School closures as a means of containing the spread of disease have received considerable attention from the public health community. Although they have been implemented during previous pandemics, the epidemiological and economic effects of the closure of individual schools remain unclear.This study used data from the 2009 H1N1 pandemic in Hong Kong to develop a simulation model of an influenza pandemic with a localised population structure to provide scientific justifications for and economic evaluations of individual-level school closure strategies.The estimated cost of the study’s baseline scenario was USD330 million. We found that the individual school closure strategies that involved all types of schools and those that used a lower threshold to trigger school closures had the best performance. The best scenario resulted in an 80% decrease in the number of cases , and the cost per case prevented by this intervention was USD1,145; thus, the total cost was USD1.28 billion.This study predicts the effects of individual school closure strategies on the 2009 H1N1 pandemic in Hong Kong. Further research could determine optimal strategies that combine various system-wide and district-wide school closures with individual school triggers across types of schools. The effects of different closure triggers at different phases of a pandemic should also be examined. Non-pharmaceutical interventions are practical methods for containing the spread of an emerging infectious disease; they are especially important during the early stages of a pandemic, as antivirals may not effectively treat new outbreaks and vaccination supplies usually require several months to develop . Among tPrevious studies have revealed that school closures are usually most effective when they result in a large reduction in contacts, when there is a low basic reproduction number , and when attack rates in children are higher than in adults . Other sOn the other hand, school closures also inevitably cause social disruptions and incur substantially higher costs than the unmitigated baseline , especiaModelling population dynamics at the more-detailed community level opens up opportunities to examine the effects of regional-level intervention strategies. Our study uses data drawn from the 2009 influenza pandemic in HK to evaluate the cost-effectiveness of various school closure strategies, particularly the closure of individual schools.The 2009 HK influenza H1N1 pandemic has provided us with great opportunities to understand the disease’s epidemiology and the effectiveness of a range of interventions on containing outbreaks –26. PrevWe developed an age-structured region-specific Susceptible-Exposed-Infected-Removed (SEIR) compartmental model  to mimicWe modelled various combinations of school closure strategies, and for each we examined the effects of school types closed, closure modes, closure triggers, and closure length. The simulation model ran various system-wide, district-wide, and individual-based school closure strategies and was set to close schools of various levels , kindergarten and primary (KP), all schools types including secondary (KPS), or any combination of those). The detailed study outcomes for the system-wide and district-wide school closures have been reported previously . The curWe attempted to incorporate realistic scenarios that would be feasible, potentially implementable, and practical into our school suspension strategies. According to a document discussing Hong Kong’s preparedness for influenza pandemic prevention and protection, the closure of schools, as a social distancing measure, may be instituted to reduce population mobility and related social contacts . Althougnumber of confirmed cases at a particular school (NCP): 1, 3, 5, 10, and 20. In the past, school closures have lasted from 1 week to several months  was achieved for the baseline scenario. The infection attack rates in each age group fell within the 95% CIs of the references values . A basicpandemic . The detpandemic . In the We first present the simulated overall attack rates for each scenario. Similar experiments were carried out previously on system-wide and district-wide school closures using different triggers . A schoofirst case is found with an onset of two weeks (FCF2-KPS); system-wide school closure once the overall number of confirmed cases reaches 200 (NC200-KPS); and district-level school closure when the number of confirmed cases in the district reaches 100 (NDC100-KPS). Our study predicted that closing individual schools would delay the epidemic peak under all of the school closure scenarios. As depicted in All of the scenarios with a school closure length of one week were compared using epidemic curves. An epidemic curve illustrates the number of confirmed H1N1 cases over time. We compared the curve of the baseline scenario with those of the mitigation scenarios to quantify the delays in the infectious disease spread. We selected the best-performing individual school closure scenario for each school type—which turned out to be NCP3-K, NCP3-KP, and NCP3-KPS. To highlight the effect of individual school closure strategies, we studied the best scenario based on other school closure strategies , includiA school closure strategy is a non-pharmaceutical intervention that can be implemented at the early phase of a pandemic. It inevitably incurs high costs as it disrupts usual societal practices so that parents may need to stay home to take care of school-age children. As a result, it is important to investigate the cost-effectiveness of different school closure scenarios so that an economically viable strategy that considers the relative costs and outcomes can be devised. Therefore, this study analysed the cost-effectiveness of various individual-based school closure strategies. We included the best scenarios from all of the closure types  and compared the results among a variety of individual school closure scenarios. Based on the proposed cost structure discussed in the economic model section, we calculated the cost of each intervention (as well as the baseline case). The cost of the baseline scenario for this city of 6.9 million people was estimated to be HKD2.55 billion (95% CI: +/- HKD4 million) or USD330 million (CI: +/- USD0.5 million); this compares with a baseline cost of about USD75 million for a North American city with a population of 650,000 people [As shown in Referring to , the basThe above results show that individual school closure strategies appear to effectively hinder the spread of disease. Based on the cost-effectiveness analysis , individCompared to the baseline case, the selected system-wide and district-wide cases achieved a meaningful drop in overall attack rates, but incurred high costs. The individual school closure strategies for kindergartens (NCP1-K-SCL1) and kindergartens and primary schools (NCP1-KP-SCL1) had slightly lower costs than individual school closure scenarios that included all school types (such as NCP1-KPS-SCL1), but the resulting overall attack rates of the former two were much higher than for the latter. Thus, closing all types of schools based on individual thresholds was found to be more cost-effective than strategies that closed only kindergartens or only kindergartens and primary schools. As not all school closure lengths decreased overall attack rates, school closure modes and triggers are important when choosing a strategy. Among the individual-based KPS scenarios, closing schools for longer periods of time was more effective, but inevitably more expensive than shorter scenarios. Furthermore, scenarios with small trigger values  were more cost effective than other scenarios.All of the intervention scenarios reduced the expected number of infected people  and the expected number of influenza deaths compared to the baseline. As a result, both the medical costs associated with illness and the costs associated with staying at home with sick children were reduced. The additional costs associated with the interventions are the costs of parents and children staying home due to school closure. Taking NCP3-KPS-SCL2 as an example, both the medical costs associated with illness and the cost associated with staying home with sick children were reduced by 80% compared to the baseline, and the cost per case prevented by the intervention was USD1,145. Although this was not the cheapest school closure strategy, it achieved the best effectiveness in terms of preventing infected cases—it resulted in an 80% decrease in patients, which is equivalent to the prevention of about 830,000 cases in a city of 7 million people.This study demonstrated that the effects of individual school closure strategies depend on (i) school types closed, (ii) closure modes, (iii) closure triggers, and (iv) closure lengths, particularly for individual school closure strategies. Perlroth et al.  commenteWe made several assumptions in this study. First, school vacations or seasonality were ignored within the simulation timeframe. Second, we assumed that school closures would not increase the per-contact probabilities of other social contact groups, such as family, household, and community. In fact, students may increase visits to public venues during school closures  and may There were scattered school suspensions throughout the HK H1N1 pandemic. We examined various strategies based on realistic scenarios that were feasible, potentially implementable, and practical. Our study provided cost-effectiveness analyses of various individual-based school closure scenarios and attempted to balance the epidemiologic and economic benefits of such interventions. Public health policy makers have demonstrated the ability to identify the number of confirmed infectious cases in schools . We foreThis paper evaluated via simulation the cost-effectiveness of various individual-based school closure intervention strategies. The estimated cost incurred in the 2009 Hong Kong H1N1 pandemic of USD330 million is used as a baseline. Our study revealed that closing individual schools based on the number of confirmed cases at the schools using small thresholds, leads to the greatest reduction in peak and cumulative ARs and is the most economically viable strategy. Such strategies could result in an 80% decrease in cases, preventing about 830,000 infections; the cost per case prevented due to intervention was estimated as USD1,145.S1 FileTable A). Cost analysis model parameters (HKD) (Table B). Probability distributions of critical cost parameters (Table C). Probabilistic sensitivity analysis (NCP3_KPS_SCL2 vs. baseline) . Probabilistic sensitivity analysis (FCF2_KPS_SCL2 vs. baseline) . Probabilistic sensitivity analysis (NC300_KPS_SCL8 vs. baseline) . Probabilistic sensitivity analysis (NDC100_KPS_SCL1 vs. baseline) .Age-stratified healthcare decision model ((DOCX)Click here for additional data file.


C-terminal extension that seems to be important for their correct recognition by the vacuole sorting machinery. The 7 amino acids long C-terminal vacuolar sorting determinant (CtVSD) of tobacco chitinase A is necessary and sufficient for the transport to the vacuole. This VSD shares no homology with other CtVSDs such as the phaseolin’s tetrapeptide AFVY  and it is also sorted by different mechanisms. While a receptor for this signal has not yet been convincingly identified, the research using the chitinase CtVSD has been very informative, leading to the observation of phenomena otherwise difficult to observe such as the presence of separate vacuoles in differentiating cells and the existence of a Golgi-independent route to the vacuole. Thanks to these new insights in the endoplasmic reticulum (ER)-to-vacuole transport, GFPChi (Green Fluorescent Protein carrying the chitinase A CtVSD) and other markers based on chitinase signals will continue to help the investigation of vacuolar biogenesis in plants.Plant chitinases have been studied for their importance in the defense of crop plants from pathogen attacks and for their peculiar vacuolar sorting determinants. A peculiarity of the sequence of many family 19 chitinases is the presence of a  N-acetylglucosamine of chitin, the major cell wall structural polysaccharide of many fungi. This activity can evidently contribute to the defenses against fungal pathogens.Over the past few decades, glycosyl hydrolases of families 18 and 19, commonly known as chitinases (EC 3.2.1.14), have been studied for their importance in the defense of crop plants from pathogen attacks and for their peculiar vacuolar sorting determinants. While family 18 chitinases are found in all organisms, family 19 chitinases are mostly found in plants, although they have been found in a few animals, fungi, protists and in many bacteria. Until January 2014, 3374 chitinase DNA sequences were present in the NCBI database while the Pfam database revealed for Viridiplantae 569 sequences from 111 species for family 18 and 1047 sequences from 204 species for family 19 chitinases. They catalyze the hydrolysis of internal β-1,4-bonds between monomers of Unfortunately, the chitinases in cultivated plants can also have negative effects on human health since they may trigger allergic responses. Chitinases are major allergens in several food plants like banana, avocado or apple, as well as in rubber. Like many allergens, they are resistant to proteolytic degradation in the acidic environment of the stomach. This resistance favors entry into the bloodstream triggering the allergic response. The use of heterologous chitinases to engineer pathogen-resistant crop plants requires that the proteins are suitably modified to be optimized for pathogen resistance as well as to be properly hydrolyzed in an acidic environment .Full understanding of the post-translational modifications (PTMs) of the engineered transgenic product is necessary both for the development of plants over-expressing defense proteins, and for the increase of nutritional value. Glycosylation certainly possesses a considerable importance, being the most abundant PTM in plant cells  and the C-terminus is extended along the surface in such a way that additional residues would stick out over the catalytic cleft [C-terminal propeptides found in many vacuolar class I chitinases are easily accessible to the sorting machinery as well as easily removed by endo- or exo-peptidases.Family 19 chitinases have been subdivided in seven classes depending on the presence of a chitin-binding domain and on the presence of four loops within the catalytic domain. X-ray structures of several plant class I and II chitinases indicate a compact fold of the catalytic domain where the ic cleft ,6. It isC-terminal 7 amino acids long propeptide of tobacco chitinase A is both necessary and sufficient for the transport to the vacuole [C-terminal propeptide to enzymatic or fluorescent reporters (rat β-glucuronidase and Green Fluorescent Protein (GFP)) confirmed these early evidences [C-terminal VSDs (CtVSD) have been demonstrated to be involved in vacuolar sorting: a barley lectin [The vacuolar sorting determinant (VSD) residing in the vidences ,9. Only y lectin , a β-1,3y lectin ,12,13, 2y lectin , pea [15y lectin .C-terminus for the sorting machinery is stressed by the fact that the C-terminal Met of chitinase A could be replaced by a Phe or a Lys or even deleted, but not replaced by a Gly [C-terminal addition of an N-glycosylation site caused a total secretion of the lectin [Contrary to ssVSDs , no specific sequence motif is needed for vacuolar sorting and different amino-acid substitutions led to different efficiencies of delivery to the vacuole. The importance of the by a Gly . Similare lectin .C-terminal targeting signal, the tetrapeptide AlaPheValTyr (AFVY) from bean phaseolin [The tobacco chitinase VSD has been less widely used than another haseolin . There ihaseolin . The reshaseolin , the prehaseolin  and the haseolin .Vacuoles are multifunctional organelles showing a high plasticity according to physiological/environmental needs ,21. The Arabidopsis thaliana root tips using trafficking inhibitors [de novo biogenesis of a lytic vacuole had already been suggested long ago [Arabidopsis plants expressing either marker, it could be seen that in nascent and expanding root hairs the central vacuole was labeled by AleuGFP, while GFPChi was excluded, while in fully expanded root hairs the latter marker also made it to the central vacuole [A recent electron microscopy study of hibitors  supportslong ago ,34. The long ago , when uslong ago . This st vacuole . This ob vacuole  showing  vacuole  was fillArabidopsis plants accumulating anthocyanins, Brefeldin A (BFA) treatment did not alter the trafficking of either anthocyanins or GFPChi while it affected the trafficking of AleuGFP [Nicotiana [trans-Golgi Network (TGN) but involved in the late events of BFA bodies formation.In  AleuGFP . Similar AleuGFP . The dru AleuGFP ) but it  AleuGFP . Recent icotiana , indirecicotiana , indeed vps45 null mutant, the vacuole forms small concentric multilayer compartments stressing the involvement of post-Golgi trafficking in provacuole release and/or provacuole fusion [N-ethylmaleimide-sensitive factor associated protein receptor) complex, composed of VAMP727, SYP22, VTI11, and SYP51. In fact it is known that the absence of both SYP22 and VAMP727 lead to vacuole fragmentation [Other studies have however convincingly argued that GFPChi follows the canonical pathway through the Golgi–TGN–PVC (PreVacuolar Compartment). The functional studies of AtVPS45  and AtVTe fusion  and openentation . Over-exentation ,44. SYP5entation  and may entation , which aentation , multiveArabidopsis based on the homology of its PA (Protease Associated) domain, with the PA domain, which is a part of the cargo-binding luminal domain of VSRs. In vitro binding assays of the luminal domain of RMRs have demonstrated an affinity for CtVSDs and not for ssVSDs, supporting their role as vacuolar sorting receptors for CtVSDs involved in sorting to the storage vacuole [in vitro binding assays of AtRMR1 with phaseolin also support an in vivo interaction between proteins with a CtVSD and some members of the RMR family [A main issue is that putative vacuolar sorting receptor(s) for CtVSD proteins have been identified, but their sorting mechanism remains unclear. The CtVSD are not specifically recognized by the well-characterized Vacuolar Sorting Receptors (VSRs), which have a strong affinity for ssVSD ,48. Thes vacuole ,51,52. CR family ). GFPChiR family ,55. The R family ), in theR family , in a neThe unclear results about sorting of markers with the chitinase A CtVSD suggests the existence of two different pathways for the chitinase: a classical pathway passing via the Golgi and the TGN and an alternative pathway in which soluble cargoes exit the ER by another Sar1-independent mechanism, delivering cargoes to the vacuoles without passing through the Golgi . This paIn vivo observation of tobacco . In vegetative tissues these dense vesicles do not seem to exist. How CtVSD proteins are sorted in these tissues remains to be determined. Combined with the use of new pathway selective inhibitors [ tobacco  or Arabihibitors , GFPChi


Depression is a common but often undiagnosed comorbid condition of people with diabetes. Mass screening can detect undiagnosed depression but may require significant resources and time. The objectives of this study were 1) to develop a clinical forecasting model that predicts comorbid depression among patients with diabetes and 2) to evaluate a model-based screening policy that saves resources and time by screening only patients considered as depressed by the clinical forecasting model. We trained and validated 4 machine learning models by using data from 2 safety-net clinical trials; we chose the one with the best overall predictive ability as the ultimate model. We compared model-based policy with alternative policies, including mass screening and partial screening, on the basis of depression history or diabetes severity.Logistic regression had the best overall predictive ability of the 4 models evaluated and was chosen as the ultimate forecasting model. Compared with mass screening, the model-based policy can save approximately 50% to 60% of provider resources and time but will miss identifying about 30% of patients with depression. Partial-screening policy based on depression history alone found only a low rate of depression. Two other heuristic-based partial screening policies identified depression at rates similar to those of the model-based policy but cost more in resources and time.The depression prediction model developed in this study has compelling predictive ability. By adopting the model-based depression screening policy, health care providers can use their resources and time better and increase their efficiency in managing their patients with depression. Clinical forecasting analyzes current and historical facts to predict clinical outcomes. Such forecasting has important applications for underdiagnosed conditions such as comorbid depression among patients with diabetes ,2, who aMachine learning methods can automatically detect patterns in data and use the patterns to predict future data . MachineWe developed the PreDICD model by using machine learning methods. Then, we compared the model-based screening policy with mass screening to evaluate the policy’s influence on provider resources and time and on the rate of depression identification. We also compared the model-based policy with 3 heuristic-based partial screening policies that assign depression screening to patients with certain risk factors (including depression history or severe diabetes or both) and assessed the implications for provider’s choice of depression screening policy.Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-4). Each question has 4 ordinal responses with assigned scores from 0 to 3; therefore, the overall scale has a possible score from 0 to 27, where the higher scores indicate more severe depression. PHQ-2 consists of the first 2 PHQ-9 questions. PHQ-2 often serves as a fast screening tool  and the Multifaceted Diabetes and Depression Program (MDDP). DCAT is a comparative effectiveness study conducted from 2010 through 2013 in safety-net clinics in the Los Angeles County Department of Health Services (LACDHS), the second largest safety-net healthcare system in the United States. DCAT tested an automated telephone depression screening and monitoring system integrated with a collaborative care management program to facilitate adoption of a collaborative depression care model ,19. MDDPThe combined data sets provided the important benefit of balancing the proportions of depressed  and nondepressed patients . In a prior analysis , we inveWe identified 20 candidate predictors from the combined DCAT–MDDP data in accordance with 2 criteria: 1) the candidate predictors were relevant to diabetes care and measure aspects that were supported by 2 prior systematic reviews ,8 as beiCorrelation-Based Feature Selection for Machine Learning. Hamilton : The University of Waikato; 1999) that searches predictors by greedy hill-climbing algorithm and targets to select a subset of predictors that are highly correlated with the outcome measure while having low intercorrelation. This predictor selection procedure was carried out by machine learning software, Weka, version 3.6.11 (Slashdot Media).From the candidate predictors we selected predictors for developing the PreDICD model. Available selection methods were variable ranking, subset evaluation, and the wrapper method . For thihttps://cran.r-project.org/bin/windows/base/old/3.1.1/), by using the whole data set.To derive the appropriate model, we trained and cross-validated (10-fold) 2 linear machine learning models, logistic regression (with Ridge parameter to improve predictive ability ) and mulThe PreDICD model can support a model-based screening policy that assigns depression screening only to patients predicted by the model to be depressed. We compared the model-based policy with mass depression screening to evaluate the influence of model-based policy on provider resources and time and on the rate of depression identification. In addition, we compared the model-based policy to 3 heuristic-based partial screening policies used by providers to save resources and time. The first heuristic, which requires depression screening for patients with a previous diagnosis of major depressive disorder, is based on the fact that depression is a highly recurrent disease . The secWe evaluated the model-based policy and compared it with mass screening and 3 heuristic-based policies under the clinical context that PHQ is used for depression screening. We assumed the scenario in which patients meeting screening policy inclusion criteria were evaluated using the 2-step PHQ screening suggested by Kroenke et al : PHQ-2 iTo evaluate the model-based policy, we trained the PreDICD model on the combined DCAT–MDDP data; however, we cross-validated (10-fold) only the DCAT data. That is, we randomly divided the samples from DCAT into 10 roughly equal parts. In each single round of validation, samples from 9 of the 10 parts of DCAT data plus samples from MDDP were used to train the prediction model; we then validated the trained model on samples from the remaining data. Mass screening and the 3 heuristic-based policies were also evaluated only on the DCAT data. Because the DCAT data included data on both depressed and nondepressed patients, they represented the LACDHS safety-net population better than the MDDP data. All comparisons were 2-sided and carried out by statistical software R, version 3.1.1.We identified 1,793 patients from the combined DCAT and MDDP data. The MDDP trial enrolled only depressed patients with diabetes (PHQ-9 ≥10 ), and the DCAT trial enrolled both depressed  and nondepressed  patients with diabetes . The comWe used a correlation-based subset evaluation predictor selection method for the PreDICD model to select 7 predictors that are highly correlated with major depression and have low intercorrelation: 1) female, 2) Toobert diabetes self-care, 3) total number of diabetes complications, 4) previous diagnosis of major depressive disorder, 5) number of ICD-9 diagnoses in past 6 months, 6) chronic pain, and 7) self-rated health status.We trained 4 machine learning models  by using the 7 selected predictors. On the basis of the 10-fold cross-validation results, we chose logistic regression as the ultimate PreDICD model because it outperformed the other 3 models in AUROC . The logistic regression model also had the highest percentage of correctly classified instances of depression  and sensitivity , and the second highest specificity  among the 4 models.P < .001), total number of complications from diabetes , a history of major depressive disorder , number of comorbidities, measured by the number of ICD-9 diagnoses in previous 6 months , and chronic pain (OR = 2.13 P < .001). Two predictors decreased the likelihood that the patient would be depressed: good diabetes self-care, measured by Toobert diabetes self-care , and self-rated good health status .The predictors of depression used for the PreDICD model are listed in The policy that assigns 2-step PHQ screening only to patients predicted by the PreDICD model as being depressed was compared with mass screening and with 3 heuristic-based partial screening policies. Results  show thaThe heuristic-based policy that assigned 2-step PHQ screening to patients with a previous diagnosis of major depressive disorder could identify only about 20% of depressed patients. Compared with the model-based policy, the other 2 heuristic-based policies had insignificantly different rates of depression identification but cost significantly more in provider resources and time.A comparison of the model-based depression screening policy using 1-step PHQ-9 with mass PHQ-9 screening  revealedThe PreDICD study developed a clinical forecasting model predicting the occurrence of depression among patients with diabetes by using data from 2 clinical trials. The study considered 20 candidate predictors and compared 4 machine learning models: logistic regression, multilayer perceptron, SVM, and random forest. The ultimate PreDICD model is logistic regression, with 7 predictors in the model: 1) female, 2) Toobert diabetes self-care, 3) total number of diabetes complications, 4) previous diagnosis of major depressive disorder, 5) number of ICD-9 diagnoses in previous 6 months, 6) presence of chronic pain, and 7) self-rated health status. Five of the 7 predictors typically can be acquired from EMR: female sex, total number of diabetes complications, previous diagnosis of major depressive disorder (ICD-9 diagnosis codes 296.2 and 296.3), number of ICD-9 diagnoses in previous 6 months, and presence of chronic pain (ICD-9 diagnosis code 338.2). Diabetes treatment guidelines recommend that health care providers collect data on 2 of the predictors during clinic visits: Toobert diabetes self-care scale, because most of the day-to-day care inherent in diabetes is handled by patients or their families , and selThree prior studies also predicted the occurrence of depression on the basis of health-related data. King et al  developeThe model-based screening policy that assigns depression screening only to patients predicted as being depressed by the PreDICD model can improve efficiency in identifying depressed patients with diabetes compared with mass screening . Such a finding is an encouraging step toward implementing a decision-support system based on available medical information that allows providers to better prioritize the use of resources and time.As health delivery systems increasingly take on responsibility for managing population health, model-based screening can help providers reach out to patients who are identified as at-risk by the model. For example, the National Committee for Quality Assurance’s standard requires patient-centered medical homes to provide depression screening . The PreThis study has several limitations. The PreDICD model combines 2 data sets with somewhat different populations recruited at different times and does not account for possible cohort and period effects on the health conditions of the study populations. Study patients were predominantly Hispanics from the safety-net population with diabetes, which may limit the generalizability of the PreDICD model to wider patient populations because underlying determinants of depression may differ by racial/ethnic group . CullingFuture work should validate and refine the PreDICD model for broader patient populations to improve its generalizability. Also, research to extend the PreDICD model from predicting current depression to forecasting future depression could help health care providers to identify patients with diabetes who are at high future risk of depression and thus warrant repeated depression screening. The model could alternatively be extended from single-level to multilevel logistic regression to account for possible cohort and period effects, and thus improve the model’s predictive ability. The model should also be tested in a clinical environment to verify the feasibility of implementing a decision-support system and to evaluate its influences on clinical outcomes and operations, including costs and cost-savings. Finally, the machine learning methods demonstrated in the study can be applied to predicting clinical outcomes related to other conditions and could be useful in future research initiatives, such as the National Institutes of Health’s recently launched Precision Medicine Initiative .Our PreDICD study developed a prediction model with compelling predictive ability for forecasting comorbid depression among patients with diabetes. Adopting such a model-based policy has the potential to outperform other heuristic approaches by better assisting health care providers to increase efficiency in managing their patients with depression and better prioritize the use of their resources and time to deliver effective care for high-risk patients.


Lung adenocarcinoma is often diagnosed at an advanced stage with poor prognosis. Patients with different clinical outcomes may have similar clinico-pathological characteristics. The results of previous studies for biomarkers for lung adenocarcinoma have generally been inconsistent and limited in clinical application. In this study, we used inverse-variance weighting to combine the hazard ratios for the four datasets and performed pathway analysis to identify prognosis-associated gene signatures. A total of 2,418 genes were found to be significantly associated with overall survival. Of these, a 21-gene signature in the HMGB1/RAGE signalling pathway and a 31-gene signature in the clathrin-coated vesicle cycle pathway were significantly associated with prognosis of lung adenocarcinoma across all four datasets . We combined the scores for the three pathways to derive a combined pathway-based risk (CPBR) score. Three pathway-based signatures and CPBR score also had more predictive power than single genes. Finally, the CPBR score was validated in two independent cohorts (GSE14814 and GSE13213 in the GEO database) and had significant adjusted hazard ratios 2.72  and 1.71 , respectively. These results could provide a more complete picture of the lung cancer pathogenesis. Lung cancer, especially non-small cell lung cancer (NSCLC), is the most common cause of cancer-associated mortality worldwideEGFR and KRAS and gene fusion detection of ALK have been widely used in clinical practiceThe current clinico-pathological staging system is not adequate5Although previous gene expression microarray studies have shown a statistically significant association of the expression of many individual genes with disease, the findings usually lack biological meaning12Classification of patient risk using a single biomarker that is strongly associated with disease outcome might not be a good strategy, as a dysregulated gene that may not show any obvious association with disease on its own may interact with others in the same pathway, resulting in carcinogenesis or drug resistance1415CSNK1A1 and MYST4) had significant HRs in all four datasets. After combining the results for the four datasets, a total of 2418 genes showed a significant HR (data not shown). When pathway mapping was used to identify survival-associated biological pathways based on these 2418 genes, 15 pathways in which these genes were enriched were identified (The study population consisted of 443 patients with lung adenocarcinoma from the University of Michigan Cancer Center (UM) n = 178), the Moffitt Cancer Center (HLM) (n = 79), the Memorial Sloan-Kettering Cancer Center (MSK) (n = 104) and the Dana-Farber Cancer Institute (CAN/DF) (n = 82)8, the Moentified .To evaluate the impact of signatures in a given pathway on survival, a pathway-based risk score was calculated for this pathway and used, together with survival analysis, to evaluate prognostic ability. Survival analysis showed that three pathways were significantly associated with survival in all four datasets . Twenty-/RAGE signalling pathway-based signature containing 21 differentially expressed genes and defining high-risk patients as those with a pathway-based risk score higher than the median, high-risk patients had a significantly shorter median survival than low-risk patients in the CAN/DF dataset , the HLM dataset , the UM dataset  and the MSK dataset .As shown in Similar results were obtained using the beta-adrenergic receptor regulation of the ERK pathway-based signature  or the cThe survival pattern for patients with each of the three pathway-based signatures in the different datasets was interesting. In the CAN/DF dataset, the high-risk group, based, respectively, on the HMGB1 / RAGE signaling pathway, beta-adrenergic receptor regulation of ERK pathway and clathrin-coated vesicle cycle pathway, had a median survival of 71, 38 and 37 months, while the low-risk group did not reach median survival. In the HLM dataset, the high-risk group had a median survival of about 20 months , while that of the low-risk group was greater than 45 months . In the UM dataset, the median survival for the high-risk group was 42, 45 and 48 months, almost 3 times lower than that in the low-risk group . In the MSK dataset, the median survival for the high-risk group was around 50 months , while the low-risk group survived longer .To evaluate the prognostic effect of the combined risk scores for these three pathway-based signatures, a CPBR score was computed by linear summation of each of the three pathway-based signature scores multiplied by the weighting coefficient for that pathway obtained by Cox proportional hazards regression. Patients in the high-risk group (risk score higher than the median) had a significantly shorter median survival than those in the low-risk group in all four datasets  .Results of multivariate Cox proportional hazards regression showed that stage effect had significant adjusted HR in each dataset and can be mentioned as an independent prognostic factor . After eMultivariate Cox proportional hazards regression was used to examine the predictive power of 2418 genes selected from the inverse-variance weighting method. Numbers of significant genes in four datasets are 820, 574, 429, and 622, respectively. Range of significant HRs was from 0.05 to 11.3 and had high variation between four datasets . ConsideThree pathway-based signatures and the CPBR score were validated in two independent cohorts (GSE14814 and GSE13213). Patients with high risk identified from the HMGB1 / RAGE signalling pathway, the beta-adrenergic receptor regulation of the ERK pathway, the clathrin-coated vesicle cycle pathway, or CPBR score had significant shorter overall survival , age, three pathway-based signatures, and CPBR score had significant HRs. Adjusted HR of CPBR score was 2.72 (95%CI = 1.80 to 4.11 and p < 0.0001). In addition, stage, three pathway-based signatures, and CPBR score had significant HRs in the second validation cohort (GSE13213). Adjusted HR of CPBR score was 1.71(95%CI = 1.45 to 2.03 and p < 0.0001) .In this study, multivariate Cox proportional regression analysis was used to identify potential survival-associated genes in each of the 4 National Cancer Institute datasets, then inverse-variance weighting method was used to combine the results from the four datasets. The use of this method increases the statistical power and provides more robust resultsCSNK1A1 and MYST4, were found to be significantly associated with survival in all four datasets. Since the results for the four datasets were not very consistent, we used inverse-variance weighting method to combine the four sets of results to increase statistical power. After pathway analysis, pathway-based risk scores were computed using the level of expression of genes in the same pathway weighted by regression coefficients. Each of the pathway-based scores was found to be a good predictor of clinical outcome. Subjects with a higher pathway-based risk score for a given pathway were classified into the high-risk group based on that pathway. The results showed that 21 of the 65 genes in the HMGB1 / RAGE signalling pathway, 22 of the 70 in the beta-adrenergic receptor regulation of ERK pathway and 31 of the 107 in the clathrin-coated vesicle cycle pathway were significantly associated with clinical outcome of lung cancer. The high-risk group defined using the risk score based on any one of the three individual pathways had a shorter overall survival than the low-risk group.In the Cox regression analysis, only two genes, We then developed a simplified CPBR score to combine the effects of these three signatures, and the results showed that the high-risk group had even shorter overall survival than the low-risk group and that the CPBR score gave a better outcome prediction of outcome of lung adenocarcinoma patients. These results showed that differentially expressed genes in the same pathway might interact with each other and contribute to a worse prognosis.Some studies have reported that cellular pathway signatures can be useful for treatment development, prognosis prediction and subtype classification in lung cancer1418162227In studies using the same strategy and similar clinical and pathological features and treatment protocol, not all patients with lung cancer show the same clinical outcome and sensitivity to treatment because of the extreme heterogeneity of tumoursThe limitation of the gene expression profiling or pathway based approach is applications in the clinical practice. The IHC method or FISH method with convenient and low cost of assays is well used in the clinical practice. However, it can be only applied in testing few genes. Several technologies including the multiplexed quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) or digital PCR may provide solutions for the clinical practice of the pathway based signatures. However, it still needs to be evaluated in the future study.Gene-expression profiling has been used to explore biomarkers associated with subtypes of lung cancer, overall survival and recurrence of cancer306323316Through risk assessment by pathway-based signatures, patients had high risk estimated from pathway based signatures may need to receive different treatments. For example, patients with completely resected stage I NSCLC was recommend for no adjuvant chemotherapySeveral limitations of the microarray data are also needed to be considered. First, the high variations of gene expression data were obtained from different microarray platforms due to different probe designs or signal detection methods. Hence, the comparisons between different microarray platforms have to be concerned. Second, the stromal cell contamination varies among dataset and samples, which would give variations in micro array-based analyses.EGFR, KRAS, and ALK fusion. It is difficult to estimate the effect of the CPBR in patients with major genomic abnormalities. Additionally, the MetaCore software used enrichment analysis to find the overrepresented pathways and pathways with the smaller size of the gene set might have more chance to reach significance. It is the weakness of the enrichment analysis. Because the false positive may be introduced from the limitation of the MetaCore software, several steps were used to reduce the bias. In the first step, the Bonferroni correction method was applied to reduce potential false positive. There were more than 1000 pathways in the database of the MetaCore. The significant level was corrected from 0.05 to 10−5. In the second step, gene-signatures of significant pathways were evaluated in four training datasets. Pathways had significant associations with survival were kept. Finally, candidate pathway-based signatures were validated in two independent cohorts.A potential limitation of this study is that the public datasets used in this study did not provide the information of treatment response and major genomic abnormalities such as Our analysis method of gene expression profiling identified pathway-based signatures closely correlated with clinical outcome of lung adenocarcinoma. We have derived a simple CPBR score which may improve the accuracy of outcome prediction for lung adenocarcinoma, as the CPBR score showed a higher correlation with clinical outcome than gene- or individual pathway-based scores. These results provide a more complete picture of the pathogenesis of lung carcinoma and provide direction for future studies. The CPBR signature may be useful in stratifying subpopulations of lung adenocarcinoma for clinical outcome prediction and individualized therapies for lung adenocarcinoma patients.https://array.nci.nih.gov/caarray/project/details.action? project.id= 182.The study population consisted of 443 patients with lung adenocarcinoma from the University of Michigan Cancer Center (UM) n = 178), the Moffitt Cancer Center (HLM) (n = 79), the Memorial Sloan-Kettering Cancer Center (MSK) (n = 104) and the Dana-Farber Cancer Institute (CAN/DF) (n = 82)8, the MoGSE14814 in the GEO database, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE14814) and dataset (n = 117) from the Nagoya University in Japan  were used to validate our findings.An additional published dataset (n = 71) from the University of Toronto , and the corresponding p value was estimated by Cox regression analysis, giving 4 HR and 4 p values for each gene, then the inverse-variance weighting method was used to combine the HRs from the four datasetsTM version 6.13 . Because more than 1000 pathways are included in the MetaCore database, the Bonferroni correction was used to avoid multiple testing issues. For each of the 15 pathways found to contain differentially expressed genes, a pathway-based risk score was calculated for each subject in each data set to determine the impact of the pathway on the prognosis. The risk score was a linear combination of the values for the expression of each gene in the pathway multiplied by a weighting value for each gene estimated using Cox proportional hazards regression. Using this approach, we found signatures in three pathways that were significantly associated with survival in lung adenocarcinoma. A combined pathway-based risk (CPBR) score was then derived by linear combination of the pathway-based risk score for each of the three pathways multiplied by a weighting value for that pathway estimated using Cox proportional hazards regression. We then verified this CPBR score using an independent cohort.Genes that were differentially expressed and associated with survival were further analysed for biological function or involvement in different pathways using pathway maps of the MetaCoreIn the pathway-based study, the pathway-based median risk score was taken as the cut-off point for high or low-risk group classification, while, in the CPBR score study, the median of the CPBR score was used as the cut-off. The Kaplan-Meier method was used to generate survival curves and the difference between survival curves was evaluated using the log-rank test. All tests were two-tailed, and p values less than 0.05 were considered to be significant.Both survival analysis and calculation method of pathway-based scores for three selected pathways and the CPBR score were the same as previous section described. HR of the CPBR score was estimated from multivariate Cox proportional hazards regression with the clinical covariates of age, sex, stage, and treatment method after operation.How to cite this article: Chang, Y.-H. et al. Pathway-based gene signatures predicting clinical outcome of lung adenocarcinoma. Sci. Rep. 5, 10979; doi: 10.1038/srep10979 (2015).


The TALEN and CRISPR/Cas9 nuclease systems have been extensively utilized in genomic engineering of model organisms. In zebrafish, the nuclease systems have been successfully applied in generating loss-of–function knockout lines. However, genome-specific knockin techniques in zebrafish are still at the beginning. In this perspective, we briefly summarize the recent progresses on knockin approaches in zebrafish with a special focus on the newly developed intron-based knockin method. LoxP insertions at interested genomic loci to generate conditional knockout animals [in vivo [Knockin animals are versatile tools for biological research . For exa animals . Knockin[in vivo . For neu[in vivo .Danio rerio) is an emerging vertebrate animal model for life science. Although loss-of-function genomic editing for zebrafish mediated by zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or the type II bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system (CRISPR/Cas9) has been developed [Zebrafish  to a specific genome loci was still challenging. It was reported that the EGFP sequence was correctly integrated at the zebrafish tyrosine hydroxylase (th) locus through TALEN-mediated double-strand breaks (DSBs) and HDR, and the germline transmission efficiency was about 1.5% [th gene was destroyed and the inserted EGFP failed to express [Previously, although HDR-mediated knockin strategies were used to insert short DNA sequences and repair mutations in the zebrafish genome , 12, theout 1.5% . However express  . This suggests that the endogenous 3′ UTR in the knockin donor is critical for the expression of the targeted exogenous gene. For the donor construction, based on our experience, the length of arms does not matter, as long as the arm sequence meets the following requirements: 1) sgRNA must be included in the intron part of the left arm. 2) 3′ intergenic region containing the full 3′UTR sequence of the mRNA must be included in the right arm. The other key point that guarantees successful knockin is the cleavage efficiency of the sgRNA. Several sgRNAs can be designed to target the intron sequence in the left arm, and the sgRNA with the highest cutting efficiency should be chosen to perform knockin experiment.In our knockin system, a sgRNA target is selected in an intron of targeted gene, and a DNA sequence spanning from the sgRNA target site to 3′ intergenic region of targeted genes is added in a donor plasmid as the homologous arm Figure . As thisin vivo. Moreover, adding more copies of fluorescent sequences in the targeted gene will decrease the transcriptional and expressional efficiencies. To solve these problems, we may integrate the intron-based knockin with other methods, such as SNAP-tag system, antibody-based fluorescent amplification system, and drug-induced fluorescent system. Taking advantage of the transparency of zebrafish larvae and super-resolution imaging, intron-based knockin approach will definitely open up a new avenue for zebrafish-relevant research.In the near future, the next-generation knockin approach should be developed for tagging endogenous proteins. As the expression level of some endogenous proteins may be very low, knockin of one or two copies of fluorescent proteins is still insufficient for observation of these proteins


Most bacteria attach to surfaces where they form a biofilm, cells embedded in a complex matrix of polymers. Cells in biofilms are much better protected against noxious agents than free-living cells. As a consequence it is very difficult to control pathogens with antibiotics in biofilm infections and novel targets are urgently needed. One approach aims at the communication between cells to form and to maintain a biofilm, a process called quorum-sensing. Water soluble small-sized molecules mediate this process and a number of antagonists of these compounds have been found. In this review natural compounds and synthetic drugs which do not interfere with the classical quorum-sensing compounds are discussed. For some of these compounds the targets are still not known, but others interfere with the formation of exopolysaccharides, virulence factors, or cell wall synthesis or they start an internal program of biofilm dispersal. Some of their targets are more conserved among pathogens than the receptors for quorum sensing autoinducers mediating quorum-sensing, enabling a broader application of the drug. The broad spectrum of mechanisms, the diversity of bioactive compounds, their activity against several targets, and the conservation of some targets among bacterial pathogens are promising aspects for several clinical applications of this type of biofilm-controlling compound in the future. Most bacteria attach to surfaces and interfaces where they form a biofilm . Here thForming biofilms is the answer of micro-organisms to hostile environments. The optimal protection of the embedded cells against noxious agents, e.g., antibiotics and the immune system, is the main reason why biofilm infections are so difficult to treat. It has been found that for the eradication of pathogens from biofilms more than 1000 times higher antibiotic concentrations were required than for the same strain living in planktonic form in the serum . The mec1–3 are probably the best studied class of them [4, a boron-bearing compound [5 [25–26, farnesol 6 [cis-2-alkenoic acids [34–41, and a variety of peptides [11–13, many of which are cyclic compounds [To form a biofilm requires coordinated gene expression of the individual cells. It has been demonstrated for a still growing number of bacteria that a strategy, called quorum sensing, regulates this process . The ind of them . Howevercompound , bradyoxpound [5 , severalrnesol 6   25 can activate the homoserine lactone biosensor although considerable higher concentrations are needed than for the natural homoserine lactone [l-4-iodo-Phe-l-Pro) 27 and cyclo(l-4-chloro-Phe-l-Pro) 28 were inhibitors of quorum sensing mediated luminescence and cyclo(l-4-chloro-Phe-d-Pro) 29 and cyclo(l-Trp-l-Pro) 30 were moderate inhibitors [l-Tyr-l-Pro) 31 and cyclo 32 reduced colony expansion in a strain of Serratia liquefaciens [Lactobacillus reuteri produces cyclo(l-Phe-l-Pro) 25 and cyclo(l-Tyr-l-Pro) 31 which strongly inhibit quorum sensing in Staphylococcus aureus [Penicillium sp. cyclo(l-Tyr-l-Leu) 33  33  has beendermidis . These s9 inhibits quorum sensing by blocking AHL-sensors [Streptococcus mutans, a membrane-localized transpeptidase possessing an important role in adherence that has been associated with cariogenicity [Looking only for inhibition of receptors of the autoinducers may be misleading because some compounds have more than one target in the biofilm. As already mentioned, curcumin -sensors  but thisgenicity ,46. It rPseudomonas aeruginosa, Escherichia coli, Vibrio cholera, Neisseria gonorrhoeae [Staphylococcus aureus [To form a biofilm gives the bacteria protection and allows the production of virulence factors. However, a biofilm can also be a trap for the inhabitants when conditions become unfavorable. To overcome this problem most microbes developed strategies to dissolve their biofilms and allow the embedded cells to move to more convenient sites. Regarding the fact that biofilms have already been established when clinical symptoms are shown, using these biofilm dissolving strategies would be of high priority to combat biofilm infections. Nitric oxide is produced by nitrite reductases in the biofilm to induce dispersal ,48. Thisorrhoeae , and Stas aureus  but alsos aureus  and evens aureus . These fs aureus  or even s aureus . This has aureus . One ways aureus . A very s aureus . For thes aureus  or sol-gs aureus . Howevers aureus .Pseudomonas aeruginosa dispersed. The compound triggering this reaction was identified as cis-2-decenoic acid 34 [cis-2-alkenoic acids have been isolated from Xanthomonas campestris, Stenotrophomonas maltophilia, and Burkholderia species and this class of compounds is termed diffusible signaling factors (DSF). In general, considerably higher activities have been observed for the cis-2-alkenoic acids compared to their saturated analogues.DSFs have a number of effects in the cells but they also mediate interspecies interactions. cis-2-Dodecenoic acid 37 increases in Burkholderia cenocepacia motility and biofilm formation but also virulence [Pseudomonas aeruginosa demonstrated a large number of genes being differently regulated by cis-2-decenoic acid 34 [Stenotrophomonas maltophila several fatty acids  have been identified which facilitate movement of its cells [Stenotrophomonas maltophila and the pathogen Pseudomonas aeruginosa. The fatty acids produced by S. maltophila increase the tolerance to cationic antimicrobial peptides in P. aeruginosa [42, not found in Stenotrophomonas maltophila, blocked swarming motility completely at 10 µg·mL−1 in Pseudomonas aeruginosa and led to reduced biofilm formation by 31% [cis-2-dodecenoic acid 37, enabled bacteria-fungus interaction as was shown between Burkholderia cenocepacia and Candida albicans [Pseudomonas aeruginosa and inhibits its type-III secretion system [When exposed to spent medium, biofilms of  acid 34   thioester of 3-hydroxydodecanoic acid which is then converted by the enzyme RpfF first to cis-2-dodecenoyl-ACP. However, RpfF catalyzes not only dehydration of 3-hydroxydodecanoyl-ACP to cis-2-dodecenoyl-ACP but also cleaves the thioester to release the free acid [The biosyntheses of the DSFs is not yet fully understood. In entified . cis-2-Dree acid .X. campestrensis. Here, synthesis and perception of DSF require products of the rpf gene cluster (for regulation of pathogenicity factors). Similar gene clusters have also been found in many but not all DSFs producing bacteria. The sensor kinase RpfC of Xanthomonas campestris, anchored in the cytoplasmic membrane, consists of a membrane spanning sensory domain, a histidine kinase domain, an ATP kinase binding domain, followed by a receiver domain, and a histidine phosphotransfer domain. After binding of DSF the histidine kinase domain is autophosphorylated requiring ATP. The phosphorelay signalling then leads to the phosphorylation of the cyclic di-GMP phosphodiesterase RpfG causing changes in the cyclic di-GMP level in the cell which in turn affects, e.g., the synthesis of virulence factors or causes biofilm dispersal [Burkholderia cenocepacia becomes a potent c-di-GMP phosphodiesterase leading to the control of several but not all DSF-influenced genes [Stenotrophomonas maltophilia clinical isolates, revealed the presence of two distinct classes of rpfC-rpfF genes. Only one class was able to form DSF while the other one seemed to permanently repress DSF-formation demonstrating that DSF-signaling is not essential for infection [The best understanding of the mechanism of action has been achieved for ispersal . The sened genes . Analysinfection .Pseudomonas aeruginosa by 2-bromoalkanoic acids was evaluated. Here stronger effects of 2-bromohexanoic acid compared to 2-bromooctanoic acid were observed, while 2-bromodecanoic acid showed the weakest effect [et al., reported dispersion of S. aureus biofilms with 10 nM of cis-2-decenoic acid but Su et al., needed about 200 µM to achieve this effect [Because of their ability to disperse existing biofilms DSFs are receiving growing attention. They have also been reported to increase metabolic activity and restore antimicrobial susceptibility of persister cells , both imt effect . It shous effect .43  biofilm formation but had no effect on established biofilms, whereas resveratrol 46 inhibited MRSA biofilm formation and could partially remove established biofilms. RNA-Seq-based transcriptome analysis of MRSA biofilm inhibition by ursolic acid and resveratrol revealed two different mechanisms. While ursolic acid inhibited biofilm formation by reducing amino acids metabolism and adhesin expressions resveratrol disturbed quorum sensing and the synthesis of surface proteins and capsular polysaccharides [44 and corosolic acid 45 are even more effective and they also increase the susceptibility of Pseudomonas aeruginosa to antibiotics [The triterpene ursolic acid 43  inhibitsr system . Ursoliccharides . The cloibiotics .Rhipicephalus microplus) were investigated. It was observed that their eggs were not infected by Pseudomonas aeruginosa and from tick eggs’ extract the cholesterol derivative boophiline 47 was isolated. Boophiline was found to be a weak antibiotic but it had not yet been connected to biofilms [Pseudomonas aeruginosa revealed that it did not interfere with the quorum sensing circuits of the pathogens. Instead, strong inhibitions of the expression of cdrA  and fliC  were found. The bactericidal effect was observed for Staphylococcus epidermidis and related Gram-positive bacteria [Following the hypothesis that the majority of all higher organisms have developed strategies to protect themselves against biofilm infections the protection mechanisms of ticks (biofilms . A detaibacteria . These o48, discovered in the sponge Agelas oroides [50, the urea 51, and the thiourea 52 analogues were demonstrated to inhibit selectively P. aeruginosa biofilm formation, while being nontoxic to Caenorhabditis elegans [53 and 54 were both active against biofilm formation of the pathogens Acinetobacter baumannii, Pseudomonas aeruginosa PA14, and Bordetella bronchiseptica [49 was active against methicillin-resistant Staphylococcus aureus and Streptococcus mutans and suppressed biofilm formation in the lower micromolar range [N-methylated derivative 55 of dihydro-oridin. Maybe some light can be shed on the mode of action of these compounds by their activity against Porphyromonas gingivalis. P. gingivalis, one of the central organisms in periodontal disease, which binds to the primary colonizer Streptococcus gordonii. Three small molecules derived from oroidin and containing 2-aminoimidazole or 2-aminobenzimidazole moieties inhibited binding of P. gingivalis to S. gordonii by reducing expression of fimbrial adhesins, necessary for P. gingivalis adhesion to S. gordonii [The alkaloid oroidin  oroides , was sho oroides . There s oroides . The sul elegans . The deriseptica . Inhibitar range . One of gordonii .21 has been reported to reduce biofilm formation of the bacteria Escherichia coli, Bacillus cereus, and Pseudomonas aeruginosa [Aspergillus niger, Penicillium citrinum, and Candida albicans [E. coli leading to increased formation of flagella has been identified as one of the mechanisms. This is probably the reaction of the cells to a stress condition caused by zosteric acid [20, lacking the p-sulfoxy-moiety, was even more active. Using an E. coli protein pull-down approach the enzyme NADH: quinone dehydrogenase was identified as the target molecule for these compounds. This is a tetrameric flavoprotein with a hydrophobic active site pocket which is most likely the interaction site for these cinnamic acids [Zosteric acid ruginosa  and the albicans . An incrric acid . Screeniic acids .Pseudomonas putida produces promysalin 56 which acts only against some Pseudomonas species, among them the pathogen P. aeruginosa [ruginosa . A recenruginosa . The mervs. antibiotic vs. pathoblocker [All higher organisms have to deal with biofilm infections and they have developed a multitude of mechanisms to control biofilm communities. These examples of natural compounds acting beyond the simple competition for the receptors of the known quorum sensing autoinducers demonstrate that there is a vast and still poorly explored field of targets for biofilm control. However, the examples show that several of these compounds have more than one target and the transition between biofilm control oblocker  seems toet al., tested patulin in Pseudomonas aeruginosa as an antagonist of acylhomoserine lactone. While patulin had no effect on the survival of P. aeruginosa cells in the bofilm and treatment of the biofilm with the antibiotic tobramycin could kill only few cells, the combination of patulin and tobramycin led to severe killing of the cells [16 reduced the formation of biofilms and virulence factors of Staphylococcus aureus but did not kill the pathogen. The combination of this quorum-quenching compound with the antibiotic tigecycline in a rat model, however, increased the sensitivity of S. aureus four-fold compared to tigecycline alone [Dissolving an existing biofilm may be sufficient to allow clearance of its inhabiting pathogens by the immune system. However, for an inefficient or overloaded immune system of a patient the release of pathogens from biofilms may even have a detrimental effect turning an infection from a chronic to an acute state and fostering the production of virulence factors . Becausehe cells . The RNAne alone .Pseudomonas auroginosa can be dissolved by cis-2-decenoic acid 34 but the bacteria are not killed. When pre-established biofilms of Staphylococcus aureus, Bacillus cereus, Salmonella enterica or Escherichia coli were treated with desinfectants or antibiotics only moderate reductions in living cells were achieved. The addition of only 310 nM cis-2-decenoic acid to these toxins caused a significant increase of cell eradication, e.g., for S. aureus and ciprofloxacin it increased from 11% for the antibiotic alone to 87% for the combination [Mycobacterium smegmatis, Neisseria subflava or Bacillus thuringiensis [Klebsiella pneumonia [Staphylococcus aureus or Acinetobacter baumannii 4,5-disubstituted-2-aminoimidazole-triazole conjugates were developed which inhibited their biofilm formation. When combined with antibiotics they also re-sensitized the pathogens for eradication with these antibiotics [Biofilms of bination . Similarngiensis  or in mineumonia . Againstibiotics .Often it has been argued that biofilm-interfering compounds will not lead to the development of resistances in the pathogens because preventing or dispersing biofilms is not lethal for the microbes . AlthougCurrently, the often high specificity of quorum quenching drugs limits their application , especiaet al., tested biofilm modulating compounds for their influence on osteoblast differentiation they found cis-2-decenoic acid 34 biocompatible but farnesol 6 induced cytotoxic responses within their biofilm inhibitory concentrations [For the application of an anti-biofilm compound it is not only important that it is active against the pathogen but also that it does not do any damage to the host. Sadly, for some of the reported compounds this aspect has been ignored but for the evaluation of a novel drug the assessment of its cytotoxicity is mandatory . Biocomptrations . Many dicis-2-decenoic acid 34, ursolic acid 43 or boophiline 47, only to mention a few. Some of the compounds so discovered have several targets allowing the control of a broader spectrum of pathogens. It is certainly not a risk to predict that in the coming years, novel mechanisms of biofilm dispersal or blocking of antibiotic resistance in biofilms will be uncovered. Combining these biofilm controlling compounds with established or novel antibiotics will add another dimension to the treatment of biofilm infections.Biofilms have complex and dynamic structures mostly composed of several microbial species. The complexity of biofilms helps them to survive under harsh conditions but it also offers a multitude of approaches for their control. To prevent or to cure biofilm infections the main focus has been set to quorum-sensing. Screening for quorum-quenching compounds can nowadays be done in high-throughput screenings because several assays targeting the formation of autoinducers or their receptors are available. However, the entire mechanism for quorum-sensing is known only for a small number of pathogens and we still know very little about interspecies cross-talks. This article had the intention to show that many organisms have developed compounds which interfere with biofilms and many do this outside the known quorum-sensing circuits. Because these natural compounds have a variety of targets or their targets are even not yet known, screening for these bioactive compounds is more laborious and slower. The reward for such an effort, however, is the discovery of new targets and new mechanisms as have been highlighted for


Ribosomal biogenesis has been extensively investigated, especially to identify the elusive nucleases and cofactors involved in the complex rRNA processing events in eukaryotes. Large-scale screens in yeast identified two biochemically uncharacterized proteins, TSR3 and TSR4, as being key players required for rRNA maturation. Using multiple computational approaches we identify the conserved domains comprising these proteins and establish sequence and structural features providing novel insights regarding their roles. TSR3 is unified with the DTW domain into a novel superfamily of predicted enzymatic domains, with the balance of the available evidence pointing toward an RNase role with the archaeo-eukaryotic TSR3 proteins processing rRNA and the bacterial versions potentially processing tRNA. TSR4, its other eukaryotic homologs PDCD2/rp-8, PDCD2L, Zfrp8, and trus, the predominantly bacterial DUF1963 proteins, and other uncharacterized proteins are unified into a new domain superfamily, which arose from an ancient duplication event of a strand-swapped, dimer-forming all-beta unit. We identify conserved features mediating protein-protein interactions (PPIs) and propose a potential chaperone-like function. While contextual evidence supports a conserved role in ribosome biogenesis for the eukaryotic TSR4-related proteins, there is no evidence for such a role for the bacterial versions. Whereas TSR3-related proteins can be traced to the last universal common ancestor (LUCA) with a well-supported archaeo-eukaryotic branch, TSR4-related proteins of eukaryotes are derived from within the bacterial radiation of this superfamily, with archaea entirely lacking them. This provides evidence for “systems admixture,” which followed the early endosymbiotic event, playing a key role in the emergence of the uniquely eukaryotic ribosome biogenesis process. Ribosomal RNAs (rRNAs) combine with structural proteins in assembly of the ribosome, the ribonucleoprotein protein synthesis complex conserved across the three superkingdoms of cellular life. While there are notable differences between bacteria, archaea, and eukaryotes, the general steps in rRNA production are shared. These broadly entail transcription of a polycistronic precursor, which is then subject to a complex series of processing events involving the interplay between distinct endo- and exo-nucleases  are also involved  recovered uncharacterized bacterial proteins from Planctomyces brasiliensis  and Parachlamydia acanthamoebae , a DTWD1-like homolog from the ciliate Tetrahymena thermophila , and a DTWD2-like homolog from the predatory mite Metaseiulus occidentalis . The above-detected relationships between these previously unlinked sets of proteins were confirmed by reciprocal PSI-BLAST searches and independently using profile-profile comparisons using the HHpred program with hidden Markov models (HMMs) constructed from multiple sequence alignments of the above sets of proteins. For example, a HHpred search initiated with a Vibrio Cholerae DTWD2 sequence (gi: 487840886) recovers the pfam DTW HMM profile  and the Pfam DUF367 HMM profile , which contains several TSR3 homologs. Given these relationships, we named this superfamily the TDD  domain.To collect all TSR3 homologs and identify more distant protein relationships, PSI-BLAST searches were run using the entire length of known TSR3 proteins as search seeds. The previously-identified archaeal and eukaryotic TSR3 homologs  the DTWD1-like family broadly present in several bacterial clades including planctomycetes, verrucomicrobia, spirochetes, and proteobacteria and also many eukaryotes including animals, plants, the amoebozoan Entamoeba lineage, and scattered presence in apicomplexa and stramenopiles, and (5) the AT1G03687 family, typified by the eponymous Arabidopsis thaliana protein, with a patchy representation in eukaryotes including land plants and several other lineages.Similarity-based clustering of all recovered sequences revealed the presence of five distinct TDD domain families  (1) the TSR3-like family universally present in eukaryotes and well-represented across archaea, (2) the previously unrecognized bacterial family named pc1599 after the protein found in In the Pfam database  rRNA/ribosomal genes in archaea and tRNA genes in bacteria, (2) RNA-binding or rRNA maturation-related domains, along with the previously reported gene-deletion and high-throughput data analysis on yeast TSR3 , Campylobacter showae , and Streptomyces camus . Reverse searches initiated with these proteins recovered a range of bacterial homologs as well as the TSR4 protein in eukaryotes. Further searches with the bacterial proteins also recovered a new set of bacterial homologs with the detected region of similarity overlapping with the Pfam model annotated as DUF1963. For example, a search initiated with the same region from the above Campylobacter showae sequence recovered TSR4 homologs in mouse  and DUF1963-containing homologs in Haliscomenobacter hydrossis . Continuing these searches we recovered the YwqG protein in E. coli and its homolog from Bacillus subtilis for which a crystal structure was solved by the Protein Structural Initiative  but also detected a significant relationship with the PDCD2_C domain , suggesting that TSR4 proteins and their homologs contain two copies of a single domain that underwent an ancestral duplication. This was supported by the recovery of the GGxP-like motif, the Q, and the pair of CxxC motifs in alignments of both the N- and C-terminal regions. In addition, both PSI-BLAST and HMM searches run with multiple full-length bacterial versions as seeds retrieved matches to eukaryotic sequences extending along the entire length of the TSR4-like protein barring the low-complexity insert. Finally, profile-profile comparisons initiated with the full-length bacterial sequence from Salmonella enterica as a seed (gi: 555248518) recovers the DUF1963 and PDCD2_C profiles at both the N- and C-terminal repeats . The duplication was further confirmed via examination of the structure of the bacterial version (1PV5) (see below). Thus, Pfam PDCD2_C and DUF1963 are models partially covering the same superfamily of proteins with the former only covering part of the C-terminal repeat. We named the unified and correctly defined superfamily of domains encompassing both repeats as TYPP  widely distributed across bacteria, including the solved crystal structure from B. subtilis, and also found in a small group of eukaryotes; (2) the PDCD2L family found across all eukaryotes and including the yeast TSR4 protein; (3) the PDCD2 family found across plants, animals, fungi, slime molds and certain stramenopiles; (4) three additional, relatively narrowly-distributed bacterial families numerically labeled 2–4 .Examination of the structure of the n Figure . A multin Figure  indicaten Figure . The twon Figure : (1) a dn Figure . SequencDrosophila, similar to the associations reported earlier for the TSR4 protein in yeast  via a chaperone-like action. A precedent for this is offered by the archease domain with an analogous two-repeat structure  of Life. Given the presence of a strongly-supported archaeo-eukaryotic clade of TDD domains, it is likely that the ancestral version of this clade acquired rRNase function. In contrast, associations of the dominant bacterial family of TDD domains, DTWD2, suggests acquisition of a tRNA-processing role. The Last Eukaryotic Common Ancestor (LECA) can be inferred as possessing two distinct versions of the TDD domain: a cognate of TSR3 closest to the archaeal cognates and a DTWD1 family representative, which is closest to the bacterial DTWD2 family. The two copies were therefore likely respectively acquired from the archaeal and bacterial progenitors participating in the primary endosymbiotic event leading to eukaryogenesis. Beyond these, additional eukaryotic versions were likely transferred later from bacteria and recruited for as-yet-uncharacterized RNA-processing events.The prediction of nuclease function for TSR3 suggests interesting possibilities for the highly-coordinated endo-/exo-nucleolytic rRNA maturation events in which it is implicated  which, upon duplication and insertion of the MYND domain, gave rise to the paralogous PDCD2 family prior to the divergence of animals, fungi, and plants from their common ancestor. Our findings suggest both of these eukaryotic paralogs are involved in ribosomal biogenesis, an avenue of research which has been largely neglected in studies on Drosophila and mammalian orthologs and a functional assignment which could account for the diverse consequences observed following its perturbation. Additional sporadic transfers of the TYPP domain from bacteria to terminal eukaryotic lineages have also been observed  is a nuclease required for rRNA processing in archaea and eukaryotes and possibly tRNA processing in bacteria. We also present the hypothesis that TSR4 might play a role in augmenting PPIs, foremost in ribosome biogenesis, and potentially in additional contexts. In evolutionary terms, we detect the first bacterial homologs of these conserved proteins. As a result, we obtain clear evidence that the provenance of the unique and complex ribosome biogenesis system of eukaryotes necessarily required the coming together of bacterial and archaeal components. This offers further support to the growing evidence that the consequences of “systems admixture” following the primary endosymbiotic event strongly contributed to the emergence of quintessential eukaryotic features.http://hmmer.janelia.org/search/jackhmmer), respectively. Queries were run against the non-redundant (nr) protein database of the National Center for Biotechnology Information (NCBI). For most sequence-based homology searches, which underlie the relationships presented in this work, a cut-off e-value of 0.01 was used to assess significance. In each iteration, newly-detected sequences included within the cut-off were evaluated via initiation of a new search with the sequence in question as the query to guard against inclusion of false positives; searches were continued with the same e-value threshold only if the profile remained uncorrupted without false positives. Postulated relationships recovered using iterative searches were further confirmed with other aids such as concordance of predicted or known secondary structural elements. Profile-profile comparisons were also used as an additional means of confirming distant relationships, these were performed using the HHpred program , using empirically-determined length and score threshold cut-off values. Multiple sequence alignments of resulting clusters were constructed using the MUSCLE alignment program . Automatic aspects of large-scale analysis of sequences, structures, and genome context were performed with the in-house TASS package, which comprises a collection of Perl scripts.Sequence-based homology clustering of TDD and TYPP proteins and associating proteins in gene neighborhoods was performed with the BLASTCLUST program  to identify related sequences in gene neighborhoods. Each resulting gene cluster is then assigned annotation based on the shared domain architecture or single domain in the encoded protein. This allows an initial annotation of gene neigborhoods which is further refined by including only genes which are unidirectional on the same strand of DNA and share a putative common promoter, identified by assigning a maximum distance between adjacent genes, with the default assigned as 150 nucleotides. “Head-to-head” gene arrangements on opposite strands are also included when potential bidirectional promoter sharing patterns are detected.For each gene of interest recovered in homology searching, the gene neighborhood was comprehensively interrogated using custom Perl scripts from the TASS package. These scripts utilize PTT files (retrieved through the NCBI ftp site) when the gene is from an assembled genome or Genbank files when the gene is from a collection of whole genome shotgun sequences. After locating the gene, a default value of the five nearest neighbors in both directions are extracted, this value is altered on rare occasions when the gene in question is part of a long, extended neighborhood. Protein sequences of all neighbors are clustered using the BLASTCLUST program (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.


Several approaches have been taken to reduce pre-antiretroviral therapy (ART) losses between HIV testing and ART initiation in low- and middle-income countries, but a systematic assessment of the evidence has not yet been undertaken. The aim of this systematic review is to assess the potential for interventions to improve or facilitate linkage to or retention in pre-ART care and initiation of ART in low- and middle-income settings.An electronic search was conducted on Medline, Embase, Global Health, Web of Science and conference databases to identify studies describing interventions aimed at improving linkage to or retention in pre-ART care or initiation of ART. Additional searches were conducted to identify on-going trials on this topic, and experts in the field were contacted. An assessment of the risk of bias was conducted. Interventions were categorized according to key domains in the existing literature.n=21) from sub-Saharan Africa. In addition, 15 on-going trials were identified. The most common interventions described under key domains included: health system interventions ; patient convenience and accessibility (i.e. point-of-care CD4 count (POC) testing with immediate results, home-based ART initiation); behaviour interventions and peer support  and incentives (i.e. food support). Several interventions showed favourable outcomes: integration of care and peer supporters increased enrolment into HIV care, medical incentives increased pre-ART retention, POC CD4 testing and food incentives increased completion of ART eligibility screening and ART initiation. Most studies focused on the general adult patient population or pregnant women. The majority of published studies were observational cohort studies, subject to an unclear risk of bias.A total of 11,129 potentially relevant citations were identified, of which 24 were eligible for inclusion, with the majority (Findings suggest that streamlining services to minimize patient visits, providing adequate medical and peer support, and providing incentives may decrease attrition, but the quality of the current evidence base is low. Few studies have investigated combined interventions, or assessed the impact of interventions across the HIV cascade. Results from on-going trials investigating POC CD4 count testing, patient navigation, rapid ART initiation and mobile phone technology may fill the quality of evidence gap. Further high-quality studies on key population groups are required, with interventions informed by previously reported barriers to care. The rapid roll-out of antiretroviral therapy (ART) programmes over the past decade in low- and middle-income countries has resulted in an estimated 9.7 million HIV-positive individuals receiving ART by the end of 2012, compared to just 300,000 in 2000 . It is eTo date, attempts have been made to quantify the losses occurring along the HIV cascade and describe the barriers to care, although data from low- and middle-income countries outside sub-Saharan Africa are scarce. Three recent systematic reviews have consistently shown major attrition occurring along each step of the HIV cascade in sub-Saharan Africa for adults. Less than half (45–46%) of individuals not yet eligible for ART were retained in pre-ART care and only two-thirds (65–68%) of ART-eligible individuals initiated ART –9. A recPatients in the period between HIV diagnosis and ART initiation can be divided into three groups: those that enter into HIV care post-diagnosis, those that remain in pre-ART care until ART-eligible, and those that initiate ART once ART eligibility is established. Rosen et al. describe these three periods as stage 1, 2 and 3, respectively . SeveralRandomized trials, comparative non-randomized observational studies, and comparative before-and-after studies were eligible for this review. Cohort studies were only included if the control (comparator) group was contemporary (delivered at the same time and in the same country). Studies were eligible if they included HIV-positive patients in low- and middle-income countries  at aSupplementary Tables 1–3. Three electronic databases were searched for on-going trials: Cochrane Central Register of Controlled Trials (CENTRAL), WHO International Clinical Trial Registry Platform and ClinicalTrials.gov. Furthermore, a total of 117 experts (Supplementary Tables 4 and 5) were contacted to identify additional studies either completed or on-going; these experts were authors of studies included in this and previous reviews  [A 2–1.18)] .A before/after study in Zambia found that patients who underwent Provider-initiated testing and counselling (PITC) had a significantly lower odds of ART initiation [aOR=0.90 (0.82–0.97)] and pre-ART retention [aOR=0.84 (0.74–0.95)] .p<0.0001)  .p=0.174).Similarly, a higher proportion of patients randomized to receive HIV testing as inpatients linked to HIV services (21%) compared to patients who were referred for outpatient HIV testing following discharge from hospital (16%) ; howeverA randomized controlled trial conducted in Uganda showed that intensified post-test counselling and monthly visits by a peer support worker almost doubled linkage to HIV care five months post-diagnosis  . A peer Visits by peer navigators also increased the likelihood of seeking care following a community-based HIV testing campaign in a observational study conducted in Kenya at 10 months for men (aHR=1.35 (0.97–1.87)] and women [aHR=1.20 (1.00–1.43)] ; howeverTwo studies from India investigated the effect of food incentives on retention in pre-ART care and ART initiation. In the first study, the introduction of monthly food supplements increased the proportion of children regularly visiting the clinic over a one year period from 81 to 93% ; however, this study was a before/after design and these findings are subject to survival bias as more children were reported dead and lost to follow-up in the control arm (9%) than the intervention arm (4%) . The secA third study, from Kenya, found that scheduled and regular visits during the pre-ART period for the purpose of co-trimoxazole refill increased pre-ART retention at 12 months post-enrolment by 20% .n=13) of trials are underway in sub-Saharan Africa, six of them in Kenya, and seek to assess interventions for injecting drug users (n=3) and pregnant women (n=5). Proposed interventions include POC CD4 count testing, rapid (same day) ART initiation, integration, peer educators, intensive counselling, assisted partner notification, home-based male partner testing and short text messages. The majority of trials will be completed by January 2015.Fifteen on-going or planned trials were identified . The majn=10) or contemporaneous comparative studies (n=7). There were three cluster randomized controlled trials [n=8) were rated as having an unclear risk of bias; four studies were classified as being at high risk of bias.Most studies included in this review were observational studies; either times series (before/after studies)  with key quality concerns related to the random sequence generation, allocation concealment and reporting. Of the 16 remaining observational studies, most had an unclear risk of bias (n=7) and four studies were rated as having a high risk of bias, with the main quality issues related to uneven distribution of baseline characteristics, no adjustment for confounders, and lack of data related to patient follow-up.Of the four peer-reviewed randomized trials assessed for study quality, the majority were categorized as having low risk of bias , there are interventions that could potentially support linkage and retention along the entire HIV cascade both in pre-ART and ART care  and thus warrants further investigation.Attending pre-ART clinic visits is a major challenge among patients in low- and middle-income countries due to high transport costs, distance to health care facilities, inability to take time off work, and long clinic waiting times  as well Apart from reducing clinic visit frequency, inflexible clinic hours have been identified as another barrier to accessing care, especially among the working population ,69,70. HSeveral studies have investigated the effect of bringing HIV care services closer to where patients reside through decentralization and service integration to overcome issues of distance and difficulties to pay for transport in the context of pre-ART care. A recent systematic review assessing the influence of various models of decentralized HIV treatment and care on initiating and maintaining ART concluded that partial decentralization can decrease attrition of patients on ART . CurrentThis review identifies that peer support and appropriate counselling may facilitate linkage to care. Stigma has been identified as a barrier to care at all steps of the HIV cascade, including HIV testing , linkageThere have been few studies on the use of incentives to improve retention in pre-ART care, including nutritional support, free medications, and conditional cash transfers. Food insecurity was highlighted as a barrier to pre-ART care in a recent systemic review  and studThis review showed a lack of studies testing interventions aimed at key populations. Men have less access to and coverage of HIV testing and ART in most settings and are at higher risk of attrition from ART and pre-ART services. No study investigating interventions specifically targeted at men were identified ,89–97. IOverall, most studies investigated the impact of individual interventions on one stage of the cascade, mainly stages 2 and 3 and few studies have investigated interventions for stage 1, linkage to HIV care.A single intervention is unlikely to solve attrition across all steps in the pre-ART period and to address the different reasons for attrition. Although, certain interventions such a POC CD4 counting are effective in improving time to linkage to HIV care and completion of ART eligibility screening, it may be a less suitable intervention for improving pre-ART retention, in contrast to food and medical incentives. The effect of interventions targeted at a single point in the pathway might be limited when assessed across the entire pre-ART period. Loss to follow-up prevented earlier on in the pathway might not guarantee retention later on. However, most of the planned or on-going trials investigate single interventions with limited follow-up time. Therefore, a combination of interventions to address the main points at which patient drop-out of care during the pre-ART period or while on ART are most likely to be successful. This package should include interventions aimed at increasing patient convenience and accessibility  as this was found to be effective in increasing ART eligibility screening and initiation rates; health system interventions such as integration of ART and ANC care as this appears to increase HIV care and ART enrolment; behavioural interventions and peer support (e.g. intensified post-test counselling and peer support) as these were found to increase linkage to care. HIV programmes should first determine the patient groups at risk for drop-out in their cohorts and reasons for drop-out  in order to inform the selection of an effective package of interventions. Programme planners should be mindful of several key factors when selecting interventions i.e.: the operational needs of specific interventions , challenges in implementing and maintaining interventions , interventions studied (e.g. incentives) could be context-specific and may be need to be tailored to the target population in their settings. Furthermore, programmes adopting the WHO guidelines supporting earlier ART initiation will likely observe a significant reduction in the number of people with HIV required to remain in the “pre-ART” period, however these programmes will need to implement interventions to improve immediate linkage to ART care as majority of people will require ART care.The main strengths of this review are the use of a broad search strategy that included an extensive expert consultation, allowing identification of both published and unpublished studies as well as information on on-going trials, a rigorous assessment of study risk of bias and quality. This assessment found that the evidence base and methodology of this current review was subject to several limitations. Firstly, the quality of studies was limited with the majority of studies being observational, using time series rather than concurrent control groups, and subject to unclear risk of bias. Outcome ascertainment was rarely adequately described and time delineation was often lacking, making it difficult to compare outcomes and calculate summary estimates. Assessment of cost-effectiveness and patient acceptability were absent from all studies. Publication bias is likely present as studies with unfavourable results are less likely to be published. In addition, HIV programme evaluation reports from resource-limited settings might not necessarily be published and thus might have been missed by this review. While the literature review was extensively conducted in four international databases including primarily studies published in peer reviewed journals, regional databases such as the African Healthline and LILAC were not searched. The review was limited to low- and middle-income countries. Thus some potential interventions from high income countries which might be applicable to low- and middle-income countries were not included in the review. Most of the studies included were conducted in sub-Saharan Africa, limiting the generalizability of our findings beyond this region and even within the region there is considerable heterogeneity of current service delivery models and approaches. Most studies assessed interventions within the ANC and PMTCT setting and thus findings may not be generalizable to general HIV care programmes. Finally, the majority of studies looked at the effect of a single intervention on a single point in the HIV continuum.The overall findings from this review suggest that streamlining services to minimize patient facility visits, providing adequate counselling, medical and peer support, and providing incentives may decrease attrition between HIV testing and ART initiation. Further implementation research, focused programme evaluation and studies with rigorous study designs investigating the impact of individual interventions and a combination of interventions across the HIV cascade, including for the various groups at high risk of attrition, is warranted in order to inform HIV policy and programmes. There is a specific need to also evaluate interventions for key populations as currently they are not only disproportionately affected by HIV but have lower access to and coverage of HIV testing and ART.