Small RNAs regulate a large set of gene expression in all plants and constitute a natural immunity against viruses. Small RNA based genetic engineering (SRGE) technology had been explored for crop protection against viruses for nearly 30 years. Viral resistance has been developed in diverse crops with SRGE technology and a few viral resistant crops have been approved for commercial release. In this review we summarized the efforts generating viral resistance with SRGE in different crops, analyzed the evolution of the technology, its efficacy in different crops for different viruses and its application status in different crops. The challenge and potential solution for application of SRGE in crop protection are also discussed. Modern plant virology commenced at the end of 19th century with the research on tobacco mosaic disease done by Russian scientist Dmittrii Iwanowski and Dutch microbiologist Martinus Beijerinck who discovered the causal agent was much smaller in size compared to other microbes because it can pass bacteria-proof filter candle . Later oPlant viruses impose serious threats to wide range of crops in modern agriculture and it is estimated that economic loss caused by viral pathogen ranks the second compared to those caused by other pathogens . DependiPlant viral disease significantly reduces crop quality and yield. It has been estimated that potato leaf role virus (PLRV) resulted in 20 million ton losses in potato production worldwide annually . In mostDue to their devastating threat to crop production, plant viruses has been studied extensively since the first virus, TMV, was discovered. The outcome of a virus infection on a plant is determined both by the genotype of the virus and that of the plant. The plant genetic architecture conferring resistance/tolerance to viruses usually includes so called recessive resistance and active defense. Recessive resistance is usually conferred by lacking positive host factors for virus propagation and accounted by many excellent reviews . In contFigures 1A,B). At the molecular and cellular level, once a virus particle, such as TMV, gets into a host cell, it has to be disassembled to release its genomic (g)RNA. The gRNA then serves as mRNA to produce viral replicase protein, which in turn transcribe gRNA into a complementary (c)RNA and further transcribe more gRNA and subgenomic (sg)RNA using cRNA as template. The amplified gRNA can participate in at least four possible pathways: replication, translation, cell-to-cell movement and assembly .Viruses are obligate intracellular parasites and complete their life cycle in living host cells. Plant viruses usually enter plant cells through wounds made by insect vectors or mechanic rubbing, replicate in the initial infected cells, move from cell to cell via plasmadosmata, and spread via phloem into newly emerged young tissue and organ, where they cause disease phenotype and became ready to exit and infect other host plants  that “Key gene products from the parasite, if present in a dysfunctional form, in excess, or at the wrong developmental stage, should disrupt the function of the parasite while having minimal effect on the host” . It is aRNA silencing refers to small interfering (si)RNAs or micro(mi)RNAs mediated sequence specific gene silencing mechanisms, which play important role in antiviral defense, development, and maintenance of genome integrity . In planArabidopsis many studies have shown that DCL2, DCL4, AGO1, AGO2, RDR1, and RDR6 are the major components in antiviral RNA silencing - triggered immunity (PTI) in plants . In lineFigures 2A; Supplementary Table S1). These efforts can be categorized into four groups based on the mechanisms by which antiviral silencing is activated, sense gene induced post-transcriptional gene silencing (S-PTGS), hairpin RNA induced PTGS (hp-PTGS), artificial miRNA induced PTGS (AMIR), and trans-acting siRNA induced PTGS (TAS) .RNA silencing has been deployed in crop improvement for viral resistance along the way it has been discovered. Successful resistance was achieved with either full length cDNA encoding functional viral products, or partial, or mutated viral cDNA  (Supplementary Table S1). Transgenic lines that accumulated high level of viral siRNA and established silencing state in absence of viral infection usually are immune or highly resistant to viral infection (Supplementary Table S1).sistance . Inspiresistance . The silgure 2B) . Protectnfection . On the nfection . Since enfection , 1997. SHp-PTGS is the second generation technology developed after dsRNA was recognized as the trigger of RNA silencing. In these practices, researchers constructed silencing vectors with pieces of both sense and antisense viral cDNA under control of plant promoters and terminators. When transformed into plants, these constructs produce transcripts that can fold into dsRNA due to the complementarity of sense and antisense viral sequences in it. The dsRNA is then processed into siRNAs and confers resistance/immunity to cognate viruses . The first example of hp-PTGS mediated viral immunity was done in tobacco against PVY, which was published at the same year as the seminar paper showing dsRNA is the trigger of RNAi in worms (Supplementary Table S1).in worms . Later oin worms  . The proof-of-concept studies for AMIR mediated viral resistance were reported in Arabidopsis and tobacco nearly 10 years ago (Supplementary Table S1).all RNAs , many smears ago , while iears ago   . The sixth strategy was using artificial trans-acting siRNA gene to express multiple tasiRNAs targeting different viruses. A stretch of synthetic sequences consists of multiple 21-nt short sequences that complementary to target viruses is inserted between a 3′-cleavable and a 5′-non-cleavable miR390 binding sites to create an artificial TAS transcript. When it is expressed in transgenic plants, the artificial TAS transcript was cleaved by miR390 and the 5′ cleavage product containing the viral sequences is turned into dsRNA by plant RDR6 and diced from its 3′ end successively by DCL4 to release 21-nt tasiRNA that will target cognate viruses . The efficacy of TAS-mediated multiple-viruses resistance has been demonstrated in Arabidopsis (echanism . The secechanism . The forgure 2E) . Transgegure 2E) . The fifbidopsis .Viral targets for SRGE include both the virus and the genes or region within a virus that are targeted by small RNAs produced by the transgene. In terms of number of studies, Potyviruses, Tospoviruses, Closteroviruses, and Geminiviruses are among the most studied virus genera (Supplementary Table S1). Viral coat protein or nuclear capsid protein are the most frequently chosen targets for SRGE; viral replicase or replication associated proteins, and VSR protein are also frequently used (Supplementary Table S1). All these targets provide essential function for virus life cycle . Besides targeting the coding region, the untranslated region (UTR) in viral genome is also targeted for efficient antiviral silencing . Nicotiana benthamiana has been widely used as a model species to study the efficacy of constructs for silencing the intended virus (Supplementary Table S1). Stable transgenic plants for a variety of crops were generated expressing small RNAs in different ways and their reactions to targeted viruses were tested in both laboratory and field condition (Supplementary Table S1). In some studies, the durability of resistance was tested for many generations (Supplementary Table S2). Potato and the United States ranks the top among different crops and countries, respectively, in terms of number of lines approved . All these commercially released crops were developed based on the first generation SRGE technology.Small RNA based genetic engineering has been applied in engineering viral resistance for many crops, including major crops of staple food, vegetables, fruits ornamentals, and some cash crop  .able S1) . Lack ofable S1) . PRSV-reable S1)  and commable S1) . Due to able S1) . Overcomable S1) . CurrentBanana, Citrus and Plum, banana is the largest tropical fruit and BBTV is the most serious viral pathogen for banana cultivation worldwide. BBTV-resistant transgenic banana was developed with hp-PTGS mechanism targeting Rep gene (Supplementary Table S1). Neither banana nor citrus transgenic lines resistant to viruses were approved for commercial release. Plum is one of the oldest domesticated fruit with versatile uses. Plum pox virus is the major viral pathogen of plum. S-PTGS mediated resistance against Plum pox virus was first demonstrated in N. benthamiana (Supplementary Table S2).Rep gene . Citrus Rep gene  while taRep gene . Early ethamiana  and latethamiana . PPV-resthamiana . The S-PSquash, cucumber, and watermelon are common vegetables and fruits belonging to the Cucubitaceae family, which suffer from a variety of viral pathogen (Supplementary Table S2).pathogen . SqMV-repathogen . Multiplpathogen . PRSV-repathogen . None ofPotato, tomato, and pepper are important vegetables belonging to Solanaceae family and potato is also a very important staple food crop. These crops suffer from a variety of plant viruses and a number of efforts to generate viral resistance with SRGE were reported (Supplementary Table S1). Supplementary Table S2).Maize, Wheat, Rice, and Cassava are the major staple food crop and supported calorie consumption for most of the human population. Maize streak virus (MSV) and maize dwarf mosaic virus (MDMV) impose the most frequent viral threat to Maize production. Transgenic maize resistant to MDMV was generated with hp-PTGS mechanism targeting P1 and CP . These viral pathogens caused significant losses in rice production in Asia and many resistant transgenic rice lines were generated using hp-PTGS mechanism . Soybean mosaic virus is the most important viral pathogen to soybean cultivation and several transgenic lines resistant to this virus were generated by hp-PTGS and S-PTGS, targeting HcPro and CP, respectively (Supplementary Table S2) while no commercial release of SRGE based viral resistant peanut and soybean were reported.argeting , howeverectively . Multiplectively . BGMV-paectively . BGMV-reNicotiana tabacum and N. benthamiana were widely used as model plants to study the efficacy of SRGE against various viruses infecting crops (Supplementary Table S1) due to their easiness in transformation. However, result obtained from tobacco is not always consistent with that in the intended crop (Agrobacterium-mediated infiltration is widely used for virus inoculation as a routine technique in the lab. It was reported that transgenic tomato showed better resistance when infected via insect than by Agro-infiltration (Tobacco including ded crop . It is pded crop . It is aded crop . Anotherltration , which mEarly application of the first generation SRGE involves expression of functional viral products, which raises concerns to the human health and the environment. These concerns were well-addressed in the application of PRSV CP transgenic papaya . In the Crop plants are often subjected to mixed viral infection. VSR from untargeted virus can suppresses the small RNA mediated silencing thus breaks the immunity to SRGE targeted virus . For theOomycete pathogen was shown to deliver effector into plant cells to suppress small RNA mediated silencing , thus poSmall RNA mediated silencing is also affected by abiotic stress, such as low and high temperature, drought and salt stress, which are often encountered in crop cultivation. Investigation of molecular mechanism by which those abiotic stresses manipulate silencing pathway, will provide solution to proper compensation strategy for SRGE application in those stress conditions.It was reported early that small RNA mediated silencing in non-cell autonomous and silencing signal is capable of both cell-to-cell and phloem dependent long distance movement . In modeFinally, plant genomes encode multiple DCL genes capable of generating miRNA and siRNAs in many ways. Fully dissection of the small RNA biogenesis mechanisms mediated by those different DCL proteins, can help design silencing constructs expressing as many as possible small RNAs, which holds the key for success of SRGE application in crop protection.Figure 1. MY contributed in literature research.AK and FL conducted literature research and analyzed the data. FL and AK wrote the manuscript. QZ contributed 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.


Abstracttree-based approach. This approach assigns anonymous OTUs to taxonomic categories based on relative placements of OTUs and reference sequences on the cladogram and support that these placements receive.Metabarcoding is becoming a common tool used to assess and compare diversity of organisms in environmental samples. Identification of OTUs is one of the critical steps in the process and several taxonomy assignment methods were proposed to accomplish this task. This publication evaluates the quality of reference datasets, alongside with several alignment and phylogeny inference methods used in one of the taxonomy assignment methods, called In tree-based taxonomy assignment approach, reliable identification of anonymous OTUs is based on their placement in monophyletic and highly supported clades together with identified reference taxa. Therefore, it requires high quality reference dataset to be used. Resolution of phylogenetic trees is strongly affected by the presence of erroneous sequences as well as alignment and phylogeny inference methods used in the process. Two preparation steps are essential for the successful application of tree-based taxonomy assignment approach.Curated collections of genetic information do include erroneous sequences. These sequences have detrimental effect on the resolution of cladograms used in tree-based approach. They must be identified and excluded from the reference dataset beforehand.Various combinations of multiple sequence alignment and phylogeny inference methods provide cladograms with different topology and bootstrap support. These combinations of methods need to be tested in order to determine the one that gives highest resolution for the particular reference dataset.Completing the above mentioned preparation steps is expected to decrease the number of unassigned OTUs and thus improve the results of the tree-based taxonomy assignment approach. Metabarcoding of living organisms is on the rise as the cost of Next Generation Sequencing goes down and processing pipelines improve . They caphylogenetic approach in de-novo, and the dataset is usually trimmed to the barcode size. The cladogram is built using one of the available phylogeny inference algorithms . Barcoding regions are purposely chosen to include hypervariable sites , which aAs will be discussed in detail elsewhere , if OTUs of marine nematodes can not be identified to species or even genus level due to incompleteness of reference databases, the largest taxon that they can be placed into, and that can still provide sufficient information for ecological studies is the family. However, before using tree-based approach to assign OTUs of marine nematodes to the families, , its possible drawbacks must be thoroughly evaluated. Such as the impact of the reference dataset, or the alignment or phylogeny inference algorithms on the quality of the results.The goal of this paper is to estimate how well the cladogram based solely on the barcoding region (in this case it is the 5' end of 18S rRNA molecule) resolves and supports families of marine nematodes. It will be accomplished by evaluating the results obtained by analyzing several reference datasets and by using different combinations of alignment and phylogeny inference algorithms. The first dataset will include all relevant sequences that fulfill specific criteria described below; the second dataset will exclude all sequences that are found to be questionable; the third dataset will also exclude all sequences that do not have sufficient coverage with the barcoding region used in Haenel et al. (unpublished) and Holovachov et al. (unpublished).Nematoda and Priapulida (to be used as an outgroup) section of it was downloaded on December of 2015. At the first step, all sequences were manually checked in order to remove animal parasitic and exclusively terrestrial nematode species, sequences already known to be incorrectly identified, unidentified sequences , and non-nematode sequences placed within Nematoda. Examples of non-nematode taxa placed among nematodes include sequences from the phylum Tardigrada, Tubulichidae (phylum Annelida), Ricinulei, Limulidae and Poduridae , Spironucleustorosa (flagellate), uncultured fungus and even Drosophilaamericana and Drosophilaauraria. Animal-parasitic taxa were not included in the current analysis with the exception of the family Mermithidae, some species of which are known from marine habitats (Rhabditidae and Anguinidae), only 3-4 species were included. Sequences were further sorted according to the following criteria:SILVA database  is regulhabitats . For theFor the same species, longer sequences were chosen over shorter sequences.Taxa identified to species level were chosen over taxa identified only to the genus level, considering that they both belong to the same genus.All fully identified species for each genus were included.For the same species (if available) no more than two sequences were included.All available genera for every family of marine nematodes were included.All families with at least two representative species were included.All sequences that were missing 40 bases and more on the 5' end  were excluded.Suppl. material Complete dataset included all selected sequences."Filtered" dataset excludes species that are likely incorrectly identified and therefore consistently had negative impact on tree topology and clade support in the first analysis of the complete dataset."Long" dataset included only those sequences that had the same length as barcoding region , or were missing no more than 10 bases on the 5' end.Dorylaimoidea and Mononchoidea will be treated as whole, without subdivision into separate families in subsequent analyses). "Filtered" dataset was created by removing all erroneous sequences from the complete dataset. It includes 276 taxa (272 nematode sequences and four outgroup taxa) belonging to 50 nematode families. "Long" dataset was created based on the "filtered" dataset by removing sequences that had insufficient coverage. It includes 212 taxa (208 nematode sequences and four outgroup taxa) belonging to 48 nematode families or superfamilies. Families Anticomidae and Phanodermatidae are not presented in the "long" dataset because only one species of Anticomidae and none of Phanodermatidae satisfied the requirement of sufficient sequence length.As a result, complete dataset includes 284 terminal taxa (280 nematode sequences and four outgroup taxa) belonging to 50 families or superfamilies  was used to root all phylogenetic trees. Monophyletic clades with bootstrap support of 70% and higher were considered well supported and fully resolved. Trees were visualized using FigTree , e.g. barcodes clustered within the clade that is equivalent to a family "A" or a genus "B" in the classification may be identified as belonging to that family "A" or genus "B". On the other hand, anonymous OTUs clustered outside well supported monophyletic clades should be treated as unassigned. Therefore, following criteria were used to evaluate the quality of the results of each individual analysis (cladogram) produced in this study:Number of nematode families resolved as monophyletic, paraphyletic or polyphyletic in each analysis. The therm "family" will be used to describe clades that are equivalent to family-level categories in nematode classification.Bootstrap support that each monophyletic clade receives. Fully resolved clades, or families, are those that receive ≥70% bootstrap support.taxa identified to species level were chosen over taxa identified only to the genus level, considering that they both belong to the same genus); #4 (for the same species no more than two sequences were included); or because these sequences are available from GenBank but not yet included in the SILVA database. They were chosen to represent both well and poorly resolved families.It is expected that monophyletic clades with high bootstrap support are likely to remain such after combining the reference dataset with anonymous OTUs in possible future studies. To confirm this, and for the final comparison, two scenarios were chosen, the "worst case"  and the "best case" . 25 pre-selected sequences  were added to both alignments to create new datasets, both were re-aligned and re-analyzed following same "worst case" and "best case" settings. These pre-selected sequences represent species, which were either not included in the original complete dataset because of the criterium #2  in all six analyses.Aphanolaimidae, Ceramonematidae and Chromadoridae) are also resolved as monophyletic, but their bootstrap support varies greatly between analyses, from the highest 94-96% to the lowest 43-60%.Three families .The family Xyalidae, Tobrilidae and Phanodermatidae) may either have very low bootstrap support, or can be resolved as paraphyletic or polyphyletic.Three families  from the family Anticomidae; the clade that includes all members of the family Thoracostomopsidae also includes three unrelated taxa, namely Parodontophora sp. (AM234630) from the family Axonolaimidae, Oncholaimus sp. (KF591739) from the family Oncholaimidae and Gammanema sp. (KF591723) from the family Selachinematidae.Five families are consistently resolved as paraphyletic: the family Terschellingia is always placed separately from the rest of Linchomoeidae; the genus Prodesmodora is consistently separated from the rest of Microlaimidae; the family Trefusiidae is always divided into terrestrial (Trischistoma and Tripylina) and marine (Trefusia and Rhabdocoma) clades; the family Oxystominidae is always split into three individual clades equivalent to the genera Halalaimus, Oxystomina and Thalassoalaimus+Litinium; the genus Syringolaimus is always placed separately from the rest of Ironidae; the family Anoplostomatidae is always split into clades represented by the genera Anoplostoma and Chaetonema. Two members of the family Diplopeltidae never form a monophyletic clade. Members of the families Oncholaimidae and Enchelidiidae are "mixed" in random manner. Paraphyly of other families is usually caused by separate placement of one or more of the sequences in the cladogram .Seventeen families are always resolved as polyphyletic. Of these, only five families are consistently divided into two or three monophyletic and highly supported clades: the genus The results were more variable between different alignments comparing to Neighbor joining analyses of the same set of data, with PRANK-based analysis resolving the maximum of 26 families, while Clustal-O, MUSCLE and SILVA-based analyses resolving only 21 each Table , 13, 14 Out of 50 nematode families and superfamilies included in this dataset, only 18 families are fully resolved as monophyletic and receive high bootstrap support (≥70%) in all six analyses.Xyalidae, Mononchoidea, Mermithidae, Enoplidae and Leptosomatidae) are also resolved as monophyletic, but their bootstrap support varies greatly between analyses, from the highest 72-90% to the lowest 44-65%.Five families  may either have very low to very high bootstrap support, or can be resolved as paraphyletic or polyphyletic.Nine families  from the family Axonolaimidae, Oncholaimus sp. (KF591739) from the family Oncholaimidae and Gammanema sp. (KF591723) from the family Selachinematidae; the family Oxystominidae is a paraphyletic "grade" that includes as one of its monophyletic clades a range of other taxa.Four familes are consistently resolved as paraphyletic: the family Diplopeltidae never form a monophyletic clade. Paraphyly of other families is usually caused by separate placement of one or more of the sequences on the cladogram .Fourteen families are always resolved as polyphyletic. Of these, five families are consistently divided into two monophyletic and highly supported clades in exactly the same way as in previous (Neighbor joining) analysis . Two members of the family Anticoma sp. (AY692344), Parodontophora sp. (AM234630), Oncholaimus sp. (KF591739), Gammanema sp. (KF591723), Cyatholaimus sp. (JN968214), Longicyatholaimus sp. (LK054720), Pomponema sp. (KF591743) and Monoposthiacostata (AY854221). Visual examination of the alignment with congeneric taxa confirmed that the identity of these sequences is likely to be incorrect. Therefore, these sequences were excluded from the "filtered" dataset.Several sequences were consistently clustered outside their family clades and are thus considered problematic: Similar to 1.1, all six alignments produced comparable results  in all six analyses.Aphanolaimidae, Ceramonematidae, Draconematidae, Selachinematidae and Tobrilidae) are also resolved as monophyletic, but their bootstrap support varies greatly between analyses, from the highest 44-94% to the lowest 19-68%.Five families  may either have very low to high bootstrap support, or can be resolved as paraphyletic or polyphyletic.Three families (Camacolaimidae and Oxystominidae) are inconsistently resolved as either paraphyletic or polyphyletic.Two families  are combined in a highly supported clade, paraphyletic in relation to each other.Two families  analyses . The family Ceramonematidae (from 74% to 69%) placed it insignificantly below the 70% threshold.Removing erroneous sequences increased bootstrap support in 12-16 clades and resolution (clades became monophyletic) in 6-7 clades (Table Between six and seven families that were non-monophyletic (paraphyletic or polyphyletic) in the Neighbor joining analysis of complete dataset (section 1.1) were resolved as monophyletic after removing erroneous sequences. Bootstrap support for such families varied between 19% and 100%. As a result, 4-5 families crossed the upper threshold (≥70% bootstrap support) and were fully resolved. Thus, depending on the alignment, between five and seven new families were fully resolved (monophyletic with ≥70% bootstrap support) in the Neighbor joining analysis of the "filtered" dataset.Similar to 1.2, the results were more variable between different alignments comparing to Neighbor joining analyses of the same set of data  in all six analyses.Aphanolaimidae, Camacolaimidae, Xyalidae, Mononchoidea and Leptosomatidae) are also resolved as monophyletic, but their bootstrap support varies greatly between analyses, from the highest 74-89% to the lowest 37-69%.Five families  may either have very low to high bootstrap support, or can be resolved as paraphyletic or polyphyletic.Eight families . The family Removing erroneous sequences increased bootstrap support in 14-26 clades and resolution in 5-8 clades Table ; 3-11 faBetween five and eight families that were non-monophyletic (paraphyletic or polyphyletic) in the Maximum likelihood analysis of complete dataset (section 1.2) were resolved as monophyletic after removing erroneous sequences. Bootstrap support for such families varied between 24% and 100%. As a result, 4-7 families crossed the upper threshold (≥70% bootstrap support) and were fully resolved. Thus, depending on the alignment, between five and eight new families were fully resolved (monophyletic with ≥70% bootstrap support) in the Maximum likelihood analysis of the "filtered" dataset.Exclusion of problematic sequences from the alignment (defined in section 1.3 above) resulted in substantial increase in resolution and support for many clades equivalent to family-level categories, because incorrect placement of each of them in previous analyses (complete dataset) affected resolution of two families, the one that they are identified with taxonomically, and the one that they are placed within in the phylogenetic analysis.Unlike in previous Neighbor joining analyses (sections 1.1 and 2.1), the results were more variable between different alignments  during the analysis of "complete" dataset, the entire clade of marine taxa (Trefusia and Rhabdocoma) was exluded, leaving the second clade of terrestrial taxa (Trischistoma and Tripylina) in the dataset.Out of 48 nematode families included in this dataset, 30 families are resolved as monophyletic and receive high bootstrap support (>70%) in all six analyses. In the case of the family Chronogastridae, Camacolaimidae, Axonolaimidae, Xyalidae, Ceramonematidae, Draconematidae, Selachinematidae and Tobrilidae) are also resolved as monophyletic, but their bootstrap support values vary greatly between analyses, from the highest 57-98% to the lowest 23-65%.Eight families (Microlaimidae is resolved as monophyletic with very low bootstrap support (19%) in one case only, polyphyletic in all other instances.The family Monhysteridae includes families Xyalidae and Sphaerolaimidae as ingroup clades; the family Desmodoridae is paraphyletic in relation to the family Draconematidae; the family Mermithidae is paraphyletic in relation to the superfamily Mononchoidea; the family Oncholaimidae is paraphyletic in relation to the family Enchelidiidae.Four familes are consistently resolved as paraphyletic: the family Terschellingia is always placed separately from the rest of Linchomoeidae; the genus Syringolaimus is always placed separately from the rest of Ironidae. The family Leptolaimidae is also consistently split in two clades, but only one of these clades is monophyletic with high bootstrap support. Two members of the family Diplopeltidae never form a monophyletic clade. Similarly, three members of the family Oxystominidae never form a monophyletic clade.Five families are always resolved as polyphyletic. Of these, only two families are consistently divided into two monophyletic and highly supported clades: the genus Removing erroneous sequences improved bootstrap support in 15-16 clades and resolution in 5-7 clades Table . ChangesBetween five and six families that were non-monophyletic (paraphyletic or polyphyletic) in the Neighbor joining analysis of "filtered" dataset (section 2.1) were resolved as monophyletic after removing erroneous sequences. Bootstrap support for such families varied between 19% and 99%. As a result, 4-5 families crossed the upper threshold (≥70% bootstrap support) and were fully resolved. Thus, depending on the alignment, between five and seven new families were fully resolved (monophyletic with ≥70% bootstrap support) in the Neighbor joining analysis of the "long" dataset.In this case PRANK-based analysis again resolves the highest number of families (36 out of 50), and Clustal-O-based analysis resolves only 29 Table . In geneOut of 48 nematode families included in this dataset, 23 families are resolved as monophyletic and receive high bootstrap support (>70%) in all six analyses.Camacolaimidae, Xyalidae, Ceramonematidae, Mononchidae, Mermithidae, Tobrilidae, Leptosomatidae and Alaimidae) are also resolved as monophyletic, but their bootstrap support varies greatly between analyses, from the highest 69-95% to the lowest 43-69%.Eight families  may either have variable (low-to-high) bootstrap support, or can be resolved as paraphyletic or polyphyletic.Nine families  analysis of "long" dataset .Removing short sequences improved bootstrap support in 13-15 clades and resolution in 2-6 clades (Table Between one and four families that were non-monophyletic (paraphyletic or polyphyletic) in the Maximum likelihood analysis of "filtered" dataset (section 2.2) were resolved as monophyletic after removing short sequences. Bootstrap support for such families varied between 41% and 100%. As a result, 0-2 families crossed the upper threshold (≥70% bootstrap support) and were fully resolved. Thus, depending on the alignment, between two and six new families were fully resolved (monophyletic with ≥70% bootstrap support) in the Maximum likelihood analysis of the "long" dataset.Exclusion of incomplete sequences from the alignment resulted in increase in resolution and support for several clades equivalent to family-level categories, although in case of Maximum likelihood analysis, a number of clades were resolved as paraphyletic or polyphyletic, or lost bootstrap support below the 70% threshold.Xyalidae from 71% to 68% and Enoplidae from 71% to 55%) and increased in three clades .Preselected 25 sequences were added to original, complete dataset and re-analyzed using Clustal-O for alignment  and Maximum Likelihood for phylogeny inference. As expected, addition of new high quality sequences did not affect the resolution of the cladogram, but affected bootstrap support for monophyletic clades  and Chromadoridae (bootstrap support of 62%).Out of 25 added sequences, only 18 could be assigned to family-level categories based on their clustering withing monophyletic clades Table Axonolaimidae from 73% to 50%, in Selachinematidae from 71% to 62% and in Achromadoridae from 98% to 66%.Similar to "worst case" scenario described in the previous section, same preselected 25 sequences were added to "long" dataset and re-analyzed using PRANK for alignment and Maximum Likelihood for phylogeny inference. Just like in the previous example, addition of new high quality sequences did not affect the resolution of the cladogram, but affected bootstrap support for monophyletic clades Fig. . ChangesCamacolaimidae, Comesomatidae, Xyalidae, Cyatholaimidae, Chromadoridae, Anoplostomatidae and Thoracostomopsidae).Out of 25 added sequences, 22 could be assigned to family-level categories based on their clustering withing monophyletic clades Table Teratocephalidae, Siphonolaimidae, Sphaerolaimidae, Desmoscolecidae, Ethmolaimidae, Achromadoridae, Haliplectidae, Rhabdolaimidae, Bathyodontidae, Cryptonchidae), or because such families are mainly freshwater/terrestrial . The latter are used here mainly to increase taxon coverage and sequence variability. The former are always represented by co-specific or co-generic taxa which monophyly is not questioned here. Both categories will not be further considered in the discussion.Results of a phylogenetic analysis are strongly determined not only by the alignment and phylogeny inference algorithms, but also by the quality of the input data. However, influence of poor quality sequences on different parts of the phylogenetic tree is not equal. Resolution and bootstrap support for some nematode families remained consistent throughout all analyses and was not affected by the presence of erroneous or short sequences. Large number of such families are unfortunately represented in current analysis by only few taxa (2-4 species), either due to limited availability of high quality sequences in the reference databases  that were always resolved as monophyletic in all analyses, independently from the alignment and phylogeny inference algorithms, are only 18S rRNA  and are Monhysterida was fully resolved (monophyletic with high support) in all analyses using Maximum likelihood inference, and in some analyses using Neighbor joining inference . Three terrestrial orders Dorylaimida, Rhabditida and Tylenchida, all of which were represented by very few sequences, were fully resolved in all analyses. Other orders were either poly- or paraphyletic, while bootstrap support for many basal dichotomies was lower than the required threshold.Higher taxa  were not fully resolved in any of performed analyses, with few exceptions. Order Various multiple-sequence alignment software naturally produced alignments of varying quality, which affected the final outcome of all analyses in this comparison. Visual examination of alignment files showed that all of them, including alignments downloaded from SILVA database, were not able to cope with hypervariable regions of rRNA molecule, evidenced by the fact that identical (very similar) segments of sequences of closely related taxa (same genera) can be aligned differently. In this test, SILVA-based alignments produced some of the worst results, alongside Clustal-O and MAFFT. On the other hand, PRANK, Clustal-W and MUSCLE-based alignments produced cladograms with higher resolution and support, but the improvements are not always significant, and may not be observed for other barcoding regions or other groups of organisms.Neighbor joining algorithm was shown to be effective in matching anonymous sequences to sequences that were preliminary identified . It is tAnticoma sp. (AY692344), Parodontophora sp. (AM234630), Oncholaimus sp. (KF591739), Gammanema sp. (KF591723), Cyatholaimus sp. (JN968214), Longicyatholaimus sp. (LK054720), Pomponema sp. (KF591743) and Monoposthiacostata (AY854221). Removing these sequences affected the resolution and support of both clades (families) that they are identified with taxonomically, and clades (families) that they were placed within during phylogeny inference. Moreover, if anonymous OTU is placed in the clade (monophyletic and highly supported) that includes problematic sequences, it might not be always possible to evaluate with confidence if it genuinely related to taxa representing majority of the clade, or if its placement is caused by similarity to a problematic sequence.Improvement in the resolution and support achieved in the "filtered" dataset should be attributed to the exclusion of problematic (erroneous) sequences, namely: Anoplostomatidae and Trefusiidae) one of the clades that defined these taxa as polyphyletic in the analyses of complete and "filtered" datasets, was completely absent in the "long" dataset, thus artificially defining Anoplostomatidae and Trefusiidae as monophyletic. In this case, it is important to find a balance between the number of incomplete sequences and completeness of the reference dataset.Removing of short sequences increased support and resolution much less significantly, and at a cost of loss of reference data. In case of two families , Desmodoridae (including Draconematidae as ingroup clade) and Oncholaimidae (including Enchelidiidae as ingroup clade). At least one of them (Desmodoridae) is similarly resolved in large scale phylogenetic studies that use nearly full-length 18S and partial 28S rRNA sequences (Tridentulus sp. (AJ966507) as an example , outside of the monophyletic ingroup clades  and in the monophyletic and highly supported subclade with identified taxa , it can be assigned the taxonomic identity of the paraphyletic clade (family Monhysteridae) with confidence.Several important and diverse families of marine nematodes are always resolved as paraphyletic in present analysis. Examples include family equences . OTUs plple Fig. . If the Often polyphyletic clades are caused by insufficient phylogenetic signal of the relatively short (barcode-size) marker. Several examples discussed in sections 1.1 and 1.2 of the Results confirm that erroneous sequences are another important culprit, affecting both resolution and support of clades. In both cases, affected clades are unlikely to be useful for the identification of anonymous barcodes that are placed within them. Polyphyly of families can also reflect genuine divergent history of the phylogenetic marker (barcoding region) that is not followed in current classification or not supported by alternative phylogenies (based on full-length gene or multiple genes). In such cases, anonymous barcodes could still be assigned to one of the subclades and classified within the family, as long as their placement in such subclades is well supported, subclades are well represented with reference taxa and have sufficient bootstrap support.A number of reference sequences were found in this analysis to be "misplaced" in the phylogenetic trees, suggesting that they are likely incorrectly identified or have sequencing errors. Public sequence databases do include erroneous sequences that will affect the results. Therefore, it is necessary to raise awareness about the importance of quality control of reference datasets for erroneous and incomplete sequences, as both will have negative impact on the results of taxonomy-assignment procedures.versus Maximum likelihood). It is thus recommended to use more then one combination of both alignment and phylogeny inference algorithms in order to be able to reliably identify anonymous sequences.The choice of alignment and phylogeny inference algorithms will affect the results. Moreover, alignment may have bigger impact on the topology of the final tree than either one of the phylogeny inference algorithms used in this study  that are monophyletic in the "full-length 18S rRNA" tree may not be monophyletic in the barcode-based tree. Nonetheless, it is still possible to assign taxonomic placement to anonymous OTUs that fall within paraphyletic and polyphyletic clades in the barcode-based tree, depending on their topology and bootstrap support.There were a number of families in our analysis that were represented only by two closely related species and were usually resolved as monophyletic. In such cases, it is difficult to forsee if they will cluster with unidentified OTUs in real-life analyses. Sequencing of more reference taxa from such families should be of higher priority than sequencing of taxa from families that are well represented in reference databases.Supplementary material 1Table S1. GenBank accession numbers and classification of sequences used in present analysisData type: listFile: oo_96341.pdfHolovachovSupplementary material 2Neighbor joining tree inferred using Clustal-O alignment of the complete datasetData type: phylogeneticFile: oo_85395.jpgHolovachovSupplementary material 3Neighbor joining tree inferred using Clustal-W alignment of the complete datasetData type: phylogeneticFile: oo_85396.jpgHolovachovSupplementary material 4Neighbor joining tree inferred using MAFFT alignment of the complete datasetData type: phylogeneticFile: oo_85397.jpgHolovachovSupplementary material 5Neighbor joining tree inferred using MUSCLE alignment of the complete datasetData type: phylogeneticFile: oo_85398.jpgHolovachovSupplementary material 6Neighbor joining tree inferred using PRANK alignment of the complete datasetData type: phylogeneticFile: oo_85399.jpgHolovachovSupplementary material 7Neighbor joining tree inferred using SILVA-based alignment of the complete datasetData type: phylogeneticFile: oo_85400.jpgHolovachovSupplementary material 8Table S2. Resolution and bootstrap support (for monophyletic clades) of nematode families based on Neighbor joining analyses of different multiple sequence alignments of complete dataset Data type: listFile: oo_95694.pdfHolovachovSupplementary material 9Maximum likelihood tree inferred using Clustal-O alignment of the complete datasetData type: phylogeneticFile: oo_85402.jpgHolovachovSupplementary material 10Maximum likelihood tree inferred using Clustal-W alignment of the complete datasetData type: phylogeneticFile: oo_85403.jpgHolovachovSupplementary material 11Maximum likelihood tree inferred using MAFFT alignment of the complete datasetData type: phylogeneticFile: oo_85404.jpgHolovachovSupplementary material 12Maximum likelihood tree inferred using MUSCLE alignment of the complete datasetData type: phylogeneticFile: oo_85405.jpgHolovachovSupplementary material 13Maximum likelihood tree inferred using PRANK alignment of the complete datasetData type: phylogeneticFile: oo_85406.jpgHolovachovSupplementary material 14Maximum likelihood tree inferred using SILVA-based alignment of the complete datasetData type: phylogeneticFile: oo_85407.jpgHolovachovSupplementary material 15Table S3. Resolution and bootstrap support (for monophyletic clades) of nematode families based on Maximum likelihood analyses of different multiple sequence alignments of complete dataset Data type: listFile: oo_95695.pdfHolovachovSupplementary material 16Neighbor joining tree inferred using Clustal-O alignment of the "filtered" datasetData type: phylogeneticFile: oo_85409.jpgHolovachovSupplementary material 17Neighbor joining tree inferred using Clustal-W alignment of the "filtered" datasetData type: phylogeneticFile: oo_85410.jpgHolovachovSupplementary material 18Neighbor joining tree inferred using MAFFT alignment of the "filtered" datasetData type: phylogeneticFile: oo_85411.jpgHolovachovSupplementary material 19Neighbor joining tree inferred using MUSCLE alignment of the "filtered" datasetData type: phylogeneticFile: oo_85412.jpgHolovachovSupplementary material 20Neighbor joining tree inferred using PRANK alignment of the "filtered" datasetData type: phylogeneticFile: oo_85413.jpgHolovachovSupplementary material 21Neighbor joining tree inferred using SILVA-based alignment of the "filtered" datasetData type: phylogeneticFile: oo_85414.jpgHolovachovSupplementary material 22Table S4. Resolution and bootstrap support (for monophyletic clades) of nematode families based on Neighbor joining analyses of different multiple sequence alignments of "filtered" dataset Data type: listFile: oo_96343.pdfHolovachovSupplementary material 23Maximum likelihood tree inferred using Clustal-O alignment of the "filtered" datasetData type: phylogeneticFile: oo_85420.jpgHolovachovSupplementary material 24Maximum likelihood tree inferred using Clustal-W alignment of the "filtered" datasetData type: phylogeneticFile: oo_85421.jpgHolovachovSupplementary material 25Maximum likelihood tree inferred using MAFFT alignment of the "filtered" datasetData type: phylogeneticFile: oo_85422.jpgHolovachovSupplementary material 26Maximum likelihood tree inferred using MUSCLE alignment of the "filtered" datasetData type: phylogeneticFile: oo_85423.jpgHolovachovSupplementary material 27Maximum likelihood tree inferred using PRANK alignment of the "filtered" datasetData type: phylogeneticFile: oo_85424.jpgHolovachovSupplementary material 28Maximum likelihood tree inferred using SILVA-based alignment of the "filtered" datasetData type: phylogeneticFile: oo_85425.jpgHolovachovSupplementary material 29Table S5. Resolution and bootstrap support (for monophyletic clades) of nematode families based on Maximum likelihood analyses of different multiple sequence alignments of "filtered" dataset Data type: listFile: oo_96345.pdfHolovachovSupplementary material 30Neighbor joining tree inferred using Clustal-O alignment of the "long" datasetData type: phylogeneticFile: oo_86262.jpgHolovachovSupplementary material 31Neighbor joining tree inferred using Clustal-W alignment of the "long" datasetData type: phylogeneticFile: oo_85428.jpgHolovachovSupplementary material 32Neighbor joining tree inferred using MAFFT alignment of the "long" datasetData type: phylogeneticFile: oo_85429.jpgHolovachovSupplementary material 33Neighbor joining tree inferred using MUSCLE alignment of the "long" datasetData type: phylogeneticFile: oo_85430.jpgHolovachovSupplementary material 34Neighbor joining tree inferred using PRANK alignment of the "long" datasetData type: phylogeneticFile: oo_85431.jpgHolovachovSupplementary material 35Neighbor joining tree inferred using SILVA-based alignment of the "long" datasetData type: phylogeneticFile: oo_85432.jpgHolovachovSupplementary material 36Table S6. Resolution and bootstrap support (for monophyletic clades) of nematode families based on Neighbor joining analyses of different multiple sequence alignments of "long" dataset Data type: listFile: oo_95718.pdfHolovachovSupplementary material 37Maximum likelihood tree inferred using Clustal-O alignment of the "long" datasetData type: phylogeneticFile: oo_86264.jpgHolovachovSupplementary material 38Maximum likelihood tree inferred using Clustal-W alignment of the "long" datasetData type: phylogeneticFile: oo_85435.jpgHolovachovSupplementary material 39Maximum likelihood tree inferred using MAFFT alignment of the "long" datasetData type: phylogeneticFile: oo_85436.jpgHolovachovSupplementary material 40Maximum likelihood tree inferred using MUSCLE alignment of the "long" datasetData type: phylogeneticFile: oo_85437.jpgHolovachovSupplementary material 41Maximum likelihood tree inferred using PRANK alignment of the "long" datasetData type: phylogeneticFile: oo_85438.jpgHolovachovSupplementary material 42Maximum likelihood tree inferred using SILVA-based alignment of the "long" datasetData type: phylogeneticFile: oo_85439.jpgHolovachovSupplementary material 43Table S7. Resolution and bootstrap support (for monophyletic clades) of nematode families based on Maximum likelihood analyses of different multiple sequence alignments of "long" dataset Data type: listFile: oo_96346.pdfHolovachov


The small GTPase Miro is best known for its regulation of mitochondrial movement by engaging with the microtubule-based motor proteins kinesin and dynein. Very recent findings have now showed that Miro also targets peroxisomes and regulates microtubule-dependent peroxisome motility. Moreover, Miro recruits and stabilizes the myosin motor Myo19 at the mitochondria to enable actin-based mitochondria movement, which is important for mitochondrial segregation during mitosis. Miro thus has much broader functions that previously known, and these new findings may have important implications on disease pathology. This domain structure is unusual amongst members of the small GTPase superfamily and is indicative of MIRO’s functional activities being regulated by Ca2+, as well as being specific to the particular sites of their membrane anchorage. Indeed, the Miro GTPases are localized to the mitochondrial outer membrane and play critical roles in intracellular mitochondria movement in metazoans, particularly over long distances along microtubule tracts in neurons [Mitochondrial Rho (Miro) is a somewhat atypical small GTPase that is best known for functions in mitochondria transport and homeostasis ,2,3. Fou neurons . Miro me neurons , the for neurons ,7,8,9. I neurons  and inte neurons . Miro tu neurons . PINK1s  neurons . On the  neurons . The mul neurons .The importance of Miro’s function in neurons can be gleaned from the phenotypes of Miro gene silencing and deletion. Silencing of the mammalian Miro paralogues, Miro1 and Miro2, in murine dorsal root ganglia (DRG) neurons resulted in altered mitochondrial distribution and disrupted axonal mitochondrial motility, respectively . Mice wiThis rather exclusive view of Miro’s major function in microtubule-based mitochondrial dynamics and trafficking could now change considerably with some very recent findings. Two of these are particularly prominent, and are discussed further in the sections below. The first pertains to the demonstration of Miro’s targeting to peroxisomes and its role in peroxisome transport along microtubules ,17. The Peroxisomes are ubiquitously present in metazoans and have important metabolic functions, particularly fatty acid oxidation and reduction of reactive oxygen species (ROS), both of which requires coordination with metabolic processes at the mitochondria  and the Okumoto et al.  identifiIn another report, Castro et al.  also shoNot only does Miro work on another organelle, more new evidence indicates that it may also engage another cytoskeletal platform for mitochondrial movement in cells. Kittler’s laboratory has shown previously that Miro1 is the principal regulator of mitochondrial movement in neurites, and observed that Miro1 deletion leads to depletion of mitochondria from distal dendrites but not axons . FurtherWhat contributed to the significant reduction in the shorter range mitochondrial displacements in Miro1/2 KOs and DKOs? The authors explored the possibility of a loss in actin engagement, particularly through the mitochondrial localized actin motor Myo19 ,24. InteThe recent findings on Miro briefly described above have important implications. Firstly, the peroxisome targeting and function of Miro1 splice variants indicate yet another layer of functional and perhaps also evolutionary links between peroxisomes and mitochondria. Initially thought to arise exclusively from the ER membrane based on studies in yeast , McBrideIt now appears that cellular mitochondria movements are dependent on both microtubule- as well as actin-based motors via Miro’s engagement of either Trak1/2 or Myo19. The relative importance of these two modes in different cell types requires further clarification, as Myo19-action-based movements in mitochondria partitioning would be more important for dividing cells as opposed to terminally differentiated, non-dividing cells. However, even within the same cell, there could be a division of labor with differential engagement of microtubule or actin-based motor by Miro. With neurons, for example, the need for long-range mitochondria transport in axons and dendritic shafts would require TRAK-microtubule based transport. However, actin-based, short-range movements would become important in specialized structures like axonal growth cones and dendritic spines see . In thisSPAST) mutations from patients with Hereditary Spastic Paraplegia [Another possibility arising from the new findings is that Miro-dependent organelle transport dysfunction-induced disease states would have contributions from both mitochondrial and peroxisomal functional deficits, in fact likely with both deficits acting in synergy. Mitochondrial functional deficiencies underlie many neurodegenerative disorders . As prevraplegia . Deficieraplegia . The abo


Cardiovascular disease remains a major health care challenge. The knowledge about the underlying mechanisms of the respective vascular disease etiologies has greatly expanded over the last decades. This includes the contribution of microRNAs, endogenous non-coding RNA molecules, known to vastly influence gene expression. In addition, short interference RNA has been established as a mechanism to temporarily affect gene expression. This review discusses challenges relating to the design of a RNA interference therapy strategy for the modulation of vascular disease. Despite advances in medical and surgical therapies, atherosclerosis (ATH), aortic aneurysms (AA) are still associated with high morbidity and mortality. In addition, intimal hyperplasia (IH) remains a leading cause of late vein and prosthetic bypass graft failure. Pathomechanisms of all three entities include activation of endothelial cells (EC) and dedifferentiation of vascular smooth muscle cells (VSMC). RNA interference represents a promising technology that may be utilized to silence genes contributing to ATH, AA or IH. Successful RNAi delivery to the vessel wall faces multiple obstacles. These include the challenge of cell specific, targeted delivery of RNAi, anatomical barriers such as basal membrane, elastic laminae in arterial walls, multiple layers of VSMC, as well as adventitial tissues. Another major decision point is the route of delivery and potential methods of transfection. A plethora of transfection reagents and adjuncts have been described with varying efficacies and side effects. Timing and duration of RNAi therapy as well as target gene choice are further relevant aspects that need to be addressed in a temporo-spatial fashion.While multiple preclinical studies reported encouraging results of RNAi delivery to the vascular wall, it remains to be seen if a single target can be sufficient to the achieve clinically desirable changes in the injured vascular wall in humans. It might be necessary to achieve simultaneous and/or sequential silencing of multiple, synergistically acting target genes. Some advances in cell specific RNAi delivery have been made, but a reliable vascular cell specific transfection strategy is still missing. Also, off-target effects of RNAi and unwanted effects of transfection agents on gene expression are challenges to be addressed. Close collaborative efforts between clinicians, geneticists, biologists, and chemical and medical engineers will be needed to provide tailored therapeutics for the various types of vascular diseases. Short interfering RNA and miRNA are useful tools to temporarily suppress target genes. Different from siRNA, miRNA are endogenously occurring short RNAs heavily involved in regulating numerous cellular functions. A recent review provides a more in-depth comparison of siRNA and miRNA, including respective transfection modalities .While siRNA usually is used to silence one specific gene, a single miRNA has the ability to affect expression of multiple genes. Approximately one-third of the genes are regulated by miRNA and it has been shown that several miRNA are able to bind to the identical 3′UTR region . VariousThe anatomy of arteries and veins reveals similarities yet also significant histological differences. The inner lining of arteries and veins (tunica intima) consists of a single layer of EC seated on a basement membrane (BM). The layers of the BM are composed of an intricate network consisting of various collagens types, glycoproteins and proteoglycans, and cell–cell, and cell–matrix interaction regulators such as integrins. Different from sinuses in the liver and spleen, which have a discontinuous BM and therefore promote extravasation of RNAi, the BM of large arteries and veins is continuous and provides an anatomical barrier. The BM is connected to a thin layer of subendothelial connective tissue. Different from veins, arteries and arterioles also have as the outer margin of the tunica intima a prominent internal elastic membrane (lamina) with elastin containing fibers. This layer presents another physical barrier for RNAi for transmural delivery.The next layer, tunica media is mostly comprised of circularly arranged smooth muscle cells with interspersed reticular and elastic fibers. This layer’s thickness varies significantly between veins and arteries. Naturally a thicker layer of SMC in larger arteries provides a greater challenge for a homogenous, transmural transfection when compared to a thin-walled vein graft. As mentioned before, VSMC appear less susceptible to RNAi compared to EC –12. If VThe outer most layer, tunica adventitia is made up of connective tissue. In large arteries an external elastic membrane may separate the tunica adventitia from the tunica media. Large arteries and veins receive additional blood supply via vasa vasorum, which enter from the adventitial site. Given the low oxygen content of venous blood, it is conceivable that large veins are more dependent on vasa vasorum for their nutrient/oxygen supply than arteries. In the setting of vein graft implantation, that supply route may be disrupted. This may contribute to the formation of IH. It is also believed that vasa vasorum play a significant role in atherosclerosis progression .Interestingly, vasa vasorum appear to cease to supply oxygenated blood to the arterial wall at the level of the renal arteries, which may help explain an increased incidence of aortic aneurysms in this region . The expAtherosclerosis (ATH) by some is considered a chronic inflammatory condition that involves a complex interplay of immune and stromal cells, cytokines and enzymes. It is characterized by a disturbed loco-regional lipid metabolism, extracellular matrix organization, and cellular homeostasis leading to lipid plaque build-up and ultimately risk for plaque rupture and vascular narrowing or occlusion , 18. HypA recent review from Welten et al. dissects the contribution of various miRNA clusters to the maladaptive processes of atherosclerosis and restenosis . PromineIn the case of IH in the setting of vein graft surgery and vascular implantation injury, synthetic VSMC migrate towards the lumen of the vessel. There, VSMC proliferate and secrete pro-inflammatory cytokines and extracellular matrix proteins forming the bulk of the eventual IH lesion .miRNA have also shown to be involved in IH formation , 25. OneSome strategies to attenuate IH aimed at attenuating VSMC proliferation after vascular injury , 26, 27.Given the immediate proximity of EC and VSMC in the vascular bed, it appears a rather difficult task to deliver RNAi exclusively to one of the two cell fractions. In addition, particularly the arterial vessel wall consists of a thick muscular layer with multiple layers of VSMC stacked on top of each other. In order to achieve a transfection success across all VSMC layers, a high dose of RNAi oligomers as well as potent transfection agents [e.g. liposomal or cationic polymer based such as polyethyleneimine (PEI)] may have to be used , 28, 29.Not surprisingly, relevant differences exist amongst the same cell type (EC or VSMC) within the separate vascular beds. A study comparing canine VSMCs within the aorta (Ao), branch pulmonary artery (bPA), main pulmonary artery (mPA) and inferior vena cava (IVC) revealed not only obvious histologic differences in layer thickness and cellular organization, but also significant protein expression differences with regards to smooth muscle actin (SMA), myosin heavy chain (MHC) and smooth muscle myosin heavy chain isoform 2 (SM2) .Separately, differential expression patterns were found with regards to Hox4. Products of the hox gene family regulate the cranio-caudad organization of the body during embryogenesis. Differential Hox4 expression has been found along the aorta in EC and VSMC of baboons. Thoracic EC and VSMC revealed higher Hox4 levels compared to corresponding abdominal aortic cells . FurtherAnother example is the protein testin, which was found to be differentially expressed in coronary and internal mammary arteries. Testin is a cytoskeleton-associated protein that localizes along actin stress fibers, at cell–cell-contact areas, and at focal adhesion plaques. Testin was also found to have tumor suppressor gene function and may be relevant for EC function , 33. TesAside from varying intrinsic protein expression profiles it could be shown that VSMC from different segments of the circulation respond distinctly to noxious stimuli. Oxidized low-density lipoprotein (OxLDL) treatment in saphenous vein graft VSMC (SV-VSMC) led to an increase in proliferation and migration as well as NF-kappa B activation. In contrast, OxLDL inhibited proliferation and migration in coronary VSMC. In addition, significant differences in cytokine, chemokine extracellular matrix protein expression were noted between these cell groups .In line with this are observations derived from in vitro experiments, which revealed that SV-VSMC were found to be significantly more proliferative and displayed a more migratory and invasive behavior than internal mammary artery VSMC .Yet in contrast, human VSMC derived from atherosclerotic lesions of infragenicular arteries displayed significantly increased rates of proliferation, adhesion, and migration as compared to human saphenous vein VSMC .Also, the response to hypoxia appears to be different in arterial and venous SMC. While both cell fractions showed a decreased proliferative response to hypoxia, arterial SMC showed a significant upregulation of vascular endothelial growth factor (VEGF-A), while venous SMC did not. VEGF-R 2 expression was found to be upregulated in hypoxic venous SMC, but not in arterial SMC . The samFurther complicating is the finding that significant differences between EC from various vascular beds exist . There aSeparately, bone morphogenetic protein (BMP) signaling has been found relevant for a variety of pathways involved in EC biology. Heterogeneous transcriptional activity seemed to correlate with vasculature undergoing hemodynamic alterations . BMP sigLocoregional differences in miRNA expression patterns as a function of flow disturbances have been investigated, coining the term “flow-sensitive miRNA” or “mechano-miRNA” . miRNAs In addition, there appear to be differences in siRNA susceptibility between EC and VSMC and possibly even between the various VSMC fractions under identical transfection conditions , 10.All these biological differences help explain the varying loco-regional responses to vascular injury including atherosclerosis, postangioplasty restenosis and vein graft disease . These dSystemic, naked siRNA administration without technology to target specific cells types leads to renal siRNA secretion within 20 min as well as rapid plasma nuclease degradation. In addition, significant degradation and phagocytosis via the reticuloendothelial system occurs which also shortens the siRNA’s systemic half-life .In parallel, several chemical modifications to the RNA have been introduced including locked nucleic acid technology (LNA), in which an additional covalent bond within the RNA backbone is formed. This has proven to improve stability to the RNA molecule and protection from phagocytosis , 48.Liposomes are spheres consisting of customizable phospholipid layers that can be used for drug delivery. Liposomes widely used to facilitate diagnostic and experimental siRNA silencing vitro and are one of the most commonly used transfection modality , 49, 50.The introduction of poly(ethylene glycol) (PEGylation) into liposomal formulations is another commonly performed process aimed at improving drug delivery efficacy. PEGylation is thought to confer some protection from mononuclear phagocytosis through the creation of a protective hydrophilic film on the surface of the liposome and decrease renal clearance due to a size increase of the liposomal complex –57.Cationic polymers such as polyethyleneimine (PEI) can also be used to complex RNA complexes and improve transfection efficiency into vascular cells in vitro and in vivo , 58–60. Another promising modality is RNAi containing nanoparticles. Some of these nanoparticles have self-assembling properties. The individual composition of these nanoparticles is highly customizable and can be adjusted to the specific needs of the therapy approach –66. ThesRNAi encoded in lentiviral and adenoviral vectors was successfully delivered to cardiovascular cells and other tissues , 70–74. Many reviews exist that provide comprehensive overviews of RNAi vectors, chemical modifications and transfection agents including detailed capabilities and current limitations of either technology , 79, 80.Depending on the target cell (EC versus VSMC), local intraluminal RNAi delivery represents a logical and simple approach. Obvious challenges are the interaction of RNAi and its delivery vehicles with the components of blood, penetration in the cellular and non-cellular layers of the vessel wall, and washout from the site of delivery.Single application can be accomplished via injection with or without distal occlusion or possibly from balloon catheters. In the setting of vein bypass grafting the RNAi solution may be infused into the graft. Similarly, brief distal arterial occlusion and target vessel distention is feasible in certain settings and may be used to enhance RNAi delivery.Utilizing above approach PEI complexed TSP-2-siRNA was successfully administered to the denuded wall of rat carotid arteries and yielded a lasting silencing effect. While this resulted in desirable changes in transforming growth factor–beta (TGF-β) and matrix metalloproteinase-9 (MMP-9) signaling, as well as anti-inflammatory M2 macrophage polarization, it did not affect intima/media ratios .In contrast, intraluminal siRNA targeting transcription factor activation transcription factor-4 (ATF-4), a downstream target of the mitogen fibroblast growth factor-2 (FGF-2) in balloon injury rat carotid arteries did lead to a measurable decrease in intimal hyperplasia .Likewise, adenovirus mediated silencing of A disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTS-7), a metalloproteinase reduced intimal VSMC proliferation in a rat model of balloon-induced vascular injury .Several studies involving miR expression modulation in injured vascular walls have yielded some encouraging results. Amongst others, miR- 24 and miR-29b were found to be decreased in balloon-injured rat carotid arteries , 83. MiRSustained, transluminal, local delivery has been attempted by using RNAi coated, implanted stents/stent grafts. Plasmid DNA has been successfully transferred to the arterial wall in a pig stent-angioplasty model . In lined,l-lactic-co-glycolic acid) (PLGA) and poly (l-lactide) (PLLA) are FDA approved as stent coatings that potentially could provide a sustained release RNAi to the vessel wall [Alternatively, various biodegradable coatings such as poly silencing after perivascular delivery of ADAMTS-7 siRNA in pluronic gel mitigated IH in injured rat arteries , 94.Silencing of STAT-3, a transcription factor of multiple receptors also inhibited VSMC and decreased IH via expression of Bcl-2 and cyclin D1 in rat vein grafts without increases in apoptosis. siRNA was encapsulated with the liposomal transfection reagent Lipofectamine and deployed in a “Bioprotein gel” that was applied onto the adventitia of the rat vein grafts .Likewise, siRNA targeting the heparin-binding growth factor midkine (MK) mixed with atelocollagen was administrated to the external wall of rat vein grafts. MK silencing decreased immune cell recruitment, and cell proliferation within MK siRNA-treated vein grafts .Primary systemic delivery requires large doses of RNAi and its delivery vehicle thus increases cost, and the chance of off-site effects and toxicity.In a mouse model of carotid injury the transcription factor GATA-2 was found to be reduced. GATA-2 regulates multiple EC specific genes as well as miR-126 and miR-221. Systemic miR-126-coupled nanoparticles enhanced miR-126 availability in the carotid artery and improved re-endothelialization of injured carotid arteries in vivo .Independently, miR-181b has been shown to inhibit TNF-α induced downstream NF-κB signaling. Including vascular cell adhesion protein 1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, and tissue factor. In a rodent model of photochemical injury-induced carotid artery thrombosis, systemic miR-181b delivery conferred some protection from thrombin-induced EC activation and arterial thrombosis .The endothelial cell biology has been well studied and various EC specific  cell surface markers have been described in the literature as targets to provide a more directed, cell specific RNAi therapy –103.Recent results showed that cationic lipoplexes can be designed that result in a predominant uptake into the vascular endothelium and thereby minimizing offsite-target effects . FurtherWhile activated, VCAM-1 expressing EC were successfully targeted by PEGylated antibody-targeted lipoplexes in vitro. However, these promising results did not result in successful silencing in vivo .While not exclusive, chemokine receptor CXCR-4 is constitutively expressed in EC. Peptide carriers as RNAi vehicles that contain a CXCR-4 ligand increased efficacy of VEGF-siRNA to EC .Another intriguing approach resulted from data of an in vivo phage display in the setting of a partial carotid ligation model of flow-induced atherosclerosis in mice .This led to the identification of two peptides (CLIRRTSIC and CPRRSHPIC) that specifically bind to EC exposed to turbulent flow in pro-atherogenic regions. These peptides were conjugated to polyethylenimine (PEI)-polyethylene glycol (PEG) to generate polyplexes containing ICAM-1 siRNA. CLIRRTSIC polyplexes carrying si-ICAM-1 specifically bound to mouse endothelium and silenced ICAM-1 expression in disturbed flow regions in a mouse model .It remains to be seen if successful transfection of a rodent arterial wall can be translated into larger animal studies or even humans given the significant differences in wall thickness. Even if a similar transmural transfection result is achieved, it may come at the risk of higher toxicity and off-site effects given as larger doses of transfection agents and RNAi oligonucleotides may be required.Vascular injury is characterized by a dramatic upregulation of thousands of genes within a matter of hours as demonstrated in a study of vein graft implantation in canine . Over thGiven the discussed issues of cell specific RNAi delivery, target gene choice becomes a conundrum. Silencing of the target gene should result in a robust alteration in the biology of the targeted cell. In contrast, inadvertent target gene silencing in bystander cells should not impede those cells’ inherent biological activities. For example, the cell cycle and apoptosis regulator survivin (SVV) is highly upregulated in SV-VSMC the setting of IH , 107. ThIn contrast, it has been shown, that overexpression of SVV in EC resulted in increased viability and migratory capability, but reduced apoptosis. SVV overexpressing EC were also associated with higher levels of angiogenesis in vivo , 107–110One of those candidate genes that differentially affects biology of EC and VSMC may be MARCKS  –114. MARConceptually an intriguing group of target genes are inflammatory cytokines and extracellular matrix proteins that are secreted by both, EC and VSMC. Arguably, silencing these targets in both cell fractions may prove beneficial.Establishing methods to reliably transfect specific cells of the vascular wall in a controlled and sustained fashion could have wide ranging clinical benefits. Naturally, atherosclerosis, aneurysmal disease, and intimal hyperplasia come to mind as primary disease entities. However, there are a variety of other clinical scenarios in which transient modulation of local vascular biology could be of great benefit. For instance it would be highly desirable to be able to increase endothelial barrier function in the setting of systemic inflammatory response syndrome and/or sepsis. Excessive vascular permeability and capillary leak lead to local edema with subsequent tissue hypoxia and acidosis. Pulmonary hypertension is another major cardiovascular condition that may be amenable to RNAi therapy , 115.Taken together, the medical community would greatly benefit from the translation of the discussed pre-clinical studies to tangible medical therapies. Based on the existing data it appears that EC and macrophages currently might be the most promising target cells with regards to cell specific RNAi delivery. Both cell-types possess characteristic surface markers that could serve as receptors/ligands for cell specific RNAi delivery. Specific next steps towards clinical application also have to involve the transition from rodent to large animal models. While these are costly, they allow crucial insight in how to address larger vessel structures, circulating blood volumes, and flow patterns, more consistent with humans.It seems pertinent to further optimize delivery of RNAi and develop methods for controlled sustained release. For a single local application, ligand-coupled nanoparticles containing RNAi or directly ligand-conjugated RNAi in combination with transcatheter administration might be a reasonable option. For systemic administrations as in the case of atherosclerosis, it might be useful to deploy receptor/ligand-RNAi constructs aimed at epitopes expressed in vascular lesions to direct RNAi preferentially to sites of high disease burden. Advances in RNAi structure design such as LNA have already yielded increased protection from enzymatic degradation and thereby increasing RNAi serum half-life . AdvanceFor sustained RNAi release approaches, perivascular and RNAi delivery from implantable cardiovascular devices may prove successful. As discussed before, several biologically compatible polymers exist that could serve as a depot for RNAi. Trapping RNAi in biodegradable materials with programmable degradation/release patterns needs to be further explored; specifically for cardiovascular applications.In the opinion of the authors, it is important to first establish RNAi as a safe and programmable tool to modulate biological processes. As we start to understand RNAi-related challenges such as sequestration in liver, lung and macrophages, it might be judicious to first investigate the effects of RNAi in the lung vasculature and macrophages. If the desired results are achieved, increased resources could be dedicated to tackle other challenges relating to RNAi delivery to the vascular wall.‘correct’ target gene choice for the individual vascular disease has yet to be made and it may be required to silence multiple genes in a synergistic fashion in order to achieve clinically relevant results. The reliable translation of promising in vitro or rodent animal data to large animal or human studies still has to occur. While multiple preclinical studies reported encouraging results of RNAi delivery to the vascular wall, it remains to be seen if a single target can be sufficient to the achieve clinically desirable changes in the injured vascular wall in humans. As discussed, the only existing clinical trial of vein graft treatment with oligonucleotides (PREVENT III) targeting E2F has failed to show clinically relevant results [As the understanding of vascular physiology and disease expanded over the past decades, it has become evident that significant loco-regional differences in vascular cell biology exist. Likewise, the technologies around RNAinterference have become increasingly sophisticated. However, anatomical barriers such as those found in large human blood vessels have not been adequately addressed. Also, off-site effects of RNAi therapy adjuncts can be significant and have to be carefully considered. Moreover, the  results , 27. Whi results . Success


We aimed to identify clinical predictors of long-term response to abiraterone (defined as >12 months drug exposure) in a retrospective cohort of metastatic castration-resistant prostate cancer patients treated in post-docetaxel setting at 24 Italian centers. The Cox proportional hazards model was used to analyze the association between clinical features and the duration of drug exposure. Results were expressed as hazard ratios (HR) with associated 95% confidence intervals (CI). A total of 143 patients met the inclusion criteria. Their median age was 73 years, median Gleason score 8 and median abiraterone exposure 20 months. At the univariate analysis, a significant correlation with the duration of abiraterone exposure was found for Gleason score , PSA  and lactic dehydrogenase levels , while the association between lower alkaline phosphatase levels and treatment duration was marginally significant . Only PSA and Gleason score were predictive of long-term treatment duration in the multivariate analysis. No other clinical factors resulted to be predictive of sustained response to abiraterone, including metastatic disease at diagnosis and visceral disease, suggesting that all subgroups of patients may derive a substantial clinical benefit from abiraterone treatment. These findings need to be validated in prospective, larger studies. Treatment options for metastatic castration-resistant prostate cancer (mCRPC) have expanded considerably over the last few years, but how to select the most appropriate drug for each patient in order to obtain maximum benefit from the available therapies still remains unclear. Although recent studies have shown that molecular biomarkers such as androgen receptor splice variant-7 (AR-V7) or ERG gene status hold promise as potential predictors of response –3, at prIn this study we retrospectively analyzed clinical records of men with mCRPC who had received abiraterone post-docetaxel in routine clinical settings in Italy, with the aim to identify potential clinical predictors of long-term response to abiraterone, by defining long-term responders as those patients receiving this drug for >12 months.We identified 143 patients who met the inclusion criteria and were evaluated. Demographic and clinical characteristics are summarized in Table Median follow-up was 34.6 months and median duration of abiraterone treatment was 19.8 months (IQR 14.3-29.4 months) , with a disease control rate of 88% (n=126).p=0.012), PSA  and LDH . The association between ALP and abiraterone exposure did not quite reach statistical significance . While increasing levels of circulating tumor biomarkers  were associated with increased risk of abiraterone interruption, a high Gleason score was found to predict decreased risk of progression. No statistically significant association with the duration of exposure to abiraterone emerged for age, M-staging at diagnosis (M0 vs M1) or site of metastases   Table .The most significant finding of this study is the considerable proportion of heavily pre-treated mCRPC patients who achieved, in a routine oncology practice setting, a relevant clinical benefit with abiraterone. The duration of response exceeded, in many cases, two years (median 20 months). This long-term response was observed also in patients with unfavorable prognostic features such as high Gleason score, synchronous metastases at diagnosis or visceral disease.A feature that sets our study apart from other similar investigations is the fact that we looked for potential clinical predictors of long-term response to abiraterone by focusing on a cohort of patients with a duration of drug exposure longer than expected (as compared with published data), rather than searching for factors that might help discriminate between responders and non/poor responders. The cut-off point of 12 months for definition of long-term response was arbitrarily selected based on the median abiraterone exposure of 7.4 months (range 0.2-25.6) reported in the pivotal abiraterone COU-AA-301 study in chemotherapy-treated patients [A significant association with the duration of drug exposure was found for Gleason score, PSA and LDH levels before starting treatment, with a marginally significant association for ALP. The correlation between rising levels of circulating tumor biomarkers and poorer response were not surprising, given that PSA, LDH and ALP are known prognostic factors for survival in CRPC across a wide range of therapies, or in the absence of life-prolonging treatments –9. Unexpin vitro and in CRPC patients [No other potential predictors of long-term response emerged from our analysis. In particular, the presence of synchronous metastases at diagnosis and visceral disease were not associated with a less favorable outcome . The lack of a significant association between presence of visceral metastases and radiological progression or survival has been observed in other recent retrospective studies in patients treated with AR-targeted therapies , 14. Thepatients .Our study had limitations that need to be taken into account when interpreting the results, including the retrospective design and the relatively small patient population. The lack of a control group of early refractory patients did not allow to discriminate between early refractory and long responding patients. Moreover, we evaluated outcome based on abiraterone exposure time rather than using standardized outcome measures such as overall survival (OS) or progression-free survival (PFS).+ Edel cancers), previously considered as unfavorable prognostic factor for survival, was associated with the greatest clinical benefit from abiraterone treatment [Several biomarkers have been recently proposed as potential predictors of treatment response in mCRPC, including tumor-associated genetic profiling parameters from biopsies, circulating biomarkers, imaging data and clinical variables. AR-V7 has been linked to primary resistance to either abiraterone or enzalutamide, and monitoring AR-V7 status in circulating tumor cells (CTCs) over the course of treatment may predict sensitivity or resistance to AR-targeted agents , 2. Analreatment . Imagingreatment . Neverthreatment , 19. A breatment . On the reatment . In a coreatment . Prelimireatment .Retrospective analysis of the Temporary Authorization for Use programme in France showed that PSA decrease at 3 months was a predictive factor for abiraterone treatment duration . In our It should be emphasized that none of the biomarkers so far investigated have yet been validated as predictors of treatment response , 6.In conclusion, our retrospective analysis in patient with mCRPC, mostly treated in post-docetaxel setting, showed that high Gleason score, lower PSA and LDH levels before starting abiraterone were significantly associated with long-term abiraterone exposure . We failed to identify more specific clinical factors predictive of sustained response to this drug, suggesting that all suitable patients may receive abiraterone therapy on the basis of clinical or tumor-related characteristics. Prospective studies in larger patient populations are needed to confirm our findings.Patients with histologically confirmed mCRPC who were treated with abiraterone for >12 months at 24 Italian cancer centers from October 2011 to July 2014 were retrospectively identified. Clinical records were collected and the following demographic and clinical parameters were analyzed: age at initial abiraterone exposure, duration of abiraterone treatment, number and type of previous anticancer therapies, serum levels of PSA, ALP and LDH before starting abiraterone, Gleason score at diagnosis, type of disease progression before starting abiraterone , M-staging at diagnosis, sites of metastases, PSA response rates and objective response (in men with measurable disease). The study endpoint was the time from the start of abiraterone treatment to abiraterone interruption from any cause. Patients' data were collected for retrospective analysis in April 2015.0: HR = 0. All statistical tests were two-sided and statistical significance was detected at the 5% probability level . For each predictor the assumption of proportional hazard was tested using a Cox's model with a constant and linear term in time. For each continuous predictor associated to abiraterone treatment duration the best thresholds were identified using the CART methodology [Descriptive statistics were used to summarize demographic and clinical features . The Cox proportional hazards model was used to detect and estimate the statistical association between the patients' demographic-clinical features and the duration of abiraterone exposure; the hazard ratio (HR) was used as population parameter and the Wald test statistic was used to test Hhodology , with reThe Kaplan-Meier method was used to estimate survival functions. The inverse Kaplan-Meier method was used to estimate median follow-up; patients still receiving abiraterone at the time of the last contact were right censored.Apart from CART methodology, the statistical analysis was performed using the SAS software , version 9.2.


Neuromyelitis optica spectrum disorders (NMOSD) are mostly relapsing inflammatory disorders of the central nervous system (CNS). Optic neuritis (ON) is the first NMOSD-related clinical event in 55% of the patients, which causes damage to the optic nerve and leads to visual impairment. Retinal optical coherence tomography (OCT) has emerged as a promising method for diagnosis of NMOSD and potential individual monitoring of disease course and severity. OCT not only detects damage to the afferent visual system caused by ON but potentially also NMOSD-specific intraretinal pathology, i.e. astrocytopathy. This article summarizes retinal involvement in NMOSD and reviews OCT methods that could be used now and in the future, for differential diagnosis, for monitoring of disease course, and in clinical trials. Neuromyelitis optica spectrum disorders (NMOSD) are autoimmune inflammatory conditions of the central nervous system (CNS) with a mostly relapsing disease course . ClinicaOCT is an interferometric technique employing low-coherent light to produce structural cross-sectional images . The ligNext to MS and NMOSD, a retinal examination by OCT is increasingly applied as a non-invasive technique to evaluate key features of various neurological disorders, e.g., in Susac syndrome, anti-NMDA receptor encephalitis, and also in neurodegenerative diseases –65. OCT The peripapillary retinal nerve fiber layer thickness (pRNFL or sometimes just RNFL or RNFLT) has become a reliable OCT marker for diagnostic evaluation in translational research and care –77. The 3), since the ganglion cells are highly concentrated parafoveally and account for about 34% of the macular volume [The ganglion cell and inner plexiform layer thickness or volume (GCIP or sometimes GCIPL) regularly complements the pRNFL as an imaging marker. The main targets of interest are the very ganglion cell bodies associated with axons in the retinal fiber layers described previously. Due to the poor differentiability in OCT imaging, the ganglion cell layer is usually measured in combination with the adjacent inner plexiform layer as GCIP Fig. . The GCIr volume , 82, 83.r volume –87. Recer volume –91. The r volume , 92, 93.Retinal measurements between OCT devices from different manufacturers are usually not comparable. Whereas, pRNFL has a reasonably good standardization and is measured similarly across devices, GCIP and INL measurements lack this standardization, thereby impeding comparability . The estON is the first clinical feature observed in about 55% of the patients with NMOSD and usually causes severe structural damage to the optic nerve and retina with resulting functional impairment . NMOSD pSo far, no published studies have investigated acute ON specifically in NMOSD. Studies investigating isolated or idiopathic acute ON, without the distinction of underlying pathologies, have shown that during clinical onset of acute ON, OCT measurements typically give a highly swollen pRNFL that is not representative of retrograde axonal damage . At thisAround 20% of the NMOSD patients have microcystic alterations of the INL after ON Fig.  89, 90,, 127. ThMueller cells are astrocytic cells of the retina residing mainly in the INL and might also play a role in NMOSD beyond MMO and ON-inflicted damage. They have multiple responsibilities, including water homeostasis, energy metabolism, and neurotransmitter recycling. Since they express AQP4 water channel proteins, they might be a direct target of AQP4-ab and a potential cause of a primary retinopathy in NMOSD –134. AniThe magnetic resonance imaging (MRI) of brain and spinal cord is an indispensable tool and a part of the diagnostic criteria for MS and NMOSD , 139–144While numerous studies exist describing brain tissue alterations in MS ), only few studies have investigated MRI characteristics in NMOSD , 151–154To date, no results from randomized controlled trials (RCTs) of disease-modifying therapies (DMTs) in NMOSD have been published. Current treatment strategies  are based on retrospective case series or uncontrolled trials , 33–37. The retina is one of the most affected CNS regions in NMOSD. The OCT is an easy-to-use diagnostic tool to assess neuroinflammatory and neurodegenerative processes in the retina and thus the visual system. An early examination of the retina by OCT in NMOSD might provide useful information on the severity of structural damage that may be predictive of functional outcomes, as well as in the long-term disease course . With re