These 4Y-PSZ suspensions could be successfully applied to our custom-made DLP machine for manufacturing 4Y-PSZ components. To this end, several processing parameters, including layer thickness of 4Y-PSZ suspension, UV illumination time for layer-by-layer photocuring process, and initial dimensions of 4Y-PSZ objects, were tightly controlled. As sintering temperature increased from 1300 °C to 1500 °C, relative density and grain size of 4Y-PSZ objects increased, and cubic phase content also increased. Thus, after sintering at the highest temperature of 1500 °C for 3 h, high mechanical properties  and reasonably high optical transmittance , evaluated by a spectrophotometer, were obtained due to a high relative density (97.2 ± 1.38%), which would be useful for dental applications.This study reports the utility of solid camphor as a novel diluent in photocurable hexanediol diacrylate (HDDA) monomer to manufacture 4 mol% yttria partially stabilized zirconia (4Y-PSZ) components for dental applications by digital light processing (DLP). The use of a 65 wt% HDDA–35 wt% camphor solution allowed 4Y-PSZ suspensions to have reasonably low viscosities (1399 ± 55.8 mPa·s at a shear rate of 75 s InHowever, to manufacture dental zirconia components, special care should be taken to optimize processing parameters for the photopolymerization-based 3D printing process since flaws and residual pores after sintering can severely deteriorate translucency ,14 and mThus, considerable efforts have been made to reduce the viscosities of zirconia suspensions, for example, by utilizing dispersants  and surf10H16O) [Recently, our group has proposed that solid camphor (C10H16O) ,31) coul10H16O) ,32, thusComponents of zirconia suspensions prepared using an HDDA–camphor solution as a liquidus vehicle for DLP process are summarized in 2) balls were used as media for vigorous mixing. After which, a predetermined amount of dispersant was then mixed with the prepared HDDA/camphene solution for 10 min. After that, a predetermined amount  of 4Y-PSZ powder was gradually added into the dispersant-containing HDDA–camphor solution and then vigorously mixed by a planetary centrifugal mixer. In order to effectively reduce viscosities of 4Y-PSZ suspensions, various dispersant contents (2–5 wt% with respect to the 4Y-PSZ content of 40 vol%) were examined. In addition, effects of 4Y-PSZ powder content  on viscosities of 4Y-PSZ suspensions were examined. At the final stage, a predetermined amount (2 wt% with respect to the HDDA content) of photoinitiator was mixed with the 4Y-PSZ suspensions for 10 min.To prepare 4Y-PSZ suspensions, an HDDA/camphor solution with a camphor content of 35 wt% was first prepared by dissolving solid camphor in liquid HDDA using a planetary centrifugal mixer  for 5 min at a rotation speed of 1000 rpm, where zirconia (ZrOη) was monitored as a function of the shear rate (dγ/dt), ranging from 75 s−1 to 150 s−1. Three suspensions were tested for each condition to obtain mean and standard deviation. In addition, rheological behaviors of 4Y-PSZ suspensions with different dispersant contents and 4Y-PSZ powder contents were examined. Three suspensions were tested for each condition to obtain mean and standard deviation.Rheological behaviors of 4Y-PSZ suspensions prepared with HDDA and HDDA–camphor solution were characterized using a cone/plate viscometer . Apparent viscosity  was utilized, whose basic concept was previously proposed by our group . In thisTo determine the optimum exposure time for complete photopolymerization of 50-micron-thick layers used in this study, a range of UV exposure times (3–15 s) were applied to layers. Their photopolymerized thicknesses were then measured by a micrometer. The degree of line broadening caused by UV scattering by 4Y-PSZ particles was characterized by measuring differences between designed diameters (200–1000 μm) and photopolymerized diameters using an optical microscope . Three and six samples were tested for measurements of photopolymerized thicknesses and diameters, respectively, in order to obtain the mean and standard deviation.To optimize processing parameters for our DLP process, 4Y-PSZ components with a relatively simple shape (a disk geometry with a diameter of 14.4 mm and a height of 1.3 mm) were first manufactured. Their microstructures, sintering behaviors, and mechanical and optical properties were characterized. To this end, 50-micron-thick layers of 4Y-PSZ suspensions were photopolymerized selectively according to predetermined 2D designs for 5 s in a layer-by-layer fashion.As-manufactured 4Y-PSZ components were then washed with ethanol several times to completely remove uncured suspensions. These 4Y-PSZ components were then heat-treated to remove organic phases  via debinding and densify 4Y-PSZ via sintering. To determine the debinding schedule, thermogravimetric analysis  was performed to monitor the weight losses of a sample during heating. The sample was heat-treated at a heating rate of 5 °C/min up to 600 °C in nitrogen, and then its weight loss was recorded. Two samples were tested to obtain reliable results. A representative TGA-DTA curve is shown in To examine the utility of 4Y-PSZ suspensions with an HDDA–camphor vehicle for the DLP process, 3D shapes and microstructures of as-manufactured components were characterized by optical microscopy and FE-SEM, respectively. In addition, their dimensions  were measured by a micrometer to evaluate the accuracy of our printing technique.The 4Y-PSZ components sintered at various temperatures  were characterized in terms of 3D shapes, microstructures, mechanical properties, and optical properties. Dimensions of sintered 4Y-PSZ components were measured to evaluate their sintering shrinkages. Sintering shrinkages were then calculated by dividing diameters of sintered 4Y-PSZ disks by those of as-manufactured disks. Five samples were tested to obtain the mean and standard deviation. To calculate relative densities of sintered components, their densities were measured using Archimedes’ principle and then divided by theoretical density of 4Y-PSZ (6.07 g/cm3 according to manufacturer’s specification). Ten samples were tested to obtain the mean and standard deviation. Microstructures of sintered 4Y-PSZ components were characterized by FE-SEM. Grain sizes of 4Y-PSZ samples sintered at 1300 °C, 1400 °C, and 1500 °C for 3 h were measured by the linear intercept method . Three sCrystalline phases of sintered specimens were examined by X-ray diffraction. An X-ray diffractometer  with Cu Kα radiation operating at 40 kV and 200 mA was used. Diffraction patterns of specimens were recorded between 2θ ranging from 10° to 80° with a step size of 0.04°. Peaks were indexed by matching their diffraction angles and relative intensities to those of tetragonal (JCPDS card no. 01-079-1767) and cubic (JCPDS card no. 01-081-1550) phases of crystalline zirconia. Contents of tetragonal and cubic phases were calculated by Rietveld refinement using X’Pert Highscore Plus software 3.0 .p) at fracture was then recorded. Biaxial flexural strength (σ) could be computed based on load (p) and specimen thickness (b) using the following equations [r1, r2, and r1 are radii of support circle, loaded area, and disk specimen, respectively. Three specimens were tested for each condition in order to obtain the mean and standard deviation.Mechanical properties of 4Y-PSZ disks sintered at various temperatures  were characterized in terms of strength and hardness for dental applications. For strength evaluations, biaxial flexural test in accordance with ISO 6872 for ceramic materials in dentistry was employed ,35. Diskquations :σ = −0.2d). Vickers hardness (Hv) was then calculated using the following equations:For hardness evaluations, Vickers hardness test was performed in accordance with ASTM C1372-2015 . A forceFive specimens were tested for each condition to obtain mean and standard deviation.W) and black backing (YB), as follows [Optical properties of 4Y-PSZ disks sintered at various temperatures  were evaluated by measuring their contrast ratio (CR) and translucency parameter (TP) values using a spectrophotometer  ,38. All CR values of 0.0 and 1.0 values correspond to transparent and totally opaque materials, respectively. TP was also calculated based on CEILAB coordinates, as follows:ectively .p values less than 0.05 were considered statistically significant.All data were expressed as the mean ± standard deviation. The statistical analysis was performed by a one-way analysis of variance (ANOVA) with MATLAB  with Tukey–Kramer Post Hoc Test . To utilize our DLP technique for manufacturing dental 4Y-PSZ components with high quality, it is crucial to prepare 4Y-PSZ suspensions with desired rheological properties for the 3D printing process and excellent densification behavior during sintering at high temperatures. Fundamentally, these properties are strongly influenced by characteristics of powders, including their size, specific surface area, and shapes ,27. For Calcined granules showed relatively large sizes in the range of 10–80 mm A. A mean−1 to 150 s−1, indicating shear thinning behaviors [To achieve excellent 3D printing capability and high densification for desired mechanical and optical properties, 4Y-PSZ suspensions should have reasonably low viscosities at high solid loadings. Thus, camphor was used as a diluent. The basic concept behind this has been reported by our group previously, in order to reduce viscosities of 4Y-PSZ suspensions with high solid loadings . The effehaviors . However−1. In addition, a higher dispersant content of 4 wt% resulted in a further reduction in viscosity to a certain degree. However, the viscosity increased at a high dispersant content of 5 wt% due to a thick layer of the dispersant. On the basis of this test, a dispersant content of 4 wt% was employed to prepare 4Y-PSZ suspensions with higher solid loadings .Effect of dispersant content on viscosities of 4Y-PSZ suspension prepared using a HDDA/camphor vehicle was also examined. Results are shown in −1, viscosities of 657 ± 26.9 mPa∙s and 1399 ± 55.8 mPa∙s were observed at solid loadings of 45 vol% and 48 vol%, respectively.To obtain high densification after sintering required for desired mechanical and optical properties, 4Y-PSZ suspensions should have reasonably high solid loadings, while their viscosities should be sufficiently low for a 3D printing process. To this end, the effect of solid loading (45 vol% and 48 vol%) on viscosities of 4Y-PSZ suspensions was examined. Results are shown in Although a range of viscosities could be successfully utilized to manufacture zirconia components by various DLP machines , a lowerTo optimize processing parameters for our DLP process, cure depth and width of a 4Y-PSZ suspension prepared with a HDDA/camphor vehicle were examined. Layers of a 4Y-PSZ suspension were photopolymerized for various time periods and their cured thicknesses measured using a micrometer. Results are displayed in To closely replicate complicated structures of dental components required for individual patients, special care should be taken to design initial dimensions, since 4Y-PSZ particles with higher refractive index than HDDA could scatter incident UV light during photopolymerization, leading to considerably expanded dimensions ,43. ThusDisk-shaped 4Y-PSZ specimens were manufactured by our photopolymerization-based 3D printing using a 4Y-PSZ suspension containing an HDDA/camphor vehicle with a solid loading of 48 vol%. The inset in After a multi-step debinding process using slow heating rates, 4Y-PSZ disks were sintered at various temperatures  for 3 h, in order to evaluate their densification behaviors. Regardless of sintering temperatures, notable defects were not observed for any disks, as shown in Microstructures of 4Y-PSZ disks sintered at various temperatures  were closely characterized by FE-SEM. Their representative FE-SEM images were taken from fracture surfaces, which were obtained after biaxial flexural strength tests, as shown in The effect of sintering temperatures on the grain growth of 4Y-PSZ disks was examined by observing their free surfaces using FE-SEM. Results are shown in Grain sizes measured by the linear intercept method increased notably with an increase in sintering temperature, as summarized in The 4Y-PSZ ceramics comprise two crystalline  phases. Their mechanical and optical properties are strongly affected by the contents of these two phases . More spv to 1371 ± 14.4 Hv with an increase in sintering temperature from 1300 °C to 1500 °C  to achieve almost full densification with high relative densities and should not be too high to avoid extensive grain growth with large grain sizes. In addition, a longer dwelling time for sintering can result in an increase in grain size. On the other hand, in terms of crystalline structure, it is important to tailor contents of tetragonal and cubic phases of 4Y-PSZ components. More specifically, yttria-doped zirconia ceramics can offer high mechanical properties since grains with tetragonal phase can transforme into those of monoclinic phase when stressed, thus hindering crack propagation . Thus, aIn order to make full use of 4Y-PSZ components manufactured in this study for dental applications, it is very important to evaluate their optical properties , since higher translucency is preferred in modern dentistry ,4,6. ThuTo more closely evaluate the optical properties of 4Y-PSZ disks sintered at various temperatures , their translucency parameter (TP) and contrast ratio (CR) were measured by CIE colorimetry. The TP and CR values computed from Equations (5) and (6), respectively, are displayed in The main purpose of the present study was to demonstrate the utility of zirconia suspensions prepared using an HDDA/camphor vehicle for photopolymerization-based 3D printing, which would be utilized to manufacture 4Y-PSZ components for dental applications, including dental crowns, abutments, and implants. Thus, dental crowns made of 4Y-PSZ were manufactured using our approach. After sintering at 1500 °C for 3 h, no notable defects, such as severe distortion or cracks, were observed even for a complicated shape, as shown in −1). Thus, 4Y-PSZ components could be successfully manufactured by our photopolymerization-based 3D printing. To obtain desired mechanical and optical properties for dental applications, several processing parameters were tightly controlled, including UV illumination time to completely photopolymerize 50-μm-thick layers of 4Y-PSZ suspensions with good bonding between deposited layers, designing of initial dimensions of 4Y-PSZ components to offer high accuracy, a debinding process to remove photopolymerized HDDA and organic phases without defects, and sintering temperature (1300–1500 °C) to obtain high densification. The 4Y-PSZ components sintered at the highest temperature of 1500 °C and high relative density of ~97.2% could be obtained. Thus, high mechanical properties  and a reasonable optical translucency (TP = ~7.77 and CR = ~0.809) could be obtained, which would be useful for dental applications.The use of a 65 wt% HDDA/35 wt% camphor solution as a photopolymerizable vehicle enabled highly concentrated 4Y-PSZ suspensions (solid loading = 48 vol%) to have reasonably low viscosities (1399 ± 55.8 mPa·s at a shear rate of 75 s


Spinal cord injury (SCI) remains one kind of devastating neurological damage, and specific molecular mechanisms involved need to be understood deeply. Currently, circular RNAs (circRNAs), as a newly discovered type of non-coding RNAs (ncRNAs), have been under active investigation. Through functional interactions with disease-associated microRNAs (miRNAs), exosome-derived circRNAs have been extensively implicated in various organ pathogenesis. Nevertheless, the functional involvement of circulating circRNAs in SCI onset, progression as well as repair remains poorly explored until now. Of note, there still lacks clinical and experimental evidence in this regard. To obtain some relevant knowledge in this field, this study was originally designed to have a general overview of differentially expressed circRNAs derived from circulating exosomes in SCI rats in comparison with the control rats. It turned out that 709 types of downregulated circRNAs and 346 kinds of upregulated circRNAs were preliminarily screened out. Functional enrichment analyses including kyoto encyclopedia of genes and genomes (KEGG) pathway and gene ontology (GO) were performed to evaluate the possible biological functions of upregulated as well as downregulated circRNAs involved in SCI. Furthermore, five types of upregulated circulating circRNAs including chr4:208359914–208362182+, chr15:20088296–20092102+, chr1:175098934– 175134845–, chr1:175099657– 175128203–, and chr1:175104454– 175134845–, and plus five kinds of downregulated circulating circRNAs including chr11:74154652– 74159524–, chr12:45412398– 45412635–, chr7:137630261– 137648924–, chr6:6280974–6281188+, and chr4:225251864–225254087+, were verified through reverse transcription-polymerase chain reaction (RT-PCR). At last, taking these differentially expressed circRNAs in the center, the circRNA-miRNA-mRNA gene interaction network was constructed to predict the possible functionalities of circRNAs in SCI through anticipating specific interactive miRNAs, giving new insights into how circRNAs contribute to this pathological process. Taken together, these findings suggest the possible involvement and functional significance of circRNAs in SCI. Spinal cord injury (SCI) remains a catastrophic injured condition for humans, and causes impaired mobility and even permanent neurological deficits, thereby bringing serious social and economic burdens . Given tDistinct from traditional linear RNAs, circular RNAs (circRNAs), as a new class of ncRNAs, are more stable, not easy to degrade, and not affected by RNA exonuclease due to their unique closed annular structure . In the On the one hand, exosomes, as mediators of cell-to-cell communication as well as vehicles of circRNAs and other ncRNAs, are key regulators in specific molecular signaling . On the For this reason, we mainly focus on circulating exosome-derived circRNAs in this study, and aim to investigate their potential role and relevant functions in the context of SCI. To this end, we utilized rat SCI model and collected exosomes, procedures and results of which have been well described in our recently published paper . Based oThe successful establishment of rat SCI model, the isolation of blood exosomes, and the identification of exosomes were detailedly described in our recently published paper . In brieAccording to the manufacturer’s protocol, Trizol reagent  was used to obtain high-quality RNA from circulating exosomes. Concentrations of different RNA samples were measured through using Nanodrop Spectrophotometer . Then the quantitative control as well as the integrity control were performed for isolated RNA prior to experiments in the next step.In the process of quantitative control, the purity of RNA was evaluated by using OD260/OD280 values. The final results would be considered as “Pass”, if these OD260/OD280 values are within the range between 1.8 and 2.1, suggesting isolated RNA has high quality and can be used further.Regarding the integrity control, 1% agarose gel  was utilized to assess the integrity of isolated RNA in this study.® Ultra™ II Directional RNA Library Prep Kit  according to the standard procedures. Furthermore, RNA library was quality-controlled and then quantified via a BioAnalyzer 2100 system . In the end, circRNA sequencing was conducted on an illumina HiSeq sequencer from Cloud-Seq Biotech . All raw data were uploaded to GEO repository as the GSE213561 study , and the link is as follows: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE213561.Prior to RNA library construction, ribosomal RNA (rRNA) needs to be completely removed from total RNA. To this end, NEBNext rRNA Depletion Kit  was used according to the producer’s protocol. Following this, the RNA library was then constructed through using NEBNext1 3′ adaptor was modified, and low-quality reads were removed in order to keep high-quality reads for further analysis  files which were obtained from the Ensembl database,3 the expression levels of circRNAs, characterized by the fragments per kilobase of exon model per million mapped fragments (FPKM), were calculated via Cuffdiff software . Through using Cutadapt software,analysis . To dispsoftware . Followi4 and GO analysis5 were performed for circulating upregulated as well as downregulated circRNAs, respectively  was calculated and presented as the enrichment score. For both KEGG pathway and GO analysis, fold change > 2 and P-value < 0.05 were taken as the threshold of the differential expression of these circRNAs.Moreover, in order to predict their probably involved pathways and functional processes, KEGG pathway analysisectively . The valCt–ΔΔ method was applied to show the relative expression of these circulating circRNAs. Given that most of differentially expressed circRNA are novel, we used their chromosome locations to name them.Next, we would like to separately verify the top ten dysregulated circulating circRNAs in SCI rats, which were revealed by RNA sequencing previously. To this end, RT-PCR was utilized here to show the relative levels of these 10 circulating exosomal circRNAs from both group rats. As mentioned in the above part, Trizol reagent was used to isolate RNA, and then cDNA was synthesized by utilizing SuperScript™ IV First-Strand synthesis kit . For RT-PCR process, SYBR Green master mix  was additionally employed. Primer 5.0 software  was used to design all primer sequences for circRNA targets as well as the housekeeping gene, i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All primers were purchased from CloudSeq Biotechnology . The primer sequences are shown in 6 based on these differentially expressed circRNAs, and then potential mRNAs were further anticipated by applying Targetscan7 as well as miRDB8  from more than three statistical independent experiments. To compare the statistical significances among different groups, The experimental design of this study was shown in P < 0.05). In a similar vein, the volcano plots demonstrated consistent changes of differentially expressed circRNAs in SCI rats  can be found below: This animal study was reviewed and approved by the Animal Care and Use Committee of Tijian Hospital.JL, CZ, and ZW designed and conceptualized this study. JL and CZ wrote the manuscript. FL and ZW further polished the manuscript. All authors prepared and performed experiments, collected, analyzed, and interpreted data, read, and approved the final manuscript.


Hepatocellular carcinoma (HCC) is one of the most malignant tumors. Immune checkpoint inhibitors (ICIs)-based therapy has recently been demonstrated to greatly ameliorate survival outcomes in advanced HCC. The objective of this research was to evaluate clinical outcomes of ICIs-based monotherapy and combination therapy as first-line treatment of adults with advanced HCC in real-world practice by conducting a systematic literature review and meta-analysis. These findings could supplement the evidence for treatment strategies of advanced HCC and inform study design in future real-world studies of HCC.Background: Immune checkpoint inhibitors (ICIs)-based therapy has recently been demonstrated to greatly ameliorate survival outcomes in advanced hepatocellular carcinoma (HCC). We aimed to evaluate clinical outcomes of ICIs-based monotherapy and combination therapy as first-line treatment of adults with advanced HCC in real-world practice by conducting a systematic literature review and meta-analysis. Methods: PubMed, Web of Science, and Embase were searched up to 25 April 2022. Retrospective or prospective real-world studies evaluating progression-free survival (PFS), overall survival (OS), objective response rate (ORR), disease control rate (DCR), and treatment-related adverse events (TRAEs) of patients with advanced HCC receiving first-line ICIs-based therapy were included. Results: Of 7805 studies retrieved, 38 were deemed eligible for inclusion. For patients receiving first-line ICIs-based therapy in real-world practice, the pooled median PFS and OS were 7.03 (95% CI: 5.55–8.51) and 14.39 (95% CI: 10.91–17.86) months. The ORR and DCR were 0.432 (95% CI: 0.327–0.538) and 0.756 (95% CI: 0.677–0.836), according to mRECIST 1.1, 0.317 (95% CI: 0.218–0.416) and 0.740 (95% CI: 0.644–0.835), judged by RECIST 1.1. The best outcomes of survival and response rate were observed in ICIs-based combination therapy of ICIs, TKIs, plus LRTs. Furthermore, ORR, DCR judged by mRECIST 1.1, and PFS could be potential prognostic factors for OS. Conclusions: This research revealed diversified first-line ICIs-based therapies for advanced HCC in real-world practice. Future studies are needed to adopt prospective, multicentric and comparative designs to test the ICIs-based combination therapies, especially triple therapies of ICIs, TKIs, plus LRTs. Liver cancer has become the second most frequent cause of cancer-related deaths globally, only after lung cancer . HepatocFor HCC at an early stage, surgical treatment; locoregional treatment (LRT), mainly consisting of stereotactic body radiation therapy; radiofrequency ablation; and trans-arterial chemoembolization (TACE) can significantly improve the progression-free survival (PFS) and overall survival (OS) of patients . HoweverIn the last decade, monotherapy and combination therapy of immune checkpoint inhibitors (ICIs) have greatly improved the survival outcomes in some solid and hematological tumors ,16,17. IHowever, the comprehensive review of clinical outcomes in real-world practice of ICIs-based therapy for advanced HCC remains underreported. Unlike clinical trials, more diversified patient samples are included in real-world practice, which not only supports data from clinical trials but may also supplement the current understanding about the clinical outcomes. The evidence from real-world practice can also provide reference to inform healthcare policy and disease management . Two preThus, the objective of this study was to evaluate clinical outcomes associated with ICIs-based monotherapy and combination therapy as first-line treatment for adults with advanced HCC in real-world practice by conducting a systematic literature review and meta-analysis. It is expected that the findings could supplement the evidence for treatment strategies of advanced HCC and inform study design in future real-world studies of HCC.This systematic literature review and meta-analysis complied with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement ,25. The The study selection criteria, according to the PICOS approach, were presented in We searched three databases, including PubMed, Web of Science, and Embase for eligible studies published from inception to 25 April 2022. The main search terms consisted of “hepatocellular carcinoma”, “immune checkpoint inhibitor”, “PD-1”, “PD-L1”, and “CTLA-4”. Details regarding search strategies were summarized in Two rounds of assessments of search results were conducted independently by two authors. Firstly, titles and abstracts were screened to exclude the irrelevant results. Secondly, potential full texts were reviewed to include eligible studies. All disagreements were discussed between the two authors until they reached a consensus.Two authors extracted the following data from each recruited study independently: first author, publication year, study type, characteristics of study patients, region, data source, follow-up time, ICIs agent, comparator, and main clinical outcomes. All disagreements were resolved by discussion between the two authors.Two authors assessed study quality independently using the Newcastle–Ottawa scale (NOS) . Each inI-square and the related p value were used to evaluate the heterogeneity. If I [p < 0.1, there was a significant heterogeneity [t test. Additionally, in order to identify the optimal strategy with superior statistical performance, we further compared subgroups under each type of classification through an independent samples t test. The subgroup with the best outcome within each type of classification was selected as the reference group in the comparisons. The relationships between ORR, DCR, PFS, and OS were evaluated by Pearson correlation analysis. Publication bias was assessed by Funnel plot and Egger’s test. Sources of heterogeneity in the estimates were determined by meta-regression analysis. Statistical analysis was accomplished using R studio version 4.1.2 and SPSS version 26.0. [Meta-analyses were performed to calculate outcomes of PFS, OS, best responses, and TRAEs. ty. If I  > 50% orogeneity . A randoon 26.0. . SensitiInitially, 7805 studies were identified through database searching, of which 2719 were excluded for duplication and 4583 were filtered for study type and language out of scope after title and abstract screening. Then, the full texts of 503 publications were screened thoroughly. A total of 114 studies, which obtained data from research settings that had many strict restrictions, such as randomized controlled trials (RCTs) rather than real-world medical institutions, families, and communities, were excluded. Finally, a total of 38 observational studies were included in the present research .The quality of all 38 studies was assessed using the NOS from three perspectives of selection, comparability, and outcome. Details regarding the assessment were provided in n = 29), with mainland China (n = 19) being the most represented region and followed by Taiwan (n = 4), Japan (n = 3), Republic of Korea (n = 2), and Hong Kong (n = 1). In addition, USA (n = 7), Germany, and Austria (n = 1) were the western countries that were involved. Only one study was conducted on a worldwide basis.n = 35), and only three were prospective in design. Data sources were mostly from traditional medical records and EMR . Specifically, studies were carried out in tertiary care settings, such as hospitals and academic centers. Comparators in most studies were of baseline (pre-drug) measures. Three studies included direct comparisons of ICIs-based combination therapies with tyrosine kinase inhibitors (TKIs), two included direct comparisons of the combination therapies with LRT (TACE) or TKI plus TACE, and four included direct comparisons between different ICIs-based therapies.A retrospective design was adopted by most studies (n = 29) mainly included Asian, and the remaining studies (n = 9) recruited Caucasian, African, and Asian.In total, 2750 patients with advanced HCC were recruited for the present research. The sample size of included cohorts ranged from 6 to 202. The mean or median age of patients varied from 49.1 to 73 years, and the percentage of male gender varied from 52.8% to 100%. In most studies, HCC patients with HBV or HCV infection were evaluated. Five studies also included NAFLD-related HCC patients, and four included alcoholic etiology. Most patients had Child–Pugh class A liver function and staged at Barcelona Clinic Liver Cancer (BCLC) C. Regarding the ethnicity, most studies (n = 17) was the most common used ICI across studies, followed by camrelizumab (n = 9), atezolizumab (n = 8), pembrolizumab (n = 8), sintilimab (n = 7), toripalimab (n = 6), tislelizumab (n = 2), durvalumab (n = 2), cemiplimab (n = 1), and AK105 (n = 1). ICIs combined with TKIs (n = 10), angiogenesis inhibitory monoclonal antibodies , LRTs (n = 6), and TKIs plus LRTs (n = 8) were evaluated. Best response was the most evaluated outcome (n = 35), followed by survival outcomes (n = 31), TRAEs (n = 29), and prognostic factors for PFS and OS (n = 14).ICIs in the setting of first-line treatment involved in this research were anti-PD-(L)1 antibodies. Nivolumab (p < 0.05 [p < 0.001) [p = 0.006) [The final results of 26 real-life studies reported the median PFS of patients receiving first-line monotherapy or combination therapy of ICIs. Three studies reported that the median PFS for first-line ICIs-based therapy (10.2–12.1 months) was better than that for TKIs therapy (5.1–8.9 months) ,31,32, op < 0.05 ,32. One < 0.001) . One rev= 0.006) .p < 0.01) in real-world practice  showed PFS benefit compared with TKIs therapy  compared with anti-PD-L1-based immunotherapy (6.66 (95% CI: 5.45–7.87) months, vs. overall PFS, p = 0.024). Most anti-PD-1 antibodies involved in this research showed comparable PFS (p > 0.05). Meanwhile, there was no statistical difference in PFS outcomes between different ICIs-based combination strategies (p > 0.05). Regarding patient characteristics, responders had significantly better median PFS than non-responders (p = 0.000), while the same results were observed in patients with or without hepatitis virus infection (p > 0.05).Next, the single-arm and statistical analyses of median PFS included 27 cohorts. The median value of the reported PFS was 6.8 months, which ranged from 2 to 16 months. The pooled median PFS of first-line monotherapy or combination therapy of ICIs was 7.03 (95% CI: 5.55–8.51) months. Subgroup analyses were conducted according to different immune checkpoint targets, ICI drugs, combination modes, and study characteristics. As shown in p = 0.105) [p < 0.001) [p = 0.004) [p < 0.001) [The median OS of first-line ICIs-based therapy was reported by 23 studies. One study reported that the median OS of ICIs + TKIs therapy and TKIs therapy were comparable  . However< 0.001) . The med= 0.004) . Moreove< 0.001) . Collectp > 0.05), indicating that the majority of ICI drugs presented great effectiveness in real-world practice (p = 0.026), even if there was no statistical difference between combination therapy and monotherapy (15.98 (95% CI: 12.63–19.33) months vs. 9.81 (95% CI: 2.18–17.45) months, p > 0.05). Concerning the treatment response, patients who had a response to ICIs-based therapy showed longer median OS than that of non-responders .In the single-arm analyses, the median value of the reported OS was 13.05 months varying from 3 to 24.8 months, and the pooled median OS for first-line ICIs-based monotherapy or combination therapy was 14.39 (95% CI: 10.91–17.86) months. Further stratified analyses found that, firstly, anti-PD-1-based immunotherapy exhibited no difference in OS outcomes with anti-PD-L1-based immunotherapy (14.54 (95% CI: 10.31–18.78) months vs. 14.9 (95% CI: 13.6–16.3) months, practice . It was Fourteen studies reported prognostic factors for PFS and/or OS of ICIs-based therapy in the setting of first line, and details were summarized in Thirty-five studies were included in the analyses of the best response of first-line ICIs-based therapy in the real world. To determine the response rates of ICIs-based therapy versus TKIs therapy in the first-line setting, meta-analysis was conducted in three studies. ICIs-based therapy showed ORR benefit compared with TKIs therapy, while the DCR was similar in both groups .p > 0.05, p > 0.05, monotherapy vs. overall, p = 0.01; DCR: combination therapy vs. monotherapy, p = 0.002, judged by RECIST 1.1). The best response rate was observed in the ICIs + TKIs + LRTs therapy, since the triple therapy was the only regimen that had a significantly higher response rate than the overall rate (ORR: 0.639 (95% CI: 0.479–0.800) vs. 0.432 (95% CI: 0.327–0.538), p = 0.043; DCR: 0.872 (95% CI: 0.800–0.944) vs. 0.756 (95% CI: 0.677–0.836), p = 0.016, judged by mRECIST 1.1).The analyses were further stratified by different ICIs-based therapy and RECIST criteria, which were presented in Twenty-nine real-world studies were concerned with TRAEs of first-line ICIs-based therapy. The pooled rate of any grade of TRAEs was 0.758 (95% CI: 0.667–0.848) A, grade p = 0.34) and DCR  judged by RECIST 1.1 had no correlation with OS. Interestingly, the correlations between ORR, DCR judged by mRECIST 1.1, and OS were 0.85 (p = 0.0037) and 0.8 (p = 0.01), respectively  for PFS and OS (p = 0.056), while it was strongly correlated with OS in ICIs-based combination therapy .Additionally, the overall correlation was 0.75 , apatinib plus camrelizumab, durvalumab plus tremelimumab, durvalumab, pembrolizumab, nivolumab, lenvatinib plus pembrolizumab or nivolumab, and FOLFOX plus camrelizumab as first-line systemic therapies for advanced HCC, since these immunotherapies have been demonstrated to improve quality of life and prolong the survival in several RCTs . HoweverIn this research, the scope of use for first-line ICIs-based therapy in real-world practice was found to expand beyond recent clinical guidelines, which provided more evidence for treatment strategies for advanced HCC. The pooled results of this research demonstrated that ICIs-based therapy in the first-line setting witnessed better outcomes in survival and response rate from real-world practice compared with TKIs therapy, which was also recommended as a first-line regimen by clinical guidelines. Furthermore, the combination therapy of ICIs, TKIs, plus LRTs deserved the optimal ICIs-based regimen in real-world practice, showing the best outcomes of OS, ORR, and DCR. Similarly, in our previous study that evaluated the efficacy of first-line ICIs-based therapy for unresectable HCC patients in clinical practice, we found longer OS in ICIs-based therapy compared with sorafenib . AlthougSeveral studies evaluated the relationships between OS and early endpoints, such as ORR, DCR, and PFS, in cancer patients treated with ICIs ,41,42. TTaken together, future studies focusing on ICIs-based regimens as first-line systemic therapy for advanced HCC are suggested to adopt prospective and multicentric design and set control group for comparison, such as classic first-line therapy or other first-line ICIs-based therapy. Regarding study regimens, ICIs-based combination therapies, particularly triple therapy of ICIs, TKIs, plus LRTs, are recommended. ORR, DCR judged by mRECIST 1.1, and PFS are determined as early endpoints, since they are accepted as potential surrogate markers for OS.Concerning the quality of included studies, since most of the study designs were a single group assignment, the scores of quality assessment were varied. However, all the studies were included in the final analyses for the reason that we aimed to consider more ICIs-based treatment strategies in real-world practice. The sensitivity analysis demonstrated that the stability of the pooled results of PFS and OS were influenced by the poor quality of some studies. Therefore, future real-world studies should attach importance to the comparability of cohorts to improve the study quality.Moreover, we identified the significant sources of heterogeneity in this research. The proportion of patients with Child–Pugh A and BCLC C, as well as the follow-up time, could contribute to the heterogeneities in pooled analyses of DCR, ORR judged by RECIST 1.1, and median OS, respectively. It, consequently, suggests that the real-world studies with large sample sizes are needed to further test the impacts of these sources of heterogeneity on the related outcomes by conducting appropriate subgroup analysis.To our knowledge, this research provided the first systematic analysis of clinical outcomes associated with ICIs-based therapy for advanced HCC in the first-line setting from real-world practice. However, several limitations still existed. Firstly, some of the recruited studies were conference abstracts, resulting in limited data of baseline and analyses of subgroups. Secondly, the majority of the included studies were retrospective studies, which presented certain levels of heterogeneity and publication bias. Thirdly, the characteristics of patients varied among different studies, which might lead to ineluctable bias for analysis. Finally, there is still a shortage of direct outcome comparisons of different ICIs-based therapies, which requires more RCTs and real-world studies in the future.This systematic literature review and meta-analysis revealed diversified first-line ICIs-based therapies for advanced HCC in real-world practice. Future studies are needed to adopt prospective, multicentric, and comparative designs to test the ICIs-based combination therapies, especially the triple therapies of ICIs, TKIs, plus LRTs, which could contribute to better survival outcomes and response rates compared with monotherapy.


Modelling both primary sequence and secondary structure preferences for RNA binding proteins (RBPs) remains an ongoing challenge. Current models use varied RNA structure representations and can be difficult to interpret and evaluate. To address these issues, we present a universal RNA motif-finding/scanning strategy, termed PRIESSTESS (Predictive RBP-RNA InterpretablE Sequence-Structure moTif regrESSion), that can be applied to diverse RNA binding datasets. PRIESSTESS identifies dozens of enriched RNA sequence and/or structure motifs that are subsequently reduced to a set of core motifs by logistic regression with LASSO regularization. Importantly, these core motifs are easily visualized and interpreted, and provide a measure of RBP secondary structure specificity. We used PRIESSTESS to interrogate new HTR-SELEX data for 23 RBPs with diverse RNA binding modes and captured known primary sequence and secondary structure preferences for each. Moreover, when applying PRIESSTESS to 144 RBPs across 202 RNA binding datasets, 75% showed an RNA secondary structure preference but only 10% had a preference besides unpaired bases, suggesting that most RBPs simply recognize the accessibility of primary sequences. A hallm% 54 of 13 of RBPsabsence)  found thng motif . Althougng motif .bona fide binding sites remains a topic of active research   . While cresearch . Additioresearch ,15. In cq (RBNS) , SEQRS (q (RBNS) , RNAcompq (RBNS)  and HTR-q (RBNS) . While Rk-mers or PFMs (RBP sequence specificities are often modelled as Position Frequency Matrices (PFMs), commonly called ‘motifs’ . The sam or PFMs ,20. Deri or PFMs ), with o or PFMs . These ak-mer model from a set of RNA sequences and associated RNA structure context probabilities, which are derived from the ensemble of possible structures for each RNA probe. GraphProt determines three possible structures for each input RNA sequence and encodes them as graphs to preserve base-pairing information along with annotating bases with specific structural contexts. These graphs are used to define the kernel for a support vector machine model that captures both sequence and structural specificity. Pysster uses the minimum free energy structure of an RNA probe along with its nucleotide sequence to encode each RNA as a string that combines sequence and structure information. These strings are then used to train a convolutional neural network to identify sequence-structure motifs. RNAcompete-S determines RNA probe centroid structure and, similarly to pysster, encodes each probe with an alphabet that defines both primary sequence and secondary structure per base. These annotations are used as input for logistic regression to identify sequence-structure motif models, which can then be concatenated to create composite motifs. Across the four methods, three different sets of structural contexts, and therefore three different structure alphabets, are used to annotate the RNA secondary structure. The alphabets are composed of 4, 6, 4 and 7 structural contexts, respectively. Furthermore, each method was evaluated on a different type of RNA binding data with different biases and properties, and consequently it is difficult to compare their performance. Finally, other than pysster, each method produces only a single representative binding motif to describe the RNA binding specificity; although RCK and GraphProt theoretically should be able to model multiple specificities simultaneously, that information is not made available to the user.The diversity of sequence-structure specificity modelling is illustrated in the following four methods: RCK , GraphPrPredictive RBP-RNA InterpretablE Sequence-Structure moTif regrESSion), a method that is conceptually related to the computational component of RNAcompete-S, but automates and significantly extends most of the process. PRIESSTESS captures sequence and structure specificity from in vitro RBP–RNA binding data and was designed specifically to address the diversity of RBP binding modes: the variation in RNA secondary structure recognition and the potential for multiple and/or bipartite motifs. Importantly, the method is designed to be modular, transparent, and flexible. It produces models that are readily interpretable, with all motifs made available to the user, provides an inherent measure of RBP structure specificity, and facilitates scanning for motifs which is less straightforward with other methods. We also describe a new data assembly, including new HTR-SELEX data for 23 proteins, which we use to show that PRIESSTESS performs comparably to other models in general while providing greater interpretability of RBP specificity. Finally, we use PRIESSTESS to gauge the structural specificity of 144 RBPs with in vitro RBP-RNA binding data and find that the dominant RNA structural attribute across all models is a propensity to bind unpaired bases.Here we present PRIESSTESS . A further 23 HTR-SELEX experiments were also performed for this paper (as described above), for which the first and fourth cycles were used as the negative and positive set, respectively. Details for all four datasets can be in found in We downloaded RNAcompete-S data  from NCBm ENCODE , along w  data.in vivo datasets can be found in We obtained matching eCLIP  experimeet al.: E—unpaired/external, B—bulge loop, L—left paired base, R—right paired base, T—internal loop, M—multi-loop, H—hairpin loop , including the minimum free energy (MFE) structure and retention of one or more suboptimal structures. To determine if these representations would better model probe structure, we identified the representation that contained the largest enrichment of the known sequence-structure binding motif for hairpin binding and complex structure binding RBPs. We folded a subset of probes from the positive and negative sets of each experiment to determine the MFE structure, the centroid structure, and 1, 2 or 3 randomly sampled suboptimal structures per probe (RNAsubopt) with Vienna tools . We counHTR-SELEX and RBNS experiments incorporate constant flanking sequences in the randomized RNA probes that are present during the binding step. For these datasets, the full-length probe including the flanks was folded and annotated; however, only the sequence and structure of the variable region of the probe was used for downstream analysis. Flanking sequences can be found in P-value of <0.01 and the -minw and -maxw options restrict motif size to between 4 and 6 bases in length, allowing the identification of shorter, partial motifs as well as motifs of standard size. For each of the seven sets of PFMs identified by STREME, we applied Bonferroni correction to the P-values and extracted up to 40 PFMs. We scanned these PFMs on the remaining one-third of the training data (the data not used for STREME), scoring the PFM at each base. For each PFM, we calculated the sum of the top four scores per probe. This was used as input to train the Logistic Regression (LR) and Random Forest (RF) models.Using two-thirds of the training data we identified enriched PFMs with STREME . This wahttps://scikit-optimize.github.io) to determine the optimal L1 (i.e. LASSO) regularization strength using 80% of the scanned probes (80% of the data not used for STREME). BayesSearchCV was set to only optimize the L1 value. The initial Area Under the Receiver Operating Characteristic (AUROC) was then computed on a validation set consisting of the remaining 20% of the data.We used the LogisticRegression function from scikit-learn ,44 alonginitialAUROC - 0.5).To simplify the model, we reiterated the process, scaling the L1 regularization strength parameter by 1.25 at each iteration, until the AUROC dropped by 10% of the predictive power . States in the model include recursive ‘begin’ and ‘end’ states, a state for each position in the PFMs used, and recursive ‘gap’ states   using th’ states . We set Due to runtime restraints, we initialized HMMs with a maximum of 10 PFMs and trained on a maximum of 10 000 RNA probes from the one-third of the positive set that was not used for STREME. If fewer than 20 000 probes were available for training, we used half. After we trained an HMM for each alphabet, we computed the log probability under the model for the remainder of the unused positive training set probes and a matching amount of unused negative training set probes. As input to a RF model, we used log probabilities for all seven HMMs. We used the RandomForestClassification function from scikit-learn along with BayesSearchCV from scikit-optimize to determine the optimal model. BayesSearchCV was set to optimize the number and depth of the decision trees in the model.k-mer size set to 6. We also ran GraphProt  enriched in the set of bound RNAs, relative to the background, using STREME , and usik-mers that must be interpreted post hoc. Furthermore, as multiple motifs can be retained, PRIESSTESS models can include components of bipartite motifs and/or multiple distinct motifs for an individual RBP. The LR step integrates the motifs into a scanning model that outputs a single score, and also provides a measure of the contribution of each motif (the model coefficient), in particular revealing the contribution of RNA structure relative to primary sequence alone. Unlike previous methods, PRIESSTESS manages variation in structural specificity across RBPs: in the annotation step, PRIESSTESS includes multiple annotations of RNA probes with differing degrees of structural representation, including no structural representation and no sequence representation. As L1 strength is increased in the LR step, unnecessary motifs are dropped from the model, thus, RNA structural information is modelled only when it is relevant. PRIESSTESS is intentionally modular and is assembled from established tools, making it amenable to modification.This series of steps was designed to solve several problems in the representation of RNA binding sites and to simultaneously facilitate both sequence scanning and interpretation of the models, including dissection of the relative weight of individual motifs in binding to RNA. Traditional motifs are easily visualized, unlike collections of in vitro binding data, for comparison , resulting in a total of 55 experimental data sets. Among the 23 proteins in the benchmarking data set, 15 have CLIP data in the literature, spanning the three categories of RBPs, and encompassing proteins with bipartite binding  RBPs that bind to a specific RNA sequence explicitly within a hairpin loop structure , ii), and i,51) Fig). RBPs wg Figure .Experiments were subsampled to a maximum of one million probes in each of the positive and negative datasets and split randomly for training  and a held-out set for testing (25%). Negative sets were constructed from the input RNA pool for RBNS and RNAcompete-S experiments, and from the first selection cycle for HTR-SELEX experiments (see Methods for details). Despite the large proportion of held-out probes, results were reproducible with different training and testing splits .The running time of PRIESSTESS is largely dependent on the number of RNA probes used to train the model and the length and construction of the RNA probes, with runtime increasing linearly with dataset size and probe length . For expWe compared the performance of PRIESSTESS to the published sequence-structure motif identification methods RCK , GraphPrP < 1E–10; paired t-test). At the task of discriminating between CLIP bound sequences and corresponding negatives, however, PRIESSTESS models trained on the in vitro benchmarking datasets outperform corresponding models generated by RCK  and display no significant difference in performance compared to GraphProt and pysster models, each method outperforming the others on some RBPs  and pysster , and performs comparably to GraphProt. Thus, while no method displays a clearly superior performance in identifying in vivo binding sites, PRIESSTESS models tend to better represent the intrinsic in vitro binding specificity. It is possible that this difference is due to the fact that RCK, GraphProt, and pysster models retain considerably more information than PRIESSTESS  and are therefore capable of learning subtle biases  that are shared by sequences within a given experimental dataset, but are different between datasets. As noted above, the PRIESSTESS models used in these analyses were obtained by a model simplification process, and we speculated that more complex models may also learn experiment specific biases that are removed by simplification. The original (i.e. unsimplified) LR models, however, display no significant difference in scoring on CLIP datasets and independent in vitro datasets, relative to the reduced (i.e. simplified) models , ) models , indicatin vitro experiments   Figure –L. Simil) Figure . This ouin vitro experiments , LR models significantly outperform RF models on CLIP data  and other in vitro data . We speculate that this outcome may reflect RF learning complex patterns that are restricted to one experimental data type , rather than to the RBP.We further tested whether any improvement was obtained by employing Random Forests (RF) in the main PRIESSTESS framework, which would have a better capacity than LR to learn non-additive relationships among motifs, such as ANDs and XORs. We note that execution of each of the steps in PRIESSTESS (and each modification tested) involves heuristic choices, and a complete exploration of parameter choices is impractical. The modular nature of the process would, however, facilitate future examination of the components.et al.  derived from different RNA sequence/structure alphabets reflected what is known about each of the proteins. Reassuringly, the vocabularies that incorporate RNA structure accounted for a large proportion of the model weight for proteins known to bind specific structures Figure . We nextet al. , and areet al. , indicatTo gain a better understanding of the PRIESSTESS models, we manually examined the motifs obtained for a set of proteins with specific characteristics of interest:QKI. QKI, or Quaking, is a well-studied KH domain containing protein named for the tremors seen in mutant mouse models .e models . QKI home models . Of the e models . The PRIHNRNPL. HNRNPL binds to two CA dinucleotides separated by 0–3 bases . The ability of the PRIESSTESS models to hold multiple representations of the binding motif presumably allows for better modelling of bipartite binding specificities.–3 bases . PRIESSTs Figure . CompariROQUIN. The protein RC3H1, or Roquin, binds the constitutive decay element (CDE) to promote mRNA decay  for PTBP1/3 and RC3H1. Importantly, these models also allow for multiple distinct motifs to be modelled when relevant (RC3H1), which can capture components of bipartite motifs as well . PRIESSTESS model motifs for all 55 benchmarking dataset experiments are compiled in k-mer in a paired RNA context, as defined in Dominguez et al., 83% also show structural context sensitivity in the PRIESSTESS model. Similarly, 72% of HTR-SELEX experiments identified as displaying hairpin loop structure specificity in the Jolma et al. analysis also displayed structural sensitivity in the corresponding PRIESSTESS model. Overall, models for 83% of RBNS datasets and 68% of HTR-SELEX datasets contain at least one motif with secondary structure information . HTR-SELEX experiment models retained a median of 6 PFMs, whereas RBNS experiments retained a median of 8 PFMs . As somePRIESSTESS represents the first systematic method that determines the appropriate number of motifs and level of structural inclusion for individual RBP models without human intervention. The models generated by PRIESSTESS have good predictive ability and are interpretable, as demonstrated using a new curated benchmarking dataset consisting of multiple datatypes for a panel of diverse RBPs that vary in RNA binding modes . This dataset represents an important resource that can be used to assess and compare computational methods and assays. PRIESSTESS also produces a measure of secondary structure contribution to RBP binding and can concurrently model multiple distinct motifs for a single RBP, facilitating modelling of complex binding specificities. The modularity of PRIESSTESS allows for alteration of various aspects of the pipeline, e.g. the method used for RNA folding or for the identification of enriched motifs. Importantly, as PRIESSTESS was not designed for a specific type of RBP-RNA binding assay, it is amenable to any experimental data that can be represented as RBP-bound and background sets of sequences.in vivo (PRIESSTESS models reveal the importance of secondary structure to the intrinsic binding specificities of a large proportion of RBPs. Most of the secondary structure motifs, however, represent preference for unpaired primary nucleotide sequence, consistent with prior observations that RBPs generally prefer unpaired sites in vivo . Discounk-mer based models. As such, it does not capture the full granularity and detail that may be achieved by methods such as RCK, GraphProt, and pysster, although it performs similarly in cross-platform comparisons. Additionally, PRIESSTESS is uniformly superior to BEESEM in our analyses, indicating that, as with transcription factors (PRIESSTESS represents an intermediate between single motifs and full  factors ,61, incl factors .https://github.com/kaitlin309/PRIESSTESS.PRIESSTESS is available in the GitHub repository: Data from HTR-SELEX performed for this paper are deposited on the European Nucleotide Archive under the accession PRJEB47428.gkac694_Supplemental_FilesClick here for additional data file.


External snapping hip syndrome (ESH) is postulated to be one of the causes of greater trochanteric pain syndrome, which also includes greater trochanteric bursitis and tendinopathy or tears of the hip abductor mechanism. However, it was not yet described what kind of bony morphology can cause the snapping and whether symptomatic and asymptomatic individuals have different imaging features.It was the purpose of this study to look for predisposing morphological factors for ESH and to differentiate between painful and asymptomatic snapping.A consecutive cohort with ESH and available magnetic resonance imaging (MRI) between 2014 and 2019 was identified. The control group consisted of patients that underwent corrective osteotomies around the knee for mechanical axis correction and never complained of hip symptoms nor had undergone previous hip procedures. The following parameters were blindly assessed for determination of risk factors for ESH: CCD (corpus collum diaphysis) angle; femoral and global offset; femoral antetorsion; functional femoral antetorsion; translation of the greater trochanter (GT); posterior tilt of the GT; pelvic width/anterior pelvic length; intertrochanteric width. Hip and pelvic offset indexes were calculated as ratios of femoral/global offset and intertrochanteric/pelvic width, respectively. For the comparison of symptomatic and asymptomatic snapping, the following soft-tissue signs were investigated: presence of trochanteric bursitis or gluteal tendinopathy; presence of surface bony irregularities on trochanter major and ITB  thickness.p = 0.025), most likely due to the higher femoral offset in the ESH group (p = 0.031). Pearson correlation analysis could not identify any significant secondary factors. No differences were found between painful and asymptomatic snapping on MRI.A total of 31 hips with ESH were identified. The control group (n = 29) consisted of patients matched on both age (± 1) and gender. Multiple regression analysis determined an increased hip offset index to be independent predictor of ESH (r =  + 0.283, A high hip offset index was found as an independent predictor for external snapping hip in our cohort, mainly due to increased femoral offset. No imaging soft-tissue related differences could be outlined between symptomatic and asymptomatic external snapping.www.springer.com/00590.This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors   Coxa saltans refers to snapping hip and involves three main locations, extra-articular  or intra-articular. The most common form of coxa saltans is the external extra-articular variety which involves either the posterior iliotibial band or the anterior aspect of the gluteus maximus as they move over the greater trochanter during hip flexion and extension or internal and external rotation . It is tDue to the distinct anatomic location and often visible snapping, coxa saltans externa is a clinical diagnosis . The snaCoxa saltans externa is postulated to be one of the causes of greater trochanteric pain syndrome, which also includes greater trochanteric bursitis and tendinopathy or tears of the hip abductor mechanism . HoweverThe pathophysiology of the condition remains uncertain, as to our knowledge, there are only few studies that examined this matter. A study by Krishnamurthy  evaluateHowever, it was not yet described what kind of bony morphology can cause the snapping and whether symptomatic and asymptomatic individuals have different imaging features.It was the purpose of the current study to evaluate radiographic parameters of bony morphology that might predispose to develop external snapping of the hip, as well as to determine associated findings on MRI imaging. Differentiating between asymptomatic or symptomatic (painful) external snapping was the second main scope of the study.Institutional review board approval and of local ethical committee was obtained. All patients that were included signed a written consent form. All clinical records and data from follow-up visits from patients with a clinical diagnosis of external snapping hip were retrospectively extracted from the local database of our institution between December 2014 and June 2019  and never complained of hip symptoms, nor underwent hip surgeries, were included. Patients with available preoperative anteroposterior whole leg radiographs, as well as CT imaging with rotational sequences were further selected. A matched consecutive population on age and gender was extracted from the consecutive cohort between October 2017 and February 2020 to include a total of 29 hips.The following parameters were independently and blindly assessed for both groups for determination of risk factors for ESH: CCD (corpus collum diaphysis) angle; femoral and global offset; surface irregularity on the trochanter major (as an indirect sign of abductor pathology); femoral antetorsion; functional femoral antetorsion; translation of the greater trochanter; posterior tilt of the greater trochanter; pelvic width/anterior pelvic length and intertrochanteric distance. In order to adjust the offset distances (mm) which are dependent of patient-specific anatomy, ratios were calculated and compared: “Hip Offset Index” was the ratio between femoral/global offset, while the “Pelvic Offset Index” was the ratio calculated between the intertrochanteric distance and pelvic width.For comparison between symptomatic (painful) and asymptomatic snapping, the following soft-tissue related parameters were compared between groups: presence of trochanteric bursitis or gluteal tendinopathy and ITB  thickness .Surface irregularity on the trochanter major was defined as when pronounced (> 2 mm) changes were visible on conventional radiographs using previous described criteria .Femoral offset was defined as the distance from the center of rotation of the femoral head to a line bisecting the long axis of the femur . Global Pelvic width or the length of the anterior pelvis, was measured as the greatest distance between the edges of the antero-superior iliac spines (ASIS) , 15. IntMeasurements of femoral antetorsion on axial images over the proximal and distal femur were performed according to previously described method on MRI , 17.Functional femoral antetorsion, posterior translation and tilt of the greater trochanter, were measured used a previously described method by Batailler et al.  Fig. 3)3). FunctITB diameter thickness was measured in the axial plane of MRI examinations. The measurement location was considered as the region of greatest thickness between the most proximal images of the greater trochanter to the most proximal sequence showing the lesser trochanter. Abductor tendinopathy was defined as presence of tendinosis, calcific tendinitis, partial or full-thickness tears of the gluteus medius and/or minimus tendons , 20.A binary model was constructed due to the dichotomous nature of the dependent variables used (presence of snapping yes/no and presence of symptoms (pain) yes/no) to assess the relationship between association with morphological parameters for external snapping hip between the study and control groups  and risk factors for pain in the study group only . A paired Wilcoxon test was used to determine statistical differences between baseline values among compared groups.2) = 0.35 (large effect size) and a desired statistical power of 0.8 . As the compared populations were already matched based on age and gender, no hierarchical analysis was necessary. The multiple regression analysis allowed the inclusion of all covariates in the regression equation for calculation of each risk factor independently to predispose to external snapping: GT translation, GT tilt, femoral antetorsion, functional femoral antetorsion, CCD angle, pelvic offset index as well as hip offset index.An a-priori power analysis for a multiple regression revealed a minimum required sample size of 41 for an anticipated effect size  with a mean age of 25 years . As such, the study groups were matched and had no significant differences in terms of age or gender (Table p = 0.86); presence of abductor tendinopathy (p = 0.59); iliotibial band thickness (p = 0.733)  Table .Table 2Mp = 0.025) in patients with external snapping hip (Table p = 0.03). A Pearson correlation could not reveal any correlation of the femoral torsion (p = 0.176) or CCD angle (p = 0.874) .No trochanter surface irregularities were observed in both groups. Multiple regression analysis revealed an increased hip femoral offset/global offset index to be an independent predictor . We have expected to see a similar finding when comparing the ratio of the intertrochanteric distance/pelvic width (pelvic offset index), that would be more representative of the biomechanical increased distance of excursion of the hip abductors, which may cause snapping. However, this expectation was not confirmed by the results of our study.The main finding of our study is that increased femoral/global offset index alone was independently associated with the presence of external snapping hip syndrome, as the regression analysis demonstrated. This has occurred mostly due to the increased femoral offset in the ESH group  as an independent predictor for external snapping hip in our cohort, mainly due to high femoral offset. This may be the base for future studies to investigate its role in the outcome of non-responders to conservative treatment and the need for operative surgery.Data were stored in a local repository RedCap with access provided to the study staff and principal investigator.


In vitro transcribed (IVT) synthetic mRNAs are in high demand due to their attractive bench to clinic translational processes. Mainly, the procedure to make IVT mRNA using bacteriophage RNA polymerases (RNAP) is relatively uncomplicated and scalable to produce large quantities in a short time period. However, IVT mRNA preparations are accompanied by contaminants such as double-stranded RNA (dsRNA) as by-products that elicit undesired cellular immune responses upon transfections. Therefore, removing dsRNA contaminants is critical in IVT mRNA preparations for therapeutic applications. One such method to minimize dsRNA contaminants is to use genetically modified thermostable bacteriophage polymerase, HiT7 RNAP that performs IVT reaction at a higher temperature than typically used. However, the cellular RNA sensor response for IVT mRNA preparations by HiT7 RNAP is not characterized. Here, we compared the cellular RNA sensor response for mRNAs prepared by HiT7 RNAP (at 50°C) and SP6 RNAP (at 37°C) in HeLa cells. We show that IVT mRNA preparations by HiT7 RNAP reduced the dsRNA levels and dsRNA specific RNA sensor response  compared to the IVT mRNA preparations by SP6 RNAP. Similarly, the incorporation of pseudouridine nucleotides instead of uridine nucleotides reduced dsRNA sensor response and increased the mRNA translation. Overall, the least dsRNA mediated RNA sensor response is observed when mRNA is synthesized by HiT7 RNAP and incorporated with pseudouridine nucleotides. In the To fill this gap of understanding, in this study we compared the cellular RNA sensor responses of mRNAs prepared by SP6 and HiT7 RNAP in non-immune cell line, Hela. For this purpose, we synthesized mRNAs with unmodified (uridine nucleoside), modified (pseudouridine nucleoside) and/or HPLC purification. We show that a combination of HiT7 RNAP and nucleotide modification optimally reduced the cellular RNA sensor responses and provided efficient reporter production in cell.2.HeLa and HEK293T cells were grown in Dulbecco’s Minimum Essential Medium (DMEM) (Biowest) supplemented with 10% foetal bovine serum (FBS) (Sigma-Aldrich), 2 mM L-glutamine and 100 mg/ml penicillin/streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5% COTo visualise the formation of dsRNA by-products during mRNA synthesis, a PCR product containing first 250 nt of the firefly luciferase coding sequence with the SP6 or T7 promoter sequence, purified with the NucleoSpin Gel and PCR clean-up (Macherey-Nagel), was used as dsDNA template for IVT reactions. A standard transcription reaction contained: transcription buffer, 25 ng/μl of dsDNA template, 1 mM ATP/CTP/GTP/UTP or ψUTP, 0.5 U/µL of ribolock ribonuclease inhibitor (Thermo) and 1 U/µl of SP6 RNAP, 1 U/µl of T7 RNAP (Thermo) or 2.5 U/µl of HiT7 RNAP (NEB). The reaction mixture was incubated 4 h at 37°C for SP6 and T7 RNAP and 4 h at 50°C for HiT7 RNAP. Following incubation, 0.025 U/µl of DNaseI (Thermo) was added and further incubated for 20 min at 37°C to remove template DNA. The transcripts were purified using the NucleoSpin RNA clean-up (Macherey-Nagel) according to the manufacturer’s instructions.27,3’−OGpppG (ARCA) (TMPrep–RNA Purification Column (ADS Biotec). A linear gradient of buffer B (25% acetornitrile in 0.1 M triethylammonium acetate pH 7.0) in buffer A (0.1 M triethylammonium acetate pH 7.0) from 35% to 55% for 20 min at flowrate 4 ml/min was applied. Fractions containing mRNA were concentrated on amicon ultra centrifugation filters (Merck), precipitated with sodium acetate and isopropanol mixture overnight at −20°C and precipitated RNA was dissolved in nuclease free water. The integrity of transcripts was checked on 1% 1X TAE agarose gel and concentration was determined spectrophotometrically.For transfection of HeLa and HEK293T cells, mRNAs were prepared in IVT reaction from a PCR template containing the firefly luciferase coding sequence with the SP6 or T7 promoter sequence, purified with the NucleoSpin Gel and PCR clean-up (Macherey-Nagel). RNA capping was carried out co-transcriptionally using cap analogue: mG (ARCA)  (molar rG (ARCA) . QualityG (ARCA) . To remoTo observe the formation of ss/dsRNAs during IVT, 0.5–1 μg of 250 nt-long mRNA was run on a native 6% polyacrylamide 1X TAE gel. The gel was stained post electrophoresis for 10 min either with SYBR™ Gold (Thermo Fisher Scientific) or with 25 μg/ml acridine orange hemi(zinc chloride) salt  in 1X TAE buffer, destained for 1 h to ON in water, and visualized on UV transilluminator.5 cells/well in 24-well plates and grown to 60%–80% confluence 24 h prior to transfection. HEK293T cells were seeded at 2 × 104 cells/well in 96-well plates coated with 200 μg/ml polyL-Lysine (Sigma-Aldrich) and grown to 60%–80% confluence 24 h prior to transfection. Cells were transfected with Lipofectamine™ 2000 transfection reagent according to the manufacturer’s protocol. Briefly, HeLa cells in each well were transfected using a mixture of 2 μl Lipofectamine™ 2000 transfection reagent and 250 ng mRNA encoding firefly luciferase in 500 μl of Opti-MEM. HEK293T cells were transfected with 100 ng RNA and 0.6 μl Lipofectamine™ 2000 transfection reagent in 10 μl of Opti-MEM per well containing 90 μl of DMEM. Cells were collected at 8 h and 24 h post transfection.In all these experiments, the passage number of HeLa cells was between 5 and 30, of HEK293T cells between 10 and 15. HeLa cells were seeded at 5 × 10HEK293T cells transfected with 250 nt-long mRNAs were lysed by addition of 20 µl per well of Cell Culture Lysis Reagent (Promega). To estimate the luciferase activity, the lysates were mixed with the luciferase substrate  in ratio 1:2 and the luminescence was measured on Synergy H1MFDG Microplate Reader . To avoid errors due to varying number of cells in each well, the luminescence values were normalized to the total protein concentration measured with Roti-Quant Protein quantitation assay according to Bradford .260/A280 ratio (DeNovix). Up to 150 ng of RNA was used to obtain cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative PCR was performed on LightCycler 480 II System (Roche). Briefly, 2 μl of cDNA (obtained from 150 ng of RNA), 4 μl of each mRNA specific primers (5 pmol of forward and reverse)  according to the manufacturer’s instructions. RNA was quantified spectrophotometrically and its quality was analyzed using Areverse) , 10 μl ot method .https://cran.r-project.org). One-way ANOVA with post–hoc Tukey HSD test was used. Statistical significance in p value was denoted in asterixes  and the data are shown as mean ± standard error for three to six independent replicates. In all experiments, the term independent replicates specify the same IVT prepared RNAs that are then used in independent cultures.All statistical analyses were performed with R software v3.6.2  incorporating unmodified uridine/modified pseudouridine nucleotides and obtained by different RNAP (SP6 and HiT7) as shown in In order to gain insight into the cellular RNA sensor responses to transfections of firefly luciferase IVT mRNAs, we prepared different mRNAs capped at the 5′ end with mIFIT1 and IFIT5) between mRNA_Pseudouridine_SP6 and mRNA_Pseudouridine_HiT7 transfections , and therefore do not recognize dsRNA contaminants in the mRNA preparations. In addition, we did not notice induction of OAS1 cytosolic dsRNA sensors 8 h post transfection with either of the transcripts  sensor showed no striking changes , collected cells at 8 h post transfection and measured the induction levels of RNA sensors . Firstlyfections . This isnscripts . Howeverdine_SP6 . This obdine_SP6   and of MDA5  when compared to the cells transfected with mRNA_Uridine_SP6  when compared to the cells transfected with mRNA_Pseudouridine_SP6 , collected the cells at 24 h post transfection and measured the induction levels of RNA sensors specific for dsRNA . In thesand MDA5 ,D  with the use of RNAPs, primarily T7 and SP6. Both of them show very high structural and functional similarity. However, some features distinguish them. Each polymerase is highly specific for its promoter, and the substrates required for mRNA synthesis are also different. While SP6 RNAP requires the complete pairing of DNA substrate, T7 RNAP only needs the pairing of a fragment of 18 bases  3′-extended RNAs that anneal to complementary sequences in either cis or trans to form duplexes  aduplexes . These bduplexes . Howeverduplexes . Althougduplexes . This widuplexes . Additioin vitro mRNA synthesis have been proposed, and HiT7 RNAP has been shown to significantly reduce the amount of dsRNA when used at elevated temperatures (MDA5, sensor of long cytosolic dsRNA, when compared to mRNA preparations by SP6 RNAP.The mechanisms for the formation of by-products during eratures . NeverthWe confirmed the presence of dsRNA by-products in the preparation of the transcripts using acridine orange dye, which emits orange fluorescence when bound to phosphate groups in ssRNA and green fluorescence when intercalated into dsRNA . As expeCypridina luciferase mRNA_Pseudouridine_HiT7 and Cypridina luciferase mRNA_Pseudouridine_HiT7_HPLC (Further, in our study we did not observe the beneficial effect of HPLC purification as post synthesis purification step when modified pseudouridine nucleotide was introduced ,D. In liiT7_HPLC . To conciT7_HPLC . Further


De novo initiation by viral RNA-dependent RNA polymerases often requires a polymerase priming residue, located within a priming loop, to stabilize the initiating NTPs. Polymerase structures from three different non-segmented negative strand RNA virus (nsNSV) families revealed putative priming loops in different conformations, and an aromatic priming residue has been identified in the rhabdovirus polymerase. In a previous study of the respiratory syncytial virus (RSV) polymerase, we found that Tyr1276, the L protein aromatic amino acid residue that most closely aligns with the rhabdovirus priming residue, is not required for RNA synthesis but two nearby residues, Pro1261 and Trp1262, were required. In this study, we examined the roles of Pro1261 and Trp1262 in RNA synthesis initiation. Biochemical studies showed that substitution of Pro1261 inhibited RNA synthesis initiation without inhibiting back-priming, indicating a defect in initiation. Biochemical and minigenome experiments showed that the initiation defect incurred by a P1261A substitution could be rescued by factors that would be expected to increase the stability of the initiation complex, specifically increased NTP concentration, manganese, and a more efficient promoter sequence. These findings indicate that Pro1261 of the RSV L protein plays a role in initiation, most likely in stabilizing the initiation complex. However, we found that substitution of the corresponding proline residue in a filovirus polymerase had no effect on RNA synthesis initiation or elongation. These results indicate that despite similarities between the nsNSV polymerases, there are differences in the features required for RNA synthesis initiation. Mononegavirales, the non-segmented, negative strand RNA viruses (nsNSVs). Like other viruses in this order, RSV encodes an RNA dependent RNA polymerase, which is responsible for transcribing and replicating the viral genome. Due to its essential role during the viral replication cycle, the polymerase is a promising candidate target for antiviral inhibitors and so a greater understanding of the mechanistic basis of its activities could aid antiviral drug development. In this study, we identified an amino acid residue within the RSV polymerase that appears to stabilize the RNA synthesis initiation complex and showed that it plays a role in both transcription and RNA replication. However, the corresponding residue in a different nsNSV polymerase does not appear to play a similar role. This work reveals a key feature of the RSV polymerase but identifies differences with the polymerases of other related viruses.RSV has a significant impact on human health. It is the major cause of respiratory disease in infants and exerts a significant toll on the elderly and immunocompromised. RSV is a member of the  The non-segmented negative strand RNA viruses (nsNSVs) are a large and diverse order of viruses, currently comprising 11 virus families. These families include, amongst others, the filoviruses, such as Ebola and Marburg virus, the paramyxoviruses, such as parainfluenza viruses types 3 and 5 (PIV-3 and PIV-5), the rhabdoviruses, rabies and vesicular stomatitis virus (VSV) and the pneumoviruses, respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) , 3. The leader (le) promoter region. The key promoter residues are contained within the first 12 nt of the 44-nt le region  ATP included as the radioactive tracer. CTP was omitted to prevent initiation from position 1U  and the α-phosphate of the incoming nucleotide (NTP2 during initiation) enabling the nucleophilic attack required for phosphodiester bond formation -GTP tracer were performed in 25 μL reactions with the following conditions: 2 μM RNA oligonucleotide (Dharmacon), 50 mM Tris, pH 7.4, 8 mM MgCl2, 5 mM DTT, 10% glycerol, 500 μM (each) ATP, CTP, GTP, and UTP with 5 μCi [α-32P]-GTP (3000 Ci/mmol). Reaction mixtures were preincubated at 30°C for 5 minutes prior to addition of L-P complexes to a concentration of 8 nM L protein. Reaction mixes were incubated at 30°C for 1 h and then heat inactivated at 90°C for 3 minutes then allowed to cool on ice for 2 minutes. RNA was extracted with phenol-chloroform and ethanol precipitated. Pellets were resuspended in RNase free water, and an equal volume of stop buffer  was added. Products were migrated alongside molecular weight ladders representing products initiated from 1U or 3C sites, prepared as described previously -CTP (3000 Ci/mmol). Reactions to detect pppGpA and elongation products generated from position 3 of the promoter were performed in a volume of 25 μL with the following conditions: 2 μM RNA oligonucleotide (Dharmacon), 50 mM Tris, pH 7.4, 8 mM MgCl2, 5 mM DTT, 10% glycerol, 10 or 500 μM ATP, 10 or 500 μM GTP, and 500 μM UTP with 5 μCi [α-32P]-ATP (3000 Ci/mmol). In all cases, reaction mixtures were preincubated at 30°C for 5 minutes prior to addition of L-P complexes to a concentration of 8 nM L protein. Reaction mixes were incubated at 30°C for 1 h and then heat inactivated at 90°C for 3 minutes then allowed to cool on ice for 2 minutes. Following inactivation of the polymerase, all reactions were adjusted to contain 500 μM GTP  and RNA was extracted with phenol-chloroform and ethanol precipitated. Pellets were resuspended in RNase free water, and an equal volume of stop buffer  was added. The samples were subjected to denaturing electrophoresis alongside T7 triphosphorylated RNA markers by electrophoresis on 25% polyacrylamide gels containing 7 M urea in Tris-taurine-EDTA buffer. Gels were analyzed directly (without drying) by autoradiography and phosphorimage analysis.Reactions to detect pppApC formation from position 1 of the promoter were performed in a volume of 25 μL with the following conditions: 2 μM 2 or MgCl2, 5 mM DTT, 10% glycerol. The pppGpC reactions contained le 1–14 2G/A RNA template, 20 μM (each) GTP and CTP, and 5 μCi [α32P]-CTP (3000 Ci/mmol) as the radioactive tracer. The pppApC reactions contained tr 1–16 RNA template, 20 μM (each) ATP and CTP, and 5 μCi [α32P]-CTP (3000 Ci/mmol) as the radioactive tracer. The pppGpA reactions contained tr 1–16 RNA template, 20 μM (each) GTP and ATP, and 5 μCi [α32P]-GTP (3000 Ci/mmol) as the radioactive tracer. Reaction mixes were preincubated prior to addition of L-P protein to a concentration of 8 nM L protein. Reaction mixes were incubated at 30°C for 1 h, heat inactivated at 95°C for 5 minutes, and allowed to cool on ice for 2 minutes. Reactions were adjusted to contain 500 μM GTP after inactivation of the polymerase and RNA was purified and analyzed as described above.Reactions were performed in a volume of 25 μL with the following conditions: 2 μM RNA oligonucleotide (Dharmacon), 50 mM Tris, pH 7.4, 8 mM MnClin vitro transcription. Annealed oligonucleotides were prepared by mixing 4 μM short oligonucleotide and 4 μM long overhang (template) oligonucleotide in Tris-EDTA pH 8.0, incubated at 90°C for 3 minutes, then allowed to cool to room temperature. 40 μL in vitro transcription reactions were prepared with the following conditions: 40 mM Tris-HCl (pH 8.0), 20 mM MgCl2, 2 mM spermidine, 10 mM DTT, 0.1 mg/mL bovine serum albumin, 2 mM ATP, 1 mM CTP, 1 mM GTP with 1 μCi [α-32P] ATP, [α-32P] CTP, or [α-32P] GTP depending on the marker being synthesized, 0.1 μM DNA annealed oligos and 1 unit/μL T7 RNA polymerase (New England Biolabs). Reactions were incubated at 37°C for 5 hours. Samples were treated with 1 μL DNase I (New England Biolabs) for 10 minutes at 37°C. 1 μL 0.5 M EDTA was added to the reactions. Products were subjected to phenol chloroform extraction and ethanol precipitation prior to use as markers.Markers consisting of 5´ triphosphorylated RNA used to characterize small RNA products were generated using a method adapted from . DNA olile 1–30 RNA oligonucleotide (Dharmacon), 50 mM Tris, pH 7.4, 8 mM MgCl2, 5 mM DTT, 10% glycerol, 500 μM (each) ATP and GTP, 10 μM CTP, and 100 μM UTP, with 10 μCi [a-32P]-CTP (3000 Ci/mmol) as the radioactive tracer. Reaction mixes were preincubated prior to addition of L-VP35 complexes to a concentration of 8 nM L protein. Reaction mixes were incubated at 30°C for 1 h, heat inactivated at 95°C for 5 minutes, and allowed to cool on ice for 2 minutes. RNA was purified and analyzed on 25% Tris-taurine gels, as described above.Reactions were performed in a volume of 25 μL with the following conditions: 2 μM MARV 2PO4, 20 mM imidazole) for 15 minutes on ice, treated with Turbo DNase (Invitrogen) for 5 minutes at 37°C, and then migrated on a 10% SDS polyacrylamide gel and analyzed by Western blot analysis using a monoclonal antibody specific for the FLAG tag (Sigma). Proteins were detected using a goat anti-mouse IgG coupled to IRDye 800CW and LI-COR analysis.To confirm that each of the mutant L proteins could be expressed in mammalian cells, BSR-T7/5 cells were transfected with pTM1 plasmids containing FLAG-tagged versions of wt or mutant L plasmids. Cells in a 6-well plate were transfected with 2 μg of pTM1 P and 1 μg wt or mutant L using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. The cells were incubated at 30°C or 37°C (as indicated) and at 48 h post-transfection, they were collected and incubated with lysis buffer Click here for additional data file.S2 Figle promoter (shown in (A) L mutant proteins containing an N-terminal FLAG tag were expressed in mammalian cells at 30°C. At 48 h post transfection, cell lysates were analyzed by Western blot analysis using a FLAG-specific antibody. The image is representative of three independent experiments. (B) Analysis of RNA products generated from the 1U and 3C sites in a minigenome containing a shown in  by mutanshown in .(TIF)Click here for additional data file.


However, a quantitative analysis of mutation rates measured by mutational profiling reveals a global 5-fold rate increase in the presence of NSP2. We demonstrate that the normalization procedure used in deriving SHAPE reactivities from mutation rates can mask an important global effect of an RNA chaperone. Analysis of the mutation rates reveals a larger effect on stems rather than loops. Together, these data provide the first experimentally derived secondary structure model of the rotavirus transcriptome and reveal that NSP2 acts by globally increasing RNA backbone flexibility in a concentration-dependent manner.Due to genome segmentation, rotaviruses must co-package eleven distinct genomic RNAs. The packaging is mediated by virus-encoded RNA chaperones, such as the rotavirus NSP2 protein. While the activities of distinct RNA chaperones are well studied on smaller RNAs, little is known about their global effect on the entire viral transcriptome. Here, we used Selective 2′-hydroxyl Acylation Analyzed by Primer Extension and Mutational Profiling (SHAPE-MaP) to examine the secondary structure of the rotavirus transcriptome in the presence of increasing amounts of NSP2. SHAPE-MaP data reveals that despite the well-documented helix-unwinding activity of NSP2  Each ds strains . This st studies . Structu studies ,10.Rotaviruses encode an RNA chaperone, NSP2, that is believed to be involved in RNA selection and stoichiometric segmented genome packaging ,11. SingRNA folding is mediated by base-pairing that forms secondary structures, which play a myriad of fundamental regulatory roles, including in gene expression ,16,17. THere, we provide experimentally validated structure models of a complete rotavirus RNA pre-genome probed by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) and mutational profiling (MaP) analysis ,22. We iin vitro transcription reactions. Sequences for the 11 segments are listed in pUC19 clones encoding for segments 1–11 of bovine rotavirus A  ,23 were A sequence-verified pET-28b-NSP2 construct  was used2, 200 mM NaCl, and 200 mM HEPES (pH 7). The diluted NSP2 and RNA were mixed in a 1:1 volume. For the no protein condition, 5 mM HEPES was added at equal volume to the RNA. The RNA:NSP2 samples were then incubated at 37°C for 30 min prior to probing. For probing, RNA:protein mix were incubated with 10% volume of 125 mM 5-nitroisatoic-anhydride (5NIA), dissolved in dimethyl sulfoxide (DMSO), to give a final concentration of 12.5 mM 5NIA. For 1M7 (1-methyl-7-nitroisatoic anhydride) treated samples, RNA was treated with a 10% volume of 100 mM 1M7 for a final concentration of 10 mM 1M7. Untreated samples were incubated with 10% DMSO. Both modified and untreated samples were vortexed and then incubated at 37°C for 5 min. RNA was subsequently purified using the Qiagen MinElute columns and eluted in 30 μl of nuclease-free water.For single RNA experiments the single RNA segments were diluted to a final concentration of 260 nM and incubated at 65°C for 5 min then slowly cooled back to room temperature for 30 min before then being kept at 4°C. For transcriptome wide experiments each RV segment was diluted to a concentration of 25 nM, incubated at 65°C for 5 min then slowly cooled back to room temperature. The segments were then pooled, resulting in an equimolar mix of all 11 RV segments. Stock NSP2 was diluted using 5 mM HEPES at pH 7 to concentrations required for the titrations described; 40 μM, 20 μM and 10 μM. The RNA was then buffered in the following conditions: 1 mM MgCl2, and 100 mM NaCl). The diluted RNA was then mixed with an equal volume of either 20 mM NSP2 and samples were incubated at 37°C for 30 min. For DMS probing, 1 M bicine pH 8.0 was added to each sample give a final concentration of 200 mM. Samples were incubated at 37°C for 1 min with 10% DMS in ethanol to give a final DMS concentration of either 0.5% or 0.1% (v/v). Control samples were incubated with an equivalent volume of ethanol. Reactions were quenched by addition of 1 M DTT to a final concentration of 167 mM. RNA was purified using the Zymo RNA Clean and Concentrator 5 kit. Samples were then prepared using our segment specific library preparation protocol as described below.Rotavirus segment 11 RNA was refolded as described above. The RNA was diluted to a final concentration of 250 nM in the RNA folding buffer , supplemented with 2 mM GlutaMAX (Gibco), 100 U/ml of Penicillin-Streptomycin (Gibco), and 10% fetal calf serum (FCS) until confluent. Cells were washed by incubating with FCS free media at 37°C for 30 min. Group A rotavirus (RVA), strain RF (bovine) was activated by incubating with 1.5 μg/ml of trypsin for 30 min at 37°C, as previously described (2 and 2 μl of SuperScript II Reverse Transcriptase (Invitrogen), supplemented with 20 U of murine RNase inhibitor (NEB) to achieve a final volume of 40 μl. The reaction was incubated at 25°C for 10 min, then 42°C for 3 h, then 70°C for 15 min. First strand cDNA was desalted using G50 columns (GE). The desalted product was then introduced into a second strand synthesis reaction. The cDNA second strand was generated using mRNA Non-Directional Second Strand Synthesis module (NEB). Double stranded DNA was then cleaned up using Ampure XP purification beads and eluted in 30 ul of water and quantified using a Qubit dsDNA HS Assay (Invitrogen). Double-stranded DNA was fragmented, repaired, and adaptor ligated for sequencing library preparation using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®. Library quality was assessed using the High Sensitivity Bioanalyzer protocol (Agilent). Libraries were then loaded on an Illumina MiSeq at 10 pM and run at 300 cycles (150 × 2) or 600 cycles (300 × 2).First stand cDNA synthesis of treated RNA was carried out as previously described . Briefly2 per sample. A total of 9 μl of the reverse transcription master mix was added to each sample. The samples were then incubated at room temperature for 2 min before the addition of 1 μl of 200 U/μl SuperScript II Reverse Transcriptase (Invitrogen). Reverse transcription was performed using the following program in a thermocycler: 10 min at 25°C, 90 min at 42°C, 10 cycles of 50°C for 2 min followed by 42°C for 2 min, and a final incubation of 70°C for 10 min. The resulting cDNA was purified using the Zymo DNA Clean and Concentrator 5 kit. A total of 30–50 ng of cDNA was combined with a final concentration of 0.2 mM dNTPs, 0.25 μM of gene segment 11-specific primer  for NSP5, NSP4 and VP6 were extracted from the NCBI virus database (07.27.2021). Pairwise alignments of all sequences of each segment with the respective segment of the bovine RF strain used in our experiments  were carried out using MAFFT with default parameters, and the Levenshtein distance for each pair was calculated ,34. Sequet al. . Multiplet al. . These set al. .2(FoldChange) of RNA structural motifs was done using the python package MatPlotLib.Mutation rate distribution analysis and establishment of linear relationships between NSP2 titration points were analyzed using R studio (version 2021.09.1 + 372) and visualized with the ggplot2 package. Box plots and histograms comparing the login vitro . We also noted that the highly conserved sequence immediately before the start codon of NSP6 encoded by segment 11, had high SHAPE reactivity both in vitro and in cells  with increasing amounts of NSP2 and carried out SHAPE chemical probing  of the mutation rate at 20 and 0 μM NSP2 respectively. As can be seen in Figure 2(FC) >0, consistent with the positive slopes we observed in the scatter plots in Figure 2(FC) for paired nucleotides (stem) compared with unpaired  in this way is that it allows quantitative comparison of nucleotides with differing mutation rates. Thus, our data show that although the effect of NSP2 on mutation rates is uniformly positive, NSP2 has a larger relative effect on the flexibility of stems versus unpaired nucleotides, consistent with its helix-destabilizing activity.To compare nucleotides in the four different structural contexts, we computed the log2Fold hange of 2(FC) analysis can detect any statistical differences at transcriptome scale when all eleven segments are incubated with NSP2. We therefore probed the entire transcriptome in the presence of increasing concentrations of NSP2. As can be seen in Figure R2 for increasing concentrations of NSP2 and report these in Table As we have previously shown, at sub-nanomolar concentrations, rotaviral segments do not appear to interact intermolecularly when NSP2 is absent . Indeed,2(FC) distributions for segments 5, 6, 10 and 11 alone and in the presence of the rest of the viral transcriptome  is systematically higher for paired nucleotides  for paired nucleotides. Remarkably, in the presence of NSP2, paired bases are significantly more reactive towards 5NIA compared to unpaired nucleotides. However, their median reactivity decreases in the presence of the remaining rotaviral transcriptome  should always be considered for careful data interpretation. Furthermore, visualization of the raw mutational data during analysis should always be carried out to reveal any changes in the RNA backbone dynamics that might be normalized out in subsequent data processing steps. Additionally, we carried RNA probing using the established SHAPE reagent 1M7 in the presence and absence of NSP2. Our results with 1M7 reagent confirm the 5NIA data, suggesting that upon addition of NSP2, the RNA backbone of segment 11 transcripts increases its reactivity as quantified by the mutation rate .2(Fold Change) in mutation rate upon addition of NSP2 was more positive for paired nucleotides across the entire RV transcriptome  for paired regions does not reflect any structural preference in binding of NSP2. Rather, our mutational profiling analysis indicates that NSP2 has a larger relative effect on paired nucleotide's flexibility, consistent with its role as an RNA chaperone that promotes RNA-RNA interactions by making sequestered complementary sequences more accessible for inter-molecular base-pairing. It is important to note that the increase in mutation rate indicates a global change in backbone flexibility, but does not change the overall SHAPE pattern. To directly interrogate any base pairing changes mediated by NSP2, we performed an analogous NSP2 titration using dimethyl sulfate (DMS), which methylates unpaired adenines and cytosines, thus directly reporting on the propensity for base-pairing. The NSP2-mediated increase in mutation rate is consistent between DMS- and 5-NIA-treated samples (2(FC) in the presence of NSP2 (Having established that we observe a very significant, global, and NSP2 concentration-dependent increase in mutation rate upon addition of the RNA chaperone, we decided to evaluate whether we observe any structural specificity. Previously, it has been suggested that with small RNAs, NSP2 preferentially binds unpaired nucleotides with sub-nanomolar affinity . Our clae Figure . Our pree Figure , therefo samples . Indeed, of NSP2 , further of NSP2 ,52, thusThe data presented here further support a model in which NSP2 is able to capture folded RNA stem–loop structures within a single RNA-binding groove, resulting in general relaxation of RNA structure without fraying of the RNA stem termini . This alRaw sequencing reads are available on the NCBI SRA database under BioProject ID PRJNA791398. All SHAPE data used in this study are available in gkac738_Supplemental_FilesClick here for additional data file.