Copidosoma floridanum, polyembryogenesis is initiated during the formation and division of the morula. In the present study, cDNA libraries were constructed from embryos at the cleavage and subsequent primary morula stages, times when polyembryogenesis is likely to be controlled genetically. Of 182 and 263 cDNA clones isolated from these embryos, 38% and 70%, respectively, were very similar to protein-coding genes obtained from BLAST analysis and 55 and 65 clones, respectively, were stage-specific. In our libraries we also detected a high frequency of long non-coding RNA. Some of these showed stage-specific expression patterns in reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. The stage-specificity of expression implies that these protein-coding and non-coding genes are related to polyembryogenesis in C. floridanum. The non-coding genes are not similar to any known non-coding RNAs and so are good candidates as regulators of polyembryogenesis.Polyembryony is a unique form of development in which many embryos are clonally produced from a single egg. Polyembryony is known to occur in many animals, but the underlying genetic mechanism responsible is unknown. In a parasitic wasp,  Copidosoma floridanum (Hymenoptera: Encyrtidae), which is an egg-larval parasitoid of plusiine moths. The embryogenesis of C. floridanum is different from that of typical insects in that the syncytial  cleavage results in the formation of the syncytial blastoderm. In C. floridanum the parasitoid egg cleaves nearly holoblastically to form a morula, which is a spherical mass of embryonic cells surrounded by an extra-embryonic syncytium C. floridanum morula into polyembryos is initiated by invagination of the extra-embryonic syncytium into the spherical mass of embryonic cells Polyembryony is known to occur in many animals. In insects it has been thought that polyembryony has evolved independently in four families of Hymenoptera and one genus of Strepsiptera C. floridanum at the cellular level have revealed that cleavage-stage development shows several analogies to mammalian embryogenesis, including the early separation of extraembryonic and embryonic cell lineages, formation of a morula and embryonic compaction C. floridanum differs from that of most other insects, both at the cleavage and primary morula stages. These unusual states during early development might have provided favourable evolutionary conditions for polyembryony in C. floridanum or in its ancestors. Therefore, the analysis of the gene expression profile during the early embryogenesis of C. floridanum may provide valuable information allowing the molecular mechanism of polyembryony in this insect to be elucidated.Developmental analyses of C. floridanum, a gene expression profile of the cleavage and the primary morula stages has been unavailable. To date, two cDNA collections of C. floridanum have been registered in GenBank, one of which is derived from polyembryos composed of proliferation-stage and early-morphogenesis-stage embryos (NCBI BioProject Accession: PRJNA65673); the other is derived from two types of larvae  by the suppression subtractive hybridization approach C. floridanum, these cDNA data may be helpful in excluding constitutive expression genes.Gene expression profiles provide a powerful basis upon which to clarify the molecular mechanisms of life phenomena in organisms without complete genomic information. However, in C. floridanum.It has been reported that non-coding RNAs (ncRNAs) act as regulators of gene expression during embryogenesis In the present study, we constructed full-length cDNA libraries of two consecutive embryonic stages of the wasp, during which polyembryogenesis is suspected to be initiated and processed. We evaluated the clones according to their similarities to known protein-coding and non-coding transcripts. Subsequently, clones of potential significance to polyembryogenesis were selected by comparing with cDNA collections and from the quantity of each clone in a library. Clones selected from both libraries were confirmed according to their expression patterns determined by RT-PCR and RT-qPCR. Here we describe the properties of stage-specific cDNA libraries and the developmental gene expression profile during the polyembryogenesis of a wasp.C. floridanum and the host Thysanoplusia intermixta were reared at 22°C under a 16 h L–8 h D photoperiodic regime The polyembryonic parasitic wasp in vitro for development into the primary morula, according to the method of Iwabuchi (1995) in vitro was recognized by the shedding of the egg chorion.Female wasps were allowed to lay eggs in 24–48 h-old host eggs for 30 min. Wasp RNAs were isolated from more than 50 cleavage-stage embryos (4–8 h post-parasitism) and from more than 50 primary morula-stage embryos (12–24 h post-parasitism). We used two different methods to avoid contamination with host RNAs. For the cleavage stage, the wasp eggs were dissected from host eggs and simply washed with culture medium (modified MGM-450 medium). For the primary morula stage, we cultured the wasp eggs from the cleavage stage singly and These RNA samples  were used for cDNA synthesis by long-distance PCR, according to the protocol of the SMART cDNA library construction Kit (TaKaRa). In cDNA construction, cDNAs were ligated into the pDNR-LIB vector and transformed into competent cells . The transformants were plated on LB agar plates supplemented with chloramphenicol  and incubated overnight at 37°C. Colonies containing individual cDNA clones were picked up randomly and used as templates for colony-PCR with Emeraldamp MAX PCR premix with dye (TaKaRa) and the M13 primer set (supplied by SMART cDNA library construction Kit) for 25 cycles under the following PCR conditions: 98°C, 10 s; 50°C, 30 s; 72°C, 4 min. The PCR products were checked by electrophoresis on a 1% agarose gel. Colonies containing inserts of over 500 bps were chosen and cultured overnight at 37°C in 2 ml LB–chloramphenicol medium. The plasmid DNA was harvested using a PureYield Plasmid Miniprep System (Promega) and stored at −40°C until use. The clones were sequenced using the M13 forward or reverse primer on an ABI3100xl capillary sequencer.Our workflow is composed of three steps : Step I −6) to known rRNA as a result of BLASTN searches on Blast2GO In Step I, all clones were sequenced in the 3′ to 5′ direction. When this was denied by a long poly (A) tail or other repeats of nucleotide, the clone was sequenced in the reverse direction. Low quality segments at the 5′ and 3′ cDNA ends and vector regions were removed from the individual sequence reads on the TraceEditor of MEGA5 −6. For performance improvements, we installed a working database to run Blast2GO in our server, according to the installation manual on the Blast2GO web page (http://www.blast2go.com/b2ghome). All of the known ncRNA sequences, which had been registered up until July 2013, are compiled into a dataset from the Functional RNA database in the Functional RNA Project (http://www.ncrna.org/). Programs from BLAST+ −6).In Step II, all sequences were evaluated with Blast2GO using the BLASTX algorithm. BLASTX hits were cut off at a threshold level of E-value = 10C.floridanum transcripts; 230 ESTs derived from larvae −6). To detect clone duplication in our libraries, we also compared our sequences with themselves by BLAST+. Each resulting cluster of clone duplication was aligned using the CLUSTALW algorithm on the Alignment Explorer of MEGA5 using the default settings. Alignment errors were removed in order to allow a fit with the consensus regions indicated by the homology search. The multiple alignment file was used, as necessary, for the prediction of RNA secondary structures by CentroidFold, an online prediction service of the Functional RNA Project (http://www.ncrna.org/).In Step III, we made a multi-fasta file containing the formally registered sequences of gapdh). gapdh has been reported as a suitable reference gene with stable expression levels in different developmental stages, castes and tissues in hymenopteran insects C.floridanum gapdh in this study (M0090) and used it for RT-qPCR as an endogenous reference gene. Results were normalized from the expression of gapdh transcript in the same sample and then illustrated as means±SDs of the expression ratios. All statistical analyses were performed using R version 2.13.0 Total RNAs were isolated in order to determine the expression patterns of clones in three replicates at various stages and in tissues. RNA isolation was performed by using NucleoSpin RNA XS (TaKaRa). Embryos at varying developmental stages  were used. Here, primary morulae had been formed by 12 h post-parasitism. In order to examine whether the selected clones were deposited maternally, the heads and abdomens were dissected from the adults seven–eight days after egress from the host carcass and treated separately. All samples were stored at −80°C until used. RNA samples were purified and then reverse transcribed according to the protocol of the SuperScript VILO cDNA Synthesis Kit (Invitrogen) with random hexamers. RT-PCR was performed using the resulting cDNAs as templates with Emeraldamp MAX PCR premix with dye (TaKaRa) and gene-specific primers  for 35 cWe constructed stage-specific full-length cDNA libraries of embryos at the cleavage and primary morula stages. From over 3000 clones preselected from each library by colony PCR, 419 and 563 clones of the cleavage-stage and primary morula-stage embryos, respectively, were sequenced in one direction. By removing the clones that consisted of only nucleotide repeats or sequences corresponding to known rRNAs, we isolated 182 and 263 clones from cleavage-stage and primary morula-stage embryos, respectively. All of these clones were resequenced in the reverse direction. Pairs of bidirectional consensus sequences derived from single clones were assembled into contigs. As a result, we acquired 63 single sequences and 137 contigs from the cleavage-stage cDNA library and 331 single sequences and 82 contigs from the primary morula-stage library. The singletons were deposited in EST division (HX954220-HX954613) and the contigs in the HTC division (AK442469-AK442687) of the DDBJ database, to be used for subsequent analyses.Nasonia vitripennis; however, only 5 clones (0.3%) showed matches with lepidopteran genes. In contrast, we found no significant similarities between all the isolated clones (182+263) from both the cleavage-stage and primary morula-stage libraries to known ncRNAs. The results of BLAST searches and GO mapping-annotation steps by Blast2GO are listed in BLASTX analyses showed that 70 clones out of 182 isolated from cleavage-stage embryos and 184 clones out of 263 from the morula-stage embryos exhibited significant similarity to known protein-coding genes. Therefore, as many as 70% of the clones isolated in the primary morula-stage library, but only 38% in the cleavage-stage library matched with no known protein-coding genes . Most ofCopidosoma sequences (15183 TSA sequences derived from polymorula and 230 EST sequences from larvae). The results of the comparison indicate that 127 clones of our cleavage-stage library and 198 of our primary morula-stage library overlapped with the sequences registered in the dataset  than in primary morula-stage embryos (12–24 h post-oviposition), but there was no significant difference in the expression ratios  and M4092 (named here Cfmemo-1) were expressed at both the cleavage and primary morula stages, but were not expressed in host eggs . Representative sequences of each CflncRNA were tested in subsequent RT-PCR and RT-qPCR analyses. The results showed that all three of the CflncRNAs were not expressed in unparasitized host eggs . Due to the higher mutation rates of ncRNAs than of protein-coding RNAs We successfully isolated 182 cDNA clones from the cleavage stage of C. floridanum have been constructed from registered data of the polyembryos (NCBI BioProject Accession: PRJNA65673) and the larvae To compare the stage-specific clones here, the gene expression profiles in Developing a molecular approach based on EST libraries implied that only a few targets were identified, compared with that based on RNA-seq data. Other very important molecular codings may be unlikely to be identified due to the limitations of EST libraries. To compensate for this bias in transcriptomic quantitative information, we performed real-time RT-qPCR analysis together with a simple RT-PCR method. The real-time RT-PCR technique provided information about precise relative expression by comparing it to the expression of a determined reference gene. Simple RT-PCR is useful for detecting the important variations observed in high intensity bands, but not adequate for detecting quantifiable accurate expression.Cftudor (C0619) and Cftp53i13 (C0663) were transcribed by C. floridanum due to their absence from the host egg (Cftudor were comparatively high (about twice that of gapdh) at both embryonic stages  may be maternally deposited in the wasp egg. In C. floridanum, early embryogenesis is potentially controlled by various previously unreported maternal factors.Two clones were chosen from the cleavage-stage-specific clones for the detailed characterization of expression by RT-PCR and RT-qPCR analyses. The results of RT-PCR analysis show that host egg . The resc stages . Therefo embryos . However embryos . TherefoCfdbr-1 (M2053) and Cfmemo-1 (M4902). Cfdbr-1 is similar to DBR-1, which has been reported to be related to RNA processing Arabidopsis thalianaCfmemo-1 is similar to MEMO-1, which is required for cell migration in animals as it relays chemotactic signals Cfdbr-1 (M2053) tends to be up-regulated during the primary morula stage (Cfmemo-1 (M4902) showed comparatively low expression in the primary morula-stage embryo , but not because of age variability (emergence of adults in the host carcass is simultaneous in a single host). Because of very small size of insects (ca. 1 mm), the quantity of total RNAs isolated were too low to apply to quality assessment. A trace elements of RNA samples are likely to show differential degradation of individual mRNAs. A hard cuticle in adults may also cause variation in RNA isolation efficiency.C. floridanum morulae orient towards the host embryo before host invasion. Along with novel odorant receptors, Cfmemo-1 may be required for host invasion during polyembryogenesis.Our previous studies have indicated that the primary morula formed from an egg laid in the yolk of the host egg secondarily invades the host embryo CflncRNA-1 was specific to our libraries (CflncRNA-1 was similar to that of CflncRNA-3 in adult tissues (CflncRNA-1 and CflncRNA-2 were most closely-related (data not shown). These results suggest that CflncRNAs have different functions. CflncRNA-1 may be responsible for cleavage- and morula-stage-specific phenomena. CflncRNA-2 is possibly related to site-specific phenomena in the primary morula, while CflncRNA-3 expression may be constantly required during the whole process of embryogenesis. To date the suggestion has been that the translation of many genes, including TP53I13 C. floridanum. Further investigations aimed at identifying the location or interaction targets of the CflncRNAs may lead to the discovery of the genes responsible for polyembryogenesis. We also believe that the cDNA sequence data in the present study will be extremely valuable to future investigations by aiding RNA-seq read mappings, because of the higher mutation rates Meteorus pulchricornis . The results suggest that other insects have varying patterns of ncRNA, which are affected by gene duplication or alternative processing, and that the ncRNAs may show tissue-specificity.We did not normalize the current libraries so that we could retain a positive correlation between the number of clones in the library and the levels of gene expression. Using the sequence clustering and alignment method, we detected novel lncRNAs, named CflncRNA, in our libraries, which showed three patterns of paralogs or splicing variants . Severalibraries , regardlibraries . In addi tissues , althougC. floridanum. We characterized four candidates for protein-coding clones and three novel lncRNAs by RT-PCR and RT-qPCR. This is the first time that the relationship between lncRNA and polyembryogenesis has been reported. These genes may be good candidates for controllers of embryo development and polyembryony in C. floridanum. Further analyses of the localization of the associated mRNAs and/or the protein products will be useful for gaining a full understanding of the roles of these genes. Among the seven candidates, CflncRNA-1 is the one with the most interesting expression profile, because it is almost exclusively expressed during the cleavage and primary morula stages.We conclude that the clones in our libraries are efficient for the elucidation of the underlying molecular mechanisms of polyembryogenesis in Figure S1Schematic representation of sequencing and analysis in this study.(PDF)Click here for additional data file.Figure S2Original gel images including molecular size markers.(PDF)Click here for additional data file.Figure S3The RNA secondary structure of CflncRNAs predicted by CentroidFold. The heat color gradation from blue to red on each predicted base-pair corresponds to the base-pairing probability from 0 to 1.(PDF)Click here for additional data file.Figure S4Alignment of three ESTs in the largest cluster of venom grand ESTs of M. pulchricornis by clustalW. ESTs of M. pulchricornis were collected from INSDC data base . The ESTs were compared with itself and the 27 ESTs of the resulting largest cluster were aligned. The result of alignment showed the ESTs in the largest cluster were classifiable as three sequence patterns . The ESTs of alignment were omitted except for three and showed in this. The alignment of three sequences was performed on the Aligment Explorer of MEGA5 using the default settings. The conservation of nucleotides was indicated by an asterisk and dashes indicate gaps. The label on the left-hand side of each sequence gives an accession number of representational ESTs. The predicted lncRNAs of M. pulchricornis have highly conserved region among them but not stable open coding flame.(PDF)Click here for additional data file.Table S1Listing of primers used for RT-PCR in this study.(PDF)Click here for additional data file.Table S2Listing of primers used for RT-qPCR in this study.(PDF)Click here for additional data file.Table S3Classification of stage-specific sequences derived from C. floridanum embryos.(PDF)Click here for additional data file.Table S4Classification of sequences derived from from C. floridanum embryos and duplicated with the formerly registered cDNA sequences derived from proliferation-stage and early- morphogenesis stage.(PDF)Click here for additional data file.Table S5Classification of sequences derived from from C. floridanum embryos and duplicated with the formerly registered cDNA sequences derived from larvae.(PDF)Click here for additional data file.Table S6Classification of sequences derived from from C. floridanum embryos and duplicated with all of the cDNA collections used for comparison.(PDF)Click here for additional data file.Table S7Listing of clones in the clusters as a result of screening for frequency.(PDF)Click here for additional data file.Supporting Information S1The run reports of RT-qPCR experiments for each target gene.(PDF)Click here for additional data file.


C) is a glycosylphosphatidylinositol (GPI)-anchored protein present at the cell surface. PrPC N-terminal moiety is intrinsically disordered and is able to interact with a variety of ligands. Physiological ligands have neurotrophic activity, whilst others, including protein toxic oligomers, have neurotoxic functions. These two opposite activities involve different interacting partners and result from different PrPC-activated signaling pathways. Remarkably, PrPC may be inactivated either by physiological endoproteolysis and release of the N-terminal domain, or by ectodomain shedding. Ligand-induced PrPC dimerization or enforced dimerization of PrPC indicate that PrPC dimerization represents an important molecular switch for both intracellular signaling and inactivation by the release of PrPC N-terminal domain or shedding. In this review, we summarize evidence that cell surface receptor activity of PrPC is finely regulated by dimerization.The cellular prion protein (PrP C is a cell surface protein with a bipartite structure: the N-terminal domain is disordered and the C-terminal domain is structured and contains three α-helices and two short β-strands  , the authors proposed that cell surface PrPC dimers induce protective signaling through an unknown transmembrane receptor. This study did not elucidate whether the formation of dimers is constitutive or depends on an unknown ligand. Also, the proportion of PrPC dimers is unknown.The assembly of natural PrPC using an antibody-induced dimerization approach activates a Fyn-dependent signaling pathway in serotonergic and noradrenergic mouse cells differenciated from the murine neuroectodermal progenitor 1C11 clone  in the 1C11 neuroectodermal precursor and its neuronal differentiated progenies, the hypothalamic GT1-7 cells, and the T lymphoid BW5147 cells  is used to mimic an extracellular signal on cell surface receptors and trigger signal transduction. Although it is unclear if such strategy mimics the interaction with a partner or dimerization of the receptor, antibody-induced dimerization is largely used to engage a receptor in the absence of its ligand and relays intracellular signals. GPI-anchored proteins associate with raft domains in the plasma membrane and activate signal transduction pathways upon engagement with ligands or via antibody-induced dimerization  transcription factor and the transcription of several genes with important function in cellular protection and neuronal plasticity  as a key molecular step during the conversion was confirmed in a subsequent study  were covalently linked with a linker and recombinantly purified. This tandem protein oligomerized after purification, and thioflavine T staining indicated that such oligomers were likely on the pathway of amyloid formation, although this was not demonstrated (Simoneau et al., Sc in animal bioassays. Hence, the biological significance of PrPC dimerization for the formation of infectious PrPSc remains speculative.Models of PrP dimers and the role of dimers in prion conversion are available (Warwicker, C forms dimers in native conditions and cell surface dimerization clearly regulates PrPC-mediated signaling and the resulting physiological neuroprotective/neurotrophic activities (Rambold et al., C dimerization likely provides PrPC with a powerful neuroprotective/neurotrophic function (Figure C dimerization may help translate these findings into novel therapeutic interventions in neurodegenerative diseases (Beland and Roucou, PrPn Figure . Howevern Figure . RegulatThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs.Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger  in vivo accumulation of extensively overlapping sets of small RNAs to target viroids. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was significantly faster than PFOR. We also describe a simple computational program that after incorporation into PFOR2 is capable of identifying viroids from deep sequencing of long RNAs instead of small RNAs. Moreover, we report the identification of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA by the computational approach. Since our new algorithms do not depend on the analysis of viroid-derived small RNAs produced in vivo, it is possible to discover viroids in a wide range of host species including plants, invertebrates and vertebrates.Viroids are a unique class of subviral pathogens found in plants, and they are difficult to identify since they are free circular non-coding RNAs and often replicate to low levels in host cells. We previously described the computational algorithm PFOR that discovers viroids by analyzing total small RNAs of the infected plants obtained by next-generation sequencing platforms. However, the algorithm written in PERL language is very slow, and viroid identification depends on the  Viroids and a group of satellite RNAs (satRNAs) have single-stranded circular RNA genomes that range in size from 220 to 457 nucleotides (nt) Viroids infect many crops and cause severe symptoms in susceptible hosts that result in economically important diseases Two major limitations of the first version of PFOR restrict its application in the discovery of circular RNAs. First, the iterative filtering of small RNAs that are not derived from a replicating circular RNA is a slow process and takes up more than 90% of the PFOR running time. Because PFOR was written in the explanatory PERL language, analyzing complex small RNA libraries using PFOR may take hours or days. Second, circular RNAs are not identifiable by PFOR if they neither replicate nor trigger Dicer-dependent siRNA production in a eukaryotic cell.In this study, a considerably improved version of PFOR was developed by adopting parallel programming in the C++ language. The use of the new version of PFOR, designated PFOR2, led to the discovery of a new viroid from grapevine and a viroid-like RNA from apple tree. Moreover, a new program was developed and incorporated into PFOR2 for the discovery of distinct classes of circular RNAs, including those that neither replicate nor induce in vivo accumulation of Dicer-dependent siRNAs. We propose that the application of PFOR2 would speed up the discovery of novel circular RNAs and expand the list of known host species that can be independently infected by viroids.The computational algorithm of PFOR has been developed to identify replicating circular RNAs including viroids by deep sequencing of small RNAs Hop stunt viroid (HpSVd), Grapevine yellow speckle viroid (GYSVd) and Grapevine hammerhead viroid-like RNA (GHVd RNA) were each identified, and their full-length genomic RNA sequences were obtained by both PFOR and PFOR2 from the grapevine tree sRNA library, which contains 4,701,135 reads of 18–28 nt in length (GEO accession no. GSM458928). However, PFOR2 required only 67 seconds and was 3.3 times faster than PFOR. The second sRNA library was from a peach tree infected with Peach latent mosaic viroid (PLMVd) and contained 7,862,905 reads of 18–28 nt in length (GEO accession no. GSM465746). Both PFOR and PFOR2 were again able to identify PLMVd and to recover the complete sequence. Instead of 2 hours by PFOR using a default k-mer of 17, PFOR2 required only 22 minutes. These results demonstrated that PFOR2 was indeed faster than PFOR in viroid discovery.To test the performance of PFOR2, two publicly available small RNA libraries, in which both known viroids and viroid candidates have been verified by RT-PCR and Northern-blot hybridization, were used to compare the running time between PFOR and PFOR2. Apple scar skin viroid (ASSVd) (accession no. KC110858) isolated previously from apple in China and was hence considered to be a new isolate of ASSVd. The second RNA species was 434 nt in length and showed no sequence similarity to any of the known entries in GenBank. Interestingly, the second predicted circular RNA also contained the conserved sequences found in hammerhead ribozymes as shown previously for GHVd RNA PFOR2 was next applied to determine whether an apple plant with typical symptoms of apple scar skin disease contained new circular RNAs. The sRNA library was constructed from this apple plant, and 15,321,500 clean reads of 18–30 nt in length with a predominant size of 21 nt were obtained after deep sequencing . Two putTo verify the predicted sequence and the circular nature of AHVd-like RNA, total RNAs from the diseased apple were isolated for divergent RT-PCR analysis. According to the sequence of AHVd-like RNA assembled by PFOR2, two pairs of adjacent primers with opposing polarities  in vivo.AHVd-like RNA did not contain the characteristic central conserved region (CCR) found in the viroid family Both strands of AHVd-like RNA could form natural hammerhead structures  containiChicory yellow mottle virus (CYMV) and Tobacco ringspot virus (TRSV) The hammerhead structures of AHVd-like RNA were carefully compared with those of other known viroids and circular satRNAs (namely viroid-like RNAs), revealing some common salient features . i) In min vitro from linearized plasmids and were found to be self-cleaved during transcription and after purification when incubated under standard self-cleavage conditions in a protein-free buffer , Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV). The presence of these three plant viruses was further confirmed by RT-PCR , which have been previously detected by RT-PCR and Northern-blot hybridization in this old grapevine tree Citrus viroid VI (CVd-VI) (accession no. AB019508). Because CVd-VI had not been previously isolated from grapevine and the sequence similarity between CVd-VI and the identified RNA molecule was below the viroid species demarcation criteria of 90% sequence similarities The obtained sRNA library contained 14,033,487 clean reads of 17–30 nt in length, with 21 nt as the most dominant size class . PFOR2 aTo confirm the viroid nature of GLVd, we first determined whether GLVd existed in a circular form in vivo. Two sets of adjacent primers of opposite polarity  Apscaviroid. The terminal conserved region (TCR) of GLVd was also similar to that found in apscaviroids  formed by the upper CCR strand and the flanking inverted repeat unpaired . Howeverunpaired . The nucunpaired . DetectiTo further verify the viroid nature of GLVd, dimeric head-to-tail transcripts of GLVd were transcribed in vitro from the constructed cDNA clones of GLVd. Virus-free grapevine seedlings (cv ‘Beta’) grown in early spring were mechanically inoculated with the GLVd transcripts by slashing the stems with razor blades. Uninoculated healthy seedlings from the same batch were kept as controls. Because GLVd was undetectable by Northern-blot hybridization 3 and 6 months post inoculation, we re-inoculated the seedlings with a higher dose of GLVd transcripts and detected weak hybridization signals from 4 of the 18 inoculated grapevine plants 3 months after the secondary inoculation. To facilitate GLVd detection in the young tissues, the apical shoots of the inoculated grapevine plants were removed, and the leaves from the young lateral branches that emerged 6 months after the secondary inoculation (or 12 months after the first inoculation) were collected for RNA extraction. We found that GLVd infection became readily detectable in 6 of the 18 inoculated grapevine plants using either Northern-blot hybridization or RT-PCR. The progeny sequence was determined via DNA sequencing of the cloned RT-PCR products and was found to be the same as the inoculated GLVd transcripts. Therefore, GLVd autonomously replicated in its natural host grapevine, fulfilling the most critical criterion to be considered as a viroid.Apscaviroid. The phylogenetic tree constructed using viroids of genus Colviroid as the out-group revealed two subgroups of apscaviroids (Diospyros virginiana L.) Apscaviroid. Interestingly, careful inspection of the apscaviroid alignments identified two types of repeat sequences between GLVd and PVd-2 , as part of PFOR2 to discover biologically active circular RNAs via the deep sequencing of long RNAs instead of small RNAs. The program cut the sequenced long RNAs into 21-nt virtual sRNAs of 20-nt overlap with their 5′ and 3′ neighboring sRNAs before PFOR2 analysis to retain only 21-nt virtual ISRs for the final assembly of circular RNAs  approaches can readily identify viral and subviral pathogens in samples of plant and animal diseased tissues that are related in nucleotide sequence or encoded protein sequence to a known pathogen. The development of PFOR for viroid discovery thus represents a conceptual advance because, unlike NGS and several available classical approaches, PFOR does not depend on sequence homology with a known viroid. The major improvements described in this study overcame the limitations of the published version of PFOR that restrict its potentially widespread applications in pathogen discovery. PFOR2 was 3.3, 5.4, and 7.1 times faster than PFOR in the analysis of the three small RNA datasets from grapevine, peach, and apple, respectively. The enhanced speed is likely to be critical for viroid discovery when targeting hosts with large genome sizes and/or abundant small RNA populations. For example, our analysis of a small RNA library from Areca catechu with 46,637,488 reads took 8 hours and 40 minutes by PFOR2 instead of 110 hours by PFOR (unpublished data). The efficacy of PFOR2 was verified with the discovery of GLVd as a novel viroid that initiates independent infection in its natural host. Moreover, the development of SLS-PFOR2 eliminates the requirement for the in vivo production and accumulation of Dicer-dependent siRNAs to target the circular RNAs to be identified. As a result, small RNA sequencing becomes unnecessary, and RNA-seq libraries depleted of either ribosomal RNAs or linear RNAs can be analyzed by SLS-PFOR2 for the discovery of both replicating and non-replicating circular RNAs in diverse organisms. In principle, SLS-PFOR2 can identify novel viroid circular RNAs in host species that replicate but do not trigger the biogenesis of viroid-specific siRNAs. Therefore, SLS-PFOR2 has the potential to expand the list of both viroids and host species that support viroid infection.PFOR and FOR2 separate small RNAs in the pool into TSR and ISR groups based on the presence of the minimal overlapping k-mer among reads and remove all TSRs progressively. As a result, the overlapping sets of small RNAs retained after the filtering process might be different when different k-mers are used, leading to the variations in the sequences assembled by PFOR and PFOR2 that may not reflect the natural heterogeneity of viroids. The successful detection of each viroid by PFOR and PFOR2 is dependent on whether the circular RNA has been completely covered by a set of overlapping small RNAs with the minimal length defined by k-mers at both ends after removing all TSRs. Because each ISR is allowed to be used only once during the assembling step, only one viroid would be revealed when two or more viroids share small RNAs with lengths defined by k-mers or longer. For example, although ASSVd was revealed by PFOR2 analysis of the apple library using k-mers in the size range of 18 to 20 nt, AHVd-like RNA was identified using k-mers of 18 or 19, but not of 20. In the grapevine sample, HpSVd was identified by PFOR2 using k-mers of 17 to 21, whereas GYSVd-1 and GYSVd-2 were each revealed with a specific k-mer most likely because the two viroids are 80% identical in sequence and share small RNAs. Therefore, it is necessary to analyze each small RNA library using PFOR2 with k-mers from 17 to 21 and to verify the assembled sequences of viroid candidates by RT-PCR and cDNA sequencing.Citrus medica L.) with mild petiole necrosis and leaf bending The evolutionary origin of viroids remains unknown Apscaviroid. Although we found that GLVd was able to independently infect grapevine seedlings (cv ‘Beta’), the accumulation of GLVd was low, and no obvious symptoms were observed in infected grapevine plants. Although future studies on biological properties of GLVd may further differentiate this viroid from those previously reported, the conserved structural elements, the low sequence identity (maximum of 79% with CVd-IV) with other members in the genus Apscaviroid, and the natural host of GLVd, strongly support the possibility of annotating it as a new species in the genus Apscaviroid.Our conclusion that GLVd is a novel viroid is supported by the molecular and biological evidence presented here including its circularity, typical structural elements of viroids, and self-replication in grapevine seedlings. The phylogenetic analysis indicates that GLVd is a member of the genus Avsunviroidae viroids It is currently unclear whether AHVd-like RNA is a viroid or a satellite RNA, in contrast to GLVd. AHVd-like RNA shared no homology with the apple genome and was not amplified by PCR without a RT step, indicating that AHVd-like RNA was exogenous. Given that AHVd-like RNA encoded self-cleavage activities and was not specifically associated with any of the viruses identified in apple trees, we propose that AHVd-like RNA is a viroid. However, we were unable to demonstrate independent infectivity in apple seedlings for either the in vitro transcripts from dimeric AHVd-like RNA cDNA clones or AHVd-like RNA purified from apple tissues. In this regard, AHVd-like RNA may be related to ASSARNA-2, a circular RNA that was previously isolated from diseased apple plants in Japan and China, known to migrate more slowly than the 330-nt ASSVd RNA and unable to establish independent infection in apple seedlings Malus pumila Mill. cv. Fuji) plant, the fruits of which showed typical symptoms of apple scar skin viroid disease, in Shandong province China, in July 2012. The grapevine (Vitis vinifera L.) leaf samples used for determination of GLVd were from Tulufan in Xinjiang China. This grapevine plant (cv. Thompson seedless) for sample collections is more than 100 years old. Young leaves of both apple and grapevine were immediately put into RNAlater stabilization solution  after collection and sent to a laboratory for deep sequencing analysis. Moreover, approximately 10 g of apple and grapevine leaves were packaged with ice, kept fresh at low temperature, and sent to a laboratory for RNA analysis using PAGE and northern-blot hybridization.For the initial identification of viroids and viroid-like RNAs from apple, leaves were collected from an apple  following the manufacturer's instructions. The integrity of the resulting RNA preparations was evaluated before preparing cDNA libraries using an Agilent Technologies 2100 bioanalyzer. For RNA analysis by PAGE and northern-blot hybridization, nucleic acid preparations were obtained with buffer-saturated phenol followed by ethanol precipitation, as reported previously To prepare templates of RT-PCR amplification performed for cloning full-length sequence of viroid-like apple RNA, the obtained crude extracts were run on a non-denaturing 5% polyacrylamide gel stained with ethidium bromide, and the region of the gel delimited by the 250-bp and 400-bp DNA markers was excised and eluted as described previously In the experiment involving the RNA-seq of the potato samples, the extracted total RNAs by TRIzol reagent were purified by depleting ribosomal RNAs and non-circular RNAs. RNA species smaller than 200 nt, such as 5S ribosomal RNA, were first removed using the RNeasy Mini Kit , and 28S and 18S ribosomal RNAs were depleted by hybridization with specific probes following the instructions for the RiboMinus Plant Kit . To enrich circular RNAs, the total RNAs from the same sample were digested with RNase R  at 37°C for 90 min to remove non-circular RNAs.The RNA extracts were separated using two-dimensional PAGE (2D-PAGE) under non-denaturing and denaturing conditions and stained with ethidium bromide, as previously described The small RNA libraries were constructed using Illumina's small RNA sample preparation Kit  following Illumina's method. The protocols for sRNA purification, adaptor ligation, RT-PCR amplification, library purification and high-throughput DNA sequencing on an Illumina HiSeq-2000 have been reported previously The RNA-seq libraries of potato samples were constructed with a modified Not Not So Random (NNSR) sequencing method PFOR in the PERL language was converted to PFOR2 initially by using the C++ language. OpenMP is an Application Programming Interface (API) that supports multi-platform shared memory multiprocessing programming Splitting Longer read into Shorter fragments) program was developed to cut longer reads into virtual sRNAs. The final number of generated virtual sRNAs was dependent on two metrics: sRNA size and overlap size between neighboring sRNAs (step size). Typically, a longer read was cut into contiguous 21-nt sRNAs covering the whole read, in which each sRNA overlapped 20 nt with its 5′ and 3′ neighboring sRNAs (step size  =  1).The SLS  to generate full-length sequences of viroids and viroid-like RNA. The products of RT-PCR amplification were ligated with additional adenine (A) at the end using Taq DNA polymerase  and cloned into pGEM-T vectors (Promega) with protruding 3′-terminal thymine (T). The recombinant plasmids were amplified by transforming DH5α Escherichia coli cells, and positive clones were randomly selected for sequencing.The primers of HpSVd, GYSVd-1, GYSVd-2 and AGVd were described previously Nco I or Sal I to generate linear plasmids. RNA transcripts in both orientations were synthesized by T7 and SP6 RNA polymerase as described previously in-vitro transcription were purified by RNeasy Mini Kit . The purified transcripts were incubated at 37°C for 1 h and then separated by 5% denaturing PAGE containing 8 M urea and visualized by ethidium bromide staining. Full-length of AHVd transcripts and the longer fragments resulting from their in vitro self-cleavage were excised from the gels and eluted, separately. The ribozyme activities of the purified transcripts were assessed according to previously described methods The sequenced recombinant plasmids containing full-length of AHVd-like cDNA amplified with primers of AHVd-88F and AHVd-87R were digested with Head-to-tail dimmers of the entire sequence of GLVd and AHVd-like RNA were prepared by ligation of unit-length inserts and cloning into pGEM-T vectors (Promega), as described previously The secondary structures with minimum free energy for GLVd and AHVd-like RNA were predicted by the circular version of the MFold program S1 FigureSize distribution of sRNA library from the diseased apple plant. The total reads and the non-redundant reads are summarized separately. For total reads, reads of 21 and 22 nt are most in the library; however, among the unique reads, reads of 24 nt are predominant.(TIF)Click here for additional data file.S2 FigureMultiple alignments of 14 variants of AHVd-like RNA. The full length sequences of AHVd-like RNA were amplified by RT-PCR with the primers AHVd-13F and AHVd-12R. The sequences forming the hammerhead structures are delimited by flags and the predicted self-cleavage sites are marked by arrowheads. Solid and open symbols refer to plus and minus polarities, respectively.(TIF)Click here for additional data file.S3 FigureConfirmation of the predicted self-cleavage sites of AHVd-like RNA by 5' RACE-PCR and sequencing. The 3′F+ and 3′F- fragments of AHVd-like RNA were eluted and purified from 5% denaturing polyacrylamide gel shown in (TIF)Click here for additional data file.S4 FigureRT-PCR detections of ACLSV, ASPV, and ASGV. Lane 1, the negative control; lane 2, the diseased apple plant for deep sequencing; lane 3, the positive control. The primers used in RT-PCR reactions are shown in (TIF)Click here for additional data file.S5 FigureSize distribution of the sRNA library from the old grapevine sample.(TIF)Click here for additional data file.S6 FigureHPI structures of some apscaviroids.(TIF)Click here for additional data file.S7 FigureEnrichment of circular RNAs from total RNA and the flow chart of the NNSR method. (A) Depletion of 28S and 18S rRNAs by hybridization using specific probes. Equal amounts of total RNA before and after rRNA hybridization were visualized by ethidium bromide staining. (B) Digestion of linear RNAs by RNase R. Equal amounts of total RNA before and after RNase R digestion were checked on agarose gels. (C) Flow chart of the NNSR method. The RNA samples after RNase R digestion or rRNA depletion were used for first-strand synthesis primed by an adaptor A-tagged random hexamer primer (shown in blue). After RNase H treatment, the second-strand cDNA was synthesized using an adaptor B-tagged random hexamer primer (shown in yellow). Then, the library was amplified with adaptor A and adaptor B, the approximate size of amplicons were selected, and the amplicons were purified for sequencing.(TIF)Click here for additional data file.S8 FigureProposed kissing loops in AHVd-like RNA. The predicted interactions between loops are indicated with red lines.(TIF)Click here for additional data file.S1 TableSequences of the primers used in this study.(XLS)Click here for additional data file.S2 TableSurvey of the occurrence of ACLSV, ASGV, ASPV, and AHVd-like RNA in the field by RT-PCR.(DOC)Click here for additional data file.


Objective. To compare the effects of combined trigger point dry needling (TrP-DN) and proprioceptive/strengthening exercises to proprioceptive/strengthening exercises on pain and function in ankle instability. Methods. Twenty-seven  individuals with unilateral ankle instability were randomly assigned to an experimental group who received proprioceptive/strengthening exercises combined with TrP-DN into the lateral peroneus muscle and a comparison group receiving the same proprioceptive/strengthening exercise program alone. Outcome included function assessed with the Foot and Ankle Ability Measure (FAAM) and ankle pain intensity assessed with a numerical pain rate scale (NPRS). They were captured at baseline and 1-month follow-up after the intervention. Results. The ANOVAs found significant Group ∗ Time Interactions for both subscales of the FAAM  and for pain : patients receiving TrP-DN plus proprioceptive/strengthening exercises experienced greater improvements in function and pain than those receiving the exercise program alone. Between-groups effect sizes were large in all outcomes (SMD > 2.1) in favor of the TrP-DN group. Conclusions. This study provides evidence that the inclusion of TrP-DN within the lateral peroneus muscle into a proprioceptive/strengthening exercise program resulted in better outcomes in pain and function 1 month after the therapy in ankle instability. Lateral ankle sprain is the most common form of ankle sprain experienced by subjects participating in athletic activities and results in substantial societal burden . LateralAnkle instability symptoms include subjective feeling of ankle giving way, pain, swelling, resprain, and/or limitations in daily life activities and sports participation . ChronicConservative management is the initial therapeutic option for these patients; however, the most appropriate strategies are still unclear. Recent studies support the use of joint mobilization techniques for the management of ankle instability symptoms –10. NeveThe role of surrounding soft tissues in the aetiology of chronic ankle instability is based on alterations in soft tissue function that may occur in the surrounding muscles. A recent meta-analysis determined that subjects presenting with ankle instability exhibit delayed peroneal reaction time when compared with the contralateral uninvolved limb or with healthy controls . This evNo studies to date have examined the efficacy of including TrP-DN combined with a proprioceptive/strengthening program in subjects with chronic ankle instability. Therefore, the purpose of this randomized clinical trial was to compare the effects of combined TrP-DN and proprioceptive/strengthening exercise program to proprioceptive/strengthening program alone on pain and function in individuals with chronic ankle instability. We hypothesized that those individuals receiving TrP-DN combined with proprioceptive/strengthening exercise program will exhibit higher improvements in pain and function than individuals receiving the proprioceptive and strengthening program alone.Consecutive individuals with unilateral ankle instability presenting to a physical therapy clinic in Madrid (Spain) from January 2013 to June 2014 were screened for inclusion in this study. To be included in the study patients had to fulfil the following criteria: (1) age between 18 and 50 years, (2) history of at least one ankle sprain, (3) at least one episode of giving away in the previous 6 months, (4) ankle pain of intensity > 3 points on an 11-point numerical pain rate scale (NPRS), and (5) score of 25 or less on the Cumberland Ankle Instability Tool . In addiParticipants were excluded if exhibited any of the following criteria: (1) fracture in the lower extremity, (2) history of surgery in the lower extremity, (3) any concomitant lower extremity pathology, for example, vascular disease and osteoarthritis, (4) pregnancy, (5) regular use of medication, or (6) previous physical therapy interventions received on the lower extremity within the previous 6 months. The study protocol was approved by the Ethical Committee of the Universidad Rey Juan Carlos and it was conducted according to the Helsinki Declaration. All participants read and signed an informed consent prior to their inclusion in the study.The primary outcome measure of this study was the Foot and Ankle Ability Measure (FAAM). A recent systematic literature has found that the FAAM is the most commonly used outcome measure for assessing function in the lower extremity . The FAAIn the secondary outcome, the intensity of ankle pain during sport practicing was assessed with an 11-point numerical pain rating scale (NPRS), where 0 is the absence of pain and 10 represents maximum pain . There iOutcomes were captured at baseline and at 1 month after the last treatment session.Following the baseline examination, patients were randomly assigned to receive proprioceptive/strengthening exercise program alone (control group) or proprioceptive/strengthening exercise program combined with TrP-DN into the lateral peroneus muscle . Concealed allocation was performed using a computer-generated randomized table of numbers created prior to data collection by an external researcher. Individual and sequentially numbered index cards with the random assignment were prepared. The index cards were folded and placed in sealed opaque envelopes. A second external researcher opened the envelope and proceeded with treatment according to the group assignment.Each group was treated by a clinician with more than 10 years of experience in the management of lower extremity injuries. A systematic review has concluded that proprioceptive exercise programs are commonly delivered over a 6-week period, with a frequency varying between 1 and 7 times weekly and with a duration ranging from 10 min to 1 h . NeverthThe experimental group also received TrP-DN in the lateral peroneus muscle of the affected extremity. TrP-DN was applied once per week for the first 4 weeks before starting any exercise on that session. Individuals were unaware of the real objective of the study in that they were aware of the clinical implications without revealing the real intervention that was being evaluated. All subjects were informed of the true nature of the study at the end of the trial.Kim et al. have recently demonstrated that combination of muscle strengthening and proprioceptive exercises is more effective than only muscle strengthening exercises for the management of ankle instability . The strThe strengthening program included a protocol consisting of the use of Thera-Bands according to the protocol described by Kaminski et al. . ParticiThe proprioceptive exercise program consisted of a number of closed kinetic chain exercises in weight bearing positions. The clinician reinforced the patient through progressive lower extremity loading from bilateral to unilateral load acceptance. The exercises applied in the current study consisted of semisquats  and one TrP-DN was applied to the lateral peroneus muscle by a clinician with more than 6 years of experience in the management of TrPs with this technique. Patients received TrP-DN with disposable stainless steel needles  that were inserted into the skin over the TrP area. TrP diagnosis was determined when all the following criteria were present : (1) hypIn this study, the fast-in and fast-out technique described by Hong was applied . Once thSample size and power calculations were performed with the ENE 3.0 software . The calculations were based on detecting a mean difference of 8.0 points (MCID) on each subscale of the FAAM , assuminAll participants were asked to report any adverse events that they experienced during all the study and the 1-month follow-up period. An adverse event was defined as sequelae of medium-term in duration with any symptom perceived as distressing and unacceptable to the individual and that required further treatment . Since TP > 0.05). Baseline demographic and clinical variables between both groups were compared using independent Student's t-tests for continuous data and χ2 tests of independence for categorical data. A 2 × 2 repeated measured ANOVA with time  as within-subjects factor and group  was used to calculate changes in the outcomes (pain and function). The main hypothesis of interest was the Group by Time Interaction at an a priori alpha-level equal to 0.05.Statistical analysis was performed using SPSS statistical software, version 18.0, and it was conducted according to the intention-to-treat analysis principle. Mean, standard deviation, and/or 95% confidence interval were calculated for each variable. The Kolmogorov-Smirnov test showed a normal distribution of all the data  were calculated by dividing the mean score differences between TrP-DN plus proprioceptive/strengthening exercise program and comparison group  by the pooled standard deviation.n = 14) or comparative group (n = 13). The reasons for ineligibility can be found in Thirty consecutive individuals with ankle instability were screened for eligibility criteria. Twenty-seven patients  satisfied the eligibility criteria, agreed to participate, and were randomized into TrP-DN experimental group : patients receiving TrP-DN plus proprioceptive/strengthening exercises experienced greater increase in function than those receiving proprioceptive/strengthening exercises program alone  with patients receiving the combination of TrP-DN and proprioceptive/strengthening exercises experiencing a greater reduction in pain intensity than those receiving proprioceptive/strengthening exercises program alone (d > 2.3) for the decrease in ankle pain in favour of the TrP-DN plus proprioceptive/strengthening exercises group.The 2 × 2 ANOVA also revealed a significant Group ∗ Time Interaction for ankle pain  assigned to the TrP-DN plus proprioceptive/strengthening exercises group experienced some muscle soreness at the lateral peroneus muscle after treatment but did not experience an increase of their ankle symptoms. TrP-DN posttreatment soreness resolved spontaneously within 24–36 hours without any intervention.The results of this randomized clinical trial suggest that the combination of TrP-DN plus proprioceptive/strengthening exercise program resulted in better outcomes 1 month after the end of therapy than when only proprioceptive/strengthening exercises were applied in individuals with chronic ankle instability. We could anticipate that the benefit of adding TrP-DN for the management of ankle instability could be clinically relevant as noted by the large between-group effect sizes in all the outcomes, although further studies are needed.Different systematic reviews had concluded that manual therapies , exercisWe found that subjects with ankle instability who received TrP-DN in addition to a proprioceptive/strengthening exercise program resulted in higher improvements in function in both ADL and SPORTS subscales than those receiving only the exercise program. It should be noted that while between-groups change scores were statistically significant and surpassed the reported MCID for both subscales of the primary outcome (FAAM), the clinical significance of these differences is not as clear given the inclusion of the MCID within the 95% CIs for these comparisons . This maOur study also observed that individuals receiving TrP-DN exhibited a higher decrease in pain intensity than those who did not receive the intervention. In this case, between-group change scores surpassed the MCID for the outcome , 26 in fThe exact therapeutic mechanism by which TrP-DN exerts its effects remains to be elucidated . Both meBecause the addition of TrP-DN resulted in statistically and potentially clinically greater improvements in pain and function in individuals with ankle instability we may hypothesize that the ankle eversor muscles can play a relevant role and may perpetuate symptoms associated with ankle instability. In fact, a recent meta-analysis has supported the presence of delayed peroneal reaction time in subjects with ankle instability . It is pFinally, we should recognize that a number of limitations existed in the current study. First, only 1 therapist provided the treatment to each group, respectively, which may limit the generalizability of the results. Second, we only assessed outcomes at 1-month follow-up and cannot be certain if these differences remained in the long term. Third, we did not also assess the perspective of the patients about the progress of their instability by using a self-reported method of evaluation like the Global Rating of Change (GROC). Finally, the influence of a placebo effect is unknown as we did not include a group receiving a sham intervention , 45 and This study provides evidence that the inclusion of TrP-DN within the lateral peroneus muscle into a proprioceptive/strengthening exercise program resulted in better outcomes in pain and function 1 month after the end of the therapy in individuals with ankle instability. Our results may anticipate that the benefits of adding TrP-DN in the lateral peroneus muscle for the management of ankle instability are clinically relevant as large between-groups effect sizes were observed in all the outcomes. Future studies should include a control group and examine the long-term effects of these interventions in this population.


The identification of oncogenic driver mutations in non-small cell lung cancer (NSCLC) has led to a paradigm shift and the development of specific molecular treatments. Tumors harboring a rearranged EML4–ALK fusion oncogene are highly sensitive to therapy with ALK-targeted inhibitors. Crizotinib is the first approved treatment for advanced lung tumors containing this genetic abnormality. In this mini review, we discuss the existing data on crizotinib as well as ongoing trials involving this medication. A brief overview of the known resistance mechanisms to crizotinib will also be presented followed by a summary of the ongoing trials involving next-generation ALK-inhibitors or other targeted therapies in patients with ALK+ NSCLC. Treatment for non-small cell lung cancer (NSCLC) has historically consisted of cytotoxic chemotherapy. Recent advances in molecular biology had led to the discovery of oncogenic driver mutations with subsequent development of oral agents that target these molecular pathways. In NSCLC, the main two driver mutations, with FDA approved targeted therapies, consist of echinoderm microtubule protein like-4/anaplastic lymphoma kinase (EML-4/ALK) translocations and epidermal growth factor receptor (EGFR) mutations. Crizotinib is the first FDA approved treatment for patients with ALK+ NSCLC. To this date, only the final data from one phase III randomized trial has been published, evaluating the use of crizotinib as a second-line therapy . MultiplThe scope of this review article is twofold. First, the existing data on crizotinib will be presented as well ongoing trials involving this medication. Second, a brief overview of the known resistance mechanisms to crizotinib will be presented followed by a summary of the ongoing trials involving newer generation ALK-inhibitors or other targeted therapies in patients with ALK+ NSCLC.in vitro studies suggested different crizotinib sensitivity for each variant of the EML4–ALK fusion protein , including ALK, c-Met, hepatocyte growth factor receptor (HGFR), and c-ros oncogene 1 (ROS1) Figure . In 2007 protein . However protein . In addiIn 2013, Shaw et al. published the first phase III randomized trial involving crizotinib in the second-line setting  has been demonstrated to be very poor . Costa eIn order to understand the rationale behind the majority of ongoing clinical trials involving ALK+ NSCLC patients, it is important to survey the currently known mechanism of resistance to crizotinib. ALK-dependant resistance mechanism occurs upon mutations in the tyrosine kinase (TK) domain, and activation of alternative signaling pathways. Alternatively, true ALK-independent resistance may arise through the outgrowth of clones that do not harbor an ALK gene fusion and contain a separate activated oncogene . Given tPast experience with the use of TKIs in chronic myelogenous leukemia as well as EGFR-mutated lung cancer teaches us that most common mechanisms of resistance to this class of medications are secondary mutations in the TK domains . This alThe first major “gatekeeper” mutation identified in the TK domain of EML4–ALK involves the substitution of leucine for a methionine at position 1196 (L1196M) of the kinase domain of ALK, thus creating a mutant bulky amino-acid side chain in the ATP-binding pocket of the receptor, ultimately interfering with the binding of crizotinib to its receptor . This isTissue analysis of harvested tumor cells from patients resistant to crizotinib has demonstrated other non-gatekeeper secondary mutations in the ALK TK receptor. ALK secondary mutations in NSCLC are distributed throughout the kinase domain, including the solvent front , ATP-binding pocket (G1269A), and N-terminal to the C-helix  .Crizotinib is an inhibitor of multiple TKIs beside ALK, including ROS1 oncogene. A recent report of a G2032R mutation in the ROS1 TK domain leading to crizotinib resistance has also been identified . AlthougActivation of alternative downstream signaling pathways, even in the setting of complete ALK receptor inhibition, is increasingly recognized as mechanisms of resistance to crizotinib. These include activation of the EGFR, heat shock protein 90 (HSP90), and the PI3K/AKT/mTOR pathways. The activation of these alternative pathways is present in up to 20% of patients .de novo mutation of the EGFR gene in patients and cell lines treated with crizotinib, accounting for resistance to this drug  is a novel and potent selective TKI targeting ALK. Results from a recent phase I/II study of this drug in both crizotinib-naïve and crizotinib-resistant patients have been published . The maxMultiple trials are nevertheless ongoing for this drug. These include three phase II trials of ceritinib for crizotinib-resistant patients  and crizotinib-naïve patients (NCT01685138). One phase II study is looking specifically at patients with ROS1 mutation (NCT01964157). Two phase III trials are comparing ceritinib with standard chemotherapy in patients previously treated with platinum-based chemotherapy (NCT01828112), and chemotherapy-naïve patients (NCT01828099). One phase I study is assessing the combination of ceritinib with a HSP90 inhibitor (NCT01772797).Alectinib  is a highly potent selective ALK inhibitor with activity against L1196M gatekeeper mutation as well as other secondary mutations such as F1174L and R1275Q , 35. ResThree phase II/III trials with alectinib are in progress in crizotinib-naïve  as well as in crizotinib-resistant  patients.AP26113  is a novel inhibitor of ALK with activity against L1196M gatekeeper mutation as well as against ROS1 and EGFR (including mutant form with the T790M gatekeeper mutation) . In an oMultiple other new-generation ALK agents are currently under phase I study. These include TSR-001, ASP3026, PF-06463922, as well as X-396 . Detailsin vitro, and led to tumor regression in in vivo models  in three NSCLC patients resulted in two partial responses and one case of prolonged stable disease . In anotThe identification of the ALK fusion protein in 2007 and the fast development and approval of a FDA targeted treatment in <5 years constitutes a remarkable feat in the field of targeted therapies. Crizotinib holds many promises from its early debut in treatment-refractory NSCLC patients. Final results from phase III randomized trials using Crizotinib as a first-line therapy are eagerly awaited for. Despite excellent response in the initial stages, most patients develop resistance to crizotinib. Elucidating resistance mechanism and subsequently developing therapeutic strategies to overcome resistance to ALK-inhibitors constitutes a priority, with the vast majority of ongoing clinical trials in this field involving new-generation ALK-inhibitors or combination therapy with other targeted agents. Key questions remain on how to correctly identify the resistance mechanism of a tumor progressing on ALK-targeted therapy given re-biopsy is often technically challenging and resource intensive, as well as on how to correctly identify and stream the correct combination of therapies to the appropriate patient populations as first-line therapy. The ongoing Q-CROC-05 multicenter phase IV clinical trial (NCT02041468) will aim to shed some light on these important clinical issues.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.http://www.frontiersin.org/Journal/10.3389/fonc.2014.00174/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.


As facial color pattern around the eyes has been suggested to serve various adaptive functions related to the gaze signal, we compared the patterns among 25 canid species, focusing on the gaze signal, to estimate the function of facial color pattern in these species. The facial color patterns of the studied species could be categorized into the following three types based on contrast indices relating to the gaze signal: A-type (both pupil position in the eye outline and eye position in the face are clear), B-type (only the eye position is clear), and C-type (both the pupil and eye position are unclear). A-type faces with light-colored irises were observed in most studied species of the wolf-like clade and some of the red fox-like clade. A-type faces tended to be observed in species living in family groups all year-round, whereas B-type faces tended to be seen in solo/pair-living species. The duration of gazing behavior during which the facial gaze-signal is displayed to the other individual was longest in gray wolves with typical A-type faces, of intermediate length in fennec foxes with typical B-type faces, and shortest in bush dogs with typical C-type faces. These results suggest that the facial color pattern of canid species is related to their gaze communication and that canids with A-type faces, especially gray wolves, use the gaze signal in conspecific communication. The facial morphology and color pattern of animals, especially those around the eyes, have been suggested to serve various adaptive functions related to the gaze signal that facilitate the detection of their gaze direction by other animals. For example, Cott Canis lupus) have facial color patterns in which the gaze direction can be easily identified, although this is often not the case in other canid species. For example, identifying the gaze direction of raccoon dogs (Nyctereutes procyonoides) appears to be difficult because of the dark facial color around their eyes. Various canid species reportedly use body postures and facial expressions in their visual communication Similar to humans, gray wolves  were collected . Facial Using these facial images, color contrasts between the following five parts of the face were analyzed as indices of the gaze signal: pupil (P), iris (I), eyelid margin (E), coat around the eyes (C), and facial area including the eyes (F) . All stuThe color contrasts were calculated assuming that the color vision of canid species closely resembles that of human deuteranopia Using these luminance values, Michelson contrasts between the following parts were calculated using the following formula,Here, we used the following four color contrast values as indices of the gaze signal: 1) contrast between the iris and pupil (IP) as an index for the conspicuousness of pupil position in the eye outline, 2) the iris–eyelid margin contrast (IE), 3) the coat around the eye–eyelid margin contrast (CE), and 4) the coat around the eye–face coat contrast (CF) as indices for the conspicuousness of eye position on the face.In the analysis of the relationship between facial color pattern and phylogeny, we referred to the phylogeny of canid species estimated by Lindblad-Toh et al. Vulpes macrotis) and swift fox (Vulpes velox) as the same species. Because gray wolves, including the high arctic wolf, red fox, and arctic fox exhibit different body color types, we analyzed the facial color pattern of each type separately.To analyze the relationship between facial color pattern and sociality in the studied canid species, we categorized the species into the following two types based on their social life in the non-breeding season: the group-living type that forms a family group of more than three individuals even during the non-breeding season and the solo/pair-living type that never forms groups larger than a pair . We alsoC. lupus), fennec foxes (Vulpes zerda) and bush dogs (Speothos venaticus) under captive conditions. Gazing behavior was defined as a behavior during which the animal fixes its face direction toward the other individual for over 1 s, keeping its body still. We also compared “face-averting” behavior in which the animal kept its face averted from the other animal in the vicinity of that animal for over 1 s. We observed these behaviors in 11 gray wolves from the same family group  at the Tama Zoological Park, in a pair of fennec foxes  at the Kyoto City Zoo, and in four bush dogs  from two family groups kept in two separate groups  in the Yokohama Zoological Gardens in Japan. Each gray wolf was observed for an average of 2.60 h (1.70–2.88 h) during the daytime (09:00–13:00) on 9 March 2011. Each of the two fennec foxes was observed for an average of 3.23 h  during the daytime (09:00–13:00) on 6 and 7 February 2014. Each bush dog was observed for an average of 2.56 h (2.41–2.66 h) during the daytime (09:30–13:00) on 12 October 2011 and in the afternoon (13:30–16:30) on 13 October 2011. All observations were conducted during the breeding season of each species We compared the gazing behaviors of the following three species that engage in group living: gray wolves .The proportion of facial color types differed marginally significant between the two types of hunting behaviors  compared to fennec foxes (three postures) or bush dogs (three postures). In particular, the pointing posture was not observed in either fennec foxes or bush dogs. The pointing posture is a characteristic behavior of gray wolves and domestic dogs in which the animal fixes its face and body toward the other animal, assuming the posture when preparing to dash or pounce, often keeping its foreleg up The mean duration time of the gazing behaviors significantly differed among the three study species  did not differ significantly among high arctic wolves , Canadian wolves , or Mexican wolves  living in arctic, temperate, and subtropical areas, respectively .Light-colored irises in A-type faces are the most important aspect of the gaze-signal in the facial color pattern of canid species. This feature results in high contrast between the iris and the dark color of the pupil (IP), making the pupil position conspicuous within the eye outline. Because most parts of the eye outline are covered by the iris and the area of exposed sclera is very small in all canid species, light-colored irises enhance the gaze signal, just as the white sclera of humans highlights the position of dark-colored irises within the eye outline. In addition, because the eyelid margin was dark-colored in all of the study canid species, light-colored irises also strongly contrast with the eyelid margin (IE), making the eye position conspicuous on the face. In contrast, the dark-colored irises of B- and C-type faces result in low contrast with the pupil and eyelid margin, thus reducing the gaze signal.Facial color patterns change with growth in many canid species, although no studies have directly examined such developmental changes. For example, all newborn gray wolves observed in the present study had dark-colored bodies and C-type faces with dark-colored irises. Their body color became lighter after 2 weeks, and their facial color patterns changed from C-types to B-types before reaching about 6 months old and to A-types with light-colored irises before reaching 1 year old . Some previously published images Although the physiological basis of eye color in canid species has not yet been examined directly, the iris color as well as the skin and hair color of humans and other mammals are reportedly pigmented by two kinds of melanin, eumelanine (black/brown color) and pheomelanin (red/yellow color), in melanocyte cells One possible adaptive function of iris pigmentation is protection against ultraviolet (UV) radiation. In general, mammals living in environments with stronger UV radiation tend to have darker body colors than relatives living in environments with less UV radiation However, from this perceptive, explaining the iris color of gray wolves and its changes with growth is difficult. Iris color did not significantly differ among high arctic wolves, Canadian wolves, and Mexican wolves living in arctic, temperate, and subtropical areas, respectively. In addition, the irises and body coat of gray wolf cubs are darkly pigmented for about 1 month after birth while they remain in the dark den (with less UV) and change to lighter colors after they begin to live outside of the den (with stronger UV). This color change with growth is opposite of what would be predicted from the “UV radiation theory.” In addition, this theory cannot explain why many species with A-type faces have a light-colored coat around the eyes (high CE and/or CF).In contrast, the relationships of facial color type with sociality and gazing behavior suggest that the A-type faces of canid species are adaptations for visual communication via the gaze signal. The fact that the studied canid species with A-type faces tended to engage in group living suggests that they use the gaze signal in communication among group members, as the need for communication is larger for group-living species than for solo/pair-living species. Group-hunting species had significantly lighter iris color  than those of solo-hunting species. These results suggest that the gaze signal is used for communication during group hunting in many of these species, although a significant relationship between facial color type and hunting behavior type was not detected.The longest duration time for gazing behavior was observed in wolves, a species with an A-type face, which also suggests that A-type faces in canid species serve as adaptations for visual communication via the gaze signal. Gazing behavior would facilitate the easy detection of the gaze signal by other conspecifics. Because wolves directed their faces toward others while keeping their entire body still during these behaviors, their gaze signal could be clearly displayed to others even from a distance. Pointing is a behavior characteristic to gray wolves that occurred for a particularly long duration (7.67 s on average). Face-averting behavior was observed in all three focal species, further suggesting the importance of the gaze signal in these species, at least at close proximities. Fox The relationship between facial color type and phylogeny  suggestsIn the wolf-like clade in which all species engage in group living, 8 of 10 study species exhibit A-type faces (African wild dogs and dingoes do not). African wild dogs with C-type faces are known to wave their white-tipped tails in an upright position like flags during group hunting Click here for additional data file.Table S2Contrast indices of the studied animals. The rank of each index was shown in the parenthesis. Indices of different body color types in gray wolves , red foxes  and arctic foxes  were shown separately. Asterisks (*) before indices means that the values were larger than the average of each index among studied species.(XLSX)Click here for additional data file.Table S3Gazing and face-averting behavior of the three group-living species. Gazing behavior was defined as a behavior during which the animal fixes its face direction toward the other individual for over 1 s, keeping its body still in standing, crouching, sitting, lying down, upside down, or pointing posture. Face-averting behavior was defined as a behavior during which the animal keeps its face averted from other animal in vicinity of that animal for over 1 s.(XLSX)Click here for additional data file.


Medication overuse headache (MOH) is a chronic headache associated with an overused of NSAIDs, combined analgesics or triptans. The pathophysiology and the risk factors of MOH are still unknown. MOH can be considered as an interaction between the chronic pain disorders and individual predisposition for the dependent behavior.The current study was conducted to determine the association between the dopamine β-hydroxylase (DBH) -1021C/T polymorphism and MOH.-1021C/T polymorphism in promoter region of DBH gene was analyzed in 44 patients with MOH with a control sample of 30 individuals without headache and without drug overuse, and with 24 patients chronic migraine without MOH and 41 episodic migraineurs. The DBH genotypes were identified by a PCR-RFLP method and clinical profile were assessed by Headache Diary and Leeds Dependence Questionnaire (LDQ)The genotype frequencies of -1021C/T polymorphism DBH gene did not differ between the MOH and other groups. In contrast, we found that the presence of the -1021T allele was significantly associated with the monthly drug consumption and LDQ total score in the MOH group. We also found an interaction between the presence of DBH -1021T genotype and efficiency serotonin-norepinephrine reuptake inhibitors in patient with MOH.These results showed the influence of the -1021T>C polymorphism DBH gene on the number of symptomatic drug doses taken and level substance dependence  in MOH, supporting a relationship between MOH and dependence-related behavior.No conflict of interest.


There is a renewed interest in MR-guided laser interstitial thermal therapy (LITT) as a minimally invasive alternative to craniotomy for local treatment of various brain tumors and epilepsy. LITT allows for focused delivery of laser energy monitored in real time by MRI, for precise ablation of the lesion. Although highly promising, the long-term effects of laser ablation as a viable treatment option for neurological disorders have yet to be rigorously studied and quantified. In this work, we present a quantitative framework for monitoring per-voxel thermal-induced changes post-LITT over time on multi parametric MRI. We demonstrate that voxel-by-voxel quantification of MRI markers over time can enable a careful and accurate (a) characterization of early LITT-related changes (if and when they are exaggerated and when they subside), and (b) identification and monitoring of MRI markers that potentially allow for better quantification of response to LITT therapy. The framework was evaluated on two distinct cohorts of patients , who were monitored post-LITT at regular time-intervals via multi-parametric MRI. On a cohort of six GBM studies we found that (a) it may be important for the initial treatment-related changes to subside to more reliably capture MRI markers relating to tumor recurrence, and (b) T1w MRI and T2-GRE may better differentiate changes that may correspond to tumor recurrence from patients with no recurrence, as compared to T2w-MRI, and FLAIR. Similarly, our preliminary analysis of four epilepsy studies suggests that (a) early LITT changes  appear to subside within 4-weeks post-LITT, and (b) ADC may be more reflective of early treatment changes (up to 1 month), while T1w may be more reflective of early delayed treatment changes , while T2-w and T2-FLAIR appeared to be more sensitive to late treatment related changes (6-months post-LITT) compared to the other MRI protocols under evaluation. There has been a recent interest in the potential utility of Magnetic Resonance Imaging (MRI)-guided Laser-induced interstitial thermal therapy (LITT) to treat brain tumors, such as glioblastoma multiforme (GBM) in vivo imaging characteristics of LITT-induced changes within and around the ablation zone for GBM patients.In the context of GBM, LITT is currently being explored for patients who, as determined by a multidisciplinary neuro-oncology board, are not candidates for standard-of-care open surgical debulking More recently, LITT has also emerged as an alternative to traditional craniotomy for epilepsy, which attempts to ablate seizure focus with minimal damage to normal surrounding tissue Currently post-treatment changes for evaluating treatment response are monitored qualitatively via comparing volumetric changes of contrast enhancement on T1w MRI protocol acquired for follow-up , with reference to pre-treatment T1w MRI  values computed from diffusion weighted imaging (DWI)) acquired at multiple time points post-LITT   for follResearchers have previously investigated some of these questions in the context of evaluating response to treatment for various neurological disorders and cancer types by qualitatively following trends of changes in MR markers over time MR intensity non-standardness across pre- and post-LITT MRI: One of the major drawbacks of MRI is known to be the lack of a quantifiable (tissue specific) interpretation of image intensities Accurate alignment of different MRI protocols and across pre-, and post-treatment at different time-points for computing voxel level absolute differences of the imaging markers (reflective of treatment changes): Accurate co-registration of MRI protocols  is essential to quantitatively compare changes in imaging markers on voxel-by-voxel basis, across different protocols, while accurate co-registration of MRI markers pre-, and at different time points post-LITT is required for careful monitor changes in MRI markers on a per-voxel basis over time, andA combination of MRI markers can capture treatment related changes over time with high sensitivity and specificity compared to a single MRI marker: A large volume of literature In this work, we address these challenges by developing a quantitative framework  that encModule 1. Correction of MRI marker drift: Pre-and post-LITT MR markers  will be quantitated by correcting for intensity drift between acquisitions using intensity standardization developed by Nyul and Udupa Module 2. Registration: An affine co-registration scheme  Module 3. Quantification of differences in MR intensities between pre, and multiple post-LITT time points: A difference map is computed between pre-LITT MRI markers , and the corresponding post-LITT MRI marker at every time-point, to capture and monitor subtle changes in MRI markers on a per-voxel basis. The changes in MRI markers monitored over time are used to quantify the relative importance of individual MRI markers in capturing treatment related changes over time.Module 4. Creating a multi-parametric MRI map by fusing difference maps obtained for different MRI protocols: A multi-parametric map is created by combining pre-, and post-MRI intensity difference maps. The combination consists of linear weighting of difference maps of MRI markers based on how responsive they are (computed in Module 3) in accurately capturing treatment related changes over time.We evaluate our quantitative framework to understand and monitor treatment changes over time for two disease types, epilepsy and GBM. Although we realize that the MRI changes for epilepsy and GBM are inherently different as different tissues are targeted during treatment , the purpose of this study is to provide a quantitative framework for monitoring changes in MR markers over time that can be used to capture LITT related changes over time. It is our intent that the analytic platform will pave the way for building novel imaging-based predictors of LITT response in neuroimaging disorders (including GBM and epilepsy), and thus enable improved understanding of patient's response to LITT.When the histograms for T1w MRI at together ), it is together  and the A 3D affine transformation ost-LITT .Post-LITT MRI at every time-point is aligned with reference to pre-LITT MRI, for every MRI protocol, T1w, T2w, GRE, FLAIR, ADC using an affine-based co-registration scheme (described above). The co-registration with reference to pre-LITT MRI allows for alignment of all protocols across different time-points in the same frame of reference for a voxel-by-voxel comparison.The range of values for pre and post-LITT MRI was normalized to have a mean of 0 and a mean absolute deviation of 1. This ensured that the different MR parameter values were in a comparable range of values when quantifying differences across pre- and post-LITT MRI. An ablation zone, Each of the difference maps, The median intensity is computed within the ablation zone of the difference maps for every MRI protocol. Median provides a measure of the most commonly occurring values within a range of values, and was used to quantify the most commonly occurring intensity changes in MRI markers within the ablation zone. A higher median value would hence reflect a high change in intensity values (between pre- and post-LITT MRI) while a low value would reflect a small change in intensity values within the ablation zone. We then extract a temporal MRI profile for every MRI protocol Post-LITT MRI at every time-point is registered with reference to pre-LITT T1w MRI, for every MRI protocol using the methods illustrated in the co-registration Section. The co-registration with reference to pre-LITT T1w MRI allows for alignment of all protocols across different time-points in the same frame of reference for a voxel-by-voxel comparison.A differences map is computed within the ablation zone as an absolute difference of imaging markers as, The median intensity is computed within the ablation zone of the computed difference maps for every MRI protocol. We then extract a temporal MRI profile for every MRI protocol For a specific time-point, each of post-LITT MRI protocol is individually aligned to pre-LITT T1w MRI via an affine co-registration scheme as described in the co-registration Section to align all MRI protocols across multiple time-points to a common frame of reference. The co-registration with reference to pre-LITT T1w MRI allows for alignment of all protocols across different time-points in the same frame of reference for a voxel-by-voxel comparison.The relative contribution of each protocol is obtained via a difference map by identifying MRI markers that change most dramatically post-LITT and hence may serve as candidate MR markers that better capture treatment related changes post-LITT. The individual contributions are obtained for each protocol depending on their ability in differentiating positive and negative treatment related changes, and are combined to develop a fused MP-MRI marker that simultaneously captures the discriminability across all individual MRI markers, and may potentially serve as a surrogate marker to distinguish successful treatment from tumor recurrence.Once a difference map is computed within the ablation zone as an absolute difference of image intensity markers between pre- and post-LITT MRI, a fused MP-MRI marker map is obtained by leveraging difference maps for each of the five MRI markers  as For a specific protocol, http://www.slicer.org/). Intensity standardization as well as modules 2 and 3 for each of the three objectives involving computation of difference maps, development of temporal signatures, optimization and integration of weights across different MRI protocols was implemented via the Matlab software package.The co-registration across different MRI sequences was implemented via the 3D Slicer software 4.1.  was employed for all LITT procedures. Details on available protocols and time-points for the two cohorts of datasets, epilepsy, and GBM are provided in The two cohorts were collected as a part of an ongoing IRB approved study "Prospective and Retrospective Database Review of Magnetic Resonance Thermometry Guided Laser Induced Thermal Therapy (LITT)" (under IRB protocol #0220110296), at Robert Wood Johnson Hospital, New Jersey. Written consent was obtained from all patients for long-term follow up. Secondary analysis was performed on the anonymized data derived from data collected under informed consent. Since de-identified data was used for analysis, IRB consent was not required.Four epilepsy patients were monitored post-LITT via MP-MRI  as a part of an ongoing study at Rutgers-RWJ Medical School between 2011-2013, after initial 1.5-Tesla multi-parametric MRI. Post-LITT, patient 1 was reimaged after 24-hours, 1-month, 3-months, and 6-months while patient 2 was reimaged after 24-hours, 2-months, and 6-months. Patients 3 and 4 only had short-term follow up available and were reimaged at 24-hours and 1-month.S)).A scheme proposed by Engel F). The remaining four were identified as successful treatment (S).Six GBM patients were monitored post-LITT via MP-MRI  as a part of an ongoing study at Rutgers-RWJ Medical School between 2009-2013, after initial 1.5-Tesla MP-MRI. Post-LITT, patients were imaged at different time-points depending on their post-operative condition and availability post-LITT. Two of the six patients had tumor recurrence within one-year of LITT treatment, and hence identified as having tumor recurrence  evaluating temporal profiles of changes in MRI markers to quantify changes post-LITT over-time, (2) identifying post-LITT MRI markers that are more sensitive to capturing treatment changes over time, and (3) evaluate when early LITT induced changes dissipate over time. Each of the experiments below corresponds to each of these three objectives and how they relate to evaluating response to LITT treatment in epilepsy and GBM patient studies.in vivo imaging characteristics of LITT-induced changes for neurological disorders. In this work, we developed a quantitative framework to identify MRI markers that evaluated early and delayed changes in MRI markers due to LITT.LITT holds tremendous potential as a minimally invasive treatment modality for clinical applications such as epilepsy Only few studies so far have investigated the efficacy of LITT in the context of GBM and epilepsy. Schulze et al. The quantitative framework presented in this work was employed to interrogate the following effects. (1) Evaluating temporal profiles of changes in MRI markers over-time, (2) identifying which post-LITT MRI marker changes most dramatically over time and is hence more sensitive to capturing treatment changes over time, and (3) identifying when LITT induced changes have subsided allowing for assessment of treatment efficacy. In the first objective, changes in MRI markers over time were monitored and compared from patients with no signs of recurrence to patients with disease recurrence. Schwabe et al. Towards the second objective, candidate MR imaging markers from multi parametric MRI that better captured different treatment related changes post-LITT were identified. Our preliminary analysis on 6 GBM studies suggested that T1w MRI and T2-GRE may be most discriminative in distinguishing patients with successful treatment and tumor recurrence across all time-points, as compared to T2w MRI, and FLAIR. Similarly, our analysis on 4 epilepsy patients suggested that ADC may be more reflective of early treatment changes (up to 3-months), while T1w may be more reflective of early delayed treatment changes  compared to the other protocols under evaluation. A weighted temporal profile was created via a combination of these candidate MRI markers, where weights are computed based on the ability of MR markers to distinguish successful LITT treatment and tumor recurrence. Most of the existing strategies on evaluating treatment changes post-treatment have so far only explored changes in individual MR imaging markers. For e.g. in a recent study involving head and neck cancers, King et al. The third and final objective of this work was to identify when LITT induced changes subside for assessment of treatment efficacy. This analysis could provide clinicians with clinical insights to more carefully monitor changes in imaging markers at the optimal time point at which early treatment changes subside and changes due to tumor recurrence may become discernible. In a related study involving prostate cancer, Foltz et al. To summarize, our preliminary analysis on four epilepsy patient studies suggested that (a) LITT related changes appeared to dissipate in a period of 4 weeks following LITT, and (b) ADC may better capture early treatment changes (up to 3-months), while T1w may better capture early delayed treatment changes , compared to the other MRI protocols under evaluation. These findings although limited to four studies appear to provide some initial insights on the ability of the different MR protocols in evaluating treatment changes at different time-points. Similarly, our preliminary results on a cohort of six GBM studies evaluated in this work suggested that, (a) trends of changes in MRI markers may serve as potential surrogate markers to distinguish patients with continued control from the ones with disease recurrence, and (b) T1w MRI and T2-GRE may be better markers for early disease recurrence, as compared to T2w MRI and FLAIR.We do acknowledge a few limitations of the current work. Only a small number of GBM patients were analyzed. These patients were all at different time points in their disease, and many known variables important for tumor control are not accounted for in this study. Three of the cases presented for treatment very late in their disease process, making any concrete conclusions about the correlation of changes with treatment response difficult. There may be an inherent selection bias in this small cohort, as the patients who survive their primary and secondary treatments and remain in a capacity to undergo this treatment, have already proven to be long survivors after diagnosis. The issue of the “crucial time point” when treatment related changes subside will need more in depth analysis with a larger group of patients, and a greater understanding of the already well-established prognostic indicators such as age, extent of resection, and MGMT methylation status. Since multiple MRIs at the same time on the same patient were not available, our framework could not be evaluated against a control population set. Additionally, per-voxel changes evaluated in this work were not correlated with extent of thermal dose, tumor morphology, shape, volume changes, and probe placement, which will a part of future work.In the absence of sufficient patient data, the temporal profiles developed in this work could not be validated within a prognostic classifier, and hence its efficacy in capturing patient's response to treatment remains to be verified in a future study. However, it is important to remember that the goal of this work was primarily to develop a quantitative framework to evaluate voxel-by-voxel changes in MR markers over time for identification of early treatment changes post-LITT.