PREDICT is a widely used online prognostication and treatment benefit tool for patients with early stage breast cancer. The aim of this study was to conduct an independent validation exercise of the most up-to-date version of the PREDICT algorithm (version 2) using real-world outcomes from the Scottish population of women with breast cancer.Patient data were obtained for all Scottish Cancer Registry (SCR) records with a diagnosis of primary invasive breast cancer diagnosed in the period between January 2001 and December 2015. Prognostic scores were calculated using the PREDICT version 2 algorithm. External validity was assessed by statistical analysis of discrimination and calibration. Discrimination was assessed by area under the receiver-operator curve (AUC). Calibration was assessed by comparing the predicted number of deaths to the observed number of deaths across relevant sub-groups.A total of 45,789 eligible cases were selected from 61,437 individual records. AUC statistics ranged from 0.74 to 0.77. Calibration results showed relatively close agreement between predicted and observed deaths. The 5-year complete follow-up sample reported some overestimation (11.5%), while the 10-year complete follow-up sample displayed more limited overestimation (1.7%).Validation results suggest that the PREDICT tool remains essentially relevant for contemporary patients with early stage breast cancer. Provided with input of a patient’s clinical characteristics, PREDICT provides personalised prognostic information displayed as 5-year and 10-year overall survival estimates, both with and without adjuvant therapies . Results are presented both in textual format using a frequency-based description of risk and graphically in the form of bar charts with percentages labelled (http://www.predict.nhs.uk/). The PREDICT online tool is popular in the United Kingdom and worldwide with 20,000 visits reported in a single month.2PREDICT is an online prognostication and treatment benefit tool for patients with early stage breast cancer.3 The first online version of the tool was published in 20101 (v1). A series of updates made since the launch have added new prognostic variables and refined the algorithm’s predictions. The first update published in 20124 added HER2 status as a prognostic marker and allowed calculation of trastuzumab treatment benefit estimates (v.1.2). In 20 14, the tumour proliferative marker Ki-67 was added as an optional prognostic variable5 (v1.3). The most recent update, in 2017, refined the model by including age at diagnosis in the breast cancer-specific death prediction as well as recoding tumour size and nodal status variables (v2).2 The aim of our study is to conduct an independent validation exercise of the most up-to-date version of the PREDICT algorithm available (v2) using real-world outcomes from the Scottish population of women with breast cancer.The algorithm behind the online tool was derived primarily from data obtained from the Eastern Cancer Registration and Information Centre (ECRIC) registry in the United Kingdom (east of England).Treatment effectiveness estimates are taken from the Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) meta-analyses of clinical trials.6 The reporting of this validation study follows the “transparent reporting of a multivariable prediction model for individual prognosis or diagnosis” (TRIPOD) guideline.7The validity of a prognostic model refers to its ability to accurately predict outcomes for patients, both in the sample from which it was derived  and in other populations to which it can be applied . This study is concerned with external validity and addresses this issue through statistical analysis of the two main performance variables, discrimination and calibration.4 Validation is confined to mortality estimates; treatment benefit is not further considered here as this would require different study designs to provide robust causal inference.The high-quality routine data available in Scotland over a long time period allow an assessment of external validity that is greater in scope than previous external validation studies of PREDICT.Patient-level data were transferred into the National Services Scotland National Safe Haven as an extract from the Scottish Cancer Registry (SCR). SCR is a population-based registry that covers all residents of Scotland (population approximately 5.5 million). National Records of Scotland provides notification of deaths for registry records. All records in the registry with a diagnosis of primary invasive breast cancer  C50) diagnosed in the period between January 2001 and December 2015 were retrieved for analysis. Vital status was recorded up to 1 February 2017 in the analysis extract. Deaths due to breast cancer were defined in accordance with the ICD-10 coding system for causes of death, recorded either as the underlying cause of death or one of three secondary causes of death on death notifications. In cases for which there were multiple records of primary breast cancer for the same individual patient, records of non-first occurrences of breast cancer were excluded.Prognostic factors available in the registry extract included: age at diagnosis, number of lymph nodes examined and number positive, tumour size , tumour histological grade , mode of detection (screen-detected or symptomatic), oestrogen receptor (ER) status and Her2 status. Treatment status was available in relation to chemotherapy use (binary) and hormone therapy use (binary). Records indicated whether treatment was started or not but did not include information about treatment completion.8 in order to allow comparison of the results. Additional sensitivity analyses, further described below, have been conducted based on unique features of the SCR data.This validation analysis followed closely the approaches taken in previous validation studies of PREDICT9The data displayed a high level of completeness of all of the variables needed as inputs for the PREDICT model with the exception of HER2 status, trastuzumab use and Ki-67 status. Ki-67 status is not recorded in these data because this marker has not been in routine use in Scotland, and therefore all cases were assigned to the “unknown” category for this variable. HER2 status is only recorded from 2009. Cases with missing data for HER2 were assigned the “unknown” category. This includes 100% of cases from before 2009. The PREDICT algorithm handles “unknown” values in these categories by averaging across the available categories weighted by their frequency in the development data. Furthermore, trastuzumab use is not routinely recorded within the SCR, and therefore it was assumed that trastuzumab was used for all cases with a recorded positive HER2 status and chemotherapy use and in no cases with a recorded negative or unknown status, or positive status with no chemotherapy use. Recent Scottish clinical audit data submitted to a national review reported transtuzumab use for eligible HER2-positive cases is greater than 90% in the majority of health boards.Chemotherapy use is recorded as a binary variable in SCR, and therefore the generation of chemotherapy is unknown. It was assumed to be second generation for all cases in the primary analysis. A sensitivity analysis varied this assumption to instead assume third-generation chemotherapy for cases which are node positive, under 70 years of age and diagnosed during or after 2006.2PREDICT 10-year prognostic index scores were calculated for each individual case based on their recorded risk factor information using the algorithms supplied by the PREDICT authors (version 2). The scores include the probability of death from all causes, probability of death from breast cancer accounting for competing risk, and adjuvant therapy benefit as the percentage point reduction in the probability of all-cause mortality for each adjuvant therapy. Details of the calculation of the prognostic index are available in Candido Dos Reis et al.1 adding an exclusion criteria that the number of lymph nodes examined in node-negative cases must be four or more;2 exclusion of T4 cases as well as neoadjuvant cases. The additional exclusion criterion in ref.1 creates a sample selected in an equivalent manner to that reported in the development and earlier validation studies of the PREDICT model.8 Data used in earlier studies were collected in a period prior to widespread use of sentinel node biopsy, and therefore compared with the patients included in this dataset a larger number of nodes were examined.Cases were excluded if the patient was male, had advanced cancer , did not receive surgery or received neoadjuvant therapy (chemotherapy or hormone therapy recorded prior to date of surgery). Two sensitivity analyses were conducted to test sensitivity of calibration results to the selection criteria applied:Cases in which neoadjuvant therapy was used were excluded in the primary analysis because neoadjuvant therapy can alter the prognostic variables recorded in registry data  and PREDICT is less relevant as a tool for estimating treatment benefit in such circumstances. Neoadjuvant therapy has become more common in recent years, and therefore we believe that this exclusion criterion is important for the validation of PREDICT, although it has not been applied in previous analyses.10 was calculated in the primary analysis sample. This is a concordance statistic which can be used with right-censored survival data such as those available in this dataset.Discrimination of the PREDICT score as a prognostic index was assessed by calculating the area under the receiver-operator curve (ROC) (AUC) for both 5- and 10-year all-cause mortality, and 5- and 10-year breast cancer-specific mortality. AUC statistics were calculated separately for ER+ and ER− cases. In addition, Harrell’s c-statistic1). The 5-year and 10-year periods of complete follow-up were considered. In each case, predicted probabilities of mortality were summed across all individuals for whom complete follow-up was available for the specified period. A third analysis considered individual-specific follow-up periods up to the time of censoring. Predicted probabilities of mortality were calculated using the PREDICT algorithm for each individual’s own potential follow-up time in this analysis. Total numbers of deaths were counted for the selected samples within each of the specified follow-up periods. Differences are reported as relative differences, predicted−observed/observed, over 5 years, 10 years and individual potential follow-up times. Absolute differences  are reported for 5-year and 10-year complete follow-up. Results are reported for all deaths, and separately for breast cancer deaths only  for survival data was calculated.A major reason to expect worse calibration in this validation sample compared to the original sample is the time periods in which the cohorts were diagnosed. All patients in the original derivation sample were diagnosed between 1999 and 2003, while this validation sample spans a period from 2001 to 2015. To explore the impact of such time trends a sensitivity analysis was conducted repeating the calculation of calibration statistics on subsets of the data including cases diagnosed from 2001–2005, 2006–2010 and 2011–2015, respectively.12 PREDICT scores were calculated for observations in all imputed datasets. The calibration analysis was repeated with the imputed datasets and the expected and observed deaths were calculated with combination of results across imputed datasets according to Rubin’s rules.13To address potential bias from missing prognostic information, multiple imputation analysis was conducted for all missing prognostic factors. Multiple imputation created 10 datasets using chained equations (MICE).A total of 63,116 records were retrieved from the registry. Following removal of “duplicate” records (see Supplementary Appendix for details) and application of the exclusion criteria, a total of 45,789 cases (72.5%) remained in the primary analysis using multiple imputation of missing data. The process is detailed in Fig. 1 Notable differences between ECRIC and SCR samples were the somewhat older age distribution, slightly higher use of adjuvant chemotherapy and a higher proportion of screen-detected cases for SCR. There was also a lower proportion of deaths attributed to breast cancer in the SCR data. This may be partly explained by a higher all-cause mortality rate in the Scottish population compared to the East of England population. The age-standardised mortality rate for females was 1025.5 in Scotland compared to 815.6 in the East of England in 2015.14The characteristics of the complete case sample are described in Table 2 The associated Harrell’s c-statistics were 0.759 for ER positive at 5 years, 0.738 for ER negative at 5 years, 0.749 for ER positive at 10 years and 0.730 for ER negative at 10 years, respectively. AUC statistics for alternative sample selections and outcomes considered across all sensitivity analyses are shown in table Across ER-positive and ER-negative cases, AUC statistics ranged from 0.75 to 0.78. Performance was similar to that reported in the original data and previous validation exercise (0.76–0.78).Calibration assessed the accuracy of the probability estimates across specific univariate groupings of individual cases. Total number of predicted and observed deaths in the full sample and in sub-groups according to the defined variables and levels are reported in Table The 5-year complete follow-up sample show a general pattern of some overestimation of mortality. Overall expected mortality was 5.44% higher relative to observed mortality. In contrast. the 10-year complete follow-up sample showed a small degree of underestimation (−1.96%). In absolute terms, the predicted mortality was 0.79% above observed over 5 years and −0.6% lower over 10 years. In the full sample, using all lengths of follow-up Table , calibraThe degree of overestimation varied between groups, usually between 10 and 25%. Expected mortality was less than observed for the over 75 year age group and grade I cases. Calibration was relatively poor in the group of patients with very large tumour size (>50 mm), cases with very large numbers of nodes involved (10+) and for younger age groups .P < 0.001) for 5-year follow-up and 13.449 (P = 0.1433) for 10-year follow-up respectively. The figure shows that calibration was very good for lower deciles of PREDICT score and less good for the highest deciles. Calibration across all deciles was good for cases with 10 years of complete follow-up.Predicted and observed numbers of deaths were also calculated by decile of PREDICT score. The results are displayed as a calibration plot in Fig. Calibration results from the sensitivity analyses are presented in the Supplementary The sensitivity analysis assessing calibration of PREDICT in cohorts diagnosed in the time periods 2001–2005, 2006–2010 and 2011–2015 displayed an apparent time trend in survival outcomes Table -S11. In In this validation exercise of the PREDICT prognostication tool using an external dataset good performance was demonstrated with regards to both discrimination and calibration, comparable to that reported in the derivation and previous validation data.The key strength of this validation study is the suitability of the data in terms of both quality and quantity for addressing the research question. The large sample size drawn from the population-based SCR, which is eight times the previous validation samples combined, improves the precision of the results considerably and also allows an assessment of the generalisability of the model to the full population in whom it may be applied. The dataset includes cases diagnosed from 2001 to 2015, allowing its performance in more recent cases to be studied. All cases were followed-up until 2017 and therefore cases diagnosed earlier in the period have a long duration of follow-up.There are some limitations to this study as a validation exercise. No data were available for so me input parameters: Her2 (before 2009), trastuzumab use, Ki-67 status and generation of chemotherapy used. The PREDICT algorithm allows for unknown Ki-67 or Her2 status. Generation of chemotherapy and trastuzumab use were based on assumptions. Alternative assumptions were explored in a sensitivity analysis which suggests these assumptions were not critical in influencing the results. This analysis cannot assess calibration with regards to Ki-67 status or the potential benefits of including this variable in relation to discrimination. A limitation of the PREDICT model is that is not suitable for providing prognostic estimates in neoadjuvant-treated patients. Exclusion of these cases from the validation sample affects the composition of sample compared to the derivation sample because neoadjuvant-treated patients are predominantly those with moderately poor prognosis. This may improve or worsen model performance depending on if the model performs relatively better or worse for these particular patients.15 Some overestimation for 10-year follow-up was observed in the Netherlands cohort, while some underestimation was observed in this cohort. These differences may largely reflect differences in age-specific all-cause mortality rates between these settings; Scotland, the Netherlands and the East of England.Calibration was more accurate for some sub-groups than others. In particular, calibration was relatively poor for younger women  and relatively good for older women. This is consistent with the results of a validation study performed in a cohort of women aged 65 years or older in the Netherlands.Missing data on individual prognostic variables create some potential for bias. This has been partially addressed by using multiple imputation. However, this relies upon assumptions regarding the pattern of missingness, assumed to be random conditional on observed covariates, and appropriateness of the imputation model. The proportion of cases with any amount of missing data was 12%, which is relatively modest.The validation results presented in this analysis provides greater confidence in the accuracy of the information given by the PREDICT online tool compared to previous validation studies. Calibration in the overall population appears to be sufficient for decision making purposes. In the Scottish context, version 2 of PREDICT is suitable for providing prognosis and treatment benefit estimates for patients with early breast cancer.6). This issue is common across prognostic models and is likely to be of particular relevance in early breast cancer because of the continuing introduction of new interventions. If clinically relevant, this could potentially be addressed by an update of the PREDICT model—a version 3—with more recent data used to derive the algorithm’s parameters. Researchers developing and validating prognostic models must strike a balance between using data with longer follow-up, necessarily from cohorts diagnosed further in the past, and using the most recent data, for which follow-up will consequently be shorter. The correct balance will need careful consideration in any future prognostic models in early breast cancer given the potential for calibration drift in this setting.There is some evidence of predictions overestimating mortality for contemporary patients. It should be noted that each percentage point overestimation of absolute mortality risk will result in a fraction of a percentage point reduction in adjuvant chemotherapy benefit estimates . Exploratory results suggest that predictions may become less well calibrated for the most recently diagnosed cohorts (an example of “calibration drift”A limitation of validation studies is that they do not provide a full investigation of the clinical utility or cost effectiveness of using the prognostic model, however accurate it may be. A decision analysis or economic evaluation of use of prognostic models in this setting should therefore be a research priority. This could help to clarify the clinical and cost effectiveness of existing and future alternative prognostic models as well as the addition of new information such as genomic data.6 and for driving continuing improvement in prognostic information. This study provides large-scale investigation of validity of the PREDICT prognostic model for early breast cancer. The results of this investigation suggest that it remains essentially relevant for contemporary patients being diagnosed and managed with invasive breast cancer.Validation of prognostic models is critical for both providing the necessary evidence for adoption into clinical practiceSupplementary Appendix


Background: Eculizumab has dramatically changed poor outcomes of complement-mediated atypical hemolytic uremic syndrome (aHUS) as first-line treatment. Discontinuation of eculizumab remains challenging, and doctor's visits every 2 weeks for intravenous injection because of standard dosing protocols is a huge burden. The Ultra-high cost of eculizumab is also an issue. We attempted to establish a personalized dosing regimen of eculizumab based on pharmacokinetics and pharmacodynamics in a 2-year-old girl with aHUS with a C3 mutation.Case presentation: She developed aHUS at 5 months of age and was successfully treated with eculizumab. At 2 years of age, we measured eculizumab concentrations and performed pharmacokinetics and pharmacodynamics analysis to optimize her dosing protocol. Her blood concentrations at every 2-, 3-, and 4-week intervals were simulated. Pharmacokinetics analysis showed that her eculizumab clearance was 40% lower than the population mean reported for aHUS. Pharmacokinetic simulation suggested that the 2- and 3-week interval regimen could be sufficient to achieve an efficient trough concentration (>100 μg/mL). We simulated her individual pharmacokinetics profile at 4 years of age with consideration of her growth, which still showed complete inhibition of the alternative complement pathway with the 3-week interval regimen. We continued the 300-mg eculizumab infusion every 3 weeks while CH50 levels were constantly maintained at undetectably low concentrations with no recurrence until 6 years of age.Conclusions: Pharmacokinetics and pharmacodynamics estimation was useful for establishing a personalized dosing regimen for eculizumab and reducing the patient's burden and high medical costs. Eculizumab  has dramatically changed poor outcomes of complement-mediated atypical hemolytic uremic syndrome (aHUS) and is a first-line and promising long-term treatment for aHUS , 2. HoweFrequent blood tests for children are unfavorable, and the measurement of ECZ concentrations is not readily available. Thus, estimation of ECZ concentrations via pharmacokinetics (PK) and pharmacodynamics (PD) analysis is a good option. There are some reports regarding PK/PD analysis in patients with aHUS; however, those data are limited to infants or young children with a low body weight –6. HereiC3 mutation  (The patient was a 2-year-old girl who was diagnosed with aHUS with a 1105Gln) . She fir1105Gln)  8). We . We C3 m1105Gln) , 10, ECZAs a result, this patient's estimated PK profile was demonstrated . The patIn 2 years, PK simulation was performed again at 4 years of age (BW 17.5 kg) with adjustment of individual PK parameters based on body weight increase according to the observed covariate relationship between PK parameters and body weight . ReanalyWe applied to establish a personalized dosing regimen of ECZ based on PK/PD analysis for a 2-year-old girl with aHUS and successfully continued maintenance therapy for 4 years. Simulated ECZ concentrations were reliable, as confirmed by measuring ECZ concentrations at 5 years old and performing blood test analyses that included CH50. These results indicate that PK/PD analysis is a very useful option for patients, even for children with aHUS. Considering the difficulty of measuring ECZ concentrations, even only CH50 monitoring may be enough for the follow-up of patients who require ECZ administration in the clinical setting.The target trough concentrations of ECZ for patients with aHUS were considered to be 50–100 μg/mL , 10. VolC3 mutation in our patient  was located in the C3b thioester-containing domain, which is exactly at the binding site of the complement factor H (CFH) and C3b, potentially disrupting CFH binding to C3  is a good option for patients with aHUS.Prolonged treatment for patients with aHUS remains a controversial issue, as does ECZ discontinuation since the clinical criteria are not exhaustive –14. Fromng to C3 . Generalutations , 16, evevariants . Poorer In order to make long-term treatment decisions for patients with aHUS, physicians must consider various factors including effectiveness, safety, adverse effect, and medical cost. In our patient, the 3-week interval administration of ECZ has been continued for 4 years after the patient was >10 kg, which is when the treatment interval is recommended to be shortened to 2 weeks from 3 weeks according to the approved standard indication. As a result, this means that the patient could skip 35 hospital visits and save ∼$200 000 in medical costs. In the chronic phase, we have the option to continue ECZ according to standard protocols or discontinue it. However, we would like to suggest the third option as presented in this study by giving due consideration to an individualized treatment regimen by measuring or estimating the dose with PK/PD analysis. This method allows for personalized treatment of patients, and reduces the patients' burden and high medical costs. This case study will facilitate PK/PD-based eculizumab precision dosing in a larger number of patients to minimize their physical and economical burdens.Optimal treatment for patients with aHUS should be discussed. Our PK/PD based-dosing strategy promises to be safe and useful for estimating individual ECZ concentrations with sparse sampling data and establishing maintenance treatment for aHUS.All relevant data is contained within the article.The studies involving human participants were reviewed and approved by The Institutional Review Board of National Center for Child Health and Development. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.KS drafts the manuscript. TF and KM performed pharmacokinetics and pharmacodynamics analysis of eculizumab made substantial contributions to the conception and design. MO and KK treated the patient while she was hospitalized, and involved in revising the manuscript. SI oversaw the project. All authors approved the final case report as submitted and agree to be accountable for all aspects of the work.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.


Mycobacterium tuberculosis isolates. Unlike the MYCOTB plate, on which the UKMYC5 plate is based, the UKMYC5 plate includes two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds.The UKMYC5 plate is a 96-well microtiter plate designed by the CRyPTIC Consortium  to enable the measurement of MICs of 14 different antituberculosis (anti-TB) compounds for >30,000 clinical  Mycobacterium tuberculosis isolates. Unlike the MYCOTB plate, on which the UKMYC5 plate is based, the UKMYC5 plate includes two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds. UKMYC5 plates were tested by seven laboratories on four continents by use of a panel of 19 external quality assessment (EQA) strains, including H37Rv. To assess the optimal combination of reading method and incubation time, MICs were measured from each plate by two readers, using three methods , after 7, 10, 14, and 21 days of incubation. In addition, all EQA strains were subjected to whole-genome sequencing and phenotypically characterized by the 7H10/7H11 agar proportion method (APM) and by use of MGIT960 mycobacterial growth indicator tubes. We concluded that the UKMYC5 plate is optimally read using the Vizion system after 14 days of incubation, achieving an interreader agreement of 97.9% and intra- and interlaboratory reproducibility rates of 95.6% and 93.1%, respectively. The mirrored box had a similar reproducibility. Strains classified as resistant by APM, MGIT960, or the presence of mutations known to confer resistance consistently showed elevated MICs compared to those for strains classified as susceptible. Finally, the UKMYC5 plate records intermediate MICs for one strain for which the APM measured MICs close to the applied critical concentration, providing early evidence that the UKMYC5 plate can quantitatively measure the magnitude of resistance to anti-TB compounds that is due to specific genetic variation.The UKMYC5 plate is a 96-well microtiter plate designed by the CRyPTIC Consortium  to enable the measurement of MICs of 14 different antituberculosis (anti-TB) compounds for >30,000 clinical  The proportion of tuberculosis (TB) cases that are multidrug resistant (MDR) is increasing worldwide. Although set against a background of a falling global incidence of TB, the net effect is that the number of MDR-TB cases continues to grow . ImproviLiquid- and solid-medium assays that measure MICs for TB exist –9 but ar–12–We designed the UKMYC5 plate, which is a variant of the MYCOTB plate, to enable the CRyPTIC Consortium  to measure the MICs of 14 different anti-TB compounds  for a large number  of clinical TB isolates that are being collected globally by participating laboratories between 2017 and 2020. Each isolate will also have its genome sequenced, and the ultimate goals of the CRyPTIC project are (i) to uncover all variations in genes known to be involved in resistance and classify the magnitudes of their effects on specific anti-TB compounds and (ii) to identify new genes that are associated with resistance. The MICs therefore need to be quantitatively reproducible and accurate.In this paper, we establish the reproducibility and accuracy of the dry-format, 96-well UKMYC5 microtiter plate for use in large-scale measurement of MICs by the CRyPTIC tuberculosis research project. We therefore assess both the reproducibility of MIC measurements obtained using this microtiter plate and their accuracy by comparing the method to a range of established DST methods. Since the UKMYC5 plate contains 14 drugs, including, in contrast to the MYCOTB plate, two new compounds (delamanid and bedaquiline) and two repurposed drugs (clofazimine and linezolid), in the future it may potentially form the basis of a new DST protocol for tailoring regimens to treat individual cases of MDR-TB.The UKMYC5 plate was designed by the CRyPTIC Consortium. The main differences compared to the commercially available MYCOTB 96-well microtiter plate are that bedaquiline, delamanid, clofazimine, and levofloxacin are included and ofloxacin, streptomycin, and cycloserine were removed. The wells for each drug are arranged in doubling dilution series. Since the plate comprises 12 columns of 8 wells and 14 drugs are included, most drugs have 6 (64× concentration range) or 7 wells, with a few having 5 or 8 wells .To validate the UKMYC5 plate for use in large-scale research susceptibility testing, it is necessary to demonstrate that the results obtained with the plate are both reproducible and accurate. Unfortunately, no standards for mycobacterial DST exist yet. We therefore borrow, where appropriate, the definitions established for aerobic bacteria. The conclusions we draw about reproducibility and accuracy should therefore be treated as tentative, as they will need revision once a standard for mycobacterial DST is established.The concentration range for each drug on the UKMYC5 plate is defined by the plate design and therefore fixed. MICs not included in the range are termed “off-scale.” Since MICs are conventionally defined as being in agreement if they lie within a doubling dilution, reproducibility and accuracy are defined exactly only for on-scale growth; including off-scale growth may alter the levels of reproducibility and accuracy.In brief, each of the seven participating laboratories  receivedMycobacterium tuberculosis in both positive-control wells was reported. As expected, the proportion of measured MICs that were off-scale, in general, fell as the incubation time increased (Table S4); however, even after 21 days, there remained large differences between drugs, with linezolid having the fewest off-scale readings  and rifabutin having the most . The exact pattern for each drug depends on the plate design and how many EQA strains are resistant to its action. A logistic mixed-effects model demonstrated that significantly fewer plates were readable at days 7 and 10 than at day 14 and that significantly more plates were readable at day 21 than at day 14  (Table S5). The proportion of results readable by use of an inverted-light microscope or mirrored box was significantly lower than that for the Vizion instrument (P < 0.001 in both instances) , the results for site F were anomalous, and the data from this laboratory were excluded from the study. For the remaining six sites, the proportion of readable results was 57.8 to 66.1% (depending on the reading method) after 7 days of incubation, which then increased to 85.7 to 93.0% after 14 days and reached ≥95.9% after 21 days (P < 0.001 for both comparisons) (Table S5). The levels of interreader agreement were similar if we considered only the strain-drug combinations for which the modal MIC was on-scale (Table S3).As stated in Materials and Methods, we define two measurements as agreeing if they are within a doubling dilution of one another. The interreader agreement was at least 92.7%, regardless of reading day or method . For VizP < 0.001) and was also lower when the results were obtained with a microscope than when they were obtained with a Vizion instrument (P = 0.010) (Table S5).As expected, the agreement between MICs measured from two UKMYC5 plates in the same laboratory, each having been inoculated from different cultures of the same strain taken from the same vial , was lower, with ≥89.7% of MICs in agreement, regardless of reading day and method  was ≥88.1%  (Table S6). Moreover, both types of reproducibility were lower at days 7 and 21 than at day 14  (Table S6).The interlaboratory reproducibility was slightly lower, as one would expect, and was ≥85.4% regardless of the reading-day and method combination, peaking again after 14 days, at 93.1% and 92.7% with the Vizion system and the mirrored box, respectively  showed the lowest interreader agreement at each reading day and the lowest intra- and interlaboratory reproducibilities across all reading days, except for day 7 . This is consistent with a previous study in which PAS had the lowest reproducibility among the 12 drugs on the MYCOTB plate . The intra- and interlaboratory reproducibilities were indistinguishable between days 10 and 14 , and the interreader agreement was lower at day 7 than at day 14 (P = 0.030).To determine if the individual drugs could be read after 14 days of incubation or whether there was large variation between compounds, we analyzed the individual MICs measured using the Vizion system for each drug and all reading days (TB plate . After 1rpoB gene (Ser450Leu) that leads to high MICs clearly distinguishable from those for wild-type genotypes (Table S9). Although there were statistically significant differences between many of the drugs and rifampin, the greatest difference was seen for para-aminosalicylic acid, for which the interreader agreement and the intra- and interlaboratory reproducibilities were both significantly lower than those for rifampin (P < 0.001). In general, although the intra- and interlaboratory reproducibilities of the drugs were similar after 10 and 14 days (Table S9), since more results were readable at day 14 than at day 10 . Since neither technique is an established reference method, we interpret the agreement here to measure the consistency of human- and software-based reading methods. Using a logistic mixed-effects model, we found that the agreement at days 7, 10, and 21 was not significantly different from the agreement at day 14  (Table S11). We later discuss the potential for software, such as AMyGDA, to further improve how microtiter plates are read.Automated plate-reading software offers the ability to add a layer of quality control to plate reading, potentially increasing both reproducibility and accuracy. MICs measured from photographs of the UKMYC5 plates after 14 days of incubation by use of AMyGDA software   were theHaving determined the optimal time and method to read the UKMYC5 plate and examined the reproducibility of this approach, we next assessed the concordance between the UKMYC5 plate and several endorsed methods. We first compared our MIC distributions for the H37Rv reference strain ATCC 27294, as measured by the Vizion system after 14 days of incubation, with MICs measured using (i) a frozen Thermo Fisher custom-made 96-well microtiter plate , 16, ii, 16, 19.For the majority of drugs, there was a good level of agreement between the distribution of MICs measured using the UKMYC5 plate and the three comparator measurements . The agrpara-aminosalicylic acid, and ethionamide were not tested). We did not observe the lack of accuracy for ethambutol observed in previous studies using the MYCOTB plate  exist for any microtiter plate, all 19 EQA strains (including H37Rv) were then categorized as resistant or susceptible by using the same CCs as those for the comparator methods , whTB plate , 21. SinTB plate . The smaTo enable quantitative comparisons to be made between the UKMYC plate and both APM and MGIT, the mode of each UKMYC5 MIC distribution was calculated by drug for each strain and then compared with the categorical results (susceptible or resistant) obtained by MGIT and APM for that strain , 22, 23.Since the genomes of all 19 EQA strains, one of which is H37Rv, are known, one can carry out a comparison between strains harboring variations in certain genes known to confer resistance to a specific compound and those with either no variation or variations with an unknown effect or known to have no effect. For all drugs, in general, strains with genetic mutations accepted to confer phenotypic resistance to specific drugs were associated with elevated UKMYC5 MICs compared to those for strains with no such mutations .rrs gene . For the rifamycins, 7 of the EQA strains contained a Ser450Leu mutation encoded within the rpoB gene. An Ala90Val mutation encoded within the gyrA gene, either on its own or in combination with a Ser91Pro mutation, was also associated with elevated MICs for fluoroquinolones (levofloxacin and moxifloxacin) on the UKMYC5 plate.This was most evident for the second-line injectable drugs  and the rifamycins (rifampin and rifabutin). For the SLID, 3 of the 19 EQA strains contained the well-known A1401G mutation in the inhA that is known to confer only a low level of resistance, whereas eight strains contained the common Ser315Thr mutation  encoded within the katG gene, which is known to confer high-level isoniazid resistance. The promoter mutation was determined to be resistant by MGIT, and APM measured the MIC to be 0.4 mg/liter—just twice the critical concentration. Not only did the strains containing either of these mutations consistently have MICs higher than those for the remaining strains, but the strain with the mutation known to confer low-level resistance was also correctly identified on the UKMYC5 plate  in the promoter of C5 plate  24)..inhA thainhA promoter mutation is known to also confer resistance to ethionamide. A second strain contains an ethA mutation that is also associated with resistance to this compound. The resulting MIC distributions for ethionamide-resistant and -susceptible strains are notably cleaner and better separated than those obtained when MGIT was used to classify the strains. No strain contained mutations known to confer resistance to linezolid. Despite some knowledge of their resistance mechanisms, the MIC distributions of the strains identified as resistant to clofazimine and para-aminosalicylic acid overlap those of the strains assumed to be sensitive.This ddn gene or the fbiC (Rv1173) gene. The ddn mutation was associated with higher MICs than those seen with the fbiC mutation . The UKMYC5 MIC distributions for these strains are higher, on average, than the distributions observed for the remaining 15 strains.Interestingly, the EQA panel included two strains that are resistant to delamanid according to APM. These have mutations in either the M. tuberculosis growth on the UKMYC5 plate at the time of reading, which not only can form a valuable audit trail but also can be analyzed by software, providing an additional set of readings.We have shown that it is optimal to read the UKMYC5 plate after 14 days of incubation by using the Thermo Fisher Sensititre Vizion digital MIC viewing system. Although the laboratory scientists were unable to read as many plates by using a mirrored box, this reading method achieved levels of reproducibility indistinguishable from those for the Vizion system and is less expensive so may be the preferred solution in low-income settings. The Vizion system has the added advantage of capturing a photograph of the para-aminosalicylic acid (PAS) performed poorly, consistent with a previous study  of ical use . ConsideAssessing accuracy is more difficult, since there are no critical concentrations for the UKMYC5 plate or any other microtiter plate, and we thus assumed shared breakpoints with the comparator methods, as done previously , 14. ThaBroth-based microdilution methods for TB DST that either directly  or indir12–Mycobacterium tuberculosis, and in particular, it appears to be able to detect genetic variants which confer low levels of resistance, which is an important subsidiary goal of the CRyPTIC Consortium. In future work, we shall assess the ability of the AMyGDA software to add some quality control and, we hope, to improve the reproducibility and accuracy of the measured MICs.This study moves the field forward: UKMYC5 is the first microtiter plate assay to incorporate both new (delamanid and bedaquiline) and repurposed (linezolid and clofazimine) drugs in a dry-well format that is convenient to transport and store. The results presented here indicate that the UKMYC5 plate is sufficiently reproducible for large-scale research susceptibility testing of In its present form, the UKMYC5 plate is not yet suitable for clinical use. The overall reproducibility and accuracy of the plate would benefit from a redesign to remove PAS and to use the vacated wells to expand the range of concentrations for drugs whose measurements were most frequently reported at the extremes of the dilution range; this redesign is under way. We hope that by the time the CRyPTIC project has built a large data set for unbiased clinical samples using the UKMYC5 plate (and its successor), an antimicrobial susceptibility testing standard for mycobacteria will have been established, which will allow a formal evaluation of the suitability of broth-based microtiter plates for clinical use in diagnosing and treating tuberculosis.The UKMYC5 plate does have several limitations that need to be acknowledged; these include the need for a preculture step, entailing a delay of up to 6 weeks before the plate is inoculated. This currently prevents the rapid turnaround of results, but we expect that this time will be reduced significantly by further development work to define the optimal inoculum from MGIT cultures. In addition, as with all liquid-based DST, a microtiter plate such as the UKMYC5 plate can be used only in a biosafety level 3 laboratory, which may affect its potential utility in low-resource settings. Despite these shortcomings, the possibility of a single microtiter plate that can provide MICs for a range of anti-TB compounds at relatively low cost should be pursued vigorously.The determination of MIC values for a range of drugs allows therapeutic decisions to be more nuanced, since they can be guided by the degree of resistance to a drug and an understanding of the tolerability of drug doses required to overcome it. Unlike those with CC-based DST methods, in which all errors are categorical in nature, MIC errors can be marginal and thereby may be potentially less disruptive to treatment decisions. In addition to these advantages, the UKMYC5 plate assays 14 drugs at once, at low cost, giving a clear advantage over MGIT and APM. Including both new and repurposed drugs is a clear advantage over other microtiter plate-based quantitative DST methods.Because different countries have different settings, it would be optimal if one could modify the design of the microtiter plate to reflect the drugs that are locally available or the makeup of locally recommended regimens. It is therefore key that while the ranges of MICs are standardized the combination of drugs included on the plate can be adjusted according to need without significantly increasing the manufacturing cost. A challenge to regulators is therefore whether the performance of the plate can be accredited for a menu of individual drugs from which country-specific plates can be constructed, rather than for a fixed plate layout.M. tuberculosis complex (MTBC) isolates or by providing detailed and timely surveillance of the prevalence of different strains by country. The Foundation for Innovative and New Diagnostics (FIND) has already expressed interest in pursuing WHO endorsement of the UKMYC5 plate. In parallel, the impact of the plate on our understanding of the effects of genomic mutations on the MICs of various drugs is likely to inform the WHO's planned assessment of WGS-based DST in 2018  TB strains were distributed by the WHO Supranational Reference Laboratory at San Raffaele Scientific Institute (SRL), Milan, Italy, to six other participating laboratories . These wMycobacterium tuberculosis  (GenBank accession number AL123456)  .H37Rv ATCC 27294 was chosen as the reference strain because it is susceptible to all antitubercular drugs on the UKMYC5 plate. The other 18 strains were chosen from clinical isolates or proficiency testing isolates stored in the repository at the SRL based on the presence of mutations known to confer resistance to specific drugs with a high degree of confidence (Table S16) and therefore could reasonably be expected to have intermediate or high MIC values on the UKMYC5 plate.Upon arrival, all strains were subcultured onto solid media as follows: H37Rv was subcultured in 10 different Lowenstein-Jensen tubes or 7H10 agar plates, while the remaining 30 strains of the validation panel were subcultured in duplicate onto solid medium (Lowenstein-Jensen or 7H10 agar plates). Therefore, each participating laboratory tested up to 70 UKMYC5 plates ; Fig. S5para-aminosalicylic acid, linezolid, delamanid, and bedaquiline) were included, at 5 to 8 doubling dilutions . Janssen Pharmaceutica and Otsuka Pharmaceutical Co., Ltd., provided bedaquiline and delamanid pure substances, respectively. Although pyrazinamide-only plates containing lyophilized substance in different stocks were tested, poor performance due to suboptimal broth pH conditions resulted in pyrazinamide being excluded from the UKMYC5 plate. Two batches of UKMYC5 plates were manufactured and distributed for this study.The UKMYC5 plate was designed by the CRyPTIC Consortium and manufactured by Thermo Fisher Scientific Inc., United Kingdom. Fourteen anti-TB drugs  from 20- to 25-day-old colonies (or no later than 14 days after visible growth) grown on Lowenstein-Jensen or 7H10 agar medium. Duplicates were tested on different days and starting from different subcultures, thus also starting from different bacterial suspensions.5 CFU/ml; approximate range, 5 × 104 to 5 × 105 CFU/ml) were dispensed into wells by use of a semiautomated Sensititre Autoinoculator . Each plate was then sealed using a manufacturer-supplied rectangle of transparent plastic that remained in place throughout the 21-day incubation period.Suspensions were then diluted 100-fold by the addition of 100 μl of suspension to 10 ml of enriched 7H9 broth. Aliquots of 100 μl of standard inoculum , at 7, 10, 14, and 21 days postinoculation . A plateP < 0.001 for all comparisons) (Table S19). Data from site F were consequently excluded from subsequent analyses. A retrospective analysis of the plate images recorded by the Vizion system revealed that readers at site F had frequently mistaken sediment from the inoculum for bacterial growth, thereby allowing us to offer targeted training to the laboratory scientists involved. This process highlights the importance of storing images of all the plates to form an audit trail.For site F, the proportion of readable results was anomalously high (≥94.2%), regardless of reading day , yet site F had an anomalously low intralaboratory reproducibility, with values ranging from 77.5% to 80.2% depending on the reading day . Site F also had an anomalously low interlaboratory reproducibility of only 72.2% to 75.3% . Logistic mixed-effects models confirmed that, for site F, interreader agreement (results being within a doubling dilution) and intra- and interlaboratory reproducibilities were all significantly lower than those for the other laboratories  the resazurin microtiter assay (REMA) (Table S21) for drugs for which the WHO has endorsed critical concentrations (CCs) (Each of the 19 strains used  was phenns (CCs) . Middlebns (CCs) . Paired-ns (CCs) , and theBoth descriptive and modeling analyses were conducted. For the latter, logistic mixed-effects models were constructed, since the data consisted of repeated measurements. A drug on a plate was defined as readable if (i) there was acceptable growth in both positive-control wells, (ii) there was no contamination in the wells for that drug, and (iii) the contents of the wells for that drug had not evaporated. We define two measurements as being in agreement if the two MICs are within one doubling dilution of each other. Furthermore, reproducibility is defined as the proportion of MICs that are within one doubling dilution of the mode. Due to the high percentage of off-scale results, results were considered to be within one doubling dilution if they were adjacent in the doubling dilution range, even if one of them was off-scale. This constitutes a best-case approach, and therefore, where appropriate, calculations were repeated using only drug-strain combinations for which the MIC mode was on-scale. To assess the intralaboratory reproducibility, the modal MIC for each drug was computed for that site, with pooling of the results across reading methods, days, replicates, and readers. To test the interlaboratory reproducibility, the mode was calculated only for each drug, with pooling of the results across sites as well as across reading methods, days, replicates, and readers. To assess the accuracy of the plate, we compared UKMYC5 MICs to the modes of the MICs obtained by other established DST methods. For both APM and REMA, two independent measurements were taken for each drug , and if there was a discrepancy , the lower value was chosen. Since no critical concentrations exist for UKMYC5 plates, we used the same critical concentrations as those for the comparator method . ReSRP068011 and SRP130092.The sequences reported in this paper have been deposited in the Sequence Read Archive of the National Center for Biotechnology Information under study accession numbers


The discovery of non-coding RNAs (ncRNAs)has been one of the central findings from early genomic sequencing studies. Not only was the presence of these genes unknown previously, it was the staggering disproportionate share of the genome that was predicted to be encoded by ncRNAs that was truly significant in genomic research. Over the years the function of various classes of these ncRNAs has been revealed. One of the first and enduring regulatory programs associated with these factors was development. In the neurosciences, the discovery of adult derived populations of dividing cells within the brain was equally substantial. The brain was hypothesized to be plastic only in its neuronal connectivity, but the discovery of the generation of new neurons was a novel mechanism of neuronal and behavioral plasticity. The process of adult neurogenesis resembles early neuronal development and has been found to share many parallels in the proper stages of specified genetic programs. Adult neurogenesis has also been found to play a role in learning and memory involved in particular hippocampal-dependent behaviors. Substance use disorders (SUDs) are an example of a behavioral condition that is associated with and possibly driven by hippocampal alterations. Our laboratory has determined that hippocampal adult neurogenesis is necessary for a rodent model of methamphetamine relapse. Due to the previous research on ncRNAs in development and in other brain regions involved in SUDs, we posit that ncRNAs may play a role in adult neurogenesis associated with this disorder. This review will cover the regulatory mechanisms of various classes of ncRNAs on the coordinated genetic program associated with adult neurogenesis with a special focus on how these programs could be dysregulated in SUDs. Caenorhabditis elegans , are often 21–22 nucleotides (nt) in length. Thus far, it has been found that miRNAs may be regulated similarly to protein-coding genes through canonical epigenetic and transcriptional pathways . They exFirst proposed to be involved in mammalian tissue-specification and lineage designation, miRNAs have been associated with developmental processes for many years . The earMechanisms involved in proliferation and differentiation can also be recruited during pathological conditions. For example, miR-124 was found to be downregulated by stroke/ischemia in the SVZ. Over expression (OE) of miR-124 repressed a Notch ligand, Jagged (Jag-1), which has been highly studied in embryonic development. Next, researchers found that miR-124a OE decreased NPC proliferation but promoted neuronal differentiation (Dcx+ cells), suggesting that miR-124 may shift NSCs out of a proliferating state toward a neuronal lineage . In contBmi1 mRNA which is part of a well-studied repressive epigenetic complex, the Polycomb Repressive Complex (PRC)  has been found to play a role in the proliferation and differentiation of adult neural stem and progenitor cells. Researchers determined if MBD1 may regulate the expression of miRNAs associated with either of these pathways . They foex (PRC) . Other mex (PRC) . FurtherNotch1 regulatory sites and inhibit its transcription   . Many yes (Piwi) . These sposition . AlthougThe most well studied function and mechanism of biogenesis of piRNAs occurs through the silencing of retrotransposons. Transposons are genomic regions that can move the position in the genome. Retrotransposons (RE) are transposons that transcribed into RNA before being reverse transcribed back into DNA and inserted into the genome. Transposons can cause genomic instability, which is especially dangerous during replication of cells or in the germline. Although transposons can be destructive they are also a natural component of many genomes and have been involved in evolution and in novel regulatory mechanisms. For example, REs are most vulnerable in the RNA state. A piRNA-loaded PIWI complex may recognize a specific RE and cause endonucleolytic cleavage of the RE to prevent reverse transcription and finally re-insertion of these sequences back into the genome. In the destruction of the RE comes production of new piRNAs which can be loaded by other PIWI proteins and either silence other complementary REs or regulate other aspects of the cells . This unSox2 repression but Sox2 downregulation during differentiation may lead to increased activity of this RE, indicating that somatic mosaicism of various sites of LINE1 re-insertion may be occurring within the brain. This process is tightly regulated by Wnt signaling and NeuroD1  is a RE that may play a role in altering neuronal gene expression and preferential differentiation of NPCs into neurons . LINE1 m NeuroD1 . Utilizi NeuroD1 . Additio NeuroD1 . REs cou NeuroD1 . Overall NeuroD1 . This maThe origin of piRNAs can be traced to other regions of the genome, such as the 3′UTR of specific genes or piRNA clusters between protein-coding genes, intergenic regions . As suchPiwi proteins were found abundantly in the brain  within mammals, let alone the mammalian CNS. For many years, it was assumed that siRNAs were an evolutionarily primitive version of miRNAs due to the mammalian interferon response to double stranded RNA (dsRNA) and were undetectable by many cloning techniques during the early discovery of miRNAs. However, it was found in  3′ ends . Later t 3′ ends . They ca 3′ ends . These n 3′ ends . SimilarTo determine if there were endo-siRNA expression patterns associated with learning, researchers trained mice in an olfactory discrimination training paradigm . In thisAbca2, Arhgef17, Camk2a, Bdnf, Gap43, Rab40b, Slc17a7, Syn1, and SynGap1. Of these loci, the SynGap1 locus was enriched in many of the upregulated endo-siRNAs. From this population of endo-siRNAs, they found that one on the sense strand was predicted to form a perfect hairpin in the sense or antisense direction of Syngap1 which could result in a high probability of destruction by Dicer. Thus, we can conclude that hippocampal-dependent learning is associated with upregulation of a set of endo-siRNAs.Next, they determined the genomic location of this population of learning-associated endo-siRNAs. They found that many of them were derived from plasticity-related protein coding genes such as NRSE/RE1. Expression of NRSE/RE1 appeared to increase over the development of a progenitor toward a neuronal state but not toward a glial fate, suggesting that NRSE/RE1 is involved in neuronal-lineage determination. Overexpression of both strands of NRSE/RE1 caused an increase in neuronal differentiation in culture as well as increased Tuj1 promoter activity corresponding with a decrease in Sox2, Gfap, and Mbp promoter activity. Interestingly, NRSE/RE1 did not diminish NRSF/REST expression. This implies that NRSE/RE1 acts through a novel regulatory mechanism compared to other dsRNAs which may be processed into endo-siRNAs. This could be considered similar to a competing endogenous RNA (ceRNA) that blocks nucleotide recognition such as a miRNA  range from 200 nt up to many kilobases in length. LncRNAs are also further subdivided by their location within the genome. LncRNAs that are antisense to a particular protein-coding gene are antisense lncRNA and can be thought of as one subset of the now mostly outdated group of natural antisense transcripts that includes endogenous siRNA precursors . Transcrcis manner, or in a trans manner by regulating distant loci. Similar to other ncRNAs, lncRNAs can post-transcriptionally regulate genes through altering the splicing, editing, and translational efficiency of target mRNA. Thus, lncRNAs have the most diverse range of regulatory modalities of known ncRNAs , active in the SVZ, and inactive in non-neuronal cell types. The mRNA from these three preparations were utilized as a positive control and did in fact illustrate that three proneuronal protein-coding gene TSS,  pattern . Ramos eDlx1 and Dlx2, validating this application to lncRNA TSS. 583 lncRNA TSSs were discovered to follow similar bivalency in both ESCs and NSCs. Thus, this population may play a role in NSC maintenance. Further, they identified lncRNA and protein-coding TSSs that are altered in a temporal fashion following differentiation and thus may play a role in regulating TSS bivalency associated with differentiation.Following the hypothesis that lncRNA TSS bivalency may be important for lineage decision, it may also be involved in maintaining tissue-specific stem cells in a semi-differentiated state . Bivalen1. They utilized in situ hybridization to identify an lncRNA that fit this criteria, lnc-pou3f2, was expressed in the SVZ but not the OB. Another lncRNA Six3os was found to be upregulated in SVZ-NSCs but downregulated in neuroblasts, possibly indicating a role for Six3os in the maintenance of stem-cells but not neuronal phenotypes.To more conclusively link stages of adult neurogenesis within the mouse SVZ, they performed a cell sorting experiment to separate activated NSCs (GFAP+EGFR+), transit-amplifying cells (GFAP-EGFR+), and migratory neuroblasts (CD24(+) in enriched populations away from “niche” astrocytes (GFAP(+EGFR-) and profiled lineage-specific lncRNA transcripts. This combined with all of the previously described data was compiled and sorted through to find lncRNAs TSSs which were bivalently modified in ESCs, repressed in non-neuronal cells, and were active in SVZ-NSCsin vitro SVZ derived monolayer of cells from adult mice grown in culture is one such model to determine differentiation of this area in vivo. Overtime these cells within the monolayer will differentiate into glia and neurons. Within the monolayer media, researchers infused lentiviruses (LV) containing short hairpin RNA (shRNA) toward Six3os were utilized to knockout expression of this lncRNA. They found that Tuj1+ and Olig2+ cells were reduced with an increase in GFAP+ cells demonstrating that Six3os plays a role in both neuronal and oligodendrocytic lineages. They next validated Dlx1as which met similar criteria as Six3os. They found that decreased Dlx1as decreased Tuj+ neuronal lineage cells but increased GFAP+ cells without any changes in Olig2+ oligodendrocytic lineage cells. Further, they found that Dlx1as was found in the SVZ and in migratory neuroblasts within the OB. Combined, this data illustrates that the lncRNA Dlx1as plays a role in neuronal lineage cells but not oligodendrogenesis. To further determine the exact mechanism of lncRNA Dlx1as, they found that KO led to decreased neurogenic transcription factors Dlx1 and Dlx2. This demonstrates that Dlx1as may work in cis to generate its effects on Dlx1/2 expression and neuronal differentiation. Thus, these lncRNAs may be involved in the in vivo regulation of SVZ adult neurogenesis. In conclusion, Next, Frm4 may play a role in cell proliferation and apoptosis which underlie the syndrome  disrupts normal embryonic neuronal development. A mutation in the associated gene loci Fragile X Mental retardation (FMR) seems to be the originator of this syndrome. Due to FMR mutations playing a role in early neuronal development, it has been found to play a role in adult neurogenesis as well . Though syndrome . This lnthan cis . This grFrm4 on the genome-wide chromatin state. They found that nearly 430 gene promoters had differential histone methylation illustrating the various targets that may be regulated by Frm4. In OE of Fmr4, they found that 92 of these promoters had increased H3K4me3 and 86 had decreased H3K27me3 implying that these are regulated directly by Frm4. In contrast, in Frm4-KD they found that 137 promoters had decreased H3K4m3 and 115 had significantly increased H3K27me3 suggesting that these promoters are regulated indirectly by Frm4. Many of these genes were found to be involved in neuron-specific processes and included brain derived neurotrophic factor (Bdnf) and Serotonin receptor 1D (5Ht1d) whose mRNA was regulated accordingly to the state of the chromatin. Thus, Frm4 regulates a number of neuron-specific genes in trans.To further investigate this, Frm4 plays a role in human ESC proliferation which is analogous to proliferation of NPCs in the DG. Using hippocampal (h)NPCs they sought to determine the impact of Frm4 overexpression on the proliferation of these cells. They used ethynyl-2′-deoxyuridine (EdU) which is an exogenous mitotic marker and taken up during the synthesis phase (S-Phase) of these cells. They found that Frm4 OE dramatically increased the number of cells with incorporated EdU, suggesting it positively influenced proliferation. Though it is mentioned that hNPCs differentiate into a heterogenous population of neurons in various states of development, astrocytes, and renewal of hNPCs after 15 days in vitro, it was not mentioned if Frm4 altered the natural proportion of these differentiated cells. In either case, Fmr4 may play a role in regulating the rate of proliferation of progenitor cells and adult neurogenesis of NPCs.Previously, Frm4 studies by in vitro to differentiate. This can allow for precise measurement of lncRNAs in the process of lineage determination, which is another important component of adult neurogenesis. Furthermore, many other processes integral to adult neurogenesis could be regulated by lncRNAs. One such case is the migration of these newly born cells to their proper location within the brain  is alternatively spliced leading to a variety of species of α subunits with varying sensitivities to alcohol-induced changes in BK channel function , which was associated with tolerance to alcohol (http://www.targetscan.org/vert_72/ and miRBase http://www.mirbase.org/, researchers were able to rapidly predict targets of specific miRNAs (in silico to target many addiction-related gene networks and was found to be associated with motivation to consume cocaine in a rat model of self-administration (As with much of the research presented before, the first ncRNA to implicated as an important regulatory factor in many disease states is the miRNA. This is much the same case in addiction-related behaviors. The first miRNA to be associated with SUDs was miR-9 in the context of alcoholism . Large pfunction . Researc alcohol . For mos alcohol . Eipper- alcohol . With thc miRNAs . Combinec miRNAs , researcc miRNAs . For exastration . Overallstration .Homer1-AS was increased within the NAc. However, these lncRNAs were found to correlate with drug exposure or changes in addiction-related behaviors and not necessarily be involved in the development or response of these behaviors to drugs of abuse. It was not until a Gomafu KO mouse was developed did researchers have more mechanistic evidence for lncRNAs in addiction-related behaviors. Although much of this paper was focused on general locomotor hyperexcitability, these Gomafu KO mice were also found to be highly sensitive to the locomotor stimulating properties of Meth after a single injection. However, it is not known if Gomafu regulation of this behavior occurs in a specific brain region or even exclusively within the brain due to the systemic KO of this lncRNA. Additionally, it is not known which targets of Gomafu may be regulated to elicit increased methamphetamine-induced locomotor activity. Thus, lncRNAs are a promising yet understudied mechanism in SUDs.Although other ncRNAs have not been studied in addiction-related contexts, lncRNAs have only recently been profiled in addiction-related behaviors, mainly as altered in response to non-contingent drug exposure . In thisNon-coding RNAs represent a new and emerging field of regulation in adult neurogenesis. Throughout this review, the potential regulatory function of ncRNAs are discussed through the lens of specific pathways involved in adult neurogenesis. They are outlined as pathways involved in proliferation or differentiation of NPCs, migration, and integration of NPCs as they mature into functional neurons Figure . There aRO wrote the review manuscript and CM and RO read the review and made final edits.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.


This enabled us to characterize the properties of the ubiquitin-positive substrates that are necessary for the SQSTM1/p62-mediated cluster formation. Our results suggest that aggrephagy is triggered by the accumulation of substrates with multiple ubiquitin chains and that the process can be inhibited by active proteasomes.The degradation of misfolded, ubiquitinated proteins is essential for cellular homeostasis. These proteins are primarily degraded by the ubiquitin-proteasome system (UPS) and macroautophagy/autophagy serves as a backup mechanism when the UPS is overloaded. How autophagy and the UPS are coordinated is not fully understood. During the autophagy of misfolded, ubiquitinated proteins, referred to as aggrephagy, substrate proteins are clustered into larger structures in a SQSTM1/p62-dependent manner before they are sequestered by phagophores, the precursors to autophagosomes. We have recently shown that SQSTM1/p62 and ubiquitinated proteins spontaneously phase separate into micrometer-sized clusters  The accumulation of misfolded proteins is a particularly dangerous threat for the cell, and multiple systems have evolved to identify and, if necessary, degrade these proteins. In the ubiquitin-proteasome system (UPS), misfolded proteins become tagged with ubiquitin chains. They are subsequently recognized by proteasomal receptors where the ubiquitin chains are removed, the substrate is unfolded and finally degraded. When the UPS is overloaded, autophagy serves as an important backup system by clustering the ubiquitin-positive proteins into larger structures that become enclosed within autophagosomes and subsequently degraded within lysosomes. This process is also referred to as aggrephagy. The autophagy cargo receptor SQSTM1/p62 plays an important role in aggrephagy as it links the cargo material to the nascent phagophore membrane due to its ability to bind to ubiquitin and Atg8-family proteins that decorate the membrane. SQSTM1/p62 is also important for the clustering of substrate proteins into larger structures upstream of autophagosome formation.in vitro system, we have recently shown that SQSTM1/p62 and certain ubiquitinated proteins spontaneously phase separate into micrometer-sized clusters. This allowed us to conduct a detailed characterization of the properties of SQSTM1/p62 and the substrates required to trigger phase separation. We found that the reaction depends on the ability of p62 to self-assemble via its N-terminal PB1 domain and to bind ubiquitin via its C-terminal UBA domain. Phase-separation activity is enhanced by a phospho-mimicking mutation in the UBA domain that facilitates ubiquitin binding and can be stimulated by the NBR1 cargo receptor. We further found that phase separation is very sensitive to the concentration of the ubiquitinated substrates and to the length of the ubiquitin chains. In particular, we discovered that the best substrates in our reconstituted system have at least 2 ubiquitin chains with more than 3 ubiquitins attached to it. Free ubiquitin chains of 4 ubiquitins or single ubiquitin chains with 4 ubiquitins attached to a substrate do not trigger clustering. Longer chains, however, may also trigger cluster formation in our system. While we conducted the initial experiments with linear (M1-linked) ubiquitin chains, the physiologically relevant substrates are likely to harbor K48- and/or K63-linked ubiquitin chains. Therefore, we conjugated these chains to a model substrate protein, revealing that these chain types are also able to support phase separation. Interestingly, K48-linked chains appear to be the least efficient in triggering cluster formation. Since these are the main targets for the proteasome, this implies that they need to accumulate above a relatively high threshold before SQSTM1/p62 can cluster them for autophagy. In addition, we found that free K48-, and to a lesser extent K63-linked chains, inhibit phase separation. This effect is mediated by a previously unknown ubiquitin binding activity of the zinc finger domain of SQSTM1/p62, which may negatively affect oligomerization of SQSTM1/p62 itself. We also found that high concentrations of free mono-ubiquitin inhibit cluster formation. Because ubiquitin chains are thought to be released en bloc by active proteasomes and subsequently hydrolyzed to individual ubiquitins, these findings may suggest that the activity of SQSTM1/p62, and by implication that of aggrephagy, can be coordinated with proteasomal activity.How the UPS and autophagy are coordinated is largely unclear, mainly due to the lack of knowledge about the nature of the substrates that trigger SQSTM1/p62-dependent aggrephagy. In a reconstituted We then went on to study the properties of the clusters resulting from the phase separation reaction. When we conducted fluorescence recovery after photobleaching (FRAP) experiments we found that the ubiquitinated substrates display fast recovery while the recovery of SQSTM1/p62 is very slow, implying that the substrates can move freely within the clusters while SQSTM1/p62 is rather immobile. The low mobility of SQSTM1/p62 was also supported by structured illumination microscopy experiments conducted with 2 differently fluorescently labelled SQSTM1/p62 proteins. When we performed FRAP experiments with human cells expressing an endogenously GFP-tagged SQSTM1/p62 protein, we also observed low recovery of the protein in cellular puncta. We then employed negative stain electron microscopy to elucidate the structural basis for the cluster formation. It has previously been shown that purified SQSTM1/p62 exists as helical filaments and we found that these filaments coalesce in the presence of a ubiquitinated substrate, suggesting that the substrates crosslink the filaments . Supportin vitro formed clusters containing SQSTM1/p62 and the ubiquitin-positive substrates are able to recruit Atg8-family proteins. To this end, we added LC3B to the clusters and found that it is readily recruited to the clusters. Interestingly, we found that the presence of LC3B inhibits the clustering reaction to some extent. LC3B binds to the LIR motif of SQSTM1/p62, which is situated in an intrinsically disordered region of the protein. In order to test if the inhibition by LC3B is caused solely by a steric effect or if the LIR motif also directly contributes to the clustering activity of SQSTM1/p62, we tested a LIR mutant version of SQSTM1/p62 and found that this mutant shows severely reduced cluster formation. Consistently, when we introduced the LIR mutation into the endogenous GFP-tagged SQSTM1/p62 protein in human cells, we found reduced puncta formation. These results suggest that the cluster reaction might be coordinated with the formation of the phagophore membrane, on which LC3B is concentrated (We finally asked if the entrated .In summary, our study shows that SQSTM1/p62 is fully sufficient to phase separate ubiquitinated proteins into larger structures and suggests how this process can be coordinated with the activity of the UPS and autophagosome formation .