Circular RNAs (circRNAs) constitute a recently recognized group of noncoding transcripts that function as posttranscriptional regulators of gene expression at a new level. Recent developments in experimental methods together with rapidly evolving bioinformatics approaches have accelerated the exploration of circRNAs. The differentiation of hematopoietic stem cells into a broad spectrum of specialized blood lineages is a tightly regulated process that depends on a multitude of factors, including circRNAs. However, despite the growing number of circRNAs described to date, the roles of the majority of them in hematopoiesis remain unknown. Given their stability and disease-specific expression, circRNAs have been acknowledged as novel promising biomarkers and therapeutic targets. In this paper, the biogenesis, characteristics, and roles of circRNAs are reviewed with an emphasis on their currently recognized or presumed involvement in hematopoiesis, especially in acute myeloid leukemia and myelodysplastic syndrome. In the last decade, the development of high-throughput RNA-seq technologies accelerated the discovery of various noncoding RNAs (ncRNAs). Although they do not encode proteins, ncRNAs account for 98% of all human RNAs  and seemCircRNAs are non-polyadenylated single-stranded closed-loop RNA structures. Naturally occurring circRNAs were first described several decades ago when uncoated covalently closed RNA molecules were discovered in plants and termed viroids . They weCommonly, circRNAs are deconvoluted and identified by next-generation sequencing (NGS), with over 10,000 human circRNAs annotated thus far . They weMyelodysplastic syndrome (MDS) represents a spectrum of hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis, peripheral blood cytopenia, and a tendency for transformation into acute myeloid leukemia (AML). Therefore, MDS is often considered a preleukemic disorder. MDS is a dynamic disease in which clonal evolution triggers disease onset and progression. Clonal evolution is assumed to be a multistep process in which several successive genetic abnormalities are acquired by normal HSCs, leading to the expansion of an MDS clone and subsequent transformation to AML. Despite considerable advances in the understanding of the roles of various classes of noncoding RNAs in MDS, the roles of circRNAs in the onset or progression of this disease remain unclear.In this review, we describe specific features and various roles of this novel class of molecules and put them into the context of hematopoiesis, with particular emphasis on myelodysplasia.Although generally expressed at low levels, global expression profiling has revealed that circRNAs comprise numerous classes of transcripts with diverse expression patterns among cell types  and are CircRNAs originate from transcripts that are joined head-to-tail via a process called backsplicing. The covalent circularization of RNA molecules results in a novel backward fusion of two gene segments . The bacCircRNAs can be formed by the circularization of a one or more exons (exonic circRNAs or ecircRNAs) , both excis and trans. Regarding cis regulation, it has been shown that parental genes of circRNAs tend to have more active promoters. For example, significantly higher levels of the histone acetylation mark H3K27Ac and lower levels of DNA methylation were observed in promoter regions of genes from which circRNAs are formed compared to genes from which linear RNAs, but no detectable circRNAs, are generated [The biogenesis of circRNAs is influenced by different factors in enerated . The bacenerated ,26.cis regulatory factors, multiple RNA-binding proteins, such as splicing factors, play important roles in backsplicing in trans. Several studies have reported that different proteins bind to pre-mRNAs as a bridge between flanking introns, thus facilitating the process of circularization. In humans, many of the repetitive structures overlap with Alu elements [In addition to elements ,27. In telements . DHX9 iselements . ADAR1 ielements .circular form of BIRC6 in human embryonic stem cells [Drosophila [Another protein that plays a role in circRNA biogenesis is MBNL1. MBNL1 binds to its own pre-mRNA to bridge two flanking introns, bringing them close together to induce backsplicing and resulting in upregulated circRNA formation from its own RNA . Furtherem cells . Splicinosophila . HoweverSeveral functions have been attributed to circRNAs. Emerging evidence indicates that circRNAs constitute one of multiple layers of posttranscriptional regulation of gene expression. They can serve as miRNA sponges ,21; inteThe most recognized mechanism of action of circRNAs is their function as a miRNA sponge. Through the formation of a circRNA-miRNA duplex, the miRNA that is complementary to a circRNA is sequestered, which allows the binding of translational machinery to the mRNA targeted by this miRNA, leading to de-repression of a silenced gene. The first circRNA observed acting as a miRNA sponge was CDR1as, which has more than 70 conserved binding sites for miRNA-7 and depletes this miRNA in neuronal tissues . Since tAlthough circRNAs are generally considered noncoding molecules, recent studies have indicated that some cytoplasmic circRNAs can be effectively translated into detectable peptides ,35. CircCurrently, the number of newly identified circRNAs is rapidly growing; however, the functions of the majority of these species remain unknown. The recent development of experimental methods for the exploration of circRNAs together with rapidly evolving bioinformatics approaches deepen our understanding of circRNA biogenesis and functionality. In this section, we summarize the methods available to detect and study circRNAs.As a convenient starting point to obtain current knowledge on circRNAs, several databases and web resources are available to explore known circRNAs, predict their binding partners, and identify putative functions in different tissues or diseases . The two+ RNA depletion. These joint methods drastically deplete linear RNAs and lead to the isolation of highly pure circRNAs [The NGS of circRNAs (circ-Seq) enables the identification and expression profiling of thousands of circRNA molecules. In contrast to mRNAs, circRNAs do not contain a poly-A tail; therefore, they are not detected by the most widely used RNA-seq methods, which are based on poly-A enrichment. Although circRNAs are retained in rRNA-depleted RNA samples, in-depth sequencing, e.g., 100–150 million reads, must be performed with these NGS libraries . As an acircRNAs . There acircRNAs ,48. AltecircRNAs . In thesIn RNA-seq data, circRNAs can be identified by their unique backsplice junctions, which result in the alignment of chimeric reads, a feature that distinguishes them from linear RNAs. Multiple computational tools have recently emerged for the detection of these backsplice junctions within sequencing data. Most of these tools were developed as open-source software by researchers and therefore differ in many regards. A list of the most relevant computational tools is presented in In addition to NGS, the microarray approach is another high-throughput expression profiling method used to detect levels of tens of thousands of established circRNAs. As it is based on probes specifically designed to recognize junction sequences, this method does not distinguish between different circRNAs that share a junction. As in circRNA-seq, usually an RNase R-treated circRNA-enriched fraction is hybridized onto an array .Another probe-based method is Northern blot analysis, where either short junction-specific or longer probes complementary to most of a circRNA sequence can be used . Under sRNA fluorescence in situ hybridization (RNA-FISH) probes targeting circRNA junctions can also be used to detect and quantify the abundance and localization of circRNAs ,62; howeTo validate the high-throughput methods, reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) analysis is usually applied ,64. For To measure circRNAs in low abundance or to quantify circRNA copies in absolute numbers, the digital droplet PCR (ddPCR) method can be advantageous. Based on this technology, template nucleic acids (cDNA) are partitioned into tens of thousands of nanoliter-sized droplets for subsequent PCR amplification, and the final ratio of positive to negative droplets is analyzed to determine circRNA concentration . This meTo better recognize the mechanisms of circRNA effects, it is essential to study the molecular networks of circRNAs with their interacting partners, such as miRNAs and RBPs. For these purposes, circRNA affinity pulldown assays are performed using biotinylated antisense oligomers specifically designed against the junction sequence of a target circRNA. Using streptavidin-coated beads, the biotinylated probes are pulled down together with the target circRNA and its interacting molecules. Then, the pulldown proteins can be analyzed by mass spectrometry and Western blotting, and the RNA components can be studied by RT-qPCR or sequencing . AnotherThe major challenge to study circRNA function is knowing the way to affect their levels specifically and effectively. The first successful steps for mimicking circRNAs have been taken. For example, circRNAs synthesized in vitro were introduced into eukaryotic cells to study the activation of the innate immune response induced by the circRNAs . FurtherOn the other hand, depleting circRNAs in a specific way without affecting existing genes and their linear transcripts is challenging. To achieve substantial circRNA-specific knockdown via RNA interference or CRISPR/Cas9 technology, siRNA molecules or CRISPR/Cas9 guide RNA sequences must be designed to the backsplice junction site, which is unique to a given circRNA. For example, Piwecka et al. successfully removed circular CDR1as by CRISPR/Cas9 from the mouse genome, and then, they deregulated the expression of miR-7 and thus affected brain functions .HSC differentiation into a broad spectrum of specialized blood cells is a tightly regulated process that depends on a multitude of transcription factors and other molecules, including noncoding RNAs. As circRNAs can drive a subset of cellular functions, their expression in different blood cell populations is being evaluated to determine their involvement in hematopoiesis. However, most roles of circRNAs in the regulation of HSC differentiation remain to be revealed.The first study on a circRNA in the context of hematopoiesis was published in 1998 by Candas et al. , who repAn RNA-seq analysis showed that whole blood is very rich in circRNAs, even comparable to the circRNA abundance in the cerebellum . HoweverDue to their high stability , circRNAOne of the first circRNAs associated with AML was the circular form of NPM1 mRNA; hsa_circ_0075001. It is highly expressed in the leukemia cells of AML patients and shows no or minimal blast maturation (M0 or M1 subtypes according to the FAB classification), whereas its expression is significantly lower in the subtypes with a more mature blast population  . SomaticAnother circRNA highly expressed in AML is circ-DLEU2. Wu et al.  reportedCirc-ANAPC7 is an additional promising biomarker for AML diagnosis. Chen et al.  showed tAnother circRNA related to AML cells is hsa_circ_100290 (circularized transcript of the SLC30A7 gene). This circRNA is downregulated not only in AML  but alsoIn de novo patients with AML, Wu et al.  observedA similar impact on the outcome of AML patients was reported for hsa_circ_0009910, a circRNA formed from a transcript of the MFN2 gene . PatientOn the other hand, hsa_circ_0004277 (circularized transcript of the WDR37 gene) is downregulated in newly diagnosed AML patients . PatientShang et al.  focused To summarize, intense research on circRNAs in AML has led to our rapidly evolving knowledge of the importance of RNA circularization in hematological malignancies in general. It clearly shows that circRNA expression is closely associated with the onset of leukemia, its progression, and resistance to treatment, and undoubtedly, these data demonstrate the high potential of circRNAs in the prediction and monitoring of the course of the disease .Significant progress in the study of MDS pathogenesis has been realized in recent years. A number of breakthrough studies have described the presence of multiple somatic mutations in MDS ,88. ThesAll of these frequently mutated splicing factors represent essential components of the spliceosome; for example, the SF3B1 protein is a core component of the U2 small nuclear ribonucleoprotein (snRNP) that is involved in the recognition of the branch point and the 3′ splice site and prevents premature nucleophilic attack at the site by the splicing machinery .These mutations frequently occur early in the pathogenesis of MDS and are also enriched in clonal hematopoiesis of indeterminate potential, suggesting their role in disease initiation . SplicinAlthough RNA splicing has been recognized as one of the key processes for MDS development, the impact of mutated splicing factors on circRNA deregulation has not been studied thus far. Given the role of mRNA splicing machinery in the formation of circRNAs, an attractive hypothesis suggests that these mutant proteins alter the amounts of circRNAs and their linear RNA cognates, thereby resulting in a myelodysplastic phenotype. Although this hypothesis has not yet been validated, a link between circRNA expression and SF3B1 depletion was reported by Liang et al. , who fouNevertheless, the degree of deregulation of circRNA expression in MDS remains a mere presumption based on its similarity in other hematological diseases, especially AML, and the exact impact of splicing mutations on circRNA expression is still unknown.The enormous progress made in the field of circRNA research highlighted a significant potential for the future applicability of circularized nucleic acids in medicine and life science technologies. Due to their high stability and specificity, circRNAs have rapidly become promising biomarkers for the development and progression of various diseases. The expression dynamics of circRNAs is different from that of currently used disease biomarkers . Thus, combination of various classes of molecular markers might increase the accuracy and specificity of present diagnostic and prognostic models. However, these expectations have raised some important challenges that need to be comprehensively considered before specific features of circRNAs confer benefits.In the context of technical challenges, the experimental methodology needs to be fine-tuned. As the expression of a circRNA is closely connected to the expression of its host gene, it is rather difficult to define the functional aspects of circRNAs using standard techniques. Further, we urgently need a unified nomenclature system for circRNAs to be able to incorporate information on circRNAs into RNA databases, such as RefSeq and the UCSC genome browser. Most likely, the naming of circRNAs in future research should be based on the host gene along with the term “circ”. However, the explicit determination of circRNA isoforms  needs to be in some form of their numbering, e.g., circNPM1–1 would denote the first isoform of a circular transcript of the NMP1 gene. Finally, more gold standard studies for data comparison are increasingly necessary to reliably understand circRNA biology.In addition to the utilization of circRNAs as helpful disease biomarkers, it is expected that they will be exploited for therapeutic purposes. One of the best understood features of circRNAs is their ability to sponge miRNAs. Numerous tumor suppressive/oncogenic miRNAs have been reported to contribute to the development and progression of leukemia. Thus, the possibility of interfering with the processes associated with leukemia via the circRNA–miRNA–mRNA axis seems very promising. A substantial benefit of the circRNA-based approach is that one circRNA generally contains multiple miRNA-binding sites (MREs); thus, affecting the expression of a circRNA may offer treatment advantages over a single miRNA/gene. Depending on the type of sponged miRNAs, circRNAs can function as oncogenes or tumor-suppressors. Depleting an oncogenic circRNA via RNA interference or CRISPR/Cas9 technology can promote the protective effect of the corresponding miRNA in suppressing oncogenes. In contrast, transfection of an artificially synthetized circRNA into leukemia cells may sponge oncogenic miRNAs, leading to impaired leukemogenesis. These potential options may lead to the development of novel therapeutic strategies through circRNA-based miRNA sponging mechanisms that prevent tumor development.Descriptions of circRNA molecules forced us to reconsider a simplistic definition of the term “gene”. This reevaluation enabled us to support the concept that genes are complex transcriptional units rather than simple pieces of a DNA sequence encoding a protein. It is now apparent that one locus can be transcribed under different conditions in considerably different ways, performing distinct biological roles.Our current understanding of HSC differentiation and the abnormalities leading to leukemogenesis mainly originated from knowledge of transcription factors. However, recent discoveries of somatic mutations in RNA splicing genes in MDS have revealed that abnormal functions related to RNA splicing may be involved in leukemogenesis. These discoveries were quite unexpected, as this type of mutation is generally uncommon in cancer. Although studies seeking to understand the effects of these mutations on RNA splicing at a global- and transcript-specific level in MDS are currently ongoing, circRNAs have largely fallen outside this line of scientific inquiry thus far. To finally understand the roles of aberrant splicing in myelodysplasia, further studies should therefore focus on questions such as, how do RNA splicing mutations affect the expression of circRNAs, and how do circRNAs contribute to the pathogenesis of MDS?


Offset restoration in total hip arthroplasty (THA) is associated with postoperative range of motion (ROM) and gait kinematics. We aimed to research into the impact of high offset (HO) and standard stems on postoperative ROM. 121 patients received cementless THA through a minimally-invasive anterolateral approach. A 360° hip ROM analysis software calculated impingement-free hip movement based on postoperative 3D-CTs compared to ROM values necessary for activities of daily living (ADL). The same model was then run a second time after changing the stem geometry between standard and HO configuration with the implants in the same position. HO stems showed higher ROM for all directions between 4.6 and 8.9° (p < 0.001) compared with standard stems but with high interindividual variability. In the subgroup with HO stems for intraoperative offset restoration, the increase in ROM was even higher for all ROM directions with values between 6.1 and 14.4° (p < 0.001) compared to offset underrestoration with standard stems. Avoiding offset underrestoration resulted in a higher amount of patients of over 20% for each ROM direction that fulfilled the criteria for ADL (p < 0.001). In contrast, in patients with standard stems for offset restoration ROM did increase but not clinically relevant by offset overcorrection for all directions between 3.1 and 6.1° (p < 0.001). Offset overcorrection by replacing standard with HO stems improved ROM for ADL in a low number of patients below 10% (p > 0.03). Patient-individual restoration of offset is crucial for free ROM in THA. Both over and underrestoration of offset should be avoided. Beside a correct intraoperative orientation of both cup and stem, restoration of biomechanics such as offset is crucial for optimal function and long-term outcome after THA4. Failure of correct offset restoration is associated with impingement, reduced hip abductor strength, altered gait kinematics and even higher wear of the artificial hip joint8.Although total hip arthroplasty (THA) is a highly successful treatment option for end-stage osteoarthritis with a positive responder rate of over 90%9. Prior to surgery the biomechanical restoration of offset is usually templated on radiographs illustrating the preferred stem design for the respective patient. However, intraoperative alterations in relation to the preoperative plan or reduced joint stability harbour the potential to complicate the right choice of femoral offset design during THA. The discussion of correlation between high offset (HO) stems and higher range of motion (ROM) is controversial in literature. Although aiming to find the balance between the risks of impingement due to offset reduction on the one hand and potential trochanteric pain syndrome in case of offset overrestoration on the other hand some studies prefer high offset designs intending to increase soft tissue tension and hip stability11 whereas others do not see a benefit in range of motion for high offset stems12. In detail, Shoji et al. report an increase in flexion of 15° and in internal rotation of 10° due to an increase in offset of 8 mm10. In contrast, Hayashi et al. found an increase of 2° regarding flexion and no difference regarding internal rotation when using a high stem geometry12. However, these studies do not account for the exact offset restoration in terms of offset over and underrestoration. To the best of the authors’ knowledge no study has analysed the impact of offset over- and underrestoration on postoperative range of motion ROM comparing a standard with a high offset stem geometry using postoperative 3D-CT so far.To address the interindividual variability of the femoral anatomy, most modern implant systems offer at least two different offset geometries of the femoral stem—a standard offset design and a high offset designThe current study aimed to compare free ROM without osseous and/or prosthetic impingement between femoral stems with standard or high offset neck geometry in minimally invasive THA using a custom made 360° hip ROM analysis software. We hypothesized that the use of stems with a high offset geometry would result in higher ROM. In particular, offset over- and underrestoration was simulated and its effect on ROM regarding activities of daily living (ADL) analysed.13. The current study is an independent analysis of the postoperative 3D-CT data.In the course of a registered, prospective controlled trial  three dimensional computed tomography scans (3D-CT) were obtained after minimally-invasive cementless THA. The investigation was approved by the local medical ethics committee of the University of Regensburg (No.: 10-121-0263). All methods were performed in accordance with the relevant guidelines and regulations. A written informed consent was obtained from all participants of this study. The original study dealt with comparing navigated to free hand THA14. In all patients press-fit acetabular components with neutral liners and cement-free hydroxyapatite-coated stems  with metal heads of 32 mm diameter were used . The offset of the femoral stem increases with stem size and ranges from 38.0 to 45.5 mm for the standard offset geometry and from 45.5 to 52.5 mm for the high offset version. For all stem sizes the difference in femoral offset between the neck configuration with standard offset and HO is 7 mm . As a reference, the non-affected contralateral side was used and offset restored with the help of digital planning software . Six weeks postoperatively, a pelvic/femoral 3D-CT was performed .According to the study protocol, eligible participants were patients between the ages of 50 and 75 with an American Society of Anaesthesiologists (ASA) score ≤ 3 who were admitted for primary cementless unilateral THA due to primary or secondary osteoarthritis. THA in all patients was performed in the lateral decubitus position using a minimally-invasive single-incision anterolateral approach by four experienced orthopaedic senior surgeons16. Based on the manually segmented bone models, a custom made 360° hip ROM analysis was performed by a previously described algorithm which automatically determines osseous and prosthetic impingement by virtually moving the leg until a collision between the 3D objects occurs17. The high accuracy of this 3D-CT method with deviations in ROM measurements of 1° was demonstrated in a previous study18. For every single patient ROM analysis was performed twice, first using the implanted stem geometry and second after replacing the original stem with the other offset configuration either standard or HO. For the alternative offset stem geometry the same size and position as obtained during the original implantation of the stem were chosen only changing the neck geometry between the standard and HO configuration. Since both stem geometries (standard and HO) share the same geometric configuration of the stem body filling the osseous femoral channel and differ only in terms of the prosthetic neck, the replacement can be easily obtained. In addition, the same head size and acetabular component were used for 3D-CT ROM calculations. Virtual 3D-ROM calculation were performed by moving the femur in all directions while the pelvis was set in a fixed position. The software recorded ROM boundaries until a collision between the femur and the pelvis occurred. In contrast to previous studies the software enabled to measure both collision between bone and or prosthesis due to the availability of the patient’s individual anatomic configuration in 3D-CT. Beside single ROM directions the proportion of patients was comparatively assessed reaching the hip joint ROM configurations without impingement for ADL as given by Davis et al., Miki et al. and Turley et al. with at least 110° of flexion, 30° of extension, 50° of abduction, 30° of adduction, 45° of external rotation during extension and 30° of internal rotation during 90° of hip flexion, respectively21 regarding combined prosthetic and osseous impingement.In total, 121 data sets were included for final analysis  or median (range), respectively. Group comparisons were performed by Wilcoxon-tests on a 5% significance level for dependent variables. Absolute and relative frequencies were given for categorical data and compared between groups by McNemar tests on a 5% significance level. IBM SPSS Statistics 25  was used for analysis.Intraoperative characteristics of stem geometry as well as implant sizes and position of the study group are demonstrated in Table Comparison of ROM analysis for each patient between the HO and standard offset stem configuration revealed higher values for the stem geometry with a HO neck regarding all ROM directions particularly of 4.6°  for flexion, 8.9°  for extension, 8.3°  for external rotation, 6.0°  for internal rotation at 90° flexion, 8.9°  for abduction and 4.7°  for adduction  for flexion, 11.5°  for extension, 12.1°  for external rotation, 8.1°  for internal rotation at 90° flexion, 14.4°  for abduction and 6.2°  for adduction Fig. .Figure 4Concentrating on the subgroup with implanted standard  stems for correct offset restoration we analysed change in ROM if the stem with the standard offset geometry had been replaced with a HO stem. Overrestoration of offset showed an increase of 3.1°  for flexion, 6.1°  for extension, 4.5°  for external rotation, 3.8°  for internal rotation at 90° flexion, 3.4°  for abduction and 3.2°  for adduction  of patients fulfilling ROM criteria required for ADL, whereas overrestoration of offset lead to negligible increase of less than 10% (p > 0.03) in patients with free ROM for ADL.22, the authors feel intraoperative assessment of the variability of dynamic pelvic tilt and spinopelvic balance has not been resolved yet23. Third, the results of the present study rely on the stem design of a single manufacturer. This stem type anchors on the metaphysis and proximal diaphysis following the anatomic twist and bow of the proximal femoral canal. The fixation and consequently the postoperative stem position might be different in other stem designs especially for short and/or anatomic stems which could potentially impact ROM differences between HO and standard geometries. For that reason, the results of this study are restricted to the current stem design, which is, however, one of the most frequently used cementless femoral components in modern THA 24. Fourth, analysis were performed by a 360° hip ROM analysis software using 3D-CT. Due to the methodology in this study, it was not possible to obtain comparative clinical data25 on outcome enabling a comparison between the offset overrestoration and underrestoration. Therefore, the results do not allow correlation to important clinical parameters or outcomes, such as dislocation, wear, or revision.There are several limitations of this study. First, the 3D-CT based ROM analysis enabled measurement of combined prosthetic and/or osseous impingement. However, it was not possible to account for soft tissue impingement. In more obese patients soft-tissue restrictions may limit ROM boundaries postoperatively. However, after THA all requirements regarding free ROM should be met independently of soft tissue restrictions, since the soft tissue situation might change over time. Second, the influence of pelvic tilt was not inlcuded in the current study. Although previous studies describe an impact of pelvic tilt on implant position and thus postoperative ROM3. Any difference with regard to individual hip joint ROM in the current analysis is due purely to the change in stem geometry varying between standard and HO stem design. Theoretically, femoral stems with a modular neck could allow control of femoral version and varus/valgus angulation of the neck thus varying ROM27. However, such modularity has been the source of serious concern due to the potential release of metal ions28.A strength of the study is the fact that postoperative implant position of both cup and stem was controlled on 3D-CT thereby minimizing confounding factors. Furthermore, biomechanical reconstruction accuracy such as leg length and offset also possibly affecting postoperative ROM was checked11. Correspondingly, a higher ROM was observed in a cadaveric study for the modular PerFix stem  with higher offset than the low offset version. An increase in offset of 8 mm resulted in 26.7° higher flexion and 21.2° higher internal rotation29. In contrast, another clinical study compared intraoperative ROM between standard offset and HO of the Summit stem  using computed tomography based navigation. The authors found no correlation between stem offset and ROM except for higher external rotation12. The implant specific natural offset design with 36–44 mm was comparable to the stem used in this study. However, the neck shaft of this stem is 5° lower with 130° compared to the stem design of the current study which might at least partly explain the different results in relation to the present study. None of the previous studies accounted for offset restoration in relation to the standard or high offset stem geometry. Beside the differences in implant design this might further illuminate why the results of ROM analysis for HO stems are so controversial in literature.In answer to the first question of the study, femoral stems with a HO neck geometry showed higher ROM values between 5° and 9° for all directions. Although no sovereign power calculation was performed, the statistical significance of the observed differences indicates appropriate power. Within the study cohort a high interindividual variability was observed. In some patients the use of a HO stem resulted in an increase in ROM of over 20°, whereas in other patients no improvement in ROM was measurable. Comparing the results to the literature, a previous saw bone study recommended high offset neck designs to improve ROM. Single increase in offset by choosing a different modular neck correlated with higher flexion up to 20° and higher extension up to 15° using a head diameter of 32 mm. Similarly, internal rotation was reported to increase with offset but the effect was smaller and depended on the neck shaft angle. However, external rotation decreased with additional neck offset of the femoral stem due to neck on liner impingement31. This differentiated perspective of HO stems regarding offset over- and underrestoration might at least partly explain the controversial data situation according to the literature available where some studies report higher ROM for HO stem geometries29 while others see no benefit in ROM for HO stems12.Therefore, we built two subgroups and analysed the effect of offset overrestoration if the intraoperatively inserted standard offset stem was replaced by a high offset neck geometry and underrestoration if the intraoperatively chosen high offset stem was replaced by a standard offset stem, respectively. In the study cohort the distribution of standard and offset stems was equal. Sixty patients required a standard stem for offset restoration compared to the contralateral side and 61 patients received a HO stem geometry to equalize offset differences. In the 61 patients, underrestoration of offset by replacing the HO stem with the standard offset stem led to a substantial reduction of ROM for all directions with a mean change between 6° and 14°. The clinical relevance of this difference in ROM was demonstrated by a lower rate of patients that fulfilled ROM boundaries required for ADL. Offset underrestoration consequently led to decrease of over 20% of patients for each ROM direction that reached the required ROM for ADL except for extension with 13%. This means offset underrestoration reduces ROM within a critical range resulting in impingement and thus instability while performing typical motions in daily life. Therefore high offset stems represent an inevitable tool to restore offset in patients with corresponding anatomic configuration and thus help to ensure free ROM without impingement in a clinically critical range. On the other hand, excessive overcorrection as simulated in the 60 patients with originally implanted standard offset stems by using a HO stem increased ROM but not clinically relevant with mean values between 3° and 6°. This was demonstrated by a low percentage of patients that gained additional ROM required for ADL of fewer than 10% for all ROM directions. The slight but not relevant higher ROM by offset overrestoration is bought with the risk of increased strain in the medial cortex and thus potential early failure of the femoral component, higher polyethylene wear of the liner, altered gait kinematics and potential trochanteric pain syndromeIn conclusion high offset stems are an essential option to restore biomechanics and enable free ROM without impingement for ADL in a substantial proportion of patients undergoing THA. However, an overrestoration of offset with HO stems does necessarily appear to improve postoperative ROM in patients in whom standard offset stems allow for adequate offset reconstruction. The patient`s individual anatomy has to be accounted for during THA to restore the patient specific biochemical offset configuration. Both excessive offset under- and overrestoration should be avoided in THA.


The transport of host proteins into and out of the nucleus is key to host function. However, nuclear transport is restricted by nuclear pores that perforate the nuclear envelope. Protein intrinsic disorder is an inherent feature of this selective transport barrier and is also a feature of the nuclear transport receptors that facilitate the active nuclear transport of cargo, and the nuclear transport signals on the cargo itself. Furthermore, intrinsic disorder is an inherent feature of viral proteins and viral strategies to disrupt host nucleocytoplasmic transport to benefit their replication. In this review, we highlight the role that intrinsic disorder plays in the nuclear transport of host and viral proteins. We also describe viral subversion mechanisms of the host nuclear transport machinery in which intrinsic disorder is a feature. Finally, we discuss nuclear import and export as therapeutic targets for viral infectious disease. For proteins to function they must have some degree of flexibility. At one end of the flexibility spectrum are ordered, globular proteins, which have a single well-defined structure stabilised by intramolecular interactions. At the other end of the spectrum are intrinsically disordered proteins (IDPs) that lack stable secondary and tertiary structure in solution and are consequently highly flexible and dynamic ,2,3. ProDespite their lack of a well-defined structure, IDPs remain functional, and are typically involved in multiple protein-protein interactions, yet they maintain specificity . Their lThe plasticity of IDRs/IDPs renders them functionally versatile proteins, capable of interacting with multiple partners , and theThe nuclear and cytoplasmic compartments of the cell are separated by an envelope consisting of a double lipid bilayer that is perforated with pores known as nuclear pore complexes (NPCs). The NPC functions as a selective permeability barrier that allows the transit of some molecules and prevents the passage of others. Generally, proteins smaller than ~40 kDa can passively diffuse through the NPC in a timely manner, and while larger cargoes can also undergo passive diffusion, their translocation efficiency is markedly reduced . AlternaWhilst the prototypical importin, importin-β, can bind and import NLS-bearing cargo into the nucleus independently, it more commonly forms heterodimers with adaptors such as importin-α . In thisExportin-1 is the primary mammalian nuclear export receptor and binds RanGTP in the nucleus to enhance its affinity for NES-bearing cargo ,18 see . Upon caThe nuclear import/export of these nuclear transport receptor–cargo interactions occurs via the NPC. Each NPC is a megadalton-sized complex that is comprised of multiple copies of ~30 different proteins, known as nucleoporins (Nups). The Nups are generally grouped into three classes including the (1) transmembrane Nups that anchor the NPC to the nuclear envelope, (2) the structural Nups that form the two outer rings and inner ring of the NPC , and (3) the phenylalanine-glycine (FG)-Nups that predominantly line the central channel of the NPC but are also localised to the filaments that project into the cytoplasm  and the nucleoplasm  . The FG-The FG-Nups collectively function as a soft barrier to passive diffusion; however, facilitated diffusion, or the active transport of molecules, can occur when molecules are accompanied by nuclear transport receptors. Specifically, the FG-repeats interact with multiple hydrophobic pockets on the surface of nuclear transport receptors ,28,29,30Regardless of the mechanism, it is clear that the FG-Nups provide a dense barrier within the NPC, and that the interaction between the FG-repeats and nuclear transport receptors is key to the timely passage of larger cargo through the NPC.As proteins that behave as IDPs, the FG-Nups are ideally suited to mediate transport through the NPC. First, IDPs can accommodate a variety of binding partners ,37, and Second, IDPs can adopt multiple states in dynamic equilibrium and can exhibit varied behaviour under different environmental conditions. For example, under appropriate conditions, multivalent IDPs can undergo liquid-gel phase separation to from reversible hydrogels that are the basis of the “selective-phase” model of nuclear transport. Additionally, IDPs can also transition between extended-disordered and collapsed-disordered states , as per Finally, nuclear transport is a selective, but fast process that occurs in milliseconds, and it is estimated that around 1000 molecules are transferred through a single NPC every second ,40,41. PStudies over the past two decades have highlighted that flexibility and dynamics are inherent features of nuclear transport receptors, including importin α/β and exportin-1. Importin-α is a ~60 kDa protein that is comprised of a short N-terminal importin-β binding (IBB) domain that is connected by a flexible linker to a larger C-terminal domain of 10 stacked helical armadillo (ARM) repeats. The ARM repeats, which form a super-helical solenoid structure, contain the major and minor NLS-binding sites of importin-α. The IBB domain, which is a stretch of around 40 predominantly basic amino acids that includes an NLS mimic, exists in an autoinhibitory conformation that is released upon binding to importin-β. The importin α/β heterodimer may then bind the NLS-containing cargo via importin-α, whereas importin-β interacts with components of the NPC. The importin α/β-cargo complex is then guided through the NPC to the nucleus. Once the importin α/β-cargo complex is imported into the nucleus, RanGTP binds to importin-β. This causes a region of importin-α to bind to the major NLS-binding site to competitively release the cargo. Thus, the flexible IBB domain is key to importin α/β binding, cargo uptake, nuclear transport and release. Accordingly, the X-ray structures of mouse and yeast importin-α revealed that the IBB domain is mostly disordered when not bound to importin-β ,46. MD sLike importin-α, importin-β forms a super-helical solenoid structure, but has 19 HEAT repeats, each comprising two antiparallel helices connected by a turn . ImportiLike importin-β, exportin-1 is comprised of 21 HEAT repeats and adopts an overall super-helical conformation A,B. The Experimentally verified importin α/β and exportin-1 cargoes have informed their consensus NLS or NES motifs, respectively, and whilst the consensus sequences vary, intrinsic disorder can be a common feature of each.Classical NLSs that are recognised by importin-α are either monopartite or bipartite. Monopartite NLSs contain one stretch of basic amino acids, whereas bipartite NLSs contain a second stretch of basic amino acids 10–12 residues downstream of the first stretch . The con2,3-ϕ2-X2,3-ϕ3-X-ϕ4, where ϕn represents a Leu, Val, Ile, Phe or Met residue and X represents any amino acid [The consensus NES is usually between 9-15 residues long and is defined as ϕ1-Xino acid . As per ino acid , but canino acid . The NESino acid .Secondary structural analysis has been conducted on known NES sequences ,73, eithThere are many potential reasons as to why NLS and NES sequences are found in IDRs of cargo proteins. The intrinsic disorder may permit the solvent accessibility of the signal sequence, allowing the exposure of the key amino acids within the motif that are key to binding the nuclear transport receptor. In this regard it may also be key to allowing varied NLS/NES sequences, that still conform to the consensus motifs, to bind the structurally invariant NLS/NES binding site, and perhaps allow them to bind to the nuclear transport receptor in an optimal configuration. Finally, this flexibility may facilitate cargoes of different shapes and sizes to translocate through the NPC which has an upper functional diameter. While these possibilities make sense in the context of what is known about IDRs, they need to be further explored in the context of nuclear transport.As a side note, 71% of 185 human nuclear proteins curated in the Nuclear Protein Database  are predGiven IDRs are hubs for protein-protein interactions and post-translational modifications, these factors, which can be sensitive to stimuli, frequently play a role in masking/unmasking NLS/NES motifs. The effect is to modulate the accessibility of cargo proteins to nuclear transport receptors, and in doing so regulate their cellular localisation. Examples of host cargo proteins whose NLS/NES motifs undergo masking/unmasking are described below.®) VL-XT [® VL-XT predicted that INI1 protein residues 356-381, which encompass the C-terminal NES masking sequence, are intrinsically disordered, suggesting that the conformational plasticity of these residues is involved in masking/unmasking events. Interestingly, INI1 is an oligomer and residues involved in oligomerisation overlap with those of the NES. Moreover, a double mutant (I264T/I268T) containing a key substitution in the NES (I268T) was both monomeric and strongly cytoplasmic, suggesting that multimerisation of INI1 masks the NES, causing the retention of INI1 in the nucleus [The integrase interactor 1 (INI1) protein, which is a subunit of the SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodelling complex, was reported to have a NES spanning residues 266-276 within the repeat 2 (Rpt2) domain. It was shown that in the steady state, the NES of INI1 is masked by its C-terminal arginine rich sequence (residues 363-378) . It was ®) VL-XT , which h®) VL-XT , to pred nucleus .® VL-XT analysis predicted to be almost entirely disordered . Accordingly, the N-terminus was absent in the structure of IκBα in complex with the NF-κB heterodimer (p50/p65) [The nuclear factor κB (NF-κB) family of transcription factors require nuclear access to regulate the expression of genes in key cellular processes . Inhibitp50/p65) . This stInterestingly, IκBα also harbours an NLS within its disordered N-terminal region (residues 45-54), and the NLSs of the NF-κB family members p50, p52 and p65 ,87,88,89Viruses are infectious agents that contain genetic material, DNA or RNA, enclosed by a protein capsid. For enveloped viruses, the capsid is surrounded by a lipid bilayer envelope that is derived from the host cell membrane. Viruses can only replicate by infecting their host, and given their viral genome is compact, they do so with a limited suite of viral proteins. For this reason, viruses interact with many host proteins during infection and depend on the host cellular machinery for their replication. Although viruses encode a limited number of viral proteins, this limitation can be mitigated by the fact that many viral proteins bear IDRs. In fact, in the manually curated Database of Protein Disorder (DisProt)  viral prMany viruses exploit nucleocytoplasmic shuttling pathways to access the nucleus of their host . VirusesDuring infection many viruses target multiple FG-Nups so as to alter the composition and function of the NPC, ultimately facilitating nuclear transport and infection. Below we list some prominent examples.Adenovirus, which is a double stranded DNA virus, docks onto the cytoplasmic side of the NPC, and releases its viral genome from the capsid into the nucleus. More specifically, the adenovirus capsid protein, hexon, binds to the NPC via an IDR of Nup214, an FG repeat-containing Nup that is localised to the base of the cytoplasmic filament  100,101101. The Dengue virus (DENV) and Zika virus (ZIKV) are both flaviviruses and single-stranded RNA viruses that replicate in the host cell cytoplasm. Despite this, during infection many of their viral proteins enter the nucleus and many host proteins are mislocalised to aid viral replication or to inhibit innate immunity. These viruses alter the integrity of the NPC to achieve this outcome. The ZIKV and DENV serine-protease complex NS23B is responsible for degrading and altering the distribution of multiple FG-Nups following DENV and ZIKV infection. While both viruses target Nup98 and Nup153, an inner ring Nup and nuclear basket Nup, respectively , the degPicornaviridae family and are single stranded RNA viruses that replicate in the cytoplasm of the host cell. As per DENV and ZIKV, poliovirus and rhinovirus also cleave multiple Nups, but do so via their 3C and 2A cysteine proteases. Rhinovirus protease 2A cleaves Nup62, Nup98 and Nup153 [Poliovirus and rhinovirus belong to the d Nup153 ,107,108,d Nup153 , and botd Nup153 ,111,112;d Nup153 . These vd Nup153 ,111,114.Picornaviridae family, infects a broad range of non-human hosts; however, human infections associated with clinical manifestations have been reported [Encephalomyocarditis virus (EMCV), which also belongs to the reported ,116. Altreported , and altreported ,119,120)reported ,121, andreported ,123. MD reported . It shouAlthough HCV is an RNA virus that replicates in the cytoplasm, it utilises both Nups and nuclear transport receptors to support infection. HCV infection leads to the recruitment and rearrangement of cytoplasmic host cell membranes into viral replication and assembly complexes. Intriguingly, NLS-bearing proteins and Nups are recruited to these HCV assembly sites during infection . This ch® VL-XT to predict disorder within experimentally validated viral nuclear import and export signals  Rev  and infl signals . Our finAs per NLS/NES motifs in human proteins, viral proteins are also subjected to mechanisms that regulate their nuclear transport. This includes the modulation of NLS/NES accessibility by intra- and inter-molecular interactions and post-translational modifications. Intrinsic disorder, which is abundant in viral proteins, plays a key role in this modulation.Rev is a HIV-1 regulatory protein that is essential to the HIV-1 life cycle. Following its translation in the cytoplasm, Rev is imported into the nucleus where it binds to Rev responsive elements (RREs) on intron-containing HIV-1 mRNA and exports them to the cytoplasm for translation. To perform this function, Rev requires access to both the nuclear and cytoplasmic compartments of the cell, and this shuttling must be tightly regulated to ensure it is in the right compartment at the right time. The structural/biophysical/cellular analysis of Rev has provided insights into how this might occur.RNA-free Rev crystal structures show that the N-terminal domain consists of two helical oligomerisation domains (residues 9-26 and 51-65) connected by a flexible linker ,140. TheRev’s intra- and intermolecular protein interactions, RRE-binding status and the conformational malleability provided by its flexible linkers and intrinsically disordered regions are proposed to dictate NLS/NES masking and unmasking events. Rev self-association within the cytoplasm is proposed to transiently mask the NES, thereby promoting its NLS-mediated trafficking to the nucleus  where it® VL-XT analysis predicts that all three NLSs (NLS1-3) within the HCV core protein are fully disordered  contains stable secondary structure and exists as an oligomer ,156. HowWe previously showed that the Hendra virus V protein shuttles between the nucleus and the cytoplasm via interaction with importin α/β and exportin-1, respectively . We expe® VL-XT analysis predicts that SV40 T-ag residues 104-139, spanning both the NLS and the upstream phosphorylation sites, are disordered (data not shown). These data suggest that the phosphorylation of residues upstream of the T-ag NLS may prompt residues 119-122 to undergo a disorder–order transition that deems the NLS more recognisable by importin-α [Phosphorylation sites in close proximity to NLS/NES motifs can often up- or down-regulate the nuclear import/export of cargoes. For example, casein kinase 2 (CK2) phosphorylates residues S111/S112 of the Simian virus 40 (SV40) large T-antigen (T-ag), which are immediately upstream of an NLS (residues 126-132). This phosphorylation event accelerates the importin α/β-mediated import of T-ag, although a crystal structure confirmed there was no direct interaction between the S112 phosphate moiety and importin-α ,160,161.portin-α ,64.Given many viruses are dependent on accessing the host nucleus to replicate their genome, or to suppress the host antiviral response, nuclear transport inhibitors that mislocalise host or viral proteins key to the viral life cycle, are emerging as promising antiviral therapeutics.Ivermectin is a small molecule that impairs viral replication by various means. Two independent in vitro studies concur that ivermectin binds to importin-α and in doing so induces structural changes that prevent the binding of NLS-bearing cargo ,163. HowLikewise, karyostatin 1A  and impoNuclear export can also be targeted by small molecules. Leptomycin B (LMB), the prototypical exportin-1 inhibitor, binds irreversibly to cysteine 528 within the NES binding groove . In doinDespite the demonstrated utility of nuclear import and export inhibitors in reducing viral replication in vitro, there are currently no cargo-specific nuclear export inhibitors. Such inhibitors will be critically important to maintain specificity and reduce the cytotoxic effects associated with globally inhibiting nuclear transport cargo. To develop cargo-specific inhibitors, extensive knowledge of the cargo-receptor interface is required. In this regard, our knowledge is most advanced for the classical NLS recognised by importin α/β and the NES recognised by exportin-1, and so there is perhaps the greatest potential to selectively disrupt NLS- or NES-bearing cargo interactions. However, as noted above, NLS and NES motifs often contain regions of disorder, and in lacking stable features, are considered challenging drug targets. Although previously considered “undruggable” targets, success stories demonstrating that small molecules can selectively bind to disordered protein targets are emerging ,187,188.The FG-Nups that reside within the NPCs that perforate the nuclear envelope provide a selective barrier that regulates the movement of molecules between the cytoplasm and the nucleus. Accordingly, nucleocytoplasmic transport is essential for many critical functions of the cell, and also to the replication strategy of viruses infecting their host. It is clear that inherent disorder/flexibility plays a role, at various levels, to the fundamental dynamics of nuclear transport. However, due to these very dynamics, understanding the consequence of protein flexibility and disorder to trafficking is considerably challenging. It is clear more approaches and methodologies are required to further clarify the role of inherent disorder in the operation, and host- and viral-mediated regulation, of the extraordinarily complex and vital nuclear transport machinery. Ultimately, an enhanced understanding is key to devising novel therapies to block the replication strategy of viruses.


Since its inception, RNA sequencing (RNA-seq) has become the most effective way to study gene expression. After more than a decade of development, numerous RNA-seq datasets have been created, and the full utilization of these datasets has emerged as a major issue. In this study, we built a comprehensive database named Grape-RNA, which is focused on the collection, evaluation, treatment, and data sharing of grape RNA-seq datasets. This database contains 1529 RNA-seq samples, 112 microRNA samples from the public platform, and 485 RNA-seq in-house datasets sequenced by our lab. We classified these data into 25 conditions and provide the sample information, cleaned raw data, expression level, assembled unigenes, useful tools, and other relevant information to the users. Thus, this study provides data and tools that should be beneficial for researchers by allowing them to easily use the RNA-seq. The provided information can greatly contribute to grape breeding and genomic and biological research. This study may improve the usage of RNA-seq. Gene expression is one of the most important processes in life activities, playing a vital role in the development and environmental adaptation of living things. The study of gene expression is fundamental for functional genomics research, especially in the post-genomic era, which began with the completion of the Human Genome Project in 2001  and sample ID (GWGT), are necessary to search for the expression level of RNA-seq. The expression level of the required genes will be listed in table format on the result page. For example, if we want to know the expression level of several genes during heat stress on grape berry, the gene ID and sample ID of 48 samples related to heat stress on grape berry should be input, and the expression level of these genes will be searched and provided on the results page. Users can also download the expression information file from a download link. This expression file can be used as the input files for the heatmap tools. Expression level of microRNA only requires the sample ID, and all microRNA information of this sample will be listed in the result page. In addition to these three search tools, we provide the search tools of GO, KEGG, NR annotation, and some related papers. These tools can supply multiple types of functional annotation of the targeted genes and give some guidance to the users, and also can do benefit to other works, such as RNA-seq analysis and gene identification.Several searching tools were developed in this database (http://www.grapeworld.cn/gt/tools.html). Some of these tools are BLAST, BLAT, GO and KEGG annotation, signal peptide identification, transmembrane finder, RT-qPCR primer design, sequence tools, and microRNA regulatory network. These tools were developed based on all existing annotation versions of 8X and 12X grape genome, and supplied the information of similar sequences, high quality primers, or the information of signal peptide, transmembrane domain, and microRNA regulation. These tools can help researchers avoid the need for complex programming languages and a huge number of computations, making the undertaking of related studies easier and more efficient. In addition, 1529 high-quality samples were used in the co-expression analysis. This analysis was based on the IGGPv2.1 annotation version and processed by WGCNA (v1.63) [Grape-RNA includes abundant tools to make the research of related studies more convenient ( (v1.63) . The thr (v1.63) . For exahttp://www.grapeworld.cn/gt/help.html) to allow users to quickly begin using this database. We provide detailed instructions with a screenshot for each section. All introduction files are in PDF and can be accessed freely through the network.In Grape-RNA, we provide a help page  and can be accessed worldwide at any time. The database will be updated when enough new sequenced RNA-seq data have been collected or a new analysis method emerges.Grape-RNA is a part of Grapeworld (Grape-RNA is currently the most comprehensive and versatile grape RNA-seq database and provides a user-friendly interface. Compared with VitisNet  and VESP


Saccharomyces cerevisiae, the telomerase RNA, TLC1, is bound by the Sm7 protein complex, which is required for stabilization of the predominant, non-polyadenylated (poly(A)–) TLC1 isoform. However, it remains unclear (1) whether Sm7 retains this function when its binding site is repositioned within TLC1, as has been shown for other TLC1-binding telomerase subunits, and (2) how Sm7 stabilizes poly(A)– TLC1. Here, we first show that Sm7 can stabilize poly(A)– TLC1 even when its binding site is repositioned via circular permutation to several different positions within TLC1, further supporting the conclusion that the telomerase holoenzyme is organizationally flexible. Next, we show that when an Sm site is inserted 5′ of its native position and the native site is mutated, Sm7 stabilizes shorter forms of poly(A)– TLC1 in a manner corresponding to how far upstream the new site was inserted, providing strong evidence that Sm7 binding to TLC1 controls where the mature poly(A)– 3′ is formed by directing a 3′-to-5′ processing mechanism. In summary, our results show that Sm7 and the 3′ end of yeast telomerase RNA comprise an organizationally flexible module within the telomerase RNP and provide insights into the mechanistic role of Sm7 in telomerase RNA biogenesis.Telomerase RNA contains a template for synthesizing telomeric DNA and has been proposed to act as a flexible scaffold for holoenzyme protein subunits in the RNP. In  Telomeres are regions of repetitive sequences at the ends of eukaryotic chromosomes that buffer against shortening caused by the end-replication problem. In most eukaryotes, telomeres are lengthened by telomerase, an RNP enzyme that adds new telomeric repeats to the ends of telomeres via reverse transcription of an RNA template . FundameSaccharomyces cerevisiae, the major isoform of telomerase RNA (TLC1) is 1157 nucleotides long and is predicted to form a Y-shaped secondary structure, with the template and TERT-binding regions in the central core and binding sites for holoenzyme subunits towards the tips of each arm  TLC1 isoform. Although poly(A)– TLC1 RNA abundance was reduced in these SmCP alleles (discussed below), Sm7 still retained partial function at all positions tested compared to negative controls. This demonstrates that Sm7 and its binding site in telomerase RNA function as an organizationally flexible module, along with the 5′ and 3′ ends, which were relocated along with the Sm site by circular permutation. Between the results presented here and the previously reported repositioning studies regarding the binding sites for the Est1 and Ku subunits [Here, we have shown that organizational flexibility of the large yeast telomerase RNP extends to include the Smsubunits ,13, it isubunits , the entsubunits . This su7 subunits can function in vivo when bound to different non-native positions within the telomerase RNP in the context of circular permutation, we next used Sm-site repositioning to investigate mechanistic hypotheses regarding Sm function in TLC1 RNA biogenesis. We tested whether the position of Sm binding defines the mature 3′ end of poly(A)– TLC1 by relocating the Sm-binding site further 5′ in the RNA compared to its native position. At positions where the 5′-shifted Sm site was functional in stabilizing poly(A)– TLC1, the stabilized species were correspondingly shorter RNAs, thus providing strong evidence that Sm7 does indeed direct 3′-end formation of poly(A)– TLC1. We also found that when wild-type Sm-binding sites were included both at the native position and at a 5′-shifted position, only the native site defined the mature 3′ end of poly(A)– TLC1, suggesting that processing proceeds in a 3′-to-5′ manner, possibly through exonucleolytic trimming, as has been previously proposed [After determining that the Smproposed .TLC1 alleles that include the endogenous promoter and terminator regions on centromeric plasmids in a tlc1Δ strain. Unlike the strictly single-copy expression of chromosomally integrated alleles, CEN plasmids are maintained from equal to a few-fold higher copy number than one per cell on average and with slightly higher variability in copy number on a cell-by-cell basis [CEN-borne controls, the results reported herein still strongly support the conclusions.All experiments in this work were performed using ll basis ,37,38. T7 did retain function when its binding site was repositioned, it did not function as well as wild type in stabilizing poly(A)– TLC1. It is possible that these defects in TLC1 RNA stabilization were caused by alterations in aspects of RNA folding. In the case of the TLC1-SmCP alleles, circular permutation may have affected local and/or global folding because of changes in the contact order of the RNA secondary structure and in the timing of when sequences emerge from RNA polymerase [7 binding or make the RNA more exposed to nucleases. In addition to effects of circular permutation on TLC1 RNA abundance, misfolding could also be deleterious to telomerase activity in the case of the TLC1-SmCP@451 allele, where the Sm site and RNA ends are very close to the template-boundary element, a structure that is essential for activity in the catalytic core of the telomerase RNP [The results of all of our Sm-site repositioning experiments—with or without also circularly permuting the RNA—show that although Smlymerase ,40,41. Arase RNP ,42. Thus7 function. This may be due to disrupted folding of the region where the Sm site was inserted. Additionally, repositioning the Sm site without circular permutation may have disrupted folding of the Sm site itself. Although the portion of TLC1 that we used in the Sm-site repositioning experiments without circular permutation was chosen for its proposed hairpin structure in poly(A)+ TLC1 [7 complex. Sm-binding sites in many other RNAs have been found to be single-stranded and preceded and/or followed by hairpin structures [Mfold secondary structure prediction of an 1195 nucleotide NNS-terminated TLC1 RNA [Mfold secondary structure predictions of tlc1-Sm–+Sm@1089, @1003, and @926 all show the repositioned Sm sites forming the same hairpin-and-loop structure seen in the published secondary structure model of poly(A)+ TLC1 – TLC1. First, as shown in A)+ TLC1 , this maructures ,25. In aA)+ TLC1 . Notably7 complex has a highly conserved function in the biogenesis of certain spliceosomal snRNAs, but why does it bind to S. cerevisiae telomerase RNA? One answer to this question may relate to an interesting theme that has emerged from the study of telomerase RNPs from a wide range of organisms. Consistent with the flexible scaffold hypothesis and the related rapid evolution of telomerase RNAs, these RNAs have independently evolved a wide variety of ways to be processed and to allow appropriate RNP biogenesis, maturation, and probably also modes of regulation [7 complex binding to the 3′ end of TLC1 may simply be another variation on this broad theme. The Sm ring is required for 3′-end maturation of some spliceosomal snRNAs in yeast, and TLC1 has apparently “borrowed” this machinery for formation and stabilization of its own mature 3′ end [S. cerevisiae telomerase RNA has evolved a different processing mechanism from snRNAs that only requires the Sm ring and the exosome. Irrespective of these details, the recurrent observation throughout evolution that telomerase RNAs coopt proteins from other RNP complexes for their own biogenesis demonstrates both the myriad ways that noncoding RNAs can be processed and stabilized and that telomerase RNAs are exceedingly flexible in which of these mechanisms are used for their own biogenesis.The Smgulation ,14,15,16e 3′ end . Howevere 3′ end ,45,46. T7 in its biogenesis. Sm7 can functionally tolerate repositioning of its binding site within TLC1 along with the RNA ends. Furthermore, the telomerase enzyme’s fundamental functions can also endure the substantial reorganization of the RNA that is caused by circularly permuting the telomerase RNA transcript in vivo, despite the fact that it globally changes the RNA-folding landscape and locally breaks the phosphodiester backbone. Sm-site repositioning also has revealed that the mature 3′ end of poly(A)– TLC1 appears to be (1) controlled by the location of Sm7 binding to the RNA and (2) generated in a 3′-to-5′ manner, lending experimental support to previously proposed models of TLC1 biogenesis. The experimental approach of repositioning the Sm-binding site in order to change the location of the mature 3′ end could be useful for next identifying the precursor of poly(A)– TLC1 and fully characterizing telomerase RNA biogenesis in budding yeasts.In summary, repositioning the Sm-binding site and the ends of yeast telomerase RNA has yielded insights about the organizationally flexible nature of the RNA and the mechanistic role of SmTLC1-SmCP alleles were constructed using a plasmid containing two tandem copies of TLC1 in a pRS414 backbone [TLC1 are fused into a single gene such that nucleotide 1134 of the first TLC1 copy is followed directly by nucleotide 1 . These circularly permuted DNAs were inserted into a separate plasmid containing the natural ends of the TLC1 gene such that sequences from nucleotide 10 upstream and from nucleotide 1130 downstream (which includes the Sm site) are retained at the ends of the new gene. As an example, the sequence of TLC1-SmCP@211 is laid out from promoter to terminator as follows: the TLC1 promoter through nucleotide 10, 211–1134, 1–210, and 1130 through the transcriptional terminator and natural end of the TLC1 gene  [TLC1 alleles were expressed from centromeric plasmids (pTLC1-TRP1-CEN) [TLC1-containing plasmids, and colonies were streaked to minimal –TRP–LYS medium. Loss of the pTLC1-LYS2-CEN cover plasmid was selected for by re-streaking cells to minimal –TRP medium containing α-aminoadipate. These cells were then serially re-streaked nine times to minimal –TRP medium and photographed after each round of growth. When estimating the number of generations at different points throughout passaging, each round of growth  after loss of the cover plasmid (including the round of growth in the presence of α-aminoadipate) is approximated as 25 generations.All experiments were performed in the strain TCy43 (plasmids  that werRP1-CEN) . TCy43 w+ Nylon membrane (GE). The membrane was UV-crosslinked and probed for both TLC1 and U1 snRNA sequence using 100-fold fewer counts of U1 probe than TLC1 probe to account for the large difference in abundance between the two RNAs. TLC1 RNA abundance was calculated by normalizing to U1 abundance, and numbers in the text and figures are expressed relative to the wild-type TLC1 condition. Lengths of poly(A)– TLC1 RNAs in Northern blotting was performed as described previously ,13,35,49+ Nylon membrane (GE) to which it was UV-crosslinked. The membrane was probed for yeast telomeric sequence and a 1621 bp non-telomeric XhoI fragment that served as a non-telomeric control band [Southern blotting was performed as described previously ,13,35,49rol band . Average


Lung cancer is a leading cause of cancer-related death. Stereotactic body radiotherapy (SBRT) is the standard treatment for inoperable early-stage non-small cell lung cancer (NSCLC). Carbon ion radiotherapy (CIRT) is a safe and effective treatment for early-stage NSCLC. However, there is no direct comparison study between these treatments. The present study aimed to compare oncologic outcomes after CIRT and SBRT for early-stage NSCLC in a single-institutional and contemporaneous cohort. We demonstrated favorable overall survival and local control in the CIRT group compared to those in the SBRT group using log-rank tests and Cox regression analyses for 89 patients. In addition, these results were validated in propensity score-adjusted analyses. The present retrospective comparison study showed a positive efficacy profile of CIRT, which is beneficial in the management of early-stage NSCLC.p = 0.0077). The 3-year LC was 87.7% in the CIRT group and 79.1% in the SBRT group (p = 0.037). Multivariable analyses showed favorable OS and LC in the CIRT group . Log-rank tests after propensity score matching and Cox regression analyses using propensity score confirmed these results. These data provided a positive efficacy profile of CIRT for early-stage NSCLC.Lung cancer is a leading cause of cancer-related deaths worldwide. Radiotherapy is an essential treatment modality for inoperable non-small cell lung cancer (NSCLC). Stereotactic body radiotherapy (SBRT) is the standard treatment for early-stage NSCLC because of its favorable local control (LC) compared to conventional radiotherapy. Carbon ion radiotherapy (CIRT) is a kind of external beam radiotherapy characterized by a steeper dose distribution and higher biological effectiveness. Several prospective studies have shown favorable outcomes. However, there is no direct comparison study between CIRT and SBRT to determine their benefits in the management of early-stage NSCLC. Thus, we conducted a retrospective, single-institutional, and contemporaneous comparison study, including propensity score-adjusted analyses, to clarify the differences in oncologic outcomes. The 3-year overall survival (OS) was 80.1% in CIRT and 71.6% in SBRT ( Lung cancer is a leading cause of cancer-related deaths worldwide . LobectoA total of 89 patients meeting the inclusion criteria were identified in our database. Sixty-two patients underwent CIRT, and 27 underwent SBRT. Patient characteristics are summarized in p = 0.75; noncancerous lung disease, p = 0.19; the other or unknown, p = 0.48). Grade 3 or worse acute toxicity was not observed in the CIRT and SBRT groups. One patient in each treatment group (1.6% for CIRT and 3.7% for SBRT) developed grade 3 radiation pneumonitis. Grade 4 or worse late toxicity related with the treatment was not observed.A total of 31 patients died during the final follow-up. The causes of death in the CIRT and SBRT groups were as follows: NSCLC, 15% (9/62) and 19% (5/27); noncancerous lung disease ) were 4.8% (3/62), and 15% (4/27); other causes or unknown were 9.7% (6/62) and 15% (4/27), respectively. Fisher’s exact tests were performed to test the difference between the CIRT and SBRT groups and showed no statistically significant difference  (p = 0.047) , 70.7–90.9%) and 73.4%  in the CIRT group, and 71.6%  and 46.3%  in the SBRT group, respectively. A log-rank test showed that CIRT was significantly associated with higher OS rates than SBRT ( 0.0077) A. A Cox = 0.047) .p = 0.14)  rates were 60.4%  and 55.3%  in the CIRT group, and 62.5%  and 29.8%  in the SBRT group B. These  = 0.14) B. A Cox  = 0.14) .p = 0.037) (p = 0.040)  and 85.5%  in the CIRT group, and 79.1%  and 55.3%  in the SBRT group C. A log-= 0.037) C. A Cox = 0.040) .Propensity scores were estimated by binomial logistic regression using available covariates, including age, sex, Eastern Cooperative Oncology Group Performance Status (ECOG PS), Charlson comorbidity index (CCI), smoking status, Brinkman index, the presence of COPD, presence of interstitial pneumonia (IP), T stage (UICC 7th edition), pathological diagnosis, and calendar year of treatment. The c-statistic value was 0.832. The distributions of propensity scores before and after matching are shown in p = 0.043)  and 71.8%  in the CIRT group, and 55.0%  and 34.4%  in the SBRT group, respectively, which were statistically significantly different (= 0.043) A.p = 0.20)  and 51.9%  in the CIRT group, and 47.2%  and 18.9%  in the SBRT group, respectively, which were not statistically significantly different ( = 0.20) B.p = 0.022)  in the CIRT group, and 70.7%  and 42.4%  in the SBRT group, respectively, which were statistically significantly different (= 0.022) C.p = 0.018; HR, 0.41 , p = 0.022; HR, 0.19 , p = 0.0051, respectively) , ctively) .To the best of our knowledge, this is the first report comparing the oncologic outcomes following CIRT and SBRT for early-stage NSCLC in a single-institutional and contemporaneous cohort. CIRT was statistically significantly associated with higher OS and LC rates than those of SBRT in log-rank tests. Cox regression analyses also showed favorable OS and LC in the CIRT group. In addition, propensity score-matching and Cox regression analyses using propensity scores as covariates certified these results. Grade 3 radiation pneumonitis was observed in one patient each in the two groups.There are several prospective studies regarding SBRT for early-stage NSCLC. In the Japan Clinical Oncology Group (JCOG) 0403 study, 48 Gy in four fractions over 4 days were delivered to patients with T1N0M0 NSCLC, and the 3-year OS and PFS were 59.9% and 54.5%, respectively . A recenCIRT for early-stage NSCLC was surveyed in a few prospective trials. In these studies, the 3-year OS and LC were 76–86% and 86–90%, respectively [p = 0.009). However, the LC rate after CIRT was significantly higher than in the SBRT group . Cox regression and propensity score-adjusted analyses were consistent with this result. Previous studies supported our findings. In CIRT, the 2-year LC of T2b–4N0M0 NSCLC was 81% [It is noteworthy that the LC rate after CIRT was favorable despite disease progression. In our baseline cohort, the number of patients with T2a disease was significantly larger in the CIRT group than in the SBRT group ) and SBRT of 48 Gy were delivered. At first glance, the higher dose of CIRT seemed to be one of the reasons for the favorable outcomes following CIRT. However, we should acknowledge that the Gy (RBE) does not always represent a biologically equivalent dose to the absorbed dose in photon radiotherapy. In clinical practice using CIRT, a fixed RBE of 3.0 is generally utilized for convenience. However, the actual RBE could be affected by various factors including linear energy transfer, tissue types, and endpoints of concern, which makes it difficult to determine the exact equivalent dose of CIRT to photon radiotherapy. Accordingly, we should not conclude that the favorable outcomes were caused by the difference in the prescribed clinical doses. In future studies, including a randomized controlled trial comparing CIRT and SBRT, the prescribed doses should be considered based on the biological and physical aspects.There are some limitations to our study. First, we cannot exclude potential sources of bias due to unmeasured confounders, even with propensity score-adjusted analyses . Second, the lack of information regarding medical operability status made it difficult to compare surgical treatment and CIRT/SBRT. We consider that these comparisons are future issues, since these are required in the management of operable patients. Finally, dose escalation may improve the prognosis of SBRT, as shown in a previous meta-analysis . In addiThe present study is a retrospective cohort study that compared oncologic outcomes after CIRT and SBRT for early-stage peripheral NSCLC. We identified patients who met the inclusion criteria from our database and evaluated OS, PFS, and LC rates after these treatments. Univariate and multivariable analyses were performed to test differences in these outcomes. In order to make these results more robust, we performed propensity score-adjusted analyses, including propensity score-matching and multivariable analyses using propensity scores as covariates.From our database, we identified patients who met the following criteria: (i) pathologically or clinically diagnosed NSCLC; (ii) clinical T1a–2aN0M0 , 7th edition) and peripheral disease; (iii) treated with CIRT or SBRT at Gunma University Hospital  between 2010 and 2015. Each patient chose between CIRT and SBRT, based on the patient’s preference after being informed. The appropriateness of the treatment was confirmed by the institutional cancer board, attended by pulmonologists, respiratory surgeons, radiation oncologists, and diagnostic radiologists.Details of the CIRT technique have been reported previously . BrieflySBRT was performed according to the protocol of the JCOG 0403 study . CT imagAfter the treatment, the patients were followed up with blood tests, including tumor markers and CT scans or chest X-rays. In the case of patients who refused regular follow-ups at our hospital, their clinical course was obtained via telephone, letters, and patient referral documents. According to the Response Evaluation Criteria in Solid Tumors guidelines version 1.1 , the tumWe calculated OS, PFS, and LC rates from the first day of irradiation using the Kaplan–Meier method. The log-rank test was used to evaluate the associations of these endpoints with the treatment modalities. These endpoints were compared between subgroups using the Cox proportional hazards regression model to estimate the HR and its 95% CI. Covariates were determined based on prognostic factors proposed in the literature and the number of events.Propensity score-adjusted analyses were performed to minimize overt selection bias. Propensity scores were estimated by binomial logistic regression using available covariates, including age, sex, ECOG PS, CCI, smoking status, Brinkman index, the presence of COPD, the presence of IP, T stage, pathological diagnosis, and calendar year of treatment. Propensity score matching was performed by one-to-one nearest-neighbor matching using a caliper width of 0.25 times the pooled standard deviation of the propensity scores’ logit without replacement. Cox regression analyses using propensity scores as covariates were also performed to make our results more robust.p-value < 0.05 was considered statistically significant. All statistical analyses and visualization were performed using R 3.6.2 [A  R 3.6.2 .In summary, a favorable efficacy profile of CIRT for early-stage NSCLC compared with SBRT was shown via this single-institutional and contemporaneous comparison study. For more reliable evidence, a randomized controlled trial is warranted in future.


Comprehending the complexity and determinants of food choices can help understand facets of the growing obesity epidemic. Focusing on consumers’ subjective sensations as determinants of food choices can provide essential insights into eating behaviors. We explored subjective sensations linked to appetite, desire, wellbeing and energy. This study aims to 1) quantify subjective temporal sensations, and 2) study the effects of these sensations on snack choice. Two-hundred and fifty-three participants (mean age 20.5) evaluated subjective sensations using a visual analogue scale. The choice of one of six snacks was offered to the participants; choices were recorded as implicit choice measures. The results demonstrated that especially sensory specific desire sensations  affected, either positively or negatively, snack choice. Furthermore, wellbeing sensations  also showed significant effects for snack choice. Health-conscious females chose healthy snacks, and males chose unhealthy snacks. Importantly, this research indicates the relevance of subjective sensations in consumer studies that focus on diverse determinants of food choice. Sensory specific desires and wellbeing sensations were notably shown to be important determinants of snack choice. The contribution of different sensations to food choice is imperative, and helps us to understand aspects of snacking behavior. This could have broader implications concerning public health issues and obesity.  We live in a multifaceted world, and the processes around food choices and eating behavior are complex and multifactorial ,2,3,4. SIn order to explain food choices, we as researchers seek to explore various factors that could play important roles in eating behavior. Psychobiological perspectives highlight the relationship between our appetite control system and food choices. Here, homeostatic processes such as hunger and satiety sensations drive eating behavior, reflecting a need or motivation to eat or not to eat ,16. Non-Research in the area of subjective appetite has moved towards a broader understanding of the concept. It points towards employing a more diverse and holistic approach by which to better understand human eating behavior ,28,29,30What subjective temporal sensations contribute to snack choice?Do other factors such as gender, time of day and health consciousness affect consumers’ snack choice?This research study thus seeks to unravel the complex area of determinants of food choice by including subjective sensations as important consumer evaluations for food choice. The overall objective of this study is to quantitatively measure consumers’ subjective sensations, and thereafter, relate these subjective sensations to actual snack choices. Snack choice is assessed with a real, actual choice, hereby applying an implicit behavioral measurement. We believe that this implicit observational measure validates and represents an actual daily snack choice very well. Specifically, the study aims to (1) quantify subjective temporal sensations such as energy, wellbeing, hunger, satiety and desires, and (2) study the effects of subjective sensations on consumers’ choice of specific snacks. Our research questions therefore include:ntotal = 253) were recruited from the campus at University of Otago in Dunedin, New Zealand. Participants included undergraduate and postgraduate students ranging from 18−30 years old. Inclusion criteria involved being above 18 years old and in general good health. Exclusion criteria involved having any food allergies. Participants  ,30,36,38® Cloud software  in randomized order. A continuous scale ranging from 0, i.e., “not at all” to 10, i.e., “very much”, was used to evaluate each sensation. Subsequently, all participants filled out a demographics and psychographics section including gender, age, weight, height, time since last food intake and health consciousness related questions. Health consciousness was evaluated by asking participants the extent to which they agree with the following two statements: “I choose food carefully to ensure good health” and “I think of myself as a health-conscious consumer”, adapted by Squirel et al. (2001) and used also by other researchers evaluating consumer health consciousness , p = 0.050), Desire-to-snack , and Salty desire . Hence, sensing Mental wellbeing, Desire-to-snack, or a high Salty desire caused consumers not to choose grapes. Salty desire had the largest effect of the three sensations. No sensations positively drove grape choice. We found that five of the subjective sensations had an effect on potato chips choice. Sensing Overall wellbeing , Fatty desire , or Salty desire  showed a positive effect on potato chips choice. On the other hand, sensing Physical wellbeing  or Sweet desire  had a negative effect on potato chips choice. Hence, sensing Physical wellbeing or a high Sweet desire caused consumers not to choose potato chips, whereas sensing Overall wellbeing, a high Fatty desire or a high Salty desire caused consumers to choose potato chips. The three desires, i.e., Sweet, Fatty, and Salty, had the largest effects. After iteratively reducing the model to increase its stability, we found that three sensations had a significant negative effect for grape choice: Mental wellbeing (Y) explained by both subjective sensations (X) and background information (Z). Interpreting the plot in ●), as expected, are represented together with the snack samples given the nature of a choice or no-choice design (1/0 binary response). The consumers are thus grouped together for one snack choice. The results further show that both subjective sensations (●) and background information (●) bring important knowledge to the overall picture, and thus provide explanations for real snack choices. Factor 1 is explained by Health consciousness, depicted to the left in the plot, as well as desire sensations , depicted to the right in the plot. Factor 2 is mainly explained by gender, with Females placed in the upper part of the plot and Males in the lower part. Clustering variables around choice, we observe that the choice of grapes was mainly explained by Health consciousness and gender Female. No sensations positively correlate and explain the choice of grapes. Around the upper left quadrant, we also see a cluster including the three presumed healthy snack options, i.e., grapes, dark chocolate, and nuts. In contrast, we see that the desire sensations and gender Male explain and cluster around the more unhealthy snack options, especially potato chips and partly cookies. Data matrix X explained in total 96% of the variance in the L-PLSR analysis, compared to data matrix Z, with 100% explained variance, and data matrix Y with 1.0% explained variance. This makes sense, considering that both the X- and Z-matrices bring new information to explain the choice and include quantitative variables that show variation amongst consumers, whereas the Y-matrix is a dummy matrix with binary variables that essentially indicates snack choice.The overall structure and relationship between subjective sensations, background variables and real snack choice were provided by the L-PLSR analysis. The preliminary full cross-validated two-block PLSR models revealed which variables were retained for the L-PLSR. Consequently, significant sensation variables were Salty desire, Fatty desire, and Desire-to-eat. Significant background variables were Health consciousness, gender female and gender male. The remaining non-significant variables were thus omitted from the L-PLSR analysis. In relation to our first research question, i.e., which subjective temporal sensations contribute to snack choice, we found that subjective desires contribute the most, both positively and negatively. The results showed that especially consumers’ sensory specific desires  discriminated between the snack choices and also had the greatest effects in general. The theory behind Sensory Specific Desires (SSD) can be described as a general desire for certain tastes  and an intrinsic motivation to eat a food that contains that characteristic ,50. Both. Sweet desire, hence, seemed to be disconnected from general appetite-related sensations. Sweet desire was not predictive of any of the presumed sweet snacks either, but instead, had a positive effect on dark chocolate choice and a negative effect on potato chips choice. Harington et al. (2016) suggest that our desire for sweet foods is partially disconnected from our appetite, hypothesizing a “dessert mentality” [Yet, Sweet desire seemed to not be related to the other desires, nor to Hunger nor Fullness, but revealed a different pattern than the other desire sensations, as illustrated in Figure 3ntality” . This contality” ,52. Harintality” . This suMultivariate analyses revealed that not only consumers’ subjective sensation state or only consumers’ background information data explained snack choice. Both subjective sensations and background data brought important knowledge to the overall picture. This underlines that we live in a multifaceted world, and that the processes around food choice are complex ,4. For tFor the included wellbeing sensations, i.e., Overall, Mental and Physical wellbeing, we observed that all three parameters were important and showed significant effects on snack choice, but in different ways. Overall, wellbeing and Physical wellbeing revealed opposite effects for potato chips choice, showing that high Overall wellbeing positively drove that choice, whereas high Physical wellbeing caused consumers not to choose potato chips. Furthermore, consumers that rated high Mental wellbeing showed a negative effect on grape choice. So, even though the three wellbeing sensations showed high positive correlations to each other independently of snack choice , it seemRegular healthy eating results in better wellbeing. Foods that are recognized as positive for wellbeing include fruits, vegetables, grains and dairy products, whereas foods high in fat, salt and sugar are perceived to be negative for one’s wellbeing ,67. ThisThe vitality- and energy-related sensations such as Sleepy, Energized and Concentration did not have any significant effects on the actual snack choice behavior in this study. Thus, in this study and in relation to the first question, these subjective sensations were not important or contributed to actual snack choice. Nonetheless, independently of snack choice measures, we observed that these sensations functioned almost as opposites in consumer perceptions , with SlThe present results contribute to the multicomplex area of understanding determinants of food choices and eating behaviors. With this study, we add new knowledge about effects of subjective temporal sensations on actual snack choice. The contribution of the different sensations to snack choice is imperative, and helps to unravel and understand some aspects of snack eating behavior.While this research study focused on snack choice, future studies should extend the investigation of the effect of subjective sensations on main meals and general food intake. The importance of subjective sensations in relation to predictions of longer-term food intake is, to date, unstudied. Therefore, additional and longer-term research is necessary to support our findings and to gain more in-depth knowledge about the importance of subjective sensations and their relevance to food choice behavior over time. Furthermore, researching subjective sensations with other and more diverse approaches such as including more implicit and indirect measures for both homeostatic and non-homeostatic sensations could add to the validation and application of predicting food choices. This entails interdisciplinary research, involving research areas such as psychology, neuroscience, human nutrition, biology and physiology. This could provide further insights into (over) eating, and have an impact in broader contexts with implications for public health issues and obesity.In the present study, the six snacks were divided into categories each representing a healthy and unhealthy version of a sensory taste profile, being sweet, salty, or fatty. It is important to consider that these categories might be a matter of interpretation, and that consumers might distinguish other sensory profiles than those that we categorized. For instance, we saw that Sweet desire showed an effect on dark chocolate, and that Fatty desire showed an effect on potato chips. Cookies represented an overall higher mean Sweet desire amongst participants than did the Fatty desire. Nuts represented an overall higher mean Salty desire than Fatty desire. These sensations do not necessarily describe the snacks by sensory attributes, but might be an indicator as to how consumers perceive them, given that desires were explanatory for specific snack choices. Furthermore, some consumers consider dark chocolates or nuts as healthy or unhealthy, or perhaps regard potato chips as overlapping in both fatty and salty. Other examples of researchers categorizing choices in consumer food studies include Muñoz-Vilches et al. (2019) , who useAnother aspect to consider when interpreting the results is the possibility that the choice outcome could be affected by the consumer’s own answers, e.g., “I answered high salty desire, therefore I should choose potato chips”. Moreover, the consumer group that chose jelly beans was the smallest of all, comprising only 5%. Therefore, any strong conclusions should not be drawn as to the effects for the choice of jelly beans. Furthermore, it is important to consider the nature and composition of the participants. All participants were relatively young adults associated with the university, whose choice behavior may differ from that of the general population. Therefore, more confirmatory studies are necessary with differing population groups in order to generalize the results from this study.This research study provided new knowledge about the effects of subjective sensations on actual snack choice. We explored subjective sensations related to appetite sensations, such as hunger and fullness, but also focused on non-homeostatic subjective sensations such as desires, wellbeing and energy sensations. Sweet desire seemed to be a unique sensation not related to appetite nor other desire sensations. The results demonstrated that especially sensory specific desire sensations  affected, either positively or negatively, snack choice. Furthermore, wellbeing sensations  also showed significant effects on snack choice, each in different ways, with Physical wellbeing contributing positively to the choice of nuts and negatively to the choice of potato chips. The vitality- and energy-related sensations such as Sleepy, Energized and Concentration did not affect snack choice. Additionally, we found that both subjective temporal sensations and background factors brought important knowledge to the overall picture. Gender and Health consciousness drove snack choice as well, with health-conscious females choosing the presumed healthy snacks and males choosing the presumed unhealthy snacks.Importantly, this study indicates the applicability of subjective sensations in food choice research that focus on diverse determinants. Notably, including desires and wellbeing sensations elucidates important knowledge. The contribution of the different sensations to food choice is thus imperative, and helps to unravel and understand some aspects of snack eating behavior. This could provide further insights into (over)eating, and have implications for public health issues such as unhealthy snacking and obesity. Future perspectives include interdisciplinary research involving implicit approaches to predict choice behavior over time.