CD86, CD40, OX40-L or ICOS-L26. In addition, inflammatory cytokines like IL-1, IL-6, and IL-12 produced by the activated DCs and/or macrophages during the priming phase provide the necessary signal 3 that further induces cell division and development of CTL effector functions30. To study the effect of SCFAs on the DC-mediated activation of antigen-specific CTLs, we studied MART-1-specific CD8+ T cells co-cultured with MART-1 peptide pulsed autologous HLA-A201+ DCs previously treated with SCFAs. We used MART-1 as model antigen since around 0.1% of naive CD8+ T cells are MART-1\u2013specific and they can be found in HLA-A201+ individuals regardless of prior antigen exposure32. We found that butyrate and propionate significantly suppressed the activation of MART-1-specific CTLs. In search for mechanism involved in this suppression, we discovered that butyrate and propionate inhibited production of IL-12 and IL-23 in the DCs and that the CTL activation could be fully reconstituted by exogenous supplementation of IL-12. Our work adds a new dowel to the understanding of the host-microbiota mutualism by revealing that SCFAs can dampen CTL activation via their effect on DCs.The gut microbiota has strong influence on the human immune system and health via the production of a variety of metabolites detectable in host circulation+ donors to generate immature DCs (iDC). The iDCs were either left untreated or were stimulated with LPS for 24\u2009h in the absence or presence of sodium acetate, sodium butyrate, or sodium propionate to generate mDC, mDC_A, mDC_B, and mDC_P, respectively, as previously described19. After 24\u2009h, the DCs were pulsed with peptides and cultured for 10 days with autologous CD8+ T cells.To study whether SCFAs affect activation of CTLs in T cell-DC co-cultures, we used monocytes isolated from healthy HLA-A0201+TNF-\u03b1\u2212, IFN-\u03b3+TNF-\u03b1+-, or IFN-\u03b3\u2212TNF-\u03b1+-producing CD8+ T cells. In order to reduce the variability among donors, we normalized the measured frequences of each subpopulation to the respective values obtained when CD8+ T cells were in co-cultures with mDC. We found that expansion of both the IFN-\u03b3+TNF-\u03b1\u2212 and the IFN-\u03b3+TNF-\u03b1+-producing MART-1 specific CD8+ T cells was significantly impaired in mDC_B  and mDC_P co-cultures   , followed by mDC_P , that also has a significant effect on the IFN-\u03b3\u2212TNF-\u03b1+-producing CD8+ T cells (P\u2009\u2264\u20090.05)  Fig.\u00a0, while t05) Fig.\u00a0. In addiTo verify that the activated CTLs were actually peptide-specific CTLs, we stained the cells with HLA-A201/MART-1-tetramers. Expansion of MART-1 specific CTLs was significantly impaired in co-cultures with mDC_B (P\u2009\u2264\u20090.05), but not if in co-culture with mDC_P, when compared to mDC as control Fig.\u00a0.+ T cells for the proliferation marker Ki67, a nuclear protein that plays a pivotal role in the regulation of T cell division33. We found that the levels of Ki67 were similar for all the groups of CD8+ T cells regardless of the SCFA used to pretreat the DCs during their maturation , HLA-pan Class I (MHC Class I) by flow cytometry Fig.\u00a0. We foun19. Here, we confirm what we have previously shown regarding IL-6   Fig.\u00a0.Figure 3+ TNF-\u03b1\u2212 -producing CD8+ T cells population with butyrate- and propionate-treated mDC co-cultures  , costimulatory molecules , and cytokines 30. Generally, expression of costimulatory molecules on DC is essentially involved in controlling T cell differentiation and the resulting immune responses. We observed that both butyrate and propionate down-regulate the LPS-induced co-stimulatory molecules CD83, CD80, and CD40 presumably inhibiting their ability to correctly interact with CD8+ T cells during the priming and activation steps. MHC class I/II expression is pivotal for activation of T cells providing the first signal needed for the activation of a na\u00efve T cells. We observed no effect of SCFA on MHC class I or II expression, indicating that SCFA do not compromise the antigens presentation via MHC molecules, thus a correct interaction between TCR and MHC can still be established. CD80 and CD86 expression on DCs probably constitute the most important costimulatory molecules in T cell activation. Signaling through binding partner CD28 on T cells confers optimal mRNA stabilization and production of IL-2, a factor that promotes expansion and survival of primary T cells44. Human iDC constitutively express intermediate amounts of CD86 and lack CD80, while mature DCs express higher amount of both45. While the down regulation of CD80 may compensate by the unaffected CD86 (considering their shared ligand), the lack of CD40 and CD83 might not. Indeed, it is known that the CD40 binds CD40L on T cells and its pathway regulates cellular and humoral immunity and plays an important role in T cell priming and differentiation46. Indeed, CD40 ligation on DCs increases expression of costimulatory, adhesion and MHC molecules and promotes the production of T cell stimulatory cytokines such as IL-1248. CD83 functions as an enhancer during the stimulation of T cells, and induces clear changes in cytokine expression during T cell priming49. Furthermore, the engagement of CD83 ligand on human T cells preferentially enriches, and significantly amplifies, the number of antigen-specific CD8+ T cells, thus by affecting its expression it results in compromised T cell proliferation52. We have observed the same tendency of butyrate-or propionate-treated mDC to decrease the numbers of IFN-\u03b3+TNF-\u03b1\u2212 and the IFN-\u03b3+TNF-\u03b1+-producing CD8+ T cells when in co-culture in all experiments regardless of the peptides used . This gives very individual responses to the different peptides, and result in a large donor variation. Taken together, this led us to consider MART-1 the most reliable peptide to use as a model for further analysis. Overall, we observed that both butyrate- and propionate-treated mDC were significantly impaired in priming/inducing the IFN-\u03b3+ TNF-\u03b1\u2212-producing-, IFN-\u03b3+ TNF-\u03b1+-producing CD8+ T cells, and only propionate to affect the IFN-\u03b3\u2212 TNF-\u03b1+-producing CD8+ T cells, while the peptide-specificity and the proliferation of the CD8+ T cells were not impaired.Impairing DCs maturation or any of the activating signals leads to a reduced functionality of the effector cells of the adaptive immune system including CD8s signal , costimu+ T cells are not merely cause by the lack of CD40, CD80, and CD83 on the DCs surface  but to a higher degree to the impairment of signal 3, that is usually represented by the cytokines produced by the antigen presenting cells.Taken together, our results indicate the the effects of butyrate and propionate on DCs ability to activate CD819. In addition, we here show that both butyrate and propionate down-regulates the LPS-induced cytokine release of IL-12p70 and IL-23; cytokines that polarize na\u00efve CD4+ T cells towards Th1 and Th17, thus eliciting pro-inflammatory properties as recently shown by Haghikia et al.42. To investigate whether reduced expression of cytokines from SCFA-treated DCs were responsible for the observed CD8+ T cells activation, we performed co-culture experiments with supplemented cytokines. Indeed, when supplementing the co-culture media with IL-12 the IFN-\u03b3+ TNF-\u03b1\u2212-producing CD8+ T cells were restored, and even enhanced, in both butyrate- and propionate-mDC co-cultures; while the IFN-\u03b3+ TNF-\u03b1+-producing CD8+ T cells population was just sufficiently restored in butyrate-mDC co-cultures, and the IFN-\u03b3\u2212 TNF-\u03b1+-producing CD8+ T cells population was just rescued in the propionate-mDC co-cultures. We did not observe the same effect with either IL-23 or IL-6 supplementation, leading to support the important role that IL-12 plays in the CD8+ T cells activation and in initiating and supporting the IFN-\u03b3 and TNF-\u03b1 production. Indeed, as previously shown30, IL-12 provides a third signal exclusively to naive CD8+ T cells, and the response to IL-12 as well as to peptide antigens depends on co-stimulation. IL-12 is known to increase CD8+ T cell cytolysis, survival, proliferation, and at the same time influence the ability of the cells to migrate to inflammatory loci. On the other hand, IL-23 has the ability to stimulate the production of IL-17 by CD4+ T cells, important for immune reactions against extracellular pathogens27, but its role in the activation of na\u00efve CD8+ T cells is still poorly understood. IL-12 and IL-23 share the p40 subunit, but most probably due to their specific and different receptors, they activate distinct sets of downstream transcriptor factors, thus that IL-12 leads to IFN-\u03b3 production while IL-23 does not induce neither IFN-\u03b3 nor TNF-\u03b1. In our study, we show that SCFAs affect co-stimulatory molecules surface expression on DCs, as well as cytokine production of these APCs and ability to prime CD8+ T cells. On the other hand SCFAs rather impair the CTLs IFN-\u03b3+ and TNF-\u03b1+ production and peptide-specificity but have no effect on proliferation. Thus, we assumed that it could be due to the down-regulation of CD40, CD80 and particularly of CD83 expression , but also due to the impairment of signal 3 by treated DCs.We have confirmed, what we have previously shown, that butyrate significantly reduce IL-6 expression and release by human monocyte-derived DCsWe here propose that SCFA, produced by our commensals, support a more tolerogenic activation of the innate immune system\u2019s component rather than a pro-inflammatory stimulation. This could be seen as a strategic way to avoid a strong immune response towards their own producers, damping a potential cytotoxic activity in the gut that could impair the gut mucosa integrity, while not affecting the immune system\u2019s ability to defeat a potential invading pathogen.PBMCs were isolated from buffy coats obtained from anonymous healthy blood donors by Ficoll-Hypaque density gradient centrifugation. Before processing, all the buffy coats were screened for HLA-A0201 positivity using HLA-A2  antibody and only those positive were chosen for further analysis. Written informed consent was obtained from blood donors at the Department of Clinical Immunology, University Hospital Rigshospitalet, Copenhagen and used without the possibility to identify case specific information: The ethical committee, Region H, Capital Region of Denmark, approved the use of these buffy coats for research that was carried out in accordance with the approved guidelines.+ monocytes and CD8+ T cells were isolated from PBMCs by positive selection using magnetic beads in accordance with the manufacturer\u2019s instructions . Monocytes were cultured at 37\u2009\u00b0C in 5% CO2 in media supplemented with 10% fetal bovine serum  GM-CSF and IL-4  for 7 days to generate iDC and mDC, the last obtained after LPS  stimulation for the last 24\u2009h in parallel with a single addition of 1\u2009mM to acetate, propionate or butyrate (Sigma-Aldrich) leading to the following groups of treatments: sodium acetate (mDC_A), sodium butyrate (mDC_B), sodium propionate (mDC_P).CD145) were washed twice and incubated in 48-wells culture plates together with autologous CD8+ T cells (0.5\u2009\u00d7\u2009106). The general informations and throubleshooting advices were taken from Wolfl et al.53. For each condition  were plated 3\u20135 co-culture replica in order to be able to exclude possible technical errors. At day 3 and 6, half of the medium was refreshed with new containing rIL-2  and rIL-7 . For experiments with supplement of exogenous rIL-12, rIL-6 and rIL-23  were added together with the medium refreshment at day 3 and 6 of the co-culture.After 7 days of culture, iDCs, mDCs, mDC_A, mDC_B, and mDC_P were harvested, the supernatants stored, and cells were washed and resuspended in X-VIVO 15 , and subsequently pulsed with 1\u2009mM MART-1 peptide (ELAGIGILTV), EBV BLMF1 peptide (GLCTLVAML), PDL101 peptide (LLNAFTVTV), or CMV pp65 peptide (NLVPMVATV)  at 37\u25e6C for 2\u2009h. Hereafter DCs , washed, resuspended in the same buffer. Isotype-matched antibodies were used to define and exclude the background staining. 7-AAD (BD Pharmigen) (for the DCs phenotype), or LIVE/DEAD\u00ae Fixable Far Near-IR stain (Invitrogen) (for the intracellular stainings) was used to exclude dead cells. DCs analysis was conducted with anti-human: HLA-pan class I (clone W6/32), HLA-DR (clone L243), anti-human CD83 (clone HB15e), CD86 (clone 2331 FUN1), CD40 (clone 5C3), CD80 (clone L307.4), all purchased at BD Bioscience.+ T cell from co-culture experiments were preformed via intracellular cytokine stainings at day nine. CD8+ T cells were harvested, washed and plated in a 96-wells round plates, and re-stimulated with MART-1 peptide for 4\u2009h with the addition of GolgiPlug  for the last 3\u2009h. Anti-human CD3 , CD8  and LIVE/DEAD\u00ae Fixable Far Near-IR stain (Invitrogen) were used for gating live CD3+CD8+ T cells, followed by the fixation and the permeabilization with the Transcription factor Staining Buffer Set  for the detection analysis of intracellular cytokines production by anti-human IFN-\u03b3 (clone B27) and TNF-\u03b1 (MAb11) (both BD Bioscience). For analysis of antigen-specificity, CD8+ T cells stained with PE-conjugated peptide-HLA-A2 MHC-tetramer  for 30\u2009min at 37\u2009\u00b0C followed by surface markers staining of CD8 and CD3 for 30\u2009min at 4\u2009\u00b0C. Data acquisition and flow cytometric analysis were done on by LSRFortessa\u2122 (BD Bioscience) at the CFFC . All data analysis was performed with FlowJo v7.0 .Functional analysis of CD8Supernatants from DC maturation cultures were collected and frozen at 80\u2009\u00b0C during DC harvest. IL-6, IL-12p70, and IL-23 production was measured in the supernatants of DC by ELISA following the manufacturer\u2019s instructions .Two-tailed paired Student\u2019s t-tests were used with a significance level of 0.05. Before each test the Gaussian distribution of the samples data was checked though D\u2019Agostino-Pearson normality test, if the test was not passed a non-parametric test was performed. All the analysis were made by GraphPad Prism 6.0 and the nomenclature for the P values is the following: *P\u2009\u2264\u20090.05, **P\u2009\u2264\u20090.01, ***P\u2009\u2264\u20090.001, ****P\u2009\u2264\u20090.0001.Each donor has been considered as independent experiment and all the flow cytometry data were performed with 3 up 5 technical replicates for each condition.Supplementary Information"}
+{"text": "The choice of surgical methods for lumbosacral tuberculosis is controversial due to the complex anterior anatomy and peculiar biomechanics of the lumbosacral junction. The objective of this study was to explore the clinical effect of posterior intervertebral space debridement with annular bone graft fusion and fixation for the treatment of lumbosacral tuberculosis.We retrospectively analysed data from 23 patients with lumbosacral tuberculosis who had undergone posterior intervertebral space debridement with annular bone fusion and fixation between January 2008 and September 2014. The mean age of the patients was 49.0\u00a0years , and the mean duration of disease until treatment was 10.2\u00a0months . The lumbosacral angle, visual analogue scale (VAS) score, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, American Spinal Injury Association (ASIA) grade and Social Functioning-36 (SF-36) score were determined to ascertain the clinical effects of the treatment.p\u2009<\u20090.05) and being maintained thereafter. The mean VAS score before operation was 8.1\u2009\u00b1\u20090.6, decreasing to 1.2\u2009\u00b1\u20090.5 (p\u2009<\u20090.05) at the final follow-up. The mean ESR and CRP values were 49.1\u2009\u00b1\u20095.6\u00a0mm and 64.9\u2009\u00b1\u200911.9\u00a0mg/L, respectively, before operation, decreasing to normal at the final follow-up. The preoperative ASIA grade was C in 6 patients, D in 12 and E in 5. At the final follow-up, all patients had an ASIA grade of E except for one patient with a grade of D. For all patients, the SF-36 score at the final follow-up was higher than the preoperative and postoperative scores.All patients underwent follow-up observation. The mean follow-up time was 34.2\u00a0months , the mean operation time was 167.0\u00a0min  and the mean blood loss was 767.4\u00a0ml . The lumbosacral angle was 21.0\u00b0\u2009\u00b1\u20092.1\u00b0 before operation, rising to 28.8\u00b0\u2009\u00b1\u20091.7\u00b0 after operation (Posterior intervertebral space debridement with annular bone graft fusion and fixation is an effective treatment for lumbosacral spine tuberculosis. The lumbosacral region includes the distal lumbar vertebrae and all sacral vertebrae and mainly refers to L3 and lower levels , 2. TubeAnterior debridement and bone grafting combined with posterior instrumentation is the leading surgical treatment for lumbosacral junction tuberculosis because it has the advantages of complete debridement under direct vision, excellent anterior column reconstruction and solid posterior fixation \u20135. HowevThis retrospective study enrolled 23 patients (12 men and 11 women) with lumbosacral tuberculosis who had undergone posterior transintervertebral space debridement with annular bone graft fusion and fixation between January 2008 and September 2014. The mean age of the patients was 49.0\u00a0years , and the mean duration of disease until treatment was 10.2\u00a0months . There were 3 patients with L3\u20134 lesions, 11 with L4\u20135 lesions and 9 with L5\u2013S1 lesions. All patients were examined with anteroposterior and lateral X-rays. Computed tomography (CT) and magnetic resonance imaging (MRI) were performed to determine local pathological changes and eliminate other diseases of the lumbosacral region. All patients had lumbosacral pain; 14 patients had low fever, sweating, emaciation and other symptoms of tubercular toxicity; 17 patients had complications of radiating pain and numbness in the lower extremity; 18 patients experienced weakness in walking and 1 patient presented with cauda equina syndrome. On clinical examination, most patients presented only with lumbosacral pain, eight patients had limited lumbar flexion and six patients had significant weakness of the lower extremity. All preoperative erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) values were above normal limits. Except for seven patients who only presented with intervertebral space narrowing, all patients were found to have various degrees of bone destruction on X-ray examination. All patients were found to have obvious bone destruction and paravertebral abscesses on CT and MRI examination.The study protocol was approved by the ethics review committee of the Shaanxi Provincial People\u2019s Hospital (No. K20070313\u2013021).The T-SPOT.TB test was used to confirm the presence of tuberculosis. All patients with suspected tuberculosis received 2\u20134\u00a0weeks of standard HREZ chemotherapy  before surgery. Anaemia and hypoproteinaemia were corrected preoperatively. If there was no significant decrease in the ESR, levofloxacin injection  was added to enhance the effect of the antituberculosis therapy.With the patient in the prone position, a posterior midline skin incision was made after induction of general anaesthesia by tracheal intubation, and subperiosteal dissection was performed up to the lateral part of the superior zygopophysis. Pedicle screws were used in the adjacent normal vertebrae Fig. . If the Breath, pulse, blood pressure and temperature were monitored postoperatively. Mobility and sensation of the lower limbs were closely observed. The drainage tube was extracted when the volume of drainage was <50\u00a0ml/d. The patients underwent straight leg raising exercises and ankle pump training during bed rest. We regularly monitored routine blood parameters, ESR, CRP and liver function, performed X-rays of the lumbar spine and, if necessary, performed CT scanning of the surgical segment. Standard HREZ chemotherapy was continued for more than 12\u00a0months. Ambulation with a brace was allowed at 6\u20138\u00a0weeks after surgery. The patients performed non\u2013weight-bearing daily activities until radiographic (X-ray or CT) evidence of fusion was observed. The patients then returned to normal weight-bearing activities.Preoperatively and at the final follow-up, the American Spinal Injury Association (ASIA) grade was used to evaluate neurological function. At the same time points, the visual analogue scale (VAS) was used to assess low back pain, and tuberculosis activity was monitored by measuring ESR and CRP. The lumbosacral angle and Social Functioning-36 (SF-36) scores were measured at preoperation, postoperation and final follow-up. Bone graft fusion and instrumentation failure were monitored by radiography.t-test was used to evaluate differences in lumbosacral angle, VAS, ESR, CRP and SF-36 among preoperation, postoperation and final follow-up. The Wilcoxon signed rank test was used to evaluate differences in ASIA grade. A p-value <0.05 was considered to indicate a statistically significant difference.The paired p\u2009<\u20090.05) and being maintained thereafter. The difference between the lumbosacral angle before and after operation was statistically significant (p\u2009<\u20090.05), and there was no significant loss of postoperative lumbosacral angle during follow-up (p\u2009>\u20090.05). Tuberculosis toxic symptoms had vanished in all patients at the final follow-up. The mean VAS score at the final follow-up was 1.2\u2009\u00b1\u20090.5 , significantly lower than the mean preoperative score (8.1\u2009\u00b1\u20090.6) , the mean operation time was 167.0\u00a0min  and the mean blood loss was 767.4\u00a0ml   higher than the preoperative scores of 77.43\u2009\u00b1\u20094.67, 27.0\u2009\u00b1\u200917.05, 24.59\u2009\u00b1\u20090.50, 68.2\u2009\u00b1\u200911.25, 72.43\u2009\u00b1\u20099.83, 69.00\u2009\u00b1\u20096.26, 59.8\u2009\u00b1\u200913.77 and 81.40\u2009\u00b1\u20093.55. The scores for each part of the SF-36 increased gradually during the follow-up period, and all scores were significantly (p\u2009<\u20090.05) higher than the postoperative scores at the final follow-up  at the final follow-up. At the final follow-up, all patients had an ASIA grade of E except for one patient with a grade of D (Table\u00a0With the resurgence of tuberculosis, the incidence of spinal tuberculosis has increased in recent years , 10. LumMycobacterium tuberculosis has migrated to and caused disease in this site [The blood supply of the lumbosacral junction is poor. Once his site . Moreovehis site .The application of antituberculosis drugs is the basis of the treatment of spinal tuberculosis . In the The aim of surgical treatment of lumbosacral junction tuberculosis is radical debridement to wipe out the factors detrimental to neural recovery and restore normal spine alignment. An anterior approach can directly remove paravertebral abscesses and vertebral sequestrum and perform effective intervertebral grafting, but due to the complex anterior anatomy of the lumbosacral junction, its ambiguous pathological structure, fragile invading blood vessels and thick, sticky adjacent tissue, the risk to the presacral vessels and the ureter is increased . AccidenPang et al.  and ZhanThe following are some notes on this technique. First, there is no problem of dura avulsion . Due to Transintervertebral space debridement with annular bone graft fusion and fixation through a single posterior approach is a good treatment for lumbosacral junction tuberculosis because of its effective debridement, bone grafting and fixation."}
+{"text": "Phyllostachys heterocycla var. pubescens) from stands of nine germination-ages, using six primer pairs which have previously been shown to yield methylation rates that reflect the age of Moso bamboo.Chronological age is the primary consideration when studying the physiological development, aging, and flowering of bamboo. However, it\u2019s difficult to determine bamboo\u2019s chronological age if the time of germination is unknown. To investigate the chronological age of bamboo from the genomic DNA methylation profile, methylation-sensitive amplification polymorphism (MSAP) was employed to analyze the genomic DNA methylation of Moso bamboo  contains supplementary material, which is available to authorized users. The perennial evergreen bamboo is a group of species in Poaceae used for building structures, biomass, and ornamental horticulture as well as panda habitat conservation efforts. Bamboo regenerates by asexual reproduction, thus a stand of bamboo may consist of shoots that emerge at different times but from the same clone/mother plant. Furthermore, bamboo stalks stop widening as they mature, therefore, unlike in trees, the chronological age of bamboo shoots cannot be determined according to annual rings.Fargesia murieliae happened across Europe in 1997\u20131998 after introduced from China, and it was also flowering in the wild in its native range  from 1996\u20132000  the emergence age reflects when an individual bamboo shoot emerged from the ground in a bamboo forest and is used to determine harvesting time; the emergence age can be determined by factors such as skin color of the bamboo stalk; and 2) the chronological age begins when a bamboo seed germinates. The chronological age considers the entire forest from seedling afforestation. Although bamboo culms in a bamboo forest of the same chronological age may emerge from the ground at different times, shoots generally exhibit synchronous developmental progress. For example, synchronous flowering of  Saarela . ChronolPinus radiata D. Don and Prunus persica (L.) Batsch  and a natural stand (with no flowering record for the past 60 years). Those stands were owned by the local forestry center, who gave the permission for the collection of material for the present study. Five bamboo plants in each age group that emerged at the year of studying were selected randomly for samples.An improved CTAB method was used to extract the genomic DNA from Moso bamboo leaves. The purity and concentration of the extracted DNA were detected using a UV spectrophotometer. The DNA quality was evaluated by performing gel electrophoresis using 0.8% agarose gel. The prepared DNA was stored in a -20\u00b0C refrigerator for later use.The enzyme digestion, ligation, and PCR amplification steps in MSAP analysis were conducted by referencing methods proposed by Xiong et al.  and the Hpa II and Msp I recognize the same tetranucleotide sequence (5\u2032-CCGG-3\u2032), but exhibit different sensitivities to methylation: Msp I cleaves methylated (C/5mCGG) and unmethylated (C/CGG) sites of the internal cytosine, whereas Hpa II cleaves only the unmethylated site (C/CGG). We used Hpa II and Msp I isoschizomers  for double-enzyme cleavage in combination with EcoR I, respectively. Each plant sample was analyzed via the two lanes, in which one lane was digested by EcoR I/Hpa II and the other by EcoR I/Msp I. Based on presence (marked as 1) or absence (marked as 0) of band, generated MSAP bands could be grouped into four types of methylation patterns. Type I (00): no band present in any lanes. This is attributed to methylation of the external cytosines (on both strands) or a full methylation of both cytosines. Type II (01): bands present in the EcoR I/Msp I lane, and these MSAP bands were caused by hypomethylation of the outer C relative to the internal C. Type III (10): bands present in the EcoR I/Hpa II lane, and these bands were associated with hemimethylation of the outer C. Type IV (11): bands present in both two lanes. These bands correspond to unmethylation. The methylation rate obtained by MSAP is generally lower than the sample\u2019s actual methylation level, for the reason that both Hpa II and Msp I can not cleave sites of the external cytosines (mCGG). To make the result more closer with the sample\u2019s actual methylation level, we calculated the methylation rate by the following formula: total rate of methylation (%)\u2009=\u2009Total number of methylation bands (Type I\u2009+\u2009Type II\u2009+\u2009Type III)/Total number of amplified bands (Type II\u2009+\u2009Type III\u2009+\u2009Type IV)\u2009\u00d7\u2009100%.Both Statistical analysis of the data, including ANOVA analysis, correlation analysis and principal component analysis were performed using the statistical program SPSS16.0 . All data were represented by an average of the five replicates . If the ANOVA indicated significant results, a Duncan\u2019s mean separation test was then performed Duncan .L.S.D multiple comparison found that variations among the DNA methylation levels in 2-, 6-, and 7-year-old Moso bamboo were insignificant, and it\u2019s also insignificant among those in 32-, 34-, and 44-year-old group. However, significant DNA metylation level variations existed among the groups of 2-year-old to 18-year-old and those of over 60 years-old. These results indicated that more DNA methylation variation exhibited between groups with larger chronological ages distances in Moso bamboo.The apparent bands obtained from MSAP analysis on the genomic DNA from nine age groups of Moso bamboo using six primer pairs were statistically analyzed. Obvious differences were found in the MSAP bands among different chronological ages, however, few clear difference can be see in the MSAP bands among the five repetitions at the same age. MSAP profiles for Moso bamboo at different chronological ages by primer pairs of E3HM6 and E4HM6 were showed in Figure\u00a0Although the genomic DNA methylation were different in the same age by different primer pairs, an overall increasing trend of the total methylation rate can be seen along with the chronological age Figure\u00a0, which iThe total genomic DNA methylation rates obtained by the six primer pairs differed within each age group Table\u00a0. HoweverThe results of principal component analysis on the six primer pairs Table\u00a0 showed ti, i\u2009=\u20091, 2, 3, 4, 5, 6), as in the following equation: Y\u2009=\u20090.191x1\u2009+\u20090.188x2\u2009+\u20090.187x3\u2009+\u20090.184x4\u2009+\u20090.178x5\u2009+\u20090.174x6.The weightings of each primer pairs on the integrated factor  were listed in Table\u00a02\u2009+\u20090.828y - 27.762, , where P(Q)\u2009<\u20090.01 and R2(Q)\u2009=\u20090.942. Based on the effectiveness and the coefficient of determination, the quadratic curve can effectively express the numerical relationship between the total genomic DNA methylation level and the chronological age of Moso bamboo.The total DNA methylation rate obtained from the six primer pairs were integrated based on the previously described integrated factor equation, it\u2019s found that the DNA methylation levels in Moso bamboo at nine chronological age were of 19.93%, 22.50%, 22.37%, 22.78%, 29.41%, 33.30%, 31.07%, 32.57%, and 41.63%, respectively. The relationship between the chronological age and genomic DNA methylation levels in Moso bamboo was represented graphically \u2009=\u20090.942), which expresses the relationship between the genomic DNA methylation level and the chronological age in Moso bamboo. In addition to see in-depth research studies concerning the chronological age of Moso bamboo, this new tool can be practically applied to anticipate the chronological age of Moso bamboo of unknown seeding or in wild stands.Relevant studies have examined genomic DNA methylation levels and patterns in plants at different developmental stages. For example, the genomic DNA methylation levels were compared in In studies on DNA methylation in rice and corn, functional genes related to stress tolerance and heterosis have been discovered by sequencing differentially-methylated fragments (Hua et al."}
+{"text": "Scientific Reports 10.1038/s41598-018-24619-1, published online 30 April 2018Correction to: The original version of this Article contained an error in the spelling of the author Vincent Blasco-Baque, which was incorrectly given as Vincent Blasco. This has now been corrected in the PDF and HTML versions of the Article, as well as the accompanying Supplementary Information file."}
+{"text": "Finnish permanent residents are covered by social security insurance administered by the Social Insurance Institution of Finland. The procedure of insurance is initiated with medical certificate written by the treating doctor. Thus, the doctor must have certificate writing skills accompanied with the knowledge of the content and goals for insurance. Quality certificates are important part of doctors\u2019 professional skills worldwide and most effective teaching methods for learning these should be investigated.N\u2009=\u2009141) and 2016 (N\u2009=\u2009142) in the medical faculty of the University of Eastern Finland. A random sample of 40 students per course was drawn for the analysis. All certificates were analyzed as one sample. This was done to obtain reliable results with internal control group on the differences between two teaching methods, the traditional approach and the flipped classroom (FC) approach, in 2015 and 2016, respectively. The medical certificates were evaluated and scored with a rubric (range: \u2212\u20094.00\u201314.25) by two independent experienced specialists.Medical certificate data were collected from two independent courses of fourth-year student taught in autumn 2015 (SD\u2009=\u20091.70) for the traditional group and 10.97 (SD\u2009=\u20091.25) for the FC group. Based on the common language effect size, a randomly selected student from the FC group had an 85% probability of receiving a higher total score than a student from the traditional group.Compared to students in the traditional classroom, students involved in the FC received significantly higher scores in all relevant sections of the assessed certificates. The mean of the total scores was 8.87  contains supplementary material, which is available to authorized users. Medical education is facing new demands from twenty-first century society and work . There hOne promising approach is the flipped classroom (FC) approach, which has several pedagogical roots in higher education . The FC Previous studies on the FC have provided differing results. These studies have mainly focused on students\u2019 experiences of the FC courses and learning results. The FC has been found to be a flexible  and enjoExamination results were significantly better when the FC model was used [In their review of the medical education context, Chen, Lui, and Martinelli  reportedwas used , 23, howwas used , 25. Baswas used , the ovewas used . Howeverwas used  focusingwas used  considerUndergraduate medical education in Finland is a continuous six-year program, where bachelor and master studies are integrated to form a licentiate degree. Medical education is based on a practical approach; for example, practical training with real patients frequently occurs as early as the first curriculum year. One important part of medical education is learning medical certificate writing skills.In Finland, medical certificates are tied to the social security insurance system. Permanent residents are covered by insurance administered by the Social Insurance Institution of Finland (SII). The insurance covers income during sickness and reimburses medication expenses. The reimbursement rate depends on the disease and its severity, as well as the products confirmed as reimbursable by the National Pharmaceuticals Pricing Board.To apply for entitlement to reimbursement of medication expenses at the special rate (\u2265 65%), a medical certificate B issued by a treating doctor is needed. The treating doctor has a key role in initiating the insurance procedure, as he or she informs the patient and works as an expert. To fulfil this role, the doctor must know the content and goals of the applicable law. The entitlement to reimbursement is assessed and granted or denied by the SII based on this certificate. Therefore in this study, we considered it important to include an evaluator from the SII as the (future) doctor\u2019s medical certificate B is evaluated by the SII and the reimbursement depends on the doctor\u2019s skills.N\u2009=\u2009141) and 2016 (N\u2009=\u2009142) in the medical faculty of the University of Eastern Finland (UEF). The participants of the module were fourth-year students. The research was explained verbally to all students. Additionally, students received written information sheet that also included informed consent form. Participation to this study was voluntary, there were not any known risk factors for participating this study and data was planned to be protected according to the national/university policies. Written informed consent forms were received from 110 students from the 2015 course and 132 students from the 2016 course. Two students from the 2015 course and four from the 2016 course did not give us permission to use their responses in the study. Twenty-nine students from the 2015 course and six from the 2016 course were not reached out. This study follows the guidelines of Finnish National Board on Research Integrity (TENK) [The data were collected from two independent courses taught in autumn 2015 (y (TENK) , 28. Accy (TENK) . This sty (TENK) . This reFrom the two courses, a random sample of 40 students per course was drawn for the analysis. The sample was blinded for the evaluators independently by another researcher. Thus, the evaluators had no knowledge to which group students belong nor the identifying information of students. After the analysis, one student from the 2016 group had to be dropped from the sample, as the medical certificate B exercise was not finalized. Thus, the total sample size is 39  for the 2016 course and 40  for the 2015 course. The gender distribution and ethnic composition (<\u20095% of other than Finnish origins) represented courses of the previous years.The study module is part of a bigger module called \u201cIntroduction to General Practice\u201d. In 2015, the medical certificate writing was taught to students during a two-hour traditional lecture including nine other kinds of medical certificates (not reported here). In 2016, the instruction method was developed as part of the ongoing evidence-based development of learning environments at UEF. The instruction method of the 2016 course was modified to follow FC principles. Thus, the content of the traditional two-hour lecture was changed to short video-based FC lectures provided to students through an electronic learning platform. The duration of the video concerning medical certificate B investigated in this study . The cases for both the 2015 and 2016 courses were presented in a similar manner for the students in electronic platform. In the instruction, the students were offered the criteria for the medical certificate B for diabetic medication. They were also told to investigate further information from the SII webpage as doctors do in real work settings. The purpose of the exercise was for the students to follow a real-life setting as closely as possible. In both years, the students also relied on peer support as they created small groups of three students to obtain feedback and correct their medical certificate B exercises. After this, each student individually returned the exercise to the teacher. Metadata from the media server right after the submission time indicated that the 2016 group had watched the medical certificate B video\u00a0263 times. At the end of the process, there were f-2-f sessions considering all the taught medical certificates.The study used the assessment rubric for medical certificate B by the UEF Department of General Medicine  read the medical certificates and assessed them independently with the rubric composed of 27 items per student. Discrepancies in the evaluations were found in five of the 79 cases. The doctors discussed these discrepancies and mutually agreed on the final scores of these five cases.U-tests for the analysis to investigate the differences between groups. We also chose to report the means (M) and standard deviations (SD) to retain the original metrics of the rubric. The common language (CL) effect size [The research used a quasi-experimental posttest-only design with nonequivalent groups  from difect size  was usedM and SD) for the 2015 group (control) and 2016 group (treatment), the results of the comparison between groups (Mann-Whitney U), and the CL effect size. The 2016 students had a significantly higher total score (p\u2009<\u2009.01) as well as higher scores in the areas of patient identification information, background information, treatment plan, and special refunds for medicines, and a lower score in non-relevant information. The effect size (CL\u2009=\u20090.85) indicates that the total score of a randomly selected student from the 2016 group had an 85% probability of getting higher score than the total score of a randomly selected student from the 2015 group. For the specific measured areas, the probabilities that the score of a randomly selected student from the 2016 group would be higher than that of a student from the 2015 group were as follows: 68% for patient identification information (CL\u2009=\u20090.68), 73% for background information (CL\u2009=\u20090.73), 71% for treatment plan (CL\u2009=\u20090.71), and 69% for special refunds for medicines (CL\u2009=\u20090.69). Moreover, a randomly selected student from the 2016 group had an 82% probability of receiving a lower score than a student from the 2015 group in non-relevant information (CL\u2009=\u20090.82). The difference was significant (p\u2009<\u2009.05) in examination findings (CL\u2009=\u20090.63), where there was a 63% probability that a student in the 2016 group would receive a higher score than a student in the 2015 group.Table\u00a0M) indicate that the 2016 group received higher scores in general for medical certificate B and a lower score in the non-relevant information scale. Wrong diagnosis can be an extra burden for the SII in an actual work context. Additionally, compared to the 2016 group, the 2015 group had a larger variation (as indicated by the SD) in all areas with significant differences, except in the treatment plan.No significant differences were found between the groups in the areas of doctor identifying information and purpose of the certificate. The mean ranks  may further increase their motivation and self-efficacy for learning. Following Kirkpatrick\u2019s  classifiThe social security insurance administered by the SII has an essential role in reimbursing medical expenses in Finland. Doctors play a key role in the system. The medical certificate B sheet is also used for different, more complicated purposes, and teaching starts with the reimbursement case. Making a good medical certificate B demands extensive knowledge from the doctor. They must also master the social security insurance system and be able to understand the medical aspects of the patient.Although this study clarifies one aspect of medical education and increases the understanding of the FC method in medical education, it has several limitations. First, only one medical education institution and its students were included in the study. In the future, more comprehensive studies with randomized experimental design may increase the understanding of FC teaching. Second, the rubric used to evaluate students\u2019 medical certificate B exercises was developed only recently; thus, there is no indication yet of the rubric\u2019s psychometric properties. However, the study used a rigorous research design with a randomized sample and found only a few discrepancies between the evaluators\u2019 assessments of the students\u2019 medical certificate B exercises. Additionally, one of the doctors evaluating the students\u2019 exercises was an official SII evaluator of certificates. These aspects increase the trustworthiness of the results. Nevertheless, in future studies, the psychometric aspects of the rubrics should be investigated. Third, the research design used only posttest data, limiting the possibilities to control the prior knowledge of the sample. Still, given the difficulty of getting into a medical education program and the fact that medical students are well educated in these programs, the variation between years can be seen as minimal and does not pose a threat to the study. Nevertheless, future research should include pretest data to its design. Fourth, these results consider only one type of medical certificate in one country; other types of certificates and countries should be included in future studies.In this study, the FC approach resulted in a statistical significant improvement in the content learning and technical quality of medical certificate writing. The results suggest that the FC approach can be applied in the teaching of medical certificate writing.Additional file 1:Medical Certificate sheet. Official Medical Certificate B1 sheet used in Social Insurance Institution of Finland (SII). (PDF 1580 kb)Additional file 2:Rubric for evaluating medical students\u2019 medical certificate B exercises. Rubric scoring follows the Social Insurance Institution of Finland (KELA in Finnish) medical certificate B (Additional file"}
+{"text": "By default, movement/action takes place in reference to the three-dimensional space: a movement thus not only involves proprioceptive information, but it also contains spatiotopic information. Therefore, the aim of this study was to investigate to what extent spatiotopic information  contributes to the acquisition and generalization of such fear of movement-related pain besides proprioception . In a between-subjects design, the location group performed joystick movements from the middle position to left and right; the movement group moved the joystick from left and right to the middle. One movement (CS+) was paired with pain, another not (CS\u2212). Feature overlap between CSs typically reduces differential learning. The endpoint of both CSs in the movement group is an overlapping feature whereas in the location group the endpoint of both CSs is distinct; therefore we hypothesized that there would be less differential fear learning in the movement group compared to the location group. We also tested generalization to movements with similar proprioceptive features but different endpoint location. Following the principle of stimulus generalization, we expected that novel movements in the same direction as the CS+ but with a different endpoint would elicit more fear than novel movement in the same direction of the CS\u2212 but with a different endpoint. Main outcome variables were self-reported fear and pain-US expectancy and eyeblink startle responses (electromyographic). Corroborating the feature overlap hypothesis, the location group showed greater differential fear acquisition. Fear generalization emerged for both groups in the verbal ratings, suggesting that fear indeed accrued to proprioceptive CS features; these effects, however, were not replicated in the startle measures.Fear of movement-related pain significantly contributes to musculoskeletal chronic pain disability. Previous research has shown that fear of movement-related pain can be classically conditioned. That is, in a differential fear conditioning paradigm, after (repeatedly) pairing a neutral joystick movement  with a painful stimulus , that movement in itself starts to elicit self-reported fear and elevated psychophysiological arousal compared to a control joystick movement (CS\u2212) that was never paired with pain. Further, it has been demonstrated that novel movements that are more similar to the original CS+ elicit more fear than novel movements that are more similar to the CS\u2212, an adaptive process referred to as  Subsequently, groups went through a crossover phase testing fear generalization; based on the principle of stimulus generalization, we hypothesized that if associative strength accrues to the proprioceptive features, generalization to novel, proprioceptively similar movements with different endpoint location would occur.In this study, we wanted to address the intriguing question as to what extent spatiotopic information contributes to the acquisition of fear of movement-related pain in addition to proprioceptive or movement-related information only. We used an adapted version of the Voluntary Joystick Movement Paradigm (VJMP) . In the t(49) = \u22122.35, p < 0.051Please note that additional analyses including the medical pain subscale of the FPQ as a covariate did not change the results, suggesting that the observed differences between the groups are not due to the higher score on this index in the movement group.. The experimental protocol was approved by the Social and Societal Ethics committee of the KU Leuven (registration number: S55434) and the Medical Ethical Committee of the University Hospital of the KU Leuven (registration number: ML9403).A total of 51 healthy, pain-free indiviuals volunteered to participate in this study. The sample size was informed by previous research using a similar paradigm and a between-subjects design . Voluntesignificantly painful and demanding some effort to tolerate\u201d was targeted. When this stimulus was selected, the experimenter asked the participant whether s/he would agree to repeatedly receive stimuli of maximally this amplitude during the experiment.The experiment was run on a Windows XP computer (Dell Optiplex 755) with 2 GB RAM and an Intel Core2 Duo processor at 2.33 GHz and an ATI Radeon 2400 graphics card with 256 MB of video RAM, using Affect 4.0 . Four prWe used an adapted version of the VJMP developed by Meulders, Vansteenwegen, and Vlaeyen  eliminatParticipants were informed both by oral and written means that painful electrocutaneous stimuli (pain-USs), tones indicating the direction of the movements and short loud noises (acoustic startle probes) would be administered during the experiment. After the participants provided informed consent, the electrodes for the eyeblink startle responses were attached. Next, the stimulation electrodes were attached and the intensity level of the pain-US was individually selected .A habituation phase was designed to prevent possible confounds in the data because startle responses to the first probes are usually relatively large. This phase contained eight trials, each lasting 17s. During each trial, one startle probe was presented (100 dBA burst of white noise) randomly, either between the 8th and 12th second or between the 13th and 17th second after trial-onset. The timing of probes during the trials were randomized across participants. After the last probe presentation a 2-s intertrial interval (ITI) was inserted before moving on to the next phase. During this phase, the headphones were put on and the central lighting of the experimental room was turned off. Small lamps provided dimmed light. Note that during this phase, no pain-USs were delivered.Before the practice phase started, we calibrated the range of motion of the joystick and participants received detailed instructions about the experimental task. They were told that at the beginning of each trial, a tone  would be presented either in their left or right ear and that their task was to move the joystick 90\u00b0 in the direction that the tone was presented. Participants were requested to move the joystick as quickly and accurately as possible when prompted by a starting signal \u201c+\u201d (fixation cross presented in the middle of the computer screen). In total, two blocks of eight trials were run: the first block comprised eight trials of the typical location group training, that is, participants performed four movements from the middle position to the left and four movements from the middle position to the right  pain-USs and startle probes were presented, (2) four blocks of one training type (movement or location group training) were run instead of two training blocks (one of each training type), and (3) instructions emphasized to pay close attention to the starting signal \u201c+\u201d and to respond as fast and accurately as possible upon its presentation.On each trial, a tone was presented either in the participants\u2019 left or right ear 1 s after trial onset. When a trial consisted of prospective questions see , the ratIn order to test whether participants transferred their knowledge about the stimulus contingencies acquired in the acquisition blocks, a generalization phase was included. During this phase one block of eight trials was run and no pain-USs were presented after the GSs. The randomization schedule of the trials and the startle probe presentation per block was the same as in the previous phase. In this phase the movement direction (to the left or to the right) remained the same, while the starting point and the endpoint location of the movements changed. That is, participants in the location group, who moved the joystick in the acquisition blocks from de middle to the left and to the right, now moved the joystick in the generalization block from the left and right to the middle. Participants in the movement group, who moved the joystick in the acquisition blocks from the left and the right to the middle, now moved the joystick from the middle to the left and to the right.\u201cTo what extent do you expect an electrocutaneous stimulus after performing the following movement?\u201d, and (2) \u201cTo what extent are you afraid to perform the following movement?\u201d on a numerical rating scale ranging from 0 to 10 with anchors \u201cnot at all\u201d to \u201cvery much\u201d. Participants were instructed to browse through the scales with the joystick using a wrist rotation  and to click on the shooting button of the joystick to confirm their answer. The Z-rotation was used to avoid possible contamination with the CS/GS movements during the experiment.During each block, pain-US expectancy ratings and fear ratings were collected once per movement type during acquisition and twice during the generalization phase . Participants answered the following questions: (1) The eyeblink startle response is a component of the reflexive cross-species, full-body defensive response mobilization, which is triggered by startle-evoking stimuli  and can be measured by the tension in the muscles underneath the eye. Startle modulation refers to the potentiation of the startle reflex during fear states elicited by the anticipation of an aversive stimulus . Orbicularis Oculi electromyographic activity (EMG) was recorded with three Ag/AgCl Sensormedics electrodes (four mm) filled with electrolyte gel. After cleaning the skin with exfoliating peeling cream to reduce inter-electrode resistance, electrodes were placed on the left side of the face according to the site specifications proposed by \u201cHow unpleasant did you find moving to the left/right?\u201d Responses were indicated on an 11-point numerical rating scale ranging from 0 to 10 with 0 meaning \u201cnot at all\u201d and 10 meaning \u201cvery much.\u201dAfter each block the following question was presented: \u201cHow painful did you find the electrocutaneous stimuli in the previous block?\u201d and (2) \u201cHow unpleasant did you find the electrocutaneous stimuli in the previous block?\u201d Responses were indicated on an 11-point numerical rating scale ranging from 0 to 10 with 0 meaning \u201cnot at all\u201d and 10 meaning \u201cvery much.\u201dAfter each acquisition block, the following questions were asked: (1) After the experiment the participants filled in a set of questionnaires to map out individual differences for descriptive purposes: the Dutch version of the FPQ , the DutParticipants were seated in an armchair (0.6 m screen distance) in a sound-attenuated and dimmed experimental room, adjacent to the experimenter\u2019s room. Further verbal communication was possible through an intercom system; the experimenter observed the participants and their physiological responses online by means of a closed-circuit TV installation and computer monitors.z-scores to account for inter-individual differences and T-scores were used to visualize the data. Averages were calculated for responding during CS/GS movements and ITI separately for the movement and the location group. Participants who failed to show an elevated peak amplitude compared to baseline on more than 30% of probe trials were considered non-responders and were excluded from statistical analyses. A total of four participants from our sample were excluded due to the absence of reliable startle eyeblink responses, therefore the statistical analyses of the psychophysiological data were run on a total sample of 47 participants.Using PSPHA , a modulp-values. All statistical analyses were carried out with Statistica 13 software .Repeated measures (RM) ANOVAs were run to examine differences in acquisition and generalization between the movement group and the location group for each dependent variable. These analyses included Group as a between-subject variable, and Stimulus Type and Block as within-subject variables. Because we had clear a priori hypotheses, data were further analyzed using planned comparisons. Significance levels were set at \u03b1 = 0.05, effect size SD = 28.23 \u00b1 21.91 mA) than participants in the movement group (mean \u00b1 SD = 21.24 \u00b1 11.41 mA), this difference was not significant, t(49) = 1.42, p = 0.16. The self-reported pain intensity of the electrocutaneous stimulus in the location (mean \u00b1 SD = 7.56 \u00b1 0.86) and movement (mean \u00b1 SD 7.48 \u00b1 0.99) group also did not differ significantly, t(49) = 0.30, p = 0.77.Although participants in the location group tended to select a more intense electrocutaneous stimulus  = 109.08, p < 0.001, F = 5.21, p < 0.01, \u03b5 = 0.64, F = 0.04, p = 0.85. Interestingly, there was a significant Stimulus Type \u00d7 Group interaction, F = 9.98, p < 0.01, F = 18.82, p < 0.001, \u03b5 = 0.76, F = 3.06, p < 0.05, \u03b5 = 0.76, Acquisition. Planned comparisons at the beginning of the acquisition phase (A1), revealed no differences in pain-US expectancy for the CS+ and CS\u2212 movements in the location group, F = 3.23, p = 0.08, nor the movement group, F = 0.96, p = 0.33. Yet, at the end of the acquisition (A4) participants in the location group gave higher pain-US expectancy ratings for the CS+ movements than for the CS\u2212 movements, F = 99.55, p < 0.001. Likewise, participants in the movement group expected the pain-US to occur more when they performed the CS+ movement than when performing the CS\u2212 movement, F = 19.82, p < 0.001. A between-group contrast further showed that the difference in pain-US expectancy between the CS+ and the CS\u2212 was significantly larger in the location than in the movement group, F = 14.49, p < 0.001.Generalization. To examine the generalization effect, within-group planned comparisons were conducted showing that participants in the location group, generalized the acquired differential pain-US expectancy ratings to the first generalization test (G1), F = 59.84, p < 0.001, and the second generalization test (G2), F = 21.25, p < 0.001. Furthermore, planned comparisons showed that in the location group the difference in pain-US expectancy between the CS+ and the CS\u2212 was significantly smaller during generalization (G1) as compared to the end of the acquisition (A4), F = 5.98, p = 0.02, which seems to be due to an increase on the CS\u2212 instead of a decrement relating to the CS+. In the movement group, the pain-US expectancy ratings were also higher when performing the CS+ movement than when performing the CS\u2212 movement in the first (G1), F = 24.96, p < 0.001, and second test of generalization (G2), F = 10.18, p < 0.01. No difference in differential pain-US expectancy ratings was observed between at the end of the acquisition (A4) compared to the first test of generalization (G1), F = 0.43, p = 0.52.F = 5.98, p = 0.02, suggesting that extinction took place. Indeed, participants did not receive any pain-USs during the generalization phase. In the movement group, however, differential pain-US expectancy ratings for the CS+ and the CS\u2212 at G2 were not significantly reduced in comparison to A4, F = 0.43, p = 0.52.To further analyze the development of generalization, planned comparisons were performed between the end of the acquisition (A4) and the second generalization test (G2). In the location group, the difference in pain-US expectancy between the CS+ and the CS\u2212 was significantly smaller at G2 compared to A4, F = 60.74, p < 0.001, F = 4.84, p < 0.001, \u03b5 = 0.78, F = 0.13, p = 0.72. The Stimulus Type \u00d7 Group interaction turned out to be significant, F = 7.32, p < 0.01, F = 13.78, p < 0.01, \u03b5 = 0.66, F  = 1.79, p = 0.15, \u03b5 = 0.66, we continued testing our a priori hypotheses.In Acquisition. Planned comparisons at the beginning of the acquisition phase (A1) revealed no differences in fear reported in response to CS+ and CS\u2212 movements in the location group, F = 1.08, p = 0.30, nor the movement group, F = 0.05, p = 0.83. At the end of the acquisition phase (A4) however, participants were more afraid of the CS+ than the CS\u2212 both in the location group, F = 49.29, p < 0.001, and in the movement group, F = 12.82, p < 0.001. Planned between-group comparisons at A4 confirmed that differential fear of movement-related pain was more pronounced in the location group than in the movement group, F = 5.57, p = 0.02.Generalization. To examine the fear generalization effect, within-group planned comparisons were conducted showing that participants in the location group, generalized the acquired differential fear of movement-related pain to the first generalization test (G1), F = 26.73, p < 0.01, and to the second generalization test (G2), F = 10.97, p < 0.01. Furthermore, the difference in fear of movement-related pain reported for the CS+ and the CS\u2212 tended to be smaller during generalization (G1) as compared to the end of the acquisition (A4) in the location group, F = 3.86, p = 0.06, which seems to be due to an increase on the CS\u2212 instead of a decrement relating to the CS+. In the movement group, the fear of movement-related pain ratings were also higher when performing the CS+ movement than when performing the CS\u2212 movement in the first (G1), F = 8.66, p < 0.01. At the second test of generalization (G2), F = 3.46, p = 0.07, this difference was only borderline significant, suggesting that extinction took place. No difference in differential fear ratings was observed between the end of the acquisition and the first test of generalization in the movement group, F = 0.34, p = 0.56.F = 14.97, p < 0.01, suggesting that extinction took place. Note that participants indeed did not receive any pain-USs during the generalization phase. In the movement group, however, the difference in fear of movement-related pain elicited by the CS+ and the CS\u2212 was not significantly reduced at G2 compared with A4, F = 3.28, p = 0.08.To further analyze the development of generalization, planned comparisons were performed between the end of the acquisition (A4) and the second generalization test (G2). In the location group, the difference in fear of movement-related pain between the CS+ and the CS\u2212 was significantly smaller at G2 compared to A4, F = 22.11, p < 0.001, \u03b5 = 0.84, F = 40.77, p < 0.01, F = 3.60, p = 0.06. None of the interaction effects were significant, nonetheless we continued testing our a priori hypotheses.Acquisition. Because startle responses elicited by few startle probes are less reliable, planned comparisons were carried out to evaluate differences in early (A1\u2013A2) and late (A3\u2013A4) acquisition. This analysis revealed that in the early acquisition participants did not demonstrate any differences in startle amplitudes in response to CS+ and CS\u2212 movements in the location group, F  = 0.01, p = 0.91. In the late acquisition, however, startle amplitudes in the location group were higher in response to the CS+ then in response to the CS\u2212, F = 4.52, p = 0.04. In the movement group, participants did not demonstrate differential startle amplitudes in the early acquisition, F = 0.06, p = 0.80, nor the late acquisition phase, F = 1.12, p = 0.30. Interestingly, during the late acquisition phase, startle amplitudes during the ITI were elevated in the movement group compared with the location group, F = 4.85, p = 0.03, suggesting that more fear of movement-related pain accrued to the context the movement group, whereas this effect was not present during early acquisition, F = 0.85, p = 0.36.Generalization. To examine the generalization effect, planned within-group comparisons were performed during the test of generalization (G). In the location group, the acquired differential startle responding did not generalize to G1, F = 0.37, p = 0.55, this was largely due to inflated responses to the CS\u2212. Because there was no acquisition effect in the movement group, we did not continue testing for generalization.F = 76.28, p < 0.001, F = 37.25, p < 0.001, \u03b5 = 0.71, F = 1.02, p = 0.32. Interestingly, there was a significant Stimulus Type \u00d7 Group interaction, F = 5.00, p = 0.03, F = 25.03, p < 0.01, \u03b5 = 0.85, In Acquisition. Planned comparisons at the end of the acquisition phase (A4) confirmed that participants in both groups found the CS+ more unpleasant than the CS\u2212, location group: F = 39.16, p < 0.001, movement group: F = 19.33, p < 0.001. There were no differences in differential unpleasantness reported for the CS+ and the CS\u2212 between both groups at A4, F = 1.54, p = 0.22.Generalization. To examine whether unpleasantness of the CSs generalized to the GSs, planned comparisons were performed between the end of the acquisition (A4) and the generalization test (G1). In the location group the difference in retrospective unpleasantness ratings in response to the CS+ and the CS\u2212 was significantly smaller at G1 than at A4, F = 26.77, p < 0.001. A similar pattern occurred in the movement group, F = 16.75, p < 0.001. Further planned comparisons at G1 revealed that participants in the location group also rated the GS+ movement as more unpleasant than the GS\u2212 movement, F = 6.54, p = 0.01. However, in the movement group the differential unpleasantness ratings for the CS+ and the CS\u2212 did not generalize to the GSs, F = 0.87, p = 0.36.F = 19.29, p < 0.001, F = 0.92, p = 0.34. None of the other main effects or interactions reached significance.Feature overlap between the CS+ and CS\u2212 has been shown to reduce differential learning  was consistently followed by the pain-US and another movement (CS\u2212) was not. In the location group, participants moved the joystick from the middle position to the left and to the right; basically, the training in this group reflected the standard VJMP procedure. Participants in the movement group moved the joystick from the left and from the right to the middle position. The middle position being the endpoint location for both CS movements therefore became ambiguous with respect the pain-US occurrence. learning . TherefoWe observed higher pain-US expectancies for the CS+ than the CS\u2212 in both the location and the movement group. In line with the feature overlap hypothesis, differential acquisition in the pain-US expectancy ratings was greater in the location group than in the movement group. A similar data pattern was observed in the fear of movement-related pain ratings. That is, self-reported fear of the CS+ was higher than for the CS\u2212, and this difference was larger in the location group than in the movement group. A more extreme pattern emerged in the startle eyeblink measures. Startle amplitudes at the end of acquisition were higher in response to the CS+ than to the CS\u2212 in the location group, but the difference between the CS+ and the CS\u2212 did not reach significance in the movement group. Interestingly, at the end of the acquisition phase, ITI startle amplitudes were elevated in the movement group compared with the location group, suggesting that in that group more fear accrued to the context. Taken together, these data provide strong support for the feature overlap hypothesis; demonstrating that differential fear of movement-related pain acquisition effects are larger when proprioceptive and spatiotopic features of the CS+ and the CS\u2212 do not overlap. With respect to the secondary outcome measures, visual inspection also suggested larger differences in unpleasantness ratings for the CS+ and CS\u2212 in the location group than in the movement group, statistical tests however failed to support this. At the end of the acquisition phase, the CS+ was found to be more unpleasant than the CS\u2212 in both groups.Testing generalization of conditioned responses to novel movements with similar proprioceptive characteristics but with a different starting point and endpoint location revealed that differential pain-US expectancy ratings generalized in both groups. In line with the feature overlap hypothesis, there was a larger generalization decrement in the location group than in the movement group. Interestingly, this was due to an increase in pain-US expectancies in response to the GS\u2212 instead of a generalization decrement reflecting lower pain-US expectancies for the GS+ in the beginning of the generalization phase. Because no pain-USs were delivered, generalized differential responses in the location group were significantly reduced by the end of the generalization phase, but the difference between the GS+ and GS\u2212 remained statistically significant. In the movement group however, this extinction effect was less manifest, most likely because there was less differential learning to start with. A similar data pattern emerged in the fear of movement-related pain ratings: generalization occurred in both groups. In the line with the expectancy ratings, extinction of generalization at least partly occurred in both the location group and the movement group. In the startle responses, no generalization was observed in the location group, this effect again seems to be due to an increase in GS\u2212 responding but not to a decrement relating to the GS+. Because of the lack of differential learning during acquisition, we did not continue to test for generalization effects in the movement group. Taken together, these data suggest that proprioceptive information alone indeed can drive generalization effects; differential learning generalized at least in two of the three outcome variables.In both the location and the movement group, the retrospective unpleasantness ratings show a large decrement in differential responding to the GSs, this is probably explained by generalization being tested under extinction and ratings collected at the end of the generalization phase. This is in contrast with the pain-US ratings and fear of movement-related pain ratings, which were collected prospectively, allowing for hindsight, experience-based corrections.Some observations deserve more in-depth discussion. First, the ITI startle amplitudes were significantly higher in the movement group compared to the location group at the end of the acquisition phase, indicating that participants were more uncertain about the non-occurrence of the pain-US during the context in the movement group and thus were in general more fearful. This could be due to the ambiguity and feature overlap between the CS+ and CS\u2212 present in the movement group. It is plausible that feature overlap did not only lead to reduced differential learning, but also created some degree of unpredictability which increased contextual fear , 2002b iSecond, the finding that generalization occurred to movements with similar proprioceptive features but another endpoint location confirms that the movement alone has gained sufficient associative strength to subsequently generate generalization effects. However, there was a generalization decrement in the location group, which seems to be due to an increase on the GS\u2212 instead of a decrement relating to the GS+. This increase in GS\u2212 responding may be explained as follows: during the generalization phase the participants in the location group lose the spatiotopic predictor for the pain-US. As a result, the CS\u2212 becomes especially \u201cuncertain\u201d or \u201cambiguous\u201d because it now has a feature in common with the GS+. Participants therefore may not fully trust the GS\u2212. Responses to the GS+ on the other hand seem to generalize perfectly when the spatiotopic predictor is lost, suggesting that when it comes to the threatening movement participants apply the \u201cbetter safe than sorry\u201d adagio. In the movement group on the contrary, the spatiotopic information was never a predictor of the pain-US in the first place and thus has not acquired any associative strength, therefore no such generalization decrement is observed in this group.Some limitations should be considered as well. First, the joystick was programmed to automatically move into the proper starting position, especially in the movement group this may serve as an additional predictor for the pain-US. In addition, this set-up may render the task easier in this group and requiring less attention to the direction signal , because the joystick is physically limited to move in only one direction. Therefore, in the movement group, the starting point  may also gain associative strength. However, based on a componential CS representation approach, late components in a (chain of) CS(s) typically gain excitatory properties by virtue of their temporal proximity with the US, whereas early components in a (chain of) CS(s) gain inhibitory properties, referred to as the law of inhibition of delay . This meAlthough this is an experimental study in healthy participants investigating the basic processes underlying the acquisition and generalization of fear of movement-related pain, we could speculate about the potential clinical implications of the current results. Previous research shows that exposure in multiple contexts  and/or tTo conclude, we successfully demonstrated that proprioceptive information is sufficient for the acquisition of fear of movement-related pain and serves a basis for the generalization of such fear at least in the verbal fear and pain-US expectancy measures. Furthermore, we showed that spatiotopic information in the standard VJMP also significantly contributes to the observed learning effects showing larger differential learning effects in the location group than in the movement group. These results further strengthen the construct validity of the VJMP and support its use to study learning processes involved in fear of movement-related pain.10.7717/peerj.6913/supp-1Supplemental Information 1The sheet \u201clegend\u201d includes the explanation of the variable names.Click here for additional data file.10.7717/peerj.6913/supp-2Supplemental Information 2The sheet \u201clegend\u201d includes the explanation of the variable names.Click here for additional data file.10.7717/peerj.6913/supp-3Supplemental Information 3The sheet \u201clegend\u201d includes the explanation of the variable names.Click here for additional data file."}
+{"text": "The gut microbiome contributes to host metabolism, protects against pathogens, educates the immune system, and, through these basic functions, affects directly or indirectly most physiologic functions of its host. Molecular techniques have allowed us to expand our knowledge by unveiling a wide range of unculturable bacteria that were previously unknown. Most bacterial sequences identified in the canine gastrointestinal (GI) tract fall into five phyla: Firmicutes, Fusobacteria, Bacteroidetes, Proteobacteria, and Actinobacteria. While there are variations in the microbiome composition along the GI tract, most clinical studies concentrate on fecal microbiota. Age, diet, and many other environmental factors may play a significant role in the maintenance of a healthy microbiome, however, the alterations they cause pale in comparison with the alterations found in diseased animals. GI dysfunctions are the most obvious association with gut dysbiosis. In dogs, intestinal inflammation, whether chronic or acute, is associated with significant differences in the composition of the intestinal microbiota. Gut dysbiosis happens when such alterations result in functional changes in the microbial transcriptome, proteome, or metabolome. Commonly affected metabolites include short-chain fatty acids, and amino acids, including tryptophan and its catabolites. A recently developed PCR-based algorithm termed \u201cDysbiosis Index\u201d is a tool that allows veterinarians to quantify gut dysbiosis and can be used to monitor disease progression and response to treatment. Alterations or imbalances in the microbiota affect immune function, and strategies to manipulate the gut microbiome may be useful for GI related diseases. Antibiotic usage induces a rapid and significant drop in taxonomic richness, diversity, and evenness. For that reason, a renewed interest has been put on probiotics, prebiotics, and fecal microbiota transplantation (FMT). Although probiotics are typically unable to colonize the gut, the metabolites they produce during their transit through the GI tract can ameliorate clinical signs and modify microbiome composition. Another interesting development is FMT, which may be a promising tool to aid recovery from dysbiosis, but further studies are needed to evaluate its potential and limitations. The gut microbiome is composed of bacteria, archaea, viruses, and eukaryotic organisms that reside in the gastrointestinal tract, and which relate with the host in a symbiotic fashion. For example, bacteria in the guts produce short-chain fatty acids (SCFA) that nourish the intestinal epithelium, while the epithelium produces mucus that feeds beneficial bacteria.The gut microbiome contributes with metabolic functions, protects against pathogens, educates the immune system, and, through these basic functions, affects directly or indirectly most of our physiologic functions. Serotonin, a neurotransmitter, is mostly produced in the intestine, which has led to the development of the gut-brain axis concept . A healt2 to 1011 colony forming units (CFU) per gram of luminal content , XI , and XIVa   , 13, 14.bacillus .Prevotella, Bacteroides, and Megamonas  are facultative anaerobes, which allows them to take advantage of the oxygen available in the small intestine. In fecal samples their increase is associated with many diseases, as will be discussed further in this review. Actinobacteria are also associated with the small intestine, and include families Corynebacteriaceae  and Coriobacteriaceae   .Dogs in their natural state are carnivorous scavengers, meaning that they thrive on a diet that is rich in meat, but will take advantage of any available food. In dogs, most microbiome studies have relied on extruded diets , which represent up to 95% of the dry dog food market. Traditionally, the extrusion process requires a high load of carbohydrates, which is achieved with the inclusion of vegetable ingredients. However, alternative industrial processes have recently become available and a percentage of the pet food market now includes kibble with reduced carbohydrate content and increased protein content. Also increasingly popular are raw diets, frozen or freeze-dried, which are typically meat based and include low to negligible carbohydrate percentages.Several studies in different species have shown that diet composition\u2014especially large macronutrient differences like those found in carnivore vs. herbivore diets\u2014is reflected in different gut microbiome profiles. In omnivore species, including humans, who can tolerate and thrive on either end of the spectrum, the short-term consumption of diets composed entirely of animal or plant products is enough to alter the microbial community structure and overwhelm inter-individual differences in microbial gene expression . In humaHowever, for dogs, the kingdom of origin of the ingredients seems to be less important than the overall macronutrient composition. Extruded diets with similar macronutrient contents, but prepared exclusively with vegetable sources of protein, do not seem to significantly alter the microbiome of dogs when compared to traditional  extruded diets .Bones and Raw Food (BARF) diets consisting of a combination of raw meat, organs, meaty bones, and vegetables. Overall, compared to the kibble-fed control group, BARF diets included more protein and fat, and less fiber and carbohydrates. Another study  (Fusobacterium), and two genera from phylum Firmicutes (Lactobacillus and Clostridium)  . Most oftridium) , 23.Clostridium perfringens and Fusobacterium varium, and a decreased abundance of Coprobacillus sp. compared to controls. However, the study  , and thehe study  includedFaecalibacterium and an increase in two Clostridiaceae strains.In another study , healthyClostridiaceae strains was later identified as Clostridium hiranonis, a bacterial species associated with normal bile acid (BA) metabolism , it has been suggested that increases in abundance of Clostridiaceae members  when protein-rich diets are fed to dogs may not be detrimental to their health . These findings suggest that Clostridiaceae may have a role in the metabolism of protein in the intestinal tract of dogs, different than the role played in the large bowel of the rat, where Clostridiaceae respond to dietary carbohydrates. In addition, Clostridiaceae had a positive correlation with fecal health score  and a negative correlation with fecal output .Despite being commonly associated with gastrointestinal disease , Lactobacillus spp. (Lactobacillaceae), and Faecalibacterium spp. (Ruminococcaceae) abundances is often investigated as they are considered beneficial in omnivores  in dogs, acetate, butyrate and propionate levels were not different from dogs fed a low-fat high-starch diet (supplemented with maize and broken rice), indicating that the production of SCFA in dogs is not exclusively dependent on carbohydrate content . SupportClostridiaceae, and in particular Clostridium perfringens, are associated with the butyrate kinase butyrate-synthesis pathway, which allows the production of butyrate from protein , a neurotransmitter, and its precursor gamma-hydroxybutyric acid (GHB) . GABA anAnother neurotransmitter, serotonin, is essential for gut health. About 90% of the serotonin produced in the body originates from the intestines, where it regulates motility, secretion and blood flow through the enteric nervous system . SerotonRegardless of the species, GI tract colonization in mammals starts even before the newborn exits the birth canal. The initial colonization varies and reflects the method of parturition and nutrition, and the establishing microbiome will increase in diversity over time . In humaFaecalibacterium spp. and Clostridium hiranonis were significantly increased, with values within the reference interval of healthy adults. In addition, adult littermates have been found to present a more similar microbiome composition than unrelated dogs, which hints to the importance of genetics and early life exposure  cell precursors can differentiate into either Treg or Th17 cells depending on the signals received from the microbiota . In home of Treg , inducin of Treg , generatIntestinal inflammation can also be triggered by gut dysbiosis through bile acid dysmetabolism, which is seen both in dogs and in humans , 27, 58.Dysbiosis is seen in many pathologies, both locally, within the gastrointestinal tract, and systemically . While oGastrointestinal dysfunctions are the most obvious association with gut dysbiosis. The gut microbiome has been found to be altered during both acute and chronic diarrhea. Like with healthy dogs, studies in dogs with GI diseases will report different taxa abundance percentages, however most taxa are consistently increased or decreased within the same disease phenotype.Much of the apparent discrepancy between studies can be attributed to the difficulty obtaining samples from well-characterized clinical cases without confounding factors like recent antibiotic administration. This difficulty, paired with budget restrictions, results in studies with small numbers of samples, which limits statistical power. New technologies are making metagenome sequencing more accessible and, with increasing numbers of samples per project, such methodology issues should become easier to overcome.Blautia spp., Ruminococcus spp., Faecalibacterium praunitzii, and Turicibacter spp. (Clostridium (In acute uncomplicated diarrhea (AD), dogs will develop a strong dysbiosis with a decrease in short-chain fatty acid (SCFA)-producing bacteria like ter spp. , and incstridium . MicrobiClostridium sp., which as previously mentioned can produce butyrate from protein using an alternative pathway, which could be another explanation for the increase of butyrate.Despite its mild clinical presentation, AD is associated with fecal dysbiosis that significantly alters not only fecal SCFA profiles but also blood and urine metabolites, suggesting that acute episodes of diarrhea have an impact on the overall metabolic profile of the host. In fact, a study  demonstrRuminococcaceae, and Faecalibacterium spp. Studies have shown an association between Clostridium perfringens and AHDS , also known as hemorrhagic gastroenteritis (HGE) . Despiteand AHDS , howeverand AHDS . The genith AHDS . In addiith AHDS , and recbundance . CombineClostridioides difficile (previously known as Clostridium difficile)   is a conne study  reportedifficile  and no iifficile .C. difficile strains isolated from dogs are capable of producing toxins in vitro that severely impair tight junctions in canine and human cell lines  has been documented in dogs following episodes of parvovirus infection , and a sChronic enteropathies in dogs are generally classified according to their response to treatment as food-responsive diarrhea (FRD), antibiotic-responsive diarrhea (ARD), and immunosuppressant-responsive diarrhea . All dogs with chronic enteropathies will present intestinal inflammation to some degree, and therefore share similarly dysbiotic microbiomes when compared to healthy dogs .Bacteroidaceae and Prevotellaceae  (Ruminococcaceae (genus Ruminococcus), Veillonellaceae (genus Megamonas), and Lachnospiraceae were observed in dogs with IBD (Enterobacteriaceae), a hallmark of dysbiosis, are overrepresented in fecal samples of dogs with CE  , 83. Witwith IBD \u201384. Due with IBD . In addi with CE , 85, 86.Blautia spp. (Class Clostridia), Faecalibacterium spp. (Class Clostridia), and Turicibacter spp. (Class Erysipelotrichia) were significantly decreased (Fusobacterium spp. (Class Fusobacteriia) and Clostridium hiranonis (Class Clostridia) were also decreased, and Streptococcus spp. (Class Bacilli) and E. coli (Class Gammaproteobacteria) were increased . Negative DI values indicate normobiosis, and a positive DI values indicate dysbiosis. The DI is the first tool that allows quantification of gut dysbiosis, and can be used to monitor dysbiosis over time and in response to treatment. Other studies have since confirmed that DI is increased in dogs with CE . Humans with IBD have been found to have increased IDO-1 expression leading to lower serum tryptophan concentrations. Similar results have been seen in cats with CE, where serum tryptophan levels inversely correlate with disease severity . IncreasTryptophan availability can also influence gut microbiota directly, as tryptophan is the precursor for the production of indole compounds. Indole compounds can only be synthesized by bacteria, and have been shown to increase expression of genes associated with improved gut homeostasis, decreased gut permeability, and increased mucin production in other species , 91. TryBacteroides, which is associated with a healthy microbiome, in the colon. However, a few specific bacterial taxa presented different abundances between FRD and IBD. Dogs with FRD had a decrease of Enterococcus spp., Corynebacterium spp., and Proteobacteria, all potential pathogens, in the duodenum after treatment. In another study focusing on dogs with FRD , and XIVa . Inulin-type fructans also increased Firmicutes but from families Erysipelotrichaceae and Turicibacteraceae (Enterobacteriaceae). Inulin and yeast cell wall were both tested in combination with a raw meat diet , which consists in administering fecal matter from a healthy donor to the patient, usually endoscopically. In humans, fecal transplants have been used with success in the treatment of recurrent  therapy , 111. Tr therapy . In huma therapy . In dogs therapy .In one of the few case-control studies in dogs so far, puppies infected with parvovirus treated with FMT had significant reduction in hospitalization time and recovered faster than puppies receiving standard treatment . HoweverWhile promising, the use of FMT to treat dysbiosis and its associated diseases still requires further research to establish the ideal methodology to be applied to dogs. Factors like donor sample preservation (freezing or additives), route (upper GI endoscopy or colonoscopy) and schedule of administration (single transplant or daily administration of capsules) can significantly affect outcomes, and data from human studies does not necessarily translates to dogs due to anatomic and physiologic differences. Hopefully, future studies will allow researchers to fully gauge the potential and eventual limitations of FMT in the treatment of gastrointestinal diseases.In conclusion, the composition of the gut microbiome in dogs is correlated with overall health. The gut microbiome is stable in adult healthy dogs, but age, diet, and many other environmental factors may influence the maintenance of a healthy microbiome. The alterations found in diseased animals however are marked, and when they impact the transcriptome, proteome, or metabolome they are termed dysbiosis. Dysbiosis should always be considered when GI tract pathologies are present. The recovery of the microbiome composition does not necessarily correlate with the clinical recovery, and the long-term consequences of such lingering alterations are so far unknown. The identification of bacterial taxa and bacteria-derived compounds involved in the pathogenesis of acute and chronic GI diseases may aid the development of new diagnostic and therapeutic tools, and should be investigated.RP did the majority of the writing and editing. JS provided significant advice on content and editing as this document evolved to its current form.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."}
+{"text": "The gut microbiota is an important modulator of immune, metabolic, psychological and cognitive mechanisms. Chemotherapy adversely affects the gut microbiota, inducing acute dysbiosis, and alters physiological and psychological function. Cancer among young adults has risen 38% in recent decades. Understanding chemotherapy\u2019s long-term effects on gut microbiota and psycho-physiological function is critical to improve survivors\u2019 physical and mental health, but remains unexamined. Restoration of the gut microbiota via targeted therapies (e.g. probiotics) could potentially prevent or reverse the psycho-physiological deficits often found in young survivors following chemotherapy, ultimately leading to reduced symptom burden and improved health.n\u2009=\u200950), and a healthy sibling, partner or friend as a control (n\u2009=\u200950). Gut microbiota composition will be measured from fecal samples using 16\u2009s RNA sequencing. Psychological and cognitive patient reported outcome measures will include depression, anxiety, post-traumatic stress disorder symptoms, pain, fatigue, and social and cognitive function. Dual-energy X-ray Absorptiometry (DXA) will be used to measure fat and lean mass, and bone mineral concentration. Pro-inflammatory cytokines, C-reactive protein (CRP), lipopolysaccharide (LPS), serotonin, and brain derived neurotrophic factor (BDNF) will be measured in serum, and long-term cortisol will be assayed from hair. Regression and linear mixed model (LMM) analyses will examine associations across time points (T1 \u2013 T3), between groups, and covariates with gut microbiota, cognitive, psychological, and physiological parameters.This longitudinal study investigates chemotherapy induced long-term gut dysbiosis, and associations between gut microbiota, and immune, metabolic, cognitive and psychological parameters using data collected at <\u20092\u2009month (T1), 3\u20134\u2009months (T2), and 5\u20136\u2009months (T3) post-chemotherapy. Participants will be 18\u201339\u2009year old blood or solid tumor cancer survivors . The digestive tract is the most densely colonized microbial ecosystem in the body. The bacteria that make up the majority of this ecosystem  are important for immune, metabolic, psychological and cognitive function. A disruption in the microbiota, termed dysbiosis may induce aberrant neurophysiological function and behaviour , such asThe incidence of cancer among young adults has risen nearly 40% in recent decades . Early an\u2009=\u2009820) of adolescent cancer with a mean current age of 30.4\u2009years [The incidence of cancer among young adults has dramatically increased in recent decades , with ov.4\u2009years . They fo.4\u2009years . Posttra.4\u2009years . Further.4\u2009years . Indeed,While the detrimental effects of chemotherapy on psychosocial and cognitive function are established, evidence for the underlying physiological mechanisms and system interactions that drive the psychological and cognitive impairments remains limited. The gut-brain axis refers to a bidirectional communication system within an organism that enables gut microbes to communicate with the brain and vice versa via neural, endocrine, immune and metabolic pathways , 16. The, Lactobacillus and E. coli [Chemotherapy has been shown to adversely affect the gut microbiota in both pediatric and adult cancer cohorts. For example, Huang et al.  investig E. coli . Further E. coli  examined E. coli , 19. Sig E. coli . While rThe gut-brain axis exerts regulatory effects on immune function and behaviour. Indeed, previous research has found that under certain conditions the protective epithelial layer in the gut can become compromised leading to increased intestinal permeability and consequently increased systemic circulation of the bacterial endotoxin, lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria , 20. ThiThe potential for gut microbiota to affect the immune system and inflammatory mechanisms is a critical consideration in the context of anxiety and depression, as inflammation has been implicated as a driving factor in depressive illness , 22. BehThe HPA-axis may also play a role in mediating gut-brain axis functions. Previous studies have shown that gut bacteria can activate stress circuits via the vagus nerve , which mChemotherapy has been associated with increased intestinal wall permeability, mucositis, and the onset of a systemic inflammatory immune response, dysregulation of the HPA-axis, and subsequent manifestation of sickness behaviours , 20, 21.Gut microbiota dysbiosis has been implicated in cognitive and psychological deficits. Previous studies demonstrate that alterations in gut microbiota can affect both anxiety and depressive behaviour in animals and humans , 28\u201331. . For instance, Prehn-Kristensen et al. [Bacteroidaceae, and both Neisseriaceae and Neisseria spec. Were identified as potential biomarkers for the ontogeny of juvenile ADHD [Lachnospiraceae species were underrepresented among depressed patients. Additionally, the Oscillibacter genus, of which valeric acid is the main metabolic end product and a homolog of the neurotransmitter GABA, a neurotransmitter implicated in major depressive disorder, was also correlated with depression [While the majority of evidence stems from animal research, some human studies have also shown associations between the gut microbiota, and psychological and cognitive functionn et al.  investigile ADHD . Additioile ADHD  examinedpression .Furthermore, in a triple-blind, placebo-controlled, randomized, pre- and post-intervention study with 40 healthy participants, Steenbergen et al.  investigChemotherapy has been associated with cognitive and psychological impairments , 15. ForFindings regarding associations between gut microbiota and obesity also hold implications related to body composition among young adult cancer survivors who tend to be more overweight and obese relative to healthy peers. For example, recent data from a sample of 49 young adult survivors of adolescent cancer found that over 57% of these survivors were overweight or obese, which is higher than the national average , and coni.Whether chemotherapy is related to long-term attenuation of gut microbiota alpha diversity ;ii.Associations between gut microbiota, body composition , inflammatory cytokine biomarkers , C-reactive protein (CRP), lipopolysaccharide (LPS), serotonin (5-HT), brain derived neurotrophic factor (BDNF), and cortisol;iii.Associations between gut microbiota, and psychological and cognitive outcomes including anxiety, depression, PTSD symptoms, pain, fatigue, and social and cognitive functioning;iv.Relationships between demographic and clinical factors, gut microbiota, and physiological, psychological and cognitive outcomes for survivors.i.Chemotherapy will be associated with significant reductions in gut microbiota alpha diversity at all time points, and relative to healthy controls;ii.Gut dysbiosis will be associated with increased risk for overweight/obesity, elevated pro-inflammatory cytokines, CRP and LPS, reduced serotonin and BDNF, and augmented levels of cortisol in survivors;iii.Gut dysbiosis will be associated with increased anxiety, depression, PTSD symptoms, pain, fatigue, and social and cognitive deficits in survivors;iv.There will be relationships between certain clinical factors , gut microbiota alpha diversity, and physiological, psychological and cognitive outcomes.We hypothesize that:Currently between 18 to 39\u2009years of ageDiagnosed with a blood cancer or solid tumorReceived chemotherapyCurrently within 6\u2009months of final chemotherapy treatmentStages I \u2013 IV including metastatic, IF patient is off chemotherapy treatment and stableEnglish speakingAccess to an electronic device  with in internet connectionCNS or colorectal cancer tumor diagnosisRecipient of an allogenic stem cell transplantWomen who are pregnantHaving taken antibiotics within 1\u2009month prior to fecal collectionDiagnosis of a significant cognitive or developmental delay that precedes cancer diagnosis Currently between the ages of 18 to 39Be a sibling, individual cohabiting with the patient , or close friendEnglish speakingAccess to an electronic device  with in internet connectionHaving taken antibiotics within 3\u2009months prior to fecal collectionPersonal history of cancerCurrent diagnosis/medication treatment for anxiety and/or depressionDiagnosis of irritable bowel syndrome/diseaseWomen who are pregnantDiagnosis of a significant cognitive or developmental condition n\u2009=\u200950 cancer survivors and n\u2009=\u200950 healthy controls were selected based on sample sizes from previous studies on gut microbiota in cancer patients [n\u2009=\u2009100 total), a medium effect size for group differences  can be detected using a one-sided test of the primary hypothesis that cancer survivors will have lower alpha diversity relative to controls [Target sample sizes of at least patients , 18 and controls .Participants will be recruited in Calgary, Alberta from the Tom Baker Cancer Center, via social media and local advocacy groups, and by mail-out through the Alberta Cancer Registry. Patient referral and consent to contact forms will be provided to oncologists. Recruitment posters will be posted at relevant sites, and a digital copy of the poster will be shared on social media platforms.Clinical and demographic factors including current age, age at diagnosis, income, occupation, ethnicity, education, living arrangements, cancer diagnosis and treatments received, history of treatment related mucositis, current diet and exercise regimes, bowel habits, glucocorticoid medication, antibiotic, and probiotic use within the last 2\u2009years, smoking, birth delivery mode , and breastfeeding during infancy will be collected as these variables are known to impact the gut microbiota.Investigators will provide participants with a stool collection kit and instructions for collecting samples. Stool samples will be collected into a sterile conical tube and placed in a biohazard bag that will be stored in the participant\u2019s freezer until the date of their appointment (up to 3\u2009days). Samples will be brought to the University of Calgary on ice and stored at \u2212\u200980\u2009\u00b0C until processed. Microbial profiling will be carried out according to our previously published protocol . BacteriBody composition will be measured using dual-energy-x-ray absorptiometry (DXA) [ry (DXA) . Specifiry (DXA) , 49.Pro-inflammatory cytokines interleukin 6 (IL-6), interleukin 1 beta (IL-1\u00df), and tumour necrosis factor alpha (TNF-\u03b1), anti-inflammatory cytokine interleukin 10 (IL-10), and C-reactive protein (CRP) will be measured in serum using MesoScale multiplexing [iplexing , in consBDNF will be assayed from blood serum using MesoScale multiplexing [iplexing , in consSerotonin will be measured using Serotonin ELISA (enzyme-linked immunosorbent assay) kits from Enzo Life Sciences [Sciences  accordinLipopolysacchide (LPS), a gram-negative bacterial endotoxin associated with intestinal permeability, systemic inflammation, and sickness behaviours, will be measured from serum using MesoScale multiplexing [iplexing .Cortisol will be analyzed using hair samples, which provide a reliable and valid measure of long-term cortisol activity [activity , 53. HaiMillisecond.com. In this version of the task, a practice block (~\u200930\u2009s) is followed by two blocks (~\u20093\u2009min each), totaling approximately 6\u2009min to complete the SART task.Post-Traumatic Stress symptoms will be measured using the Impact of Events Scale. Depression, anxiety, pain behaviour, fatigue, cognitive function, and social isolation will be measured via the NIH Patient-Reported Outcomes Measurement Information System (PROMIS). PROMIS is a set of person-centered measures that evaluates physical, mental, and social health, and is a valid measure of psychosocial outcomes among young adult cancer patients . DetailsOnce identified, participants will be contacted by telephone or email to further verify inclusion and exclusion criteria, to obtain written informed consent, and schedule their first appointment. We will aim to assess participants at 3 different time points: <\u20092\u2009months (T1), 3/4 (T2) and 5/6 (T3) months post chemotherapy, with healthy controls ideally being tested on the same day or within 5\u2009days from when the patient is tested.Participants will then receive a package via mail or in person, with instructions and materials for stool sample collection, and a link to fill out the self-report questionnaires, and demographic and clinical data online using REDCap. REDCap is a free, secure, browser-based application designed to support Electronic Data Capture for research studies provided through the Clinical Research Unit in the Cumming School of Medicine at the University of Calgary. Medical charts will also be accessed to confirm information regarding cancer diagnosis, treatment, glucocorticoid and antibiotic medication use, and mucositis.Participants will be instructed to collect stool samples at home which can be stored in a freezer up to 3\u2009days prior to their appointment, and to complete their online questionnaires prior to the appointment. On the day of their appointment, we will collect their stool sample from them. Participants will then be taken to have a hair sample collected for cortisol analysis (approximately 10\u2009min), complete the cognitive task (i.e. the SART) on a computer (approximately 6\u2009min), and complete their DXA scan (approximately 10\u2009min), and have their blood drawn for serum biomarker analyses (approximately 10\u2009min). This whole process should take approximately 45\u2009min. Participants\u2019 T2 and T3\u2009month follow-up appointments will be scheduled at each subsequent visit and the procedure will be the same with respect to psychological and cognitive measures completed online, followed by their in person testing and biological sample collection. Participants will be provided a voucher for parking, and may choose from compensation in the form of either $30 at the end of the study or a pass to a fitness class after each appointment (they will have options to choose from in advance). If desired, participants will also receive a package with their individual results summarized and interpreted at the end of the study.Objective 1 will examine whether chemotherapy is related to long-term gut dysbiosis as indicated by reduced alpha diversity in patients relative to controls, and from time points 1 through 3. Gut microbiota alpha diversity will be evaluated with the Shannon and Chao1 indexes using QIIME. To further examine differences in bacterial composition between the groups, LEfSe  will be used to evaluate differential abundance between patients and controls, allowing us to identify the key bacteria that contribute most to the chemotherapy group having different bacteria relative to healthy controls. Linear Mixed Modelling (LMM) analyses will be used to examine differences across time and between groups. Group  and time (T1 \u2013 T3) will be fixed factors and individual subject will be a random factor.Objective 2 will first test group differences in gut microbiota, cytokines, LPS, 5-HT and BDNF, cortisol, and body composition using LMM as described above. We will test the interplay between the physiological systems of our model by examining associations between the gut microbiota, cytokines, LPS, 5-HT and BDNF, cortisol, and body composition using univariate correlations. Then multivariate regression models regressing cytokines, cortisol and body composition on gut microbiota diversity will be conducted, including variables that showed significant univariate correlations in the modelling. Covariates included theoretically based on the literature will include type of cancer diagnosis (i.e. blood or solid tumor), history of mucositis, glucocorticoid medication, antibiotic, and probiotic use, birth delivery mode, and breastfeeding in infancy.Objective 3 will first test group differences in cognitive and psychological parameters, gut microbiota diversity, and levels of 5-HT and BDNF using LMM as described above. We will test the relationships between gut microbiota, 5-HT and BDNF, and cognitive and psychological parameters using univariate correlations. Then multivariate regression models regressing psychological and cognitive parameters on gut microbiota diversity will be conducted, including variables that showed significant univariate correlations in the modelling. Covariates included theoretically based on the literature will as specified in objective 2.Objective 4 will explore relationships between clinical factors and all dependent variables, first using univariate correlations, followed by multivariate regression modelling to examine associations between cancer survivors\u2019 clinical and demographic factors, and the dependent variables of gut microbiota alpha diversity and differential species composition, physiological parameters , body composition, and psychological and cognitive outcomes.Using the Microbiota-Gut-Brain Axis model and the biopsychosocial framework, this research aims to elucidate the long-term effects of chemotherapy on gut microbiota, and psychological, cognitive, metabolic, and immune outcomes in young adult cancer survivors. Moreover, this study will allow us to systematically explore and validate aspects the novel model proposed here; the Chemotherapy Driven Dysbiosis of the Microbiota-Gut-Brain Axis Model.The design of this study is not without its limitations and we have taken steps to reduce potential confounds and bias. Although the sample size of this study is relatively small, it is comparable to other studies examining the effects of chemotherapy on gut microbiota, and taking into account feasibility, this sample size will produce adequate statistical power. Furthermore, while most studies typically require participants to have not taken antibiotics for at least 3\u2009months prior to fecal collection, we have set the required time at 1\u2009month. This is to account for the health status of the clinical population, tight timing between follow-up appointments, and to reduce potential attrition. Although colorectal cancers do frequently occur in young adults, we have chosen to exclude this specific diagnosis type from our sample as previous research suggests that specific changes in gut microbiota may be involved in the onset and/or progression of colorectal cancer , 57, andKnowing what bacterial species are depleted after chemotherapy, how long these effects last, and the physiological mechanisms that may drive psychological and cognitive issues among survivors is a crucial step forward in gut microbiota and young adult cancer research. Understanding the bio-behavioural mechanisms that drive psychological and cognitive dysfunction among survivors will allow for tailored interventions to be developed. Future studies can aim to aid cancer patients and survivors by co-administering specific health promoting bacteria (i.e. probiotics), with the potential of preventing or reversing physical and mental health issues that many young survivors face."}
+{"text": "Dengue presents with a variable clinical course, ranging from mild illness to potentially fatal hemorrhage and shock. We aimed to evaluate the capabilities of various hematological parameters observed early in the course of illness for predicting the clinical outcomes of illness.  We retrospectively analyzed the records of children admitted in the pediatric inpatient services of the institute with dengue between 2017 and 2019. We determined the relationships between the hematological parameters observed during the first evaluation and the various clinical outcomes.  We evaluated data from 613 patients . Of these, 29.85% exhibited fever with warning signs, and 8.97% had severe dengue. Lower values of hemoglobin, platelet count, mean corpuscular volume, mean corpuscular hemoglobin concentration, and mean platelet volume, and higher values of total leukocyte count (TLC), hematocrit, and red cell distribution width variably correlated with numerous clinical outcomes-duration of hospital stay, development of complications, requirement of blood component transfusion, inotropic support, and mortality. Among the parameters, TLC \u226520,000/mL and initial platelet count \u226420,000/mL significantly associated with mortality, with odds ratios  of 11.81 (4.21-33.80) and 5.53 (1.90-16.09), respectively.  Hematological parameters observed early during dengue infection may predict its clinical outcomes in infected children. Initial high TLC and low platelet count are potential predictors of fatal outcomes in the course of disease. For the last three decades, dengue has continued to pose a major public health problem worldwide. Globally, there are approximately 100 million infections reported each year with up to 2% resulting in a fatal outcomeDengue targets people from a wide range of sociodemographic characteristics and contributes to considerable morbidity and mortality in the pediatric populationAs hematological derangements are among the most frequently observed manifestations in severe dengue infection, we theorized that detailed exploration of hematological parameters, including red cell indices and platelet size, observed early in the course of illness might predict various clinical outcomes. Being commonly available and routinely performed as part of investigations, these predictive factors could assist clinicians in resource-limited settings to identify children with anticipated severe illness. This study was conducted at a tertiary care pediatric teaching hospital in northern India. We retrospectively analyzed the records of children with laboratory-confirmed (NS1 antigen or anti-dengue IgM antibody) dengue infection, admitted between 2017 and 2019. The study protocol was approved by the institutional ethics committee (No 2019-01-IM-01). We recorded various parameters-(1) demographic: age, sex; (2) clinical: weight for age, presenting symptoms, signs observed during first examination, duration of illness at first blood sampling, and duration of illness at admission; and (3) hematological parameters observed in the first blood sample: hemoglobin level (Hb), total leucocyte count (TLC), hematocrit (Hct), platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW-CV), and mean platelet volume (MPV). The minimum platelet count during the illness was also documented. The weight for age was recorded as \u2018z\u2019 scores according to the Indian Academy of Pediatrics anthropometric data. For the hematological parameters, we used reports generated by the 5-part automated analyzer, CELL-DYN Ruby System . We extracted various clinical outcomes, including duration of hospital stay, requirement of packed red blood cell (PRBC) or platelet transfusions, need for inotropic support, occurrence of complications, and mortality, from patient records. .The observations were entered into Microsoft Excel , and statistical analysis was performed using the SPSS Statistics software . For descriptive statistics, continuous measures were presented as means and standard deviations (SD), while categorical variables were presented as absolute and relative frequencies. Parameters were compared using \u2018z\u2019 statistics. Correlations among various parameters were explored using Pearson\u2019s coefficient of correlation. Multivariate analysis was performed for various hematological parameters to calculate odd ratios ) for risk of various dichotomous clinical outcomes. The distribution of relevant parameters was graphically depicted using boxplots. For parameters significantly associated with mortality, logistic regression and receiver operating characteristic (ROC) curve analyses were performed to identify their cut-off levels for predicting mortality. Taking reference from the observations of Malhi et al.A total of 1195 samples were reported to be positive for dengue (NS1 antigen or anti-dengue IgM antibody) during the 3-year study period. Details from 613 admitted patients were available for analysis following retrieval from the record repository. At the initial evaluation, 28 (4.56%) patients had Hb level \u22648 g/dL. The TLC was \u226510,000/mL and \u226520,000/mL in 122 (19.90%) and 25 (4.07%) patients, respectively. Most of the patients had leucopenia. The initial platelet count was \u226420,000/mL among 37 (6.03%) patients, while only 3 (0.5%) children had platelet count \u226410,000/mL. During the course of illness, 97 (15.82%) children had a minimum platelet count falling below 20,000/mL. In the first sample, 120 (19.57%) children showed hemoconcentration with Hct \u226545% and 116 (18.9%) had MPV \u22649 fL. Overall, the group showed low MCV and MCHC and high RDW-CV. The relationship between sex and mortality was not significant. We observed a statistically significant correlation between occurrences of any type of complication and sex. There was no significant difference in mean weight for age among the two sexes. It was not associated with any other clinical outcome. Bivariate correlation analyses revealed no significant correlation between age or weight for age and any of the clinical outcomes. History of bleeding from any site and presence of jaundice on examination significantly correlated with duration of hospital stay, requirement of platelet transfusions, need for inotropic support, and mortality. The presence of jaundice also significantly correlated with the requirement of PRBC transfusion during the clinical course. Palpable liver or spleen significantly correlated with the development of complications in children. Among the hematological parameters, the requirement of PRBC transfusion correlated with all studied parameters except minimum platelet count and MCV. While initial platelet count showed a significant inverse correlation with most outcomes, the minimum platelet count during hospital stay failed to associate with any outcome. Higher TLC significantly correlated with unfavorable results for all clinical outcomes. The MPV showed a significant inverse relationship with all outcomes except the duration of hospital stay. Both mortality and requirement of inotropic support significantly correlated with TLC, initial platelet count, and MPV. Among the survivors and non-survivors, the  TLC , initial platelet count , and MPV  were significantly different (p<0.05), and the distribution is presented in 2 \u221249.62, p<0.05), explained 30% (Nagelkerke R2) variance in the risk of mortality, and correctly classified 96% of the cases. Higher TLC along with lower platelet count, Hb level, and Hct level were associated with higher likelihood of mortality.The retrospective analysis of 613 patients admitted with dengue infection showed variable derangements in clinical and laboratory parameters. The female sex experienced a significantly higher number of complications. History of bleeding from any site and presence of jaundice or palpable liver or spleen was associated with various poor outcomes. The lower values of Hb, platelet count, MCV, MCHC, and MPV and higher values of TLC, Hct, and RDW-CV correlated with various clinical outcomes, such as duration of hospital stay, development of complications, requirement of blood component transfusion, inotropic support, and mortality. Among the observed hematological parameters, higher TLC at presentation emerged as the most important predictor for mortality. As our institute caters to children from a wide spectrum of socioeconomic backgrounds, our study population reasonably reflects the general pediatric population of northern India. The methods used in our analyses are routinely available, even at medical facilities located in resource-limited settings. Hence, our results can be easily extrapolated for any establishment providing care to sick children. ,,The classical hematological derangements described in dengue infection are thrombocytopenia, leucopenia, and hemoconcentrationOur study fairly evaluates the proposed hypothesis and provides some simple markers for projecting clinical outcomes for children admitted with dengue infection. Although the retrospective nature of data collection is a limitation, our results provide information that may help clinicians, especially in resource-poor settings, in their decision-making process and attract researchers for further exploration of this issue.Hematological parameters observed early in the course of dengue infection may predict its clinical outcomes in infected children. Initial high TLC, low initial platelet count, and possibly low MPV are potential predictors of shock and fatal outcome in the course of disease."}
+{"text": "In the twentieth century, a conspicuous lack of effective treatment strategies existed for managing several retinal disorders, including age-related macular degeneration; diabetic retinopathy (DR); retinopathy of prematurity (ROP); retinitis pigmentosa (RP); uveitis, including Beh\u00e7et's disease; and vitreoretinal lymphoma (VRL). However, in the first decade of this century, advances in biomedicine have provided new treatment strategies in the field of ophthalmology, particularly biologics that target vascular endothelial growth factor or tumor necrosis factor (TNF)-\u03b1. Furthermore, clinical trials on gene therapy specifically for patients with autosomal recessive or X-linked RP have commenced. The overall survival rates of patients with VRL have improved, owing to earlier diagnoses and better treatment strategies. However, some unresolved problems remain such as primary or secondary non-response to biologics or chemotherapy, and the lack of adequate strategies for treating most RP patients. In this review, we provide an overview of the immunological mechanisms of the eye under normal conditions and in several retinal disorders, including uveitis, DR, ROP, RP, and VRL. In addition, we discuss recent studies that describe the inflammatory responses that occur during the course of these retinal disorders to provide new insights into their diagnosis and treatment. In the last 2 decades, advances in the interdisciplinary collaboration of the fields of molecular biology, biochemistry, genetics, and biomedicine have resulted in tremendous breakthroughs in the treatment of refractory ocular disorders. Infliximab (IFX), a chimeric antibody of the tumor necrosis factor (TNF)-\u03b1, is a biologics that is used for treating ocular symptoms of Beh\u00e7et's disease that have not been adequately controlled . Anti-vaThe eye, just like the brain and the testes, is an immune-privilege site . Ocular Immunological responses to various environmental stimuli have been associated with the pathogenesis of uveitis and retinal vascular diseases such as diabetic retinopathy (DR) and ROP \u201313. RetiIn this review, we focus on the roles of immunological responses in a normal conditions and in several major retinal disorders including non-infectious uveitis (NIU), DR, ROP, RP, and VRL. In addition, we contemplate new approaches for the diagnosis and treatment of these intractable retinal disorders from an immunological point of view.A properly elicited and regulated immune response by the human body is necessary for eliminating threats due to infectious microbes and tissue trauma to avoid irreversible tissue damage . Acute iOcular immune privilege is believed to elicit self-limiting immune responses . Several+ dendritic cells (DCs) also transport antigens to the thymus  , 25. ACAe thymus . APCs bee thymus .+ hyalocytes are distributed over the retinal surface  is a sight-threatening disorder associated with systemic autoimmune diseases such as Behcet's disease, sarcoidosis, and Vogt\u2013Koyanagi\u2013Harada disease . NIU is Experimental autoimmune uveitis (EAU) is an animal disease model of a T cell\u2013mediated autoimmune disease that mimics many of the pathological features of human uveitis , 46. EAU+ cells, and CD11b+ cells. This effect was specific to microglia, given that it was not reproduced by other immune cell types such as monocytes-macrophages. Therefore, immunomodulatory therapies targeted at microglia are a primary focus in the development of new treatments for patients with uveitis.The important role of microglia in the regulation of inflammatory cell infiltration into the retina associated with the initiation of retinal autoimmune uveitis has recently been demonstrated . During We previously explained that NKT cells suppress the induction of Th17 cells and the ocular infiltration of hIRBP-specific T cells in EAU. However, in contrast to the ameliorating effects of NKT cell activation that is apparent during the initiation phase of EAU, the activation during the effector phase exacerbates disease pathology. This finding suggests that NKT cells have a dual role in EAU, depending on the phase of the disease .+CCR7+ NK cells induced by interleukin (IL)-18 released from DCs promote EAU (CD83mote EAU . NK cellmote EAU . In addimote EAU .+CD25+CD4+ Treg cells require programmed cell death 1 (PD-1) stimulation through a melanocortin-adenosine pathway to suppress EAU. These Treg cells did not induce suppressor activity in APCs through the PD-1 pathway  is a key chemotactic signal for the recruitment of innate immune cells to sites of brain injury . ATP is + Treg cells and inhibiting the activation of naive T cells induced by splenic DCs and antigens  in EAU . Histolo T cells . Cytokin T cells , 79. We n of DCs . MoreoveThe global prevalence of diabetes mellitus (DM) tends to increase yearly, and the number of DM patients is estimated to reach 592 million within 20 years . DiabetiPDR is characterized by the development of preretinal neovascularization and epiretinal fibrovascular membranes (FVMs) . ProlongIn a previous study with an experimental animal model, we previously reported that intravitreal injection of an anti-VEGF agent can attenuate the infiltration of leukocytes, especially macrophages, into the retina and can suppress preretinal neovascularization . Esser eBlood vessels in the central nervous system (CNS), including the retina have a vascular barrier function and maintain a proper neural microenvironment through strict control of vascular permeability . RetinalVEGF is the most studied molecule that can increase vascular permeability . RegularTo identify a new target molecule or a novel biomarker for predicting anti-VEGF resistance, many researchers have examined the relationship between the response to anti-VEGF treatment and the concentration of intraocular inflammatory cytokines/chemokines. Hillier et al. showed that an increase in baseline aqueous ICAM-1 is associated with a favorable anatomic response, whereas an increase in baseline aqueous VEGF is associated with an unfavorable anatomic response . ShimuraThe results of animal experiments have revealed that VEGF induces endothelial ICAM-1 expression in the early stage of DR , and thaRetinopathy of prematurity (ROP) is a retinal vasoproliferative disorder that can lead to childhood blindness . BlencowCurrent primary treatments for ROP are retinal photocoagulation and anti-VEGF therapy. Large clinical trials such as the Early Treatment for Retinopathy Of Prematurity study and the Bevacizumab Eliminates the Angiogenic Threat of Retinopahty of Prematurity (BEAT-ROP) study have proven that both methods are effective , 110. HoThe recurrence rate of ROP after IVR monotherapy is ~30% . The proRetinitis pigmentosa refers to a subgroup of inherited retinal degenerations (IRDs) that cause progressive rod-cone degeneration . More thin vivo is still challenging, and (3) many patients are first diagnosed in the mid- to late-stages of the disease when the rod cells are mostly lost. Therefore, the elucidation of the biological mechanisms that underlie retinal degeneration, especially in the secondary cone cell death phase, will be critical in developing novel treatments for RP, in addition to individualized gene therapy.Recent advances in gene therapy have shed light on the treatment of IRDs. Supplementation of the retinal pigment epithelium-specific 65 kDa protein (RPE65) gene in patients with Leber congenital amaurosis due to RPE65 mutations improves their light sensitivity and performance in the multi-luminance mobility test . This thIn RP, rods are expected to be injured because of gene mutations that are exclusively expressed or critically function in rod cells. However, why and how cones also die subsequent to rod cell death is puzzling. Accumulating evidence suggest that microenvironmental changes associated with rod cell death such as loss of trophic factors , metabolCell death and inflammation have a tight interaction with each other. Dying or dead cells stimulate phagocytes to mediate their clearance and maintain tissue homeostasis, whereas excessive activation of inflammatory cells can exert cytotoxicity and exacerbate the disease .vivo in slit-lamp biomicroscopy, and found that RP patients with a higher number of inflammatory cells had worse visual acuity and lower central retinal sensitivity  are increased in the vitreous with more significant fold changes . Lu et aThe inflammatory cytokines/chemokines elevated in RP are related to innate and acquired immunity. IL-1\u03b2, IL-8, and MCP-1 are pivotal molecules for activating and recruiting monocytes/macrophages and neutrophils to the inflammatory loci. IFN-\u03b3, IL-2, IL-4, and IL-10 are produced primarily or partly by T lymphocytes, and they mediate the differentiation and polarization of Th cells and macrophages. These profiles of inflammatory cytokines/chemokines in RP are consistent with the infiltration of a variety of inflammatory cells into the vitreous, as described above . To deveHigh sensitivity CRP (hs-CRP) is a serum inflammatory marker, and an increased hs-CRP is associated with age-related macular degeneration, DR, and uveitis , 129. ThThe findings outlined previously suggest that innate and acquired immunity are activated and involved in the pathology of RP. However, the function of each inflammatory cells  and its regulatory mechanisms remains unclear and is a topic of interest.Microglia, a resident macrophage in the CNS that derived from the embryonic yolk sac progenitors, are the most prominent immune cells in the retina . MicroglSeveral reports have demonstrated the detrimental function of microglia/macrophages in retinal degeneration in experimental RP. In a previous study, suppression of activated microglia/macrophages with minocycline or toxin-induced depletion of CX3CR1-positive microglia/macrophages protected rod cells against cell death in retinal degeneration (rd) 10 mice , 136. HoOxidative stress significantly contributes to cone cell death in RP. Campochiaro et al. postulated that rod cell loss in RP substantially reduces oxygen consumption in the retina, and the remaining cone cells are exposed to a high-level of oxygen and resultant ROS . They shOxidative stress may have a direct harmful effect on cone cells, but it also affects microglial/macrophage activation in RP. In a previous study, we showed that treatment with anti-oxidant N-acetylcystein (NAC) substantially suppresses microglia/macrophage activation with reduced MCP-1, IL-1\u03b2, RANTES, and TNF-\u03b1 expression . The antLymphocytes and lymphocyte-related cytokines are increased in the eyes of RP patients. In addition, several studies suggest the possible involvement of an autoimmune response and antiretinal autoantibodies in the progression of RP . HoweverScid or Rag1\u2212/\u2212 mice, both of which lack functional T and B lymphocytes, and showed that the deficiency of lymphocytes did not change rod cell death , which were previously termed as intraocular lymphomas, are related to high-grade non-Hodgkin's lymphoma, which is a subset of primary central nervous system lymphomas (PCNSL) . In JapaThe clinical ocular features of VRL, termed \u201cmasquerade syndrome,\u201d are often similar to those of chronic uveitis; therefore, a misdiagnosis of VRL sometimes leads to the administration of anti-inflammatory agents such as corticosteroids and thereby cause a delay in reaching a definitive diagnosis. The interval between the onset of the ocular or neurological findings and a definitive diagnosis is variable, and ranges from 4 to 40 months . The invThe overall survival of VRL patients has improved over the decades because of earlier diagnosis of the disease, which is a result of advances in molecular biological or genetic techniques. In addition, intense systemic chemotherapy and/or radiotherapy have also increased the overall survival rate. Several prospective studies have recently demonstrated that high-dose methotrexate (HD-MTX)-based chemotherapy with intravitreral MTX injections, subsequent whole brain radiotherapy (WBRT), and/or consolidation chemotherapy could improve overall survival and prevent CNS progression \u20137. KaburMost cases of PVRL can be classified as diffuse large B-cell lymphoma (DLBCL), whereas very few cases are classified as a T cell lymphoma or NK cell origin PVRL . Based oMYD88 L265P), is the most common mutation and accounts for more than 60% of VRLs  and CD79B mutations have been characterized in PCNSL . A singl of VRLs . MYD88 i of VRLs . The B-cFurthermore, in cases of PCNSL, because of the HLA locus mutation (chromosome 6q21.32), a shortage in HLA class I and II expression on tumor cells leads to escape from T or NK cell-mediated immune surveillance against tumor cells , which sPD-1, which is expressed on activated T-cells such as cytotoxic T lymphocytes (CTLs), interacts with its ligands (PD-L1 and PD-L2). These ligands are commonly expressed on tumor cells and upregulated in the tumor microenvironment (TME), thereby promoting inhibitory signaling of T cell receptors (TCRs) in CTL and subsequent tumor growth , 164. InCytological examination of the intraocular fluid or tissue is the gold standard for a definitive diagnosis of VRL. However, cytology alone can have a low diagnostic yield (40\u201360%) because of the limited amount of specimen that can be obtained, necrosis, and the fragility of VRL cells , 166. ThSeveral supplementary diagnostic methods can improve the definitive diagnosis of VRL. They include cytokine analysis to determine the ratio of IL-10 to IL-6  , moleculMYD88 L265P can be screened with new genetic techniques, including allele-specific polymerase chain reaction and next generation sequencing (NGS) using an oncogene gene panels, which allows for lower cellularity or a smaller volume of samples to confirm the definitive diagnosis of VRL  is associated with PCNSL, specifically in immunocompromised patients such as individuals with acquired immune deficiency syndrome (AIDS) . EBV, whith PVRL .On account that PVRL has selective tropism to CNS lesions, a theory has been proposed that chemokines and their receptors encourage the attraction and maintenance of VRL cells in intraocular tissues. In patients with B-cell chemokines, CXCL12, and CXCL13 are specifically expressed in retinal pigment epithelial cells and/or in the vitreous cavity of patients with VRL. As a consequence, B-lymphoma cells [which express CXCR4 and CXCR5 ] are recruited into intraocular tissues such as the retina, vitreous body, and subretina . CXCL13 VRL cells can evade attacks by CTLs and NK cells because the eye is an immune-privileged site that possesses an immunosuppressive ocular microenvironment composed of soluble and cell surface inhibitory molecules. TGF-\u03b2 is abundant in the vitreous humor to maintain an anti-inflammatory state in the eye . IL-10, Based on the outcome of the genetic studies described previously, several new agents are currently being investigated as a salvage therapy in clinical trials assessing in patients with refractory or relapsed PCNSL and PVRL. In a prospective French multicenter phase II trial, monotherapy with ibrutinib, which targets Bruton's tyrosine kinase downstream of BCR, was effective and had objective response rates (ORRs) of up to 70% . Among 1Furthermore, increased expression of proinflammatory cytokines related to CTLs, such as IFN-\u03b3, granzyme A, and IP-10 occurs in the aqueous humor and/or vitreous of VRL patients , which sA profound understanding of the intricacies of immune responses will raise innovations for the management and treatment of these intractable retinal disorders. The generation of biologics, including IFX or ADA, has dramatically changed the treatment of NIU in the past few decades. However, during long-term treatment of NIU patients, a decreased response or adverse events to the biologics has emerged because of the development of antidrug antibodies or paradoxical effects. The development of selective small-molecule therapies is expected to resolve these problems. From the results of our analysis of EAU, the induction of regulatory DCs may be useful for the treatment of NIU.In retinal vascular diseases, low-grade inflammation can destroy vascular integrity by the action of VEGF and some cytokines/chemokines from infiltrated leukocytes. Resistance to anti-VEGF therapy is sometimes observed in DR (including DME); therefore, developing a new therapy associated with low-grade inflammation as a \u201cbeyond VEGF\u201d therapy for retinal vascular diseases, including DR and ROP, may be useful.Immunological responses also affect the pathogenesis of RP, despite differences in genetic backgrounds. Targeting cytokines/chemokines associated with immunological responses against RP may be an attractive target for the treatment of RP, in addition to gene therapy.The recent introduction of molecular profiling technologies, including NGS, can exhibit the molecular characterization of several cancers to provide information on tumor diagnosis and specific targeted therapy. Several agents, which were selected on the basis of the molecular characterization, have been assessed in clinical trials of cases of refractory/relapsed PCNSL; however, the utility of these molecular profiling technologies has not been established. Considering the rarity of VRL, large-scale collaborative registries, tumor molecular profiling programs, and clinical trials in institutions across the world are necessary to enhance diagnosis, prognostication, and treatment outcomes in the future.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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."}
+{"text": "Chronic obstructive pulmonary disease (COPD) is characterised by progressive airflow limitation and chronic inflammation. Predicting exacerbations of COPD, which contribute to disease progression, is important to guide preventative treatment and improve outcomes. Blood eosinophils are a biomarker for patient responsiveness to inhaled corticosteroids (ICS); however, their effectiveness as a predictive biomarker for COPD exacerbations is unclear.This post hoc analysis pooled data from 11 Boehringer Ingelheim-sponsored Phase III and IV randomised COPD studies with similar methodologies. Exacerbation data were collected from these studies, excluding patients from the ICS withdrawal arm of the WISDOM\u00ae study. Patients were grouped according to their baseline blood eosinophil count, baseline ICS use and number of exacerbations in the year prior to each study.Exacerbation rate data and baseline eosinophil count were available for 22,125 patients; 45.6% presented with a baseline blood eosinophil count of \u2264\u2009150 cells/\u03bcL, 34.3% with 150\u2013300 cells/\u03bcL and 20.1% with >\u2009300 cells/\u03bcL. The lowest exacerbation rates were observed in patients with \u2264\u2009150 cells/\u03bcL, with small increases in exacerbation rate observed with increasing eosinophil count. When stratified by exacerbation history, the annual rate of exacerbations for patients with 0 exacerbations in the previous year increased in line with increasing eosinophil counts . A similar trend was identified for patients with one exacerbation in the previous year, 0.62, 0.66 and 0.67 respectively. For patients with \u2265\u20092 exacerbations, exacerbation rates fluctuated between 1.02 (\u2264\u2009150 cells/\u03bcL) to 1.10 (150\u2013300 cells/\u03bcL) and 1.07 (>\u2009300 cells/\u03bcL). Higher exacerbation rates were noted in patients treated with ICS at baseline (range 0.75 to 0.82 with increasing eosinophil count) compared with patients not on ICS (range 0.45 to 0.49).We found no clinically important relationship between baseline blood eosinophil count and exacerbation rate. Hence, the current analysis does not support the use of blood eosinophils to predict exacerbation risk; however, previous exacerbation history was found to be a more reliable predictor of future exacerbations.ClinicalTrials.gov Identifiers: NCT00168844, NCT00168831, NCT00387088, NCT00782210, NCT00782509, NCT00793624, NCT00796653, NCT01431274, NCT01431287, NCT02296138 and NCT00975195. Doctors can prescribe these treatments together (long-acting muscarinic antagonist/long-acting \u03b22-agonist) if needed. Other medicines called inhaled corticosteroids can be added as required. Measuring the number of eosinophils, a type of white blood cell, in the blood can help to predict which people will benefit most from inhaled corticosteroid treatment.People with chronic obstructive pulmonary disease often start treatment with either a long-acting muscarinic antagonist or a long-acting \u03b2People with chronic obstructive pulmonary disease sometimes experience a worsening of their symptoms, known as an exacerbation. Eosinophil levels may be useful to predict the risk of exacerbations. We studied the results from 11 clinical trials, involving 22,125 people with chronic obstructive pulmonary disease. We looked at how many exacerbations people had during these trials and whether this was linked to the level of eosinophils in their blood, their previous history of exacerbations, or whether they had been treated with inhaled corticosteroids before.Overall, we found that a previous history of exacerbations predicted the future rate of exacerbations. We did not find a clear link between the rate of exacerbations and eosinophil levels.Characterised by progressive airflow limitation and chronic inflammation, chronic obstructive pulmonary disease (COPD) is a major source of morbidity and mortality that mainly affects individuals above the age of 35\u2009years , 2. COPD1) and persistent cough have been shown to be predictive of higher exacerbation rates  or \u2018frequent exacerbators\u2019 [\u2265\u20092 moderate exacerbations or\u2009\u2265\u00a01\u2009severe exacerbation in the previous year]), and ICS use at baseline or according to both ICS use at baseline and exacerbation history. COPD exacerbations were counted from the start to the end of treatment. Patients were followed for the duration of each clinical trial (a minimum of 48\u2009weeks).Annual rates of exacerbations were assessed using negative binomial models with treatment exposure as offset, adjusted for treatment, study, ICS use at baseline, region, GOLD stage, smoking status, baseline eosinophil count, and number of exacerbations in previous year as covariates.From the pooled population, baseline eosinophil data were collected from 22,125 patients who also had exacerbation rate data . When compared with patients with a baseline eosinophil count of \u2264\u2009150 cells/\u03bcL, patients in the >\u2009150\u2013\u2264\u2009300 cells/\u03bcL and\u2009>\u2009300 cells/\u03bcL subgroups had exacerbation rate ratios (RRs) of 1.05  and 1.09 , respectively  compared with non-ICS users (range 0.45 to 0.49)  and 1.09 , respectively. For non-ICS patients, when compared with the \u2264\u2009150 cells/\u03bcL group, patients in the >\u2009150\u2013\u2264\u2009300 cells/\u03bcL and\u2009>\u2009300 cells/\u03bcL subgroups had exacerbation RRs of 1.07  and 1.09 , respectively  [Some cohort studies have also shown an association between blood eosinophil counts and exacerbation risk. Vedel-Krogh et al. found that individuals with a blood eosinophil count above 340 cells/\u03bcL had a higher risk of severe exacerbations compared with patients below this value  . In addi\u00a0=\u20090.93) . Similar\u00a0=\u20090.93) .As eosinophil levels are known to vary over time, this may lead to patients fluctuating between different eosinophil categories, despite the patient experiencing only a small absolute change , 37. ThiWhen assessing eosinophils in categories, the current study did not show a strong relationship between blood eosinophil counts and risk of future exacerbations. Although there is a paucity of data evaluating blood eosinophil count as a continuous value, Bafadhel et al.  modelledOther candidate biomarkers have been studied as predictors of increased risk of COPD exacerbations , 9\u201311, iIn RCTs, blood eosinophil counts >\u2009300 cells/\u03bcL are associated with the greatest effect of ICS on exacerbation prevention. In this analysis, 20.1% had eosinophil counts above this threshold. These results are in line with other studies that suggest that around 25% of patients have eosinophil counts higher than 300 cells/\u03bcL , 19, 39.Our analysis has some advantages. Since the pooled studies were all conducted by one pharmaceutical company (Boehringer Ingelheim), the methodologies between these studies are generally compatible. Due to the pooled population, this analysis has a very large sample size and is sufficiently powered to detect even modest changes in exacerbation frequency. However, similar to observational studies, post hoc analyses should be treated with caution as these may be subject to bias. Secondly, the inclusion and exclusion criteria between the studies are mostly generalisable; however, additional factors such as comorbidities may be present in some of the pooled patient population, which were not controlled for and may introduce variability. Thirdly, the pooled studies included a number of active-controlled, or placebo-controlled studies. In COPD, active-controlled studies compare the drug of interest against a leading comparator in the field. Although more ethical, the results of the active-controlled trials could impact on the overall exacerbation rates. It is important to note that this pooled analysis was not designed to assess a relationship between blood eosinophil count and response to ICS use. The scope of this study was to determine the use of eosinophils as a predictor of exacerbations.What are the practical implications of our study? For many of the currently studied biomarkers of exacerbation risk, the data are conflicting . Whilst In this pooled analysis of 22,125 patients with COPD, we did not find a clinically important relationship between baseline blood eosinophil count and exacerbation rate. This analysis, coupled with other studies on this topic , 19, ind"}
+{"text": "Previous studies have shown that forgiveness is associated with the ability of self-control. However, whether self-control can modulate interpersonal forgiveness remains unclear. In the current study, we aimed to explore the relationship between self-control and the process of forgiveness using a behavioral measure of forgiveness during which participants distributed money between themselves and unknown others who had previously treated them fairly or unfairly in an adapted decision-making task. Seventy-two participants with low or high self-control were recruited based on their scores on the self-control scale (SCS). Results showed that participants exhibited increased anger and decreased happiness after experiencing unfair treatment. Participants with high self-control distributed more money to opponents who previously treated them unfairly compared with those with low self-control, whereas no such difference was observed to opponents who previously treated them fairly between the two groups. A significantly positive correlation was also found between the forgiveness rates and participants\u2019 self-control scores. These findings suggest that self-control modulates interpersonal forgiveness responses. Individuals with high self-control expressed an increased prosocial response toward people who previously offended them, which is similar to the process of forgiveness. Experiencing conflict, offense, or unfairness is inevitable in a social situation; forgiveness is regarded as a good approach to reduce these threats and increase harmony in society . PsycholAfter being offended, the dominant response toward a perpetrator is anger, hostility, or revenge rather than forgiveness . When chPrevious studies have also explored the relationship between self-control and forgiveness. Research has indicated a positive correlation between self-control and forgiveness . Finkel In the present study, we aim to explore whether self-control can modulate interpersonal forgiveness using a behavioral measure of forgiveness. This study is the first to explore the relationship between self-control and interpersonal forgiveness using the behavioral measurement paradigm combined with adapted \u201cultimatum game\u201d (UG) and \u201cdictator game\u201d (DG) to measure forgiveness. The behavioral measurement paradigm can better conceal the purpose of the experiment, obtain more real responses from participants, and avoid the limitation of self-report scales as a social expectation effect . In addiSD = 1.03), whereas the low self-control group consisted of 36 students . Chi-square tests of gender and self-control group showed that no significant interaction existed between the self-control group and gender (x2(1) = 1.71, p = 0.19). In comparison with the low self-control group, the high self-control group reported significantly higher level of self-control . This study was conducted in accordance with the recommendations of the SHNU Ethics Review Board. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the SHNU Ethics Review Board.Two-hundred seventy participants were selected from a pool of undergraduate students at a university in China based on their scores on the 13-item brief self-control scale SCS; . Scores Before the experiment was conducted, the subjects were told that they would play a money allocation game online with other opponents, and their performance in the task would influence their final payment. The behavior experiment included two stages . In the The specific experimental process of UG was as follows. First, participants saw the name and picture of their opponents (viewing period for 3000 ms), and this was followed by a jittered 800\u20131200 ms of anticipation period (random fixation \u201c+\u201d appears on the screen). Then, the opponent\u2019s proposal was presented for 3000 ms in the decision-making period. During this period, the subjects had the option to accept or reject by pressing the \u201cj\u201d button for rejection, which would result in both sides receiving nothing, or the \u201cf\u201d button for acceptance, which would result in 10 yuan beeing split according to the offers. Finally, in the feedback phase for 1500 ms, the amount of money each side received appeared on the screen.In the second stage, the subjects\u2019 roles were reversed to be dictators , and participants were asked to distribute 10 RMB with four previous opponents (two fair opponents and two unfair opponents) in the DG each trial. The receiver had no right to refuse and could only passively accept the dictator\u2019s proposal unlike in UG . If partThe experimental process of DG is similar to UG. After the viewing period of 3000 ms and the anticipated period of jittered 800\u20131200 ms, the subjects were asked to decide how much money (0\u201310 RMB) to allocate to their previous fair or unfair opponents. Finally, the participants could see how much money they and their opponents had received in the feedback phase of 1500 ms. The experiment comprised 144 trials, 72 in UG, and 72 in DG. During the experiment, the interval between each trial was jittered 2000\u20133000 ms. All measures, conditions, and data exclusions were been reported. Details on each experimental condition can be found in the First, 270 undergraduates were tested using the brief SCS. Subsequently, participants who met the requirements were invited to the laboratory for experiments according to their score (score in the highest 27% and the lowest 27%). Prior to the experiment, the subjects filled in the informed consent form and demographic information. Moreover, the subjects\u2019 basic emotional states  were measured using a five-point scale. Then, the subjects completed the adapted UG and re-evaluated their emotional states. Finally, participants completed the adapted DG as well as the measurement of the basic emotional states. Then, the subjects were asked how they felt about the experiment, some expressed doubts about the manipulation of the experiment and did not trust the cover story that they were playing with real opponents . Thereafter, we explained the purpose and method of the experiment to the subjects, including the purpose of deceiving the real opponents of the subjects to obtain objective and real experimental results. Finally, the participants expressed their understanding and accepted payment of 25\u201330 yuan.p < 0.01, d = 0.49) and less happy  compared with the baseline state. In comparison with the baseline state, participants felt less fear  and less happy  after Phase 2. No other significant differences were found in other emotional states at different time points compared with other time points.We conducted a repeated-measures ANOVA at three time points  for each emotional state to explore the changes of emotional states before and after each stage. The results showed that, after Phase 1, subjects felt more anger  and unfair proposals . However, participants in each group accepted more fair proposals than unfair proposals .Low self-control subjects accepted 75.84% fair proposals and 17.14% unfair proposals , whereasF = 49.484, p < 0.001, \u03b72 = 0.414) and a significant interaction effect of allocation proposals \u00d7 self-control groups  and opponents \u00d7 allocation proposals . A simple effect analysis showed that subjects in the high self-control group made more fair distributions  and fewer unfair distributions  than subjects of the low self-control group. Moreover, a significant three-factor interaction of opponents \u00d7 allocation proposals \u00d7 self-control groups  was observed. Individuals with high self-control carried out more fair distributions (p < 0.01) and fewer unfair distributions (p < 0.01) to the previous unfair opponents compared with those with low self-control. However, no significant difference was found between the allocation proposals of the high and low self-control groups to the previous fair opponents (both ps > 0.05).The results showed that for the former unfair opponents, the rate of fair distribution was 47.38% in the low self-control group and 52.62% in the unfair distribution, whereas the rate of fair and unfair distribution in the high self-control group was 69.14% and 30.86%, respectively . Howeverr(72) = 0.26, p < 0.05) and a significant negative correlation with retaliation rate (r(72) = \u22120.26, p < 0.05).To further explore the relationship between self-control and interpersonal forgiveness, we examined the correlation between self-control scores and forgiveness rates  and retaliation rates . The results showed a significant positive correlation between self-control and forgiveness rate . The measurement and analysis of anger can reveal whether the subjects\u2019 behaviors are due to forgiveness or forbearance. Additionally, the finding helps us ensure that participants forgive their previous unfair opponents to some extent. Future research needs to collect self-report data when using the behavioral paradigm of forgiveness. Finally, our study was a cross-sectional one, and we did not manipulate self-control; thus, the causal relationship between self-control and interpersonal forgiveness could not be determined. Future studies may use longitudinal research or manipulate self-control to examine the causal relationship between self-control and interpersonal forgiveness.This study has some limitations. One is that some subjects questioned the authenticity of economic decision-making tasks, which may affect the experimental results. Thus, these subjects were excluded from the subsequent analyses. Moreover, this study repeated previous research findings and found that self-control could modulate interpersonal forgiveness. Nevertheless, different behavioral measurement paradigms for measuring forgiveness involve different forgiveness-related psychological processes, and future research requires the use of other behavioral measurement paradigms to verify the relationship between self-control and interpersonal forgiveness. Third, participants were not required to report their intention when giving fair distribution toward a prior unfair opponent, which makes the forgiving conclusion negotiable. However, we found no significant difference in anger levels after DG between the high and low self-control groups (In sum, the current study extends previous findings concerning self-control and interpersonal forgiveness and provides a different perspective of behavioral measure to further explore the relationship between self-control and interpersonal forgiveness. This study has been the first to explore the relationship between self-control and interpersonal forgiveness using the behavioral measurement paradigm combined with adapted UG and DG. Our findings suggest that, when confronted with interpersonal conflicts, individuals with high self-control ability could suppress the negative emotions generated by their instincts and show increased prosocial behaviors consistent with their long-term goals toward transgressor, such as forgiveness.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by the Shanghai Normal University Ethics Review Board. The patients/participants provided their written informed consent to participate in this study.HJL and HL conceived and designed the experiments. HL collected the data and performed the statistical analysis. HJL and HL contributed to the writing of manuscript.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. The handling Editor is currently editing co-organizing a Research Topic with one of the author, HJL and confirms the absence of any other collaboration."}
+{"text": "Their interaction has been recently reported to be relevant during physical and psychological trauma. However, literature reports on H2S in physical trauma and OT in psychological trauma are abundant, whereas available information regarding H2S in psychological trauma and OT in physical trauma is much more limited. This review summarizes recent direct and indirect evidence of the interaction of the two systems and their convergence in downstream nitric oxide-dependent signaling pathways during various types of trauma, in an effort to better understand biological correlates of psychosomatic interdependencies.The purpose of this review is to explore the parallel roles and interaction of hydrogen sulfide (H You et hannels) . They alal cells . Finallyal cells . The paud volume ,13, blood volume ,15,16,17d volume ,19.2S and OT systems in the regulation of fluid homeostasis [2S has been reported to depolarize magnocellular neurons of the PVN in a dose\u2013response fashion, hinting at its possible role in regulating autonomic and endocrine functions [2S), another H2S-releasing salt, led to increases plasma concentrations of OT and a decrease of hypothalamic nitrate/nitrite [2S regulates the roles of both the behavioral and neuroendocrine responses to water deprivation independently of nitric oxide (NO) and stimulates OT secretion by inhibiting the NO system [2S was also shown to be a (positive) regulator of OT in response to acute osmotic stimulus, whereas NO played a key role as a negative neuroendocrine modulator of OT [The hypothalamus is the central integrative structure for the regulation of blood and body fluid volume and osmolality . In respeostasis . In traneostasis ,22. H2S unctions . Moreove/nitrite . The autO system . In a moor of OT . Additioor of OT . This omor of OT . A furthor of OT ,22,23,24or of OT , suggest2S can also stimulate hypothalamic OT release [2S stimulating the release of OT as a consequence of the dramatic fluid shifts generated by the hemorrhagic shock . Recently, in a porcine model of acute subdural hematoma-induced acute brain injury with resuscitation and neuro intensive care maintenance, Denoix et al. characterized the spatial expression pattern for the OT/OTR and the endogenous H2S-producing enzymes [2S systems in osmotic regulation than what was reported previously [In a clinically relevant porcine hemorrhagic shock study , variabl release . Thus, thock see B. Finall enzymes . The aut enzymes . Interes enzymes . CBS, OTeviously ,24.2S has been reported to improve the integrity of the barrier and attenuate cerebral edema, and the reduction of CSE expression close to injury sites coincided with increased Alb extravasation and barrier dysfunction [Furthermore, Hfunction ,30,31. Dfunction . The prefunction ,32.2S producing enzyme in the cardiovascular system, chronically exposed to cigarette smoke prior to undergoing acute thorax trauma, displayed decreased OTR expression in the heart, which was attenuated by the administration of the slow-H2S-releasing compound GYY4137 [Recently, in a mouse model of acute-on-chronic disease, Merz et al. showed that for mice with homozygous global deletion of CSE (CSEko) (generated by ), the ma GYY4137 . Further GYY4137 ,17,19.2S and its protective effects in the cardiovascular system [2S production or expression of H2S-producing enzymes (primarily CSE) has been reported in the following cell types of the cardiovascular system: smooth muscle cells, cardiomyocytes, endothelial cells and immune cells [2S has been shown to play a decisive role in the modulation of the cardiovascular system, i.e., as an endogenous activator of angiogenesis [2S-dependent vaso-active effects activate KATP-channels and stimulate Akt-dependent endothelial eNOS. Furthermore, H2S can induce antioxidant effects via the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which can be also regulated by OT [2S administration in rodents ameliorated myocardial fibrosis and oxidative stress in hypertension [2S donor AP39 during the resuscitation period and reduced lung tissue iNOS expression; however, it aggravated circulatory shock-induced hypotension due to its vasodilator capacities [2S and NO in inflammation has been reviewed previously [2S and OT systems share downstream signaling cascades that converge on the same NOS/NO-dependent pathway, which is further support of their interaction/relationship [There are a plethora of fairly recent reviews on Hr system ,37,38,39ne cells ,25,40,41ogenesis  via hypoogenesis ,44. Downed by OT ,46,47. Hrtension , and hadrtension ,50,51,52rtension . A murinpacities . The comeviously . The H2Stionship .2S administration have been less robust than those seen in the rodent models. Sulfide administration in long-term porcine hemorrhage and resuscitation reduced mortality and attenuated organ dysfunction and injury, but its effectiveness in this model was restricted to a narrow timing and dosing window [2S did not induce hypothermia or improve survival from hemorrhagic shock in pigs [2S-based therapies, which might potentially also affect OT/OTR signaling, further research is warranted.In large animal models, in general, the effects of Hg window . Other a in pigs ,59. Thus2S, which does not require any membrane receptor [2S) [2+ homeostasis, necessary for cardiomyocyte function [2S, e.g., increase of glucose uptake in cardiac cells, anti-inflammatory and antioxidant activity [2S [In contrast to the highly-diffusible, gaseous Hreceptor , the OT tor [2S) . OT can,function . OTR expfunction ,65,66,67function ,68, and activity ,70,71, bactivity , negativactivity ,68,73,74activity ,76,77,78vity [2S .Subcutaneous OT administration in myocardial infarct resulted in reduced inflammation, apoptosis, and ultimately ameliorated heart function ,80,81,822S [2S release is reported to be a key mediator with regard to the development of chronic cardiovascular pathology [2S and OT signaling converge in atherosclerosis and cardioprotection . OT, CSr OT [2S ,96. H2S -\u03b2-cells ; the OT -\u03b2-cells . OT has diac OTR . Hypergldiac OTR  and suppdiac OTR ,100. Lowsistance ,95. It iPsychological stress, e.g., early life stress, is well established as a risk factor for developing cardiovascular diseases ,103,104.2S systems [OT plasma levels were positively related to improved cardiovascular and sympathetic responses to stress . In mice systems . Interes systems . This le systems .2S [2S/CSE systems in the context of psychological trauma was highlighted by the protective effect of exogenous H2S on early life stress-related colonic inflammation [2S and OT are beyond the scope of this work, but have been reviewed recently [This provides evidence that the type of stress has an important impact on the OT ligand and its receptor modulation. In addition to the fact that the loss of trauma-related OTR in cardiac tissue was attenuated with administration of exogenous H2S , the intammation . In turnammation . The therecently ,117,118.2S and OT system: there are plenty of reports on the role of H2S in physical trauma and OT in psychological trauma, whereas investigations of the role of H2S in psychological trauma and OT in physical trauma are rather limited. The current knowledge is not sufficient to speculate about treatment options; thus, this imbalance in studies should be addressed by researchers in the future. H2S and OT have parallel effects suggesting an interaction of the two systems. Both the H2S/CSE and OT/OTR systems assume major importance in the regulation of blood pressure and circulating blood volume. Moreover, there is evidence for their interaction with one another in peripheral organs, particularly in the heart. Finally, the two systems share signaling cascades that converge on the same signaling pathway. The interaction of the two systems seems to regard both psychological disorders and cardiovascular disease, and, hence, understanding more of the way that the H2S/CSE and OT/OTR systems work as mediators in stress may contribute to tackling the mutual interplay between body and mind.When reviewing the literature, it is striking that there is a disparity of studies of the H"}
+{"text": "Casino-going has been acknowledged as a common leisure activity for older adults, but what having a casino in the community means for the local older population has been understudied. Previous research has focused on problem gambling among older adults, but little is understood about how older residents perceive having a casino nearby and further how it impacts relevant senior services. This mixed-methods study gathered perspectives from 14 senior center directors and older residents (N = 411) of communities in Massachusetts that surround Plainridge Park Casino, the first casino that opened in the state in 2015. We conducted qualitative interviews with senior center directors and distributed a quantitative survey to older residents of the surrounding communities and those who visited the casino during the study period. We found that while most senior centers did not engage in trips to this \u201chometown\u201d casino, many had other creative interactions with the casino, such as using casino space to host senior center events or seeking funding support from the casino. Older residents exhibited low rates of problem gambling risk, preferred to go to casinos outside of the state as an excursion, and attributed their reasoning to go to casinos to socializing rather than gambling aspects. While we must be aware of risks for problem gambling, this study also highlights the possibility of reframing a local casino as a potential asset that could contribute back to the community by providing resources for senior service providers and expanding social engagement opportunities for the older population."}
+{"text": "Taken together, targeting ROS systems has a great potential to treat cancer patients with chemoresistance.Anti-cancer chemo-drugs can cause a rapid elevation of intracellular reactive oxygen species (ROS) levels. An imbalance in ROS production and elimination systems leads to cancer cell resistance to chemotherapy. This study aimed to evaluate the mechanism and effect of ROS on multidrug resistance in various human chemoresistant cancer cells by detecting the changes in the amount of ROS, the expression of ROS-related and glycolysis-related genes, and cell death. We found that ROS was decreased while oxidative phosphorylation was increased in chemoresistant cells. We verified that the chemoresistance of cancer cells was achieved in two ways. First, chemoresistant cells preferred oxidative phosphorylation instead of anaerobic glycolysis for energy generation, which increased ATPase activity and produced much more ATP to provide energy. Second, ROS-scavenging systems were enhanced in chemoresistant cancer cells, which in turn decreased ROS amount and thus inhibited chemo-induced cell death. Our  ABCB1) gene, and the inhibition of cell apoptosis, etc Chemotherapy is the main treatment approach for most patients with advanced cancer. However, patients often experience chemoresistance and recurrence GLUT1) HK2) PKM2) GAPDH) LDHA) in vitro and in vivo.In general, cancer cells tend to obtain energy through anaerobic glycolysis, which is called the Warburg effect All cells used in this study were human cancer cells. Ovarian cancer cell line A2780 and its paclitaxel (PTX)-resistant counterpart A2780/PTX , breast cancer cell line MCF-7 , its PTX-resistant counterpart MCF-7/PTX and DDP-resistant counterpart MCF-7/DDP  were cultured in DMEM  supplemented with 10% fetal bovine serum . Lung carcinoma cancer cell line A549 (ATCC) and its PTX-resistant counterpart A549/PTX (ATCC), myelogenous leukemia cell line K562 (Keygen Biotech), its doxorubicin (ADR)-resistant counterpart K562/ADR (Keygen Biotech), PTX-resistant counterpart K562/PTX and DDP-resistant counterpart K562/DDP (Meixuan Biotech) were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS. The pH value of the culture medium was determined by a pH meter.4 cells/well with 100 \u00b5L of medium containing various concentrations of chemo-drugs in quadruplicate. The cell viability was detected using the CCK-8 kit . The optical density (OD) of each well was measured by a microplate analyzer  at 450 nm wavelength. The IC50 value was calculated using GraphPad (V5.0).Cells were seeded in a 96-well plate at a density of 1\u00d7105 cells/well. After incubation for 16 h, cells were treated with or without a chemo-drug at a concentration equal to the IC50 for 6 h and conducted for ROS detection. The cells were trypsinized, centrifuged, and stained by using a ROS assay kit  according to the product instructions. Subsequently, 500 \u00b5L PBS was added to each sample and a ROS level was determined by flow cytometry . The data were analyzed using FlowJo software .Cells were seeded in a six-well plate at a density of 2\u00d710Cells were seeded in a 35 diameter mm confocal culture dish with 20 mm of glass-bottom at a volume of 0.5 mL/dish. After confluence reached 60-80%, cells were stained using a ROS assay kit (Beyotime Biotechnology) according to the product instructions, further stained with Hoechst 33342 (Beyotime Biotechnology), washed with PBS for 3 times, and imaged by a laser confocal microscope .Total RNA was extracted using an RNA-Quick Purification Kit  according to the manufacturer's instructions. PCR was applied using a qPCR RT kit . The PCR primer sequences were shown in Supplementary \u00ae RNA Sample Preparation Kit  according to the preparation guide. After the products were purified, the cDNA libraries were generated by enriching the products with PCR and quantified by the Agilent 2100 bioanalyzer . Molar concentration was determined using a Qubit\u00ae 2.0 Fluorometer . Subsequently, sequencing was performed on an Illumina HiSeq system  and the analyses were conducted using HISAT2 (https://daehwankimlab.github.io/hisat2/) and Stringtie (http://ccb.jhu.edu/software/stringtie/) software. Gene Set Enrichment Analysis (GSEA) was performed using the Molecular Signature Database .Total RNA was extracted from chemoresistant and chemosensitive cells using an RNA-Quick Purification Kit  according to the manufacturer's instructions. mRNA sequencing was performed by Gene Denovo Co. Ltd. . In brief, a paired-end library was synthesized using a TruSeqCells were lysed with sodium dodecyl sulfate (SDS) lysate containing 1% benzyl sulfonyl fluoride and 1% phosphatase inhibitor. After carrying out ultrasonic cracking, the total proteins were run in SDS-polyacrylamide gel electrophoresis. The primary antibodies of the rabbit anti-P-gp , pro-apoptotic protein Bax , anti-apoptotic protein Bcl-2 , and mouse anti-\u03b2-actin  and the secondary antibodies of goat anti-rabbit IgG and anti-mouse IgG labeled with horseradish peroxidase  were used. The protein bands were photographed by the chemiluminescence imaging system .6/100 \u03bcL was transferred into a 5 mL tube, followed by adding 1 \u03bcL of Annexin-V-FITC and/or 3 \u03bcL of PI. After incubation with 400 \u03bcL of 1\u00d7 binding buffer in the dark for 15 min, cells were detected by flow cytometry .Cells were stained with Annexin-V conjugated with fluorescein (FITC) and propidium iodide (PI) according to the product instructions . In brief, cell suspension at a density of 1\u00d710+K+ and Ca++Mg++ ATPase decomposition. After adding a phosphorus fixing agent for 30 min at 37 \u2103, the OD value of each sample was measured by a microplate analyzer at 660 nm wavelength. A 0.5 \u00b5mol/ mL of phosphorus storage solution was used for calibration. ATPase activity was calculated according to the following formula: ATPase activity (\u00b5 molPi/gHb/hour) = (ODexperment-ODcontrol)/ODcalibrator \u00d7 Calibrator concentration \u00d7 Dilution ratio of sample \u00d7 6 \u00f7 Protein concentration.ATPase activity was determined according to the ATPase kit instructions (Keygen Biotech). Briefly, cells were cultured in a 6-well plate for 24 h. After washing twice with saline, the cells were lysed with a solution containing SDS, followed by ultrasonication. After centrifugation, the suspension was collected. The activity of ATPase was evaluated by measuring the amount of inorganic phosphorus produced by Na2 for 0-120 sec. Subsequently, the ROS concentrations were detected using a ROS assay kit, cell viability was determined using a CCK-8 kit, and apoptotic cells were measured using an Annexin-V FITC apoptosis detection kit.Protoporphyrin  was dissolved in dimethylsulfoxide (DMSO) at a concentration of 4 mg/mL. Cells were seeded in 48-well or 96-well plates  and incubated for 24 h. After replacement of fresh medium containing 0~5 mg/mL of protoporphyrin and incubation for 4 h, the cells were irradiated using a laser device  with a wavelength at 638 nm, 300 mW/cm6 A549/PTX cells in 100 \u00b5L of serum-free RPMI-1640 medium on the right side. After 2 weeks, the tumor-bearing mice were randomly divided into 2 groups (7 per group). Each mouse was injected intratumorally with either 100 \u00b5L of normal saline (control) or 100 \u00b5L of protoporphyrin  once every 2 days for a total of 5 times. The body weight and tumor volume were monitored during the treatment. The calculation formula of tumor volume is 2/2V = ab, where V, a, and b indicate the volume, tumor length, and tumor width, respectively. At the end of the treatment, the mice were sacrificed and their tumors were excised and photographed. Detection of the production of hydroxyl radicals in tumor tissue lysates was performed according to the hydroxyl radicals production kit instructions (Keygen Biotech). The tumor tissue was fixed with formalin and paraffin-embedded tumor tissue specimens were conducted for hematoxylin-eosin (H&E), proliferating cell nuclear antigen (PCNA), and Tunel assays at Shenghua Biological Technology Co., LTD .The ethics of animal experiments was approved by the Ethics Committee of Shanghai Public Health Clinical Center. Four-week-old female BALB/C nude mice were purchased from B&K Laboratory Animals Co., LTD.  and raised in a standard feeding condition with adequate food and water. After 7 days, each mouse was injected with approximately 3\u00d71050 values and qRT-PCR. Animal statistics for analyzing the difference between two groups were performed using Student's t-test. Statistical results with P < 0.05 were considered statistically significant.The experimental data are shown as mean \u00b1 standard deviation (SD) for cell viability, relative fluorescence intensity, and ATPase activity, and as the mean \u00b1 standard error (SEM) for ICABCB1) , the levels of ROS were elevated after exposure to a chemotherapeutic drug PTX . Next, ws Figure G-L.G6PD in A2780/PTX and K562/ADR cells, GCLC in A2780/PTX, A549/PTX, K562/DDP, and K562/PTX cells, TXN in A2780/PTX, MCF-7/PTX and MCF-7/DDP, A549/PTX and K562/PTX cells, SOD1 in A2780/PTX, MCF-7/PTX and MCF-7/DDP, and K562/PTX cells, NQO1 in A2780/PTX cells (Figure FTL in A2780/PTX, K562/DDP, K562/ADR and K562/PTX cells, MCOLN1 in A2789/PTX and K562/ADR cells, HIF1A in MCF-7/PTX and MCF-7/DDP, K562/DDP and K562/ADR cells (Figure ABCB1 mRNA was confirmed to be increased in K562/PTX resistant cells (Because the ROS levels were elevated after chemotherapeutic drug treatment and were lower in chemoresistant cells than chemosensitive cells, we examined the ROS-scavenging system by detecting ROS elimination-related mRNAs using qRT-PCR. We found that the expression of some reductive or oxygen-free radical scavenging enzymes was increased in chemoresistant cells, including s Figure . Accordis Figure . The expnt cells .GLUT1, HK2, PKM2, and GAPDH were slightly upregulated, and LDHA were downregulated (Figure Generally, cancer cells tend to obtain energy through anaerobic glycolysis rather than oxidative phosphorylation. However, in chemoresistant cells, glycolysis-related mRNAs including d Figure , which pd Figure E-G. In ad Figure H. The pHd Figure I-J. ThesABCG2 in chemoresistant cells. We found that the ABCG2 level was not increased in most chemoresistant cells compared with their sensitive cells, except for A2780/PTX (Next, we used protoporphyrin as a photosensitizer for photodynamic therapy Figure A. As pro2780/PTX , indicat2780/PTX B-E, whil2780/PTX F-H, alon2780/PTX I-5L. Flo2780/PTX . These dGAPDH is a glycolysis-related gene and was differentially expressed between chemoresistant and sensitive cells, 18S was applied as an endogenous control gene to normalize the interested genes. To confirm the reliabilities of qRT-PCT, another endogenous control gene Actin was applied. Indeed, the results of relative mRNA expression of targeted genes were reproducible after normalizing to Actin. (Because o Actin. .in vivo effect of photodynamic therapy on the inhibition of chemoresistant cancer cells. A549/PTX bearing mice were injected intratumorally with normal saline or protoporphyrin and irradiated with a 638 nm laser (Figure Animal experiments were conducted to evaluate the Patients with advanced cancer often face a big challenge of chemoresistance during chemo-drug treatment. An imbalance of ROS level may play a critical role in cells resistant to a chemo-drug. The present study examined the value of ROS, the expression of ROS-related genes, and glycolysis-related genes in various chemoresistant cancer cells. Here, we observed interesting results that the intracellular level of ROS was significantly lower in chemoresistant cancer cells than chemosensitive cancer cells, while a chemo-drug elevated the intracellular ROS level in both chemoresistant and chemosensitive cancer cells. It leads to a hypothesis that the decrease of ROS concentration in chemoresistant cells is due to the enhancement of the ROS-scavenging system during chemoresistant processes. Indeed, we confirmed that an increase in the ROS elimination together with the restraint of aerobic glycolysis conducts cancer cell resistance to a chemo-drug.The current study demonstrated that ATPase activity was enhanced in chemoresistant cells. Unlike chemosensitive tumor cells that mainly rely on glycolysis in vitro and in vivo experiments using photodynamic therapy showed that an increase in ROS can promote chemoresistant cell death. Because protoporphyrins are widely used to produce ROS efficiently We did observe an increase of ROS in chemoresistant cells after a chemo-drug treatment, which is consistent with the previous report that ROS was elevated in cisplatin-resistant ovarian cancer cells It has been known that chemoresistant cells can inhibit apoptosis G6PD, TXN, and GCLC was upregulated. This series of genes that constitute the ROS scavenging system indeed lead to the decrease of ROS levels in chemoresistant cells. TXN as a key protein of the thioredoxin system was also significantly increased in the chemoresistant cells, indicating that the thioredoxin system may play an important role in the reduction of ROS in chemoresistant cells. Therefore, increasing intracellular ROS concentration may be a therapeutic strategy to reverse chemoresistance. Thus, photodynamic therapy has a great value to treat cancer patients with chemoresistance.Our results showed that chemoresistant cells indeed activated the ROS-scavenging system as the expression of In summary, the intracellular level of ROS is significantly lower in chemoresistant cancer cells than chemosensitive cells. Chemoresistant cancer cells prefer oxidative phosphorylation instead of anaerobic glycolysis for energy generation. The ROS-scavenging system is enhanced and aerobic glycolysis is restrained in chemoresistant cells. The reduction of ROS by oxidative phosphorylation results in the inhibition of chemotherapeutic drug-induced apoptosis, and in turn, leads to the progression of chemoresistance in cancer cells Figure . The sigSupplementary figures and table.Click here for additional data file."}
+{"text": "The application of graph theory to model the complex structure and function of the brain has shed new light on its organization, prompting the emergence of network neuroscience. Despite the tremendous progress that has been achieved in this field, still relatively few methods exploit the topology of brain networks to analyze brain activity. Recent attempts in this direction have leveraged on the one hand graph spectral analysis (to decompose brain connectivity into eigenmodes or gradients) and the other graph signal processing (to decompose brain activity \u201ccoupled to\u201d an underlying network in graph Fourier modes). These studies have used a variety of imaging techniques  and connectivity estimators to model brain networks. Results are promising in terms of interpretability and functional relevance, but methodologies and terminology are variable. The goals of this paper are twofold. First, we summarize recent contributions related to connectivity gradients and graph signal processing, and attempt a clarification of the terminology and methods used in the field, while pointing out current methodological limitations. Second, we discuss the perspective that the functional relevance of connectivity gradients could be fruitfully exploited by considering them as graph Fourier bases of brain activity. Modern attempts at understanding brain function have leveraged the use of graph theory to grasp complex properties of neuronal networks, giving rise to the field of network neuroscience  and discrete networks associated with a particular condition or task. Exploiting the topology of brain networks, this framework allows for a decomposition of brain activity or structure as a The goal of this paper is first to summarize recent contributions that have used connectivity gradients and GSP for neuroimaging. In particular, we aim to clarify terminology, compare different methodologies, and provide resources see  and key Box 1.\u2003Toolboxes and resources for gradient analysis and GSP in neuroscienceNilearn: A widely used Python toolbox for machine learning in neuroimaging. It also includes useful functions for brain connectivity computation and visualization that can be easily adapted to plot gradients and signals on brain networks. https://nilearn.github.io/#BrainSpace: A Matlab/Python software package that allows connectivity gradients computation and analysis specifically adapted to neuroimaging and connectome datasets  that also scale to very large graphs. https://pygsp.readthedocs.io/en/stable/index.htmlNetworkX: A Python package to analyze network structure, build network models, and visualize networks. https://networkx.github.io/Congrads: A Python package to compute and map connectopies (connectivity topography) of a predefined region of interest. https://github.com/koenhaak/congradsVB toolbox: A pair of packages (available in both MATLAB and Python) including connectivity gradient analysis pipelines and the computation of the Vogt-Bailey index  is a potential edge in the graph. In many cases, relations are weighted with the convention that a weight 0 corresponds to the absence of an edge, and any nonzero value can be used otherwise. A concise way to represent a weighted graph consists of using its (weighted) W, indexed by nodes. As such, Wij is 0 if and only if there is no edge between nodes i and j, and Wij represents the weight of the edge  otherwise. By summing a row of W, we obtain the strength (or weighted degree) of the corresponding node, which can be thought of as the importance it has, compared with the other nodes .Graphs are tools that are ubiquitous in many fields of science, thanks to their generic nature and expressivity. They make it possible to efficiently represent relations between items, called graph signals. In the light of this formalism, it is possible to define ad hoc graph Fourier modes, tied to the specific considered graph structure. These Fourier modes simply consist of the eigenvectors of L. The corresponding eigenvalues \u03bbl exhibit behaviors that can be interpreted in terms of spatial frequencies over the graph structure , authors are interested in manipulating graph matrices together with vectors indexed by the nodes of the graph, called ture see .In short, the graph Laplacian  revealed a macroscale cortical organization spanning from unimodal sensory areas to transmodal association areas. Atasoy and colleagues  connectivity and showed that they can predict resting-state functional network activity. Results indicated that visual, sensory-motor, and limbic networks more closely matched low-frequency harmonics, while higher cognitive function networks spanned over a broader range of brain modes. Another recent work on brain structure/function relation  to extract anatomically relevant features. In Germanaud et al. , Laplaciconnectopies) using Laplacian eigenmaps  is presented in Ortega et al.  for the analysis of brain dynamics is an approach that complements (rather than excluding) the more classical hard boundaries parcellation. Some processes may be best characterized in terms of nonoverlapping fixed regions, others in terms of delocalized, overlapping eigenmodes. In this sense, this graph modal approach may be more appropriate than modular analysis in describing complex cognitive states depending on multiple overlapping phenomena. Neural patterns of ongoing activity could be seen as location in a multidimensional state-space constructed out of large-scale brain gradients , thus exploiting complementary measurements of brain properties. Only a few studies have analyzed fMRI and EEG signals at rest using graph Fourier modes of the underlying structural graph . In the future, this framework could be applied to jointly decompose electrophysiological and functional time series using underlying structural topology, thus integrating different temporal and spatial scales in a multimodal analysis, with potential to shed new light on the complex interplay between brain function and structure. In addition, GSP could be extended to the analysis of brain signals during different tasks and  conditions, holding promise for developing more sensitive markers of disease.\u03bb0 = 0) is not constant as for the combinatorial Laplacian, which is less intuitive for an interpretation of eigenvalues in terms of spatial frequency  because of a series of advantageous properties for brain connectivity analysis. Future work should explore how different choices could affect results and which metrics are more adapted to the analysis of specific graph structures or brain signals.As described in Another point to clarify is the impact of the choice of parcellation on the connectivity gradient topography. In the pioneering work of Margulies et al. , high-reOne important limitation of the studies we have reviewed is the lack of accounting for temporal dependencies in the graph model. Indeed, in most of the literature \u201cconstant\u201d spatial dependencies between brain regions are considered . Several theoretical breakthroughs have been made to address dynamic properties , and their application to neuroimaging data could prompt the understanding of cognitive process dynamics. Some promising frameworks to model time-varying aspects in graphs include graph slepians , Award ID: SAD-2019.Nicolas Farrugia, R\u00e9gion Bretagne ("}
+{"text": "A 33-year-old female visited the outpatient department of Acharya Vinoba Bhave Rural Hospital (AVBRH), Sawangi with chief complains of painful ulcer over lower lip. On taking proper history the patient said she was apparently alright 4 months back when she noticed a painful non healing ulcer over lower the front region of jaw which was initially small in size and gradually increased to the present size of 5.3 x 2.3cm approximately. Patient had history of pain which was gradual in onset, dull aching, intermittent and localized in nature. Pain aggravated on mastication and relieved after taking medications. Patient also had history of change in quantity and consistency of saliva from normal to thick and ropy with intermittent bleed and pus discharge since 3months. On clinical examination face was asymmetrical due to soft tissue defect present over lower lip with irregular everted margins. On incisional biopsy of lesion done under local anaesthesia on 15/09/20 revealed squamous cell carcinoma of lower lip."}
+{"text": "The presence of hyperphosphorylated microtubule-associated protein tau is strongly correlated with cognitive decline and neuroinflammation in Alzheimer\u2019s disease and related tauopathies. However, the role of inflammation and anti-inflammatory interventions in tauopathies is unclear. Our goal was to determine if removing anti-inflammatory interleukin-10 (IL-10) during an acute inflammatory challenge has any effect on neuronal tau pathology.Il10-deficient (Il10\u2212/\u2212) versus Il10+/+ (Non-Tg) control mice using a single intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) to examine microglial activation and abnormal hyperphosphorylation of endogenous mouse tau protein. Tau phosphorylation was quantified by Western blotting and immunohistochemistry. Microglial morphology was quantified by skeleton analysis. Cytokine expression was determined by multiplex electro chemiluminescent immunoassay (MECI) from Meso Scale Discovery (MSD).We induce systemic inflammation in Il10 promotes enhanced neuroinflammation and tau phosphorylation. First, LPS-induced tau hyperphosphorylation was significantly increased in Il10\u2212/\u2212 mice compared to controls. Second, LPS-treated Il10\u2212/\u2212 mice showed signs of neurodegeneration. Third, LPS-treated Il10\u2212/\u2212 mice showed robust IL-6 upregulation and direct treatment of primary neurons with IL-6 resulted in tau hyperphosphorylation on Ser396/Ser404 site.Our findings show that genetic deletion of These data support that loss of IL-10 activates microglia, enhances IL-6, and leads to hyperphosphorylation of tau on AD-relevant epitopes in response to acute systemic inflammation. Alzheimer\u2019s disease (AD) is one of the tauopathies and the most common neurodegenerative disease involving the abnormal phosphorylation and accumulation of the microtubule-associated protein tau (MAPT or tau). Tau is a neuron-enriched protein known to bind and stabilize microtubules under homeostatic conditions and has non-microtubule-binding functions , 2. HoweIl10 is associated with abnormal hippocampus-dependent memory function [Interleukin-10 (IL-10) is a well-establishedanti-inflammatory cytokine with an important role in preventing unrestrained inflammatory responses from activated immune cells , 14. Thefunction  and incrfunction \u201322. Therfunction , 24. HowIl10-deficient (Il10\u2212/\u2212) versus Il10+/+ (Non-Tg) control mice using a moderate dose of LPS to induce systemic inflammation. We examined microglial activation and phosphorylation of tau in the hippocampi after 24 h of LPS treatment. We predicted that lack of IL-10 would exacerbate an ongoing inflammatory response to promote hyperphosphorylation of tau.In the current study, we sought to determine if IL-10 suppresses inflammation leading to tau pathology in a model of acute systemic inflammation induced by lipopolysaccharide (LPS). We have shown that LPS-induced microglial activation promotes hyperphosphorylation of tau in non-transgenic mice through toll-like receptor 4 (TLR4) signaling. In our previous study, 1 mg/kg b.w. of LPS did not induce significant increases in AT8 or AT180 tau phosphorylation compared to vehicle-treated controls, and even 10 mg/kg b.w. of LPS had only a moderate increase in pTau in the wild-type mice . This suIl10tm1Cgn/J (Il10\u2212/\u2212) [B6.129P2-Il10\u2212/\u2212)  and C57BEscherichia coli 055:B5 was administered  to 5\u20136-month-old non-transgenic or Il10\u2212/\u2212 mice on C57BL/6J background. After 24 h, the mice were sacrificed, and brain tissue was collected as described below. LPS concentration is based on previous papers showing the efficacy of this dose to induce tau phosphorylation in the hippocampi of wild-type mice [Vehicle  or lipopolysaccharide  from ype mice .Mice were anesthetized, transcardially perfused with 0.125 M phosphate buffer (PB) and brains were removed. Left hemispheres were fixed in 4% paraformaldehyde in PB (4% PFA/PB), incubated in cryoprotection solution for 24 h, sliced into 30\u03bcm thick sagittal brain sections, and stored at -20\u00b0C in cryostorage solution until use in TUNEL and immunohistochemical analysis. Right hemispheres were micro-dissected into cortex, hippocampus, and the rest of the brain, wet weights were recorded, and the tissues were snap frozen in liquid nitrogen. The cortices and hippocampi were used in biochemical protein analysis.g at 4 \u00b0C for 10 min. Protein concentrations of cortical lysates were determined by Bradford protein assay (Biorad). A total of 200 \u03bcg of protein/sample in duplicate was used to determine cytokine levels using multiplex electrochemiluminescent immunoassay (MECI). The V-PLEX Plus Proinflammatory Panel 1 (mouse) electrochemiluminescent immunoassay platform  was used to detect multiple cytokines, including IL-10, IL-12p70, IL-1\u03b2, TNF-\u03b1, IL-6, and the chemokine, CXCL1. Plates were read on a Quickplex SQ 120 Imager.Cortical tissue sections were homogenized in a buffered sucrose solution containing protease and phosphatase inhibitors and processed for protein analysis as previously described \u201330. Tissg at 4 \u00b0C for 30 min. The soluble fraction was diluted with NuPage LDS Sample Buffer and Sample Reducing Agent, run on 4\u201312% Bis-Tris Novex NuPage gels (Invitrogen) for 1 h. Proteins were transferred from the gel to PVDF membranes overnight. Membranes were washed and blocked in 5% milk/PBS blocking buffer and incubated overnight in primary antibodies. Membranes were washed and incubated for 1 h in corresponding HRP-conjugated secondary antibodies . Membranes were incubated with ECL reagent (ThermoScientific) and exposed to X-ray film. Immunoreactive bands on the developed film were scanned and quantified with AlphaEaseFCTM Software (Alpha Innotech Corporation).Hippocampal tissues were homogenized in Tissue-Protein Extraction Reagent, TPER  with protease and phosphatase inhibitor cocktails. The tissue homogenates were sonicated and spun at 13,700\u00d7For in vivo experiments: Western blots were probed for phosphorylated tau antibodies: PHF-1 ; AT180 (1:5000), AT8 , and total tau Tau5  (Thermo Scientific); and activated phospho-p38 MAPK (1:500) and total p38 MAPK (1:1000) ; and loading control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) .For in vitro experiments: Western blots were probed for phosphorylated tau antibodies: PHF-1 ; AT8 ; Tau5 ; GAPDH , activated phospho-p38 MAPK (1:500) and total p38 MAPK (1:1000) ; and activated phospho-STAT3 and STAT3  to verify intracellular neuronal signaling downstream of IL-6.2O2 in PBST, and 5% normal goat serum in Triton/PBS was used as blocking buffer. Primary antibody incubation was overnight at 4\u00b0C. Incubation with corresponding Biotinylated IgG secondary antibodies  was for 1 h. Sections were then incubated with Avidin:Biotinylated enzyme complex reagent and developed using SIGMAFASTTM3,3\u2032-Diaminobenzidine (DAB) tablets  without metal enhancer to reveal immunoreactive signals. IHC brain sections were probed for phosphorylated tau, AT180 , and microglia, ionized calcium-binding adapter molecule 1 (Iba1) .Free-floating sections were processed via standard immunohistochemical methods. Briefly, a sodium citrate solution was used for antigen retrieval, endogenous peroxidases were blocked with 3% HFor terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, staining was performed using the protocol supplied with a Roche in situ cell death detection kit (Roche Applied Science # 11684809910). Sections were co-stained with NeuN (1:250)  to visualize neurons. The percent area of TUNEL+ staining of pyknotic neuronal bodies was imaged and quantified using fluorescent microscopy and threshold analysis in ImageJ.ImageJ Skeleton Analysis was based on procedures previously described . Briefly2 incubator prior to any treatment. DIV 16 cultures were treated with 25 ng/mL recombinant mouse IL-6 (Biolegend) and collected in LDS/RA solution 12 h after treatment. Collected cell lysates in LDS/RA were sonicated and boiled before use in western immunoblot assay.Primary neuron cultures were prepared following an established protocol . Brieflyl-glutamine) every other day. One week after neuronal isolation, neurons were treated with 25 ng/mL recombinant mouse IL-6 (Biolegend) and collected in LDS/RA solution 12 h after treatment. Collected cell lysates in LDS/RA were sonicated and boiled before use in western immunoblot assay.Dissociation of adult mouse brains was performed using the \u201cAdult Brain Dissociation Kit for mouse and rat\u201d  and \u201cNeuron Isolation Kit for mouse\u201d  following the user manual, in combination with the gentleMACS Dissociator (Miltenyi Biotec). Mice were anesthetized and the brains removed then cut into 8 sagittal slices. Brain slices were homogenized using the gentle MACS Dissociator in C Tubes  with the recommended volume of enzyme mixes. Tissue was processed with no modification to the protocol in the kit. Isolated neurons from the adult mouse brains were cultured for one week changing half the media  with Sidak\u2019s multiple comparison post hoc test. All statistical analyses were performed using GraphPad Prism\u00ae (Version 8).Unless otherwise indicated, comparisons between the two groups were done via unpaired Il10\u2212/\u2212 and Non-Tg mice was performed to determine the levels of tau phosphorylation at AT8, AT180, and PHF-1 epitopes 24 h following one dose of intraperitoneal LPS injection  of sagittal brain sections was performed. Quantification of AT180+ area, 24 h post-LPS treatment, confirmed significantly enhanced pTau in the dentate gyrus of Il10\u2212/\u2212 LPS mice compared to the Veh and Non-Tg LPS groups  in the hippocampal lysates  and significantly increased the number of junctions compared to the other groups  and decreased the number of junctions compared to Non-Tg microglia post-LPS  levels were significantly elevated in IL-6-treated neurons  in the sk of AD , 47. Forsk of AD , 48. Parsk of AD  and is isk of AD , 47. In sk of AD . Our stuIl10\u2212/\u2212 mice, which are deficient in IL-10 throughout their life, seemingly had an intermediate stage of activation (increased Iba1 and hypertrophic processes) in the hippocampus CA1, but not the CA3, region. Thus, discerning the effects of dysregulated IL-10 on AD may require an understanding of intermediate activation states of microglia, the microglial microenvironment, and the dosage as well as the timing of IL-10.Second, IL-10 regulates microglial phenotype and immune processes, which are implicated in AD. According to a recent genome-wide association study\u00a0(GWAS) by the International Genomics of Alzheimer\u2019s Project, some of the most influential risk factors for AD compared to controls were in genotypes associated with microglia and the immune system , 52. FurIl10\u2212/\u2212 mice compared to Non-Tg mice at 24 h after LPS administration. This may suggest another compensatory mechanism limited the ongoing expression of IL-1\u03b2 within 24 h in our model. However, we do not know the relative expression levels of IL-1\u03b2 at earlier timepoints and cannot rule out a role for IL-1\u03b2 in promoting tau hyperphosphorylation in this model. Potentially, there are multiple routes leading to AD-relevant tau phosphorylation if left unregulated. In our study, TNF\u03b1 and IL-6, were particularly elevated by LPS in the absence of IL-10. Interestingly, IL-6 remained significantly elevated in the Il10\u2212/\u2212 mice, which alludes to clinical correlations between genotypes associated with low IL-10 and high IL-6 production as a risk factor for AD [Finally, dysregulation of cytokines, including IL-10, is implicated in AD. For example, inflammasome signaling molecules are increased in AD brains  and color for AD , 68, 69.r for AD . InteresThe role that inflammation plays and whether it contributes to AD has increasingly become an important area of research due to increased evidence that microglial activation and abnormal expression of cytokines are associated with AD. Understanding inflammatory pathways that promote tauopathy is necessary to find novel targets for AD. Here, we provide evidence that IL-10 deficiency enhances a pro-inflammatory environment in the brain after a peripheral immune challenge leading to hyperphosphorylation of tau on AD-relevant epitopes. Our model involves inducing inflammation through TLR4 signaling using LPS as the stimulus. Immune activation through TLR4 is relevant in AD as amyloid beta (A\u03b2), is known to trigger TLR4 signaling to secrete inflammatory factors , 72. TLRIl10 promoted enhanced pro-inflammatory cytokine expression, activated microglial morphology, and evidence of neuronal loss. Exacerbated neuroinflammation in the IL-10-deficient environment corresponded with increased tau-kinase p38 MAPK activation and significantly increased tau phosphorylation. These data support the significance of IL-10 in modulating pro-inflammatory microglial activation, dysregulated cytokine production, and hyperphosphorylation of AD-relevant tau epitopes.Our study shows that IL-10 deficiency is sufficient to promote tau hyperphosphorylation and dysregulated neuroinflammation in the context of an acute peripheral inflammatory insult. Genetic deletion of"}
+{"text": "Aedes albopictus), 2017 (Culex pipiens complex), 2018 (Cx. pipiens complex), and 2019 (Cx. pipiens complex), respectively, was positive for USUV. The 2018 and 2019 positive pools shared 99.31% nucleotide homology within the USUV NS5 gene and both clustered within USUV Europe 2 lineage. The next-generation sequencing of one mosquito pool (Cx. pipiens complex) collected in 2018 in Zagreb confirmed the presence of USUV and revealed several dsDNA and ssRNA viruses of insect, bacterial and mammalian origin.In the period from 2015 to 2020, an entomological survey for the presence of West Nile virus (WNV) and Usutu virus (USUV) in mosquitoes was performed in northwestern Croatia. A total of 20,363 mosquitoes were sampled in the City of Zagreb and Me\u0111imurje county, grouped in 899 pools and tested by real-time RT-PCR for WNV and USUV RNA. All pools were negative for WNV while one pool each from 2016 ( Flavivirus, family Flaviviridae. In Europe, both viruses have been detected in humans, horses, mosquitoes, and birds [West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne arboviruses of the genus nd birds ,2.Culex are the main vectors [WNV is sustained in an enzootic cycle between birds and mosquitoes while humans and horses represent \u201cdead-end\u201d hosts. Numerous bird species are competent amplifier hosts for WNV, and mosquitoes of the genus  vectors . After t vectors . In the  vectors ,5. So fa vectors .Accipiter gentilis) from the same aviary in northwestern Croatia [In Croatia, first human cases of WNV neuroinvasive disease (WNND) were reported in 2012 in eastern counties . In the  Croatia ,11.Culex pipiens, Cx. perexiguus, and Cx. modestus [Cx. pipiens, Ochlerotatus caspius, and Cx. modestus in Italy [Cx pipiens and Cx. modestus in Greece [Cx. pipiens and Cx. perexiguus [Cx. modestus and Cx. pipiens complex in Slovakia [Cx. pipiens, Aedes vexans, and Culiseta annnulata in Serbia [In Europe, the most important vector species of WNV are modestus ,13. WNV in Italy ,15,16,17n Greece ; Cx. piprexiguus  in SpainSlovakia ; and Cx.n Serbia .Culex mosquitoes as the main vectors [Cx. neavei mosquito caught near the Usutu River in Swaziland [USUV is a mosquito-borne arbovirus, genetically closely related to WNV. The natural cycle of USUV is similar to that of WNV and involves birds as the main amplifying hosts and  vectors ,23,24. H vectors ,25. Humawaziland . Since twaziland . In the waziland ,25.Turdus merula) in Zagreb County [In Croatia, the first serologic evidence of USUV was reported in 2011 in two seropositive horses detected in northwestern Croatia . In 2012b County ,25.Cx. pipiens mosquitoes from Catalonia, Spain [Cx. perexiguus collected in southern Spain [Cx. pipiens, Ae. albopictus, Cs. annulata, Oc. detritus, Anopheles maculipennis s.l., and Oc. caspius in Northern Italy [Cx. pipiens in Serbia [Cx. pipiens/Cx. torrentium and Ae. japonicus in Austria [Cx. pipiens pools in Belgium [Cx. modestus in Czech Republic [Cx. pipiens in France [Cx. pipiens is the main vector for the virus [The first isolation of USUV in Europe was made in 2006 in a, Spain . Since trn Spain ; Cx. piprn Italy ,31,32,33n Serbia ,35; Cx.  Austria ,37; in o Belgium ; Cx. modRepublic ; and Cx.n France  and Germn France . Althoughe virus .Cx. pipiens mosquitoes. Phylogenetic analysis of WNV strains from mosquitoes in different geographic areas in Europe showed circulation of WNV lineage 1 and 2 [Both viruses were most frequently detected in  1 and 2 . Analysi 1 and 2 .Ae. albopictus and Ae. japonicus) [So far, 52 mosquito species have been detected in Croatia, of which two are invasive (ponicus) . In the ponicus) ,44, seveCx. pipiens complex pools were negative for WNV RNA [The first screening of mosquitoes for flaviviruses in Croatia was conducted during the 2012 WNV outbreak in three northeastern counties. Mosquitoes were sampled within the area where WNV human infections occurred and all tested  WNV RNA . The aimThe research area was the northwestern part of Croatia. It included the City of Zagreb and Me\u0111imurje County. WNV and USUV were previously identified in that Croatian region, based on testing of human samples, horses, poultry and wild birds ,29.In the period from 2015 to 2020 a total of 618 mosquito sampling occasions were conducted using three methods , 613 in 2) as an attractant (CO2-baited traps) and used to collect adult mosquitoes of various species. They were placed approximately 1.5 m from the ground and set in the late afternoon, before sunset, left overnight, and removed after sunrise (07:00\u201310:00). Over the last three years (2018\u20132020), the CO2-baited traps were set at the same eight collection sites , the mosquito sampling occasions were conducted over six years (2015\u22122020). Mosquitoes were collected by three different traps and methods, including CDC Mini Light traps , BG-Sentinel traps , and aspirator collection. CDC Mini Light traps were equipped with dry ice , mosquito samples were collected only once, between August 17 and 18 2017, from the late afternoon to the morning, at five different locations  [The mosquitoes sampled by traps with CO. (2010)  and Scha. (2010) . Specimehttp://www.ncbi.nlm.nih.gov (accessed on 28 February 2021)). Maximum likelihood phylogenetic analysis was conducted, and an evolutionary analysis was performed by using MEGA7 [Viral RNA was extracted using a High Pure Viral Nucleic Acid Kit (Roche Applied Science). TaqMan real-time RT-PCR assays for detection of WNV and USUV RNA were performed according to the protocols of Tang et al. (2006)  and Nikong MEGA7 .Cx. pipiens pool. The RNA was extracted from sample suspensions with Trizol reagent . The cDNA was synthesized from the extracted nucleic acids using the cDNA Synthesis System  and Random Hexamer Primers  and fragmented with a Covaris M220 targeting peak fragment lengths of 400 bp. The library was prepared with the GeneRead\u2122 DNA Library L Prep Kit , according to the manufacturer\u2019s instructions and sequenced on the Ion PGM platform using the Ion PGM\u2122 Hi-Q\u2122 View Sequencing Kit reagents . Sequenced reads were quality checked and trimmed using the Ion Torrent Suite v.5.6.0. Additionally, low quality bases were trimmed with Geneious software suite v.11.0.5 . Clean reads were subjected to BlastX and BlastN search. The BlastX and BlastN results were analyzed with MEGAN6 [Ion Torrent high-throughput next-generation sequencing (NGS) technology was used to determine the virome in one selected h MEGAN6  for the Oc. sticticus, followed by Ae. albopictus (23.5%), Ae. vexans , and Cx. pipiens (20.7%). Other species were represented by less than 2%. Out of a total of 72 mosquitoes from Me\u0111imurje County, 52.8% belong to Cx. pipiens complex, followed by Ae. vexans (41.7%) and Oc. sticticus (5.5%) (During a six-year period (2015\u20132020) a total of 20,363 mosquitoes were collected and identified in northwestern Croatia, of which 20,291 from the City of Zagreb, and 72 mosquitoes from Me\u0111imurje County. The mosquitoes belong to 11 species. Of these, in the City of Zagreb, 31.1% of mosquitoes were identified as s (5.5%) .Cx. pipiens complex pool in 2017 from Me\u0111imurje County (Prelog). All USUV positive mosquitoes were trapped between July 19 and September 7. The USUV-positive Ae. albopictus pool contained 15 mosquitoes which were collected by human landing collection on September 2 and September 7, 2016, in two adjacent yards in Zagreb. The other three positive pools comprised Cx. pipiens complex mosquitoes collected by the CO2-baited trap. In 2017, one pool of mosquitoes collected on August 19 in Prelog (Me\u0111imurje County) next to a horse stable. In 2018, a positive pool was trapped on July 19 in Zagreb, in a wood near the Jarun lake. The last Cx. pipiens complex positive pool was collected on 1 August 2019, in the center of Zagreb, near the Botanical garden  in Austria in 2016 (accession number MF063042) and Czech Republic in 2017 (accession number MN419913), respectively. In contrast, the 2019 USUV detection shared the highest nucleotide homology (99.71%) with USUV detected in 2016 in Cx. modestus mosquito from Czech Republic.Conventional RT-PCR yielded a USUV positive result for  lineage  with 99.Cx. pipiens , was whole virome sequenced by using the NGS method and a total of 817,573 (mean length of 184 nucleotides) clean reads were obtained from the sample. The BlastX and BlastN search identified 16,888 reads  belonging to viruses. Other reads belonged to cellular organisms, to other sequences and to unassigned sequences. Most of the virus sequences were assigned to viruses infecting mosquitoes. Virus sequences belonging to six different virus families and to unclassified viruses were identified, including dsDNA and ssRNA viruses of insect, bacterial and mammalian origin , sequenced in complete genome within Picornaviridae family. A closely related strain in Genbank was Culex picorna-like virus 1 strain 16-0168/ROK/2016 (MH703059) with 96.56% nucleotide identity to the determined virus. Other identified ssRNA (+) virus sequences revealed the complete genome (2409 reads) of Alphamesonivirus 1 with 99.36% nucleotide identity with Alphamesonivirus 1 strain 11/2008 (MF281710) from the Mesoniviridae family. The identified Culex mosquito virus 4 (1171 reads) which belongs to the family Nodaviridae and had 96.03% nucleotide identity with strain CMos/Santa Clara (MH188031). The presence of USUV was also confirmed (31 reads). Virus sequences with most homology to an unclassified ssRNA (\u2212) virus (916 reads) and to an unclassified RNA virus (191 reads), both of mosquito origin, were also detected in the sample.The previously determined RT-PCR USUV positive sample, which consisted of n origin . BacteriAe. albopitus pool comprising 15 mosquitoes was collected in Zagreb, the same area where the first human cases were detected in 2013, i.e., two years before the start of the mosquito screening in Croatia. In 2017, there was a small WNV outbreak involving 8 human cases from five Croatian counties. Human USUV infections were not reported at that time; nevertheless, one pool of Cx. pipiens complex mosquitoes collected in north-west Croatia  tested positive for USUV RNA. In addition, one pool of Cx. pipiens complex mosquitoes collected in Zagreb in 2018 also tested positive for USUV RNA. In 2018, additional three cases of human neuroinvasive USUV infections were reported during the largest WNV outbreak in Croatia. One USUV infected patient was a resident of Zagreb County. Furthermore, USUV RNA was detected for the first time in one dead blackbird (Turdus merula) from the same geographic area (Zagreb County) [Cx. pipiens complex pool collected in Zagreb. Phylogenetic analysis of NS5 gene of USUV detected in the two Cx. pipiens pools collected in 2018 and 2019, respectively, showed their high nucleotide homology (99.31%), both of them clustering within USUV Europe 2 lineage. Detected sequences from a fatal human case  and a dead blackbird  also belonged to the USUV Europe 2 lineage [Human neuroinvasive USUV infections in Croatia were reported in 2013, and the first detection of USUV in mosquitoes was recorded in 2016. The USUV positive  County) . USUV in lineage . This in lineage ,39.Although outbreaks or sporadic WNV infections as well as seropositivity in humans and sentinel animals (horses and poultry) have been continuously recorded in continental Croatian counties ,10,11, WAe. albopictus and Ae. japonicus [Aedes albopictus is established in Zagreb and neighboring counties [Ae. albopictus pool, whereas the other USUV positive pools were Cx. pipiens mosquitoes. Of all the tested pools, 25% were Ae. albopictus and 21% Cx. pipiens complex. Aedes albopictus is an invasive species that is rapidly adapting to temperate regions, already known as a vector of dengue, chikungunya, Zika and other arboviruses [Ae. albopictus seems to have low vector competence for USUV [Ae. albopictus pools sampled through the surveillance program of the Emilia-Romagna region  in the period 2009\u20132012 tested positive to this virus [Ae. albopicus pool in this study, it is necessary to monitor the development of this vector-pathogen association.In the Zagreb area, 32 species of mosquitoes have been recorded so far, two of them are invasive species, aponicus ,44. Aedecounties . The preoviruses ,55. It ifor USUV , numerouis virus ,31,32. TCx. pipiens complex includes two morphologically indistinguishable biotypes, Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus. Members of the Cx. pipiens complex, as well as their hybrids, represent the most important vectors of arboviruses including WNV and USUV. In the city of Zagreb, the presence of both biotypes and their hybrids is confirmed by DNA barcoding . During the research in Zagreb and Me\u0111imurje area, Cx. pipiens complex individuals were sampled by CO2-baited trap and by aspirator from walls in apartments, underground shelters, and cellars. Approximately 20 pools were overwintering mosquitoes collected during November, December, and January in underground shelters and cellars. All three USUV positive Cx. pipiens pools were sampled in open area by CO2-baited trap and all overwintering mosquitoes were negative. Nevertheless, the high nucleotide homology (99.31%) between two USUV sequences found in Cx. pipiens in 2018 and 2019 in Zagreb raises question on USUV overwintering in mosquitoes in Croatia.The Cx. pipiens pool revealed, beside USUV, several dsDNA  and ssRNA  viruses as well as two unclassified RNA viruses, namely, Hubei chryso-like virus 1 and Wuhan Mosquito Virus 8. Although these viruses were not our primary research aim, this finding presents a contribution to the knowledge on a geographical variation of the viromes in medically important mosquito vectors.The NGS results of a Detection of USUV in mosquitoes during the four consecutive transmission seasons (2016\u20132019) indicates the virus has become endemic in northwestern Croatia. Although WNV infections in humans and sentinel animals have been continuously recorded, WNV has not been detected in mosquitoes in Croatia so far."}
+{"text": "We have co-designed a tailored blended physiotherapy intervention for people with progressive multiple sclerosis (PwPMS) who often struggle to access support for physical activity. Underpinned by self-management principles, the Lifestyle, Exercise and Activity Package for people with Multiple Sclerosis (LEAP-MS) intervention incorporates face-to-face or online physiotherapy coaching sessions with an accompanying online physical activity platform. The LEAP-MS platform is a multi-user system enabling user and physiotherapist to co-create activity plans. The LEAP-MS platform consists of an information and activity suite, interactive components enabling selection of exercises into an activity programme, goal setting and activity logging. The platform also facilitates online remote support from a physiotherapist through an embedded online messaging function. We aim to evaluate the LEAP-MS platform in a feasibility trial.LEAP-MS will be evaluated within a single-arm feasibility study with embedded process evaluation. After registration and initial eligible screening, 21 participants will be required to complete baseline self-completion measures. This will be followed by an initial home-based or online coaching session with a physiotherapist  and access to the online intervention for an initial 3-month period. During this period, participants are given the option to request up to five further home-based or online physiotherapy coaching sessions. Follow-up questionnaires and semi-structured interviews will be administered 3 months after baseline with participants and intervention physiotherapists. The LEAP-MS platform will be available to participants for a further 3 months. Usage of the LEAP-MS platform will be tracked during the full 6-month period and final follow-up will be conducted 6 months after baseline.Feasibility outcomes  will be reported. The process evaluation will be undertaken to identify possible mechanisms for any observed effects. The data will inform full-scale evaluations of this co-produced, blended physiotherapy intervention.ClinicalTrials.gov, NCT03951181. Registered 15 May 2019The online version contains supplementary material available at 10.1186/s40814-021-00852-w. Multiple sclerosis (MS) is the most common disabling neurological disease among young adults  affectinIn the UK, it is estimated that 10\u201315,000 have primary progressive MS  and 38,0Physiotherapists play a central role as part of a multidisciplinary team of healthcare professionals who support people with progressive multiple sclerosis in the management of their symptoms . PrimariPhysical activity is \u2018any bodily movement produced by skeletal muscles that requires energy expenditure\u2019  and relaRegular physical activity is generally regarded to be an important component of the long-term management of MS. Positive outcomes of regular physical activity include improved mobility, strength and cognition and reduced fatigue \u201322. TherEngaging with physical activity significantly enough to benefit from such outcomes, however, relies on changing behaviours. Behaviour change theories  are therefore often selected to underpin the development and testing of physical activity interventions\u2014known as \u2018behaviour change interventions\u2019 .Although physical activity interventions can be based on a plethora of behaviour change theories, aspects of social cognitive theory and self-regulation theory , 29 are Various physical activity interventions for people with MS have been reported in the literature, ranging from group-based to digital versatile disc (DVD) and web-based interventions \u201334. A reDespite the potential benefits of physical activity and the value placed on supporting people with MS to remain active, there remains little evidence about the benefits of physiotherapy or physical activity for PwPMS who have more advanced disability . Most reDespite the limited evidence about the benefits of physical activity for PwPMS, we know that people with MS, including those with progressive MS, want to keep physically active and moving , 43. HowHere we present the protocol for the LEAP-MS single-arm feasibility trial and embedded process evaluation. Underpinned by social cognitive theory and self-regulation theory, taking a self-management approach\u2014LEAP-MS is a co-designed blended physiotherapy digital intervention . Our priThis is a single-arm feasibility study with an embedded process evaluation. Those who are eligible and consent to participate in the study will complete a series of self-completed assessments at baseline, 3 and 6 months; during this time, they will also have access to the LEAP-MS blended physiotherapy intervention  and an online platform. The online platform consists of a series of co-produced resources and functions. These include online interactive education, an activity selection and planning tool, specifically developed for PwPMS and a participant-physiotherapist messaging system. The activity selection and planning tool includes tailored physical activity ideas and interactive functions enabling the development of personalised activity programmes, goal setting and activity logs. The online platform works in conjunction with remote or face-to-face coaching sessions and facilitates remote support (via the online platform messaging system) from trained physiotherapists.We will recruit 21 participants with either primary or secondary progressive multiple sclerosis (as defined by the Lublin classification)  who are The sample size is based on the 95% confidence interval for an adequate proportion of eligible subjects being recruited (70%). The lower 95% confidence interval is 50% which is the minimum acceptable recruitment proportion.Eldrix HealthContact is a tertiary centre MS database where PwPMS who meet the inclusion criteria and who have also given consent to be contacted about research will be identified by authorised Eldrix HealthContact users. A selection of those identified  will be sent a study information sheet.Physiotherapists at the two participating Health Boards will screen all MS outpatients for eligibility during the recruitment phase. Those eligible and interested in participating will be provided with an information sheet about the study.Information about the study will be made available via the local branch of the MS Society  and the UK MS Register inviting interested participants to complete the online expression of interest.There will be three routes for informing potential participants about the study: (1) Eldrix HealthContact, (2) outpatient physiotherapy services or (3) MS Society branch and national MS register publicity in the local region.Those interested in participating will be required to complete an online expression of interest form and eligibility checklist via the LEAP-MS website. The expression of interest includes confirmation of MS diagnosis and EDSS self-completion. All participants recruited via the Eldrix HealthContact will have a consultant confirmed diagnosis of MS prior to entry into the database but recruitment via other routes relies on self-reporting. Having a self-enrolment option however is aimed at enhancing accessibility and inclusivity.We will monitor population characteristics of those who express an interest in participating . The expression of interest form will remain open until such time as eligibility has been confirmed for the entire recruited participant cohort. This will take the form of a two-stage process. First, prior to the initial target sample size being recruited, those who submit an expression of interest will receive an automatically generated response from the system. The message will thank them for their interest and explain that eligibility will be assessed in the order of expression of interest receipt and that the study team will contact them in due course. Second, once eligibility has been confirmed for the entire participant cohort and the study has closed to recruitment, the expression of interest page will disabled; however, interested individuals will be able to provide their contact details to receive study updates and results.Those who complete an online eligibility checklist and are deemed potentially eligible will receive a telephone call from the research team, to discuss what participation in the study involves and be given the opportunity to ask any questions. Their eligibility will be checked during this call and fully confirmed by their physiotherapist at the first coaching session. Those participants who are interested in the study will be directed back to the LEAP-MS website and provided with individual user details to complete an online consent form. Once the consent form has been submitted online, participants will be directed to complete the baseline assessment battery.All participants will be required to complete a range of patient-reported outcome measures directly online at baseline, 3 and 6 months post-baseline  , at 3 moAutomatic prompts will be provided to the registration email address at the start of the follow-up data collection window (2 weeks before and 2 weeks after the expected assessment completion date). Participants will receive a telephone reminder if they have not logged on in the 2 weeks prior to the expected assessment completion date. Electronic data capture will be standardised across all study remote processes using an online platform developed by the Centre for Trials Research using a bespoke Structured Query Language (SQL) database. A study-specific data management plan has been developed to ensure the security and confidentiality of all participant data and that high-quality data is available for ongoing analyses.At the end of the initial intervention period (3 months), participants and their treating physiotherapists will be asked to participate in a semi-structured interview aimed at eliciting experiences and reflections on the intervention, and the process of its delivery . Given the small sample size of this feasibility study, everyone who consents to being interviewed will be interviewed, even if they withdraw from the intervention see the \u2018\u2019 sectionThe aim of the LEAP-MS intervention is to provide improved awareness of achievable, relevant and interesting activities and exercises for PwPMS. It will also provide an opportunity for sharing experiences of participating and enable shared management and monitoring (self and physiotherapist) of activities and exercises. It is a blended physiotherapy intervention made up of (1) a co-produced, encrypted multi-user web-based platform accessible to participants and physiotherapists and (2) coaching sessions with intervention physiotherapists delivered via a secure web video conferencing system, or in person, in the participant\u2019s home. Intervention physiotherapists are trained on self-management principles and practice, use of technology in coaching sessions and physical activity and exercise guidelines for neurological conditions. As this is a blended intervention including a web-based platform, participants will require computer skills (or a carer companion who can assist them is needed) and Internet access for the duration of their study participation.The platform is specifically developed for PwPMS and includes regularly updated multimedia education about being active with PMS, tailored physical activity ideas and interactive functions enabling the development of personalised activity plans, goal setting and the monitoring of activity through activity logs. Activities are displayed in an \u2018activity suite\u2019 grouped into categories, e.g. cardiovascular, strengthening, balance, flexibility, pelvic health etc., and include a large range of exercise videos from aerobics to seated boxing to tai chi. These sit alongside other, more specific exercises described or demonstrated through text and images. Each exercise or activity suggestion can be selected and added to an \u2018activity plan\u2019, enabling participants to select individual activities to try. In addition, participants are able to set and monitor personalised goals using a goal-setting function and input all activity undertaken through the use of an activity log. A fuller description and images of the intervention are described elsewhere .The LEAP-MS platform enables access and data input options via desktop computers, laptops, tablets or smart phones. Participants, physiotherapists and the research team all have different access rights to and editing permissions for the LEAP-MS platform. Participants use the platform to register; complete eligibility forms, consent, baseline and follow-up measures; input safety information; and access the interactive education and activity selection and planning tool. They are also enabled to contact their intervention physiotherapist via a messaging function to ask questions, seek guidance or request coaching sessions. Physiotherapists use the platform to record coaching session notes, respond to participant questions and requests and view participant activity selections and goal setting. The platform is also used by the study team to evaluate participant engagement with the intervention and to manage data throughout the study.LEAP-MS participants will meet with an intervention physiotherapist up to six times, remotely or face-face in their own homes. During coaching sessions, participants will be shown how to navigate and use the LEAP-MS online platform as required, select suitable activities, form an activity plan and set and review activity goals. Drawing on both their professional knowledge and training underpinned by the Bridges self-management approach , people In the initial 3-month intervention period, participant use of the LEAP-MS platform works in conjunction with support from intervention-trained physiotherapists and includes a user pairing facility where patient users are paired with an intervention physiotherapist. The physiotherapist can view the activity selections and goals set by the patient participant and provide coaching and support to engage with participants in setting small targets and incorporating physical activity into their everyday life. The web-based platform also has an in-built messaging function to enable participants to contact their physiotherapist in the initial 3-month period to ask questions, seek guidance about activity engagement and request up to six coaching sessions. Coaching sessions will be conducted at participant\u2019s homes or online dependent on participant preference whilst accommodating any local restrictions . Paper-based learning materials traditionally accompanying face-face training workshops and video-recorded films from the training have been digitised and housed in an online learning platform for use by intervention physiotherapists to refer back to as required. Given the emergent challenges in rehabilitation service delivery during the COVID-19 pandemic, and the anticipated move to greater use of remote intervention delivery, further resources (https://www.bridgesselfmanagement.org.uk/covid-19-resources/) to help structure remote interactions were also made available as part of the final training package to ensure standardisation of coaching interactions regardless of the mode of delivery (online or face to face in the home). In addition, an accompanying online training resource included video-recordings with experienced physiotherapists with expertise in the use of digital technologies in practice to provide guidance on the use of technology in coaching sessions and videos with an expert in exercise prescription for people with neurological conditions to teach core principles of exercise physiology and prescription [All intervention physiotherapists will receive bespoke LEAP-MS training, which delivers (1) real-time training (either face-face or remotely) about self-management principles and how to integrate these into contacts with patients, (2) how to use online conferencing as a method of communication and providing consultations, (3) how to introduce/instruct patients in the use of online conferencing and online resources (in this case the LEAP-MS platform) and (4) a clinical update on physical activity and exercise guidelines for use with people with neurological conditions. The training package consists of real-time training days (delivered face-face or remotely) and an online self-study resource. The real-time self-management training workshops are delivered by experienced facilitators from Bridges Self-Management . They will also have the opportunity to practice coaching conversations and receive peer review. They will be asked to take part in interviews to share their experience of intervention training and intervention delivery.We will assess and record any adverse events that may be reported.In this patient population, hospitalisation due to MS, acute illness resulting in hospitalisation, new medical problems and deterioration of existing medical problems are expected. This information will be self-reported by patients online and will not be subject to expedited reporting; however, it will be reviewed on a monthly basis by the study team. The physical activity intervention does not specifically involve any heavy load-bearing exercise or heavy eccentric muscle activity. However, some minor muscle soreness or muscular strain may occur in the few days following the initiation of a new exercise programme or increased physical activity. This would normally resolve spontaneously and would not require any specific interventions or additional medical care but will be noted as a potential expected related AE if reported during the 3-month intervention period. Falls and fatigue are an expected AE as part of the clinical condition but will be monitored for the duration of the intervention.Participants will be asked to use the LEAP-MS platform to self-report any incidents of falls, fatigue, increased muscle soreness or sprain, or other incidents they feel are relevant, and whether the incident required medical intervention. Selecting that medical intervention was required will trigger an automated prompt to the paired physiotherapist. Similarly, no activity on the online platform for 3 weeks will also trigger an automated prompt to the paired physiotherapist. Once prompted, the paired physiotherapist will contact the participant to discuss the incident. All serious adverse events (SAEs) that occur between the time of consent and the 3-month follow-up must be reported immediately to the Centre for Trials Research (within 24 h of knowledge of the event) by the intervention therapist using a dedicated SAE form, unless the SAE is specified as not requiring immediate reporting.Analyses will be guided by the CONSORT extension for pilot and feasibility studies . The priIf at the end of the study the feasibility progression criteria are achieved, then the recommendation would be to move to a randomised evaluation. Modifications in the trial processes or the intervention may be required if progression criteria are not fully achieved. If there is not an identifiable reason or remediable action that can be taken, then progression to a full trial would not be recommended.Intervention uptake and safety are not formal progression criteria in this single-arm feasibility study but will be closely monitored and considered in any final recommendations for further evaluations. Intervention uptake will be reflected by (1) the percentage of initial coaching sessions completed, the number of additional physiotherapy coaching sessions requested and completed and the number of remote physiotherapist contacts recorded and (2) frequency and duration of weekly logged physical activity. Website log in rates and length of time between each log in episode will provide supplemental information on intervention uptake. Safety will be assessed using an online process of self-reporting by the participant and from any SAE forms completed by the intervention therapist.The process evaluation will enable an understanding of the acceptability and fidelity of the intervention, identify possible mechanisms for any observed effects and learn about any adaptations made by the participants (PwPMS and physiotherapists) in undertaking the programme. Acceptability assessment will focus on content, design, language and adaptability to personal needs. It will be assessed through the analysis of any remote contact between participants and physiotherapists as well as semi-structured interviews (conducted either face to face or via the secure web video conferencing system) with participants who completed the intervention, those who withdraw from the study and physiotherapists who deliver the intervention. In physiotherapist interviews, we will collect detailed demographic information so we can understand how their characteristics  influence their approach, experience of the training and delivering the intervention.Fidelity of the LEAP-MS intervention delivery will be assessed using independent analysis of audio-recorded and/or observed (sampled) sessions, analysis of participants\u2019 online activity logs and participant-physiotherapist communications. Six initial coaching sessions  and between 5 and 10 follow-up coaching sessions (dependent on participant consent) will be observed and audio-recorded if conducted face-face. Coaching sessions conducted via web video conferencing will be recorded using an in-built recording function.Actions observed during face-face observations will be captured using basic proxemic sketches (stick people drawings) and kinesics , alongside standard ethnographic notations (the description of actions/happenings as the observer sees them) . The proThis range of data collection methods has been selected to enable a comprehensive and multi-faceted description of the experience of those taking part, a nuanced understanding of intervention delivery and usage. The multiple methods selected will also act as a robust form of triangulation.All qualitative data collection for the process evaluation will be carried out by a research team member with qualitative research experience, not involved in intervention delivery. All data will initially be separated into their \u2018type\u2019, i.e. speech, text or action (observation), with appropriate methods of analysis applied to each type of data. Interviews, recorded coaching sessions and observations will be analysed thematically initially . Should Due to the focus of this process evaluation on the use of each component of the intervention, the personal documentation will be initially subjected to content analysis\u2014with a more detailed thematic analysis applied if required , to assiFindings will be separated into process and outcome data ready for reporting but with consideration being given to where/if the doing of any process is a major contributor to the outcomes or perceived experience of participants. At this point, we will explore possible mechanisms for any observed effects (both for the person with MS and the intervention physiotherapist) so as to validate change objectives, behavioural outcomes and patient-reported intermediate and longer-term outcomes as depicted in the proposed intervention logic model Fig. . There aThe LEAP-MS platform is a multi-user system enabling participants and physiotherapist to co-create activity plans. The LEAP-MS platform consists of an information and activity suite, interactive components enabling selection of exercises to create an activity programme, goal setting and activity logging. The platform also facilitates remote support from a physiotherapist through an embedded online messaging function. Our experience here lays the basis for the development of multi-user platforms that can be adapted according to population and trial design.As the secure, encrypted multi-user web-based platform will be accessible to participants, physiotherapists and researchers with data input options via desktop computers, laptops, tablets or smart phones, participants are able to use the platform to register; complete eligibility forms, consent, baseline and follow-up measures; and access the intervention. Participants, physiotherapists and research administrators all have different access and editing level permissions within the platform. It is also used by the study team to evaluate participant engagement with the intervention and to manage data throughout the study.Our ambition has been to co-design a model for physical activity self-management support for PwPMS that is patient-, family/carer- and community-centred with physiotherapists providing a unique role as a coach and partner throughout the whole disease trajectory . Self-maUnlike other physiotherapy-based online activity platforms for other conditions or general education platforms, the LEAP-MS platform has a paired account function in which people with MS can be paired with their physiotherapist. Critically, rather than the physiotherapist selecting and prescribing activities, the person with MS has complete choice and control of this process. The physiotherapist can view participant activity logs, but advise only as required by the person with MS. Furthermore, the patient-facing element of the LEAP-MS intervention platform combines multimedia educational content, activity provision, activity monitoring and goal setting. It includes an online hub for physiotherapists, which draws together self-management training and provides a space for multimedia exercise in long-term neurological conditions.Evaluation of feasibility, including intervention uptake as measured by login rates and duration, and acceptability in terms of content, design, language and adaptability to personal needs will inform modification and future evaluation. Findings from the feasibility study will be disseminated to participants, healthcare professionals and the public via a series of outputs. These include a lay summary of findings to be sent to participants and published on university and funder websites for public viewing, formal research reports, peer-reviewed publications and conference papers to share findings with healthcare professionals.ClinicalTrials.gov and in peer-reviewed literature.The trial is sponsored by Cardiff University (resgov@cardiff.ac.uk) and is set up. Recruitment will commence on 01.06.2020 and is anticipated to end on 30.10.2020. This manuscript has been drafted according to version 1.1 (12/05/2020) of the trial protocol. The protocol has been written according to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) statement  Checklist"}
+{"text": "The LEAP-MS  study has developed an individualised supported self-management approach for physical activity for people with progressive multiple sclerosis (MS) and severe disability. The intervention has been evaluated in a single-arm feasibility study with embedded process evaluation. The feasibility study was due to open to recruitment during the COVID-19 2020\u20132021 pandemic, 1\u00a0month into the first UK-wide lockdown. We worked rapidly to implement adaptions to the trial procedures and intervention delivery that we believe are applicable to randomised controlled trials.Recruitment became predominantly via self-referral. Electronic consent was employed, with consent discussions occurring over the telephone. Registration, consent, eligibility assessment and data collection as well as the intervention  were via a secure, encrypted multi-user web-based platform for participants, physiotherapists and researchers accessible via various hardware. Physiotherapy consultations, as well as the process evaluation, were conducted remotely using video conferencing software or the telephone. A remote training package for physiotherapists and site initiations was also developed and electronic site files employed.Our adaptions are extremely topical given the COVID-19 situation, and whilst not what we had originally planned, have enabled successful delivery of the feasibility study and are relevant to conducting randomised controlled trials and meeting the needs of people with MS who are far more isolated than ever before.ClinicalTrials.govNCT03951181. Registered on 15 May 2019. LEAP-MS  aims to develop and evaluate an individualised supported self-management approach for physical activity with a specific focus on people with progressive multiple sclerosis (PwPMS) and severe disability.Multiple sclerosis (MS) is a long-term deteriorating condition which causes a range of symptoms like blurred vision and problems with how people move, think and feel affecting an estimated 107,000 people in the UK  and 38,0Regular physical activity is generally regarded to be an important component of long-term management of MS , 7. PhysDespite the possible benefits, we know that people with MS want to be physically active , 17. In In MS there is an additional need for health professionals to really understand the condition and have the knowledge and skills in relation to self-management of physical activity . As suppIn phase 1 of the LEAP-MS study, we collected information about the barriers to and facilitators of physical activity that PwPMS experience, their current levels and type of physical activity and their perceptions of the role physical activity plays in managing MS symptoms from both them and their families\u2014or people that support them. This provided us with important information about why physical activity might be important for PwPMS, the challenges they face in doing physical activity or accessing it, and ways which they have found to overcome any barriers. We also collected information from physiotherapists about their understanding of self-management and their needs for training about using self-management approaches with PwPMS.We used this information gathered in Phase 1 to work with PwPMS and physiotherapists to co-design the LEAP-MS personalised intervention to facilitate on-going physical activity for people with PwPMS. The outcomes achieved through the collaboration have exceeded expectations through the co-production of an online education and activity platform, a patient-led approach to consultations, an online hub providing resources for clinicians delivering research protocols and a novel online-only evaluation method (paperless) for clinical evaluation of the co-produced interventions.A multi-user web-based online physical activity toolUp to six physiotherapy consultations (coaching sessions)A training package for physiotherapists about self-management with PwPMSThe intervention is made up of:Further details of the intervention development are\u00a0published separately .We sought to evaluate the feasibility and acceptability of the intervention and trial procedures in a single-arm feasibility study with embedded process evaluation . The stuThe study had ethical approval  and sought R&D approval from three NHS Health Boards in Wales to recruit 21 participants via three recruitment routes, namely (1) a MS research database hosted in the NHS; (2) referral from NHS outpatient physiotherapy services; (3) self-referral from within Health Boards. Recruitment routes 1 and 3 required the potential participant to register their interest in the study via the LEAP-MS website, self-complete an online consent form, initial eligibility screen and baseline self-completion measures. For participants recruited via route 2 in physiotherapy outpatient clinics, written informed consent, eligibility assessment and baseline measures would be undertaken at the clinic in person. In all cases, consent and eligibility would be reconfirmed at the initial home-based (face-face) coaching session by a physiotherapist, who had received tailored intervention and study procedure training. Eligible patients had either primary or secondary progressive multiple sclerosis (as defined by the Lublin classification) , were agModified form of the Fatigue Impact Scale (MFIS)  to assesMultiple Sclerosis Impact Scale (MSIS-29) to measure the physical and psychological impact of MS from the patient\u2019s perspective ;EQ-5D-5\u2009L to provide an indication of health-related quality of life ;Oxford Participation and Activities Questionnaire (OxPAQ) to assess the impact of ill-health on participation, activities and autonomy ;University of Washington 6-item short form self-efficacy scale (UW-SES-SF) (MS specific) to indicate self-efficacy ;Modified Patients\u2019 Global Impression of Change (PGIC)  at 3\u00a0monFollowing the initial coaching session, the participants would have been given access to the online intervention for an initial 3-month period. During this period participants would be able to request up to five further home-based physiotherapy coaching sessions, after which the LEAP-MS platform without physiotherapy support would be available to participants for a further 3\u00a0months. Follow-up was scheduled for 3-months and 9-months post-baseline. The following outcome measures were to be self-completed by the participants and collected using the online study platform at baseline and both follow-up timepoints:Semi-structured interviews were going to be conducted in person with participants 3\u00a0months after baseline. Consenting intervention physiotherapists were also going to be interviewed in person once all their participants had received the intervention for the initial 3-month period. Usage of the LEAP-MS platform was going to be tracked during the 6-month intervention period. Intervention physiotherapy notes were intended to be completed on paper, as was the eligibility assessment undertaken by the physiotherapist and any safety reporting case report forms. The intervention coaching sessions were going to be observed in person by the study\u2019s qualitative researcher. Costs associated with intervention delivery (therapist travel and contact time) were also going to be recorded by the physiotherapists.The COVID-19 2020/2021 pandemic  and the Given the impending start of recruitment we worked rapidly to implement amendments and succeeded in securing a 6-month no-cost extension from the funder (MS Society). The following amendments to the feasibility study protocol were classed as non-substantial amendments under the guidance issued by the Health Research Authority  and the As the intended research sites were unable to open to recruitment due to staff redeployment and revised NHS priorities, non-NHS research sites were set up. Physiotherapists were utilised from within the project team to deliver the intervention, so not to draw resources from the NHS. NHS research sites were able to open to recruitment once recruitment to non-COVID-19 clinical trials and studies resumed.http://www.bridgesselfmanagement.org.uk/), in which ten physiotherapists participated in prior to the COVID-19 pandemic. The workshop was video recorded and utilised as part of the final online training package. Further resources to help structure remote interactions (https://www.bridgesselfmanagement.org.uk/covid-19-resources/) were made available to standardise coaching interactions regardless of mode of delivery. Site initiation training in study procedures was delivered remotely via Zoom [Time was invested in developing a bespoke LEAP-MS physiotherapy training package that focused on the provision of self-management support to participants, the use of technology in consultations, updates on physical activity and exercise guidelines for long-term neurological conditions, what to expect when using the LEAP platform along with potential challenges and solutions. The initial training package was developed through a two-day face-face interactive workshop underpinned by Bridges Self-Management principles and delivered by Bridges Social Enterprise , save a local copy of the consent form and email it to the LEAP-MS email address. A copy was also saved in the electronic investigator site file.Physiotherapists who attended the intervention training and site initiation were emailed the physiotherapist information sheet and a link to the online consent form. They were asked to complete an informed consent form using Cardiff online survey software  coaching session and recorded by the physiotherapist on an electronic case report form accessed from the online platform.Virtual physiotherapy consultations using web conferencing software (Zoom ) or teleThe follow-up period was reduced from 9-months to 6-months and the economic evaluation was removed to streamline data collection.We developed a secure, encrypted multi-user web-based platform for participants, physiotherapists and researchers accessible via desktop computers, laptops, tablets or smart phones. Participants were able to use the platform to register, complete eligibility forms, consent, baseline and follow-up measures as well as to access the intervention. The physiotherapist-completed case report forms were incorporated into the online platform, with the added functionality of being able to download the therapy notes to facilitate incorporation into the patients\u2019 NHS medical records. Participants, physiotherapists and administrators all had different access and editing level permissions. As participants entered their own data most of the data could not be queried; to limit errors and ensure quality data inbuilt database validations were used including using input masks, restricted data formats and mandatory data fields. Effort was made to make the platform visually appealing, easy to navigate and allowed one measure/case report form to be completed and saved at a time. The platform was also used by the study team to evaluate participant engagement with the intervention and to manage study data.The process evaluation also had to be undertaken remotely; recorded Zoom coaching sessions were used for the observations and the qualitative interviews were conducted over Zoom or telephone.The use of an electronic study master file and electronic investigator site files were also employed. Microsoft One Drive and password protected files on secure shared drives on University servers were used.Efficiencies have been achieved in multiple areas relevant to the delivery of the LEAP-MS study. Most notably flexibility in intervention delivery reduced burden on the NHS and ensured that we were able to successfully recruit, consent, deliver assessments and evaluate outcomes in a group of PwPMS who were increasingly isolated given the COVID-19 lockdown restrictions. In our view, the availability of an online training package and the conduct of remote site initiation was key to the success we observed in rapidly opening sites. Some of the physiotherapists who were relatively new to research required additional assistance and support when undertaking the study processes. This may not have been the case had training been delivered face-face. Whilst remote training may reduce site buy-in and rapport due to the limited personal contact, we would recommend that the mode of delivering the training and site initiations should be considered on a study-by-study basis.Advertising the study to potential participants via MS Society communication channels proved highly efficient providing greater reach as expressions of interest became independent of Health Board and gave scope for increased generalisability. It was however limited in that the study team were not able to ensure clinical confirmation of the EDSS score. This was dealt with through an additional eligibility screen conducted by the central study team followed by a further physiotherapist eligibility assessment at the first physiotherapy consultation.We used an electronic trial master file and site files and developed a system to enable the electronic registration and consent process. A separate study visit was thus not required to obtain informed consent thereby reducing both staff and participant burden. We found that approximately a third of participants required explicit instructions and a link to the website/consent form area, before they were successfully able to access the website and complete the online consent form. This may have been compounded by the patient population as impaired cognitive and memory function are common symptoms of MS; however, we would recommend building an automated email function into the online platform to notify participants when the consent form is released to them for completion.The LEAP-MS database acts as the source consent documentation and will be retained for the required archiving period in line the requirements for archiving the electronic data set, and thereby also being accessible in case of a statutory inspection(s). The LEAP-MS patient information sheet explained that University staff would have access to their data and the completed consent form, and explicit consent was sought for this as one of the statements on the consent form. A copy of the completed consent form was downloaded and saved in the electronic investigator site file for retention by the site. The participant had access to their completed consent form through the online platform. We are considering how to enable the participant to have long-term access to the completed consent form. The intention for future iterations of the platform is to enable participants to download, save and/or print their completed consent form. For this study, we intend to download and post the completed consent form to the participant\u2019s home so they have a copy once access to the platform is removed.The e-consent process and system used in LEAP-MS exceeds the ethical and legal requirements for e-consent recommended for \u2018other types of research\u2019 as described in the joint statement on seeking consent by electronic methods ; it alsoDue to the COVID-19 pandemic there has been an expedited move towards telemedicine and remotely delivered rehabilitation , 41. ThiTechnical issues were experienced in a number of consultations relating to internet connectivity, sound and video quality as well as user capability and confidence; two participants ended up having their first consultation over the telephone, one of these participants also had their second consultation over the phone. Attend Anywhere  was initParticipants experienced a shorter waiting time (mean of 3\u00a0weeks) for their initial physiotherapy appointment compared to typical home visit waiting times of up to 8\u00a0weeks . This waThe online intervention platform has been future-proofed in that recommendations for activities not allowed due to COVID-19 pandemic social restrictions have been blocked but were included in the build so that access can be easily allowed in future. LEAP-MS has also featured in the NIHR remote delivery guidance as an example of remote delivery of complex interventions .Although this study is a single-arm feasibility study, the database has been developed so that it can be efficiently repurposed for a main randomised controlled trial. The participant self-report outcome measures used in LEAP-MS were intended to be collected remotely prior to the COVID-19 pandemic using the online platform. The database had in-built validations to ensure high-quality data and efficient data management. Limited data querying and data cleaning could be conducted as participants\u2019 self-completed many of the measures. Data management tasks focused primarily on prompting participants to complete the forms. Initial prompts were conducted by email 2-weeks and 1-week before follow-ups were due, however, the number of completed forms improved when the email prompts were accompanied by telephone calls. This may in part be due to the patient population but could also be due to external factors effecting participants\u2019 mood, such as the follow-ups coinciding with increased COVID-19 related social restrictions and the local and national lockdowns of autumn and winter 2020 , 32.Some technical issues were experienced with the database, mainly that the data collection forms were locked part way through two participants completing their baseline measures, and three participants were able to complete their 6-month follow-up prior to their intended follow-up window. These errors were rectified as soon as the central team became aware. Although standard database testing was employed, the scheduling of access to forms was not initially tested, and this experience highlights the necessity for thorough testing prior to database release, especially when participants are entering their data themselves.The physiotherapist-completed case report forms were incorporated into the online platform due to the COVID-19 implications. A benefit of this, in addition to reduced data entry and reduced printing and stationary costs, was the added functionality of being able to download the therapy notes. Although this function was not used during this study, it will facilitate incorporation into the patients NHS medical records in the future.In LEAP-MS, we have developed an entirely novel approach to remote participant enrolment, consent and assessment as well as a more comprehensive, interactive and adaptable intervention than was originally planned, which can be delivered remotely. Both of which are very topical given the COVID-19 situation and our evaluation, whilst not what we had originally planned, is highly relevant to meeting the needs of people with MS who are far more isolated than ever before. Pending our evaluation in this single-arm study, we propose a follow-on randomised feasibility trial  or a plan to progress to a full effectiveness evaluation with internal pilot to assess willingness to be randomised should only minor modifications be required."}
+{"text": "The aim of this cross-sectional study was to explore the relationship between quality of life (QoL) and body image distress in patients with head and neck cancer (HNC), considering relevant psychological variables . We also aimed to explore gender differences in patients with HNC in terms of relevant psychological variables in HNC.Fifty-one HNC patients  completed self-report questionnaires to assess body image distress, physical and mental QoL, and relevant psychological variables in HNC  before undergoing treatment. Pearson\u2019s correlations and four-step hierarchical regressions were performed to assess the relationship between body image distress, QoL, and the abovementioned psychological variables, while one-way analyses of variance and one-way analysis of covariance were employed to assess gender differences.Physical QoL was associated with body image distress above and beyond disease duration, distress, coping strategies, pain, mental QoL, and self-esteem, while mental QoL was associated with pain above and beyond distress, coping strategies, physical QoL, self-esteem, and body image distress. Concerning gender differences, females scored higher than males on most of the explored psychological variables, except for physical QoL and intolerance of uncertainty, and showed lower mental QoL and self-esteem than males.Body image distress and pain emerged as negatively associated with QoL, and almost all the explored psychological variables differed among genders. Psychological interventions targeting body image distress and pain should be promoted in patients with HNC to increase their QoL, while keeping gender differences in mind.The online version contains supplementary material available at 10.1007/s00520-022-07334-6. Body image is the internal representation that individuals have of their own body and physical appearance . In receBody image distress due to HNC is associated with a variety of life changes   and may In accordance with the conceptual framework of Rhoten and colleagues , patientGender differences pertaining to body image distress and other relevant psychological variables before undergoing HNC treatment deserve more attention as well. Indeed, few studies are available on this topic and inconsistent results emerged from studies conducted after HNC treatment . The stuThe first aim of this cross-sectional study was to investigate the relationship between QoL and body image distress in patients with HNC. To explore this relationship, we focused on the preoperative period to obtain estimates of body image distress associated with the anticipation of a potentially disfiguring treatment . PreviouTo shed light on gender differences before HNC treatment, the second aim of the study was to explore gender differences in body image distress, QoL, and associated psychological variables . In accordance with evidence points to higher rates of certain psychological conditions in women, especially related to body image , we hypoTo our knowledge, no previous studies have considered all these relevant psychological variables to deepen the relationship between QoL and body image distress in patients with HNC. A better understanding of the relationship between body image, physical and mental QoL, and relevant psychological variables in HNC will allow clinicians to better understand how patients react to disfigurement and dysfunctions related to HNC and its treatment. Findings of this cross-sectional study may therefore have important implications for early identification and treatment of body image distress in patients with HNC that can potentially improve the QoL of these patients .This is a cross-sectional study: participants completed self-report questionnaires before undergoing a medical treatment for HNC (no other assessments occurred).Patients with HNC who were about to receive a medical treatment approximately in a month  were asked to take part in the study. Eligible patients were identified during multidisciplinary HNC visits at the Radiotherapy Unit of the Veneto Institute of Oncology (IOV) in Padua . Eligible criteria included patients who 1) were older than 18\u00a0years, (2) were diagnosed with a HNC, (3) were about to receive a medical treatment for HNC , (4) were not receiving medical treatments for other cancer diseases at the time of the research, and (5) were competent to provide informed consent. No restriction was placed on the type of treatment participants received/tumor sites for inclusion in the study. The only exclusion criterion was the presence of a benign neoplasm localized on the head and neck anatomical district . Based on such criteria, 51 patients  were considered eligible and were enrolled for the study , or at home. Two modalities of compilation were offered since most patients were willing to participate in the study but were distress after the HNC visit. When patients preferred the in-person modality, they completed self-report questionnaires in a quiet room at the IOV, specifically arranged for the self-report questionnaires compilation; the clinical psychologist was available to answer any inquiry during the filling process. The time for the compilation was approximately 40\u00a0min. When patients preferred the at-home modality, the clinical psychologist provided self-report questionnaires and a sealing envelop and instructed patients to complete the questionnaires at home and to take them back in the sealing envelop the day of the next HNC visit (approximately 2\u00a0weeks after). Patients did not receive any compensation for their participation. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Psychological Research of the University of Padua.ocio-demographic information form: employed to assess socio-demographic information of participants such as sex , gender , age, education, and self-reported psychological disorders.SPersonal medical history: information about participants\u2019 medical history was collected through the electronic medical record of each patient, including cancer history (presence of recurrence and previous treatments for HNC), time of diagnosis, stage and localization of disease, and medications.Body Image Scale : se: seBody Short Form-12 Health Survey : a brief version of the SF-36 health survey. The SF-12 evaluates eight dimensions related to an individual\u2019s life that can be influenced by the presence of a disease. Answers can be provided through dichotomous yes/no answers, or through items on a 3- or a 5-point Likert scale. In addition to the eight dimensions, the SF-12 produces two summary scores evaluating physical and mental health. Higher scores are associated with higher QoL. The Italian version of the SF-12 showed good content, construct, and criterion validity [validity . For theRosenberg Self-Esteem Scale : measure made up of 10 items rated on a 4-point Likert scale (from 1\u2009=\u2009\u201cstrongly disagree\u201d to 4\u2009=\u2009\u201cstrongly agree\u201d) assessing global self-esteem, with higher scores indicating greater self-esteem. The Italian version of the RSES showed good psychometric properties: its internal consistency was \u03b1\u2009=\u20090.84 [SES; 32; ): measurIntolerance of Uncertainty Scale-12 : 12-item self-report measure evaluating the tendency to find uncertainty upsetting and distressing. Individuals are asked to rate the extent to which each statement applies to themselves on a 5-point Likert scale , with higher scores indicating greater IU. The IUS-12 demonstrated excellent internal consistency (Cronbach\u2019s \u03b1\u2009=\u20090.80), convergent, and discriminant validity in its Italian version [-12; 34; ): 12-iteSocial Interaction Anxiety Scale : 19-item self-report questionnaire designed to assess social interaction anxiety on a 5-point Likert scale , with higher scores indicating greater social anxiety symptoms. The Italian version of the SIAS proved to be highly reliable (Cronbach\u2019s \u03b1\u2009=\u20090.86) [IAS; 36; ): 19-iteCoping Response Inventory Adult Form : self-report questionnaire made up of 48 items assessing approach  and avoidance coping . Each subscale is composed of six items rated on a 4-point Likert-type scale . Participants had to think specifically about how they cope with the diagnosis of HNC when replying to the items of the questionnaire. The administration of the Italian version of the CRI-Adult showed internal consistency values ranging from \u03b1\u2009=\u20090.58 to \u03b1\u2009=\u20090.68 for the approach scales, and ranging from \u03b1\u2009=\u20090.57 to \u03b1\u2009=\u20090.66 for the avoidance scales [ult; 38; ): self-rDepression Anxiety Stress Scale-21 : 21-item self-report questionnaire assessing depression, anxiety, and stress on a 4-point Likert scale , with higher scores indicating greater distress. The Italian version of the DASS-21 proved to be highly reliable : 21-ite-21; 40; ): 21-iteBrief pain inventory : 15-item self-report questionnaire designed to measure pain intensity and impact of pain on daily functioning. The BPI allows to calculate a total score and two specific scores (pain severity and pain interference) on a 10-point Likert Scale , with higher scores indicating higher pain severity and interference. The Italian version of the BPI showed good psychometric properties [BPI; 42; ): 15-iteTo investigate the relationship between body image distress (BIS) and physical (PCS-SF-12) and mental (MCS-SF-12) QoL, preliminary Pearson\u2019s correlation analyses were performed. Correlations between age, disease duration, and self-report measures  were performed on the whole sample to identify variables to include in the regression model see . Based op value of 0.006 for the CRI-Adult questionnaire and of 0.025 for the SF-12. Partial Eta Squared (\u03b7p2) values were reported to evaluate the magnitude of effects. Cohen [\u03b72\u2009=\u20090.01), medium (\u03b72\u2009=\u20090.06), and large (\u03b72\u2009=\u20090.14) effects when partial eta squared are computed.To assess gender differences on socio-demographics , HNC-related variables, psychological variables, Chi-squared analyses \u03c72), one-way analyses of variance (ANOVAs), and one-way analysis of covariance (ANCOVA), one-ways. Cohen  has prov2) of 0.13, with a type I error of 0.05.A post-hoc analysis of the sample size was established using the G*Power 3.1 software . The varLess than 0.5% of the total dataset was missing. Given the minimal missing data, these were replaced using the mean replacement method .All statistical analyses were conducted using IBM SPSS statistics , versionF\u2009=\u20091.81, p\u2009=\u20090.18). The inclusion of coping strategies in the second step of the model did not explain an additional variation (13.6%) in physical QoL (PCS) , despite alternative reward coping strategy (CRI-Adult) emerged as significant (p\u2009=\u20090.04) . The inclusion of mental QoL (MCS) and pain (BPI) in the third step of the model did not explain an additional variation (10.3%) in physical QoL (PCS) , despite the step emerged as significant (p\u2009=\u20090.03). Finally, the inclusion of self-esteem (RSES) and body image distress (BIS) explained an additional 17.3% of the variation in QoL (PCS) . Results showed that body image distress (BIS) was the only variable significantly associated (negatively) with physical health (PCS), whereas all the other variables were not  of the variance in physical QoL (PCS). Disease duration and general distress DASS-21) were entered in the first step, but were not significantly associated with physical QoL (PCS) \u2009=\u200918.95, p\u2009<\u20090.001). The inclusion of coping strategies in the second step did not significantly explain an additional variation (4.1%) in mental QoL (MCS) . Physical QoL (PCS) and pain (BPI), entered in the third step of the regression model, significantly accounted for mental QoL (MCS), F change\u2009=\u20095.48; p\u2009=\u20090.01, explaining an additional 15.6% of the variation. Finally, the inclusion of self-esteem (RSES) and body image distress (BIS) did not significantly explain an additional variation (1.2%) in mental QoL (MCS) . Results showed that pain (BPI) was the only variable significantly associated (negatively) with mental QoL (MCS), whereas all the other variables were not  of the variance in mental QoL (MCS). General distress (DASS-21), entered in the first step of the regression model, emerged as significantly associated (negatively) with mental QoL (MCS) , while age differed: males were older than females (p\u2009=\u20090.02). In terms of HNC-related variables, no gender differences emerged .No significant differences between genders with respect to years of education emerged (ps\u2009>\u20090.05). As a covariate, age was significant for both CRI-Adult positive reappraisal \u2009=\u20098.46, p\u2009=\u20090.01, \u03b7p2\u2009=\u20090.15) and alternative rewards \u2009=\u20095.73, p\u2009=\u20090.02, \u03b7p2\u2009=\u20090.11) , social anxiety symptoms (SIAS), general distress (DASS-21), CRI-Adult emotional discharge, and pain (BPI) and scored lower than males on mental QoL (MCS) and self-esteem (RSES). No significant differences emerged with respect to physical QoL (PCS), intolerance of uncertainty (IUS-12), and other CRI-Adult subscales . WithinDespite such interesting results, our research is not free from limitations. First of all, the small number of participants may have affected the accuracy of the results and does not allow the generalization of the results obtained. Therefore, results of the current study should be interpreted with caution. However, HNC is a rare oncological disease, affecting 18 people per 100,000 inhabitants in Italy . Future Despite the abovementioned limitations, the current study is, to our knowledge, the first of its kind, since no previous research has analyzed neither the relationship between body image distress and QoL in HNC while considering relevant psychological variables, nor exhaustively examined gender differences in psychological variables among patients with HNC. Indeed, psychological research in the area of HNC is considered to be in its infancy, and this study provides important insights into psychological variables that might be related to QoL in HNC patients. At the same time, findings of this study may have important clinical implications for early identification and treatment of body image distress and pain in patients with HNC, with the ultimate goal of enhancing the QoL of these patients, guiding the development of a patient-tailored care.Supplementary file1 (DOCX 22.3 KB)Below is the link to the electronic supplementary material."}
+{"text": "COVID-19 infection in the pediatric population usually leads to a mild illness; however, a rare but serious complication of MIS-C has been seen in children. MIS-C usually presents 2\u20134 weeks after COVID-19 infection or exposure, and rare reports have been documented in neonates. Vaccinations for COVID-19 have been approved for children aged 6 months and above in the United States, and recent reports suggest significantly low prevalence and risk of complications of Multi-organ Inflammatory Syndrome (MIS-C) in vaccinated children compared to unvaccinated children. Vaccinations for COVID-19 are safe and recommended during pregnancy and prevent severe maternal morbidity and adverse birth outcomes. Evidence from other vaccine-preventable diseases suggests that through passive transplacental antibody transfer, maternal vaccinations are protective against infections in infants during the first 6 months of life. Various studies have demonstrated that maternal COVID-19 vaccination is associated with the presence of anti-spike protein antibodies in infants, persisting even at 6 months of age. Further, completion of a 2-dose primary mRNA COVID-19 vaccination series during pregnancy is associated with reduced risk for COVID-19\u2013associated hospitalization among infants aged 6 months or less. Therefore, it can be hypothesized that maternal COVID-19 vaccination can reduce the risk of and severity of MIS-C in infants. In this article, we review the literature to support this hypothesis. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes relatively mild illness in children as compared to adults, and children usually recover without any adverse clinical course ,2. DurinAn increasing number of MIS-C cases in infants 0\u20136 months of age are being reported and most of the data is emerging in the form of case reports and case series. As per the latest systematic analysis, a total of 90 cases of MIS-C and MIS-N were identified in 0\u20136-month-old infants . HoweverThe COVID-19 vaccine is approved for children 6 months of age and above in the United States and it decreases the severe complications of SARS-CoV-2 infection, hospitalization, and death . There iOur search was aimed at identifying studies (1) evaluating transplacental transfer of antibodies to the newborn following maternal COVID-19 vaccination, and (2) evaluating the impact of COVID-19 infection-related hospitalization and MIS-C incidence in vaccinated children and infants born to mothers who were vaccinated during pregnancy. Data for this review were identified by searches of MEDLINE, EMBASE, SCOPUS, Google Scholar, Science Citation Index, and references from relevant articles using the search terms \u201cmaternal\u201d, \u201cvaccine\u201d, \u201cvaccination\u201d, \u201cBNT162b2\u201d, \u201cPfizer-BioNTech\u201d, \u201cmRNA-1273\u201d, \u201cmRNA\u201d, \u201csevere acute respiratory syndrome coronavirus 2\u201d, \u201cSARS-CoV-2\u201d, \u201cCOVID-19\u201d, \u201c2019-nCoV\u201d and \u201ccoronavirus\u201d. Only articles published in English from inception to 18 May 2022 restricted to humans, and directly related to this review were included.Our search yielded 19 studies that have thus far evaluated the transplacental transfer of antibodies after maternal vaccination . Most ofThe next question relates to the durability of these antibodies. Two of the studies evaluated the duration for which the transplacentally transferred maternal anti-S IgG antibodies were present in the infant. Shook et al. (2022) studied 77 vaccinated pregnant women and 12 pregnant women with SARS-CoV-2 infection during pregnancy and tested the durability of these antibodies in both of these groups . VaccinaOur search yielded 10 studies in this section, including 5 case-control studies, 3 population-based surveillance studies, 1 prospective cohort, and 1 patient registry. Most of the studies are case-control studies from the United States.Between June 1 and September 30, 2021, during Delta variant predominance, 2 doses of BNT162B2 were effective in reducing 93% of COVID-19-related hospitalizations among patients aged 12\u201318 years . CompariReceipt of 2 doses of BNT162b2 vaccine showed protection against MIS-C, which is a life-threatening complication of SARS-CoV-2 infection in children aged 12\u201318 years (July\u2013December 2021) with estimated effectiveness of 91% against MIS-C, and along with this, MIS-C complications were also decreased in vaccinated children, and all MIS-C patients requiring life support were unvaccinated . StudiesApproved vaccine for children 12\u201318 is available in the USA since May 2021, and for children 5\u201311 years of age it has been available since November 2021 . VaccinaAt the time of writing this paper, no study is available that has examined MIS-C in vaccinated younger children aged 5\u201311 years, although the vaccine has been available for this group since November 2021 in the United States. Risk of MIS-C has also not been examined yet in infants born to vaccinated mothers.CDC defines MIS-C as an individual aged less than 21 years presenting with fever, laboratory evidence of inflammation, and evidence of clinically severe illness requiring hospitalization, with more than two organ involvement with no alternative plausible diagnoses, and with current or recent SARS-CoV-2 infection or COVID-19 exposure within the 4 weeks prior to the onset of symptoms.  Most casMIS-C is temporally associated with current or prior SARS-CoV-2 infection, but the exact pathogenesis of MIS-C is not fully understood. Available literature proposes the multilineage activation of the immune system, which includes both innate and adaptive signatures . Viral lThe safety and efficacy of COVID-19 vaccination in children > 5 years of age has been well documented. Further, a multistate study from the US hospital network by Zambrano et al. (2022) revealed that receipt of 2 doses of the Pfizer-BioNTech vaccine was associated with protection against MIS-C in patients aged 12\u201318 years. The likelihood of developing MIS-C was 91% less in vaccinated children, and vaccinated children who developed MIS-C were less likely to develop respiratory or cardiac complications . SimilarOur review identified several studies that have documented transplacental transfer of antibodies following maternal COVID-19 vaccination and a reduced risk of hospitalization related to COVID-19 in infants 0\u20136 months of age. Further, these antibodies can persist in infants up to 6 months of age, after which, a decline in their titers was reported ,28. It iVaccination for children in the 6 months through 4 years age group was recently approved, but so far, it is unknown if and when a vaccine for infants 0\u20136 months will be available. Our review supports the evidence that maternal COVID-19 vaccination could provide protection to the infant in the first 6 months of life, as seen with other recommended vaccines during pregnancy, e.g., Tdap and influenza, which have been successful in reducing the morbidity and mortality associated with these infections in infants .Our review has many limitations. Due to heterogeneity of the available studies, a quantitative analysis could not be performed. Since the search was limited to English only and was not systematic, some studies could have been missed. Most of the available studies were case reports or case series, and thus, the level of evidence is low. Further, well-conducted randomized clinical trials can strengthen the evidence and provide clearer information about the risk of MIS-C in infants following maternal vaccination for COVID-19.Maternal vaccination for COVID-19 may be effective in rendering passive immunity to infants under 6 months of age. There is emerging evidence that it can prevent SARS-CoV-2 infection and its complications, and hence, the life-threatening consequence of MIS-C in infants < 6 months. The data is still premature and further studies can elicit the exact impact and timing of vaccination during pregnancy on the risk of MIS-C in infants."}
+{"text": "Cyanocitta stelleri) occupy different ecoregions in western North America, which vary in climate and landcover. These morphotypes  differ in size, plumage coloration, and head pattern. We sampled 1080 Steller's Jays from 68 populations (plus 11 outgroups) to address three main questions using data on morphology, plumage, genetics , and ecological niches: (1) How do phenotypic and genetic traits vary within and among populations, morphotypes, and ecoregions? (2) How do population\u2010level differences in Steller's Jays compare with other sister species pairs of North American birds? (3) What can we infer about the population history of Steller's Jays in relation to past climates, paleoecology, and niche evolution? We found substantial morphological, genetic, and ecological differentiation among morphotypes. The greatest genetic divergence separated Coastal and Interior morphotypes from the Rocky Mountain morphotype, which was associated with warmer, drier, and more open habitats. Microsatellites revealed additional structure between Coastal and Interior groups. The deep mtDNA split between Coastal/Interior and Rocky Mountain lineages of Steller's Jay (ND2\u2009~\u20097.8%) is older than most North American avian sister species and dates to approximately 4.3\u00a0mya. Interior and Rocky Mountain morphotypes contact across a narrow zone with steep clines in traits and reduced gene flow. The distribution of the three morphotypes coincides with divergent varieties of ponderosa pine and Douglas fir. Species distribution models support multiple glacial refugia for Steller's Jays. Our integrative dataset combined with extensive geographic sampling provides compelling evidence for recognizing at least two species of Steller's Jay.The relationship between ecology and morphology is a cornerstone of evolutionary biology, and quantifying variation across environments can shed light on processes that give rise to biodiversity. Three morphotypes of the Steller's Jay ( Cyanocittastelleri) occupy different ecoregions in western North America, which vary in climate and landcover. We found substantial morphological, genetic, and ecological differentiation among morphotypes. Our integrative dataset combined with extensive geographic sampling provides compelling evidence for recognizing at least two species of Steller's Jay.Three morphotypes of the Steller's Jay ( Similar integrative studies have identified ecomorphological divergence and genetic differentiation in other species of western birds, e.g., Dusky Grouse Dendragapus obscurus and Sooty Grouse D. fuliginosus  occur from the Pacific Coast to the Rocky Mountains  is a common resident bird species of forests and woodlands in western North America and Central America. As many as 16 recognized subspecies are divided into three main groups  Group with four subspecies that range from southwestern Alaska through western British Columbia (including Haida Gwaii) and the United States west of the Cascade Mountains and the Sierra Nevada; some authors  Group with four subspecies that occur east of the Cascades and Sierra Nevada to the northern and southern Rocky Mountains, extending into northern Mexico; and (3) the Central American (coronata) Group with eight subspecies that range from the highlands of northern Mexico to northern Nicaragua. These three groups and associated subspecies are distinguished primarily by plumage coloration , crest length, and facial patterning . In addition, subspecies also differ in body size within and among groups, with a trend of larger birds in the north to smaller birds in the south  forest, suggesting an ecomorphological correspondence between sites.In another study, Bay\u00a0 comparedCyanocitta cristata), and together they form a lineage that is part of a clade that includes the Pinyon Jay  and scrub\u2010jays  of North America; also related to this clade are Mexican to South American species of jays in the genera Calocitta, Cyanocorax, and Psilorhinus  found high levels of genetic variation among populations, with the highest divergence between the Haida Gwaii endemic C. s. carlottae and mainland subspecies C. s. stelleri and C. s. annectens  and Interior  subspecies groups , C. s. ridgwayi from Mexico (1) and Guatemala (2), C. cristata , Aphelocoma californica , Aphelocoma coerulescens , and Gymnorhinus cyanocephalus . Of the 1091 total individuals sampled for this project (1080 ingroup samples of C. stelleri plus 11 outgroup samples), 1053 specimens are housed in the Museum of Vertebrate Zoology (MVZ), University of California, Berkeley. Data for another 33 specimens were obtained from specimens at the Burke Museum of Natural History and Culture, the University of Washington , and the Museum of Southwestern Biology, University of New Mexico . We also made use of five sequences sourced from GenBank. Details for the total dataset are given in Table\u00a0We sampled 1080 2.2We used Fowler Ultra\u2010Cal digital calipers to measure standard morphological characters on 1035 specimens Table\u00a0 preparedWe recorded the sex and age of each bird from the specimen label data. Sex was based on examination of reproductive organs during preservation and was scored as male, female, or unknown/questionable. Age was recorded in two ways: (1) extent of skull ossification noted when the specimen was prepared; and (2) examination of tail shape, which can be used to distinguish first\u2010year from older birds. Steller's Jays have a partial first prebasic (first year) molt, which involves the replacement of body but not wing and tail feathers  The color of frontal (forehead) streaking. Birds were scored as having either blue or white frontal streaks. Although Pacific Slope and Northwestern Interior birds have mostly blue streaking, close examination of the feathers revealed varying amounts of white mixed with blue. This contrasts with Rocky Mountain birds that have white streaking with no blue. Thus, if the streaks contained any amount of blue, they were scored as being \u201cblue.\u201d (2) The extent of the white superciliary line above the eye. We measured the length and width of the line using digital calipers and then multiplied width by length to estimate the eye line area. Birds with no white line were scored as \u201c0\u201d for both length and width. We recognize that a more quantitative approach to measuring plumage coloration  and patterning  in Steller's Jays would be beneficial  for each linear mixed model to assess whether including sampling locality as a random effect impacted the fit of the model  using the function princomp from base R  for 15\u2009s, and 72\u00b0C elongation for 15\u2009s. We isolated 2\u00a0\u03bcl of the 10\u00a0\u03bcl PCR products for visualization on 1% agarose gels stained with ethidium bromide. We then mixed the PCR products with a GS\u2010500 LIZ size standard (Applied Biosystems) and formamide, denatured the mixture at 94\u00b0C for 2\u00a0min, and loaded the mixture on an ABI 3730 DNA Analyzer. For 5 of the 12 markers, we cleaned the PCR product with T4 DNA polymerase (0.21\u2009U) prior to loading to remove peaks of 3\u2032 A nucleotide additions. We scored the resulting alleles for all 12 loci and 1075 individuals using GeneMapper 4.0 (Applied Biosystems).We used 12 polymorphic microsatellite markers developed specifically for 2.4n\u00a0=\u00a060 individuals). We determined the best\u2010fitting model of nucleotide substitution with the programs PartitionFinder v2.1.1 , we used an admixture model with correlated allele frequencies and performed 10 independent runs of 5\u00a0\u00d7\u2009105 MCMC iterations after a burn\u2010in period of 1\u00a0\u00d7\u2009105 iterations, using sampling locations as priors (LOCPRIOR). We assessed the likely number of K based on the inspection of plots ; Pritchard et al.,\u00a0K  and number of pairwise differences (\u03c0); and (3) Fu's FS  and tested for departure from Hardy\u2013Weinberg equilibrium at each of the 12 microsatellite loci .For each model, we ran a chain of 1\u00a0\u00d7\u2009102.7Aphelocoma californica and A. wollweberi, Gowen et al.,\u00a0To place our study in a broader context, we extracted the raw percent divergence in ND2 sequences for a set of 30 North American avian species pairs Table\u00a0 and comp2.82.8.1GBIF.org, 26 April\u00a0To examine how Steller's Jays are distributed across environmental space, we downloaded 1,032,960 specimen\u2010based and observational occurrence records from 118 datasets accessed through the Global Biodiversity Information Facility  to 1 . We then sampled 100,000 points in proportion to these grid cell values, such that coordinates near roads were more likely to be sampled. This set of points was used as pseudo\u2010absences in subsequent species distribution modeling.Spatial sampling bias, which is pervasive in museum collections and observational databases  with the environmental dataset using the MASS package v7.3\u201051 in R  between sex and age groups, with males being larger than females  showed strong geographic patterning among populations that corresponds to the three morphotypes Figure\u00a0. FrontalDiscriminant Function Analysis of the morphological data showed strong separation among the three morphotypes, and cross\u2010validation led to high accuracy in predicting the correct morphotype Table\u00a0. Specifi3.2The ND2 phylogeny and haplotype networks showed congruent patterns of mitochondrial DNA variation within and between the three morphotypes Figure\u00a0. In bothN\u2009=\u00a0683) and the most unique haplotypes (N\u2009=\u00a0198). Although Interior and Rocky Mountain morphotypes had equivalent sampling, Interior populations had approximately 1.6 times the number of unique haplotypes. Similarly, nucleotide diversity and both genetic diversity estimates (\u03b8) were highest in Coastal populations, intermediate in the Interior, and lowest in Rocky Mountain populations. Fu's\u00a0 varied from 0.25 to 1.0. When populations were grouped by morphotype (Table\u00a0HO) varied from 0.72\u20130.92 across the three morphotypes.We successfully scored all 12 nuclear microsatellite loci for 1069 of the 1075 Steller's Jay individuals analyzed. One individual was successful for only 10 loci, and five individuals were successful for 11 loci Table\u00a0. All 12 pe Table\u00a0, the nump\u2010value\u2009<\u2009.001, Table\u00a0The only significant departure from Hardy\u2013Weinberg equilibrium occurred in locus Cst58 in Rocky Mountain populations after Holm\u2013Bonferroni sequential correction for multiple comparisons (adjusted K\u00a0=\u00a02 using the using Delta K (\u0394K) method  values showed support for a higher K\u00a0=\u00a04 across all populations  had an optimal value of K\u00a0=\u00a03 that distinguished San Francisco Bay Area  and southern California  populations from each other and the remaining Coastal populations except for Monterey (11), which shared microsatellite alleles with both Contra Costa and San Diego  that occurs only from the San Francisco Bay region south to Monterey Bay .These results prompted us to further examine the genetic substructure within each of the Coastal, Interior, and Rocky Mountain populations separately. Within the Coastal morphotype, both \u0394o Figure\u00a0. Populats Figure\u00a0 do not iK\u00a0=\u00a03. Individuals from both Coastal and Rocky Mountain populations were predicted to be in the correct group with high accuracy  based on the 12 microsatellite loci showed strong discrimination of the three morphotypes along the two discriminant function (DF) axes Table\u00a0. The firThe map of nuclear genetic diversity for Steller's Jays Figure\u00a0 showed tThe nuclear genetic diversity results are consistent with the private allele analysis Table\u00a0, which s3.4HZAR fits to the data showed strong and geographically coincident clines Figure\u00a0 in ND2 h3.5Collinearity reduction of climatic variables decreased the set of predictors from 33 to 12. When landcover variables were included, the initial set of 42 predictors (33 climates plus 9 landcovers) was reduced to 22. A list of predictor variables is given in Table\u00a0Species distribution models for the three morphotypes Figure\u00a0 showed sProjections to the Last Glacial Maximum  consisted of 13 climatic predictors plus closed habitat landcover. Variables that contributed most to the discrimination included Climate Moisture Index (CMI), mean monthly temperature range (BIO2), isothermality (BIO3), annual temperature range (BIO7), and minimum temperature of the warmest month Figure\u00a0. Examina44.1Junco hyemalis) have shown that local adaptation driven by environment can promote rapid differentiation , a southern clade found in the Rocky Mountains from eastern Nevada and northern Utah through the southern Rocky Mountains to Arizona and New Mexico , and a previously unrecognized northern clade in the northern Rocky Mountains and Columbia Plateau from British Columbia to southern Idaho . The northern and southern Rocky Mountain clades of grouse meet in the same general area as Interior and Rocky Mountain morphotypes of Steller's Jay, although a detailed study of that contact zone is lacking. Other examples of western North American bird species that occupy coniferous forests and show three geographically congruent divisions include Hairy Woodpecker  have similar morphologies in different geographic regions Bay,\u00a0, provideBrown\u00a0 suggestehttps://www.mbr\u2010pwrc.usgs.gov/bbs/ra2015/ra04780.htm; Sauer et al.,\u00a0Population density and social behavior also may influence visual signaling in Steller's Jays. In addition to habitat openness, Brown\u00a0 noted thFinally, the role of vocalizations for signaling is a critical component of understanding lineage divergence and ecomorphological associations. Song complexity is significantly associated with habitat openness in New World sparrows, which also show stronger phylogenetic signal in behavioral traits  compared with morphological traits . Steller's and Blue jays occupy different bioclimatic regimes in western and eastern North America, respectively, with Blue Jays breeding primarily in more humid hardwood habitats east of the Rocky Mountains including in the Great Plains  and Douglas fir , two conifer species dominant in Steller's Jay habitat. Populations of ponderosa pine within the Coastal lineage show further subspecific differentiation  and Rocky Mountain (P. p. var. scopulorum) lineages of ponderosa pine is closely associated with climate and especially winter versus summer precipitation regimes, respectively  that dates to approximately 4.3 mya, which places their divergence in the mid\u2010Pliocene during a period of warmer conditions  were isolated in different Pleistocene refugia with their preferred conifer species (black spruce Picea mariana and white spruce Picea glauca) to account for range\u2010wide phylogeographic variation. Molecular and fossil evidence for Douglas fir suggests two coastal refugia and three to four in the Rocky Mountains  on Haida Gwaii (formerly known as the Queen Charlotte Islands) has been interpreted similarly as evidence for isolation on an island refugium during the Pleistocene . Specifically, the guidelines state that for \u201ctrue phylogenetic daughter species formerly treated as a single parental species, the usual policy is to create a new name for each daughter species. This practice is designed to prevent confusion in the literature as to what taxonomic entity the parental name\u2026references.\u201d Prior to 1957, polytypic species of North American birds had English names assigned to subspecies rather than to the species itself  and Long\u2010crested Jay (Cyanocitta macrolopha) for Coastal/Interior and Rocky Mountain morphotypes, respectively.The splitting of species requires consideration of alternate English names, as outlined by the Guidelines for English Bird Names published by the American Ornithological Society's Committee on Classification and Nomenclature of North American Birds  from those to the north in the Rocky Mountains\u2014a value higher than the median (2.15%) for other North American avian species that we compared. Steller's Jays from those southern populations are phenotypically distinct Brown,\u00a0, most noCarla Cicero: Conceptualization (lead); data curation ; funding acquisition ; investigation ; methodology ; project administration (lead); supervision (lead); visualization ; writing \u2013 original draft (lead); writing \u2013 review and editing . Nicholas A. Mason: Data curation ; formal analysis ; methodology ; visualization ; writing \u2013 review and editing . Zheng Oong: Data curation ; formal analysis ; investigation ; methodology ; visualization ; writing \u2013 review and editing . Pascal O. Title: Data curation ; formal analysis ; investigation ; methodology ; visualization ; writing \u2013 review and editing . Melissa E. Morales: Investigation . Kevin A. Feldheim: Investigation ; writing \u2013 review and editing . Michelle S. Koo: Investigation ; writing \u2013 review and editing . Rauri C. K. Bowie: Conceptualization ; formal analysis ; funding acquisition ; investigation ; methodology ; supervision ; writing \u2013 review and editing .The authors declare that they have no conflict of interest.https://doi.org/10.6078/D14Q5N.This article has earned an Open Data badge for making publicly available the digitally\u2010shareable data necessary to reproduce the reported results. The data is available at Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.Table S7Click here for additional data file."}
+{"text": "Mytilus galloprovincialis for 24 h to 1, 10, and 100 nM chromium (VI) on the properties of Protamine-like (PLs) and their gene levels in the gonads. Specifically, we analyzed, by AU-PAGE and SDS-PAGE, PLs extracted from unexposed and exposed mussels. In addition, via EMSA, we evaluated the ability of PLs to bind DNA and also verified their potential to protect DNA from oxidative damage. Finally, we assessed possible alterations in gonadal expression of mt10, hsp70, and genes encoding for PLs-II/PL-IV and PL-III. We found that for all experimental approaches the most relevant alterations occurred after exposure to 1 nM Cr(VI). In particular, a comigration of PL-II with PL-III was observed by SDS-PAGE; and a reduced ability of PLs to bind and protect DNA from oxidative damage was recorded. This dose of chromium (VI) exposure was also the one that produced the greatest alterations in the expression of both mt10 and PL-II/PL-IV encoding genes. All of these changes suggest that this dose of chromium (VI) exposure could affect the reproductive health of Mytilus galloprovincialis.Chromium (VI) is the most dangerous oxidation state among the stable forms of chromium. In this work, we evaluated the effect of exposing  This mussel is a sessile, filter-feeding organism capable of accumulating many of the pollutants  contained in seawater within its tissues. Mussels also have a wide geographical distribution, which allows these organisms to be studied in large coastal regions [Mytilus galloprovincialis, partial replacement of somatic histones by protamine-like (PL) occurs. In the mature spermatozoa, three PL  are present in this organism [Mytilus galloprovincialis sperm chromatin structuring as they account for 76% of the nuclear basic protein components [Mytilus galloprovincialis PL [Mytilus galloprovincialis PL by exposing mussels to 1, 10, and 100 nM Cr(VI). We chose these exposure doses considering that the concentration of total dissolved chromium in seawater and open-ocean surface waters are about 0.1\u20130.55 and 0.1 \u00d7 10\u2009\u22123\u20130.55 \u00d7 10\u22123 \u03bcg L\u2009\u2212\u20091, respectively, and usually decreases with depth [Mytilus galloprovincialis. To assess this, after exposure of mussels to these three doses of chromium (VI), we extracted PLs from spermatozoa and analyzed them by polyacrylamide-acetic acid gel electrophoresis (AU-PAGE) and SDS-PAGE to reveal changes in the electrophoretic pattern. In addition, by EMSA, we assessed the ability of PLs to bind DNA and determined their potential to protect DNA from oxidative damage. Finally, we evaluated, by RTqPCR, the possible alterations in the expression of gonadal hsp70, mt10, PL-II/PL-IV, and PL-III genes following exposure of mussels to chromium (VI), given that other heavy metals have been shown to cause alterations in some stress genes and in those encoding Protamine-like PL-II and PL-IV [Heavy metals are defined as metals with a particular density of greater than 5 g/cmrganisms . Among hrganisms . It can rganisms . Cr(VI) rganisms . Once itrganisms . Such sprganisms . Chromiurganisms . Reprodurganisms . In addirganisms ,11 and trganisms  has beenrganisms ,15,16,17rganisms . There ao Cr(VI) . Accumulo Cr(VI)  and in to Cr(VI) . Finallyo Cr(VI) . In moni regions . During organism . These pmponents . Since sialis PL ,30,31,32th depth . Therefond PL-IV ,34M. galloprovincialis , which is not protected by any environmental agency in Italy. This study was conducted in strict accordance with European (Directive 2010/63) and Italian (Legislative Decree n. 116/1992) legislation on the care and use of animals for scientific purposes.This research was performed on the marine invertebrate M. galloprovincialis adult mussels were provided by Eurofish Napoli S.R.L. Baia, in Naples, and used in this research. The mussel had medium size of the shell length 4.93 \u00b1 0.17 cm. Fifteen mussels of undetermined sex were exposed to Cr(VI) concentrations of 1, 10, and 100 nM; a standard aqueous solution of K2Cr2O7 50.0 mM was used to realize these exposure doses. Mussels were exposed in laboratory plastic tanks (36 cm \u00d7 22 cm \u00d7 22 cm) holding 6 L of 33\u2030 artificial sea water (ASW) with the following composition for 1 L: NaCl 29.2 g, KCl 0.60 g, MgCl2 1.2 g, NaHCO3 0.20 g, and CaCl2 1.08 g, as previously stated for the experiment conducted with other heavy metals [y metals . Musselsg for 2 min at 4 \u00b0C to remove debris. In order to collect the spermatozoa, the supernatant obtained was centrifuged at 9000\u00d7 g for 10 min at 4 \u00b0C. The sperm-containing pellets of approximately 200 mg were recovered and stored at \u221280 \u00b0C for further investigation. Gonads of male organisms were stocked and stored at \u221280 \u00b0C. Spermatozoa were used for acid extraction of PL proteins. In particular, PL proteins were extracted from spermatozoa using 5% perchloric acid (PCA), as described earlier in Notariale et al., 2018 [After the exposure for 24 h to the three different concentrations of Cr(VI), mussels were opened by forcing the valves with the use of a knife, being careful not to cut the soft tissues. Mussel\u2019s sex was identified by gonad and gametes examination under a light microscope. The sex of the mollusk was identified by observation of the gametes under a light microscope. Gametes were obtained after stimulation of the male gonads with a Pasteur pipette and the use of seawater, as previously described in Piscopo et al., 2018 . In briel., 2018 . For thiEscherichia coli HB 101 cells using the standard protocol of the QIAGEN Plasmid Midi Purification kit , but with the precautions explained in Carbone et al., 2012 [In order to obtain high amounts of supercoiled pDNA, the pGEM3 plasmid (2867 bp) was extracted from transformed l., 2012 . Gel eleMytilus galloprovincialis specimens unexposed to metals, as described in Piscopo et al., 2018 [Sperm DNA was extracted from spermatozoa of l., 2018 . In briew/v) acrylamide (acrylamide/bis-acrylamide 30:0.15), and the separating gel was 18% (w/v) acrylamide (acrylamide/bis-acrylamide 30:0.15). The gels were stained with Coomassie Brilliant Blue at the end of both AU-PAGE and SDS-PAGE. Quantity One v.4.4.0  software was used to acquire gels using a Gel-Doc system . Densitometric analysis on gel bands was conducted using the software ImageJ ver. 1.50 d (https://imagej.nih.gov/ij/ (accessed on 7 March 2022)) supported by the National Institute of Health .To analyze protein samples, two types of electrophoretic analyses were used: AU-PAGE, and SDS-PAGE as earlier described by Piscopo et al., 2018  and by PM. galloprovincialis after exposure to Cr(VI) on DNA Electrophoretic Mobility was analyzed by Electrophoretic Mobility Shift Assay (EMSA). The procedure published [Mytilus galloprovincialis unexposed (sperm DNA). The modality of preparation of the same was similar to that for plasmid DNA with the difference that a fixed amount of genomic DNA (450 ng) and increasing amounts of PLs were used for a range of wt/wt protein/DNA ratios from 0.2 to 0.5. The electrophoretic run was performed on a 0.6% agarose gel at 60 V for approximately 1 h. After electrophoresis, gels were stained with ethidium bromide (2 mg/mL) to observe DNA migration. Quantity One v.4.4.0  software was used to collect gels using a Gel-Doc system . The program ImageJ ver. 1.50 d , 1997\u20132018) was used to do a densitometric analysis of the bands on the gel.The effect of PL proteins of ublished  was foll2O2 and 5 \u00b5M CuCl2 was evaluated by using plasmid DNA (pGEM3) and PLs extracted from the spermatozoa of mussels exposed at the three different concentration of Cr(VI). The samples were prepared using EMSA protocol described in the paragraph \u201cAnalysis of the Effect of M. galloprovincialis PL Proteins on DNA Electrophoretic Mobility\u201d, with slight modifications. In particular, 150 ng of plasmid DNA (pGEM3) and proteins/DNA wt/wt ratios in a range from 0.4 to 0.8 were used. H2O2 and CuCl2 were added after 5 min of room temperature interaction between DNA and PLs, and the samples were incubated for 30 min in the dark in a Thermoblock set at 37 \u00b0C. To minimize EDTA coordination of subsequent metals, sample buffer 10\u00d7  was added right before electrophoresis analysis at the conclusion of incubation. The electrophoretic analysis of the samples was conducted on 1% agarose gel at 100 V for 30 min in TBE 1\u00d7. The assay was also conducted using genomic DNA extracted from spermatozoa of Mytilus galloprovincialis unexposed (Sperm DNA) in the presence of 70 \u00b5M H2O2 and 50 \u00b5M CuCl2, using a fixed amount of genomic DNA equal to 450 ng and increasing amount of protein considering the different wt/wt protein/DNA ratios. A range of ratios between 0.4 and 0.8 was used. Samples were loaded on 0.6% agarose gels. The run was performed at 60 V for 1 h in 1\u00d7 TBE buffer. After electrophoresis, agarose gels were stained with ethidium bromide (2 g/mL) to observe DNA migration, and the gels were acquired using the GelDoc Biorad .PLs ability to protect DNA from oxidative damage in the presence of 30 \u00b5M Hmt10, hsp70, PL-III, and PL-II/IV in mussels was compared to that of control mussels.Total RNA was isolated from the gonads of unexposed mussels (control) and exposed to 1, 10, and 100 nM Cr(VI) using Trizol reagent , as indicated by the manufacturer. A UV-Vis spectrophotometer was used to determine the quantity of RNA extracted  . An electrophoretic analysis was done on 1% agarose gels under denaturing conditions to determine the quality of RNA. The DNA-free kit from Ambion  was used to eliminate genomic DNA from the samples. M-MLV reverse transcriptase was used to obtain cDNA from 1 \u00b5g of RNA from each sample . The RT-qPCR was carried out as described in Lettieri et al., 2021 . To evalv/v). Five subjective motility score classes were estimated: class 1 corresponded to no motility to 20% motility, class 2 from 20 to 40% motility, class 3 from 40 to 60% motility, class 4 from 60 to 80% motility, and class 5 to maximum motility (from 80 to 100% motility) [Sperm motility was assessed using sperm suspensions prepared by diluting the sperm collected from the mantle in ASW (1:100 otility) . A sampl2O milliQ was performed in the range from 420 to 600 nm after excitation at 350 nm. Spectra were acquired in presence of increasing concentrations of chromium (VI) in the range from 0 to 100 nM using 0.02 mg/mL concentration of PLs in H2O milliQ. Each spectrum was signal averaged at least three times and smoothed with the software Spectra Manager ver. 2.09 . All measurements were performed at least three times at room temperatures.The fluorescence analysis was carried out in a 0.5 mL volume cuvette  (STARNA) using a Jasco spectrofluorimeter model FP 8200, using 8-anilino-1-naphthalenesulfonic acid (ANS) as a probe. Fluorescence emission of ANS (5 \u03bcM) in absence and in presence of 0.01 or 0.02 mg/mL concentration of PLs in HMytilus galloprovincialis exposed to 1 nM, 10 nM, and 100 nM chromium (VI) were analyzed by AU-PAGE and SDS-PAGE to evaluate possible differences in the electrophoretic pattern compared to PLs extracted from spermatozoa of unexposed mussels. 4 \u00b5g of proteins were loaded into each well. By AU-PAGE, shown in the Chaetopterus variopedatus sperm H1 histone, in lane 2 were loaded PLs extracted from unexposed mussels (CTR), from lane 3 to 5 were loaded PLs extracted from exposed mussels to 1 nM, 10 nM, and 100 nM chromium (VI), respectively.PL proteins extracted from spermatozoa of AU-PAGE  showed nOn the same samples showed in For this analysis, 6 \u00b5g of PLs were loaded on the gel for each condition. As in the previous gel, PLs extracted from unexposed mussels (CTR) were loaded in lane 1 panel a, , whereasIn these EMSA, a fixed DNA concentration (150 ng) and PL/DNA ratios (wt/wt), ranging from 0.1 to 1.8, were used . The proIn the assay conducted with PLs extracted from unexposed mussels, as shown in panel a, the DNA saturation was reached at a wt/wt Protein/DNA ratio of about 0.8. A similar situation was observed for the PL from mussels exposed to 10 nM and 100 nM chromium (VI), . Differently, saturation was not reached even at very high ratios of wt/wt Protein/DNA (1.8) with PL from mussels exposed to 1 nM chromium (VI) (panel b).For the EMSA performed with sperm DNA a fixed amount of sperm DNA (450 ng) and increasing amounts of PL from mussels unexposed and exposed to the three different concentrations of chromium (VI), in order to reach wt/wt protein/DNA ratios ranging from 0.4 to 0.6. .Under all four conditions, almost complete DNA saturation at the w/w protein/DNA ratio 0.6 was observed both for unexposed mussels (lane 3) and in mussels exposed to 10 and 100 nM chromium (VI) (see lanes 7 and 9). In contrast, for mussels exposed to 1 nM chromium (VI), the fraction of saturated DNA was lower, and a greater fraction of DNA resulted in high mobility compared to previous conditions (see lane 5).2O2 and CuCl2 as specified in Materials and Methods in order to cause the Fenton reaction. The experiments were conducted using both plasmid DNA and genomic DNA extracted from unexposed mussels (sperm DNA). Below we first show the results obtained using plasmid DNA . The effects on DNA of adding PLs to the mixture composed by DNA, H2O2 and CuCl2 were evaluated for both exposed and unexposed mussels at w/w protein/DNA ratios of 0.4, 0.6 and 0.8.In the wells indicated with number 3 of the gels, is shown the result obtained from a DNA damage condition that we induced by treating the DNA at appropriate concentrations of HThe assay performed with PLs from unexposed mussels reveals that these proteins have DNA protective capacity as can be seen in samples at protein/DNA ratios of 0.6 and 0.8 . Under these conditions, the DNA does indeed appear organized with the proteins being mostly near the well, and the band of relaxed DNA is very light and no more supercoiled DNA is observed. A trend towards DNA protection is also observed for PLs from mussels exposed to 10 and 100 nM chromium (VI) (see panel b lanes 5\u20136\u20138\u20139), although less pronounced than for PLs from unexposed mussels. In contrast, no protection against DNA is revealed for PLs from mussels exposed to 1 nM chromium VI . Indeed, in this condition, the addition of PLs do not significantly change the electrophoretic pattern of plasmid DNA compared to that shown in well 3. The protective assay performed by using sperm DNA from unexposed mussels gave the results shown in 2O2, and CuCl2 allowed almost complete DNA protection, at the 0.6 protein/DNA ratio . In contrast, PL extracted from mussels exposed to 1 nM chromium (VI) (see lanes 6 and 7) showed a lower capacity to protect DNA from oxidative damage; in fact, at protein/DNA ratio of 0.4 most of the DNA is degraded (lane 6) and DNA protection obtained at 0.6 protein/DNA ratio is lower than previous conditions (well 7).Additionally in this case, a condition of damage was purposely generated for the spermatic DNA, which, as can be seen in well 3, appears degraded. For sperm DNA, only the addition of PL from mussels unexposed and exposed to 10 and 100 nM chromium (VI), to the mixture containing sperm DNA, Hhsp70 and mt10 in the gonads of mussels exposed to the three different concentrations of chromium (VI)  and of the genes encoding for PL-II/PL-IV and PL-III were evaluated (Quantitative polymerase chain reaction with reverse transcription (RT-qPCR) was used to evaluate the expression of some genes as a response to possible stress. The gene expression level of the stress genes valuated .hsp70 gene (see panel a), no significant changes in expression were observed in the three different exposure conditions compared to unexposed mussels. For the mt10 gene (see panel b), there was an increase in expression of two-fold and about one-fold following 1 nM and 10 nM chromium (VI) exposure, respectively, compared to unexposed mussels. In mussels exposed to 100 nM chromium (VI), instead no change in expression was observed compared to unexposed mussels. For the PL-II/PL-IV gene (see panel c), a down-regulation of expression was observed after exposure, which was more relevant in the 1 nM chromium (VI) exposure condition, resulting in about 1.3-fold lower than in unexposed mussels. For the PL-III gene (see panel d) for all exposure conditions no significant changes in expression were observed compared to unexposed mussels.For the M. galloprovincialis unexposed and exposed to 1, 10, and 100 nM chromium (VI). For all the exposure doses, a reduced number of motile spermatozoa was observed. In particular, it is interesting to note that just following 1 nM chromium (VI) exposure, the number of motile spermatozoa was very low in comparison with unexposed and exposed mussels to 10 and 100 nM chromium (VI). In addition, always at this exposure dose, the lowest score was recorded. The exposure at 10 and 100 nM chromium (VI) produced the same score (3).Given the alterations found at protein and gene level after the exposure of mussels to chromium (VI), we evaluated the sperm motility. In In the presence of the three PLs mixture, we observed a fluorescence increase and a blue shift in the maximum of ANS fluorescence emission from 521 to 498 nm a, indica3+), which occurs naturally, and hexavalent chromium (Chromium VI or Cr6+), which also occurs naturally due to erosion of chromium deposits but is found more commonly as a result of industrial pollution. Cr(VI) is much more bioavailable to aquatic organisms than Cr(III), therefore in aquatic ecosystems, Cr(VI) exposure poses a significant threat to aquatic life [Mytilus galloprovincialis. This was done to examine whether even at these doses of chromium (VI) there were any adverse effects on spermatogenesis in this organism, since reproductive capacity is the basis for the survival of the species. A concentration of 1 nM chromium (VI) is normally present in marine waters [M. galloprovincialis to CdCl2 [2 [C. variopedatus PL protein [2O2 and CuCl2. In fact, although the toxicity mechanisms of heavy metals are not well understood, some common mechanisms underlying their toxicity have been recognized. The production of reactive oxygen species (ROS) and oxidative stress are the major strategies that result in the alteration of proteins, lipid peroxidation, and DNA damage [mt10 and hsp70 and PL encoding genes. This because the literature reports an alteration in mt10 gene expression in Mytilus galloprovincialis digestive gland after exposure to metals such as copper, zinc, mercury and cadmium [mt10 and hsp70 expression in Mytilus galloprovincialis spermatozoa and gonads. For example, mercury causes an increase in mt10 gene expression in Mytilus galloprovincialis spermatozoa after exposure of mussels to 10 pM and 100 pM HgCl2 [mt10, and the highest level of overexpression of this gene was observed just after 1 nM of chromium (VI) exposure. In contrast, for hsp70, we found no significant changes in its expression after exposure to chromium (VI). RTqPCR analyses to assess any alterations in the expression of PL-II/PL-IV and PL-III genes in the gonads of mussels exposed to chromium (VI) showed a hypo-expression of PL-II/PL-IV genes in all three conditions of exposure compared to unexposed mussels. Additionally, in this case, the greatest differences were found for exposure to 1 nM chromium (VI). For the PL-III gene, instead, we found no significant changes for the three exposure conditions compared to unexposed mussels. Analyzing all the data obtained, the condition of exposure to chromium (VI) 1 nM appears to be the one that causes the greatest number of alterations at the protein and gene levels. It is also intriguing to note that as chromium (VI) concentration increases, but particularly at the 100 nM value, the ability of PL to both bind and protect DNA from oxidative damage is restored. In addition, at this dose of chromium (VI) exposure, there were no greater changes in the expression of mt10 than in unexposed mussels. Thus, low concentrations of chromium (VI) appear to induce greater stress in Mytilus galloprovincialis gonads and PL proteins properties than the higher concentrations of the same metal tested in this work. This result might suggest a possible hormesis effect. Hormesis is a dose/response relationship characterized by a biphasic effect. Many biological organisms/systems, exposed to a wide range of stimuli, produce opposite responses depending on the dose [Mytilus galloprovincialis following exposure to NiCl2 [Lonicera japonica Thunbriporta induced by cadmium on growth and photosynthetic performance [Mytilus galloprovincialis because affects the DNA binding of PLs that are in charge of the proper compaction of the sperm chromatin and consequently the protection of DNA from oxidative damage. Considering the alterations found in the properties of PL following exposure to Cr(VI) in Mytilus galloprovincialis, it would be interesting in the future to evaluate any conformational changes in these proteins following exposure of mussels to these doses of chromium (VI) by further fluorescence spectroscopy and circular dichroism experiments. The preliminary data we obtained from fluorescence spectroscopy measurements are encouraging and thus prompt further investigation. As a further perspective, it will be interesting to make measurements using the SEC-MALS technique, which allows the determination of molar mass and size in solution, to highlight possible PL aggregates that could be formed following exposure of mussels to these doses of chromium (VI). Finally, to understand with certainty if these doses of chromium (VI) can induce alterations in the fertilizing capacity of spermatozoa, in vitro fertilization experiments have been planned.Reproductive success is a key determinant for the survival of the species. In the last decade, the greatest risks affecting reproduction success occur for those species that are externally fertilized, such as several marine invertebrates, since many marine ecosystems have been contaminated with different types of xenobiotics. This is because the process of external fertilization is extremely susceptible to marine xenobiotics in seawater resulting from industrial and agricultural activities . Externatic life . The biotic life . In thise waters . Values e waters . In addito CdCl2  and HgClCdCl2 [2 . The difCdCl2 [2 , as sper protein ,52. SimiA damage . In addiA damage ,54. ConsA damage  to invesA damage . As expe cadmium . On the pM HgCl2 . Regardithe dose . We prevto NiCl2 . Incidenformance . In conc"}
+{"text": "Loss of TDP-43 protein homeostasis and dysfunction, in particular TDP-43 aggregation, are tied to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 is an RNA binding protein tightly controlling its own expression levels through a negative feedback loop, involving TDP-43 recruitment to the 3\u2032 untranslated region of its own transcript. Aberrant TDP-43 expression caused by autoregulation defects are linked to TDP-43 pathology. Therefore, interactions between TDP-43 and its own transcript are crucial to prevent TDP-43 aggregation and loss of function. However, the mechanisms that mediate this interaction remain ill-defined. We find that a central RNA sequence in the 3\u2032 UTR, which mediates TDP-43 autoregulation, increases the liquid properties of TDP-43 phase separation. Furthermore, binding to this RNA sequence induces TDP-43 condensation in human cell lysates, suggesting that this interaction promotes TDP-43 self-assembly into dynamic ribonucleoprotein granules. In agreement with these findings, our experiments show that TDP-43 oligomerization and phase separation, mediated by the amino and carboxy-terminal domains, respectively, are essential for TDP-43 autoregulation. According to our additional observations, CLIP34-associated phase separation and autoregulation may be efficiently controlled by phosphorylation of the N-terminal domain. Importantly, we find that specific ALS-associated TDP-43 mutations, mainly M337V, and a shortened TDP-43 isoform recently tied to motor neuron toxicity in ALS, disrupt the liquid properties of TDP-43-RNA condensates as well as autoregulatory function. In addition, we find that M337V decreases the cellular clearance of TDP-43 and other RNA binding proteins associated with ALS/FTD. These observations suggest that loss of liquid properties in M337V condensates strongly affects protein homeostasis. Together, this work provides evidence for the central role of TDP-43 oligomerization and liquid-liquid phase separation linked to RNA binding in autoregulation. These mechanisms may be impaired by TDP-43 disease variants and controlled by specific cellular signaling. Tardbp autoregulation  are hallmarks of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD)  and limbgulation . CollectThe cellular organization of RNA binding proteins, which in turn impacts protein function, involves condensation into ribonucleoprotein (RNP) granules. This process is mediated by liquid-liquid phase separation (LLPS) into often dynamic, liquid-like complexes . The higWe sought to define the molecular mechanisms in TDP-43 autoregulation and determine the effect disease-associated conditions have in this process. Our results strongly suggest that autoregulation is mediated by TDP-43 self-assembly and LLPS upon RNA binding. In addition, specific TDP-43 binding to RNA sequences that mediate autoregulation promotes liquid properties of TDP-43 condensates or liquid droplets. In addition, we find that TDP-43 posttranslational modifications and disease-associated variants may alter autoregulation by modulating TDP-43 condensation. Together, these findings highlight the potential of developing therapies based on controlling pathways linked to TDP-43 autoregulation as well as specific TDP-43-RNA binding interactions.The HA tagged TDP-43 sequence was cloned into pcDNA5\u2122/FRT/TO (Thermo Fisher) between BamHI and NotI restriction sites. Quikchange- Site directed mutagenesis protocol (Agilent) was performed to create single-site and multiple-site mutations. The \u0394CR deletion missing a.a. 316\u2013346 was generated from gBlock sequence. pcDNA5 HA-mEGFP-TDP constructs were created by subcloning TDP-43 cDNA into the mEGFP-C1 vector (Addgene) between XhoI and HindIII. HA-mEGFP-TDP-43 was subcloned into pcDNA5\u2122/FRT/TO using EcoRV and NotI. Oligonucleotides and gBlock sequence are listed in the Recombinant TDP-43 expression and purification was carried out as previously described .Absnorm=(Abs\u2212Absblank)/(Absmax\u2212Absblank), where Absmax is the condition being normalized to 1. The RNA oligonucleotides used in these experiments are listed in The protocols developed in our lab for these assays were recently described . Microscapp50,. The apparent IC50  is the protein concentration in which 50% of the maximal anisotropy change is observed. Data analysis were performed using GraphPad Prism 9.Purified TDP-43 WT and mutants previously purified were serially diluted in a 1:2 ratio in a final range of 0\u20132 \u03bcM protein into a 300 mM NaCl, 10 mM Tris (pH 8.0), 5% glycerol, 5% sucrose, 0.5 mM TCEP buffer solution. The protein dilution was mixed with CLIP34 (IDT) 3\u2032 labeled with FITC (fluorescein isothiocyanate) in a final concentration of 100 nM and added in triplicate in a 384-well black flat bottom plate (Corning) in a total reaction volume of 30 \u03bcL protected from light. The anisotropy measurements were performed in a Spectra Max i3 plate reader (Molecular Devices) with excitation and emission wavelengths of 480 and 520 nm, respectively. To assess the binding, the anisotropy data was fitted in a nonlinear fit in 4-parameter logistic model to calculate the apparent IC2. Stable cell lines were achieved through co-transfection of pcDNA5\u2122/FRT/TO/HA or mEGFP constructs and pOG44 using Lipofectamine 3000\u2122 (Invitrogen). Cells were selected using 75 \u03bcg/mL hygromycin (Sigma).HEK293 Flp-In\u2122 T-REX\u2122 cells (Thermo Fisher) were maintained in DMEM  with 10% FBS  and incubated at 37\u00b0C and 5% COThis protocol was modified from WT and M337V HA-TDP stable cells were plated and induced with 1 \u03bcg/mL of tetracycline for 72 h to reach confluency of 85\u201390%. Media was then changed to DMEM, 10% FBS, 100 \u03bcg/mL cycloheximide. Samples were taken at 0, 4, 8, 16, 32, and 40 h. Each sample was washed once with PBS and collected by centrifugation. Cell pellets were immediately lysed in cell lysis buffer and sonicated before being prepared for immunoblotting.g for 30 min at 4\u00b0C. The pellet was then rinsed with RIPA buffer and centrifuged again. The resulting pellet was resuspended in urea buffer . Fractions were quantified by western blot as described above.WT and M337V stable cell lines were plated and induced for 72 h before being harvested by centrifugation. Cell pellets were then resuspended in RIPA Buffer  and sonicated. Total fraction samples were taken after sonication and the samples were then centrifuged at 98,400 Immunoblotting was carried out using standard western blotting techniques and the antibodies for TDP-43 , FUS/TLS , matrin-3 , and GAPDH . Membranes were imaged using Odyssey scanning (LI-COR) and quantified using ImageStudioLite software (LI-COR).Trizol Reagent (Thermo Fisher) was used for RNA extraction and was carried out according to manufacturer instructions. M-MLV reverse Transcriptase (Thermo Fisher) was used for cDNA production according to the manufacturer instructions. Each primer mentioned in Tardbp), spanning approximately 500 nucleotides, during autoregulation . The mechanisms proposed to follow TDP-43 recruitment to TDPBR are based on analyses of the endogenous Tardbp transcript and derived gene reporters. These studies support the engagement of different processes during autoregulation: alternative polyadenylation, alternative splicing producing isoforms targeted for degradation via nonsense mediated decay (NMD), and nuclear retention of specific transcripts. Detailed studies by 18. We measured the size of the condensates in the different conditions to quantify the changes. The condensates mediated by CLIP34 are more than twofold significantly larger than control conditions . Our findings suggest that at physiological salt conditions (150 mM NaCl) TDP-43 binding to CLIP34 specifically increases the liquid and dynamic properties of TDP-43 condensates. Fluorescence recovery after photobleaching (FRAP) of the TDP-43-CLIP34 condensates showed rapid recovery of Oregon green-labeled TDP-43 indicating a dynamic exchange between the light and dense phases. We observed approximately 40% recovery in TDP-43 condensates formed with specific-binding RNA (CLIP34), while condensates without RNA or in the presence of non-binding RNA showed virtually no recovery (18 (TDP-43 binds to an extended region in the 3\u2032 UTR of its own transcript (gulation . This TDnditions . Additiorecovery . This surecovery . Our resvery (18 . Collect18 RNA and observed a marked decrease in phase separation compared to CLIP34. The condensates observed with A(CA)18, which is not expected to bind TDP-43 specifically 18 to F4L lysate did not significantly increase turbidity compared to control. This data suggests that in the context of cellular lysate, the remaining TDP-43-RNA contacts of F4L are sufficient for condensate formation, albeit with lower efficiency relative to WT. Collectively, these results indicate that CLIP34 binding specifically increases the liquid properties of TDP-43 condensates and promotes phase separation of TDP-43 in cell-like conditions.To test whether condensation of CLIP34-bound TDP-43 may be observed in a cellular environment, we adapted a recently developed protocol to observe biomolecular condensates in human cellular lysate . We prepifically , may be ifically . However  for the interaction between WT and mutant TDP-43 and CLIP34, as an estimate of the binding affinity. ICapp50, is the protein concentration in which 50% of the maximal anisotropy change is observed. Our results indicate that NTD mutations and \u0394CR do not significantly affect CLIP34 binding (Based on our results showing modulation of TDP-43 phase separation by CLIP34, we next asked whether LLPS plays a role during TDP-43 autoregulation in human cells. For this, we introduced site directed mutations in different TDP-43 domains that disrupt self-assembly and phase separation . We targ binding . As expe binding . Based oTo test the role of TDP-43 oligomerization and LLPS on autoregulation, we compared autoregulatory function in human cells expressing each of the LLPS-defective mutants. Stable HEK293 cells were generated expressing a single copy of the tagged TDP-43 transgene upon tetracycline induction, as previously described . To dete18 or no RNA control (18 RNA did not significantly change the LLPS behavior of the mutants compared to control, as seen with WT TDP-43. Previous studies of the isolated CTD showed that these mutations disrupt LLPS through alterations in \u03b1-helix structure and helix-helix interactions (Tardbp produces sTDP-43, which lacks the CTD and is replaced by a unique 18 amino acid long sequence (18 RNA did not significantly alter properties compared to control. The stark difference between sTDP-43, WT and \u0394C suggests that the short peptide at the C terminus dramatically alters the conformation of the rest of the protein by conferring aggregation-prone properties. Alternatively, the short tail, unique to sTDP-43, may drive intermolecular interactions that promote fibrilization.We and others have shown that specific ALS-associated TDP-43 mutations alter the dynamic properties of TDP-43 condensates . Here, w control . We founractions . Q331K aractions  may due ractions , alters sequence . This vasequence . We analsequence  and prevsequence . In contsequence . AdditioTardbp mRNA processing and increased TDP-43 protein and transcript levels (50, suggesting a moderate decrease in binding affinity of sTDP-43. The spontaneous fibrilization of sTDP-43 may account for this reduction in binding and should be further investigated. Nevertheless, based on previous studies, we predict that a decrease of binding affinity in this range is unlikely to alter cellular RNA processing function (We then investigated whether changes in condensate behavior seen with the ALS mutations affect autoregulation. We created stable HEK293 cell lines expressing a single copy of each mutant transgene upon tetracycline induction as for t levels . Howevert levels . The obst levels . These rt levels . Becauset levels . While \u0394function . We alsofunction . This mafunction . Importafunction .in vitro condensation assays. These intriguing findings also suggest that the defects caused by M337V alter general pathways of cellular proteostasis, affecting aggregation-prone proteins that are relevant to disease.We found that M337V did not affect autoregulatory function when analyzing mRNA transcript levels . These fin vivo models of TDP-43 pathology and patient-derived tissue show aberrantly increased levels of TDP-43 expression (The autoregulatory activity of TDP-43 is central to its function in RNA processing as even moderate changes in TDP-43 levels may significantly alter gene expression. The importance of autoregulation for TDP-43 metabolism is further highlighted by two lines of evidence. First, pression . Second,pression . These oBased on our results, TDP-43 autoregulation requires TDP-43-self-assembly and condensate formation. We find that disruption of TDP-43 LLPS activity through targeted mutations strongly impairs autoregulatory activity . These rTo explore possible links between the condensation-autoregulation process and TDP-43-associated disease, we investigated the function of TDP-43 mutations causative of ALS. Among the disease mutations within the CTD conserved region, we found that A321G and M337V were able to form condensates on their own, similar to WT. However, CLIP34 RNA did not confer liquid properties to the condensates formed by these mutants, in contrast to WT and the other mutations tested . These dOur present studies led to intriguing observations of disease-associated factors disrupting normal TDP-43 homeostasis. We found significantly increased accumulation of endogenous TDP-43 protein in M337V expressing cells, compared to wild-type . These rOur findings show that TDP-43 binding to CLIP34, specifically modulates liquid-liquid phase separation properties of TDP-43 . We prevThe original contributions presented in the study are included in the article/LK, ZG, AB, and YA designed the study. ZG and LM generated the recombinant protein and performed the phase separation analysis. LK carried out the cell biology experiments and analyses. AB measured the RNA binding affinity. TH participated in the design, analysis and interpretation of the RNA binding assays. LM generated the DNA constructs and mutagenesis. YA wrote the manuscript. LK, ZG, AB, LM, and YA reviewed and edited 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": "Drosophila strain that mimics ALS upon TDP-43 expression,\u00a0the mRNA levels of the HspA5 homologue (Hsc70.3) were significantly increased. Similarly\u00a0we observed upregulation\u00a0of HspA5 in prefrontal cortex neurons from human ALS patients. Finally, overexpression of HspA5 in Drosophila rescued TDP-43-induced toxicity, suggesting that upregulation of HspA5 may have a compensatory role in ALS pathobiology.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure or effective treatment in which TAR DNA Binding Protein of 43\u00a0kDa (TDP-43) abnormally accumulates into misfolded protein aggregates in affected neurons. It is widely accepted that protein misfolding and aggregation promotes proteotoxic stress. The molecular chaperones are a primary line of defense against proteotoxic stress, and there has been long-standing interest in understanding the relationship between chaperones and aggregated protein in ALS. Of particular interest are the heat shock protein of 70\u00a0kDa (Hsp70) family of chaperones. However, defining which of the 13 human Hsp70 isoforms is critical for ALS has presented many challenges. To gain insight into the specific Hsp70 that modulates TDP-43, we investigated the relationship between TDP-43 and the Hsp70s using proximity-dependent biotin identification (BioID) and discovered several Hsp70 isoforms associated with TDP-43 in the nucleus, raising the possibility of an interaction with native TDP-43. We further found that HspA5 bound specifically to the RNA-binding domain of TDP-43 using recombinantly expressed proteins. Moreover, in a  Any interference in proteostasis leads to accumulation of misfolded proteins, a central pathological hallmark of several neurodegenerative diseases including Alzheimer\u2019s disease and amyotrophic lateral sclerosis (ALS)3. In over 95% of ALS patients, TAR DNA-binding protein of 43\u00a0kDa (TDP-43) is mislocalized from the nucleus to the cytoplasm where it misfolds and aggregates in affected neurons and glia2. Several fragments from the C-terminal region of TDP-43, traditionally referred to as CTFs, have been detected in\u00a0post-mortem\u00a0tissue from\u00a0 patients with TDP-43 proteinopathies6, but their exact nature and abundance seem to vary between tissues, patients and/or mode of detection7. TDP-43 pathology has been observed across several neurodegenerative disorders including frontotemporal degeneration (FTD), Alzheimer\u2019s disease, and limbic-predominant age-related TDP-43 encephalopathy (LATE)10. Although the causative factors that lead to TDP-43 aggregation are still not fully understood, studies implicate proteostasis mechanisms such as impaired autophagy and the ubiquitin proteasome system (UPS)12 as well as compromised endolysosomal function15. TDP-43, a DNA/RNA-binding protein, consists of a folded N-terminal domain (NTD) linked by a flexible loop to two tandem RNA recognition motifs (RRMs)\u2014RRM1 and RRM2\u2014and a predominantly unfolded C-terminal prion-like domain that harbors the majority of disease-associated mutations in ALS16. TDP-43 functions primarily in RNA metabolism including splicing, translation and the cytoplasmic stress granule response17. Thus, in ALS, TDP-43 aggregation leads to repression of TDP-43-controlled pathways as well as a dysregulation of proteostasis19.Proteostasis is the proper equilibrium between the biogenesis, folding, trafficking and degradation of proteins within the cellular milieu21. One major chaperone subfamily is the evolutionarily conserved Hsp70s, which consists of 13 gene products )23. The canonical Hsp70 proteins share high sequence identity and have diverse cellular localizations and functions22. All canonical Hsp70 proteins have an N-terminal nucleotide binding domain (NBD) and a C-terminal substrate-binding domain (SBD) that allosterically communicate in an ATP-dependent manner to recognize and bind client proteins24.Central to proteostasis are the chaperones; a large family of proteins that typically bind to exposed hydrophobic sequences to assist in protein misfolding, degradation, and the clearance of aggregated protein25 and expression of disordered proteins28. Intriguingly, in motor neurons, the primary cells affected in ALS, there appears to be an incomplete stress response, as inferred from the lack of Hsp70 upregulation in response to several stress paradigms30. Moreover, overexpression of chaperones, including Hsp70s, prevented TDP-43 aggregate formation, more specifically CTF-25  aggregation31 and injection of recombinant human Hsp70 was effective in improving motor defects as well as increasing lifespan of a superoxide dismutase type 1 (SOD1) mouse model of ALS32. Collectively, these findings may partially explain why strategies to boost Hsp70 have been touted as neuroprotective in neurodegenerative diseases, particularly ALS. In support of this, Arimoclomol, a co-inducer of heat shock protein expression, has been under investigation in a clinical trial for ALS patients but recently failed in phase II/III . Arimoclomol is known to prolong heat shock factor 1 (HSF1) binding to the heat shock element (HSE) localized in the promoter of inducible Hsp70 isoforms, and it induces expression of a certain subset of heat shock proteins in neuronal cell lines33. As not all Hsp70s are controlled by the HSE, this might indicate that only a precise Hsp70 isoform subset is able to mitigate ALS toxicity.Typically, high levels of Hsp70 can be produced by cells in response to hyperthermia, oxidative stress, changes in pH, chemical disruption of proteostasis34. It was later hypothesized that Hsp70s could be constitutively bound to TDP-43. Upon a heat shock event, Hsp70 could be released from its interaction with TDP-43 as misfolded proteins accumulate, which could thereby promote the formation of TDP-43 aggregates35. More recently, it was shown that in cells, several Hsp70 isoforms accumulate within mutated TDP-43 phase separated anisosomes 36. To date, potential direct binding between the Hsp70 isoforms and TDP-43 has not been investigated. Here, we interrogated the association of TDP-43 with specific Hsp70 isoforms using BioID, a technique that leverages the activity of a promiscuous biotin ligase to biotinylate proteins based on proximity37. We found that HspA5 and HspA8 were enriched in the nuclear, but not cytoplasmic, fraction of TDP-43. We further tested direct binding of TDP-43 with the Hsp70 isoforms HspA1A, HspA5 and HspA8 and found that the TDP-43 RRM domains selectively bind HspA5. Moreover, the mRNA levels of the\u00a0HspA5 homologue (Hsc70.3) in Drosophila melanogaster (Drosophila)\u00a0were significantly increased upon TDP-43 expression and we observed an upregulation of\u00a0HspA5 in prefrontal cortex neurons of human ALS patients. Finally, we discovered that upregulation of Hsc70.3 in Drosophila protects against TDP-43-induced toxicity while the ATP binding-deficient mutant Hsc70.3K97S variant35 had no effect. Our data underscore an Hsp70 isoform preference by TDP-43 and thus position induction of HspA5 binding to TDP-43 as a novel therapeutic strategy for mitigating TDP-43 toxicity.It is still unclear how and which Hsp70 isoforms regulate TDP-43. Previous studies demonstrate that at least three Hsp70 isoforms immunoprecipitate with TDP-43: HspA1A, HspA5 and HspA8To characterize nuclear versus cytoplasmic localization as well as possible Hsp70 isoform specificity of TDP-43, we performed proximity-dependent biotin labeling (BioID) of TDP-43 in the nucleus or the cytoplasm. BioID2 was fused to the N-terminal domain of TDP-43, and either a 3\u2009\u00d7\u2009tandem nuclear localization signal (3xNLS) or a nuclear export signal (NES) was added to localize TDP-43 to the nucleus or cytoplasm, respectively. BioID2-3xNLS-TDP43, BioID2-NES-TDP43 or the BioID2 control were stably expressed in human neuroblastoma SH-SY5Y cells, and its localization was verified using immunofluorescence Fig.\u00a0. It is w38. Although HspA5 is mostly known for its ER localization, several studies have shown the presence of HspA5 in the nucleus40, including in SH-SY5Y cells41. Thus, our data suggest that in the SH-SY5Y cells and in the absence of stress, HspA5 and HspA8 selectively associate with nuclear but not cytoplasmic, TDP-43.Surprisingly, HspA5 and HspA8 were found as highest confidence and good confidence associations respectively in the nuclear TDP-43 sample (BioID2-3xNLS-TDP-43) Table . No Hsp733, but the exact Hsp70/TDP-43 interface was never investigated. We thus set out to characterize the binding of TDP-43 to different Hsp70 isoforms. To this end, we selected HspA5 and HspA8 (identified from BioID) and HspA1A, an Hsp70 isoform implicated in TDP-43 binding34.The BioID data hinted towards TDP-43 binding selectively to Hsp70 isoforms, as demonstrated by the fact that only HspA8 and HspA5 were found to be significantly enriched. Another previous study showed via immunoprecipitation that Hsp70 interacts with TDP-43 primarily through its RRMs42. LIMBO is based on a position-specific scoring matrix (PSSM) trained from in vitro peptide binding data and structural modelling and predicts the binding of bacterial Hsp70 homolog DnaK, which shares\u2009~\u200950% identity with human Hsp70 isoforms. For Hsp70 prediction, TDP-43 was divided into three fragments: aa 1\u2013120 ), aa 101\u2013269 (the two RNA recognition motifs (RRM)) and aa 270\u2013414   Fig. A. While ns) Fig. B,C. Thus1\u2013102, a construct corresponding to TDP-43-NTD. The SBD of these Hsp70 isoforms is approximately 200 amino acids long and is composed of a two layered twisted \u03b2-sheet and a C-terminal \u03b1-helical subdomain. The SBD and its binding to the client peptide are allosterically modulated by the ATP binding site. However, binding of ATP to the TDP-43 RRM domains has also been shown to enhance the stability of TDP-4343. Thus, we reasoned that this may inhibit Hsp70 isoform binding, and opted to use an Hsp70 construct that lacked the N-terminal nucleotide binding site but retained the ability to recognize client peptides.Using microscale thermophoresis (MST), we measured the binding of the substrate binding domain (SBD) of HspA1A, HspA5 and HspA8 to TDP-431\u2013102 with a similar affinity calculated to be in the high nanomolar to low micromolar range , we found that HspA8 did not bind at all, and that HspA5 (298\u2009\u00b1\u2009150\u00a0nM) bound with greater affinity than HspA1A (2.35\u2009\u00b1\u20091.39\u00a0\u03bcM)  to HspA5, we set out to experimentally map, in greater resolution, potential HspA5 binding sites within TDP-43. To do this we synthesized a peptide array of 15-mer peptides with an overlap of 5 amino acids that spanned the RRM region of TDP-43. The peptide array was incubated with HspA5-SBD protein and peptide binding was detected using an antibody directed against HspA5 6 RNA (PDB code: 4BS244)  HspA5 might recognize only a portion of the peptide, sufficient for initiating binding, and (ii) there might be structural elements at play in the HspA5/TDP-43 interaction.We next mapped these potential HspA5-binding regions on TDP-436 RNA, the canonical binding sequence of TDP-4345, and we detected a decreased affinity of the HspA5/TDP-43102\u2013269 interaction from 0.9\u2009\u00b1\u20090.3\u00a0\u00b5M to 28.3\u2009\u00b1\u200923.7\u00a0\u00b5M  model of TDP-43 toxicity51. Compared to the expression of a normal control (si.mCherry), expression of human TDP-43 in the Drosophila eye disrupts the external surface  had no effect on the Drosophila eye. However, by contrast, downregulation of Hsc70.3 altered the structure of the external eye indicating that loss of Hsc70.3 is detrimental to the Drosophila eye 35 in the Drosophila eye and selected the variant that conferred no toxicity  . Co-exse) Fig.\u00a0, indicatConsidering the interaction between TDP-43 and HspA5, as well as the mislocalization of TDP-43 in ALS, we next set out to determine if in our Drosophila model of TDP-43 disease Hsc70.3 levels were upregulated. Due to the lack of antibodies available to Hsc70.3 we opted to measure the levels of Hsc70.3 mRNA in Drosophila expressing either a normal control (si.mCherry) compared to Drosophila expressing TDP-43. This revealed that Hsc70.3 mRNA levels, relative to Tubulin, were significantly increased upon TDP-43 expression ((compare 0.79\u2009\u00b1\u20090.13 (SD) to 1.17\u2009\u00b1\u20090.13 (SD), control vs TDP-43, respectively) Fig.\u00a0. It is e52, recently failed phase II/III clinical trials for the treatment of ALS . A greater understanding of which HSP family members control TDP-43 will be crucial for insights into the mechanisms that propagate disease as well as in developing more nuanced therapeutic strategies. Here we show that the Hsp70 isoform HspA5 specifically binds to the RNA-binding domain of TDP-43, that there is an apparent increased expression of cytoplasmic HspA5 in the prefrontal cortex of ALS patients and that upregulation of the HspA5 homologue mitigates TDP-43-induced toxicity in Drosophila, identifying HspA5 as a potential target in TDP-43-associated disease.Targeting the molecular chaperone pathway is a potential therapeutic strategy in neurodegenerative disorders such as ALS. Arimoclomol, a compound that increases Hsp70 proteins as well as other Hsp chaperones58 and affinity pull-down methods60. Here, we used BioID, a unique technique that leverages promiscuous nature of biotin ligase to biotinylate proteins based on proximity37, on a dividing neuroblastoma cell population (SH-SY5Y cells) expressing TDP-43 to which we added either an extra NES  or an NLS . We have no information on normal folding as well as sub-nuclear/cytoplasmic localization of such TDP-43 constructs. While our BioID data was able to reproduce, to some extent, several TDP-43 interactions with known partners including HspA8 and HspA5, ribosomal proteins, and other proteins of RNA metabolism59, the cell type used, and the expression of modified TDP-43 could have impacted the binding partners that were found. Nevertheless, our data indicate that HspA5 and HspA8 were found to bind TDP-43 in the nucleus and in the absence of exogenous stress. HspA5 and HspA8 are constitutively expressed, contrary to HspA1A expression, for example, that is only induced by different stressors. Moreover, even though both HspA5 and HspA8 are commonly known to reside within the endoplasmic reticulum and the cytoplasm respectively, HspA5 is actively translocated to other cellular locations, including mitochondria and the nucleus62, and cytoplasmic HspA8 shuttles between cytoplasm and nucleus, which enables it to import client proteins into the nucleus63. Moreover, several Hsp70 isoforms, including HspA5 and HspA8, were found accumulated within mutated TDP-43 phase separated anisosomes 36. Overall, this supports the possibility of a TDP-43/Hsp70 isoform interaction in the nucleus, and while an interaction with Hsp70 chaperones in the cytoplasm was not observed here it is possible that such an interaction may happen upon activation of the stress response.Although a plethora of proteins and protein families have been reported to interact with and control TDP-43, they have generally been identified using indirect measures such as genetic interaction screens20. It is thus possible that the recognition of the RRM domains of TDP-43 by HspA5 needs structural elements in addition to the predicted Hsp70 binding sites and perhaps may be involved in an alternative function to chaperone activity. In the absence of stress, HspA5 maintains the three transmembrane UPR sensors  in an inactive state through direct binding to the respective proteins64. Upon ER stress, accumulated misfolded proteins titrate HspA5 away from PERK/IRE1/ATF6, leading to their activation and subsequent stimulation of the UPR65. Here, we show that HspA5 binding to TDP-43 is inhibited by RNA. The importance of RNA binding to TDP-43 in maintaining TDP-43 solubility has been previously reported67, and our data suggest that HspA5 may recognize the non-RNA bound version of TDP-43 to ensure proper folding and/or prevent misfolding or to trap TDP-43, similarly to HspA5 binding to UPR sensors. Another interesting client peptide is 246-EDLIIKGISV-255, encompassing E246 and D247 residues, which exposition is a marker of misfolded TDP-43, as well as a cleavage site generating TDP-43 CTF68. In line with this, Hsp70 overexpression prevented TDP-43 aggregate formation of CTF-25 but was unable to disassemble or solubilize those inclusions31. However, it is important to note that TDP-43 CTFs may not be imperative for neurodegeneration70 since studies have detected much less TDP-43 CTFs than the entire protein in ALS spinal cords72.Using in vitro binding approaches, we further established that HspA1A, HspA5 and HspA8 bind directly to TDP-43. Our data indicate that while the Hsp70 isoforms HspA1A, HspA5 and HspA8 bind to the partially or fully unfolded N-terminal domain of TDP-43 with equal affinities, binding to the conformationally stable RRM domains of TDP-43 is highly selective for HspA5. Using a peptide-binding array we identified HspA5 binding sites in each RRM of TDP-43. The HspA5 binding regions in the RRMs are only partially exposed, which is surprising since chaperones typically recognize hydrophobic stretches of amino acids in unfolded proteins73. Moreover, the neuronal pathology caused by expression of mutant SOD1 (SOD1-G93A) was exacerbated in mice deficient in the HspA5 co-factor SIL1, while SIL1 overexpression induced significant neuroprotection related to improved ER proteostasis and reduced SOD1 aggregation74. It is worth noting that previous work showed that in Drosophila down regulation of tankyrase 1 and tankyrase 2 (Tnks-1/2), which physically interact with TDP-43, reduces TDP-43 toxicity while their upregulation enhances TDP-43 toxicity75. This further suggests that not all binding partners of TDP-43, when upregulated, ameliorate TDP-43 toxicity.Expression of TDP-43 in the eye during development leads to adult Drosophila with a disrupted external eye and vacuolization and loss of tissue in the retina. Our data indicate that TDP-43 expression in the developing eye\u00a0recapitulates HspA5 pathology observed in human ALS as we observe an upregulation of Hsc70.3 mRNA. Furthermore, upregulation of Hsc70.3 mitigates the toxicity of TDP-43 when expressed in the developing eye, implicating upregulation of Hsc70.3/HspA5 as a potential therapeutic strategy. Further studies are needed to address how Hsc70.3 upregulation may mitigate TDP-43 toxicity in aging adult neurons. HspA5 has also been implicated in regulating the toxicity and aggregation of the ALS-causing protein superoxide dismutase (SOD1). For example, knock-in mice expressing HspA5 that lacks the ER retention signal, KDEL, display age-related motor problems, loss of motoneurons and aggregation of wild-type SOD176 but cellular stimuli such as ER stress and ER-associated degradation can lead to the localization of HspA5 to the mitochondria and the cytosol77. Our data indicates a nuclear interaction between TDP-43 and HspA5 but, conversely, HspA5 was largely localized to the cytoplasm in ALS and aged matched control patients  where it controls protein folding during ER-associated stressnts Fig. . While w82. Moreover, Arimoclomol, was shown to induce the expression of only HspA6 and HspA1A in human SH-SY5Y cells33. The failure of Arimoclomol in phase II/III of clinical trial for ALS patients might be partly explained by a lack of specific Hsp70 isoform targeting.Finally, it is worth noting that Arimoclomol is a co-activator that prolongs the binding of activated HSF1 to heat shock elements in the promoter region of many chaperones including Hsp70 family members. Notably, HspA5 expression is not under the control of Hsf1Overall, the observations in this study suggest that upregulation of HspA5 in ALS may have a compensatory role, prolonging the survival of neurons by preventing TDP-43 misfolding and subsequent toxicity. Elucidating the stimuli and the underlying cellular mechanisms that control HspA5 binding to TDP-43 will provide the platform for investigating HspA5 as a potential therapeutic target in\u00a0TDP-43-associated disease.102\u2013269 and TDP-431\u2013102 were obtained as previously described84. The TDP-43 expression strain was described previously51. The Hsc70.3-WT or Hsc70.3K97SDrosophila strains were obtained from the Bloomington Drosophila stock center, Indiana, USA. All fly experiments were carried out at 25\u00a0\u00b0C in standard cornmeal molasses agar.All reagents were purchased from Sigma  and Fisher Scientific  unless otherwise indicated. TDP-43All BioID plasmids were made using In-Fusion Recombination. mycBioID pBabe (Addgene #80901) was used as the control plasmid. TDP-43 was amplified via PCR from a pDuet TDP43 WT  with an AgeI restriction enzyme (RE) site built into the 5\u2019 primer upstream of TDP-43. Amplified PCR product was inserted into mycBioID pBabe (Addgene #80901), using XhoI and SalI RE sites. The SV40 nuclear localization signal (NLS\u2013PKKKRKV) was inserted in tandem (3x) into the newly made BioID2-TDP43 pBabe using XhoI and AgeI. Similarly, the classic protein kinase inhibitor nuclear export signal (NES- NELALKLAGLDI) was inserted into BioID2-TDP43 pBabe using XhoI and AgeI.600 of 0.6 at 37\u00a0\u00b0C before being shifted to 16\u00a0\u00b0C. Expression was induced once the OD600 reached 0.8\u20131.0 with 0.5\u00a0M IPTG overnight. Cells were then harvested, lysed, and protein was purified using cobalt IMAC resin (Gold bio). The His6 tag was cleaved using TEV protease overnight in dialysis into buffer A 2, and 1\u00a0mM DTT). After complete cleavage, the DTT was dialyzed out for 4\u00a0h, and the TEV protease was recaptured with cobalt resin. Protein was then concentrated, flash frozen on liquid nitrogen and stored at \u2212\u00a080\u00a0\u00b0C.BL21-Codon plus bacteria (Agilent) were transformed with pSpeedET vectors containing Hsp70 isoform. Cells were grown to and OD102-269-His was labelled using the Monolith Protein Labeling Kit RED-NTA  according to the manufacturer\u2019s instructions. Briefly, 50\u00a0nM of labeled protein was mixed with ranging concentration of Hsp70 isoforms in MST buffer. The thermographs were recorded using MST premium capillaries at 40% LED and medium MST power. Data analysis was performed with the MO Affinity Analysis software (Nanotemper).Purified TDP43Peptides of TDP-43 (15 amino acids in length) were spotted on nitrocellulose on glass slides. Peptides were synthesized using standard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, in 30\u2009\u00d7\u200920 spot arrays using a Multipeptide synthesizer adapted for SPOT synthesis . Membranes were blocked for at least 1\u00a0h in Tris-buffered saline containing 0.5% Tween 20 (TBST) with 5% semi-skimmed milk powder before an overnight incubation with 2.5\u00a0\u03bcM of HspA5 and 1\u00a0mM ADP at 4\u00a0\u00b0C with gentle shaking. Following a series of washes in TBST, the blot was probed for an hour with an HspA5 antibody at 4\u00a0\u00b0C. The following day, blots were washed three times for 10\u00a0min each time in TBST, incubated in secondary antibody (IgG (H\u2009+\u2009L) Cross Adsorbed Secondary Antibody, DyLight 800  at dilutions 1:5000) for 45\u00a0min at room temperature, and washed in TBST three more times for 10\u00a0min each time before visualizing SPOTs by exposing the membranes.51. The UAS-Hsc70.3WT, UAS-Hsc70.3K97S, UAS-Hsc70.3D231S, si.Hsc70.3 and si.mCherry lines were obtained from the Bloomington Drosophila stock center, Indiana, USA. All Drosophila experiments were carried out at 25\u00a0\u00b0C on Bloomington cornmeal food.The full genotypes and source of all Drosophila stocks are described in Table 85. Eight micrometers paraffin sections were cut and mounted onto glass slides. Three sections per head were imaged at the same anatomical position and the retinal width and vacuolization was quantified using image J software. Graphpad 6 was used to determine statistical significance.For external eye imaging, female Drosophila\u00a0were imaged with a Leica Z16 Apo A microscope, DFC420 camera and 2.0\u2009\u00d7\u2009planapochromatic objective. For paraffin sections, Drosophila heads were fixed, processed and quantified as previously described57. Briefly, TDP-43 or LacZ was expressed in the eye with gmr-GAL4, protein was extracted from 5 to 10 male (TDP-43) or female (LacZ) heads in 10\u00a0\u03bcl/head of 2X Laemelli buffer with 5% (v/v) \u03b2-mercaptoethanol, denatured at 95\u00a0\u00b0C, chilled on ice for 5\u00a0min and centrifuged at 5000\u00a0rpm for 5\u00a0min at 4\u00a0\u00b0C. Half a fly head (5\u00a0\u03bcL) was electrophoresed on a 4\u201312% bis\u2013tris gel and transferred onto nitrocellulose by wet transfer (30\u00a0V for 65\u00a0min). Blots were blocked in 5% milk in TBST . Primary antibodies made up in TBST were: TDP-43 , \u03b1-Tubulin-HRP  and \u03b2-galactosidase . Horseradish peroxidase (HRP)-coupled secondary antibodies made up in TBST were goat anti-rabbit-HRP  and goat anti-mouse-HRP . All experiments were carried out on three or more biological replicates, blots were quantified with ImageJ86 and statistical analysis was carried out using Graphpad prism 6 software.Immunoblotting was performed as previously described57. Briefly, heads were homogenized in 1\u00a0ml of Trizol (ThermoFisher). After adding 200 \u00b5L of chloroform (Thermo Scientific), the tube was shaken for 15\u00a0s, centrifuged for 10\u00a0min at 4\u00a0\u00b0C, and the aqueous phase was transferred to a fresh tube. RNA was precipitated in ethanol and 3\u00a0M sodium acetate pH 5.2 (ThermoFisher) on ice for 25\u00a0min. Samples were centrifuged at maximum speed at 4\u00a0\u00b0C for 30\u00a0min. The RNA pellet was washed in 70% ethanol and centrifuged at maximum speed at 4\u00a0\u00b0C for 15\u00a0min. The pellet was dissolved in RNase-free water (ThermoFisher). Genomic DNA was digested with DNA-free DNase (ThermoFisher). First-strand DNA was synthesized using 300\u00a0ng of RNA and Superscript III (ThermoFisher) and random primers. Luna Universal qPCR Master Mix (NEB) was used for real-time PCR analysis. Standard curves were performed to test primer efficiency. Each experiment was carried on 3 independent fly crosses each with 3 technical repeats. Statistics were calculated using Graphpad prism 9 software. Primers to Hsc70.3 designed by the fly primer bank were used (https://www.flyrnai.org/flyprimerbankused). Primers were:RNA was prepared from\u2009~\u200950 Drosophila heads as previously describedHsc70.3 Fw: 5\u2019 GATTTGGGCACCACGTATTCC 3\u2019.Hsc70.3 Rv: 5\u2019GGAGTGATGCGGTTACCCTG 3\u2019.\u03b1-Tubulin Fw: 5\u2019 CATCCAAGCTGGTCAGTG 3\u2019.\u03b1-Tubulin Rv: 5\u2019 GCCATGCTCATCGGAGAT 3\u2019.2 at 37\u00a0\u00b0C in DMEM/F12 1:1  supplemented with 10% fetal bovine serum. All cells were tested monthly for mycoplasma contamination.SH-SY5Y cells were obtained from the American Type Culture Collection . BioID stable cell lines for were generated using retroviral transduction. HEK293 Phoenix cells  were transfected with each construct using Lipofectamine 3000 (Thermo Fisher Scientific) per manufacturer's recommendation. The transfected cells were incubated at 37\u00a0\u00b0C for 6\u00a0h. After 6\u00a0h incubation, the transfected cells were replenished with fresh medium and further incubated at 32\u00a0\u00b0C for 72\u00a0h. The culture media was filtered through a 0.45-\u03bcm filter and added to SH-SY5Y cells along with Polybrene . At 72\u00a0h after transduction, puromycin  was added to the target cells. Stable cells lines were verified for fusion-protein expression and proper localization using immunofluorescence and western blot. The stable cell lines were maintained in 5.0% COCells grown on glass coverslips were fixed in 3% (wt/vol) paraformaldehyde/phosphate-buffered saline (PBS) for 10\u00a0min and permeabilized by 0.4% (wt/vol) Triton X-100/PBS for 15\u00a0min. For labeling fusion proteins, a chicken anti-BioID2 antibody was used . The primary antibody was detected using Alexa Fluor 568\u2013conjugated goat anti-chicken . Alexa Fluor 488\u2013conjugated streptavidin  was used to detect biotinylated proteins. DNA was detected with Hoechst dye 33342. Coverslips were mounted using 10% (wt/vol) Mowiol 4\u201388 (Polysciences). Confocal images were obtained using a Nikon A1 confocal microscope (60\u2009\u00d7\u2009/1.49 oil APO TIRF Nikon objective) with a charge-coupled device camera  linked to a workstation running NIS-Elements software . Epifluorescence images were captured using a Nikon Eclipse NiE (20\u2009\u00d7\u2009/0.75 Plan Apo Nikon objective) microscope.6 cells were lysed in SDS\u2013PAGE sample buffer, boiled for 5\u00a0min, and sonicated to shear DNA. Proteins were separated on 4\u201320% gradient gels  and transferred to nitrocellulose membrane (Bio-Rad). After blocking with 10% (vol/vol) adult bovine serum and 0.2% Triton X-100 in PBS for 30\u00a0min, the membrane was incubated with chicken anti-BioID2 antibody  overnight, washed with PBS and detected using horseradish peroxidase (HRP)\u2013conjugated anti-chicken . The signals from antibodies were detected using enhanced chemiluminescence via a Bio-Rad ChemiDoc MP System . Following detection of BioID2, the membrane was quenched with 30% H2O2 for 30\u00a0min. To detect biotinylated proteins, the membrane was incubated with HRP-conjugated streptavidin  in 0.4% Triton X-100 in PBS for 45\u00a0min.To analyze total cell lysates by immunoblot, 1.2\u2009\u00d7\u20091056 with four 10\u00a0cm dishes per sample instead of two. In brief, four 10\u00a0cm dishes at 80% confluency were incubated with 50\u00a0\u03bcM biotin for 18\u00a0h. Cells were lysed in 8\u00a0M urea 50\u00a0mM Tris pH 7.4 containing protease inhibitor  and DTT, incubated with universal nuclease , and sonicated to further shear DNA. Lysates were precleared with Gelatin Sepharose 4B beads  for 2\u00a0h and then incubated with Streptavidin Sepharose High Performance beads  overnight. Streptavidin beads were washed four times with 8\u00a0M urea 50\u00a0mM Tris pH 7.4 wash buffer and resuspended in 50\u00a0mM ammonium bicarbonate with 1\u00a0mM biotin.Large-scale BioID pulldowns were performed as described inBeads were resuspended with 8\u00a0M urea, 50\u00a0mM ammonium bicarbonate, and cysteine disulfide bonds were reduced with 10\u00a0mM tris(2-carboxyethyl)phosphine (TCEP) at 30\u00a0\u00b0C for 60\u00a0min and cysteines were then alkylated with 30\u00a0mM iodoacetamide (IAA) in the dark at room temperature for 30\u00a0min. Following alkylation, urea was diluted to 1\u00a0M urea, and proteins were subjected to overnight digestion with mass spec grade Trypsin/Lys-C mix . Finally, beads were pulled down and the solution with peptides collected into a new tube. Affinity purification was carried out in a Bravo AssayMap platform (Agilent) using AssayMap streptavidin cartridges (Agilent). Digested peptides were then desalted in a Bravo AssayMap platform (Agilent) using AssayMap C18 cartridges and dried down in a SpeedVac concentrator.m/z) of 350\u20131700 with a resolution of 70,000 at m/z 400. Automatic gain control target was set to 1\u2009\u00d7\u2009106 with a maximum injection time of 100\u00a0ms. Up to 12 MS2 spectra per duty cycle were triggered, fragmented by HCD, and acquired with a resolution of 17,500 and an AGC target of 5\u2009\u00d7\u2009104, an isolation window of 1.6\u00a0m/z and a normalized collision energy of 25. The dynamic exclusion was set to 20\u00a0s with a 10\u00a0ppm mass tolerance around the precursor.Prior to LC\u2013MS/MS analysis, dried peptides were reconstituted with 2% ACN, 0.1% FA and concentration was determined using a NanoDrop\u2122 spectrophometer (ThermoFisher). Samples were then analyzed by LC\u2013MS/MS using a Proxeon EASY-nanoLC system (ThermoFisher) coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific). Peptides were separated using an analytical C18 Aurora column  at a flow rate of 300 nL/min (60\u00a0\u00b0C) using a 120-min gradient: 1% to 5% B in 1\u00a0min, 6% to 23% B in 72\u00a0min, 23% to 34% B in 45\u00a0min, and 34% to 48% B in 2\u00a0min . The mass spectrometer was operated in positive data-dependent acquisition mode. MS1 spectra were measured in the Orbitrap in a mass-to-charge (Homo sapiens Uniprot protein sequence database (downloaded in January 2020) and GPM cRAP sequences (commonly known protein contaminants). Precursor mass tolerance was set to 20\u00a0ppm and 4.5\u00a0ppm for the first search where initial mass recalibration was completed and for the main search, respectively. Product ions were searched with a mass tolerance 0.5\u00a0Da. The maximum precursor ion charge state used for searching was 7. Carbamidomethylation of cysteine was searched as a fixed modification, while oxidation of methionine and acetylation of protein N-terminal were searched as variable modifications. Enzyme was set to trypsin in a specific mode and a maximum of two missed cleavages was allowed for searching. The target-decoy-based false discovery rate (FDR) filter for spectrum and protein identification was set to 1%. Interaction candidates were those proteins enriched at least 3\u2009\u00d7\u2009over control samples (BioID2-only) and identified in at least two of the three experimental triplicate samples (N\u2009>\u20092).All mass spectra were analyzed with MaxQuant software version 1.6.11.0. MS/MS spectra were searched against the Samples from the prefrontal cortex and spinal cord of ALS and control patients were obtained from the University of Michigan Brain Bank. Consent for autopsy was obtained in accordance with guidelines from the University of Michigan Brain Bank who reviewed and confirmed that protocols met the criteria for human-subjects research. Immunostaining was accomplished using the Dako Autostainer Link 48 . Anti-HspA5 antibody (Abcam ab21685) was used at 1:1000 with the Dako High pH Target Retrieval Solution   and the Dako Envision Flex Plus Mouse Link Kit  to detect the antibody along with the Dako DAB . The images were analyzed using free, open-access QuPath (v.0.3.2) software and were analyzed by a blinded experimentator.Supplementary Information 1.Supplementary Table S1."}
+{"text": "TDP-43 is the primary or secondary pathological hallmark of neurodegenerative diseases, such as amyotrophic lateral sclerosis, half of frontotemporal dementia cases, and limbic age-related TDP-43 encephalopathy, which clinically resembles Alzheimer\u2019s dementia. In such diseases, a biomarker that can detect TDP-43 proteinopathy in life would help to stratify patients according to their definite diagnosis of pathology, rather than in clinical subgroups of uncertain pathology. For therapies developed to target pathological proteins that cause the disease a biomarker to detect and track the underlying pathology would greatly enhance such undertakings. This article reviews the latest developments and outlooks of deriving TDP-43-specific biomarkers from the pathophysiological processes involved in the development of TDP-43 proteinopathy and studies using biosamples from clinical entities associated with TDP-43 pathology to investigate biomarker candidates. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are diagnosed based on clinical criteria. Often, this leads to a delay between the onset of symptoms and diagnosis up to one year in ALS and 5 years in FTD, narrowing the window where potentially disease-modifying therapies would likely be most effective ,2,3,4,5.Ubiquitinated cytoplasmic aggregates of the nuclear 43 kDa transactive-response, DNA-binding protein, TDP-43, is the core pathobiology in almost all cases 97%) of ALS 7% of ALS,11,12. TThe clinical heterogeneity ranging from pure motor (ALS) to cognitively impaired (ALS-FTD) or dementia  phenotypes that are associated with TDP-43 proteinopathy is striking and likely associated with a distinct topographical distribution of TDP-43 pathology in the brain ,17. PathHere, we will review the latest developments and outlooks of deriving TDP-43-specific biomarkers from: (1) pathophysiological processes involved in the development of TDP-43 proteinopathy, and (2) biofluid studies using biosamples from clinical entities associated with TDP-43 pathology. We identified the existing evidence through author knowledge and PubMed searches from the database\u2019s inception to April 2022.TDP-43 belongs to a family of heterogeneous nuclear ribonucleoproteins (hnRNP) . StructuPathological processes that disturb the selective binding mechanism of TDP-43 enhance homonymous binding and self-aggregation, disturb its autoregulation, and lead to increased concentrations of TDP-43-aggravating protein accumulation b 47,48],4847,48]The pathology of TDP-43 in ALS and FTD is characterized by its mislocalization from the nucleus to the cytoplasm of neurons and glia cells, where it is found post-translationally modified by phosphorylation and ubiquitination, as well as N-terminally cleaved into smaller C-terminal fragments (CTF) . NeuropaUltimately, a major breakthrough in neuropathology was achieved when the serial fractionation of post mortem brain tissue led to the extraction of insoluble proteins, which specifically recovered pathological TDP-43 from the affected brain regions . A charaWhile cytoplasmic aggregation with a toxic gain of function is a proposed mechanism of TDP-43 pathobiology, a second mechanism of toxicity is the nuclear loss of TDP-43 leading to altered RNA/DNA metabolism ,76. It iA further example of using a transcriptome-driven approach to develop non-invasive prognostic biomarkers for disease has been demonstrated for ALS . In thisIt is known that TDP-43 specifically interacts with mitochondrial proteins and alters mitochondrial dynamics . PatholoTaken together the differential expression patterns of the chitinases in the ALS-FTD spectrum suggests that other factors, such as the severity of the disease, progression, or neurodegeneration pattern may account for differences rather than simply reflecting a reliable signal for TDP-43 proteinopathy.TDP-43 pathology has been identified in a wide range of neurodegenerative diseases, both in cases inherited in a Mendelian pattern or sporadic patients, providing an exciting link between the pathophysiology of apparently sporadic and familial diseases. Generally, ~97% of all ALS and ~45% of FTLD cases involve TDP-43 aggregation ,116. In The discovery of key proteins might lead to the identification of candidate ALS-FTD genes, a prominent example being the identification of missense variants in TARDBP. Mutations in the gene encoding TDP-43 have, however, only being associated in less than 5% of ALS patients . This isAs alterations in RNA metabolism have emerged as one critical driver of ALS/FTD pathogenesis, the role of microRNA, small non-coding RNAs and key determinants of mRNA stability, has recently been studied ,129. BesIn the future, however, additional types of non-coding RNAs could also be explored as ALS biomarkers. These include circRNAs that originate from back-splicing events during precursor mRNA processing and may act as miRNA sponges or to sequester RBP proteins, thus affecting protein and gene expression. Their usefulness as a biomarker mostly resides in the fact that circRNAs are very resistant to RNA exonucleases and are thus highly stable in cells. For example, a recent promising study of circRNAs in blood leukocytes of sporadic ALS patients has allowed us to identify four circRNAs whose expression may be differentially affected in patients compared to controls .Finally, long noncoding RNAs (lncRNAs) could also represent another possible type of RNA that could be easily detected in blood and CSF of patients. As the name suggests, lncRNAs are long RNAs that are not normally translated but can affect gene expression by affecting epigenetic and transcriptional profiles of their target genes. To this date, lncRNAs have not received a very high level of attention in ALS, but differential expression of several lncRNAs has been reported to occur in peripheral blood monocytes of ALS patients . It shouThe lack of studies aiming to develop TDP-43 disease-specific in vivo biofluid biomarkers by searching for common changes in TDP-43 pathology relevant patient groups is striking . Especian = 8 for Tau and n = 12 for TDP-43) were investigated. In total, ten biomarkers were observed to be differentially expressed between FTD-TDP and FTD-Tau, which were later validated. Specifically, Ubiquitin-like protein 3 was upregulated in FTD-TDP versus FTD-Tau, while \u03b1-Galactosidase A, Heat shock protein 8 and Kallikrein 7 were all downregulated in FTD-TDP compared to FTD-Tau. As a result, ten biomarkers were differentially regulated between the control group and FTD-TDP patients. However, only one candidate, catalase activity, was also found downregulated in FTD patients compared to the control group in the validation experiments, while other candidates failed. A second study investigated CSF samples from asymptomatic (n = 14), symptomatic (n = 14), ALS mutation carriers , and sporadic ALS patients (n = 12) [A recent proteomic study compared CSF samples from FTD patients with TDP-43 or Tau proteinopathy, confirmed by autopsy or genetic testing to a group of patients with subjective memory complaint . For the(n = 12) . In thes(n = 12) ,142,143.(n = 12) .Specific biomarkers have been investigated across clinical cohorts of ALS-FTD spectrum disorders in comparison to other neurodegenerative diseases, such as AD and PD. Here we review biomarker studies in light of their potential to differentiate ALS, FTD-TDP, and eventually LATE, from FTD-Tau and AD patient cohorts .A promising biomarker in neurodegenerative diseases is the light and phosphorylated heavy chain of neurofilaments (NfL and pNfH). Neurofilaments (Nf) are abundant cytoskeletal proteins of myelinated central and peripheral neurons . IncreasIt is now widely accepted that neuroinflammation contributes to neurodegeneration in ALS and FTD, although it is unresolved whether the immune response causes, aggravates, or counteracts neurodegeneration . SpecifiVariants in GRN are among the most frequent genetic causes of FTD, most leading to progranulin haploinsufficiency. Progranulin is a ubiquitous protein involved in several biological pathways, including growth factor-like activities, neuroinflammation, and neuronal survival . Levels Neurogranin is a neuron-specific dendritic protein regulating synaptic plasticity and learning. Considering the central role of synaptic pathology in AD pathogenesis, neurogranin is particularly interesting as a biomarker in AD. Large studies showed that neurogranin levels in CSF specifically increased in AD compared to several other neurodegenerative diseases, including PD, FTD, ALS, and vascular dementia ,158, conAnother biomarker candidate is transthyretin, which is required for the transport of thyroxine and retinol but also binds with \u03b2-amyloid, and thus, is considered to prevent formation of senile plaques. Proteomic studies have demonstrated an increase in transthyretin concentration in CSF from both AD and FTD patients ,161,162.Clusterin is a potent chaperone that can inhibit protein aggregation, and therefore, might protect against neurotoxicity. Importantly, it has been shown that clusterin protects against TDP-43 aggregation and mislocalization . In initp < 0.01 and p = 0.005) [Tau, the major microtubule associated protein of a mature neuron, is regulated by its phosphorylation state. In AD and other tauopathies, brain tau is hyperphosphorylated (p-tau) and becomes aggregated, resulting in neurofibrillary tau pathology. The CSF biomarkers for AD, particularly (p-) tau and amyloid b-42, mostly appeared to be of limited value for the diagnosis of FTD and its pathological subtypes ,199. How= 0.005) ,200. Int= 0.005) ,168.As already described, TDP-43 is the pathological protein found aggregated in neurons and glial cells of patients with ALS, FTD, and LATE. While the pathological form of TDP-43 can be detected in human post mortem brains, the detection in patient biofluids, such as CSF and serums using antibody-based assays, have been difficult , mainly In summary, none of the biomarkers described above yet have the ability to differentiate between clinical phenotypes of TDP-43 pathology across the ALS/FTLD spectrum diseases. Most studies show potential biomarkers of fast progressive ALS. Only a few studies have investigated biomarkers in different FTD clinical subtypes, of which some, such as bvFTD, are more likely to have underlying TDP-43 proteinopathy. However, most studies do not compare biomarkers across all clinical cohorts of ALS, FTD, and AD sufficiently. Rather worryingly, some studies have also included clinical entities that often harbour TDP-43 pathology but without a definite proof of its pathology. In addition, definite FTD-TDP patient cohorts are usually mixed cohorts of autopsy-confirmed or genetically proven FTD-TDP and limited to small numbers. Furthermore, at the moment there have been no studies that separate confirmed LATE-NC from typical AD biosamples. This should be considered of high priority because an important development of biomarkers for AD would be to allow them to discriminate AD from definite FTD-TDP or LATE. Indeed, an initial step towards developing such a biosample cohort could be to already separate samples that lack typical AD profiles, such as TREM2, Tau, p-Tau, and Amyloid-\u00df changes.Thus far, the development of TDP-43 as a biomarker to detect TDP-43 proteinopathies has not been successful for several reasons. In general, low concentrations in CSF and serum, as well as the detection of pathology unspecific forms are limiting such developments. Therefore, to advance TDP-43-specific biomarker development it will be necessary to further develop techniques that are aimed at detecting the pathological forms of TDP-43. As proteomic studies have yet failed to detect such forms in vivo in CSF ,73,74, ihttps://patentscope.wipo.int/search/en/detail.jsf?docId=WO2021198698, accessed on 8. February 2023).E.F. has a patent filed for a method for diagnosing a condition characterized by TDP-43 proteinopathy (PCT/GB2021/050821;"}
+{"text": "Transactivation response DNA binding protein 43 kDa (TDP-43) and tau are major pathological proteins of neurodegenerative disorders, of which neuronal and glial aggregates are pathological hallmarks. Interestingly, accumulating evidence from neuropathological studies has shown that comorbid TDP-43 pathology is observed in a subset of patients with tauopathies, and vice versa. The concomitant pathology often spreads in a disease-specific manner and has morphological characteristics in each primary disorder. The findings from translational studies have suggested that comorbid TDP-43 or tau pathology has clinical impacts and that the comorbid pathology is not a bystander, but a part of the disease process. Shared genetic risk factors or molecular abnormalities between TDP-43 proteinopathies and tauopathies, and direct interactions between TDP-43 and tau aggregates, have been reported. Further investigations to clarify the pathogenetic factors that are shared by a broad spectrum of neurodegenerative disorders will establish key therapeutic targets. Neuronal and glial aggregates of misfolded proteins, as well as systematic neuron loss and astrogliosis, are pathological hallmarks of neurodegenerative disorders. Transactivation response DNA binding protein 43 kDa (TDP-43/TARDBP) is a pathological protein observed in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) ,2,3, andNumerous basic studies reproducing pathological aggregates of TDP-43 and tau have revealed the neurotoxicity of the aggregates or loss of physiological functions of these proteins, although it is often controversial which of them is the more prominent pathway in patients . By contInterestingly, patient autopsies have revealed comorbid TDP-43 pathology in tauopathy patients, and vice versa. For example, a subset of AD patients showed TDP-43 pathology in the limbic system ,11. ComoTDP-43 is an RNA-binding protein that is coded on the chromosome 1p. Systemic organs, including the central nervous system, pancreas, and spleen, abundantly express TDP-43 ,12. ThisAnother pathological finding of ALS-TDP is Bunina bodies. Bunina bodies are highly eosinophilic granules, mainly found within the motor neuron cytoplasm, and which show immunoreactivity for cystatin-C and transferrin, but rarely for TDP-43 . Althougvalocin-containing-protein (VCP) gene [VCP mutation are quite diverse among families or even within a single family [TDP-43 is also known as a pathological protein of FTLD; each of TDP-43 and tau accounts for nearly 50% of total FTLD patients. The term \u201cFTLD\u201d encompasses the pathological entity corresponding to a clinical term \u201cfrontotemporal dementia (FTD)\u201d. FTLD-TDP is characterized by a prominent TDP-43 pathology in the frontotemporal cortices, and the limbic system, hippocampus, neostriatum, and substantia nigra are also vulnerable ,17,18. PCP) gene . The clie family .C9orf72 [progranulin (GRN), which is clinically associated with the pure FTD form with an absence of motor neuron degeneration [TDP-43 mutation is also found in familial ALS; the TDP-43 mutations typically manifest as ALS [TDP-43 are mostly located on the C-terminal side, including the prion-like domain.TDP-43 pathology is also common in familial ALS and FTLD. The most frequent gene mutation among familial ALS/FTLD-TDP is hxanucleotide (intronic GGGGCC) expansion of C9orf72 ,23; apprC9orf72 . Anotherneration ,25. The t as ALS ,27 and at as ALS . The knoALS-TDP and FTLD-TDP seem to have a close relationship. It is well known by neurologists that ALS sometimes co-occurs with FTD in patients, which has been termed FTD-MND or ALS with dementia. Neuropathological observations of autopsied patients have also suggested a relationship between ALS and FTLD. Studies have reported that TDP-43 pathology extends across multiple systems in the brains of ALS and FTLD patients, and the distributions of the lesions often overlap between these diseases. For example, the prefrontal cortices, primary motor cortex, limbic system, hippocampus, parahippocampal gyrus, and neostriatum are involved in, not only FTLD-TDP, but also a subset of ALS-TDP . FurtherTDP-43 pathology can be found in some aged people. Aging-related TDP-43 pathology is typically prominent in the limbic system, hence being termed limbic-predominant age-related TDP-43 encephalopathy (LATE); a recent neuropathological guideline defines LATE stages as stage 1 , 2 (spreading to the hippocampus), and 3  . This maBasic research has accumulated evidence that the cytoplasmic inclusions of TDP-43 are neurotoxic. Neuronal death or axonal dysfunction has been reported using models with TDP-43 overexpression , transfeBy contrast, the loss of nuclear TDP-43 has also been suggested to be a TDP-43-mediated pathomechanism. Transgenic mice expressing human TDP-43 with a mutated NLS displayed neuronal loss and tract degeneration in association with the downregulation of endogenous nuclear TDP-43, but with sparse cytoplasmic inclusions; loss of nuclear TDP-43, rather than cytoplasmic inclusions, was correlated with neuronal dysfunctions in this study . ConditiMicrotubule-associated protein tau (MAPT) gene encodes tau protein, and the alternative splicing of exons 2, 3, and 10 in MAPT generates six isoforms. These isoforms are largely subclassified into two isoform groups: 3-repeat (3R) and 4-repeat (4R) tau. MAPT exon 10 is located in microtubule-binding repeats, and the 4R isoform contains exon 10, whereas the 3R isoform lacks this. The microtubule-binding repeats physiologically interact with the C-terminal end of tau, forming a folding structure, even in a soluble state [Tauopathies are defined by neuronal or glial aggregates of hyperphosphorylated tau and are subclassified into 3R-tauopathies and 4R-tauopathies, according to the prominent isoforms of tau aggregates  Figure . Tau is le state . The reple state . Cryo-elle state , whereasle state ; these sle state ,52. Misfle state  and presle state .MAPT gene were identified as a cause of chromosome-17-related familial frontotemporal dementia with parkinsonism (FTDP-17) [GRN. Approximately 50 mutations have been identified, which can be associated with diverse 3R-tauopathy and 4R-tauopathy phenotypes, according to the mutation site [MAPT mutations and tau aggregation have not been fully clarified, most mutation sites have been found in exons 9\u201312 and adjacent introns and associated with altered microtubule assembly abilities [Mutations of the FTDP-17) , althougion site . Althougbilities . It has bilities .Patients with PiD show prominent brain atrophy and neuron loss in the prefrontal area and the bottom sides of the temporal lobes. The primary motor cortex, the most posterior region of the frontal lobes, is less involved than the prefrontal area, representing a contrast with other FTLDs. A characteristic neuropathological finding of PiD is Pick bodies, which appear as spherical inclusions within the neuronal cytoplasm, typically nearby the apical dendrites. Pick bodies are aggregates of 3R-tau and strongly labeled with silver staining methods. Neurons having Pick bodies also show diffuse immunoreactivity for hyperphosphorylated tau in the cytoplasm. The hippocampal granule cells often demonstrate numerous Pick bodies in association with severe neuron loss. Ballooned neurons that display swelling of cytoplasm with deviated nuclei are also abundant in PiD.PSP systematically involves the globus pallidus, subthalamic nucleus, and tegmentum of the midbrain and pons, in combination with impairment of the cerebellar efferent system, including the dentate nucleus and upper cerebellar peduncle. Lesions show globose neurofibrillary tangles in the neuronal cytoplasm and tufted astrocytes that are labelled using anti-4R-tau immunohistochemistry and silver staining methods. White matters display coiled bodies that are tau aggregates in oligodendrocytes.CBD grossly shows para-sylvian atrophy, which is often asymmetrical, in association with atrophy of the basal ganglia. The most striking microscopic finding is dense and numerous neuropil threads that are immunopositive for hyperphosphorylated tau in the deep cortical layers and white matters. Neuronal cytoplasm shows pretangles that are more fuzzy and more diffusely occupy the cytoplasm than NFTs. Ballooned neurons are often observed. Astrocytic plaques, tau aggregations within the distal portions of astrocytic processes, are characteristic glial inclusions of CBD; this is in contrast with the tufted astrocytes of PSP that are prominent in cytoplasm and proximal processes.AGD is characterized by 4R-tau-immunopositive and argyrophilic granules in the neuropil. AGD preferentially involves the anterior portion of parahippocampal gyrus and subiculum, showing atrophy of the medial temporal lobes. In a small subset of patients, the argyrophilic grains are extended to the limbic systems, basal ganglia, and frontal cortices. Ballooned neurons with tau-immunopositivity are often observed in the parahippocampal gyrus and hippocampal granule cells.GGT is a recently-identified 4R-tauopathy. Although several subtypes have been published for this disease, the typical findings are thick and globular aggregates of 4R-tau in oligodendrocytes, which are clearly distinguishable from the coiled bodies in other tauopathies.Neuropathological findings of AD comprise neurofibrillary tangles (NFTs) and senile plaques. NFTs are argyrophilic flame-like structures that are composed of 3R-tau and 4R-tau aggregations; it has been reported that 4R-tau is prominent during the early process of tau aggregation, whereas 3R-tau is increased during the maturation of NFTs in AD . It is wA subset of neurologically healthy people show local deposition of hyperphosphorylated tau in the brain. Age-related tau aggregation includes primary age-related tauopathy (PART)  and aginPostmortem studies have reported that TDP-43 aggregates can be observed in brain tissue under non-ALS/FTLD disorders, including AD ,65 Figu, PSP 6666,67 Fi, CBD 6868,69 Fi, Lewy boVarious neuropathological studies have emphasized that the comorbid TDP-43 pathology in tauopathies, including AD, PSP, and CBD, has disease-specific characteristics. A study revealed that TDP-43 pathology was observed in 195 (57%) out of 342 AD patients . TDP-43 The characteristic TDP-43 pathology has also been found in 4R-tauopathies, including PSP and CBD. It was reported that 47 out of 945 PSP patients (5%) showed TDP-43 aggregates in the hippocampus . A studyC9orf72 hexanucleotide expansion was associated with more abundant tau aggregates in the limbic system than GRN mutation or sporadic ALS/FTLD-TDP [Conversely, the comorbidity of tau aggregation has been reported in ALS-TDP and FTLD-TDP. A study reported that 24 (59%) of 41 ALS patients, seven (44%) of 16 FTD-MND patients, and 10 (43%) of 23 FTD patients showed tau pathology . Tau patFTLD-TDP . These sFTLD-TDP .Several studies have addressed the colocalization of TDP-43 and tau aggregates in tauopathies. A study of the lower motor neuron of PSP and CBD patients reported that TDP-43 pathology was fundamentally observed in the neurons without tau aggregates, and colocalization of TDP-43 aggregates with tau aggregates were rare, even when they were present within the same neuronal cytoplasm . AnotherC9orf72 hexanucleotide expansion than in nonmutated ALS/FTLD-TDP patients or age-matched controls [C9orf72 hexanucleotide expansion and its haploinsufficiency are not the same. In particular, GVD granules intensely express charged multivesicular body protein 2B (CHMP2B), which is a marker of endosomal vesicles. Mutations of the CHMP2B gene are known to cause familial FTD and ALS [C9orf72 hexanucleotide expansion, found that GVD expressing necrosome markers was spread with the extension of TDP-43 aggregates, as well as tau aggregates [Granulovacuolar degeneration (GVD) is defined as the presence of clusters of slightly basophilic granules within fused vacuoles, which are typically observed in the cytoplasm of pyramidal neurons  Figure . Granulecontrols . C9orf72controls . It is i and ALS ,94, and gregates . Taken tgrn increased hyperphosphorylated tau in P301L-Tg mice [grn and tmem106b in mice exhibited more severe TDP-43 aggregates than single knockout of grn [c9orf72 insufficiency reproduced the aggregation of p62-immunopositive protein aggregates via an altered endosomal trafficking ability [-Tg mice . Furthert of grn . An in v ability . Deregul ability , and in  ability . Moreove ability . Thus, amapt exon 2/3 splicing, which are critical regions for the differentiation of tau protein isoforms. Mapt splice variant expression (1N/0N) was significantly altered in tardbp Q331K homozygous mice compared to heterozygous or wild-type mice. The study concluded that TDP-43 binds to an intronic sequence upstream of mapt exon 2 [TDP-43 M337V and MAPT T175D co-expression demonstrated increased aggregation of hyperphosphorylated tau in the hippocampus, when compared to subjects expressing wild-type tau, suggesting a synergistic effect of these human-derived aggregates toward tau pathology [In vivo studies have also indicated a tau-seeding activity is demonstrated by misfolded TDP-43. A study found that human mutant-TDP-43-knock-in mice showed alterations in t exon 2 . It has athology . An in vathology . Data frTMEM106B rs1990622 A/A genotype, a known risk allele for ALS/FTLD-TDP, often show comorbid neuroglial aggregates of 4R-tau [C9orf72 is associated with risk of CBD [TMEM106B genotypes or hexanucleotide lengths of C9orf72 between PSP/CBD patients with and without TDP-43 pathology [Patient-based, genetic studies have emphasized the particular relationship between ALS/FTLD-TDP and 4R-tauopathies. Genome-wide association studies revealed selective genetic overlaps among ALS, FTLD, PSP, and CBD, which were absent in patients with AD and Parkinson\u2019s disease and healthy controls ,130. ALSf 4R-tau . Intermek of CBD . A laterathology ; this mamapt exon 10 splicing in mice expressing human-strain 3R- and 4R-tau isoforms, and impaired interaction of these molecules results in a 4R-tau-dominant condition [Translational research also supports a mechanistic link between ALS/FTLD-TDP and 4R-tauopathies . We haveondition . It is iondition ,134, andondition ,136. Supondition ,137; henondition . SFPQ anondition , in contondition ,139. It GRN, a causative mutation of FTLD-TDP, was positively correlated with more severe tau aggregation in patients having AD pathology but not with A\u03b2-amyloid deposition [Postmortem studies have suggested neuropathological overlapping between AD and LATE . As discposition .Autopsy-based research has revealed that comorbid pathology often has a disease-specific manner, in terms of the biochemical properties, morphological characteristics, and spatial distributions of aggregates, and has an impact for clinical phenotypes. Genetic studies have identified overlapping genetic risk factors between LATE and AD or between ALS/FTLD-TDP and 4R-tauopathies. These facts indicate that comorbid pathology is not an incidental bystander, but a part of the disease pathogenesis. It will be important to determine when a TDP-43 or tau pathology is comorbid during disease pathogenesis; antemortem studies using functional neuroimaging targeting aggregated proteins will be useful in the future.Basic research findings have suggested that the molecular pathways are partially overlapped between TDP-43 proteinopathies and tauopathies. In vivo studies have revealed that aggregated TDP-43 altered the splicing of tau or exacerbated tau aggregation. SFPQ, FUS, and TDP-43 were suggested to be commonly be involved by pathogeneses among ALS/FTLD-TDP, PSP, and CBD. Moreover, perturbation of the autophagosome-lysosome system-related molecules has been reported in both TDP-43 proteinopathy and tauopathy models. However, it currently seems to be difficult to reproduce the condition of double proteinopathy comprising TDP-43 and tau pathologies by altering one of molecules or genes shown above. This fact suggests that pathogeneses of TDP-43 proteinopathies and tauopathies arise from multifactorial and polygenetic processes. Further investigations to clarify the pathogenetic factors that are shared by a broad spectrum of neurodegenerative disorders will establish key therapeutic targets."}
+{"text": "TAR DNA binding protein 43 (TDP-43) is a DNA/RNA binding protein involved in pivotal cellular functions, especially in RNA metabolism. Hyperphosphorylated and ubiquitinated TDP-43-positive neuronal cytoplasmic inclusions are identified in the brain and spinal cord in most cases of amyotrophic lateral sclerosis (ALS) and a substantial proportion of frontotemporal lobar degeneration (FTLD) cases. TDP-43 dysfunctions and cytoplasmic aggregation seem to be the central pathogenicity in ALS and FTLD. Therefore, unraveling both the physiological and pathological mechanisms of TDP-43 may enable the exploration of novel therapeutic strategies. This review highlights the current understanding of TDP-43 biology and pathology, describing the cellular processes involved in the pathogeneses of ALS and FTLD, such as post-translational modifications, RNA metabolism, liquid\u2013liquid phase separation, proteolysis, and the potential prion-like propagation propensity of the TDP-43 inclusions. TARDBP gene encoding TDP-43 in both ALS cases and rare patients with FTLD have demonstrated that TDP-43 is fundamentally involved in the pathogenesis of ALS and FTLD-TDP [Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease characterized by progressive muscle weakness due to loss of upper and lower motor neurons, leading to death from respiratory failure, usually within four years . FrontotFTLD-TDP ,9,10,11.FTLD-TDP ,13,14.TDP-43 was initially identified as a repressor protein associated with HIV-1 transcription, and it is a highly conserved and ubiquitously expressed RNA/DNA-binding protein belonging to the heterogeneous nuclear ribonucleoprotein (hnRNP) family . TDP-43 TARDBP gene on chromosome 1 (1.p36.22). The structural domains comprise the following: an N-terminal region (aa 1\u2013102) with a nuclear localization signal ; two RNA recognition motifs: RRM1 (aa 104\u2013176) and RRM2 (aa 192\u2013262); a nuclear export signal , a C-terminal region (aa 274\u2013414) harboring a prion-like glutamine-/asparagine-rich (Q/N) domain (aa 345\u2013366) and a glycine-rich region (aa 366\u2013414) (TDP-43 contains 414 amino acids (aa) and is encoded by the 366\u2013414)  [21,22,2 NES, aa 9\u2013250, a 2 aa 192\u20132; a nuclUnder physiological conditions, TDP-43 is natively dimeric or exists in a monomer-dimer equilibrium . The N-tRRM1 and RRM2 domains in TDP-43 are indispensable for RNA/DNA binding to regulate the transcription, translation, splicing, and stability of mRNA . These dTARDBP gene mutations and phosphorylation sites, strongly contributes to the pathological behavior of TDP-43 and is intrinsically disordered and aggregation-prone [The C-terminal region of TDP-43, which harbors most of the ALS-associated on-prone . This reon-prone .As a DNA/RNA binding protein, TDP-43 is involved in multiple aspects of RNA metabolism, including splicing, microRNA (miRNA) biogenesis, RNA transport and translation, and stress granule formation by interacting with numerous hnRNPs, splicing factors, and microprocessor proteins . Given tIn contrast to the relatively stereotyped pathology of TDP-43 aggregates in the vast majority of sporadic ALS and FTLD-TDP, the underlying mechanisms are varied and remain uncovered .Numerous pathogenic mutations in the TARDBP gene (>50 mutations) have been identified in both sporadic and familial cases of ALS and FTLD-TDP, accounting for six percent of global familial ALS (FALS) patients. The mutations are exclusively found in the C-terminal glycine-rich region ,15. MutaVarious post-translational modifications of TDP-43, including cleavage, ubiquitination, and phosphorylation, have been implicated in neurotoxicity in the TDP-43 proteinopathies. The aberrant mislocalization of TDP-43 triggers various post-translational modifications, although the precise mechanisms remain elusive .The generation of 25\u201335 kDa C-terminal fragments (CTFs) of TDP-43 through proteolytic cleavages by the caspase and calpain proteases is reported as one of the prominent pathological processes in ALS and FTLD-TDP. The C-terminal region of TDP-43 harbors most of the ALS-associated mutations and phosphorylated sites, and it is intrinsically disordered and aggregation-prone . The CTFUbiquitinated TDP-43 inclusions are a pathological hallmark in the ALS- and FTLD-TDP-affected brain ,6. The EIn addition to ubiquitination, hyperphosphorylated TDP-43 is a pathological feature in ALS and FTLD-TDP ,6. WhethThe covalent attachment of small ubiquitin-like modifier (SUMO) proteins to specific proteins, termed SUMOylation, is a reversible pathway that competes with ubiquitin to alter subcellular localization and protein turnover. SUMOylation regulates the functional properties of the specific proteins in the nucleus and cytoplasm of neurons, playing a role in the cellular responses to hypoxia, oxidative stress, glutamate excitotoxicity, and proteasome impairment, pivotal processes linked to motor neuron degeneration in ALS . The mutLysine acetylation is a major covalent modification controlling diverse cellular processes and has been implicated in Alzheimer\u2019s disease and other neurodegenerative diseases . Among 2TDP-43 functions as a DNA/RNA binding protein and is predominantly located in the nucleus of cells. It shuttles between the nucleus and the cytoplasm, engaging in diverse cellular functions within both compartments . This nuTDP-43 interacts with several proteins involved in the mRNA metabolisms in the nucleus and the cytoplasm . For thiTDP-43 is involved in all aspects of RNA metabolism ranging from splicing, transcription, transport, storage into RNA/protein granules, and translation. Accumulating evidence has demonstrated that dysregulation of RNA metabolism contributes to ALS pathogenesis ,74. WithKcnip2, Abhd14a, Ctnnd1, and Atp2b1, demonstrating a loss of normal TDP-43 function. In contrast, the Q331K mutant enhances the splicing of other target RNAs, such as Eif4h and Taf1b [TDP-43 has been shown to function as a splicing regulator and its nuclear depletion results in the mRNA splicing aberrations of multiple RNA targets . In addind Taf1b .The neuronal growth-associated factor, stathmin-2, is an attractive candidate for the splicing target of TDP-43. Stathmin-2 is necessary for normal axonal outgrowth and regeneration, and lowered TDP-43 levels reduce its binding to sites within the first intron of stathmin-2 pre-messenger RNA and produce a truncated and non-functional mRNA. Of note, reduced stathmin-2 expression is associated with poor axonal regeneration, which is rescued by normalization of stathmin-2 expression ,79.TDP-43 also regulates a cryptic exon UNC13A splicing event. TDP-43 depletion from the nucleus induces robust inclusions of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression ,81.Mapt in TDP-43 Q331K knock-in mice [Kcinp2, Sort1, and Sema3f is deregulated in the spinal cords of TDP-43 M337V knock-in mice [The gain of splicing function has been recently demonstrated in knock-in mice carrying TDP-43 missense mutation: TDP-43 autoregulation is perturbed, leading to altered splicing of -in mice ; TDP-43 -in mice ; mRNAs s-in mice .The examination of splicing patterns of TDP-43 target genes in lower motor neurons of postmortem ALS cases has revealed the widespread dysregulations of mRNA splicing that specifically affected genes involved in ribonucleotide binding . MoreoveTDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons and facilitate the delivery of target mRNA, such as neurofilament light (NEFL) mRNA, to distal axonal compartments; notably, TDP-43 mutations (A315T and M337V) impair this mRNA transport function . LikewisTARDBP genes, implicating TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS and FTLD-TDP [While TDP-43 is predominantly located in the nucleus, it continuously shuttles between the nucleus and cytoplasm in a transcription-dependent manner, engaging in diverse physiological cellular functions . TDP-43 FTLD-TDP .Eukaryotic cells have developed mechanisms that protect cells against exposure to multiple cellular stresses such as heat shock, oxidative stress, hyperosmolarity, viral infection, and chemical exposure. Non-membranous cytoplasmic foci with a size range of 0.1\u20132.0 \u03bcm are formed as stress granules (SG) in response to these diverse environmental conditions ,90. The Liquid\u2013liquid phase separation (LLPS) is a process that mediates the formation of membraneless, spherical, liquid droplet-like organelles, in which proteins containing prion-like domains are involved. This process is a vital pathway in various neurodegenerative diseases . The lowThe oligomer formation of TDP-43 is an intermediary step that occurs during the formation of large aggregates. The TDP-43 oligomerization stage is initiated by its RNA-binding region. Specific binding to GU-rich RNA strongly intercepts TDP-43 oligomerization and the resultant aggregate formation, suggesting that RNA is a crucial regulator in maintaining TDP-43 solubility . TDP-43 Drosophila model, thereby suggesting an imbalance of the mitochondrial dynamics [Mitochondrial dysfunction has been implicated in the mechanism of TDP-43 toxicity. Since post-mitotic neurons have high demands for mitochondria due to synaptic homeostasis, mitochondrial dysfunction in neurons significantly affects cellular function and survival ,112. Mitdynamics . In the dynamics . Fractiodynamics . Interesdynamics , althougdynamics .2O2) and the superoxide radical anion [Reactive oxygen species (ROS) arise as by-products of aerobic metabolism. Most cellular ROS originate from the leaked electrons from the mitochondrial respiratory chain. The oxidative phosphorylation unavoidably produces ROS, such as hydrogen peroxide  are stored in the ER, crucially involved in the ER-mitochondrial calcium cycle, which may link mitochondrial energy production and ER protein processing with neuronal synaptic activity [2+ homeostasis [2+ signaling from the ER compared with WT TDP-43 in cell lines, suggesting that ER plays a crucial role in Ca2+ signal homeostasis in ALS [C. elegans (worm) and D. rerio (zebrafish) models expressing mutant TDP-43 (G348C), Vaccaro et al. showed that ER stress suppression with pharmacological compounds is neuroprotective against TDP-43 proteinopathy [Most secretory proteins in eukaryotes enter the endoplasmic reticulum (ER) after translation and are folded and assembled. The ER responds to the burden of unfolded proteins in its lumen (ER stress) by activating various intracellular signaling pathways, termed the unfolded protein response (UPR) . Large aactivity . Inapproactivity ,127. TDPeostasis . Mutant s in ALS . In C. einopathy , and theThere are two major proteolytic pathways in eukaryotic cells: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway, which control protein quality and maintain cellular homeostasis. The frequent presence of cytoplasmic aggregates of TDP-43 is tightly linked to proteostasis dysfunction in ALS ,131,132.In neurodegenerative diseases, the UPS overwhelming may result in inappropriate protein degradation and aberrant protein accumulation, which can lead to cellular apoptosis . A recenTDP-43 functions as an autophagy regulator by associating with the mRNA for autophagy-related 7 (ATG7) and some of the ALS-linked TDP-43 mutations lose their ATG7 mRNA-binding ability . TDP-43 UBQLN2 gene encoding the ubiquitin-like protein ubiquilin 2 cause X-linked ALS/FTLD, and UBQLN2-positive inclusions are identified in ALS with UBQLN2 mutations as well as in cases of both familial and sporadic ALS without UBQLN2 mutations [UBQLN2 gene impair autophagic protein degradation and promote TDP-43 aggregation in neuronal cells [Ubiquilin (UBQLN), a member of the ubiquitin-like (UBL)-ubiquitin-associated (UBA) family, is a dual regulator of both the proteasomal and autophagic protein degradation system. Mutations in the utations . Mutatioal cells . Moreoveal cells .Drosophila motor neurons, primary murine cortical neurons, and stem-cell-derived motor neurons, which facilitate the delivery of target mRNA to distal neuronal compartments. ALS-associated TDP-43 mutants (M337V and A315T) impair the mRNA transport function [Axonal transport is the process whereby motor proteins actively navigate microtubules to deliver cargoes, such as organelles, cytoskeletal elements, and growth factors, from one end of an axon to the other and is widely regarded as essential for nerve development, function, and survival ,148. Impfunction . Furtherfunction ,114. TheAccumulating evidence indicates that the spatial progression of neurodegenerative diseases is mediated by prion-like cell-to-cell propagation of disease-related proteins, in which the template-induced misfolding of normal endogenous proteins to pathological conformations occurs . The conGlial cells such as astrocytes, microglia, and oligodendrocytes crucially maintain multiple neural homeostatic functions, including synaptic function, supply of metabolites and neurotrophic factors to neurons, and repairment of damaged neural tissue ,163,164.Although pathologically ubiquitinated and phosphorylated TDP-43 inclusions are commonly linked to neurodegeneration in ALS and FTLD-TDP ,6, TDP-4Strikingly, nuclear depletion and the cytoplasmic accumulation of TDP-43 inclusions are evident in up to 97% of all ALS cases ,6. PathoVCP gene and has many lentiform neuronal TDP-43 intranuclear inclusions throughout the cortical layers [C9orf72, GRN, CHMP2b, and TARDBP genes are also associated with FTLD-TDP [GRN gene typically correlate with type A pathology. C9orf72 mutations are generally associated with FTLD-TDP type B [TDP-43 inclusions in frontal and anterior temporal lobe regions are detected in a subgroup of FTLD, named FTLD with TDP-43 (FTLD-TDP). FTLD-TDP accounts for ~45% of all FTLD cases and is distinguished from other FTLD subgroups with pathological tau (FTLD-tau), fused in sarcoma (FTLD-FUS), and other proteins . FTLD-TDl layers . The genFTLD-TDP . AlthougP type B ,182.Interestingly, TDP-43 pathology can also be detected in the cells related to the clinical profiles of ALS/FTLD other than motor and cognitive dysfunctions. ALS/FTLD patients often show unusual eating behavior , sleep , and eneGRN, TMEM106B, ABCC9, KCNMB2 and APOE, is associated with LATE [Limbic-predominant age-related TDP-43 encephalopathy (LATE) is clinically associated with an amnestic dementia syndrome that mimics Alzheimer\u2019s-type dementia, and its neuropathological change is defined by a stereotypical TDP-43 proteinopathy in older adults, with or without co-existing hippocampal sclerosis pathology. In LATE, TDP-43 inclusions are predominantly confined to the limbic system, the middle frontal gyrus, and the medial temporal lobe. The genetic variation in five genes, ith LATE .TARDBP, SOD1, SQSTM1, VCP, and CHCHD10 have been reported in relation to FOSMN syndrome, and TDP-43-positive intraneuronal inclusions are identified in the brain, spinal cord, and dorsal root ganglia, suggesting that FOSMN is most likely to be a TDP-43 proteinopathy within the ALS-FTLD spectrum [DCTN1 encodes the largest subunit of the dynactin complex, which associates with the microtubule-based motor protein dynein and is required for dynein-mediated long-distance retrograde transport [Facial onset sensory and motor neuronopathy (FOSMN) is a rare neurodegenerative disease of motor and sensory neurons, initially developing paresthesia and numbness in a trigeminal nerve distribution and motor manifestations, with limb and bulbar muscle weakness developing later in the course of the illness . Mutatiospectrum ,193,194.spectrum . TDP-43 spectrum . Furtherspectrum ,198. In spectrum . TDP-43 spectrum . TDP-43-spectrum ,202. Recspectrum . G-PDC, spectrum ,205. Perspectrum . The cauransport . Interesransport . ConsideTDP-43 has emerged to function as a pivotal protein for cellular homeostasis. Overall, TDP-43 dysfunction due to various factors, such as imbalance of nucleo-cytoplasmic distribution, dysregulations of RNA metabolism, genetic mutations, aberrant post-translational modifications, aggregation, and gain of cytotoxicity, induces the collapse of cellular homeostasis and leads to TDP-43 proteinopathy in ALS and FTLD-TDP. Although the findings do not conclusively prove whether the aberrant TDP-43 protein is fundamentally involved in the pathophysiology of other neurodegenerative diseases, the expanded neurodegenerative spectrum of TDP-43 proteinopathy, including LATE and Perry\u2019s syndrome with TDP-43-positive inclusions, suggests that TDP-43 may, at least in part, mediate neurodegeneration. Therefore, the revelation of the mechanisms involved in TDP-43 homeostasis and dysfunctions will yield novel therapeutic targets against ALS and FTLD-TDP and multiple neurodegenerative diseases."}
+{"text": "After the pregnant rats were exposed to hypoxia (10.5% oxygen) from embryonic day (E) 5 to E21, PH offspring were generated. All animals maintained normoxia during lactation. The number of follicles was counted in female offspring at 3\u00a0months under an optical microscope. The expression of Nobox, Gdf9, and Tets was detected by quantitative real-time polymerase chain reaction (PCR) and Western blot. Global DNA hydroxymethylation was measured by dot blot. The hydroxymethylation level of the Nobox gene was evaluated with an NGS-based multiple targeted CpG hydroxymethylation analysis method. Body weight and ovary weight were significantly decreased in the PH group compared with the control group. PH offspring have abnormal estrous cycle, decreased serum anti-Mullerian hormone (AMH), and increased serum follicle-stimulating hormone (FSH), and follicular atresia, which are consistent with the clinical manifestations in patients with ovarian dysfunction. In terms of mechanism, the expression of Nobox was significantly decreased in the PH group. Subsequent high-throughput sequencing results showed that the level of hydroxymethylation in the candidate region of the Nobox gene was reduced. Cultured cells treated with hypoxia exhibited lower levels of both 5hmC and Nobox, while vitamin C, a coactivator of Tets, rescued hypo-hydroxymethylation and increased the expression level of Nobox. This study indicated that PH could cause hypo-hydroxymethylation of Nobox through epigenetic regulation and may consequently contribute to ovarian dysfunction in adult rat offspring.Prenatal hypoxia (PH) is a common feature of a suboptimal intrauterine environment affecting the development of fetuses. Whether PH leads to abnormal ovary development is not yet clear. This study investigated ovarian function in offspring exposed to PH and the potential underlying molecular mechanisms. SD female rats (The online version contains supplementary material available at 10.1007/s43032-022-00866-6. The intrauterine environment plays an important role in shaping fetal development and impacting later health . PrenataAMH) and antral follicles (AFC), and by increased follicle-stimulating hormone (FSH). DOR has multiple etiologies including autoimmune, genetic, iatrogenic, and idiopathic etiologies [GDF9) and newborn ovary homeobox gene (NOBOX) [GDF9 is an oocyte-secreted factor that plays an important role in follicular development [GDF9 can inhibit the apoptosis and atresia of cumulus cells and regulate the growth and differentiation of early follicles [GDF9 are associated with DOR [NOBOX is an important transcription factor for follicular development [NOBOX is an oocyte-specific gene and is expressed in primordial and germ cell cysts [Ovarian reserve refers to the total number of primordial follicles remaining in two ovaries, which is established in the fetal period and vulnerable to adverse intrauterine environments. It has previously been observed that ovarian follicle and oocyte development is sensitive to oxygen tension . Disturbiologies . In addi (NOBOX) \u201327. Base (NOBOX) . GDF9 iselopment . GDF9 caollicles , 30. In with DOR , 31. NOBelopment . It can elopment . In addill cysts \u201336. Noboll cysts . In addill cysts . WhetherThe occurrence of FOAD is related to increased susceptibility to certain adult diseases and is closely related to epigenetic modification. It is known that the adverse intrauterine environment can lead to epigenetic changes that would be sustained throughout one\u2019s lifetime, and potentially be passed on to offspring. Epigenetic regulation includes DNA methylation/demethylation, noncoding regulatory RNAs, and histone modifications . EpigeneAt present, there is litter research on the effect of prenatal hypoxia on the reproductive function of offspring, and the specific mechanism remains unclear. Our aims are to study the effect of hypoxia during pregnancy on the Nobox gene of the ovary of offspring and to explore the relationship between the Nobox gene and hydroxymethylation.This work investigates the epigenetic mechanisms of prenatal hypoxia on Nobox gene functions in ovary development in female offspring. The data may help to further understand the mechanism of the prenatal hypoxia-induced increase in risks in the development of ovarian dysfunction later in life .2 concentration corresponds to high altitude living  at which more than 140 million people currently reside. The hypoxia chamber was fabricated in which the oxygen concentration can be arbitrarily set. The nitrogen generated from the N2+ gas generator was mixed with ambient air at an arbitrary ratio using a N2+ air blender to generate hypoxia. In this study, we generated a hypoxia model using 10.5% oxygen which has been set up in our lab and demonstrated to be stable [n\u2009=\u200912 per group) give birth naturally, then we selected one female offspring at 3\u00a0months from each litter to perform Western blot and q-PCR. As for 5-hmC sequencing, we chose 1\u20132 female offspring per litter to get more sample size for the experiment. Body weight after birth and at 3\u00a0months of age was measured. Each 3-month-old female offspring rat was anesthetized intraperitoneally with chloral hydrate (4%) and sacrificed. Rats underwent cuts into the abdominal cavity with scissors in a midline incision. One ovary of each rat was stored in a\u2009\u2212\u200980\u00a0\u00b0C freezer, and the other ovary was fixed in formalin-paraldehyde. Unified incineration treatment of animals occurred after euthanasia.All procedures were approved by the ethical committee of Soochow University. One female rat was mated with one male rat. When vaginal plugs were observed, the day was recorded as embryonic day 1 (E1). The control group  and hypoxia group  were enrolled in the experiment. A modest 10.5% Oe stable . All aniThe estrous cycle was observed at 8\u00a0weeks of rats for consecutive 10\u00a0days. Vaginal cells were collected via saline lavage and then fixed with absolute ethyl alcohol and stained with hematoxylin and eosin (HE). The stages of the estrous cycle were determined based on vaginal cytology.\u00a0g for 10\u00a0min immediately after collection. The upper layer of plasma was drawn for later use. Separated plasma samples were stored at\u2009\u2212\u200980\u00a0\u00b0C until use. The plasma levels of AMH, FSH, luteinizing hormone(LH), and testosterone (T) were measured by a rat AMH ELISA kit , a rat FSH ELISA kit , a rat LH ELISA kit , and a rat T ELISA kit  respectively. Assays were performed according to the manufacturer\u2019s instructions. The absorbance at 450\u00a0nm was determined using Multiskan Spectrum.The rat offspring were anesthetized by intraperitoneal injection of chloral hydrate. After exposing the abdominal cavity, approximately 4\u20135\u00a0ml of arterial blood was drawn from the abdominal aorta with a 5-ml syringe. Blood samples were centrifuged at 3000Ovaries were stained with HE according to standard protocols. To determine ovarian morphology and follicles, every 12th section was mounted on a glass slide, and the number of follicles was counted. Each section was 8\u00a0\u00b5m. Only follicles with a visible nucleus were counted.l-ascorbic acid under hypoxic condition for 48\u00a0h. The expression of Nobox, Gdf9, and Tets was examined by q-PCR and/or Western blot.Currently, we do not have a suitable ovarian-related cell line. 3T3 cells are mouse embryonic fibroblasts derived from mouse embryos, and we verified that Nobox and Tet were expressed at high levels in 3T3 cells. Therefore, we chose 3T3 cells to investigate the potential relationship between Nobox and Tet in vitro. 3T3 cells may not be the best cell for the study. Nobox and Tet were expressed in 3T3 cells so that they can be used for investigation. 3T3 cells were cultured at 37 \u2103 in Dulbecco\u2019s modified Eagle\u2019s medium  containing 10% (v/v) heat-inactivated fetal bovine serum  and 100\u00a0mg/ml penicillin G and 100\u00a0mg/ml streptomycin sulfate. After seeding in 6-cm dishes overnight, cells were treated with 0.5\u00a0mM \u00a0g for 30\u00a0min at 4\u00a0\u00b0C, part of the supernatant was extracted, and the protein concentration was measured according to the instructions of the Detergent Compatible Bradford Protein Assay Kit . The rest of the supernatant was denatured at 96 \u2103 for 10\u00a0min and subjected to Western blot analysis. Protein (20\u00a0\u03bcg) was loaded onto 10% SDS-PAGE gels and transferred to PVDF membranes. NOBOX and \u03b2-actin protein abundance were measured using primary antibodies . Blots were visualized using chemiluminescence detection . Imaging signals were digitized and analyzed with a UVP imaging system . The membrane was incubated with a secondary antibody; HRP-conjugated goat anti-mouse IgG  or goat anti-rabbit IgG  was applied in TBS-T for 1\u00a0h at room temperature. The relative density of bands was normalized to actin as a control. The gray value was analyzed by Image J software.Ovary tissues (50\u00a0mg) or 3T3 cells were disrupted in 500-\u03bcl of RIPA buffer supplemented with PMSF and then held on ice for 30\u00a0min. After centrifugation at 13800\u2212\u25b3\u25b3CT method.Total RNA was extracted from offspring ovaries using RNAiso Plus (TaKaRa). The extracted RNA (1000\u00a0ng) was reverse-transcribed with a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer\u2019s instructions. qRT-PCR was performed with gene-specific primers, cDNA and SYBR Premix Ex Taq (TaKaRa), using a Bio-Rad IQ icycler. Primer sequences are listed in Table Ovaries were used for genomic DNA extraction. Genomic DNA was extracted using phenol: chloroform: isoamyl alcohol . Dot blot analysis was performed to detect hydroxymethylation levels. Ten microliters of 100\u00a0ng/\u03bcl genomic DNA was denatured with 10\u00a0\u03bcl of 2\u00a0M NaOH for 60\u00a0min. Two microliters of genomic DNA was spotted on an NC membrane (Millipore). The membrane was dried at RT for 30\u00a0min and incubated for 60\u00a0min at 75\u00a0\u00b0C. The membrane was then blocked with 5% BSA in TBS-T for 1\u00a0h and incubated overnight with a rabbit anti-5-hmC polyclonal antibody  in TBS-T at 4\u00a0\u00b0C. The membrane was incubated with a goat anti-rabbit IgG secondary antibody  in TBS-T for 1\u00a0h at RT. The dot blot intensity was quantified by a UVP imaging system . The gray value was analyzed by Image J software.Five candidate regions of Nobox were selected and sequenced. The selection was based on a previous experiment. We previously performed genome-wide hydroxymethylation sequencing of the rat forebrain. According to the results, we found that the Nobox gene has abundant hydroxymethylation modification sites, so we selected five enriched peaks to carry out target region hydroxymethylation sequencing. The DNA hydroxymethylation level was evaluated at Shanghai Genesky Biotechnology Company , with an NGS-based multiple targeted CpG hydroxymethylation analysis method. Briefly, genomic DNA was quantified by Qubit 3 (Invitrogen). With 0.2% synthetic fully methylated and fully hydroxymethylated control DNA spiked in the genomic DNA was treated using T4 \u03b2-glucosyltransferase , and APOBEC3A (NEB) in sequence. Enzyme-treated genomic DNA was used as a template for multiplex PCR amplifications with HotStarTaq polymerase (TaKaRa). The PCR program included 95\u00a0\u00b0C for 2\u00a0min; 11 cycles of 95\u00a0\u00b0C for 20\u00a0s, 62\u00a0\u00b0C for 40\u00a0s with a decreasing temperature step of 0.5\u00a0\u00b0C per cycle, and 72\u00a0\u00b0C for 1\u00a0min; then 24 cycles of 95\u00a0\u00b0C for 20\u00a0s, 64\u00a0\u00b0C for 30\u00a0s, and 72\u00a0\u00b0C for 1\u00a0min; and finally 72\u00a0\u00b0C for 2\u00a0min. PCR products were diluted and amplified using indexed primers with HotStarTaq polymerase (TaKaRa) to generate a sequencing library. The PCR program was 95\u00a0\u00b0C for 2\u00a0min; 12 cycles of 95\u00a0\u00b0C for 20\u00a0s, 60\u00a0\u00b0C for 40\u00a0s, and 72\u00a0\u00b0C for 1\u00a0min; and 72\u00a0\u00b0C for 2\u00a0min. PCR products were separated by agarose electrophoresis and purified using a TIANgel Midi Purification Kit (TIANGEN). Libraries from different samples were quantified, pooled, and sequenced on an Illumina sequencer according to the manufacturer\u2019s protocols, generating 2\u2009\u00d7\u2009150\u00a0bp paired-end reads with an average depth of 1000\u2009\u00d7\u2009at the target region.t tested, we first tested whether the data between the two groups (CON group and PH group) had homogeneity of variance. For the data with an F value greater than 0.05, we performed a parametric t test, and for the data with an F value less than 0.05, we performed a nonparametric t test. Data were determined for the difference between 3 groups  using 2-way analysis of variance (ANOVA) and P\u2009<\u20090.05 was considered significant. Data were expressed as the mean\u2009\u00b1\u2009SEM. The sample size was chosen on the basis of similar previous studies, and not on statistical methods to predetermine sample size.The statistical analysis was performed using GraphPad Prism 8. For the statistical data that needed to be P\u2009>\u20090.05, t test). Female survival ratios of the control group were 98.3% (57/58). Female survival ratios of the hypoxia group were 94.5% (52/55). Female survival ratios were not different between the control and hypoxic groups . Birth body weight was measured at birth. Bodyweight and ovary weight were measured at 3\u00a0months. Birth body weight was reduced by 20.3% in the PH group . Bodyweight at 3\u00a0months was reduced by 3.82% in the PH group . Ovary weight at 3\u00a0months was reduced by 5.27% in the PH group  ; however, there was no significant difference in the ratio of ovary weight to body weight between the two groups , whereas the serum FSH level was significantly increased in the PH group . In addition, the LH level and T level were not significantly different between the two groups . In addition, the number of atresia follicles was increased in PH rats . The numbers of other follicles were similar in PH and control rats. These data indicated that ovarian function in the offspring of the PH group was obviously abnormal.Litter size was not different between the control and hypoxic groups (t) Table . BodyweiP\u2009<\u20090.000, t test.P\u2009<\u20090.000, t test.P\u2009<\u20090.000, t test.P\u2009<\u20090.000, t test.P\u2009<\u20090.05, t test) and Gdf9  in the ovary were decreased in the PH group   Fig.\u00a0b. These P\u2009<\u20090.01, t test)   Fig.\u00a0a. The dost) Fig.\u00a0b.Fig. 3TP\u2009<\u20090.05, t test). These results suggested that Nobox was hypo-hydroxymethylated in the ovary exposed to PH. Figure\u00a04To identify whether Nobox gene expression is regulated by hydroxymethylation modification, high-throughput sequencing was performed on the Nobox gene. Five candidate regions of Nobox were selected and sequenced Fig.\u00a0a. The th4P\u2009<\u20090.001, t test) , Tet2 , and Tet3  were decreased in 3T3 cells treated with hypoxia    in a concentration range from 0.05\u00a0mM to 1\u00a0mM under hypoxic conditions for 48\u00a0h. Tets mRNA expression was evaluated by q-PCR to determine the best working concentration of Vitc. The rational dosage of 3T3 cells was 0.5\u00a0mM under hypoxic conditions for 48\u00a0h as the expression of Tet1 increased significantly . The q-PP\u2009<\u20090.01, ANOVA) and Tets  was increased in 3T3 cells treated with vitamin C    Fig.\u00a0c, t testst) Fig.\u00a0d.Fig. 6EIn the present study, we used a rat model to show the effects of PH on the ovary. Birth weight was reduced by 1.28\u00a0g and 20.3% by PH , which iOvarian function includes reproductive function and endocrine function, which begin to form in the fetal period. A favorable in utero environment is a necessary condition for embryo development and ovarian development. Oxygen tension is important for ovarian follicular reserve at 3\u00a0months of gestation, because primordial germ cells migrate to the genital ridge and follicular development depends on oxygen tension . ExposurNOBOX is essential for the early development of the ovary and is involved in the regulation of genes at the transcription level during the follicle stages [NOBOX was reported in women with POI and infertility [e stages . Mutatioertility . Nobox kertility , 36. Theertility , 49. In ertility , 50\u201353. Ten-eleven-translation 5-methylcytosine dioxygenase (Tets) is involved in epigenetic regulation of hydroxymethylation. The Tet family of enzymes catalyzes the conversion of 5mC to 5hmC, a modified cytosine base that facilitates gene expression. It has been demonstrated that Tet is essential for viability and fertility. Meanwhile, the enzymatic activities of Tets require oxygen. In our study, Nobox changed dynamically with Tets in ovaries exposed to PH. To identify whether Nobox gene expression is regulated by hydroxymethylation modification, high-throughput sequencing was performed with the Nobox gene. Because the Nobox gene does not have a CPG island, methylation sequencing of the Nobox gene was not performed. The sequencing results suggested that Nobox was hypo-hydroxymethylated in the ovary exposed to PH. In addition, an in vitro experiment using 3T3 cells treated with hypoxia indicated significant decreases in Tets and Nobox. The dominant form of vitamin C is ascorbate. It serves as a direct cofactor for Tets that catalyzes the oxidation of 5mC into 5hmC. Treatment with vitamin C enhanced Tet expression and increased the expression of Nobox and the known downstream gene Gdf9. Therefore, vitamin C was useful to reverse the decreased 5hmC following exposure to adverse in utero environments and prevent adverse outcomes caused by abnormal pregnancy. This strongly suggested that the Nobox gene could be regulated by 5hmC modification. Prenatal vitamin C supplementation may have a long-term effect on FOAD in general. In the future, more downstream genes of Nobox will be explored to clarify the effect of hypoxia on ovarian function.In summary, PH could affect gene expression through epigenetic regulation in a long turn, and abnormal 5hmC levels of Nobox caused by PH were a major cause of ovarian dysfunction in rat offspring exposed to PH Fig.\u00a0. VitaminThe present findings support the hypothesis that Nobox and Tets play a vital role in the onset of ovarian dysfunction. Further work on the involvement of Nobox and Tets in ovarian dysfunction pathogenesis may be promising to inspire novel therapies, particularly in patients with ovarian dysfunction and infertility.We did not check the pregnant rats for body weight, water, food intake, cross-fostering, litter size, or glucocorticoid levels, which were fundamental flaws in the study design and might induce differences in energy intake during the lactation period. In addition, no ovarian cells were used in the study, which may limit the generalizability of these results. Finally, further experiments are clearly warranted to study the exact mechanism by which Nobox acts.In conclusion, prenatal hypoxia significantly affected ovary function in offspring and caused downregulation of Tets, which might decrease Nobox/Gdf9 through DNA hydroxymethylation. The evidence showed that epigenetic regulation of Nobox could be one of the possible mechanisms contributing to the dysfunction of ovaries exposed to prenatal hypoxia. The results from prenatal hypoxia experiments indicated impaired hydroxymethylation levels of the genomic DNA in the ovary. This view contributed to further understanding the mechanisms underlying diminished ovarian reserve and suggested the benefits of further investigation of the early prevention of ovarian dysfunction.Supplementary file1 (PDF 91 KB)Supplementary file2 (PDF 84 KB)Below is the link to the electronic supplementary material."}
+{"text": "Drosophila centrocortin (cen) is required for normal centrosome separation, although a role in centriole duplication was not closely examined. Through time-lapse recordings of rapid syncytial divisions, we monitored centriole duplication and the kinetics of centrosome separation in control vs cen null embryos. Our data suggest that although cen is dispensable for centriole duplication, it contributes to centrosome separation.Centrosomes are microtubule-organizing centers that duplicate exactly once to organize the bipolar mitotic spindle required for error-free mitosis. Prior work indicated that  Centrosomes are the microtubule-organizing centers (MTOCs) of most eukaryotic cells and promote error-free mitosis through organization of the bipolar mitotic spindle. Centrosome function as a MTOC is instructed by the pericentriolar material (PCM), a matrix of proteins that encircles the central pair of centrioles .Drosophila embryogenesis, where they coordinate rapid S-M abridged nuclear division cycles and cortical migration of the nuclei to give rise to the syncytial blastoderm embryo  mRNA localized to centrosomes is similarly required for error-free mitosis. Here, the cen coding sequence is necessary and sufficient for cen mRNA localization to pericentrosomal RNA granules, which likely function as sites of translational regulation , and crosses were maintained at 25\u00b0C in a light and temperature-controlled incubator chamber. To examine maternal effects, P[Ubi-RFP-PACT] was a gift from Nasser Rusan (NIH) and was generated by introducing amino acids 2,479\u20133,555 from PLP-PF (Fragment 5), into the vector pURW  to generate an RFP fusion, as described for the GFP fusions in For live imaging, dechorionated 1\u20132\u2009h embryos were adhered to a 22 \u00d7 30 mm #1.5 coverslip using glue extracted from double-sided tape (3M), covered with Halocarbon oil 700 , and inverted onto a 50-mm gas permeable dish (Sarstedt) with broken #1 coverslips used as spacers. Images were captured on a Nikon Ti-E inverted microscope fitted with Yokagawa CSU-X1 spinning disk head using a motorized stage, Nikon LU-N4 solid-state laser launch , Hamamatsu Orca Flash 4.0 v2 digital CMOS camera, and a Nikon 100x, 1.49\u2009NA Apo TIRF oil immersion objective. Images were acquired at 300\u2009ms exposure times over 0.5\u2009\u03bcm Z-intervals through 3.5\u2009\u03bcm of tissue at 20\u2009s time intervals for one or more complete embryonic nuclear cycles (NCs).Images were randomized, and the experimenter was blinded to genotype during analysis. We measured the time elapsed from when centrioles were duplicated, visible as 2 proximal RFP-PACT signals, until they were fully separated. We define full separation as the moment when centrioles remain at distal sides of the nucleus, approximately late prophase. Time-lapse recordings from age-matched nuclear division cycle 13 samples were used. Once centrosomes were fully separated, the distance between centrosomes was measured using the line tool. The time between successive centriole duplication events was monitored between NCs 12 and 13. Image analysis was completed in FIJI NIH; . Images t-test with Welch\u2019s correction, or a chi-square test, as indicated in the figure legend. Data shown are representative results from at least 2 independent experiments.Data were unblinded, plotted, and statistical analysis was performed using Microsoft Excel and GraphPad Prism software. Normal data distribution was tested using a D\u2019Agostino and Pearson normality test, followed by a 2-tailed nonparametric Mann\u2013Whitney test, 2-tailed cen contributes to centriole duplication, we examined the kinetics of centrosome duplication and separation in live control vs\u2002cen mutant Drosophila embryos. For these studies, we used embryos collected from homozygous cenf04787 mothers. The cenf04787 allele is a PBac transposon insertion disrupting the cen locus . Examination of the frequency distribution of centrosome separation velocities confirmed that the centrosomes in cen mutant embryos require more time than controls (>400\u2009s) to separate (P\u2009=\u20090.01081 by chi-square test). These data indicate cen is required to complete efficient centrosome separation.NC 13 control and croscopy . Similarcen mutant embryos. On average, control centrosomes were separated 4.4\u2009\u00b1\u20090.7\u2009\u00b5m in NC 13 late prophase embryos  3\u2019-UTR demonstrated that local cen mRNA is required to recruit Cen protein to distal centrosomes and support spindle morphogenesis, genome stability, and embryonic viability  show embryos expressing RFP-PACT and GFP-Cnn. Supplemental Material included at figshare: https://doi.org/10.25387/g3.17096684.Strains are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables. Supplemental data are available on Figshare: time-lapse recordings Supplementary Video 1 (control) and Supplementary Video 2 ("}
+{"text": "We further show that ADO is critical for thermogenic mitochondrial respiratory function as its ablation in adipocytes significantly reduces taurine levels, which leads to declines in mitochondrial oxygen consumption rates. Finally, we demonstrate via assay for transposase-accessible chromatin with sequencing (ATAC-seq) that taurine supplementation in beige adipocytes has the ability to remodel the chromatin landscape to increase the chromatin accessibility and transcription of genes, such as glucose-6-phosphate isomerase 1 (Gpi1), which are critical for NST. Taken together, our studies highlight a potential mechanism for taurine in the activation of NST that can be leveraged toward the treatment of obesity and metabolic disease.Non-shivering thermogenesis (NST) has strong potential to combat obesity; however, a safe molecular approach to activate this process has not yet been identified. The sulfur amino acid taurine has the ability to safely activate NST and confer protection against obesity and metabolic disease in both mice and humans, but the mechanism of this action is unknown. In this study, we discover that a suite of taurine biosynthetic enzymes, especially that of cysteamine dioxygenase (ADO), significantly increases in response to \u03b2 It is well established that obesity is linked with comorbidities such as cardiovascular disease, type 2 diabetes mellitus, and dyslipidemia [3 agonist CL 316,243, practical and safe approaches to stimulate NST in humans are still challenging [Obesity is defined as having a body mass index (BMI) greater than or equal to 30 kg/mipidemia . Accordiipidemia ,5,6. Oneipidemia ,8,9. In ipidemia ,11,12,13llenging ,15. Intr5U) becomes incorporated at the wobble position of the anticodon loop of human mitochondrial tRNAs critical for the synthesis of mitochondrial proteins [Ucp1) and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (Pgc1a) in the inguinal and white adipocytes. Consistent with elevated standard thermogenic markers, taurine supplementation also increased oxygen consumption and body temperature, demonstrating the beneficial molecular mechanism of taurine in energy metabolism [PPAR-1a), peroxisome proliferator-activated receptor gamma (PPAR-\u03b3), and CCAAT/enhancer binding proteins alpha (C/EBP\u03b1) [3 agonist CL 316,243 can significantly elevate taurine levels in the inguinal and white adipocytes [Taurine is a naturally occurring amino acid derived from animal protein or by endogenous synthesis. Taurine can be synthesized from two major pathways. Synthesis can be driven by oxidizing cysteine to cysteine sulfinic acid catalyzed by the enzyme cysteine dioxygenase (CDO) which is then subsequently converted into hypotaurine via the cysteine sulfinic acid decarboxylase (CSAD) to finally generate taurine. Alternatively, taurine can be synthesized from cysteamine via an oxidation reaction catalyzed by the cysteine dioxygenase enzyme (ADO) ,19,20,21proteins ,26. In aproteins ,27,28,29proteins ,31,32,33tabolism ,34,35. M(C/EBP\u03b1) . The relipocytes . OverallIn this present study, we demonstrate that pharmacological activation of NST with CL 316,243 robustly increases the taurine biosynthetic enzymes predominately in the inguinal adipose tissue compared to other adipose depots. We further define the dependency of the cysteamine dioxygenase (Ado) biosynthetic enzyme and demonstrate that the loss of Ado significantly blunts intracellular taurine levels and impedes mitochondrial respiration in thermogenic adipocytes. Finally, we show that taurine supplementation can alter the chromatin landscape of primary inguinal adipocytes to upregulate genes linked to enhance NST and metabolic health. Collectively, our study provides novel mechanistic insight into the role of taurine in NST and positions taurine as a potential treatment option to combat obesity and metabolic disease in the future.n = 4, per treatment). Following euthanasia with carbon dioxide, brown, inguinal, and white adipose depots, along with liver tissue, were collected. Each sample was immediately flash frozen in liquid nitrogen and stored at \u221280 \u00b0C for further protein or RNA extractions.Studies on mice were performed according to the permission from the Cornell University Institutional Animal Care and Use Committee. Four-week-old wild-type male C57BL/6J mice were purchased from Jackson Laboratory (#000664) and housed at room temperature (25 \u00b0C) with 12 h cycles of darkness and light. Mice were fed ad libitum food and water. To conduct pharmacologically induced thermogenesis experiments, 5-week-old mice were acclimated to a thermoneutral environment (30 \u00b0C) for 7 days. Subsequently, mice were daily intraperitoneally injected (IP) for 7 consecutive days with either saline or 1 mg/kg of CL 316,243  , and 1.5 unit/mL collagenase D) in a shaking water bath at 37 \u00b0C for 15 min. Lysates were filtered through a 100 \u03bcm cell strainer and spun down for 5 min at 600 g at 4 \u00b0C. After removing the digestion buffer, the stromal vascular fraction (SVF) was resuspended in adipocyte culture media  and filtered through a 40 \u03bcM cell strainer, followed by centrifuging for 5 min at 600\u00d7 g at 4 \u00b0C. Subsequently, cells were resuspended with adipocyte culture media and plated on polystyrene cell culture plates, coated with 2% gelatin. After 48 h, cells were gently washed with PBS two times and replenished with fresh adipocyte culture media. All primary inguinal and immortalized brown adipose cell cultures were grown at 37 \u00b0C with 5% CO2. For adipocyte differentiation, cells were seeded on polystyrene cell culture plates with 2% gelatin coating. The following day, cells were differentiated with DMEM/F12 . After 48 h, the medium was replaced with maintaining media (5 \u03bcg/mL Insulin and 1 \u03bcM Rosiglitazone) and replenished every two days until day 6. On the seventh day, cells were treated with 1 \u03bcM CL 316,243, PBS, or 1 mM taurine for different experimental designs.Primary inguinal adipose tissue was harvested from 3-week-old male wild-type C57BL/6J mice, followed by thoroughly chopping with scissors for 5 min and digesting with 15 mL of lysis buffer . The sgRNAs sequences are as follows: sgAdo forward: 5\u2032-TTCCCGGGCCGAGTACACCG-3\u2032, sgAdo reverse: 5\u2032-CGGTGTACTCGGCCCGGGAA-3\u2032. The sgRNAs were cloned into the LENTICRISPR v2.0 (Plasmid #52961) plasmid vector developed by Zhang\u2019s group. The vector V2 CRISPR DNA Plasmid (1 \u00b5g) was co-transfected in 293T cells along with 3 mg of the viral envelope PMD2 (Addgene # 12259) and 4 mg of the viral packing PsPAX (Addgene #12260) plasmids using the Polyfect reagent according to the manufacturer\u2019s instructions. The empty vector CRISPR DNA Plasmid was used as a control. After 48 h, the lentiviral supernatant was collected from the 293T cells and transduced into the immortalized brown fat cell line D.E 2.3 . Stable transduced cells were then established via puromycin selection (1 \u00b5g/mL).The single guide (sgRNAs) of the CRISPR-Cas9-based Ado knockout cells were designed according to the following database g at 4 \u00b0C for 10 min. The supernatant was then collected and centrifuged for 10 min at 8500\u00d7 g at 4 \u00b0C to pellet the mitochondria. The supernatant was then removed, and the mitochondrial pellet was washed with 1 mL 1\u00d7 MAS buffer  and centrifuged for an additional 10 min. The mitochondria pellet was then resuspended with 100 mL MAS buffer, and the protein concentration was quantified by BCA Protein Assay Kit . The mitochondrial oxygen consumption rate (OCR) value was assayed by the Agilent Seahorse Bioanalyzer. For this, 10 mg of mitochondria was loaded into XFe24 cell culture plates followed by 20 min of spinning at 2000\u00d7 g at 4 \u00b0C and ultimately refilled 450 \u00b5L MAS buffer into each well. The mitochondrial stress test compounds were then administered as follows : Port A: Pyruvate (9 mM) and Malic acid (9 mM) (for pyruvate-driven respiration), Port B: Rotenone/Antimycin A (135 \u03bcM each). Respirometry data were collected using the Agilent Wave software v2.4. To measure extracellular acidification rate (ECAR), cells were plated and differentiated on XFe24 Seahorse cell culture plates. Cells were then washed and incubated for 30 min in unbuffered Dulbecco\u2019s Modified Eagle\u2019s Medium (DMEM) supplemented with 2 mM L-glutamine. We then performed the cellular glycolysis stress test by administering the following compounds: Port A: Glucose (25 mM), Port B: Oligomycin (3 \u00b5M), Port C: 2-Deoxy-D-glucose (2-DG) (50 mM). After each experiment, cells were stained with Hoechst and the cell numbers were measured using the Lionheart FX automated microscope .The brown fat cell line DE 2.3 cells were differentiated as described above. Cells were then scraped with digestion buffer  and lysed with a pre-chilled glass-Teflon homogenizer (13 strokes). Cell lysates were centrifuged at 800\u00d7 Tissues were lysed with 2% Sodium Dodecyl Sulfate (SDS) supplemented with protease inhibitor  and homogenized with metal beads for 30 min at 4 \u00b0C. Lysates were then centrifuged at maximum speed for 20 min and the protein supernatant was then retained. To extract protein from cells, adipocyte cultures were scraped with 2% SDS lysis buffer and rotated at 4 \u00b0C for an hour. The cells were then subjected to ultrasonic treatment for 10 min with 30 s on/30 s off  followed by 15 min of centrifugation at 4 \u00b0C at maximum speed. The protein concentrations of the both the tissue and cell lysates were assessed by BCA Protein Assay Kit  and protein samples were supplemented with 4\u00d7 laemmli blue  and heated at 37 \u00b0C for 10 min. Prepared samples were resolved on SDS-polyacrylamide gels and then transferred to PVDF membranes. A membrane was blocked with 5% milk for one hour and then washed with TBST before being incubated with primary antibodies overnight. The following day, membranes were washed with TBST and then targeted by secondary goat anti-mouse or anti-rabbit antibodies. Following the secondary staining, the membranes were washed with TBST and imaged by FluorChem system. Densitometry was conducted by applying Image J software version 1.530. The list of primary antibodies has been included in the Tissues were homogenized with Qiagen TissueLyser II in Trizol reagent (Invitrogen), and cells were scraped with Trizol reagent . The RNA was extracted according to the manufacturer\u2019s protocol. The concentration and quality of RNA were analyzed using Nanodrop  and the reverse transcription reaction was performed by qScript cDNA Synthesis Kit (Quanta Bio). Gene expression analyses were performed by the CFX384 Real-Time PCR System using SYBR Green  for the real-time polymerase chain reaction (RT-PCR). The list of primers has been included in the Differentiated brown fat DE 2.3 cells were washed with 2 mL PBS and processed according to the manufacturer\u2019s protocol  to measure the intracellular taurine concentrations.n = 3, per treatment). Mice were euthanized with carbon dioxide, and inguinal tissues were homogenized with 2 mL cold methanol and 4 mL of cold chloroform. Samples were then mixed with 2 mL molecular-grade water. After 5 min of incubation, samples were centrifuged at 4 \u00b0C for 10 min at 3000\u00d7 g. The polar metabolites were transferred into new tubes and dried under nitrogen flow. Metabolites were resuspended in 30 \u00b5L 30% acetonitrile and analyzed by liquid chromatography/ mass spectrometry (LC-MS) on a Vanquish LC coupled with the ID-X MS  conducted by the Harvard Center for Mass Spectrometry. Samples or standards were injected into ZIC-pHILIC PEEK-coated columns . The mobile phase was HPLC grade water, 20 mM ammonium carbonate, 0.1% ammonium hydroxide, and 97% acetonitrile. The flow rate at the first 30 s ramped from 0.05 to 0.15 mL/min and was maintained at 0.15 mL/min. All data were acquired in the ID-X polarity switching at 120,000 resolutions. MS1 data were acquired in polarity switching for all samples. MS2 and MS3 data were acquired by the AquirX DeepScan function for pooled samples. Results were analyzed in Compound Discoverer 3.3 . Identifications were based on MS2/MS3 matching with the mzVault library and corresponded to retention time built with pure standards (Level 1 identification), or on mzCloud match (Level 2 identification). For taurine-targeted metabolomics, primary inguinal cells were separately treated with 1 \u00b5M CL (Cayman #17499) or PBS for 24 h. Subsequently, cells and media were processed with the same biphasic extraction described previously. Taurine concentrations were then measured via LC-MS and analyzed the area of the exact mass for the corresponding ions of the targets as well as the intracellular labeled isotopic taurine levels .For untargeted metabolomics, six-week-old C57BL/6J mice were injected daily via intraperitoneal injection (IP) with either saline or 1 mg/kg CL 316,243 (Cayman #17499) for consecutive seven days .Differentiated primary inguinal and brown DE 2.3 adipocytes were washed with PBS and then scraped with digestion buffer  and lysed with a pre-chilled glass\u2013Teflon homogenizer (10 strokes). Cell lysates were centrifuged at 1200\u00d7 g at 4 \u00b0C for 5 min and the nuclear pellet stored at \u221280 \u00b0C for the protein process.Differentiated primary inguinal and brown DE2.3 adipocytes were washed with pre-cold PBS and scraped with 10 mL PBS. After spinning at 450 g at 4 \u00b0C for 5 min, the nuclear pellet was resuspended with lysis buffer  and incubated on ice for 15 min. Subsequently, the sample suspension was mixed with 25 \u03bcL detergent solution NP  and spun at 10,000 g at 4 \u00b0C for 5 min. After removing the supernatant, the pellet was resuspended with the lysis buffer, followed by centrifuging at 10,000\u00d7 DE 2.3 brown adipocytes were plated in 96-well plates and differentiated as described previously. After 7 days, differentiated cells were washed with PBS three times and subsequently stained with 30 nM of MitoTracker Red CMXRos dye  for 20 min. The nuclei were then stained with Hoechst for 45 min as an indicator of total cell number. Cells were then imaged and quantified using the Lionheart FX automated microscope  as described previously.The genomic DNA from DE 2.3 cells was isolated using the QIAamp DNA Micro Kit  following the manufacturer\u2019s instructions. A quantitative PCR reaction was then performed using mitochondrial DNA and nuclear DNA-specific primer sequences (detailed below in g to generate cell pellets. Cell pellets were then resuspended by 200 mL homogenization buffer (60 mM Tris (pH 7.8), 30 mM CaCl2, 18 mM MgAc2, 0.1 mM PMSF, 1 mM \u00df-mercaptoethanol, 320 mM sucrose, 0.1 mM EDTA, 0.1% NP40) and homogenized by using plastic pestles for one minute. The samples were then mixed with 1.8 mL washing buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.001% Tween-20) and spun down at 4 \u00b0C for 10 min at 500 g. After removing supernatants, the cell pellets were homogenized by the plastic\u2013Teflon in 300 \u00b5L washing buffer and filtered through 70 \u00b5m nylon filters, followed by spinning at 4 \u00b0C for 10 min at 500 g three times. The supernatant was removed, cell pellets were resuspended with transposition mix TD buffer  and filtered through 40 \u00b5m nylon filters. For nuclei quantification, the samples were treated with DAPI  and trypan blue in a 2:1:1 ratio, counted on a hemacytometer. The library preparation and data processing were performed in the laboratory of Dr. Paul Soloway, as described previously [Two million primary inguinal cells were seeded in a 10 cm culture plate and scraped with 1 mL PBS after fully differentiating. Subsequently, cells were centrifuged at 4 \u00b0C for 10 min at 200\u00d7 eviously . The nucp-values less than 0.05.Shiny GO v0.741 was used to perform the gene ontology analysis (GO) on the top 50 upregulated inguinal gene accessibilities profiles with t-tests. Compound Discoverer 3.2 was used to run the analysis of variance (ANOVA) and Tukey\u2019s HSD post hoc test on the LC-MS data (Thermo Fisher). The significance level was set at p < 0.05. Each image legend depicts the value of n together with the statistical characteristics.All data were expressed as mean \u00b1 SEM, unless other specified. GraphPad Prism 9 was used for the statistical analysis to determine the difference between two independent groups by two-tailed unpaired Student\u2019s 3 adrenergic receptor agonist CL 316, 243 (CL), or saline vehicle control for a period of seven consecutive days to activate thermogenesis. We then extracted brown, beige, and white adipose tissue depots in addition to liver tissue and profiled the taurine biosynthetic enzymes Cdo, Csad, and Ado to determine their response to activated thermogenesis , ubiquinol\u2013cytochrome c reductase core protein 1 (Uqcrc1), and succinate dehydrogenase complex flavoprotein subunit a (Sdha) [Taurine biosynthesis can originate from two biological starting points, cysteine-driven synthesis through the Cdo enzyme or cysteamine-driven synthesis through the Ado enzyme. The enzyme Cdo has been well studied and it has been shown that the loss of Cdo in adipocytes significantly reduces taurine levels and impairs mitochondrial function by blunting mitochondrial respiration and inhibiting mitochondrial gene expression, such as cytochrome c oxidase subunit 5b (a (Sdha) ,46. Our a (Sdha) . In addia (Sdha) ,49. Addia (Sdha) . Indeed,a (Sdha) . Therefoa (Sdha) ,54,55,56a (Sdha) . This wiIn our studies, we noted that the taurine biosynthetic enzyme Ado migrates on immunoblot analyses as a doublet, and we confirmed using CRISPR Cas9-mediated ablation that both bands are indeed Ado G. It is 18FDG) uptake significantly increased in young healthy volunteers, while decreasing in elderly or patients diagnosed with metabolic diseases, who have been considered to have lower NST capacity [It still remains enigmatic why taurine biosynthesis is elevated following either environmental or pharmacological activation of NST A,B 16,3,316,37. capacity . It is ucapacity ,56. Thiscapacity ,66. ThisIn conclusion, we provide evidence to a potential mechanistic role for taurine in the regulation of NST. Following NST activation, taurine levels significantly accumulate in the inguinal adipose depots and its synthesis is driven by all three taurine biosynthetic enzymes of which Ado appears to be the most responsive to NST stimulation. We demonstrate that Ado is critical not only to maintain taurine levels in the cell, but that it is also critical for mitochondrial bioenergetic capacity. Finally, we share our findings that taurine supplementation either directly or indirectly alters the chromatin landscape to enhance a suite of genes that promote functional thermogenesis, which will lead to protection against obesity and metabolic disease. Taurine can be purchased as an over-the-counter supplement and supplementation is not reported to have adverse effects in humans ,68. Inde"}
+{"text": "BackgroundOrthopedic surgery has become an increasingly competitive specialty. With a pass-fail Step 1, an even greater emphasis on research has been placed to allow candidates to better distinguish themselves. This study analyzes the scholarly activity of accepted orthopedic residency applicants during medical school, assessing what factors, including the novel altmetric attention score, may be associated with greater research productivity.MethodsA list of orthopedic residency programs was obtained from the Electronic Residency Application Service (ERAS). A total of 688 orthopedic residents from 180 programs who matriculated in 2020 from allopathic medical schools were identified.\u00a0Resident demographic information and bibliometric data \u00a0of publications published from July 1, 2016, to\u00a0September 1, 2020, were collected. Descriptive statistics were calculated. Kruskal-Wallis tests analyzed the association between medical school characteristics and research productivity using Stata\u00ae 17.0 .ResultsPostgraduate-Year-3 orthopedic residents (N=688) published 2,600 articles during medical school, averaging 3.8 articles per resident. The residents from a top 25 medical school for research had publication counts, altmetric scores, and h-indices, on average, that were higher than those from non-top 25 medical schools for research. Over 150 residents had no publications, and ~10 residents had more than 30 publications.ConclusionsThe results illustrate that medical school research status influences the research productivity of applicants. Also, given the average number of publications, most research listed on applications are abstracts and presentations. Utilization of the altmetric score may not yet be the best way of examining research experience because orthopedic applicants do not appear to use social networks for academic research. Orthopedic surgery has become an increasingly competitive specialty, with a match rate of 65.8%\u00a0in the 2021-2022 cycle . FurtherThe NRMP reports the mean number of Research Experiences and the mean number of abstracts, presentations, and publications (APPs). The NRMP notes that \u201cAbstracts, Presentations, and Publications\u201d include peer-reviewed articles, abstracts, poster sessions, and invited national or regional presentations. However, peer-reviewed articles or publications tend to be emphasized more and are more easily verifiable than posters and abstracts ,8. Over Still, there is no consensus on the best way to evaluate both the research quality and quantity of residency applicants. Research involvement, considering only verifiable publications, can generally be characterized by the number of publications, authorship rank, h-indices, journal impact factor, and the impact and quality of the research. The incorporation of the novel altmetric attention score with all these other parameters may give us a more comprehensive assessment of the productivity of orthopedic surgery applicants. The altmetric attention score provides data on how much a scholarly article has been shared or interacted with on social media feeds . This scIn this study, we aim to determine whether there are significant differences in research productivity, focusing on publications, among certain groups of residents using parameters such as: attending a US News top 25 medical school for research, attending a US News top 25 medical school for primary care, the number of total papers applicants published in medical school, the number of orthopedic papers published in medical school, the h-index of each resident, and the altmetric attention score of each paper ,14. It iStudy populationA list of orthopedic surgery residency programs participating in the match was obtained from the Electronic Residency Application Service (ERAS) Directory from the Association of American Medical Colleges (AAMC) website. Then, from the website of each residency program, a list of current residents who matriculated in 2020 was compiled to obtain 688 residents from 180 programs . The matriculation year 2020 was chosen to gather recent data and to ensure all residency programs would have this group of residents listed on their websites. The residents were confirmed using LinkedIn, Doximity, and ResearchGate. Once all the residents for analysis were compiled, a database of demographic information and research productivity, focusing on publications, was created.Demographic information on each resident included sex, medical school attended, medical school type , state of the current residency program, IMG  status, and PhD holder status. Information about the rankings of the medical school they each graduated from, such as if it was considered a Top 25 medical school for primary care or Top 25 medical school for research (according to US News & World Report) was included. US News & World Report criteria for research rankings were calculated from weighted averages of the following components: peer assessment score, residency directors' assessment score, median MCAT score, median undergraduate GPA of the entering class, acceptance rate, faculty resources, total federal research activity, and average federal research activity per faculty member . US NewsFor research productivity, each resident's publication count was collected by first searching each resident in PubMed to analyze their publications from July 1, 2016, through September 1, 2020. These dates were chosen to ensure that only publications produced during their time in medical school were analyzed and not their work done after matriculating into residency. Then, the number of total and orthopedic-related publications for each resident was noted. However, publications listed in PubMed that were erratum, or corrections of errors by the publisher, were not counted as additional publications. The h-index of each author was calculated by determining the number of citations in each publication. The altmetric attention score of each publication was also gathered for each author . The altDO n = 100), PhD (n = 5), and IMG (n = 9) residents were excluded from the database . Descriptive statistics were calculated. Kruskal-Wallis tests analyzed the association between medical school characteristics and research productivity. Tests were conducted with and without outliers based on the total number of publications. Outliers (n = 34) were defined as being greater than the 95th percentile of resident statistics and were not included in our analysis. The outliers had more than 14 publications in total.Individual research productivityPostgraduate-Year-3 (PGY-3) orthopedic residents (N = 688) published 2,600 articles during medical school, averaging 3.8 articles per resident Table .Research productivity as a function of medical school research productivityp < 0.001, \u03b72\u00a0= 0.04), published significantly more orthopedic-related articles , accrued significantly higher total AS  and average AS per article , and had a significantly higher h-index  than those who graduated from non-Top-25 schools for research , and h-indices of residents from the top 25 medical schools for research were higher than those from non-top 25 medical schools for research. Additionally, many residents did not have any publications while they were in medical school. On the other hand, there were at least 10 residents with more than 30 publications. At the same time, our effect sizes were less than 0.06, indicating that there are factors influencing an individual\u2019s research productivity other than school research status alone. It\u2019s unknown what the effects are when more demographic information such as race and ethnicity, data we didn\u2019t have access to, are factored in.What do our findings tell us about the aggregate match data published for ERAS?Table Additionally, there have been concerns about the reliance on the quantity versus quality of research activities in selecting desirable candidates. A study found that the number of publications for matched orthopedic surgery applicants was much lower than the reported national average of APPs . Ngaage Another finding seen during data collection was the fact that a few residents who had over 30 publications took a research gap year (RGY) as confirmed by the research year program\u2019s website or LinkedIn. This is far greater than the average number of APPs reported by the NRMP (Table Are students with the hopes of matching into orthopedics engaging in scholarly activity through social networking?To our knowledge, this is the first study that analyzes altmetric attention score of orthopedic surgery residency applicants. When assessing research impact, the use of the non-traditional altmetric attention score proves advantageous in its immediate availability, measurement of initial reactions towards publications, and longitudinal tracking of influence and attention over time . An advaWhat is the best metric?Currently, the number of APPs is being used to assess research productivity. The use of this number by programs is not inherently bad if combined with the understanding that the bulk of APPs are abstracts and presentations rather than publications for most applicants. Applicants should be aware that other students are not publishing as much as they might think. Additionally, while h-index can be considered, it lacks meaning for applicants without publications.The altmetric attention score could be used more as more applicants increase their use of social media to disseminate their research findings. Other bibliographic metrics can take years to accumulate, while diffusion of publications on social media can happen much quicker and can be a better reflection of applicant publication impact for applications. While altmetric attention score can serve as a good indicator as to how much social interest a paper has generated, it should be interpreted with caution. Altmetric attention score measures the amount of social media attention a paper gets but it does not disclose information on the quality of a paper or differentiate between good attention versus bad attention. For example, if a paper receives a high volume of social media attention for being an inaccurate article, altmetric only sees that as having a high number of web-driven interactions regardless of reason. However, negative press is subjective in research as this could stimulate a productive discussion among readers because the paper is found to be controversial rather than inaccurate. Either way, this wouldn\u2019t be clear from the number alone. Altmetric attention score is a metric that should be kept in mind for future use for more accurate analysis of applicants when dispersion and discussion of publications through social media becomes a more popular form of engagement in this field.What is a possible consequence of an increased focus on publications in the wake of a pass/fail Step 1?One possible consequence of an increased focus on publications is applicants from a non-top 25 school for research being disadvantaged when applying for orthopedic surgery residency. US News Report stated that two of the factors implemented when ranking the Top 25 Medical Schools for Research are \"Faculty Resources\"\u00a0and \"Average federal research activity per faculty member\", which calculates research activity based on medical school financing data . This meLimitations and future workWhile our analysis focuses on the publication aspect of research productivity to better understand aggregate data, there are some limitations that deserve additional evaluation. First, we were restricted to resident publications only rather than residents\u2019 full research activity. We understand that publications do not tell the full story of an applicant's research involvement. However, publications are more easily verifiable and are often promoted as being vital to matching while\u00a0the aggregate APPs still don't give insight into the number of publications. Since only published manuscripts could be searched for, we could not account for submitted papers that had not yet been accepted or papers under revision. It is not unlikely that some applicants were unable to publish their projects in time for the residency application cycle. Additionally, despite many efforts to verify applicant identity and check various platforms to ensure our study analyzed the correct candidate and all their publications, there is no guarantee that all publications could be accounted for. Applicants may have undergone name changes during medical school. This would have affected the publication counts in our analysis, but it is unlikely that the overall trends would be significantly different. Also, our study was not able to evaluate the same data for applicants who applied to orthopedic surgery residencies and did not match. This data would have been beneficial in further assessing the value of publications to programs when determining which candidates to accept.Lastly, another limitation of this study includes candidate self-inflation of altmetric attention scores through self-advertising on social media. This is similar to the limitation of self-inflation faced with using the h-index, where candidates can self-cite\u00a0in subsequent publications. Additionally, the altmetric attention score is time-dependent and recalculated daily. This means that altmetric attention scores were possibly lower at the time of residency applications than when we collected altmetric attention scores. This depends on the publication date and the fluctuating attention each publication receives. If residency programs were to consider altmetric attention scores during future application cycles, it would be important for them to try to review the scores of all applicants' research within a shorter window\u00a0of time to limit variability.In summary, our analysis of 688 orthopedic residents showed that residents from the top 25 medical schools for research had more research publications, higher average altmetric attention scores, and higher h-indices than those from non-top 25 medical schools for research. Our findings give insight to future orthopedic surgery applicants as to the different research parameters residency programs can examine when evaluating applicants and what the average applicant looks like.\u00a0This can help guide these applicants on ways to demonstrate their interest in orthopedics throughout the medical school while informing them that other applicants are not publishing as much as they may think. Students should continue to be encouraged to not overlook the quality of their research projects, and programs should be increasingly encouraged to consider the quality of applicant research and an applicant's research setting. Additionally, the altmetric attention score may not yet be the best way of qualifying research experience. However, it has the potential to be a useful metric if orthopedic surgery applicants increase their use of social media as a way of disseminating their scientific findings."}
+{"text": "For the periodic tableof elements as well as small molecules, our model accurately reproducesatomic  polarization potentials and multipolar dispersioncoefficients, elucidating the high promise of the presented modelin the development of next-generation quantum-mechanical force fieldsfor (bio)molecular simulations.The quantum Drude oscillator (QDO) is an efficient yetaccuratecoarse-grained approach that has been widely used to model electronicand optical response properties of atoms and molecules as well aspolarization and dispersion interactions between them. Three effectiveparameters  fully characterize the QDOHamiltonian and are adjusted to reproduce response properties. However,the soaring success of  The many-bodyextension of the QDO model (the coupled QDO model)has been widely employed to study both molecules and materials, includingtheir electronic12 and optical13 properties, polarization,15 dispersion,24 and exchange27 interactions, as well as a wealth of nonadditivefield effects in quantum mechanics28 and quantumelectrodynamics.29 Coupled QDOs are also extensivelyused in the development of van der Waals (vdW) density functionals,31 quantum mechanical6 and polarizable force fields,36 as well as recent machine learning force fields.38 Despite such a wide applicability of the coupled QDO model, itssuccess in describing real atoms remains fundamentally unexplained,and the optimal mapping between atoms and oscillators has not beenestablished. In this Letter, we develop an optimized parametrization(OQDO) in which the parameters are fixed by using only the well-knownatomic dipolar properties. Remarkably, the OQDO reproduces spatialatomic polarization potentials and atomic multipolar dispersion coefficients.Our OQDO model for atoms and small molecules also paves the way tothe development of next-generation quantum-mechanical force fieldsfor (bio)molecular simulations.The development of predictivemodel Hamiltonians that can describe various properties of realisticmolecules and materials is a cornerstone of modern physicsq, \u03bc, \u03c9} fullydefine the QDO, and three atomic response properties could be chosento fix them, meaning that the choice of QDO parameters is not unique.In addition, all QDO response properties, multipolar polarizabilitiesand dispersion coefficients, are uniquely fixed by the three parameters via closed-form relations.6 Thestatic dipole polarizability of a QDO, \u03b11 = q2/\u03bc\u03c92, conveniently combines all three parameters, and it is natural toset this expression to the reference atomic \u03b11. TheQDO expression for the dipole\u2013dipole dispersion coefficient C6 and \u03b11 are given. Since \u03b11 and C6 are accurately known forall elements in the periodic table,41 they form a baselinefor the QDO parametrization. However, one more condition is requiredto obtain {q, \u03bc, \u03c9}, for which differentconstraints can be imposed. A reasonable idea is to fix q = 1 au since a QDO should reproduce the response of electrons. Thisresults in the fixed-charge QDO (FQDO):q and usingQDO recursion relations for high-order response usually yields largeerrors in the multipolar response properties  parametrization scheme:44 However, the C8 dispersion coefficient is not directly measurable, and accurate ab initio calculations of quadrupole (\u03b12) and octupole (\u03b13) polarizabilities and C8\u2013C10 dispersioncoefficients are currently technically feasible only for closed-shellspecies  or alkali and alkaline-earthatoms with s valence shells.48 For other open-shell atoms ,convergence of quantum-chemical response calculations becomes a technicalhurdle49 . Thus, usinghigher-order atomic response properties does not lead to a parametrizationthat would be universally applicable across the periodic table aswell as for small molecules.The three parameters {ties see  and 43).1, C6} to the oscillator parameters. The third parameteris fixed by using the force balance equation for vdW-bonded dimersderived recently.27 Two equations for q and \u03c9follow the JQDO scheme, whereas the third one is replaced with a transcendentalequation for a product \u03bc\u03c9 to be solvednumerically (vide infra)fsc = e2/4\u03c0\u03b50\u210fc is the fine-structure constant and a0 is Bohr\u2019s radius. The vdW radius (RvdW) is calculated via the universalformula connecting it with the dipole polarizability26 delivers \u03bc\u03c9.Here, we introduce an optimizedQDO parametrization (OQDO), wherewe effectively map dipolar atomic quantities {\u03b1i and j and usinginteratomic perturbation theory,51 the interactionenergy can be written as the integrated product of the electron densityof moiety i with the electric potential generatedby moiety j:5252 and it was shown that exchange53 anddispersion56 interactions can be represented using the form of V being effective quantities differentfrom free-atom counterparts. The argument that dispersion interactionscan be written using 54 which was further elaborated by Hunt55 with a focus on dispersion forces and finallyextended to dispersion energies with a demonstration of its validityfor real molecules and materials.58The QDO Hamiltonianeffectively captures the integrated atomicresponse. However, when modeling molecules or solids, coupled QDOsmust properly describe noncovalent interactions between atoms. Consideringtwo fragments Eint asE, which can also model the effect of environment, \u03b4\u03c1(r) = \u03c1E(r) \u2013 \u03c1(r), where \u03c1E(r) is the electron density under the externalfield. Then the dominant contribution to \u03b4Vj(r) is generated by the corresponding \u03b4\u03c1j(r\u2032) via the polarization potential5960 In the Supporting Information, we present VpolQDO(r) in comparison to Vpol(r) calculated for 21 atoms (between H\u2013Ca andKr) within hybrid density functional theory DFT-PBE065 shown to yield a highly accurate description of electronic response66 comparable to coupled-cluster calculations (seethe Supporting Information). Here, we remarkthat the strength of the electric field was chosen individually foreach element depending on its reference static dipole polarizability41 so that the field-induced dipole moment is set as d = \u03b11E = 0.01 au for all atoms.67Responseproperties are given by variations of Vpol(r) for real atoms with different QDO flavors,it is instructive toconsider which atomic properties can be faithfully captured by a QDO.First, the QDO does not aim to describe static properties of the atomicelectron density but rather its response under applied static andfluctuating fields, as demonstrated also by the insets in q determinesits magnitude. This explains why VelFQDO yields good agreement with VelDFT for hydrogen. However, the QDO model does not describe Vel for many-electron atoms because q \u22481 au, while the ESP of atoms scales nonlinearly with Z (see the example of carbon in the inset of VpolQDO(r) in Vpol curves of real atoms and VpolQDO(r) employingthe three QDO models discussed above. We used the accurate ab initio reference data on \u03b11 and C6(C8(Vpol(r) curves obtained from DFT calculationswithout any adjustments. In fact, the performance of the OQDO is sensitiveto variations in the QDO parameters (solutions A or B in Vpol(r) underline the importance of optimal mapping between atomicresponse properties and QDO parameters.Before comparing \u03b11 and C6,41,68 toand C6(C8\u221247 to paSupporting Information) and their connection to real atoms.The starting point is 26\u03bc\u03c9 in RvdW as from 1 = \u03b11(RvdW). For simplicity, we rewrite x asa and b given byRvdW in termsof \u03b11. For all elements in the periodic table, wefound that 1 \u2248400 au) of Cs.41 The existence of two solutionsextends beyond the employed QDO model. We obtained an analogous resultby using the Tang-Toennies potential69 withthe repulsive interaction treated by the Born-Mayer form (see the Supporting Information).We discuss now the technical aspects of derivingthe two solutionsof the OQDO model ( labeled as OQDO(A) and OQDO(B) in \u03bc\u03c9 differ both in mass and charge, yielding quite different results.First, VpolQDO(r) constructed from solutionB does not resemble DFT potentials, while A is in good agreement withthem  and 12 small molecules. Overall, our results showthat the OQDO parametrization improves the dispersion coefficientscompared to the FQDO one, reducing the MARE from 31% to 25% for C8 and from 68% to 33% for C10 when averaged over all 28 (26 for C10) systems. The OQDO model consistently surpasses theFQDO in accuracy for all systems except for alkaline-earth metalswhere the FQDO gives more accurate results. Moreover, C8 and C10, but roughly for one-third of them, the dispersioncoefficients are overestimated. The maximal errors of FQDO are observedfor Xe in the case of both C8 (66%) and C10 (190%). In contrast, the OQDO consistentlyunderestimates both dispersion coefficients for all systems, exceptfor C8 of Li as well as C10 of Li and Cs. The maximum errors of the OQDO are observedfor CO2 (46%) in the case of C8 and CO (58%) in the case of C10, whichare significantly smaller than maximum errors of the FQDO. The consistencyof the OQDO errors allows a straightforward rescaling of dispersioncoefficients: with our best rescaling factors, 1.3 for C8 and 1.5 for C10, one candecrease the MARE of the OQDO to 15% and 22%, respectively, whichis commensurate with the uncertainty of the reference molecular C8 and C10 values.A more detailed analysis of the dispersion coefficients  can be foundin the Supporting Information, where wealso discuss static polarizabilities \u03b12 and \u03b13. The latter becomes less important in the QDO approach wherethe dispersion coefficients, determining the dispersion energy, aredirectly expressed in terms of QDO parameters. It is important tomention that the effects of three-body interactions are captured bythe OQDO scheme on an equal footing with the JQDO scheme. The accuracyin determination of C6 and C9 coefficients is known to be comparable.70 In ref molecular simulations.We presented the OQDO model based on robust parametrizationthatsolely employs dipolar \u03b1"}
+{"text": "Metabolism is a hallmark of cancer and it involves in resistance to antitumor treatment. Therefore, the purposes of this study are to classify metabolism-related molecular pattern and to explore the molecular and tumor microenvironment characteristics for prognosis predicting in prostate cancer.The mRNA expression profiles and the corresponding clinical information for prostate cancer patients from TCGA, cBioPortal, and GEO databases. Samples were classified using unsupervised non-negative matrix factorization (NMF) clustering based on differentially expressed metabolism-related genes (MAGs). The characteristics of disease-free survival (DFS), clinicopathological characteristics, pathways, TME, immune cell infiltration, response to immunotherapy, and sensitivity to chemotherapy between subclusters were explored. A prognostic signature was constructed by LASSO cox regression analysis based on differentially expressed MAGs and followed by the development for prognostic prediction.A total of 76 MAGs between prostate cancer samples and non-tumorous samples were found, then 489 patients were divided into two metabolism-related subclusters for prostate cancer. The significant differences in clinical characteristics  and DFS between two subclusters. Cluster 1 was associated with cell cycle and metabolism-related pathways, and epithelial-mesenchymal transition (EMT), etc., involved in cluster 2. Moreover, lower ESTIMATE/immune/stromal scores, lower expression of HLAs and immune checkpoint-related genes, and lower half-maximal inhibitory concentration (IC50) values in cluster 1 compared with cluster 2. The 10 MAG signature was identified and constructed a risk model for DFS predicting. The patients with high-risk scores showed poorer DFS. The area under the curve (AUC) values for 1-, 3-, 5-year DFS were 0.744, 0.731, 0.735 in TCGA-PRAD dataset, and 0.668, 0.712, 0.809 in GSE70768 dataset, 0.763, 0.802, 0.772 in GSE70769 dataset. Besides, risk score and Gleason score were identified as independent factors for DFS predicting, and the AUC values of risk score and Gleason score were respectively 0.743 and 0.738. The nomogram showed a favorable performance in DFS predicting.Our data identified two metabolism-related molecular subclusters for prostate cancer that were distinctly characterized in prostate cancer. Metabolism-related risk profiles were also constructed for prognostic prediction.The online version contains supplementary material available at 10.1186/s12894-023-01275-w. Prostate cancer (PC) is most malignancy in men, with a globally estimated of approximately 1.4 million new cases and 375,000 deaths in 2020 . The impMetabolic reprogramming becomes a hallmark of solid cancer, and closely related to the tumor development and progress , 21. MetTaken together, the purposes of this study are exploring the significance of MAGs in prostate cancer and constructing metabolism-related gene signatures for survival prediction. In the present study, prostate cancer patients were classified into two metabolic-related subclusters based on differentially expressed metabolism related genes (DE-MAGs). The molecular characteristics, tumor microenvironment characteristics, and responses to therapy in subclusters were analyzed. Besides, we also constructed and validated the prognostic gene signature and predictive nomogram based on glucose, fatty acid, and amino acid metabolism MAGs.https://portal.gdc.cancer.gov/) and the cBioPortal database . Clinical data contained age, pathological stages, Gleason score, prostate-specific antigen (PSA) value, and disease-free survival (DFS). The TCGA-PARD dataset was used as the training set in this study. Moreover, mRNA expression files and corresponding clinical information of GSE70768 and GSE70769 datasets, including age, pathological stages, PSA value, Gleason score, and biochemical relapse (BCR) survival time, were obtained from GEO (https://www.ncbi.nlm.nih.gov/gds). GSE70768 dataset included 199 prostate cancer samples and 111 of them with BCR information (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE70768). GSE70769 dataset included 94 prostate cancer samples and 92 of them with BCR information (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi). GSE70768 and GSE70769 datasets were performed by Illumina HumanHT-12 V4.0 expression beadchip and were used as the testing sets. A total of 948 MAGs were collected from c2.cp.kegg.v7.4.symbols.gmt which was downloaded from Molecular Signatures Database , the search used the keywords \u201cmetabolism\u201d.Transcriptome expression profiles and corresponding clinical data for 489 prostate cancer tissues and 52 non-tumor tissues were obtained from the TCGA-PARD dataset in the TCGA  were obtained by intersecting the DEGs and MAGs. And the differentially expressed MAGs were visualized using pheatmap package in R script.In the present study, differentially expressed genes (DEGs) between prostate cancer tissues and non-tumor tissues were screened using Limma package in R script with the criteria of absolute log2 \u2009>\u20091 and adjust Based on the differentially expressed MAGs, 489 prostate cancer samples from TCGA-PARD dataset were classified into different molecular subclusters using unsupervised non-negative matrix factorization (NMF) clustering via NMF R package. The optimal number of clusters was determined by k value at which cophenetic correlation coefficient began to decline. Then, t-distributed stochastic neighbor embedding (t-SNE) were performed to verify the classification performance using the mRNA expression data of DE-MAGs. Kaplan\u2013Meier (KM) DFS curves were drawn using survival R package to validate the correlation between prognosis and classification. The differentially expressed MAGs between molecular subclusters were shown using pheatmap package in R.Estimate package in R script was performed to evaluate the EISTTIMATE score, immune score, and stromal score of each sample, with differences between molecular subgroups subsequently detected by the Wilcoxon rank sum test. Besides, single-sample gene set expression analysis (ssGSEA) was conducted based on the mRNA expression data to estimate the immune infiltration by calculating the enrichment score of each gene in a special immune cell marker gene set . SsGSEA https://www.gsea-msigdb.org/gsea/msigdb/). Then, GSEA was performed using the GSEA software (version 4.2.2) to explore the potential molecular mechanism between molecular subclusters. In addition, GSVA was carried out using the GSVA R package to estimate the score of above certain pathways and signatures based on the mRNA expression data. And the distinctions between subclusters were detected by the Wilcoxon rank-sum test. To explore the potential molecular characteristics of TME between the two subclusters, GSVA was performed using GSVA R package between two subclusters. The annotated gene set list, c2.cp.kegg.v7.4.symbols.gmt, and TME related gene set , were selected as the reference gene set from MSigDB and previous article [The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway gene set \u201335 . And the different TIDE score between subclusters was detected by the Wilcoxon rank-sum test. Besides, Submap mapping was used to investigate the response of anti-CTLA4/PD1 immunotherapy. Considering the distinction in chemotherapeutic sensitivity in prostate cancer patients, the IC50 values of 138 antitumor drugs in Genomics of Drug Sensitivity in Cancer  were calculated by ridge regression using pRRophetic package in R script.The Tumor Immune Dysfunction and Exclusion (TIDE) score was calculated to estimate the likelihood of response to immunotherapy based on the TIDE database  regression cox analysis to construct a metabolism-related risk signature for prognosis. To validate the prognostic value of the risk signature in training (TCGA-PARD) and testing datasets (GSE70768 and GSE70769). The risk score of each sample was calculated according to the following formula, risk score\u2009=\u2009The clinical characteristics  and risk score were subsequently integrated into univariate and multivariate cox analyses to identify the independent risk factors for prostate cancer. Forest plots were constructed to show the independence of the independent risk score. ROC curves for each factor were assessed using the survival ROC package in R.The independent risk factors (risk score and Gleason score) which were obtained from univariate and multivariate cox analyses were incorporated into a nomogram to predict the DFS using rms package in R script. The score of each variable was calculated, and then all scores were added up to predict the probability of the outcome of each patient. The higher total score indicated the lower survival rate of each patient. The predictive efficacy of the nomogram was evaluated using calibration curves.P-value\u2009<\u20090.05 \u2009>\u20091 and adjusted P\u2009=\u20090.0434), pathological T stage (P\u2009<\u20090.001), pathological N stage (P\u2009=\u20090.0013), and Gleason score (P\u2009<\u20090.001). KM curve also indicated significantly different DFS between the two subgroups , Gleason score, and PSA value. As shown in Fig.\u00a0Integration of the clinicopathological characteristics and risk score, univariate and multivariate cox analyses demonstrated that the risk score and Gleason score could independently predict the DFS of prostate cancer patients Fig.\u00a0A, B. TheIn recent years, numerous studies have demonstrated that metabolic reprogramming is required for tumor initiation, malignant transformation, development, resistance to antitumor therapy , and unfavorable outcome \u201340. MetaThen, biological function analyses indicated that cluster 1 is mainly associated with cell cycle and metabolic pathways, such as aminoacyl tRNA biosynthesis, base excision repair, glyoxylate, and dicarboxylate metabolism, homologous recombination, mismatch repair, and protein export, and spliceosome. Previous study has demonstrated that lipid metabolism-associated pathway, including de novo lipogenesis through steroid hormone biogenesis and \u03b2-oxidation of fatty acids is related to the prognosis of prostate cancer . LikewisMeanwhile, we found cluster 2 is associated with three cardiac diseases, such as ARVC, dilated cardiomyopathy, and HCM. Although the phenomenon is rare, the similarity has been found in previous research . BesidesIn addition, GSEA analysis also indicated that hallmarks of tumor set were enriched in cell cycle-related pathways, such as MYC targets V1 and V2, E2F targets, and G2M checkpoint in the cluster 1, and cluster 2 enriched in apical surface, apical junction, myogenesis, KRAS signaling downregulated, EMT, UV response downregulated. MYC targets V1 and V2, E2F targets, and G2M checkpoint are the four typical hallmarks of the cell cycle. MYC gene is one of the most frequently deregulated driver genes in human cancer and usually acts as a potential anticancer target . E2F famFurthermore, we investigated the TME characteristics between two subclusters. And we found cluster 1 was associated with cancer progression-relevant signaling, and cluster 2 was associated with immune activation and response-related pathways. These findings have been supported by the ESTIMATE algorithm, which was a higher ESTIMATE score, stromal score, and immune score in cluster 2 compared with cluster 1. The abundance of immune cell population in cluster 2, including B cells, CD8 T cells, cytotoxic cells, DCs, eosinophils, iDCs, macrophages, mast cells, neutrophils, NK cells, pDC, T cells, T helper cells, Tem, Tgd, Th2 cells. And Th2 and Treg cells enriched in cluster 1. There are shown significant immune heterogeneity between two metabolism-related subclusters. Whether is there any different responses for antitumor therapy between two subclusters. Firstly, we found HLA family and immune checkpoint-related genes are upregulated in cluster 2. The above results indicated that cluster 2 is associated with stromal and immune activation. And cluster 2 showed a significant response to anti-CTAL4 treatment. Moreover, cluster 1 showed more sensitivity to chemotherapy drugs, such as ABT.888, PF.4708671, GW.441756, and RO.3306. It suggested that the patients in cluster 1 are more suitable for individualized chemotherapy.Moreover, we constructed a prognostic signature based on MAGs and divided prostate cancer patients in TCGA and two GEO datasets (GSE0768 and GSE0769) into high-risk and low-risk groups. We found higher risk scores associated with age, higher T and N stages, higher Gleason score and PSA value, and poor prognosis. The risk score and Gleason score could be used as independent factors for DFS prediction. Although MAG signature can be used as potential biomarkers for DFS prediction in prostate cancer, it is still a lack of extensive clinical validation. Gleason score remains the most reliable prognosticator in men with prostate cancer , neverthTaken together, we constructed the metabolism-related molecular patterns with 79 MAGs in prostate cancer in prostate cancer with the different phenomena, which also showed significant differences in biological function, immune characteristics, and prognosis. MAGs have prognostic value in prostate cancer, and with constructed prognostic signature based on MAGs, patients were subsequently divided into two high- and low-risk score groups with different oncological outcomes. Our finding provided useful tools to manage prostate cancer. Although MAG signature has been demonstrated that revealed the reliable predictive ability for prostate cancer based on the bioinformatics analysis, the results also needed more experimental evidence to prove. Thus, the relationship of MAGs and clinicopathology parameters will verify in a larger in-house cohort and the function of these MAGs in cell phenotypes will be discussed.Additional file 1.\u00a0Supplemantary file 1 NMF.Additional file 2.\u00a0FIGURE S1 IC50 values of 32 drugs between two subclusters.Additional file 3.\u00a0Table S1_TCGA-PRAD_limma_diff.Additional file 4.\u00a0Table S2_venn_intersectGenes."}
+{"text": "Metabolism reprogramming is a hallmark that associates tumor growth, metastasis, progressive, and poor prognosis. However, the metabolism-related molecular patterns and mechanism in clear cell renal cell carcinoma (ccRCC) remain unclear. Herein, the purpose of this study was to identify metabolism-related molecular pattern and to investigate the characteristics and prognostic values of the metabolism-related clustering.We comprehensively analyzed the differentially expressed genes (DEGs), and metabolism-related genes (MAGs) in ccRCC based on the TCGA database. Consensus clustering was used to construct a metabolism-related molecular pattern. Then, the biological function, molecular characteristics, Estimate/immune/stomal scores, immune cell infiltration, response to immunotherapy, and chemotherapy were analyzed. We also identified the DEGs between subclusters and constructed a poor signature and risk model based on LASSO regression cox analysis and univariable and multivariable cox regression analyses. Then, a predictive nomogram was constructed and validated by calibration curves.A total of 1942 DEGs (1004 upregulated and 838 downregulated) between ccRCC tumor and normal samples were identified, and 254 MRGs were screened out from those DEGs. Then, 526 ccRCC patients were divided into two subclusters. The 7 metabolism-related pathways enriched in cluster 2. And cluster 2 with high Estimate/immune/stomal scores and poor survival. While, cluster 1 with higher immune cell infiltrating, expression of the immune checkpoint, IFN, HLA, immune activation-related genes, response to anti-CTLA4 treatment, and chemotherapy. Moreover, we identified 295 DEGs between two metabolism-related subclusters and constructed a 15-gene signature and 9 risk factors. Then, a risk score was calculated and the patients into high- and low-risk groups in TCGA-KIRC and E-MTAB-1980 datasets. And the prediction viability of the risk score was validated by ROC curves. Finally, the clinicopathological characteristics (age and stage), risk score, and molecular clustering, were identified as independent prognostic variables, and were used to construct a nomogram for 1-, 3-, 5-year overall survival predicting. The calibration curves were used to verify the performance of the predicted ability of the nomogram.Our finding identified two metabolism-related molecular subclusters for ccRCC, which facilitates the estimation of response to immunotherapy and chemotherapy, and prognosis after treatment.The online version contains supplementary material available at 10.1186/s12894-023-01317-3. Renal cell carcinoma (RCC) is a malignant solid tumor that accounts for 2.2% of the total cancer cases and 1.8% of the cancer deaths in 2020 . CcRCC iMetabolic reprogramming is a cancer hallmark that supports tumor cell proliferation and growth in nutrient-poor settings . There aIn the present study, we comprehensively identified metabolism-related molecular patterns, the molecular characteristics of metabolism-related patterns, the landscape of immune cell infiltration, and the prognostic values of metabolism-related genes based on the TCGA-KIRC from The Cancer Genome Atlas (TCGA) database, GSE73731 dataset from Gene Expression Omnibus (GEO), E-MTAB-1980 dataset from ArrayExpress database.https://portal.gdc.cancer.gov/), which contains a total of 533 KIRC samples and 72 paracancerous samples. According to the data integrality, 526 KIRC samples and 72 paracancerous samples were ultimately involved in the subsequent analysis. Meanwhile, the gene expression profiles GSE73731 dataset which contained 265 ccRCC samples were obtained from Gene Expression Omnibus , performed on Affymetrix Human Genome U133 Plus 2.0 Array. And the accession number of ArrayExpress is E-MTAB-1980, including 101 who had follow-up information obtained from the ArrayExpress database (https://www.ebi.ac.uk/arrayexpress).In this study, the mRNA expression profiles and corresponding clinical information were obtained from The Cancer Genome Atlas | > 1. The results were visualized by the ggplot2 R package. Then, a total of 254 metabolic-associated genes (MAGs) expressions were screened and visualized by pheatmap R package \u2009<\u20090.5 and logge Table .After combining the expression of MAGs with survival time, then, the prognostic MAGs were identified by univariable Cox analysis using the Survival package in R.Unsupervised hierarchical clustering was performed using the ConsensusClusterPlus R package to group the prognostic MAGs in the TCGA database. The optimal number of clusters  was determined according to the cumulative distribution function (CDF) reached an approximate maximum. Besides, t-Distributed Stochastic Neighbor Embedding (t-SNE), which is a non-linear dimensionality reduction method, was performed to detect the accuracy of clustering. Meanwhile, the unsupervised hierarchical clustering was also performed using the ConsensusClusterPlus R package and validated using t-SNE in the GSE73731 dataset. Finally, the Subnetwork Mappings in Alignment of Pathways (SubMAP) matrix was conducted to investigate the similarity of a subset from TCGA and GEO datasets.https://www.gsea-msigdb.org/gsea/msigdb/).GSEA was performed to explore the potential molecular mechanism between the two clusters based on Hallmark gene sets from Molecular Signature Database  algorithm was performed using the GSVA package in R to quantize the relative abundance of each immune cell type.https://www.gsea-msigdb.org/gsea/msigdb/). The enrichment score of pathways in each sample was calculated and the differences between subclusters were detected using the Wilcoxon rank-sum test. The differential pathways were screened with the criteria of FDR\u2009<\u20090.05 and |log2 (FC)| > 0.2.Gene set variation analysis (GSVA) algorithm was used to explore the distinct signaling pathways between subclusters based on the gene expression profiles. The gene sets associated with TME-related pathways were downloaded from The Molecular Signatures Database v7.2  and the SubMap algorithm were used to predict the likelihood of response to immunotherapy. Human leukocyte antigen (HLA) genes and immune checkpoints exert crucial roles in response to immunotherapy. The differences in TIDE scores and differential pathways were determined by Wilcoxon rank-sum test.Tumor immune dysfunction and exclusion . The half-maximal inhibitory concentration (IC50) value was assessed by ride regression using the pRRophetic R package. The smaller the IC50 value indicated the stronger inhibitory effects on cancer cells.The sensitivity of each sample to chemotherapy drugs was decided by Genomics of Drug Sensitivity in Cancer  Cox regression analysis was used to identify risk gene signature based on prognostic associated DEGs using the glmnet package in R. Then, the multivariable cox regression analysis was used to assess the independence of the risk gene signature via the coxph package in R. According to the risk gene signature, the risk score was calculated as the following formula, risk score = The DEGs between subclusters were obtained using the Limma package in R with the criteria of FDR\u2009<\u20090.05 and |log2 (FC)| > 1. Then, the prognostic associated DEGs were screened using univariable cox regression analysis in the TCGA database. Hazard ration (HR)\u2009>\u20091 indicated the poor survival outcomes, while HR\u2009<\u20091 indicated the good survival outcomes. Gene with The clinicopathological factors and risk score incorporated into the nomogram to construct the predictive model for prognosis using the rms package in R. Calibration curves were established to evaluate the predictive accuracy of the nomogram.In this study, all statistical analyses and visualized were performed using R software version 3.4.4 according to previous manuscript from Assel et al. . The conThe design of this study was shown in Fig.\u00a0We further incorporated the survival data and MRGs into a univariable cox regression model to identify the prognostic related genes. And the results showed that 117 MRGs were associated with ccRCC prognosis Table\u00a0, Table\u00a02We further investigated the TME characteristics in subclusters, ESTIMATE algorithm results indicated that the stromal score, immune score, and ESTIMATE score of cluster 2 higher than cluster 1, suggesting immune activation characteristics in cluster 2 Fig.\u00a0A. Then, Considering the differences between the two subclusters, we investigated the different responsiveness of two subclusters to immune checkpoint blockade (ICB) therapy. The results showed a significantly higher TIDE score of cluster 1 than cluster 2 Fig.\u00a0A. And thHere, we also explored the differences in the chemotherapeutic sensitivity between the two subclusters. Based on the GDSC database, we screened the sensitivity between the two subclusters to 138 common chemotherapeutic drugs, the results were shown in Fig.\u00a0Previous data indicated the heterogeneity of each sample and discrimination of response for antitumor therapy. Thus, we further investigated the prognostic risk signature between metabolism-related subclusters. First of all, we screened 295 DEGs between two metabolism-related subclusters Table A-B. WithWe confirmed the molecular pattern, risk score, and clinical characteristics, including age and stage, were independent prognostic variables of the OS in the training set Fig.\u00a0A-B. We sMetabolism programming has become a central feature of ccRCC, which involves tumor initiation, progression, resistance to antitumor treatment, and poor survival rates in ccRCC patients \u201325. In tBiological function and molecular characteristics analyses indicated that cluster 2 showed a poor survival rate and was associated with beta-alanine metabolism, fatty acid metabolism, glycerolipid metabolism, histidine metabolism, peroxisome, and PPAR signaling pathway, and starch and sucrose metabolism. Beta-alanine is not an essential amino acid and exerts as a sports supplement to increase anaerobic endurance and athletic performance, beta-alanine can be used as a potential antitumor agent in malignant breast epithelial cells, renal tumor cells, and cervical tumor cells , 27. AnoWe also investigated the TME infiltration feature of two subclusters. The stromal score, immune score, and ESTIMATE score of cluster 2 were higher than in cluster (1) at CD8 T cells, gamma delta T cells, activated NK cells, macrophages M1, resting DCs, resting mast cells were abundant in cluster 1, while memory activated CD4 T cells, Tregs, Macrophage M0, neutrophils were increased in cluster (2) Furthermore, we found the stromal activation associated with biological processes, such as EMT, WNT targets, angiogenesis, and Pan TBRS significantly enriched in cluster 1 than in cluster 2. And the effector immune cells, such as CD8 T cells , and actPrevious sections analyzed the biological function, molecular characteristics, TME infiltration feature, responsiveness to ICB, and target therapy of molecular subclusters. Here, we further investigated the prognostic values of metabolism-related clustering. We identified the 295 DEGs between two subclusters. And a 15-gene signature was constructed, including ANK3, WDR72, PLS1, SLC16A12, ASPA, EMX2, SMIM24, EMCN, FLRT3, LAMB3, PLG, IL20RB, MDK, CXCL5, PDK4. Then, SLC16A12, ASPA, SMIM24, FLRT3, LAMB3, PLG, IL20RB, CXCL5, and PDK4 were identified as risk factors for ccRCC patients. The prognostic values of those risk factors were verified by ROC curves and previous studies. Such as, SLC16A12 is a creatine transporter for creatine and guanidinoacetate in the kidney , and itsIn conclusion, a metabolism-related molecular pattern for ccRCC was constructed, and we also investigated the biological function, molecular characteristics, TME infiltration feature, responsiveness to ICB and target therapy, and prognostic values between two subclusters. Based on the differences, a prognostic signature and a risk model were constructed for survival predicting in ccRCC. Our finding suppled a novel insight for ccRCC diagnosis and prognosis prediction. However, more experimental evidence is needed to validated in a larger internal cohort, and the function of these MAGs in cellular phenotypes will also be discussed.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6Supplementary Material 7Supplementary Material 8Supplementary Material 9"}
+{"text": "Urethral stricture refers to the narrowing of the urethral lumen. While previous studies have hinted at inflammation as the initial driver of this condition, the reasons and mechanisms behind its progression remain largely unknown. By Atomic force microscope (AFM), researchers measured the matrix stiffness of urethra to be 5.23\u2009\u00b1\u20090.37\u00a0kPa for normal tissue and 41.59\u2009\u00b1\u20092.48\u00a0kPa for stricture urethral scar. Similar results were observed in rat urethral stricture models, where the matrix stiffness of normal urethra was 4.29\u2009\u00b1\u20090.82\u00a0kPa, while 32.94\u2009\u00b1\u20097.12\u00a0kPa for urethral stricture scar. Notably, the matrix stiffness increased in rat models over time. To further investigate, polyacrylamide hydrogels were employed to mimic different levels of stiffness for normal and stricture condition. Interestingly, higher matrix stiffness led to an increased fibroblast-to-myofibroblast transition (FMT) in rat urethral fibroblasts, indicated by enhanced expression of \u03b1-SMA and Collagen I, as well as changing in the morphology of fibroblast. RNA-seq analysis suggested that Igfbp3/Smads might regulate the progressive FMT in urethral stricture. In the experiment where the expression of Igfbp3 was inhibited, increasing matrix stiffness lose the potential to stimulate FMT progression and the expression of p-Smad2/3 decreased. On the contrary, overexpression of Igfbp3 promoted the process of FMT in urethral fibroblasts. In conclusion, Igfbp3/Smad pathway appeared to be involved in the progression of urethral fibrosis. This finding suggested that Igfbp3/Smad might be an promising target for future research and treatment in this filed. As urethral stricture advances, it can give rise to bladder decompensation and vesicoureteral reflux, and even more severe complications such as renal dysfunction and hydronephrosis, which pose significant threats to the well-being of affected patients4. Despite the most appropriate treatments, the recurrence and progression of urethral stricture present daunting challenges for urologists5. Similar to the management of various solid organ fibrosis, such as pulmonary fibrosis and renal fibrosis, there is currently no pharmaceutical intervention capable of completely reverse urethral fibrosis. Therefore, studying the progressive pathogenesis of urethral stricture and exploring novel therapeutic targets and medications hold promise as a novel avenue for preventing urethral stricture, delaying recurrence, and improving the quality of life of affected patients.Urethral stricture refers to the narrowing of the urethral lumen, accompanied by the presence of fibrous scar lesions in the mucosa and surrounding cavernous tissue. Urethral stricture is a common disease, with an incidence rate ranging from 0.2 to 1.2%9. The heightened secretion of extracellular matrix (ECM) by myofibroblasts leads to escalating tissue stiffness, thereby contributing to the progression of fibrosis11. Research has indicated that under physiological conditions, the stiffness of lung and liver tissues ranges from 0.5 to 1\u00a0kPa. However, in tissues affected by pulmonary fibrosis, where an elevated FMT level has been observed, the matrix stiffness exhibits a significant increase, reaching values between 25 and 100\u00a0kPa, in a time-dependent manner13. Similar outcomes have been observed in hepatic fibrosis14, myocardial fibrosis15, renal fibrosis16, ect. Although several studies have demonstrated the association between fibrosis progression and increasing stiffness, the mechanism remained largely unknown, especially in urethral fibrosis23. Therefore, further investigation is warranted to elucidate the intricate processes governing this phenomenon in urethral fibrosis and enhance our understanding of its pathogenesis.Fibroblast-to-myofibroblast transition (FMT) plays a pivotal role in the pathogenesis of fibrosis, encompassing various conditions such as myocardial fibrosis, lung fibrosis, renal fibrosis etcIn this study, we measured the precise stiffness of normal and stricture urethra in both human and rat models. Further, we investigated the potential of increased matrix stiffness, occurring subsequent to initial fibrosis, to exacerbate the fibrotic process via the Igfbp3 pathway. Consequently, the primary focus of this investigation is to examine the impact of matrix stiffness on the advancement of urethral stricture, thus establishing a fundamental groundwork for the subsequent identification of potential drug targets for this condition.2O2 solution was used to remove endogenous catalase, sodium citrate was used for antigen retrieval, goat serum was used for blocking, primary antibody treated overnight at 4\u00a0\u00b0C. After adding the secondary antibody, the DAB color was developed.The normal urethral tissues of the human were from penile cancer , the patient underwent partial penectomy, and the urethral tissue was used as the control group; the stricture urethral tissue was obtained from urethral stricture patients who suffered from external urethral trauma during the periods from June 30, 2021 to June 30, 2022. Only males aged between 18 and 65 were selected for this study. These patients were diagnosed with posterior urethral stricture based on clear diagnostic criteria. The underlying cause of this condition was found to be pelvic fractures resulting from car accidents. This study was approved by the Medical Ethics Committee of Xiangya Hospital, Central South University (202112612). After deparaffinization and hydration, 3% HThe rats were anesthetized with 1% sodium pentobarbital solution. The urethral tissue or urethral stricture tissue of the rats were extracted and placed on the AFM microscope. On the holder, calibrate the probe in the air to obtain the spring constant of the probe: 92.04\u00a0N/nm, place the calibrated probe in a water environment, and perform the optical lever sensitivity calibration of the probe again, Amp InvOLS: 38.56\u00a0nm/V. In contact mode, force curve measurement, force curve speed: 1\u00a0Hz. In Asylum software, Young's modulus was automatically fitted to the needle insertion curve of 256 force curves within the mapping range in Hertz model, in which the ball material: polystyrene ball with a diameter of 12\u00a0\u00b5m; the elastic coefficient of the probe: 0.05 N/m; the probe frequency in the air: 22\u00a0kHz.The animal experiments were approved by the Animal Ethics Committee of Central South University, and conformed to the relevant regulations on animal ethics such as animal protection, animal welfare and ethical standards (No: 2021921). The rats were provided by Changsha Tianqin Biotechnology Co., Ltd., and were 10-week-old male Sprague Dawley (SD) rats weighing 350\u2013450\u00a0g. Rats were cultured in a specific pathogen free (SPF) barrier with constant temperature and humidity , and all rats could eat and drink freely.24.Twelve male SD rats were randomly divided into 2 groups, namely the control group and the urethral stricture group, with 6 rats in each group. Rats were marked respectively, and anesthetized with 1% sodium pentobarbital (50\u00a0mg/kg). The epidural catheter was inserted into the rat urethra which help to visualize the urethra when the ventral penile skin was removed. Rats underwent 4 partial incisions of the penile urethra with a 23G needle. The rats in the urethral stricture group were injected with TGF\u03b21 (1\u00a0\u00b5g) dissolved in 100\u00a0\u00b5L of saline around incisions, however, the rats in the control group were injected with saline and underwent sham operation. In order to reduce the mortality of the rats, a cystostomy  was performed in each rat. The epidural catheter was then removed and the penile skin was closed with 5\u20130 absorbable suture. Rats were fed for 1\u00a0monthFurthermore, all animal experiments were performed in accordance with relevant guidelines and regulations and reporting in the manuscript follows the recommendations in the ARRIVE guidelines 2.0.The rats were anesthetized with 1% sodium pentobarbital (50\u00a0mg/kg), and the loach guide wire was inserted from the external urethra of the rat to determine the position of the urethra. As for cysto-urethrography, the Iohexol contrast agent was injected through the ostomy tube. All above were finished by professional radiologists.4 cells from one rat and all those cells were plated in one well of a 6-well plate. 8\u00a0h after first plating, un-attached cells  were removed by replacing with new medium. And these cells could be passaged 3 times (in 1:3 ratio).For fibroblasts isolation, we usually use 60\u201380\u00a0g rats. Simply put, after separation, cutting, digestion, and filtration, we collected about 1025. Place the cell slide on the bottom, gently drop an appropriate amount of polyacrylamide hydrogel solution onto the cell slide, and quickly cover the top of the droplet with another slide, solidify for 60\u00a0min. The polyacrylamide film surface was rinsed several times with 10\u00a0mM Hepes. Use 4\u00a0mL of 50\u00a0mM Hepes solution to dissolve 2\u00a0mg of Sulfo-SANPAH, and irradiate with UV light for 10\u00a0min. Take 0.2\u00a0mg/mL type I rat tail collagen, pipette an appropriate amount and evenly drop it on the surface of the treated polyacrylamide gel, and incubate at 4\u00a0\u00b0C overnight26.Immerse the cell slides in a 6\u00a0cm dish containing 0.1\u00a0mol/L NaOH, and after drying, place them in a 6\u00a0cm dish containing 3-aminopropyltrimethoxysilane, and then place the slides in a 6\u00a0cm dish containing 0.5% NaOH. In a 6\u00a0cm dish of glutaraldehyde, prepare the solution according to previous study5 rat urethral primary fibroblasts were added to each culture dish, pictures were taken at 0, 6\u00a0h, 12\u00a0h and 24\u00a0h. For trans-well test, rat urethral primary fibroblasts were digested with trypsin, and the cell concentration was 3\u2009\u00d7\u2009105\u00a0cells/mL in culture medium containing 1% fetal bovine serum (FBS). The upper chamber of the trans-well chamber was inoculated with 100\u00a0\u00b5L of cell suspension, and then 600\u00a0\u00b5L of culture medium containing 10% FBS was added to the lower chamber. And crystal violet staining was performed after culturing the cells for 24\u00a0h.For would healing test, 5\u2009\u00d7\u200910\u2212\u0394\u0394Ct method and data were expressed as a ratio of the control gene GAPDH. Information on PCR primers is in Table RNAiso Plus reagent  was used to extract total RNA and PrimeScript\u2122 RT reagent kit  was used to synthesized cDNA. SYBR Green Reagent  was used to perform the two-step real-time RT-PCR. The results were calculated by the 2After trypsin digestion to extract the protein, the protein concentration was determined by Bicinchoninic Acid Assay (BCA) kit , electrophoresed on 10% color gel, transferred with PVDF membrane, and then blocked with 5% BSA (BioFroxx. Germany). Incubate the primary antibody at 4\u00a0\u00b0C overnight, incubate the corresponding secondary antibody, and develop ECL imaging. The antibodies used were as follows: Igfbp3 (Invitrogen) 1:1000; \u03b1-SMA (abcam) 1:1000; smad2 (CST) 1:1000; p-smad2 (CST); smad3 (CST) 1:1000; p-smad3 (CST); GAPDH (Abcam) 1:5000; \u03b1-tubulin (CST) 1:1000.p value\u2009<\u20090.05).Total RNA was isolated and purified using TRIzol reagent . NanoDrop ND-1000  and Bioanalyzer 2100  was used for quality control. After fragment processing, the 2\u2009\u00d7\u2009150\u00a0bp paired-end sequencing (PE150) on an illumina Novaseq 6000  was performed following the vendor's recommended protocol. The differentially expressed mRNAs were selected with fold change\u2009>\u20092 or fold change\u2009<\u20090.5 and with parametric F-test comparing nested linear models (Igfbp3 (NM_012588-HA), the GV707 vector, the BamHI/XhoI cloning site, and the control CON522 were used for plasmid conduction. After digestion, the Igfbp3 fragment was amplified by PCR, transformed with competent E. coli, sequenced, and the plasmid was extracted Table . The GV Virus-infected cells were transfected into primary rat urethral fibroblasts with 100 MOI of virus solution, and siRNA was transfected with Lipofectamine 3000 .p\u2009<\u20090.05 was considered statistically significant, p\u2009<\u20090.05 (*); p\u2009<\u20090.01 (**) ; p\u2009<\u20090.001 (***); p\u2009<\u20090.0001 (****).All data were analyzed by SPSS 20.0 and Image J 1.8.0 software. According to the results of 3 or more independent repeated experiments, measurement data were expressed as mean\u2009\u00b1\u2009standard deviation (mean\u2009\u00b1\u2009SD), paired student\u2019s T or one-way analysis of variance (ANOVA) was used for analysis, All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients for being included in the study. All institutional and national guidelines for the care and use of laboratory animals were followed.We enrolled three samples of urethral fibrosis from patients who had undergone surgery for urethral stricture, while normal urethral samples were collected from patients who underwent partial penile resection due to penile cancer, with their informed consent. Histological examination using H&E staining revealed distinct differences between the two groups. The urethral tissue in the normal group exhibited intact and well-organized epithelium, whereas the urethral stricture group displayed disordered tissue structure, with an unclear demarcation between the urothelial layer and the submucosa  to analyze both normal and stricture urethra samples. The results demonstrated a notable contrast in matrix stiffness between the two groups. Specifically, the normal urethra exhibited a matrix stiffness of 5.23\u2009\u00b1\u20090.37\u00a0kPa, whereas the stricture urethra displayed significantly increased stiffness, reaching as high as 41.59\u2009\u00b1\u20092.48\u00a0kPa  analysis of differentially expressed mRNAs and qPCR validation of sequencing results . These findings provide valuable insights into the stiffening process during the progression of urethral fibrosis in the rat model and offer a significant comparison to human stricture tissues.34. Among them, the investigation of matrix stiffness in relation to fibrosis has garnered the most extensive and in-depth attention. The definition of matrix stiffness in biology and biomedicine is the ability of an object to resist deformation by external forces34. Extensive research has explored the interplay between matrix stiffness and fibrosis in various tissue and organ settings, such as pancreatic fibrosis, myocardial fibrosis, liver fibrosis, and wound healing. In these studies, higher matrix stiffness was consistently associated with enhanced fibroblast proliferation, increased migratory capabilities, and elevated extracellular matrix (ECM) expression37. Our study yielded similar findings, confirming the influence of matrix stiffness on fibrosis progression. Moreover, by using cellular and animal models, we observed a time-dependent advancement of fibrosis, closely correlated with the increasing stiffness of the micro-environment. The evidence from our study, in conjunction with existing literature, further substantiates the significance of matrix stiffness as a key modulator of fibrosis. Understanding the intricate relationship between tissue mechanics and fibrotic processes holds promising implications for devising targeted therapeutic approaches aimed at mitigating fibrosis progression in various pathological conditions.Tissue mechanics, encompassing aspects such as matrix stiffness, tension, hydrostatic forces, tensile forces, and osmotic pressure play a crucial role in fibrosis. These forces together regulate various cellular processes, including the proliferation of fibroblasts and other cell types, the activation of growth factors in damaged tissue as well as the structure and forces of the matrix38. In the present research, there are four main pathways for the molecular mechanism of ECM stiffness to enhance the degree of fibroblast fibrosis and to activate fibroblasts. (1) Integrin-related pathways18; (2) focal adhesion kinase (FAK) related pathway19; (3) RhoA/Rho-ROCK related signaling pathway20; (4) YAP/TAZ related signaling pathway21. In addition, mechanosensitive ion channels such as PIEZO1, transient receptor potential ion channels vanilloid receptor 4 (TRPV4) and the lysyl oxidase (LOX) family have also been shown to play an important role in matrix stiffness promoting fibrosis23. Although numerous studies have shown that extracellular matrix stiffness can activate fibroblasts, including fibroblasts in multiple organs such as lung, heart, kidney, and skin, the specific impact and underlying mechanism of matrix stiffness on urethral fibroblasts remain an area necessitating further investigation.Studies have shown that matrix stiffness plays a critical facilitating role in regulating TGF\u03b21-stimulated lung fibroblast contraction, suggesting that stiff fibrotic lung tissue may promote myofibroblast activation through contraction, whereas normal lung tissue compliance could prevent formation of lung myofibroblastsThrough bioinformatics analysis, Igfbp3 might contributed to regulating FMT and promoting the progression of urethral fibrosis. FMT contributed to the pathogenesis of fibrosis. Compared with fibroblast, myofibroblast secreted more ECM component and \u03b1-SMA. Extra ECM increased the stiffness of matrix and higher expression of \u03b1-SMA increased the contraction of scar, which stimulate FMT in turn. We also observed a stiffness-depend advanced progression of FMT in our study.39. Igfbp3 is found to be high expression in urethra and bladder40. Igfbp3 can combine with insulin-like growth factor and play a role in synergistic regulation of cell proliferation and apoptosis. Igfbp3 is closely related to fibrosis. Studies on patients with idiopathic pulmonary fibrosis have revealed a significant increase in Igfbp3 content in bronchoalveolar lavage fluid, lung tissue, and lung primary fibroblasts, where it exerts a promoting effect on extracellular matrix deposition41. Similarly, our investigation unveiled elevated Igfbp3 levels in human urethral stricture tissue.Bioinformatic analysis has implicated Igfbp3 as a potential regulator of fibrosis through its involvement in TGF-\u03b2 signaling pathwaysIn cellular and animal models, we evaluated the expression of Smads proteins, key components of the classical TGF-\u03b2 signaling pathway. Notably, when we inhibited Igfbp3 expression, p-Smad2/3 levels decreased, while overexpression of Igfbp3 in fibroblasts led to increased p-Smad2/3 levels. This finding led us to believe that Igfbp3 is involved in fibrosis through the regulation of Smad2/3.However, the precise mechanism through which Igfbp3 senses mechanical stimulation remains incompletely elucidated in this study. It is conceivable that Igfbp3 might interact with proteins that are sensitive to matrix stiffness, such as FAK and LOX, both of which serve as potentially crucial regulators of gene expression in cancer, influencing cell function and shaping the surrounding tumor microenvironment. Addressing this aspect is an avenue that will receive focused attention in our future studies. By further investigating the interplay between Igfbp3 and these mechanosensitive proteins, we aim to gain a more comprehensive understanding of how matrix stiffness influences fibrosis and related cellular responses.In sum, Igfbp3/Smad pathway participates in driving the progressive exacerbation of urethral fibrosis and Igfbp3 might be a novel therapeutic target for urethral stricture.Supplementary Information."
\ No newline at end of file