However, their functions in cancer genome instability and the consequent clinical effect as the response to chemotherapy have not been fully elucidated. In this study, we utilized multidimensional TCGA data and the known miRNAs involved in DDR to identify a miRNA-regulatory network that responds to DNA damage. Additionally, based on the expression of ten miRNAs in this network, we developed a 10-miRNA-score that predicts defects in the homologous recombination (HR) pathway and genome instability in ovarian cancer. Importantly, consistent with the association between HR defects and improved response to chemotherapeutic agents, the 10-miRNA-score predicts the outcome of ovarian cancer patients treated with platinum agents, with a surprisingly better performance than the indexes of DNA damage. Therefore, our study demonstrates the implication of miRNA expression on cancer genome instability and provides an alternative method to identify DDR defects in patients who show the best effect with platinum drug treatment. Mutations and chromosomal alterations often occur in human tumours. To preserve the genomic integrity, cells have evolved an elaborate cellular system to sense and repair DNA lesions2. DNA double-strand breaks (DSBs) are one of the most severe types of DNA damage and are repaired by the error-free homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Other types of DNA damage are processed through mismatch repair (MMR), base excision repair (BER) and nucleotide excision repair (NER)3. Hereditary and somatic defects in DDR genes could lead to genome instability and various human cancers. For example, mutations in NER genes predispose to skin cancer, and mutations in HR genes predispose to various cancers, including ovarian and breast cancer, lymphomas and leukaemia6. However, high-throughput sequencing studies in recent years have revealed that genome instability in most sporadic human cancers is not due to mutations targeting caretaker genes in these pathways, raising the need to consider aberrant post-transcriptional regulation that can also inactivate DDR genes1.Genome instability is one of the hallmarks of human cancers and is often associated with cellular deficiency in the DNA damage response (DDR)7. Increasing evidence has shown that cells deficient for miRNA biogenesis have abnormal cell cycle checkpoints and DNA repair8. Additionally, many miRNAs, such as miR-24, miR-21, miR-146, miR-421 and miR-373, have been implicated in DDR9. miR-24 attenuates H2AX expression, resulting in higher sensitivity to IR and reduced DNA repair ability10. miR-421 down-regulates ATM, leading to altered cell cycle checkpoint and increased radio-sensitivity in cells11. miR-146 targets BRCA1 and is involved in the response to DSBs12. These findings demonstrate that DDR genes are regulated by miRNAs and suggest that the aberrant expression of miRNAs would affect the DDR process. In fact, several recent studies have revealed that aberrant expression of miRNAs in human cancer affects the sensitivity of DNA damage-based therapy. For example, overexpression of miR-151 by the gain of chromosome 8q24.3 facilitates tumour cell migration and spreading through the downregulating DDR gene RhoGDIA13. The miR-183-96-182 cluster, whose expression is increased in many human cancers, increases the sensitivity to platinum agents and PARP inhibitors by downregulating RAD51, REV1 and BRCA115.miRNAs that are involved in the highly coordinated network of DDR processes might play important roles during the development of genome instability and might determine the cellular response to DNA damaging agents16. For example, the standard treatment of epithelial ovarian cancer, a cancer type characterized by frequent HR deficiency, is aggressive surgical debunking, followed by platinum-based chemotherapy, delivered concurrently with other agents . The chemotherapy is most effective with patients exhibiting severe DDR deficiency. Improved survival has been observed in ovarian cancer patients with mutations of BRCA1 or BRCA2 when treated with platinum agents19. However, there are various genomic alterations of DDR genes that may cause different extents of DDR deficiency, whereas a standardized signature for assessing the severity of DDR deficiency has not been established. Realizing that caretaker genes are under exquisite regulation by miRNAs, we utilized multidimensional TCGA data and the known miRNAs involved in DDR to identify the miRNA-regulatory network responding to DNA damage in ovarian cancer. Based on the expression of these miRNAs, we have developed a miRNA-score that is associated with genome instability and predicts the outcome of ovarian cancer. We found that our miRNA-score reflects HR deficiency and can identify chemo-sensitive samples with or without mutations of caretaker genes.Due to the key role of DDR in sensing and repairing DNA damage, the combined use of agents targeting DDR and DNA damaging agents is becoming a very attractive strategy in cancer treatment20 and at least one of the five miRNA prediction software programs ; (3) we searched Pubmed for each interaction using the name of the miRNA and gene symbol as keywords, and manually checked the publications to ensure that the target gene expression is repressed by the miRNA. The resulting miRNA-regulatory network involved in DDR consists of 142 miRNA-target interactions between 75 miRNAs and 55 DDR genes. Specific information, including the miRNA-gene interactions, DNA repair pathway of the target gene and supporting references, is provided in Supplementary Table To collect a high-quality set of miRNAs in the regulatory network involved in DDR, we developed the following strategy: 1) we obtained 522 DDR genes involved in the “cellular response to DNA damage stimulus” according to Gene Ontology annotation; (2) we downloaded all the miRNA-gene interactions between miRNAs and the 522 DDR genes from the starBase 2.0 database and only kept the interactions supported by both CLIP-Seq experiments we obtaiTP53 and its inhibitor MDM2 were found to be the most regulated gene targets. Fourteen genes of this network, including ATM, CHEK1, CHEK2, H2AX, MCL-L, BRCA1, RAD23B, RAD51, RAD52, SIRT1, RhoGDIA, PTEN, AKT1 and AKT3, are in pathways known to be activated in response to platinum19. To further demonstrate the importance of this network in the DDR, we investigated the expression change of genes in the network upon DNA damage. We found that approximately 40% of these genes showed a significant expression change in 36M2 ovarian cancer cells after treatment with carboplatin, and approximately 60% of these genes showed a significant expression change in MCF7 cells after treatment with doxorubicin  by analysing 1,405 candidate miRNAs23. Notably, 23 of these 41 differentially expressed miRNAs are included in our miRNA-regulatory network, which is highly enriched . This includes the key p53 target miR-34 that showed the most dramatic expression change upon DNA damage. It has been shown that miR-34 may also be involved in the DDR by targeting SIRT1 and UNG225.All the DDR genes have been classified as DNA damage signalling (DDS) genes or genes belonging to one of the following DNA repair pathways: HR, NHEJ, MMR, NER and BER. Genes involved in other pathways—i.e., cross-link repair, RecQ helicase pathway and translation synthesis—were clustered together into one group due to the small number of genes within each group. For genes involved in multiple pathways, such as ATR and ATM, we classified them into the most appropriate pathway according to our knowledge. Thus, miR-29 and miR-16 were identified as the most connected miRNAs, and 28. Here, we propose that the mutation frequency is a quantified measurement of genome instability regarding ovarian cancer, which is characterized by frequent HR deficiency29. To verify this, we compared tumours with known BRCA1/2 deficiency and wild-type tumours using 319 tumours with level 3 mutation data of the TCGA dataset. Four overall indexes of genome instability, the fraction of the altered genome, TAI, frequency of LOH and frequency of somatic mutation, were analysed, whereas only the LOH and mutation frequencies showed a significant difference between the two groups of tumours in the TCGA dataset. The frequency of mutation is significantly correlated with the frequency of LOH but shows a broader distribution that is more suitable to further stratify different tumours  of copy number variation and loss of heterozygosity (LOH) frequency, have been used in previous studies* and miR-193a, for which lower expression is associated with a high frequency of mutation.We then evaluated the association between the expression of miRNAs and mutation frequency of the cancer genome as an index of genome instability. By analysing the expression of 723 human miRNAs across 319 TCGA high-grade advanced-stage ovarian tumours, we identified 17 miRNAs whose expression showed a significant association with the frequency of mutation , with ten of them in our miRNA-regulatory network . We also found that the targets of miR-211, which is not included in the curated miRNA-regulatory network, are significantly enriched in DDR genes . In addition, the higher expression of miR-211 is associated with increased mutation number  in ovarian cancer, which suggests that this miRNA is likely involved in genome instability regulation. The miRNA-target regulations between platinum-responsive genes and the 8 miRNAs, as well as miR-211, are plotted in Fig. To highlight the potential clinical relevance of these miRNAs in the treatment of ovarian cancer, we collected 80 DNA repair genes from previous studies that showed a response to platinum drugs  of a patient that show higher expression than the median value across the dataset and the number of negatively correlated miRNAs  that show lower expression than the median value across the dataset. In other words, for each patient, we assigned a point for each of the 6 miRNAs  if the patient showed a higher than median expression of miRNA and assigned a point for each of the 4 miRNAs  if the patient showed a lower than median expression of miRNA, and then summarized these points as the 10-miRNA-score. As expected, the 10-miRNA-score was strongly correlated with the level of DNA mutation in the cancer genome of the training set of 319 ovarian cancers  and high 10-miRNA-score group (6–10). Analysis of the frequency of these pathways harbouring mutations, copy number changes or epigenetic silencing events showed that the HR pathway was the most significantly altered pathway in the high-versus-low 10-miRNA-score group. Genomic alterations in HR-related genes that might have clinical relevance in ovarian cancer are shown in Fig. The TCGA group has identified several pathways that are commonly altered in ovarian cancer, including the RB1 signalling pathway, PI3K/RAS signalling pathway, FOXM1 transcription factor network and HR pathwaysFOXM1 signalling though TP53 to control cell proliferation. However, in ovarian cancer, a high frequency of TP53 mutation (87%) causes the constitutive overexpression of FOXM1 and its target genes. Despite the different frequencies of DDR defects and different levels of genome instability between the high- and low 10-miRNA-score groups, we did not find a significant difference in the upregulation of FOXM1-related genes  of the 319 ovarian cancer patients were platinum sensitive, and 58% (186) of patients achieved a complete response (CR) to platinum-based chemotherapy. Plotting the percentage of platinum-sensitive patients and percentage of patients achieving CR against an increasing 10-miRNA-score revealed strong correlations  and divided 180 patients into the high-mutation and low-mutation groups with almost the same distribution of the 10-miRNA-score . We found that patients within different 10-miRNA-score groups with a similar genomic mutation frequency still showed significantly different outcomes  are small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. miRNAs possess multiple attractive features to allow clinical use, such as in the diagnosis and prognosis of diseases. These attractive features include stability and resistance to degradation, relatively easy detection and quantitation. Additionally, because they are small and regulate gene expression via a “seed sequence” only consisting of 6 to 7 nucleotides, a miRNA could target multiple genes. For example, let-7 has been revealed to play central roles in regulating cell metabolism via targeting multiple genes involved in this process, such as InsR, IGFR, IRS, PDK1, AKT and Rictor37. We hypothesize that the deficiency in the DNA damage response leads to genome instability that can be measured by high-throughput sequencing technology. In ovarian cancer, chromosome instability based on copy number variation has been successfully applied to identify patients responsive to chemotherapy in recent studies38. In this study, we propose that the accumulation of somatic mutations is probably the most direct index of HR deficiency in ovarian cancer. This is also supported by a recent whole-genome sequencing study of ovarian cancer that revealed that chemo-sensitive tumours showed obviously more somatic mutations than chemo-resistant tumours39. The accumulation of indels (insertion-deletion) might be a more direct reflection of HR deficiency. However, according to the TCGA data, ovarian cancer only showed on average 2.3 indels per tumour, and approximately 40% of tumours do not show any indel at all, thus restricting its predictive power.In this study, we first curated a miRNA-target list involved in the DDR, providing a hypothesis-driven signature using the expression of known miRNAs implicated in the regulation of the DDR. Unlike most previous studies, we deliberately avoided machine learning methods and the clinical information of patients to develop the signature to overcome the over-fitting that is commonly seen in most prognostic models37, all of the selected miRNAs for further study were examined prospectively by text-mining and extensive literature studies. Next, 10 miRNAs were extracted and used to generate a 10-miRNA-score in a learning process independent of any connections with clinical data. We demonstrated that this 10-miRNA-score not only predicts the genome instability and HR deficiency in ovarian cancer but also predicts the outcome of ovarian cancer patients treated with platinum agents. Thus, this miRNA-regulatory network involved in the DDR can serve as a useful resource to search for better biomarkers based on DDR deficiency.To avoid the rising concern of reproducibility that stems from the incorporation of unrelated genesIn conclusion, this study strongly suggested that the 10-miRNA-score based on the expression of ten extensively studied miRNAs is correlated with the severity of HR deficiency and provides a prognostic tool to predict the chemotherapeutic effect of ovarian tumours to platinum.http://firebrowse.org). Next, 469 raw MAF files of mutation data were downloaded for ovarian cancer, in which 319 samples have level 3 validated mutations. Samples with level 3 validated mutations were used as the training set to find the miRNAs correlated with genome instability, whereas additional samples with level 2 mutation data were used as an independent validation set. In total, the miRNA microarray data of 570 samples, miRNA-Seq expression of 453 samples, methylation data of 594 samples, clinical information of 591 samples and gene expression of 574 samples were downloaded from the same resource. For the identification of the 17 miRNAs whose expression showed a significant association with the frequency of mutation, the batch-adjusted Agilent 8*15k human miRNA-specific microarray data were used that detects the expression of 723 human miRNAs. Copy number alterations of genes were determined by the GISTIC algorithm and were downloaded together with the results of LOH analysis from the standard analysis of the Broad institute. Germline mutations of BRCA1/2 were obtained from the TCGA publication32. Next, 1,078 miRSeq expression profiles and 977 MAF files of TCGA breast cancer were downloaded from the GDSC Broad institute at 07/Jul/2017.In this study, 602 ovarian cancer samples and eight normal control tissues of ovarian cancer  were used. All the TCGA data were downloaded from the GDSC Broad Institute . If the rank of a sample from the high-score (high-mutation) group was followed by the rank of a sample from the low-score (low-mutation) group, the two samples were selected and divided into the high-score (high-mutation) group and low-score (low-mutation) group separately. Samples that did not meet this criterion were discarded. This resulted in two equal groups of patients with different scores (numbers of mutations) but similar mutation numbers (scores), which we further evaluated by the Mann-Whitney U test.This study was approved by the Ethics Committee of the Harbin Medical University. Informed consent was obtained. All methods were performed in accordance with the relevant guidelines and regulations.The association between the number of mutations or LOH and BRCA1/2 deficiency was evaluated by the non-parameter Wilcoxon rank-sum test or Mann-Whitney U test. To minimize the retrospective optimization, we deliberately used a simple strategy to develop the signature by assigning a point for a miRNA if a tumour shows a higher or lower than median expression of that miRNA. To be consistent, the miRNAs associated with genome instability were identified by partitioning tumours into two groups according to the median value of miRNA expression, and then evaluated the difference of mutation frequency between the two groups using the Mann-Whitney U test. Survival analyses were conducted by the Kaplan-Meier method using the log-rank test. The univariate and multivariate analyses with Cox regression were used to determine the proportional hazards. All statistical analyses were performed using R software. All the statistical analyses in this study were two-sided, or otherwise specified. Significance was defined when p < 0.05.This study was approved by the Ethics Committee of the Harbin Medical University, and informed consent was obtained.All data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplementary InformationSupplementary Dataset 2


To review the evidence on the cost-effectiveness of heart valve implantations generated by decision analytic models and to assess their methodological quality.A systematic review was performed including model-based cost-effectiveness analyses of heart valve implantations. Study and model characteristics and cost-effectiveness results were extracted and the methodological quality was assessed using the Philips checklist.Fourteen decision-analytic models regarding the cost-effectiveness of heart valve implantations were identified. In most studies transcatheter aortic valve implantation (TAVI) was cost-effective compared to standard treatment (ST) in inoperable or high-risk operable patients  and in all studies surgical aortic valve replacement (SAVR) was cost-effective compared to ST in operable patients , but the results were not consistent on the cost-effectiveness of TAVI versus SAVR in high-risk operable patients (ICER range: dominant to dominated by SAVR). Mechanical mitral valve replacement (MVR) had the lowest costs per success compared to mitral valve repair and biological MVR. The methodological quality of the studies was moderate to good.This review showed that improvements can be made in the description and justification of methods and data sources, sensitivity analysis on extrapolation of results, subgroup analyses, consideration of methodological and structural uncertainty, and consistency  of the models. There are several opportunities for future decision-analytic models of the cost-effectiveness of heart valve implantations: considering heart valve implantations in other valve positions besides the aortic valve, using a societal perspective, and developing patient-simulation models to investigate the impact of patient characteristics on outcomes.The online version of this article (doi:10.1007/s10198-017-0880-z) contains supplementary material, which is available to authorized users. The first cost-effectiveness analysis on heart valve implantations was published by Wu et al. . They esIn 2010, the first model-based cost-effectiveness analysis of TAVI compared to ST and SAVR concluded that TAVI had high potential to be cost-effective for inoperable patients, but the cost-effectiveness of patients with lower operable risk was uncertain , 4. BaseSince then almost every cost-effectiveness analysis investigating TAVI based their clinical effectiveness parameters on the PARTNER trial. There are two trial-based cost-effectiveness analyses , 7; the We are currently developing a decision-analytic model to estimate the cost-effectiveness of current and future heart valve interventions  . In thisThe goal of this study is to investigate the opportunities for new decision-analytic models in the field of heart valve interventions and to learn from the methodological choices made by previous model developers. Therefore, and in contrast with previous reviews –13, we fThis systematic review was conducted according to PRISMA guidelines . On May Study and model characteristics and cost-effectiveness results were extracted. Costs were inflated to 2015 and converted to euros(€) using purchaser power parities and exchange rates .Reported cost per quality adjusted life years (QALY) ratios were compared to thresholds used in individual studies and thresholds based on the WHO-CHOICE approach where interventions are highly cost-effective when they have incremental cost-effectiveness ratios (ICER) below the gross domestic product (GDP)/capita, cost-effective if the ICER is 1–3 times the GDP/capita, and not cost-effective when the ICER is more than 3 times the GDP/capita , 18.The ‘Drummond checklist’  and ‘EveThe literature search resulted in 1019 studies, of which 14 studies were included  in three studies , 26, 30,SAVR gains more QALYs at higher costs than ST. According to thresholds used in individual studies and the WHO-CHOICE approach SAVR is (highly) cost-effective compared to ST in all studies.One study evaluated heart valve implantations in the mitral valve position . They foThe assessment of methodological quality of studies using the Philips checklist is reported in Table A2.1 in the Electronic supplementary material. The total score represents the percentage of criteria that were fulfilled, corrected for criteria that were not applicable, and ranged from 49 to 87%. The lowest percentage was found in the study on mitral valve interventions .Even though most studies compared the same heart valve implantations, cost-effectiveness results varied substantially between studies. Based on thresholds from individual studies or using the WHO-CHOICE approach, TAVI was cost-effective compared to ST in inoperable or high-risk operable patients in most studies and in all studies SAVR was cost-effective compared to ST in operable patients. The results were not consistent on the cost-effectiveness of TAVI versus SAVR in high-risk operable patients, ranging from TAVI being dominant to being dominated by SAVR. However, the cost-effectiveness thresholds were relatively high. The thresholds used in individual studies ranged from £20,000/QALY to CDN$100,000/QALY and thresholds based on the WHO-CHOICE approach ranged from 123,264 €/QALY for France to 168,198 €/QALY for the US. When we apply the threshold of the UK , TAVI is cost-effective compared to ST in seven instead of eight  or all (according to WHO-CHOICE approach) studies. Just as with the individual studies' and WHO approach thresholds, SAVR is cost-effective compared to ST in all three studies. Using the UK threshold does not influence our conclusion on the cost-effectiveness of TAVI versus SAVR; it remains not cost-effective in all but one study.Our results did not reflect a clear trend in the cost-effectiveness of heart valve implantations over time; probably due to the short time frame in which the studies were performed (>80% in 2012–2013).There was no correlation between methodological quality scores and ICERs of the included studies (Spearman’s rank correlation coefficients: TAVI vs ST (12 studies) = 0.000, TAVI vs SAVR (7 studies) = −0.126, SAVR vs ST: correlation not determined because there were only three studies in this subgroup). The methodological quality assessment showed that the decision-analytic models were of moderate to good quality. However, authors did not always justify their choices and assumptions and major improvements can be made in the description of methodology. The following discusses our assessment of the methodological quality, structured according to the Philips checklist .Most studies used a healthcare perspective  and two studies used a healthcare payer perspective  , 26. SimThe ICERs are generally the lowest in the UK and the highest in the US. Comparisons of studies within the US showed that the costs of TAVI in Gada et al. , 26 are Many studies combined a short- (often 1 month) and long-term model, mostly decision trees and Markov models. Health states were based on treatment , ward orOnly one study described who was involved in developing the model structure . Two stuSeveral structural assumptions were not reasonable and some might have impacted the cost-effectiveness results. For instance, four studies assumed that valve prosthesis functionality and/or complication rates were similar for TAVI and SAVR , 26, 33 Further, Orlando et al.  assumed Many studies evaluated TAVI, but the implantation routes differed. Most studies investigated transfemoral TAVI (through the leg), while others investigated transapical TAVI (through the chest cavity), or combinations of implantation routes. Further, almost all studies investigated balloon-expandable transcatheter valve prostheses, while one study  evaluateThe definition of ‘standard treatment (ST)’ or ‘medical management (MM)’ differed between studies. In studies based on the PARTNER trial , 31–33 SThe appropriate time horizon when evaluating the cost-effectiveness of heart valve implantations is lifetime, because the interventions affect mortality rates . AlthougCommon practice after heart valve implantations is to schedule follow-up visits at least once a year . TherefoSeveral studies failed to describe their data sources in such detail that replication of the study using the same data would be possible , 25, 26.Since TAVI is a relatively new treatment,  clinical effectiveness data are limited. Therefore, many studies used the PARTNER trial as source for clinical data. This trial consists of two cohorts: PARTNER-A comparing TAVI with SAVR in high-risk operable patients  and PARTThree studies comparing TAVI with SAVR included high-risk operable patients based on the PARTNER-A trial definition , 24, 29.Besides differences between studies, there were differences in baseline characteristics between groups within studies that might have influenced the cost-effectiveness outcomes , 30. ForThe time horizon of most models included in this review is  lifetime, while the follow-up of the clinical trials that are used as input for mortality and complication rates is limited to a few years. Therefore, the included studies needed to make assumptions about survival beyond the trial data, or needed to extrapolate the available data using survival analysis techniques. The extrapolation technique of survival data was reported in most studies  of differences in costs and utilities [The quality of a decision-analytic model does not only depend on the methods of determining the point estimate of the ICER, but also on how uncertainty surrounding this outcome is considered . Paramettilities , 32, 33.tilities  the difftilities , 32, 33 tilities , 32, 33.The studies did not pay much attention to consistency of their models. Only three studies , 23, 32 This review revealed several opportunities for future economic models regarding heart valve implantations.Firstly, gaps in the literature on model based economic evaluations of heart valve implantations can be filled by evaluating cost-effectiveness of heart valve implantations in valve positions other than the aortic valve and by comparing the cost-effectiveness of SAVR with mechanical or biological valves. Both valve types have their own strengths and limitations and there are differences in healthcare use which might influence cost-effectiveness. Further, it would be interesting to investigate how including costs outside of healthcare , such as productivity and informal care costs, would influence the cost-effectiveness of heart valve implantations.Secondly, there are methodological alternatives to the frequently used decision trees and Markov models, such as patient-simulation models. Advantages of patient-simulation models are their ability to incorporate recurrent events and to ‘remember patient history’ without producing unmanageable numbers of health states, resulting in greater flexibility in examining the impact of patient characteristics on outcomes , 49.Thirdly, improvements can be made in the methodological quality of studies by describing and justifying chosen methods and data sources in more detail, performing sensitivity analysis on extrapolation of results, performing subgroup analyses, and considering methodological and structural uncertainty and consistency  of the model.Finally, in this review only two studies used real-world data from patient registries instead of clinical trials , 22. In This study has several limitations. Firstly, we experienced difficulties in using the Philips checklist to assess the methodological quality of the studies. Some criteria are umbrella-criteria that should be assessed differently for different types of data . For many criteria the methods were described but not explained or justified. In these cases we decided that the study fulfilled the criteria but we added a remark that there was no justification reported. Sometimes criteria were partially fulfilled which made it difficult to decide if the criteria should be assessed as fulfilled or not. Therefore, we did not exclude studies with low scores on the Philips checklist. Secondly, it was often difficult to fully understand the details of a decision-analytic model because of space limits on papers.This review provided an overview of the existing decision-analytic models regarding the cost-effectiveness of heart valve implantations. Our results showed that in most studies TAVI was cost-effective compared to ST in inoperable and high-risk operable patients and in all studies SAVR was cost-effective compared to ST in operable patients, but the results were not consistent on the cost-effectiveness of TAVI versus SAVR in high-risk operable patients. This review showed that future models can improve their methodological quality and that there is room for patient-simulation models considering the cost-effectiveness of heart valve implantations in other valve positions besides the aortic valve, performed from a societal perspective.Supplementary material 1 (PDF 305 kb)Below is the link to the electronic supplementary material.


Alu elements are a highly successful family of primate-specific retrotransposons that have fundamentally shaped primate evolution, including the evolution of our own species. Alus play critical roles in the formation of neurological networks and the epigenetic regulation of biochemical processes throughout the central nervous system (CNS), and thus are hypothesized to have contributed to the origin of human cognition. Despite the benefits that Alus provide, deleterious Alu activity is associated with a number of neurological and neurodegenerative disorders. In particular, neurological networks are potentially vulnerable to the epigenetic dysregulation of Alu elements operating across the suite of nuclear-encoded mitochondrial genes that are critical for both mitochondrial and CNS function. Here, we highlight the beneficial neurological aspects of Alu elements as well as their potential to cause disease by disrupting key cellular processes across the CNS. We identify at least 37 neurological and neurodegenerative disorders wherein deleterious Alu activity has been implicated as a contributing factor for the manifestation of disease, and for many of these disorders, this activity is operating on genes that are essential for proper mitochondrial function. We conclude that the epigenetic dysregulation of Alu elements can ultimately disrupt mitochondrial homeostasis within the CNS. This mechanism is a plausible source for the incipient neuronal stress that is consistently observed across a spectrum of sporadic neurological and neurodegenerative disorders.The online version of this article (10.1007/s10577-018-9573-4) contains supplementary material, which is available to authorized users. Alu elements are primate-specific SINEs that are approximately 300 nucleotides in length and are abundant in the human genome, with over 1.3 million elements accounting for at least 11% of overall DNA sequence , transcriptional regulation, and creation of novel response elements ; Dixon et al. Alus are enriched within both TADs and super-enhancer domains (SEDs) supports the hypothesis that Alus directly influence a wide range of critically important processes within primates across multiple levels, from overall genome stability to tissue-specific gene regulation  and a 2.2-fold higher rate in humans when compared to chimpanzee and bonobo  and those without (non-LTR)  and specific Alu subfamilies contain retinoic acid response elements which help to regulate neural patterning, differentiation, and axon outgrowth  modification in order to control their expression and de novo retrotransposition  which help to regulate oxidative stress and metabolic processes in the CNS  are generally conserved  residues to inosine (I) thereby recoding pre-mRNAs  and have greater levels of intranucleotide variation. When considering the functional mechanics of A-to-I editing , and these proteins have distinct tissue-specific expression patterns  A-to-I editing. The GRIA2 protein forms a critical subunit of α-amino-3-hydroxy-5methyl-4-isoxazole propionate (AMPA) receptors, which regulate synaptic calcium and are involved with synaptic plasticity, memory, and learning , and schizophrenia . The interference of A-to-I editing processes associated with the KCNA1 gene  is hypothesized to underlie episodic ataxia type-1 disorder, a disease of the CNS characterized by seizures, stress-induced ataxia, and myokymia  are active throughout neurogenesis and contribute to mosaic neuron genomes that ultimately form the human brain connectome  are major contributors to neurogenesis, perhaps serving to establish the neuronal and biochemical diversity that underlies the ~ 100 billion neuron brain connectome. Remarkably, emerging data suggests that a third Alu-centric process is associated with the formation and function of neurological networks, this being the production of circRNAs that are enriched in the brain and concentrated at synaptic junctions . In light of this observation, it is interesting to note that another, seemingly unrelated, feature of many neurological disorders is mitochondrial dysfunction   Table . KnowingAlu activity concerns metabolic pathways. The efficient processing of glucose and lipids across the CNS is critical for the stability and function of neurons, and the disruption of mitochondrial-mediated metabolic pathways has been linked to many neurologic disorders including AD and peripheral neuropathies  and superoxide dismutase 2 (SOD2) during hyperglycemic conditions , a gene which encodes for a glycoprotein that mediates cholesterol and lipid transport . Although the “amyloid cascade hypothesis” has dominated Alzheimer’s research for decades  homopolymer repeat, a variable stretch of deoxythymidine (T) located within TOMM40 intron 6  are distributed throughout MAPT introns, and A-to-I editing is occurring at 315 Alu-related sites with elevated levels at the 3′ end of MAPT  and evidence of Alu RNAs interacting with tau proteins  A-to-I editing has contributed to non-synonymous amino acid changes. We identified 57 mitonuclear genes with A-to-I editing occurring within putative Alu exons, and in 52 of these genes, the post-transcriptional modification resulted in non-synonymous amino acid changes , it is presently unknown whether or not these processes are actively influencing mitochondrial function. What evidence is there indicating that post-transcriptional modification of mitonuclear genes can alter gene expression or function? Are there particular neurological or neurodegenerative disorders that are associated with mitonuclear genes that have elevated levels of A-to-I editing? To provide insights into these questions, we searched the REDIportal A-to-I editing database , it is possible that these phenotypes arise from deleterious activity operating across tissue-specific gene networks. If correlated with eroding or fluctuating epigenetic control mechanisms of retrotransposons that are associated with aging, cellular senescence, and/or cellular stress .Enhanced somatic retrotransposon throughout neurogenesis contributes to the mosaic brain; however, such activity likely contributes to mosaic pathways leading to disease .


Their mechanism of action is primarily based on the imperfect matching of a seed region located at the 5′ end of a 21-23 nt sequence with a partially complementary sequence located in the 3′ untranslated region of target mRNAs. This leads to inhibition of mRNA translation and eventually to its degradation. Individual miRNAs are capable of binding to several mRNAs and several miRNAs are capable of influencing the function of the same mRNAs. In recent years networks of miRNAs are emerging as capable of controlling key signaling pathways responsible for the growth and propagation of cancer cells. Furthermore several examples have been provided which highlight the involvement of miRNAs in the development of resistance to targeted drug therapies. In this review we provide an updated overview of the role of miRNAs in the development of melanoma and the identification of the main downstream pathways controlled by these miRNAs. Furthermore we discuss a group of miRNAs capable to influence through their respective up- or down-modulation the development of resistance to BRAF and MEK inhibitors. Malignant melanoma is a neoplasm of melanocytes and its incidence has increased dramatically over the past few decades . SurgeryApproximately 50% of patients harbor v-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600 mutations , 8. BRAFAcquired resistance to BRAF inhibitors is usually characterized by reactivation of the MAPK pathway , 14. Furde novo mutations, mutant BRAF V600E amplification or its alternative splicing, MEK1/MEK2 mutations or CDKN2A loss at the basis of resistance [Several studies have been directed to understand the molecular mechanisms of acquired resistance. The first studies conducted on acquired resistance to BRAFi monotherapy identified NRAS or KRAS sistance , 23–25. sistance . Geneticsistance . The scesistance . A more sistance . The anasistance , i.e. sasistance . These sIn our opinion the answer can be found in a better understanding of adaptive epigenetic and/or post-transcriptional mechanisms of resistance. In a significant percent of cases of drug resistant melanomas about 26%) no new mutations have been found % no new . Recent We believe that a variety of post-transcriptional adaptive changes orchestrate the development of drug resistance, which involve also non-coding RNAs. In this context since microRNAs are important multifunctional post-transcriptional modulators of gene expression, which play key-roles in various human cancers , 34 it iDuring the last two decades small non-coding RNAs have been described as the undisputed protagonists of the eukaryotic post-transcriptional machinery regulation . Among tmiRNAs are classified as “intergenic” or “intronic” or on the basis of their genomic location and are mostly situated in clusters . The firCalin and colleagues were the first to report in 2002 miRNA deregulation in human cancer identifying miR-15a/16-1 cluster deletion in chronic lymphocytic leukemia . This deIn the last years several studies have analyzed the involvement of miRNA in the progression of metastatic melanoma –53. We wvs normal melanocytes, which were considered to be associated to the early progression of melanoma. In a second type of analysis the same authors compared the miRNAome of HMB2 melanoma cells before and after stable transfection of an antisense melanoma inhibitory activity (MIA) construct. Here they observed a reverse trend, namely 12 up- and 34 down-regulated miRNAs respectively. Comparison of the first with the second list revealed 18 miRNAs inversely regulated, namely upregulated in early progression and downregulated when MIA was knocked down and viceversa. In a third type of analysis comparison of miRNA expression between primary melanomas and metastatic samples led to the identification of a smaller set of dis-regulated miRNA, namely 11 up-regulated and 2 down-regulated which were thus considered to be involved in metastatic colonization of melanoma cells.Mueller and colleagues were the first to study differential miRNA expression between melanocytes and melanoma cell lines through a microarray-based profiling . Using svs melanocytes. A significant analysis of microarray (SAM) analysis identified 32 most important deregulated miRNAs, of which 13 overexpressed and 19 down-regulated. In addition the Prediction Analysis of Microarrays (PAM) method revealed a minimal miRNA signature composed of 10 miRNAs capable to distinguish the two groups with a prediction accuracy of 100%. Of note, among these miRNAs, miR-126 had been identified in the previous study by Mueller et al [A similar study was carried out by Caramuta et al , who comer et al . Furtherer et al . Among ter et al .versus non non-acral melanomas [Chan et al analyzed the expression of a panel of 384 miRNAs between 42 patient derived primary melanomas samples with three normal melanocyte control samples and found eight miRNAs to be differentially expressed . Of theselanomas . Among telanomas . Of notielanomas .3, which has a well-known role in melanoma progression and invasion. Through experiments of transient in vitro overexpression and luciferase assays, was it possible to link the up-regulation of integrin β3 occurring in melanoma cells to the amount of let-7a present in the cell, thus demonstrating that this miRNA has an oncosuppressive role in melanoma [in vitro, but also in a model of lung colonization in T-cells deficient mice [In addition to large profiling data above described some specific miRNAs have been the object of further investigations during the last years. Let-7a, for example, which is a member of a known family of oncosuppressor microRNAs, has been reported to be downregulated in melanoma cells compared to melanocytes . Moreovemelanoma . Moreovemelanoma . Anothermelanoma . This mimelanoma . In addient mice . Moreoveent mice  whose inConversely the following human poly-cistronic miRNA clusters have been considered to play oncogenic roles in melanoma. The miR-17/92 cluster, for example, codes for miR-17, which has been shown to increase the motility of melanoma cells. miR-17 targets ETV1, which belongs to the ETS (E-twenty six) transcription factor family, which has a suppressive role in melanoma . mir-514The RAS-RAF-MAPK signaling pathway has a central role in sustaining the oncogenic and proliferative phenotype of melanoma cells, as well as in other human cancers . For thiin vitro and in vivo, thus confirming its effect on the inhibition of these two potent oncogenic members of the MAPK pathway [The most thorough analysis of the relationship between the oncogenic BRAF signaling and miRNAs expression and function in metastatic melanoma was carried out by Couts at al . The hig pathway .In conclusion, deregulation of the MAPK pathway and miRNA epigenetic control strongly influence tumor cell fate disrupting normal homeostasis in tumors addicted to this pathway. We represent in the diagram in Figure Both comprehensive miRNAome profiling studies –53, 70 avs melanocytes by Caramuta et al and Chan et al. [Table n et al. , 45; iiin et al. –65; iv) n et al. . In Tabln et al. , 78. As In order to verify if the deregulated miRNAs, reported in Table In summary, our analysis demonstrates that, while in different melanoma samples and cell lines there is a limited or no overlap in the number of individual miRNAs upregulated or downregulated, on the contrary the same miRNAs converge on a constant set of metabolic pathways, which are key to the development of malignancy.Therapy of metastatic cancer is mostly based on the use of chemotherapy and targeted therapies . HoweverGarofalo et al. for example, first showed that the oncogenic miR-221/222 induce TRAIL resistance in aggressive non-small cell lung cancer through the activation of the AKT pathway, by targeting PTEN and TIMP3 tumor suppressors . Furtherin vitro” cell proliferation assays [vs “drug resistant” tumor samples derived from patients.Some miRNAs act as “facilitators” of drug resistance. One such example was provided by Stark et al. who suggested the potential involvement of mir-514a in the modulation of sensitivity to BRAF inhibitors in melanoma cells . These in assays . Howevervia cytokine production with a strong cross-talk with miRNAs deregulation, which act as facilitators of drug resistance. Vergani and colleagues proposed that BRAFi-resistant melanoma cells up-regulate chemokine monocyte chemoattractant protein-1 (CCL2), which in turn activate the expression of miR-34a, miR-100 and miR-125b [Very recently, it has been shown that BRAF inhibitors induce adaptive cell response miR-125b . These mmiR-125b , which imiR-125b . In linemiR-125b . VerganimiR-125b .An opposite mechanism of action has been attributed to miR-200c, member of a family of known oncosuppressive miRNA . This miFinally, our group, which studies the mechanisms responsible for the establishment of drug resistance in melanoma , 32, 113All together these studies point to the involvement of a number of miRNAs either as promoters or as antagonists of resistance  allowed the identification of four genetic subtypes of cutaneous melanoma, BRAF mutant (the most common condition responsible for more than 50% of cases), RAS mutant, NF1 mutant, and Triple Wild-Type . MutatiomiRNA deregulation, albeit heterogeneous in different patients and tumor samples (see Figure in vivo delivery technologies for miRNA mimics capable to restore their normal expression profile in the tumor [Most important for its therapeutic and diagnostic implications is the increasing evidence that miRNA deregulation is heavily responsible for the development of resistance to target therapies. During the development of resistance melanoma select cells with low expression of selected miRNAs, such as miR-200c and miR-579-3p, two potent oncosuppressors acting apparently on different and complementary pathways , 112. Rehe tumor . Finallyhe tumor , 121, dehe tumor –125.


We sought to develop a surgical navigation system using magnetic resonance angiography (MRA) and a three-dimensional (3D) printer for robot-assisted radical prostatectomy (RARP). Six patients with pathologically proven localized prostate cancer were prospectively enrolled in this study. Prostate magnetic resonance imaging (MRI), consisting of T2-weighted sampling perfection with application-optimized contrasts using different flip-angle evolutions (SPACE) and true fast imaging with steady-state precession (true FISP), reconstructed by volume rendering, was followed by dynamic contrast-enhanced MRA performed with a volumetric interpolated breath-hold examination (VIBE) during intravenous bolus injection of gadobutrol. Images of arterial and venous phases were acquired over approximately 210 seconds. Selected images were sent to a workstation for generation of 3D volume-rendered images and standard triangulated language (STL) files for 3D print construction. The neurovascular bundles (NVBs) were found in sequence on non-contrast images. Accessory pudendal arteries (APAs) were found in all cases in the arterial phase of contrast enhancement but were ill-defined on non-contrast enhanced MRA. Dynamic contrast-enhanced MRA helped to detect APAs, suggesting that this 3D system using MRI will be useful in RARP. Prostate cancer is the most prevalent of all cancers in men in Western nations. The incidence and prevalence of prostate cancer have risen steadily across the globe, accounting for 28% and 22.2% of new cancer diagnoses in American men in 2010 and European men in 2008, respectively .Robot-assisted radical prostatectomy (RARP) was first conducted using the da Vinci system in 2000. Since then, RARP has rapidly become the preferred surgical procedure for clinically localized prostate cancer across the globe . While RWhile the gold-standard imaging modality for assessment of vessels is multidetector-row computed tomographic angiography (CTA) , Whang, In this study, we aimed to develop a surgical navigation system using MRI for RARP.Materials and methodsPatient Selection and Clinical CharacteristicsThis prospective study was approved by the institutional review board , and informed consent was obtained. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. In this single-institution study, between August 2016 and January 2017, six patients scheduled for RARP were enrolled  with a spine matrix coil and body matrix coil for signal reception. The sequences were created for navigation and differ from the diagnostic sequence described in the Prostate Imaging-Reporting and Data System (PI-RADS) . All pat3D Workstation and 3D ModelAll MR data were sent to a workstation . Volume rendered (VR) images were created using T2-SPACE, True-FISP, and T1-weighted VIBE with contrast enhancement. Images of the periprostatic blood vessels were generated using True-FISP and T1-weighted VIBE. Standard triangulated language (STL) files were created for 3D printing using a MUTOH personal 3D printer MF 2000 .ResultsFigure The NVBs were detectable in five patients on non-contrast-enhanced MRI scans and in one patient on a dynamic contrast-enhanced MRI. The accessory pudendal arteries (APAs) were detectable in all patients on dynamic contrast-enhanced MRI and in three patients on non-contrast-enhanced sequences. Identification of NVBs and APAs on MRI is shown in Table The images and 3D models of the bladder neck and NVBs were very useful and helped the surgeons to know the shape of the prostate and the course of the NVBs as they performed bladder neck dissection, as well as nerve sparing. However, it was impossible to identify the APAs intraoperatively in these cases. The vascularity of 3D models was overestimated, but the position of the blood vessel could be confirmed and visualized before the operation, which helped surgery.Case 4 had a relatively large prostate. In two patients, the prostate protruded into the bladder (Cases 3 and 5). Case 2 had a history of transurethral resection of the prostate. Four patients required no urinary pads at six months after RARP. Two remaining patients wore one pad per day due to mild incontinence at three months after RARP. In addition, no patients developed severe complications related to imaging and RARP. In Cases 2 and 6, bilateral NVB preservation was planned. However, the erectile function of Cases 2 and 6 was already abnormal before RARP, and it had not recovered six months after surgery.RARP is a widely accepted surgical approach for the treatment of prostate cancer. Due to advances in optics, video technology, and specialized instrumentation, the use of RARP has the advantage of minimal invasiveness while clearly displaying the periprostatic anatomy. The use of video-endoscopy allows better preservation of the structures around the urinary sphincter, such as APAs, improved apical dissection, and preservation of the NVBs -13The proportion of men with APAs varies according to the method used to detect them. Incidence has ranged from 4%–75% in radiological and pathological studies , 13-18. Non-contrast MRA has a longer imaging time than dynamic contrast-enhanced MRA and is inferior to dynamic MRA in the detection of NVBs and APAs due to the influence of motion artifact due to rectal peristalsis. In our study, the NVBs and APAs were detected on dynamic MRI in all six patients. Dynamic MRI has been widely used to detect prostate tumors and to evaluate tumor stage . FurtherThis study has several limitations. First, we can’t identify APAs intraoperatively. Since 60% of APAs are the apical type, it is often impossible to confirm APAs during surgery . HoweverPreoperative dynamic contrast-enhanced MRA provided good surveillance of the tissues surrounding the prostate, the NVBs, and APAs with the use of a 3D model and is a useful diagnostic option that may improve potency outcomes when used with RARP.


Host–guest assembly at the interface of microfluidic droplets offers a versatile strategy to construct supramolecular hydrogel microcapsules with “smart” cargo release. e.g. agrochemicals, nutraceuticals or cosmetics.Host–guest assembly in droplet-based microfluidics opens a new avenue for fabricating supramolecular hydrogel microcapsules with high monodispersity and controlled functionality. In this paper, we demonstrate a single emulsion microdroplet platform to prepare microcapsules with supramolecular hydrogel skins from host molecule cucurbit[8]uril and guest polymer anthracene-functionalized hydroxyethyl cellulose. In contrast to construction of microcapsules from a droplet-in-droplet double emulsion, here the electrostatic attraction between charged polymer and surfactant facilitates formation of defined supramolecular hydrogel skins in a single emulsion. Furthermore, by taking advantage of dynamic interactions and the tunable cross-linked supramolecular hydrogel network, it is possible to prepare microcapsules with triggered and UV-controlled molecular permeability. These could be potentially used in a delivery system for  Although powerful, the requirement for multiple immersion-centrifugation/filtration-washing steps render the preparation process both time and material intensive.22There has been a significant growth in the study of microcapsules, no doubt inspired by their numerous applications in fields ranging from biotechnology and biopharmaceuticals, the food industry and even incorporation within electronic displays.25 The monodisperse nature of droplet production in a microfluidic device provides a unique reaction environment for the formation of microcapsules with very low polydispersity and high fabrication throughput, with efficient use of reagents.26 However, the reported microfluidic methods are mostly based on engulfment of templates by a hydrogel shell, generation of ‘droplet-in-droplet’ double or triple emulsions, followed by solidification of the outer layer of the droplets.28 Introduction of colloids to produce a Pickering emulsion, where colloids can adsorb to the water/oil interface, for subsequent polymeric cross-linking is an alternative way to construct colloidal hydrogel microcapsules.30 However, the hydrophobicity, shape, and size of the colloids must be carefully designed.Droplet microfluidics is a microchip-based emulsion technique which has been extensively applied over the past decade in the preparation of hydrogel microcapsules.33 The supramolecular polymer hydrogels exhibit properties of traditional polymer hydrogels such as high water content and environmental sensitivity.35 Moreover, in view of the dynamic nature and reversibility of non-covalent interactions, the supramolecular hydrogels possess new functionality with potential for processability, recycling, and self-healing.38In this paper, we employ single emulsion droplet microfluidics to fabricate supramolecular hydrogel microcapsules using macrocyclic host–guest complexation, and further extend their stimulus responsive properties. Recent developments of supramolecular assembly have offered a framework to design advanced functional hydrogels.2 homoternary complexes with monocationic guests or 1 : 1 : 1 heteroternary complex with both a dicationic and a neutral guest.47 The head-to-tail arrangement of two anthracene moieties within the cavity of CB[8] enables multiple non-covalent cross-links to form between adjacent anthracene-functionalized polymer chains,44 forming supramolecular polymer hydrogel skins at the water-in-oil interface of single microdroplets. This interaction can be disrupted by the introduction of a competitive guest, that displaces anthracene from within CB[8] giving a method to rupture the microcapsule and release encapsulated cargo.Herein, we utilize supramolecular host–guest assembly between the macrocyclic host cucurbit[8]uril (CB[8]) and the guest of anthracene-functionalized hydroxyethyl cellulose (Ant-HEC) polymers  to fabriSignificantly, exposure of the pre-organized anthracene guests within the CB[8] host to UV light, stimulates photo-dimerization to generate a fully covalent microcapsule architecture. In this way, the supramolecular assembly can be used for fabrication of chemically stable, covalent microcapsules with different storage profiles compared to the dynamic non-covalent system. Adding the ability to determine the extent of covalency present in the microcapsule skin, we illustrated the use of precisely-controlled single emulsion microdroplet templating for tailored supramolecular cross-links. It is expected that this strategy would expands the potential of an already powerful microcapsule platform with new capabilities in the storage and delivery of a range of cargoes.To fabricate supramolecular hydrogel microcapsules, monodispersed aqueous microdroplets containing a water soluble Ant-HEC and the macrocyclic host, CB[8], were generated in perfluorinated oil within a microfluidic device consisting of three inlets meeting at a single flow focus. This produced water-in-oil microdroplets in a single step at a frequency of 30 Hz . Flourin–1 and 60 μL h–1 respectively. The microdroplets exhibited a low level of polydispersity The microdroplets were collected and air dried, forming stable dehydrated microcapsule structures that can be stored  as molecular probes. After rehydrating FITC-dextran-loaded microcapsules in water, they were studied by fluorescence microscopy (excitation at 470–495 nm and detection above 520 nm to avoid overlap with the fluorescence of the anthracene moiety). Upon rehydration in water, the size of the microcapsules encapsulating FITC-dextran (500 kDa) approximately doubled  due to o49 thought to be close to the pore size of the microcapsule skin and therefore not able to readily diffuse out of the microcapsule.To evaluate the molecular permeability of the supramolecular hydrogel microcapsules, the release of FITC-dextran cargoes of different molecular weights was studied over time. The 150 kDa and 250 kDa FITC-dextran were released rapidly and quantitatively, with 80% and 70% release, respectively, of the total loading within the first hour . In cont2 microcapsules can be released by treatment with a higher affinity ligand for CB[8] such as 1-adamantylamine , hydrated microcapsules undergo rupture after 3 minutes (2 hydrogel complexes.The cargo in the CB[8]·(Ant-HEC)ine ADA, .50 On ex minutes  accompan minutes . This tr2 microcapsules offer triggered release of their cargo, their chemistry also allows for the formation of progressively covalently crosslinked hydrogels with the ability to control the molecular permeability of the microcapsules. This is achieved by taking advantage of the [4 + 4]-photo-dimerization of the two anthracene moieties pre-organized as guests in the CB[8] (Not only do CB[8]·(Ant-HEC)he CB[8] . Fig. 6EWe have demonstrated a host–guest assembly approach to prepare supramolecular hydrogel microcapsules through a homoternary complex from CB[8] and Ant-HEC polymers in microfluidic droplets. The electrostatically-directed attraction between the supramolecular polymer in water and the surfactant in the surrounding oil facilitates fabrication of defined supramolecular hydrogel skins from a single emulsion. Disruption of the supramolecular interaction leads to rupture of the hydrogel network, achieving triggered molecular permeability. In contrast, irradiation with UV light resulting in [4 + 4]-photo-dimerization of the anthracene guests. This converts the supramolecular hydrogel network from a non-covalent to covalent structure post-fabrication, offering a mechanism to control the permeability of the microcapsule. This increases the range of potential applications for these self-assembled hydrogel microcapsules with dramatically different architectures produced from small changes in simple parameters during fabrication or with post-processing.


Six female patients with bilateral medial knee OA and 6 healthy controls were recruited. Patients with knee OA received a 6-week physiotherapist-supervised and home-based exercise program. Outcome measures, including the Western Ontario and McMaster Universities Arthritis Index and Short Form-36 Health Survey as well as objective biomechanical indices were obtained at baseline and follow-up. After treatment, no significant difference was observed in the knee abductor moment (KAM), lever arm, and ground reaction force. We, however, observed significantly improved pain and physical function as well as altered gait patterns, including a higher hip flexor moment and hip extension angle with a faster walking speed. Although KAM was unchanged, patients with bilateral knee OA showed an improved walking speed and altered the gait pattern after 6 weeks of supervised exercise. This finding suggests that the exercise intervention improves proximal joint mechanics during walking and can be considered for patients with bilateral knee OA. Non-weight-bearing strengthening without external resistance combined with stretching exercise may be an option to improve pain and function in individuals with OA who cannot perform high resistance exercises owing to pain or other reasons. Because of the aging of the population, the prevalence of symptomatic knee osteoarthritis (OA), a common degenerative joint disease in the elderly population, has increased . ApproxiTherapeutic exercise not only reduces OA symptoms but also delays disease progression and is thus beneficial to patients with knee OA , 11. TheAn increased knee abductor moment (KAM) is strongly correlated with increased compressive medial compartment load , knee paThe quadriceps and hamstrings, which act primarily in the sagittal plane, probably generate minor effects in the frontal plane; thus, targeting hip abductor musculature may be another biomechanically based exercise option for medial knee OA . This coBecause alteration in knee joint mechanics because of a disease and/or intervention may cause biomechanical changes in the adjacent joints, changes in joint mechanics at the hip and ankle are also crucial to gait improvement. Similar to Bennell et al. , Lim et In the current study, 6 patients with medial knee OA were recruited by a physician (LFH) and were included in the OA group. These patients met the following inclusion criteria: (a) bilateral medial knee OA, (b) Kellgren/Lawrence grade  2 or 3 aThe Western Ontario and McMaster Universities Arthritis Index (WOMAC) was used to assess symptomatic severity in patients in the OA group. The WOMAC is a disease-specific, self-administered questionnaire for patients with knee OA that is composed of 3 dimensions (with 24 items): pain (5 items), stiffness (2 items), and physical function (17 items). The results are scored on a 0–20 scale for pain, 0–8 scale for stiffness, and 0–68 scale for physical function. Higher scores indicate a higher degree of pain, stiffness, or physical dysfunction. The quality of life of patients in the OA group was measured using the 36-item Short Form-36 Health Survey (SF-36) , which iThe objective of the exercise program was to improve lower extremity strength and flexibility . During The gait of patients was analyzed at baseline and at 6 weeks after the completion of the exercise program (follow-up). Patients walked at a self-selected pace on an 8 m walkway while infrared retroreflective markers were attached to the posterior superior iliac spine, anterior superior iliac spine, greater trochanters, midthighs, medial and lateral femoral epicondyles, tibial tuberosities, fibular heads, medial and lateral malleoli, heels, navicular tuberosities, and 2nd metatarsal bases [x-axis was directed anteriorly, the positive y-axis superiorly, and the positive z-axis to the right [z-x-y) [For dynamic analysis, the pelvis-leg apparatus was modeled as a 7-link system, with each link embedded with an orthogonal coordinate system. Following the recommendation of the International Society of Biomechanics, the positive he right . The rot [z-x-y) . By perf [z-x-y) . The froThe maximal value of KAM during early stance phase was defined as the first peak KAM. The maximal value of KAM during late stance phase was defined as the second peak KAM. The peaks of KAM during early (first peak at P1) and late stance phases (second peak at P2) and the corresponding magnitude of the frontal GRF and its lever arm available at the knee were extracted. The joint angles and moments at the hip, knee, and ankle joints when the first and second peak KAM occurred were also extracted for subsequent statistical analysis. U test was used to investigate the differences between patients with OA and controls at baseline and follow-up. All statistical analyses were performed using SPSS . All significance levels were set at α of 0.05.A Wilcoxon test was used to investigate the effect of 6-week exercise on SF-36 and WOMAC scores and on all calculated biomechanical variables at baseline and follow-up. A Mann-WhitneyPt < 0.05) and pain (Pt < 0.05) and unchanged scores for RP, BP, VT, SF, RE, and MH . After treatment, the results of WOMAC showed unchanged scores for stiffness, but significantly lower scores for pain and physical function were found compared with those obtained before treatment .The mean percentage of home-based exercise sessions completed was 84%. None of the participants received medication during the evaluation and training periods. Only one participant sought a cointervention , and the data of this patient were excluded from statistical analysis. After the 6-week exercise program, the results of SF-36 showed significantly lower physical functioning (Pg = 0.03, Pg > 0.05, Baseline evaluation of temporal-spatial parameters of walking revealed no significant between-group differences in step length and step width, and the OA group displayed a longer walking time and a significantly slower walking speed (For the group effect at baseline and follow-up, a significant difference was observed in the first KAM  and its Baseline evaluation revealed no between-group difference in the hip, knee, and ankle joint angles in the frontal plane or in the hip and ankle joint moments in the sagittal plane at P1  and P2 . Follow-This study evaluated the effect of a 6-week supervised exercise program that included strengthening and stretching in patients with bilateral medial knee OA. The results showed that specific gait adaptations, including a significantly increased hip flexor moment and faster walking speed, were observed after the intervention, even though knee loading was not reduced compared with that at baseline. Significantly lower scores of physical function in both SF-36 and WOMAC as well as decreased pain suggested that patients with knee OA responded favorably to the exercise program used in the current study.Performing resistance lower extremity exercises can aggravate OA knee symptoms , 33. AltExercise is routinely prescribed for patients with knee OA. A systematic review reported that a general exercise program for patients with knee OA should include strengthening and stretching . Hence, For the temporal-spatial variables, the comparison between pre- and postevaluation time in the current study revealed an increased step length and walking speed . At baseAlthough harmful KAM was not reduced after 6 weeks of exercise, specific gait adaptations, including an increased hip flexor moment and hip extension angle, were found at P2 , which mEvidence has indicated that the level of patient self-reliance required to sustain the exercise program through a home-based program determined the exercise effect . Hence, This study had several limitations. First, the lack of a control OA group may have limited the validity of the conclusions of the current study. Nevertheless, the biomechanical endpoints were nonsubjective, and these are known to be stable over time in untreated individuals; hence, the objective gait adaptation was likely attributed to the biomechanical exercise intervention rather than to a placebo effect. The current findings showed some significant changes in gait after 6 weeks of exercise. For the first time in the literature, the observed gait adaptation indicated the effect of exercise on 3-dimensional joint mechanics at P1 and P2 and at the lever arm of the GRF at the knee in patients with knee OA, comprehensively illustrating the gait pattern before and after exercise. Additional studies are required to determine how long the acquired gait patterns can be maintained if exercise is discontinued. Future studies should use larger sample sizes. Moreover, because we only included level walking when assessing gait patterns, additional studies that include more challenging tasks such as staring and obstacle crossing should be conducted. Nevertheless, based on the various exercise types proposed in the literature, the current study was the first to provide beneficial evidence of non-weight-bearing strengthening without external resistance combined with stretching exercise for patients with knee OA.The effect of exercise on walking was investigated in patients with bilateral medial knee OA. Although peak KAM was unchanged, our 6-week exercise program improved pain and physical function and altered gait patterns, producing results such as a higher hip flexor moment and hip extension angle with faster walking speed. This finding indicated that the exercise program had positive effects on patients with bilateral medial knee OA. Therefore, a supervised exercise program that monitors the overall lower extremity muscular conditions and includes strengthening and stretching 5 days per week over 6 weeks may generate biomechanical benefits with clinical improvement. Non-weight-bearing strengthening without external resistance combined with stretching exercise improves pain and function with a higher walking speed and altered gait patterns at the hip in individuals with OA. Such an exercise program should thus be encouraged in patients with bilateral medial knee OA.


Retinal vein occlusions are the second most common form of retinal vascular disease. Previously, laser treatment for branch retinal vein occlusion and intravitreal triamcinolone acetonide for central retinal vein occlusion were the standard of care. Recent studies have demonstrated that anti-vascular endothelial growth factor (anti-VEGF) agents have a superior safety and efficacy profile for the treatment of both branch and central retinal vein occlusions. The use of wide-field fluorescein angiography has also allowed better visualization of the retinal periphery. Despite the better documentation of retinal non-perfusion, laser photocoagulation to the areas of non-perfusion does not seem to result in a reduction of macular edema or reduction in treatment burden and has been relegated to patients who develop rubeosis or neovascularization of the retina. More recently, several studies have demonstrated the use of a long-acting dexamethasone implant administered intravitreally or triamcinolone administered in the suprachoroidal space as a viable approach to treat retinal vein occlusion. There are two major anatomic forms of retinal vascular occlusions: branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO). Furthermore, retinal vein occlusions (RVOs) can be classified as ischemic and non-ischemic occlusions, depending on the degree of non-perfusion based on the fluorescein angiogram2. Wide-field angiography has become more available and is an enhanced method of determining non-perfusion.Retinal vascular occlusions are the second most common form of retinal vascular disease after diabetic retinopathy3.BRVO is three to four times more common than CRVO and often occurs at the crossing of an artery and a vein. CRVO has a poorer prognosis than does BRVO, and ischemic CRVO has a poorer prognosis than does perfused CRVO. CRVO usually occurs from a thrombus in the central retinal vein at the level of the lamina cribrosa in the optic nerve5. BRVO treated in the SCORE study with 1 or 4 mg of triamcinolone versus focal laser showed that all groups gained three lines of vision in a similar fashion. CRVO treated in the SCORE study showed three lines of improvement with both 1 mg (21%) and 4 mg (26%) triamcinolone acetonide versus 7% with observation. The complication rates of cataracts and increased intraocular pressure were higher in the 4 mg triamcinolone group versus the 1 mg group. Despite the results of the SCORE study, intravitreal anti-vascular endothelial growth factor (anti-VEGF) therapy has replaced corticosteroids as the treatment of choice for patients with CRVO because of a more favorable side effect profile.A National Eye Institute-sponsored SCORE  study compared 1 and 4 mg of preservative-free intravitreal triamcinolone acetonide versus observation in patients who had both BRVO and CRVO7. At 180 days, 41% and 40% of eyes receiving 0.7 or 0.35 mg of dexamethasone, respectively, improved 15 letters versus 23% of sham.The dexamethasone implant  is a biodegradable form of micronized dexamethasone that releases 700 mg of dexamethasone over several months and is inserted in the office through a 23-gauge needle puncture. Two multi-center trials included patients with CRVO and BRVO8. An important learning point from this study was that frequent injections and early treatment led to better visual acuity results with all anti-VEGFs.Although there have not been head-to-head studies comparing all three anti-VEGF medications, the SCORE2 study recently investigated six monthly injections of bevacizumab or aflibercept and found no significant difference in the visual result when both were administered monthly for six months9, a six-month study that randomly assigned patients with macular edema due to BRVO to 0.5 mg of ranibizumab or 1.25 mg of bevacizumab. At six months, the mean gains in visual acuity were 18.1 letters for the ranibizumab group and 15.6 letters for the bevacizumab group.The SCORE2 study confirmed the MARVEL study10. Several studies have examined retinal photocoagulation to the areas of peripheral non-perfusion in eyes with CRVO and macular edema and showed no improvement in macular edema11.Laser photocoagulation therapy was previously considered a treatment of choice for macular edema secondary to BRVO11.In the RELATE trial, subjects received 0.5 or 2 mg of ranibizumab for six months and then were randomly assigned to ranibizumab with laser photocoagulation or ranibizumab only. There was no long-term benefit in visual acuity, macular edema, or number of injections needed by the addition of laser treatment to ranibizumabPanretinal photocoagulation therapy (PRP) is still being used for the treatment of neovascular complications of CRVO such as neovascularization of the retina, iris, or angle. Owing to the emergence of anti-VEGF therapy, PRP is being used less frequently.Several surgical treatments have been attempted, but none has undergone strict scrutiny in randomized clinical trials. In addition, most studies were done before the availability of anti-VEGF drugs.Radial optic neurotomy. In this approach, an incision is made in the optic nerve and adjacent retina with a microvitreoretinal (MVR) blade during pars plana vitrectomy12. The original idea was to reduce the congestion of the optic nerve by opening the scleral canal, although it has also been hypothesized that this procedure may allow the formation of a retinal-choroidal anastomosis. There have been mixed results of its safety and efficacy13, and this procedure has fallen out of favor and is rarely performed.Surgical formation. Surgical formation of retinal-choroidal anastomosis, either by laser14 or directly at the time of vitrectomy, has also been advocated for perfused CRVO and has shown favorable results in some patients. The potential complications, however, include vitreous hemorrhage and neovascularization. No long-term follow-up or randomized clinical trials have been performed.Vitrectomy. Vitrectomy with internal limiting membrane and panretinal endophotocoagulation for macular edema secondary to CRVO has not shown any improvement16.Arteriovenous sheathotomy. Arteriovenous sheathotomy for BRVO has been advocated by some to release the arteriovenous adhesion and decompress the vein at an arteriovenous crossing point18. Hypertension and atherosclerotic changes to the arteriole may impinge the vein and lead to thrombus formation. The surgery involves performing a vitrectomy and then using either scissors or a bent MVR blade to cut the sheath at an arteriovenous crossing point until the artery becomes mobile. If done early before there is complete sclerosis of the vein, the procedure has shown an improvement of visual acuity in some patients20. Arteriovenous sheathotomy has shown functional and anatomic outcomes similar to those of intravitreal triamcinolone in a comparative trial21.Tissue plasminogen activator. Tissue plasminogen activator (tPA) has also been used, both intravitreally as well as through direct injection into a cannulated retinal vein25. This approach has not been compared with medical management in a comparative trial.26, or hemodilation27, have shown variable results, not allowing a recommendation. Unfortunately, no significant improvements have been found.Case reports of patients with RVO treated with anti-thrombotic or thrombolytic medications, including clopidogrel, tPA , heparin, aspirin, low-molecular-weight heparin31.Several systemic conditions have been associated with retinal vascular occlusions, including hyperviscosity and hypercoagulability conditions: specifically, protein C and protein S deficiency, prothrombin gene mutation, anti-thrombin abnormalities, anti-phospholipid syndrome (anti-cardiolipin and lupus anti-coagulant), factor V Leiden deficiency, and hyperhomocysteinemia32, and blood lipid disorders have been identified as risk factors36. In a meta-analysis, however, only hyperhomocysteinemia and anti-cardiolipin antibodies were significantly associated with RVOs. With RVO and no other history of vascular occlusions, it is rare to find a coagulation defect. In younger patients (<50 years old), a work-up for underlying hypercoagulable disease should be considered.Hypertension, diabetes mellitus, renal disease, atherosclerosis, glaucoma37. Similarly, a meta-analysis association was found for an increased risk of acute myocardial infarction38. It is prudent to recommend that patients with retinal vascular occlusions have their primary care providers evaluate and optimize their cardiovascular risk factors.Recently, a meta-analysis of patients with both BRVO and CRVO showed an increased risk of stroke, especially in subjects between 50 and 69 years of age39. Anti-VEGF therapy is now the treatment of choice for retinal venous occlusive disease.Currently, there are three anti-VEGF drugs that are available to treat both CRVO and BRVO. Both ranibizumab and aflibercept are US Food and Drug Administration-approved, while bevacizumab is an off-label use of an anti-VEGF drug approved for the treatment of metastatic colon cancer41. Bevacizumab has been shown to be an effective off-label anti-VEGF for controlling macular edema associated with RVOs.Bevacizumab  is the off-label use of the humanized monoclonal antibody that binds all forms of VEGF-A and has been approved only for the treatment of metastatic colon cancer. The drug is compounded in small aliquots and is used in the treatment of vein occlusions as well as diabetes and age-related macular degenerationRanibizumab  is a monoclonal Fab fragment which binds all forms of VEGF. Randomized phase III clinical trials using ranibizumab have been performed to assess the safety and efficacy of ranibizumab for the treatment of macular edema secondary to RVO.pro re nata (prn) treatment showed stabilization of vision, although laser treatment was added in almost 50% of patients42.Ranibizumab for the treatment of macular edema following BRVO has been studied in the Efficacy and Safety of Ranibizumab Injection in Patients with Macular Edema Secondary to Branch Retinal Vein Occlusion (BRAVO) trial. In this study, 55% and 61% of patients receiving 0.3 and 0.5 mg ranibizumab, respectively, experienced a three-line improvement in vision compared with 29% in the control group. Continued43.Ranibizumab for the treatment of macular edema following CRVO has been studied in the Ranibizumab for the Treatment of Macular Edema after Central Retinal Vein Occlusion Study: Evaluation of Efficacy and Safety (CRUISE). A total of 392 patients with macular edema secondary to CRVO were randomized to 0.3 mg of ranibizumab, 0.5 mg of ranibizumab, or sham injection. At 6 months, 46% and 48% of patients in the 0.3 and 0.5 mg groups, respectively, showed three lines of improvement of vision versus 17% in the control group44.Aflibercept  is a fusion protein with portions of VEGF receptor 1 and 2 bound by a fragment crystallizable (FC) portion. Intravitreal aflibercept binds to the isoform of human VEGF-A and placental growth factor (PIGF) with a higher affinity than does ranibizumab. The VIBRANT trial evaluated the efficacy of aflibercept over grid laser treatment in patients with BRVO and macular edema. A total of 57% of affected eyes treated with aflibercept gained three lines or more of vision. The study also showed that after monthly injections for six months, less frequent injections could still maintain vision46.The VEGF Trap-Eye: Investigation of Efficacy and Safety in Central Retinal Vein Occlusion (COPERNICUS) and the General Assessment Limiting Infiltration of Exudate in Central Retinal Vein Occlusion with VEGF Trap-Eye (GALILEO) studies both evaluated the use of aflibercept in the treatment of macular edema from CRVO. In both studies, over 50% of treated eyes compared with 12% of control eyes gained three lines of vision47. Smaller studies have confirmed or demonstrated that delay in treatment is associated with decrease in visual improvement compared with prompt treatment.There have been few recent advances in the treatment of BRVO. The sustained benefits of ranibizumab for 24 months in the BRIGHTER study confirmed visual superiority of ranibizumab or ranibizumab plus laser (14.8 letters) versus laser for six months and then ranibizumab as needed after (+6 letters)48. Disorganization of the retinal inner layers is a predictor of subsequent visual acuity improvement or decline following the first three monthly injections in patients with macular edema49.Several studies have evaluated predictors of macular edema recurrence and visual acuity in patients with BRVO. Recurrence of macular edema has been shown to be associated with the degree of non-perfusion of the central 1 mm ETDRS (Early Treatment Diabetic Retinopathy Study) circle or with an initial central retinal thickness of more than 57050.RVO-associated macular edema may be refractory to treatment with an anti-VEGF agent. Risk factors for suboptimal response include older age, shorter occlusion distance from the optic nerve, longer pre-treatment duration, and larger areas of non-perfusion. A study of eyes with macular edema from RVO that were refractory to treatment with an anti-VEGF agent revealed that treatment with a long-acting dexamethasone implant showed a small improvement in both optical coherence tomography (OCT) and vision51.A small study investigated ranibizumab, aflibercept, or dexamethasone implant injections in patients with suboptimal response to bevacizumab. Patients were assessed every three months for a year. At month 12, vision improved in 59% of patients. There was no difference between each therapy, but the number of injections varied from 3.30 for dexamethasone to 6.50 for aflibercept and 8.27 for ranibizumab52. Other studies have suggested that the deep capillary plexus may be more severely affected than the superficial plexus53. It has been shown that eyes with a reduction in vessel density of the deep plexus compared with the superficial plexus did not recover after a dexamethasone implant54.Optical coherence angiography has given us an opportunity to study the superficial and deep capillary plexus in patients with retinal vascular diseases. The degree of perifoveal capillary non-perfusion has been correlated with visual functionet al. showed that combination therapy with an anti-VEGF agent and dexamethasone implant led to a mean re-injection interval of 135 ± 36.4 days for patients with macular edema secondary to CRVO and BRVO as well as improvements in visual acuity and central foveal thickness55. Unfortunately, there was no control group. However, combination therapy remains a possibility for difficult-to-treat eyes.Combination of an anti-VEGF and a corticosteroid drug for the treatment of RVO has been advocated, but there is a paucity of long-term studies supporting this approach. A study by Singer56. This finding supports the results of the aflibercept COPERNICUS and GALILEO studies showing that a delay in treatment results in a decrease in eventual visual improvement compared with early treatment.Recent studies have shown that VEGF suppression reduces macular edema and prevents vessel closure by leukocytes57. The use of wide-field fluorescein angiography has allowed us to better determine capillary non-perfusion and the risk of developing neovascular complications.The degree of retinal non-perfusion may be prognostic for the chance of developing new vessels. The odds of developing neovascularization go from 0% with less than one disc area (DA) of non-perfusion to an 80% risk with 75 to 150 DA of non-perfusionThere is ample hope that new anti-VEGF drugs will be coming to market in the near future.58. Conbercept blocks all VEGF-A isoforms as well as VEGF-B, VEGF-C, and PIGF.Conbercept (KH902) has already shown significant improvements in patients with RVO in ChinaBrolucizumab  has shown preliminary efficacy and safety and appears to be a strong anti-VEGF drug designed for the treatment of neovascular age-related macular degeneration. A vein occlusion trial for this drug will be initiated soon.Nanoparticles, liposomes, and other drug delivery systems hopefully will allow less-frequent injections of anti-VEGF agents or corticosteroids. Special needles and devices to allow more predictable penetration of a retinal vein are being developed with the hope that they may be used to inject tPA and other agents directly into the area of occlusion.59. At three months, the combination arm showed an increase in visual acuity and improved OCT compared with the aflibercept-alone cohort. A total of 74% of the combination-treated group did not receive any additional treatment over nine months.Clearside Biomedical Inc.  has introduced a novel approach to the treatment of RVOs, using a combination of a suprachoroidal delivery system for the delivery of corticosteroids and intravitreal aflibercept, to improve vision and decrease the treatment burden. In the TANZANITE study, 46 treatment-naïve patients with RVO received intravitreal aflibercept alone or the combination of aflibercept and concomitant suprachoroidal delivery of triamcinolone acetonideIn conclusion, RVOs continue to be a commonly encountered retinal condition. With the advent of improved therapies, we now have the ability to treat the secondary complications of neovascularization and macular edema. Future advances will allow for more effective treatments while hopefully minimizing the treatment burden for our patients.