The transfer of charges through double helical DNA is a very well investigated bioelectric phenomenon. RNA, on the contrary, has been less studied in this regard. The few available data report on charge transfer through RNA duplex structures mainly composed of homonucleotide sequences. In the light of the RNA world scenarios, it is an interesting question, if charge transfer can be coupled with RNA function. Functional RNAs however, contain versatile structural motifs. Therefore, electron transport also through non‐Watson–Crick base‐paired regions might be required. We here demonstrate distance‐dependent reductive charge transfer through RNA duplexes and through the non‐Watson–Crick base‐paired region of an RNA aptamer. RNA charge transfer: Reductive charge transfer through regular RNA duplexes and through a RNA aptamer is demonstrated. Charge transfer is induced by photochemical activation of 5‐dimethylaminopyreneuridine, incorporated in RNA as an electron donor. Charge transfer proceeds in a distance‐dependent manner and was monitored by fluorescence quenching and dehalogenation of 5‐bromodeoxyuridine, used as an electron trap. The strictly ordered structure of the DNA double helix has proven to be capable of transferring charges in the form of an electron over long distances.For our studies we chose the flavine mononucleotide (FMN) aptamer, which was previously identified by in vitro selection. It is a rather small RNA structure comprising an 11 nucleotide bulge segment as the binding site for FMN,We started our investigation with a series of model duplexes and aptamer structures as shown in Figure i, pH 7) showed a differing impact of the pyrene moiety on the duplex versus aptamer stability .First, we characterized our models regarding the influence of the pyrene moiety on duplex and aptamer stability. It has been shown previously that pyrene modifications attached to the sugar residue differently affect DNA and RNA duplexes,ty Table . The insng Table . This maAnother interesting observation was made through UV/Vis measurements. The pyrene specific absorbance between 330 and 400 nm of the RNA duplex structures showed differently pronounced first maxima at 347 nm . The extent of this first maximum appears to be dependent on the exact location of the electron acceptor and can be related to the achievement of the charge separated state of 5DMAPyU. Such differences in the absorbance pattern were not seen in previous studies with the sugar‐bound pyrene moiety.i, at an excitation wavelength of 350 nm , which is significantly smaller than that determined for a pyrene moiety attached to the sugar backbone of a RNA homoduplex used in previous studies.For the investigation of charge transfer, we first looked at quenching of the pyrene fluorescencem Figure . Duplex n_ds (with n=number of nucleotides that separate donor and acceptor) in relation to the reference Dpy_ds. Fluorescence quenching was 52 % for n=0 (in D0_ds), 38 % for n=2 (in D2_ds), and 19 % for n=3 (in D3_ds)  in the different duplexes Figure . The res) Figure . For theDbr_ds  in Na‐Pi, was irradiated at 365 nm for 40 min, followed by enzymatic digestion and analysis of the resulting nucleoside mixture by RP‐HPLC  was taken as a measure of the 5BrdU content in the individual samples. Guanosine was chosen, because it has the smallest reducibility of the four nucleobases,[D0_ds, D2_ds and D3_ds, followed by enzymatic digestion and RP‐HPLC analysis, reduction of the 5BrdU peak area in relation to the peak area of G is clearly visible, thus indicating debromination of 5BrdU and thereby confirming successful charge transfer. Moreover, as already seen in the fluorescence quenching assay, charge transfer occurs in a distance dependent manner, as Q5BrdU/G is most significantly reduced for D0_ds, followed by D2_ds and D3_ds in relation to Dbr_ds  and 19 % (for A2_ds) in relation to the reference aptamers ApyL_ds and ApyH_ds is clearly visible, indicating successful charge transfer through both aptamer structures.After having successfully shown distant dependent charge transfer through RNA duplexes with 5DMAPyU as electron donor, we were interested in investigating this phenomenon with the FMN aptamer. Looking at the recorded spectra in the fluorescence quenching assay, the fluorescence intensity appears to be strongly dependent on the specific location of 5DMAPyU as electron donor, as seen with aptamer structures s Figure . FluoresA3_ds is significantly higher compared to ApyH_ds, which, with 5DMAPyU in the helical region, is the closest system to serve as standard. The relatively high fluorescence observed for A3_ds implies that excess electron transfer does not occur. This result is confirmed by the results of the dehalogenation assay (see below), and is most likely a consequence of the nucleotide sequence in the neighborhood of 5DMAPyU in A3_ds.The observed fluorescence of A2_ds, charge transfer occurs through an adenosine and uridine, both nucleosides having a medium to high reduction potential, in A3_ds the electron needs to be transferred through an adenosine and two guanosines  in comparison to the Abr_ds reference (Q5BrdU/G=0.09) is observed .Owing to the long irradiation times used in the dehalogenation assay, the extent of 5BrdU degradation is remarkably similar in the duplex and aptamer structures, as can be seen when converting Q5BrdU/G value for A3_ds is the same as for the reference system Abr_ds (Q5BrdU/G=0.09). This implies that, upon irradiation of A3_ds, 5BrdU remains undamaged, which allows to conclude that transfer of an electron from the pyrene modified uridine derivative to 5BrdU did not occur. This is, as discussed above, most likely a consequence of the two guanosine residues between electron donor and acceptor, since charge transfer with donor and acceptor separated by 3 nts, as well as in 5′→3′ direction was clearly shown in the RNA duplexes , making a superexchange mechanism possible, but do not exclude the hopping mechanism completely. The strong FQ effect of the donor moiety and nearly complete degradation of the electron trap in D0_ds make a superexchange mechanism very likely. On the other hand, the distances in the RNA aptamer structure are not clearly to obtain, since only the FMN bound structure is available. The A11 position folds out, to let the FMN ligand into the loop structure. Therefore, the FMN position within the FMN aptamer was used to obtain the distances between the donor and acceptor moiety . The distances for n=0, 2, 3 correspond to a distance of 5.0 Å, 10.4 Å and 11.7 Å in the aptamer region, which would explain the less efficient transfer rate of the excess electron for A0_ds and A2_ds.The here described electron transfer distances in the RNA duplex for Moreover, together with the above described similar degradation of 5BrdU in duplex and aptamer structures , a hopping mechanism is very likely for the aptamer structures, since obtaining a charge separated state in a multi‐step transfer reaction involves several equilibrium reactions with the possibility of a back electron transfer.However, the rather medium reduction potential of the adenosine nucleobase sets a rather high barrier for the hopping mechanism. The here used electron donor 5DMAPyU could play a crucial role, since the charge separated state is reached by a TICT mechanism.A3_ds aptamer, our results clearly show that reductive charge transfer in a distant dependent manner proceeds not only through regular RNA duplex structures, but also through an RNA aptamer. To the best of our knowledge, this is the first example of charge transport through a non‐Watson–Crick base paired RNA region. There is no structure of the FMN aptamer in the absence of the ligand available. However, the NMR structure in the presence of FMN shows that base stacking is a characteristic structural feature of the aptamer 11 nucleotide bulge region.Independent of the results for the The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


UpE and N genes as a panel that combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF). This test panel was designed to improve the diagnostic accuracy through dual-target screening after referencing laboratory testing guidance for MERS-CoV. The limit of detection was 1.2×101 copies/μl viral RNA for the UpE assay and 1.2 copies/μl viral RNA for the N assay, with almost consistent with the sensitivity of the RT-qPCR assays. The two assays exhibited no cross-reactivity with multiple CoVs, including the bat severe acute respiratory syndrome related coronavirus (SARSr-CoV), the bat coronavirus HKU4, and the human coronaviruses 229E, OC43, HKU1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the panel does not require sophisticated equipment and provides rapid detection within 30 min. This panel displays good sensitivity and specificity and may be useful to rapidly detect MERS-CoV early during an outbreak and for disease surveillance.Since its first emergence in 2012, cases of infection with Middle East respiratory syndrome coronavirus (MERS-CoV) have continued to occur. At the end of January 2020, 2519 laboratory confirmed cases with a case-fatality rate of 34.3% have been reported. Approximately 84% of human cases have been reported in the tropical region of Saudi Arabia. The emergence of MERS-CoV has highlighted need for a rapid and accurate assay to triage patients with a suspected infection in a timely manner because of the lack of an approved vaccine or an effective treatment for MERS-CoV to prevent and control potential outbreaks. In this study, we present two rapid and visual nucleic acid assays that target the MERS-CoV  Symptoms of MERS are atypical, including fever, cough, shortness of breath and occasionally pneumonia and gastrointestinal symptoms. As a result, it is difficult to distinguish from other respiratory pathogens based on the clinical symptoms. MERS-CoV is routinely diagnosed using real-time RT-PCR. Real-time RT-PCR has great advantages in sensitivity and specificity compared with other molecular techniques; nevertheless, it is only suitable for well-equipped or central laboratories due to the demand for sophisticated alternating temperature and fluorescent capture instruments. Here, we developed and evaluated the MERS-CoV RT-RPA-VF assay. Compared to the gold standard real-time RT-PCR, the former was less time consuming and had lower equipment requirements. Thus, this panel may be preferred to rapidly detect MERS-CoV in low-resource settings, even as a point-of-care approach for the timely prevention and control of a pandemic. For the N gene, the sense primer N-F (5’-Biotin-CCCTTGAGTCTGGCAAAGTAAAGCAATCGC -3’), reverse primer N-R (5’-AGGAGCAAGCTCAGCAATTTGGGGCCAACGTG-3’) and probe N-P (5’-FITC-AGCAATTTGGGGCCAACGTGGGTCCTCAGT[THF]CCGAGTTTATTCAAT-C3 Spacer-3’) were designed to target a 233 bp section. The probes were labeled with FITC at the 5′ end, THF in the internal region, and a blocking C3-spacer at the 3′ end. The reverse primer for the UpE gene and the forward primer of the N gene were labeled with biotin at the 5′ end.The conserved sequences of the UpE assay and at 35°C for the N assay. Therefore, 33°C and 35°C were deemed the optimal temperatures for the RT-RPA-VF assays to detect the UpE and N genes, respectively.The reaction conditions of the RT-RPA-VF assays were optimized to improve sensitivity. First, 10-fold dilutions of synthesized RNA transcripts were used as a template for the RT-RPA-VF assays, and the reaction process was performed at different temperatures  for 30 min. At the optimal reaction temperatures described above, 10-fold dilutions of the synthesized RNA transcripts were used as the templates to evaluate the amplification efficiency at different times . According to the highest dilution at which 100% of replicates were positive, the optimal reaction time for the two assays was 25 min, as shown in UpE gene described in a previous report to quantify the concentration of the MERS-CoV RNA in a total RNA sample containing 5 × 106 PFU/ml virus in the culture [UpE-RNA transcripts were used to establish the standard curve, 100-fold dilutions of total RNA from virus-infected cell suspensions were used as the test template, and the RNA of uninfected cell suspensions was used as the negative control template. Linear amplification was achieved from 2.6 × 104 to 2.6 × 108 copies/μl, with a calculated linear correlation coefficient (R2) of 0.999 and efficiency value of 100.1%  and a specificity of 100% . The RT-RPA-VF assay for the N gene had a sensitivity of 100%  and a specificity of 100% . When we compared the gold standard with our assay panel, two gold standard RT-qPCR assays detected concentrations as low as 1.2 copies/μl viral RNA, and these results were consistent with previous reports [UpE gene was 1.2 × 101 copies/μl viral RNA, which was lower than RT-qPCR for the UpE gene (UpE gene at 87.5% (kappa = 0.6). The limit of detection of the RT-RPA-VF assay targeting the N gene was 1.2 copies/μl viral RNA, with a sensitivity equivalent to the RT-qPCR assay (N gene and gold standard was 100% (kappa = 1), indicating the two assays displayed high consistency  and highly sensitive , and no sophisticated equipment was needed. Thus, this assay is promising for the detection and active surveillance of MERS-CoV in low-resource areas. Furthermore, this assay can minimize risk of false positives and the requirement to ship specimens. The design of the RT-RPA-VF assay can also be used to develop methods for the rapid detection of other infectious diseases. According to previous reports, fluid samples can be quickly heated directly to release RNA without RNA purification and used in the RT-RPA assay [On the other hand, the two RT-RPA-VF assays targeting the MERS-CoV PA assay ,38. We s


Lipoptena cervi has intensified in Poland in recent years. The population genetics of this ectoparasite in Poland has never been described in the literature. The objectives of this study were to investigate the population genetics of L. cervi in selected regions of Poland, to evaluate molecular differences between L. cervi populations, and to determine phylogenetic relationships with other L. cervi sequences obtained in previous studies. In 2019, louse flies were sampled in natural mixed forests in five Polish voivodeships. Seven samples of L. cervi were collected from each voivodeship, and a total of 35 insects were analyzed molecularly. In the first step, Lipoptena spp. were identified to species level under a stereoscopic microscope. A fragment of the rRNA 16S gene was used as a marker to identify L. cervi by the PCR assay. The sequences were assigned accession numbers MT337409 to MT337416. A total of eight haplotypes were identified, two of which were dominant. In the obtained sequences, intraspecific pairwise genetic distances varied between 0.000 and 0.0496 . Mean interpopulation diversity was d = 0.0135 (SE = 0.0027). The acquired nucleotide sequences were highly similar to the sequences from the Czech Republic , Lithuania (MN889542-MN889544) and Poland (MF541726–MF541729). The similarity with GenBank sequences ranged from 97.24% to 100%. This study revealed two dominant haplotypes of L. cervi in Poland, MT337410 and MT337413. Fragments of the analyzed sequences were detected in only one voivodeship. These findings suggest that the two dominant sequences are the oldest sequences that gave rise to the locally identified haplotypes. The lack of significant correlations with the sequences obtained in regions situated west of the research sites suggests the presence of other genetic populations in Europe.The activity of   •Eight new haplotypes with 0.000–0.0496 intraspecific pairwise genetic distance.•Similarity with sequences from GenBank ranged from 97.24% to 100%.•L.cervi were observed in Central Europe.Two dominant haplotypes of •Part of the sequences have been discovered in single studied location.•The lack of significant connections with sequences obtained from West Europe. Measurements were carried out with the use of Leica Application Suite 4.4 . Deer keds were identified to species level based on body dimensions, wing venation, length and structure of palpi, and the number of erect hairs on the mesonotum .2.3L. cervi samples (n = 35) from each voivodeship were randomly selected for molecular analyses. In the first step, keds were air-dried for 15 min and then individually crushed with a sterile rod in sterile Eppendorf tubes. Genomic DNA was extracted from each specimen with the Sherlock AX universal kit  according to the manufacturer's instructions. DNA was eluted in 40 μL of TE buffer, and the concentration of the extracts was checked using the Nano Drop 2000 spectrophotometer . The extracted DNA was stored at −20 °C until analysis.Seven 2.4Lipoptena spp. with an estimated length of 412 bp was amplified by PCR. The PCR primers designed by L. cervi DNA template, 0.125 μl of Taq DNA Polymerase , and 19.875 μl of double distilled water as a negative control. The PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide and viewed under a UV transilluminator.A fragment of the mitochondrial 16S rRNA gene of 2.5After the PCR reaction, DNA samples were purified by the ethanol precipitation method described by 2.6L. cervi obtained in this study were deposited in the GenBank database of the National Center for Biotechnology Information. The sequences were assigned accession numbers MT337409 to MT337416.The representative sequences of 2.7m), standard deviation (SD) and variation (V), were calculated in the Statistica 13.3 program . Intraspecific pairwise genetic distances, mean interpopulation diversity, mean genetic distances, standard error (SE), mean evolutionary distance, and nucleotide frequencies were calculated in MEGA 10.1.17  were detected in the Greater Poland voivodeship. Four haplotypes were found in the Kuyavia-Pomerania voivodeship . Three haplotypes were acquired in the Lubusz voivodeship . Three haplotypes were identified in the Pomerania voivodeship , and two  – in the Warmia-Masuria voivodeship. The detailed distribution of haplotypes in the examined locations is presented in m = 0.0135; SD = 0.0149; SE = 0.0006; V = 110.11). Mean interpopulation diversity was d = 0.0135 (SE = 0.0027). Mean genetic distances within voivodeships were: d = 0.013 (SE = 0.003) for the Greater Poland voivodeship; d = 0.004 (SE = 0.002) for the Kuyavia-Pomerania voivodeship; d = 0.018 (SE = 0.004) for the Lubusz voivodeship; d = 0.021 (SE = 0.005) for the Pomerania voivodeship; and d = 0.007 (SE = 0.003) for the Warmia-Masuria voivodeship. Mean genetic distances between voivodeships are presented in In the obtained sequences, intraspecific pairwise genetic distances varied between 0.000 and 0.0496 . No similarities were found between the identified sequences and the haplotypes from Denmark and Germany (Eight sequences (MT337409- MT337416) were characterized by 97.24%% to 100% similarity with  Germany . Detaile4L. cervi (MT337410 and MT337413) in Poland. These haplotypes were found in nearly all studied regions. Some sequences were detected in only one voivodeship. These findings suggest that the two dominant sequences are the oldest sequences that gave rise to the locally identified haplotypes. These haplotypes probably spread throughout Poland and evolved independently, by natural selection, into the sequences obtained in this study. This observation is supported by the differences in the analyzed gene. The expansion of louse flies is boundless in the temperate climate ; therefore, the population and diversity of keds could be nearly identical in the studied locations . New local haplotypes are likely to be discovered in the future due to low reproductive rates and habitat limitations resulting from host preferences  across countries. The above could also be attributed to limited panmixia because reproduction occurs only among individuals colonizing the same host. The presence of stable L. cervi populations in some areas probably induced genetic selection that was influenced by environmental factors and the availability of host species. The above suggests that L. cervi easily adapt to new ecosystems. Very close similarities were observed between the studied sequences of L. cervi and the sequences from the Czech Republic, Lithuania and Poland deposited in GenBank  gene of L. cervi in Poland. An analysis of the correlations between COX1 and 16S rRNA gene sequences could also deliver new insights.Molecular differences have been observed between nd ticks . These sL. cervi is an underestimated vector of veterinary importance. This parasite is not fully understood from the epidemiological point of view. The potential flow of pathogens across regions could be predicted be examining the results of population genetics studies and the transfer of infectious agents by selected haplotypes. A thorough knowledge of the genetic profile of L. cervi combined with pathogen detection would support the identification of potential high-risk areas. Further research is needed due to the steady increase in L. cervi population. The presence of L. cervi on non-specific hosts or in non-specific locations (such as cities) could also indicate that the species rapidly assimilates to new environmental conditions. The above could be correlated with specific haplotypes that better adapt to non-specific ecosystems. Migratory processes could contribute to the spread of new pathogens to previously uncolonized areas, including human settlements. Previous studies of Hippoboscidae confirmed that pathogens could be horizontally transmitted. Bacteria of the genera Bartonella spp., Borrelia spp. and Coxiella-like bacteria have been identified in Lipoptena spp., but the ectoparasite's ability to transmit pathogens has been confirmed only for Bartonella spp.  in the search of a host, which is why an increase in the density of the wild ungulate population can also reduce mortality associated with the lack of hosts. Cervid migrations play a significant role in the spread of L. cervi. This ectoparasite can survive for more than a year on the host (L. fortisetosa has been identified in Central Europe (L. cervi and L. fortisetosa, and to explore potential hybridization between these two closely related species. Due to the apparent physiological similarities and similar activity patterns of L. cervi and L. fortisetosa (The population of the host ; therefol Europe . In the tisetosa  incorrec5L. cervi population in selected Polish voivodeships. The studied regions were colonized by two dominant haplotypes of L. cervi that have been identified in other Central European countries, and local haplotypes were detected in individual cases. The lack of significant correlations with the sequences obtained in regions situated west of the research sites suggests the presence of other genetic populations in Europe. In the future, the population genetics of L. cervi should be investigated in greater detail to contribute to the development of preventive programs aiming to reduce ectoparasitic populations in the environment and minimize the negative impact of L. cervi on wild animals, livestock and humans.The present study reveal genetic variation in the 16S rRNA gene of the 10.13039/501100004569Minister of Science and Higher Education under the program entitled "Regional initiative of Excellence" for the years 2019–2022, Project No. 010/RID/2018/19, amount of funding PLN 12,000,000. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Project financially co-supported by the The authors declare that they have no conflict of interest.


The discovery that transcription is pervasive with the vast majority of the genome encoding transcripts not translated into proteins, has transformed our understanding of the basic unit of genetic information  are powerful tools for gene expression profiling and can sequence large numbers of DNA fragments in parallel producing millions of short reads in a single run Metzker, . These mHoldt et al. report on the latest findings on circRNA in CVDs, the potential therapeutic approaches based on either modulation of native circRNAs by therapeutic knockdown or by ectopic expression and the prospect of engineering non-native  circRNAs. The major hurdles for therapeutic strategies targeting circRNA in terms of design, delivery, and side effects are considered.Bioinformatic analysis of RNA-seq data also revealed a novel class of lncRNA transcripts, the circular RNAs (circRNAs) that emerge by RNA “backsplicing,” whereby the spliceosome fuses a splice donor site in a downstream exon to a splice acceptor site in an upstream exon . Their interaction with RNA binding proteins (RBPs) is thought to be crucial for very diverse cellular functions  and lncRNAs and the large body of evidence demonstrating their pleiotropic effects in pathological processes in vascular diseases. The potential of ncRNAs as effectors and biomarkers in vascular pathology is critically evaluated and insights into the technical limitations in establishing a standard protocol to ensure robust reproducibility for circulating ncRNAs as biomarkers in vascular diseases are provided.Apart from diabetic vasculopathy, ncRNAs have a profound effect in the vasculature at baseline and in disease. Hobuß et al. summarize the function of lncRNAs in the development and progression of cardiac diseases with a particular emphasis on their molecular mode of action in pathological tissue remodeling. They also examine the challenges that have to be overcome to establish lncRNA based therapies and effective intervention strategies in the heart. In addition, the prognostic and diagnostic value of lncRNAs in biological fluids as a novel class of circulating biomarkers for heart diseases and the prospect of using these molecular fingerprints to replace protein-based indicators of disease is discussed.In the heart, an increasing number of studies highlight the critical regulation of lncRNAs in cardiac disorders. Laina et al. highlight the two main designs to target RNA and modulate gene expression the double-stranded small interfering RNAs (siRNAs) and single stranded antisense oligonucleotides (ASOs) their advantages and limitations. The review also summarizes results from the clinical trials of RNA-targeting interventions and elaborates on the advances and hurdles for RNA based therapeutic applications. The future prospect of RNA therapeutics to empower precision medicine implementation and fulfill the promise of patient specific therapeutics is also evaluated.Extending beyond ncRNA, RNA therapeutics focus on RNA as a prime target for therapeutic applications. in vivo delivery of targeting agents are required to bring RNA based therapeutics closer to the clinic.In conclusion, this Research Topic elucidates the current understanding about the mechanisms of function of ncRNA and its role in CVDs and highlights the major advancements and promising developments in RNA therapeutics. Despite the significant progress this is a new field of research and several challenges remain. Better understanding of the mechanisms of function of ncRNAs and integration of innovative approaches to enhance target binding affinity, cellular uptake and efficient AZ and LM wrote and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As a major human pathogen, RSV affects 3.4 million children worldwide annually. However, no effective antivirals or vaccines are available. An in-depth mechanistic understanding of the RSV RNA synthesis machinery remains a high priority among the NNS RNA viruses. There is a strong public health need for research on this virus, due to major fundamental gaps in our understanding of NNS RNA virus replication. As the key enzyme executing transcription and replication of the virus, the RSV RdRP is a logical target for novel antiviral drugs. Therefore, exploring the primer-dependent RNA elongation extends our mechanistic understanding of the RSV RNA synthesis. Further fine mapping of the promoter sequence paves the way to better understand the function and structure of the RSV polymerase. de novo and primer-based RNA synthesis. We defined the minimal length of the RNA template for in vitro de novo RNA synthesis using the purified RSV polymerase as 8 nucleotides (nt), shorter than previously reported. We showed that the RSV polymerase catalyzed primer-dependent RNA elongation with different lengths of primers on both short (10-nt) and long (25-nt) RNA templates. We compared the sequence specificity of different viral promoters and identified positions 3, 5, and 8 of the promoter sequence as essential to the in vitro RSV polymerase activity, consistent with the results previously mapped with the in vivo minigenome assay. Overall, these findings agree well with those of previous biochemical studies and extend our understanding of the promoter sequence and the mechanism of RSV RNA synthesis.Respiratory syncytial virus (RSV) is a nonsegmented negative-sense (NNS) RNA virus and shares a similar RNA synthesis strategy with other members of NNS RNA viruses, such as measles, rabies virus, and Ebola virus. RSV RNA synthesis is catalyzed by a multifunctional RNA-dependent RNA polymerase (RdRP), which is composed of a large (L) protein that catalyzes three distinct enzymatic functions and an essential coenzyme phosphoprotein (P). Here, we successfully prepared highly pure, full-length, wild-type and mutant RSV polymerase (L-P) complexes. We demonstrated that the RSV polymerase could carry out both IMPORTANCE As a major human pathogen, RSV affects 3.4 million children worldwide annually. However, no effective antivirals or vaccines are available. An in-depth mechanistic understanding of the RSV RNA synthesis machinery remains a high priority among the NNS RNA viruses. There is a strong public health need for research on this virus, due to major fundamental gaps in our understanding of NNS RNA virus replication. As the key enzyme executing transcription and replication of the virus, the RSV RdRP is a logical target for novel antiviral drugs. Therefore, exploring the primer-dependent RNA elongation extends our mechanistic understanding of the RSV RNA synthesis. Further fine mapping of the promoter sequence paves the way to better understand the function and structure of the RSV polymerase. Respiratory syncytial virus (RSV) infection is a leading cause of severe lower track respiratory diseases in young children, older adults, and immunocompromised individuals in the United States and worldwide , 2. Curr3–7–12–The RSV genome contains 10 genes that encode 11 proteins . The RSVin vitro assays with recombinant polymerase have provided insights regarding NNS RNA synthesis (–in vivo cell-based minigenome assays (de novo RNA synthesis at positions 1 and 3 (in vitro characterization of RNA synthesis of vesicular stomatitis virus (VSV) and RABV polymerases (Rhabdoviruses (in vitro assay for EBOV and Nipah virus (NiV) polymerases  complex and its catalytic mutant, which allowed us to perform in-depth structural and biochemical analyses. We integrated and adapted the RNA polymerization assays from protocols developed for RSV and other NNS RNA viruses –40. We u–reported , 34, 38.me assay . This wome assay –42.2+-nitrilotriacetic acid (NTA), removed the 6×His tag through TEV protease cleavage, and then ran the sample through a heparin column, followed by size exclusion chromatography. We used this procedure to prepare mostly pure wild-type (wt) L-P complex in good quantity. Previous studies reported that HSP70 or HSC70 usually comigrates with L-P complex  to determine the size distribution of the L-P complex. Histogram analysis revealed that the purified L-P complex had a relatively narrow peak with a measured hydrodynamic radius (Rh) of 11.27 ± 0.17 nm (98.5%), suggesting a homogeneous conformational distribution of L-P . The RSVin vitro RSV RNA synthesis, we adapted the protocols developed for RSV and other NNS RNA viruses  , lane 1.template . As expetemplate , lane 2.template . We invetemplate , lane 3.Further, we checked several RNA templates with varied lengths and sequences. The RSV polymerase also synthesized RNA for longer (TrC50) and shorter (TrC21 and TrC14) RNA templates, as shown in abortive products , 52, 53.32P]GTP and [α-32P]ATP to compare the polymerase activity .Having shown that the RSV polymerase was active, we characterized the template length requirements for RSV RNA synthesis. First, to further investigate the specificity of the promoter sequence, we examined the polymerase activities with the addition or deletion of the first nucleotide(s) at the 3′ terminus of the promoter sequence. Previous studies indicated that the first nucleotides of the promoter sequence are critical for the polymerase to initiate RNA synthesis , 31. We activity , lane 3 32P]ATP , and it seemed that [α-32P]GTP gave stronger signals. This may be due to the specific sequences to be added to the nascent RNA products. For simplicity, we focused on TrC10 as the minimum template for effective de novo initiation and used [α-32P]GTP as the labeling nucleotide for further testing.Next, we examined the minimum length requirement of the RNA templates for RNA synthesis by the RSV polymerase. Based on our results, we knew that TrC14 could serve as the template. We further trimmed the size of the RNA and tested whether TrC13, TrC12, or shorter templates represented the minimal lengths. The results showed that TrC7 could not generate RNA products , lane 1 The RNA synthesis products of each template revealed a specific pattern, showing an accumulation of products predominantly around positions 8, 9, and 10 when the templates were copied. To further characterize the RNA template specificity, we compared the activities of the RSV polymerase using Le19 from RSV, RABV, VSV, NiV, and EBOV, as well as TrC19 from RSV . As expeIt is interesting to note that several longer RNA oligonucleotides but not shorter RNA oligonucleotides were generated using NiV-Le19 and RABV-Le19. When we checked the potential self-dimer based on the sequence secondary structure prediction, both RNA oligonucleotides formed a partially overlapped duplex. It is possible that the longer RNA bands generated represented extension of the RNA templates using a back-priming mechanism. Together, although a uridine-rich sequence is commonly found in the 3′ end of those NNS RNA viruses, our results suggest that the specificity of the RNA template resides in the specific promoter sequence.32P]ATP and ATP only for TrC25. We preannealed the primer with TrC25 and then incubated it with the RSV polymerase. As expected, TrC25 itself did not generate any product (Previous studies reported primer-dependent elongation on a short (11-nt) mutant RNA template , 54, 55. product , lane 1, product . It is a product , 38. One product , 38. The32P]GTP and NTPs. Interestingly, as shown in de novo RNA synthesis, the RSV polymerase could catalyze primer-dependent template elongation.To further examine whether the RSV polymerase uses other primed RNAs as a template, we compared other terminal sequences of the RSV genome, such as Tr25, Le14, and LeC14, and supplied them with a short (5-nt) primer using GTP and NTPs for the assay. The generated products are shown in 32P]GTP, elevating our measured activity level. Interestingly, the U-to-C mutation at position 8 had the lowest (10%) activity. This result highlights the critical position of the promoter. Compared to positions 1 to 5, there was no enhancement of the polymerase activity, compared to the wt activity, suggesting those promoter sequences might be conserved through evolution.To map the essential promoter sequence, we performed additional single mutations at positions 6, 7, and 8 based on TrC10 . In VSV and RABV, the L proteins can be isolated independently from the P proteins , 48. In ectively . The difectively , 40, 53.ectively , and we ectively , we founectively . Regardiin vitro reconstitution of de novo NNS RNA synthesis was successfully studied in RSV and several other systems  but RSV L initiates de novo RNA synthesis at both the 3′ end (+1) and internal (+3) positions  and primer dependent . Like the primer-dependent elongation in EBOV and NiV  to pair with the 5′ end of the RNA templates to examine the RNA elongation. Then we added the incoming nucleotide to the preannealed primer-template for RNA synthesis. This study used longer wt templates (25 nt and 14 nt), compared to the shorter mutant template (11 nt) reported previously  was used to transfect the bacmid DNA into Sf21 cells to obtain the recombinant baculoviruses. Sf21 cells were infected by the recombinant baculoviruses in suspension culture and harvested 72 h postinfection by centrifugation for 15 min at 1,000 × g. Cells were resuspended in lysis buffer , lysed with an homogenizer, and clarified through centrifugation for 60 min at 16,000 × g. The clarified lysate was incubated with Co2+-NTA agarose resin (GoldBio) and washed with wash buffer , and the RSV L-P complexes were eluted with elution buffer . The eluted sample was then treated with TEV enzyme and applied to Co2+-NTA agarose resin again. The flowthrough sample was applied to a heparin column and further purified by size exclusion chromatography with gel filtration buffer [25 mM HEPES [pH 7.4], 300 mM NaCl, 6 mM MgSO4, 0.5 mM tris(2-carboxyethyl)phosphine hydrochloride [TCEP]] using a Superose 6 Increase 10/300 GL column . SDS-PAGE analyzed the quality of purified proteins. The bands migrating at ∼250 kDa and ∼35 kDa were confirmed to be the RSV L and P polypeptides by liquid chromatography-mass spectrometry (The helper plasmids of codon-optimized sequences of the RSV (strain A2) L and P proteins were provided as a generous gift from Martin Moore (Emory). The L and P genes were subcloned into the pFastBac Dual vector (Invitrogen) with the RSV L gene at open reading frame 1 (ORF1) and the RSV P gene at ORF2. A 6×His tag was added to the N terminus of the RSV L protein, separated by a TEV protease cleavage site. The RSV L(D811A)-P mutant was generated using PCR-based site-directed mutagenesis, with the plasmid encoding the wt RSV L-P complex as the template. Then, the recombinant pFastBac Dual vector was transformed into trometry . The pur32P]GTP, [α-32P]ATP, [α-32P]UTP, and [γ-32P]ATP were purchased from Perkin Elmer. The reaction mixtures contained 2 μM RNA template (without or with 2 μM primer), the RSV L-P complexes (∼300 ng RSV L), NTPs  (figure legends indicate the details for each reaction mixture), and reaction buffer  in a final volume of 20 μl. The reaction mixtures were incubated at 30°C for 3 h and heated to 90°C for 3 min, and then 5 μl of the stop buffer  was added to each reaction mixture. Other radioactive isotope-labeled nucleotides  were incubated similarly as [α-32P]GTP (figure legends indicate the details for each reaction mixture). The isotope-labeled nucleotides with the same concentration were freshly purchased and used for the reactions. For clarity, we directly compared only the reaction mixtures containing the same radioactive isotope-labeled NTPs. The RNA products were analyzed by electrophoresis on a 20% polyacrylamide gel containing 7 M urea in a Tris-borate-EDTA buffer, followed by phosphorimaging with a Typhoon FLA 7000 scanner . The quantification of the images was carried out with an analysis toolbox from ImageQuant TL software . We analyzed the images using area- and profile-based tools and selected the corresponding area of each lane with a box for calculation by the software. The molecular weight ladders were generated by labeling Tr7, Tr14, Tr21, and Tr25 with [γ-32P]ATP using polynucleotide kinase and following the protocols according to the manufacturer (NEB).The terminal sequences of the genome or antigenome, such as the Le and TrC promoter sequences, were used in the RNA synthesis assay. All RNA oligonucleotides were chemically synthesized by IDT or Dharmacon. Radioactive isotope-labeled nucleotides [α-DLS experiments were performed on a DynaPro plate reader II (Wyatt Technologies). Measurements of the RSV polymerase (L-P complex) samples (1 mg/ml) were obtained in gel filtration buffer at 25°C and analyzed using Dynamics software (Wyatt).−/Å2.Samples were prepared on continuous carbon films supported by 400-mesh copper grids (Ted Pella). A 3-μl drop of the RSV L-P was applied to a freshly glow-discharged grid, blotted to a thin film with filter paper, and immediately stained with 1% (wt/vol) uranyl formate. EM was performed using an FEI Talos L120C electron microscope, operating at 120 keV, equipped with an FEI Ceta 4,000 × 4,000-pixel charge-coupled device (CCD) camera. Images were collected at nominal magnifications of ×73,000 (1.97 Å/pixel). The images were acquired at defocus values of −1.2 to −2.0 μm and electron doses of ∼25 e


Most who study virus–host interactions are familiar with 5′-triphosphate (5′-PPP) RNA, a well-established pathogen associated molecular pattern (PAMP). 5′-PPP RNA activates the pattern recognition receptor (PRR) RIG-I leading to production of the antiviral type I interferons ,2. Less DUSPs are a subfamily of protein tyrosine phosphatases (PTPs) that contain a highly conserved phosphatase catalytic domain (HCXXGXXR), which generally enables dephosphorylation of both phospho-serine/threonine and phospho-tyrosine . DUSP11 Transcriptome-wide analyses reveal that a subset of RNAP III transcripts , Alu short interspersed nuclear element (SINE), and Y RNAs) are not only more likely to be triphosphorylated but are also more abundant in cells lacking DUSP11 ,17. ThisInduction of the cellular pool of 5′-PPP RNA accessible to RIG-I can be triggered by various biological stimuli and mechanisms and can increase the propensity of a cell to undergo an antiviral response. Herpes simplex virus-1 (HSV-1) infection depletes the levels of RNA binding proteins (RBPs) (thiosulfate sulfurtransferase (TST), mitochondrial ribosomal protein L18 (MRPL18)) that typically shield pseudogene 5S rRNA (RNA5SP141). This results in activation of RIG-I . Infecti5′-PPP RNAs are a molecular hallmark of RNA virus replication that are produced during the course of infection via viral RNA-dependent RNA polymerases (RdRPs) . Cells aSeparately, some viral 5′-PPP RNAs have a role in mammalian RNA gene silencing. Select noncanonical viral precursor microRNAs (pre-miRNAs) transcribed by RNAP III utilize DUSP11 phosphatase activity to promote RNA silencing activity . DUSP11 Although DUSP11 is beneficial to some viruses, DUSP11 activity is not always pro-viral. For viruses such as the hepatitis C virus (HCV) that are sensitive to 5′-3′ exoribonuclease (XRN)-mediated restriction, the triphosphatase activity of DUSP11 can be antiviral. Mechanistically, DUSP11 converts the 5′-PPP HCV RNA to 5′-P and renders it susceptible to the monophosphate-specific XRN exonuclease ,14. In aAlthough multiple groups have contributed to the understanding of triphosphate RNA balance and the role of DUSP11 –17 key qThe bulk of DUSP11 protein is nuclear. Yet, a small fraction of DUSP11 is cytosolic in all situations that have so far been examined ,13,17. TAnother open question regarding DUSP11 is the possibility that it has substrates other than RNA. DUSP11 shares a common ancestor with a family of protein phosphatases . RecentlThe chronic low-level interferon response of DUSP11-deficient mice presents an opportunity to explore how such a state alters the antiviral response and the plausibility of inducing or preventing such a state for therapeutic benefit. Such therapies could involve reducing DUSP11 or the activity of other RNA phosphatases for defense against pathogens and cancers. Alternatively, enhancing RNA phosphatase activities may ameliorate inflammatory disease. To pursue these ideas, it would be beneficial to define all the relevant players involved in triphosphate RNA balance and develop a deeper understanding of the mechanisms by which they function. The emerging understanding of triphosphate RNA balance and its relevance to the host response to infection promise a fruitful near future, replete with new insights into controlling infection and the innate immune response.


A′ retains the Watson–Crick base‐pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4‐thiouridine (4sU) into C, the combination of both modified nucleosides in dual‐labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC‐seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA‐lifetime evaluation with unprecedented precision.Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH Novel purine‐nucleoside conversion chemistry paves the way for mRNA‐lifetime evaluation with unprecedented precision. The technique, termed TUC‐seq DUAL, uses two modified nucleosides, 6‐thioguanosine (6sG) and 4‐thiouridine (4sU), in subsequent pulses for metabolic labeling. Chemical treatment induces G‐to‐A and U‐to‐C mutations that are simultaneously analyzed upon RNA sequencing. Thus, synthesis and decay of mRNA can be clearly distinguished. These two pyrimidine derivatives give rise to apparent U‐to‐C mutations in RNA‐seq analysis although one has to keep in mind that downstream processing can be complicated or even impaired by the modification. All three 4sU‐based RNA‐seq methods have been validated independently by meaningful applications, including detection of endogenous 4sU in tRNAs,N6‐methyladenosine,RNA maintains many important functions in the cell with most of the RNA‐involving processes being tightly regulated. Whole transcriptome sequencing (RNA‐sequencing) reveals the presence and quantities of RNAs at a given time.These new developments have demonstrated the power of chemical nucleoside conversion to study cellular RNA dynamics upon RNA‐seq.Under these prospects, we set out to develop a concept for dual metabolic labeling and appropriate conversion chemistry for RNA‐seq applications. We envisaged purine nucleoside transformations with the aim to generate apparent G‐to‐A mutations Figure . Such an4/NH4Cl at pH 8.9 for 2 hours at 40 °C . Similarly, 2,2,2‐trifluoroethylamine (TFEA) used previously in TimeLapse‐seq experimentations as the nucleophile,We selected 6‐thioguanosine (6sG) as a potential convertible nucleoside in RNA. Like 4sU, 6sG is amenable to metabolic labeling.3− moiety into 6‐hydrazino‐2‐aminopurine (A′). Importantly, this two‐step procedure was mild enough to leave the RNA intact  at 40 °C for 2 hours, we observed quantitative substitution of the SOEncouraged by these findings, we thoroughly characterized the novel A′ modification. As expected, the thermodynamic stability of an A′–U base pair was comparable to native A–U base pairs in short RNA oligonucleotides Figure . FurtherNext, we tested the impact of the modifications on reverse transcription in a primer extension assay using Superscript III reverse transcriptase Figure  C. AlthoEscherichia coli tRNAPhe) both containing a single 6sG . Detection of G‐to‐A conversion was performed by reverse‐transcription PCR (RT‐PCR) followed by subcloning and Sanger sequencing. Indeed, for both RNAs, more than 95 % of the original 6sG nucleosides were decoded as A . Notably, when the same RNAs were treated with OsO4/NH4Cl only, the majority of 6sG sites were detected as G , which can be explained by biased PCR amplification of unconverted 6sG‐containing molecules due to transcription termination of 6soG‐containing RNAs, as shown in Figure Next, we examined if 6sG is also cleanly converted to A′ under our reaction conditions when it resides in a longer and structured RNA. To this end, we tested a 47 nt RNA oligonucleotide as well as a synthetically generated 76 nt tRNA . We found that 25 and 50 μm 6sG in the labeling medium caused only a mild reduction in cell numbers at 72 h after labeling for 30 min and slightly stronger inhibition of proliferation when the labeling duration was 60 min. By contrast, 100 μm 6sG had a more pronounced negative effect even at the shorter labeling time . Analysis of mRNA expression patterns of selected cell‐cycle‐related genes after a 60 min labeling period with 100 μm 6sG, however, did not reveal perturbed transcript levels .We then investigated the suitability of 6sG for metabolic labeling. Although cells can take‐up 6sG and incorporate it into RNA and DNA,m 6sG, mRNA was isolated, digested to mononucleosides, and analyzed by RP‐HPLC along with nucleoside standards . We found a clear peak of 6sG in mRNA samples  that was absent in mRNA from unlabeled cells  indicating successful incorporation. Finally, we examined whether OsO4‐hydrazine conversion treatment affects the integrity of cellular mRNA preparations. We observed only moderate RNA degradation in converted samples in comparison to untreated mRNA samples . Altogether, the results suggest that 6sG is a suitable nucleoside for metabolic labeling experiments provided low concentrations and short labeling times are used.Because 6sG has reduced base‐pairing strength with cytidine  and might therefore be less frequently incorporated during transcription in the cell, we next determined whether it is detectable in cellular RNA. Cells were labeled for 2 h with 100 μ4‐hydrazine chemistry using the endogenous mRNAs cyclin E1 (CcnE1) and p21, which we had previously used in our TUC‐seq experiments with 4sU, as examples. HEK293T cells were exposed to 100 μm 6sG for two hours before the mRNA was isolated, subjected to OsO4‐hydrazine treatment, and subsequently processed by cDNA synthesis and deep sequencing of CcnE1 and p21 PCR amplicons. As controls, we sequenced OsO4‐hydrazine‐treated mRNA from unlabeled cells as well as untreated mRNA from cells that were 6sG labeled . Importantly, very low mutation frequencies of A, C, and U nucleotides with no differences between test and control samples were detected  transcripts containing 6sG that were synthesized during the initial 6sG pulse, ii) transcripts containing 6sG and 4sU transcribed in the early phase after 6sG wash‐out and 4sU addition, iii) transcripts containing only 4sU that were generated during later time points of 4sU incubation, and iv) unlabeled transcripts corresponding to the preexisting pool.To test this, we performed a two‐step labeling experiment, in which we first incubated the cells with 100 μIndeed, bioinformatic analysis revealed that all four expected transcript types could be detected simultaneously based on increased G‐to‐A and U‐to‐C mutation frequencies Figure  B. We al4/NH4Cl/hydrazine conversion chemistry is fully compatible with the conversion of the frequently applied metabolic label 4‐thiouridine (4sU) into C (TUC‐seq),In this study, we have developed the selective and quantitative conversion of 6sG‐ into A′‐containing RNA, where A′ constitutes a 6‐hydrazino purine derivative that is cleanly decoded as adenosine in RNA sequencing experiments. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the new OsOR.M., A.L., C.G., I.D. have applied for intellectual property rights for this work.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Humans adjust their behavioral strategies to maximize rewards. However, in the laboratory, human decisional biases exist and persist in two alternative tasks, even when this behavior leads to a loss in utilities. Such biases constitute the tendency to choose one action over others and emerge from a combination of external and internal factors that are specific for each individual. Here, we explored the idea that internally-mediated decisional biases should stably occur and, hence, be reflected across multiple behavioral tasks. Our experimental results confirm this notion and illustrate how participants exhibited similar choice biases across days and tasks. Moreover, we show how side-choice behavior in a two alternative choice task served to identify participants, suggesting that individual traits could underlie these choice biases. The tasks and analytic tools developed for this study should become instrumental in exploring the interaction between internal and external factors that contribute to decisional biases. They could also serve to detect psychopathologies that involve aberrant levels of choice variability. The number of participants used for each task's analysis is illustrated inside parentheses in the corresponding figure panels.We used t-tests, parametric and non-parametric tests for statistical comparisons, and repeated measures ANOVA (RM-ANOVA) tests with Bonferroni’s or Wilcoxon Signed Rank edictors . With thst visual choice (2AFC), participants had to identify which of the two stimuli was identical to a target stimulus (SD), by responding with a joystick down-left or down-right. We recorded the choices and response times  of the participants by connecting the joystick to a standard computer. Feedback for the visual task involved waiting longer times after incorrect choices (3000 ms) than after correct ones , participants responded either up-left or up-right to advance to the next trial, which involved visualizing the target stimulus for another 750 ms, regardless of the chosen side , and side-choice biases  during consecutive phases within each trial (see sen side .k = 0) or zero  discriminability conditions. We found similar  visual performance across two consecutive days for both groups of participants , and they were ~ 23% smaller in high discriminability conditions . To visualize the side-choices of the participants (2nd choice from task#1), we employed colormaps to illustrate left and right choices with white or black rectangles, respectively (P(Bias) = P(Bias|left) + (P(Bias|right)), or in alternation (P(Alternation); see k = 0; Prob. Bias | Repeated Measures [RM] ANOVA test, F = 0.49, P = 0.87; Prob. Alternation | Repeated Measures [RM] ANOVA test, F = 1.13, P = 0.29; k = 1; Prob. Bias | Repeated Measures [RM] ANOVA test, F = 0.13, P = 0.96; Prob. Alternation | Repeated Measures [RM] ANOVA test, F = 0.22, P = 0.92, k = 0, P = 0.13; k = 1, P = 0.10; Prob. Alternation | k = 0, P = 0.35; k = 1, P = 0.08). As a reference, we calculated the probability distributions for a random binomial process  increased it's overall difference relative to P(rnd) when using zero discriminability. These results illustrate how human side-choice behavior is stable over time. Intra-individual  stability in free side-choice behavior was present in both groups irrespectively of the discriminability conditions.Next, we explored the stability of visual and side-choice behavior along consecutive days using either high (ectively . We thenk = 1) followed by a second orientation discrimination task .We next wondered whether participants’ side-choices would manifest similarly across different tasks. For that, in a new group of naïve participants, we conducted the free side-choice task  with their side-choice behavior extracted using the original free side-choice task (task #1). In ). In particular, predictors 37–42 from task #2, which directly quantified sensory and decisional biases, had strong and significant interactions with practically all response variables from task #1. This reveals a direct link between the decisional and sensory biases measured in task #2 and the side-choice bias and alternation probabilities extracted from task #1. We illustrate four relevant sample pair-wise linear regressions in We then applied a multivariable linear regression model (MLRM) to predict the response variables from task #1 . The MLRFig 2Hk = 0.8 to k = 1. We also changed the 2nd side-choice phase to a negatively reinforced schedule to favor particular sides during training , or all-left (L), or all-right (R) sequences, respectively. We repeated each of these blocks four times so that all participants experienced all combinations of possible transitions across conditions , with performance dropping when k → 1  by calculating the probability that the side-choice (2nd choice) involved repeating the same side as with the visual choice (1st choice). Interestingly, in these conditions, we found no apparent sequential effects between choices , with the observed side-choices following a random binomial process  and reward (r) rates, we fitted an optimal Bayesian Learner (BL) model [r depends on v, and will change from trial to trial. For example, if a current trial is preceded by many trials with incorrect responses, then the model will estimate a high v, which will increase the change in r. In contrast, lower v values will lead to more stable r probabilities. Thus, when r is stable, then v will be small, which in turn implies that r will not get much influence by previous trials. Volatilities were similar with left and right reinforcements , but they were ~35% higher with randomized reinforcement . Likewise, the estimated reward rates from the BL were similar with left and right reinforcement , but were ~43% smaller with randomized reinforcement . Interestingly, participants with higher volatility exhibited a reduced performance (area under the curve) during the single side-reinforced , but not during the randomized-side reinforced blocks .To estimate the volatility (L) model  to the si.e., Shannon) and transition  entropies [As secondary metrics to detect and quantify overall choice biases, we included the stationary  and 'final' predictors extracted from the entire 60 trials/block . We then calculated the cumulative match characteristic curves by using increasing epoch durations  | five best predictors (increasing strength): 22, 12, 13, 3, 1; five worst predictors (increasing strength): 24, 17, 4, 7, 11; P(Alternation) | five best predictors: 3, 7, 18, 12, 24; five worst predictors: 20, 13, 19, 1, 11; these numeric IDs correspond to predictor numbers displayed in Because the MLRM from the previous section revealed that behavioral metrics extracted from multiple tasks strongly co-varied, we explored the possibility of using the predictors to identify the participants. We implemented a simple linear classifier that minimized the Euclidean distance between observed (lculated ). As expnd free side-choice phase) tended to be more similar across participants under high discriminability conditions (task #1). This was reflected as a strong group choice coherence and could be explained by at least two factors. The first one is that all participants were visually tested with precisely the same sequence of pseudo-randomized stimuli. The second one is that side-choices in task #1 exhibit strong sequential effects with respect to choices produced during the discriminative phase [We used three tasks aimed to explore perceptual and internally-guided choice biases . The tasve phase . In agreOne of our main results is that behavioral attributes extracted from the participants solving the different tasks showed a strong intra-individual consistency that could not be explained by chance , 34–36. In humans, evidence suggests that individual differences influence learning about the predictive value of multiple cues , and difBy using reversing contingencies, we found that side and alternating choice sequences could be effectively reinforced. The premise here was that participants with more flexible choices had greater choice variability (lower bias) whereas those with less flexible choices had lower variance (higher bias)i.e., action volatility), and reward estimations were beneficial to subjects to discount information with passing trials. Usually, volatility refers to blocks of trials in which the optimal solution is continually changing . Volatility in positive reinforcement studies has been widely explored . Some sdelayed; . At the delayed; , 21, 52.delayed; . There idelayed; , and givdelayed; ), a sterdelayed; , a propodelayed; , Tourettdelayed; , 58. Othdelayed; . Thus, iTo summarize: we showed that human decisional biases were similar across tasks and days. Although choice stereotypy probably emerges under a variety of environmental conditions, we here described how this behavior could be precisely characterized in the laboratory using quantitative metrics. Finally, we showed how side-choice biases could be used as a ‘behavioral signature’ to identify participants.


Vegetable amaranth is a source of natural phytopigments and functional components of the commercial food industry for sustainable health benefits across the globe. It is guessed that recently identified amaranth (drought-tolerant) genotypes may contain ample phytopigments and phytochemicals suitable to extract juice as drinks. Hence, phytopigments and phytochemicals content of amaranth were assessed in detail for suitability as drinks to feed the phytochemicals deficient community across the globe.The selected amaranth contained adequate carbohydrates, protein, moisture, and dietary fiber, phytopigments, minerals, phytochemicals including the ability to scavenge radicals. Nine flavonoids compounds were estimated in amaranth genotypes including six flavonols, one flavanol, one flavone, and one flavanone. It is the first effort in which we identified one flavonol such as myricetin, one flavanol, such as catechin, one flavone i. e., apigenin, and one flavanone, like naringenin in drought-tolerant vegetable amaranth. Across six flavonols, quercetin and rutin were the most noteworthy compounds followed by myricetin and isoquercetin. Across the accessions, AT7 and AT15 had abundant phytochemicals, and radical quenching ability including considerable proximate, nutraceuticals, and phytopigments in comparison to the accessions AT3 and AT11. AT15 demonstrated the maximum total flavonols including the highest rutin and hyperoside. AT7 showed high total flavonols including the highest quercetin, isoquercetin, myricetin, and kaempferol. The association of values revealed that studied phytopigments and phytochemicals of vegetable amaranth accessions demonstrated good radical quenching ability of 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and 2,2- Diphenyl-1-picrylhydrazyl equivalent to Trolox.These advance lines AT7 and AT15 had abundant nutraceuticals, phytopigments, and phytochemicals including radical quenching ability. These lines might significantly contribute to the promotion of health benefits and feeding the community across the globe deficit in nutraceuticals and antioxidants. Identified flavonoid compounds open the new route for pharmacological study. Vegetable amaranths are low-cost vegetables containing abundant nutraceuticals, minerals –7, phytoRecently, natural antioxidants, especially from vegetables attain the interest of researchers and consumers. Phytopigments containing betacyanins, betaxanthins, chlorophylls, carotenoids, and phytochemicals including vitamin C, β-carotene, flavonoids, and phenolics are available natural antioxidants in vegetable amaranth , 12. TheBrassica species , and ODs = The optical density of samples tested. The data were measured as μg TEAC g− 1 DW.Where, RQA = radical quenching capacity, ODThe leaf samples were extracted following the protocol described by Sarker and Oba , 49.− 1 FW.Flavonols, flavanols, flavones, and flavanones in the leaf sample were estimated using a Shimadzu SCL10Avp  HPLC equipped with a detector, binary pump, and degasser following the method of Sarker and oba , 49. FlaThe sample data were averaged replication-wise to obtain the replication mean. Statistix 8 software was used to analyze the data for analysis of variance (ANOVA) , 53. DunThe ANOVA revealed that amaranth demonstrated huge variations across the traits studied.− 1 FW. The maximum content of moisture was noted in the advance line AT11 (88.56 g 100 g− 1 FW) followed by AT15 (86.68 g 100 g− 1 FW) and AT3 (86.57 g 100 g− 1 FW). On the contrary, the minimum moisture content was recorded in AT7 (81.79 g 100 g− 1 FW). Amaranth demonstrated considerable protein content that significantly varied regarding accessions albeit variability was not prominent (5.05 to 5.55 g 100 g− 1 FW). The maximum protein content was recorded in AT11 (5.55 g 100 g− 1 FW) which was statistically similar to AT3 and AT7. Conversely, the minimum protein content was noted in AT15 (5.05 g 100 g− 1 FW). Higher protein content was obtained from AT11, AT3, and AT7. In this investigation, amaranth demonstrated low-fat content.The composition of the proximate of selected amaranth is given in Fig. − 1 FW). AT7 demonstrated the maximum carbohydrates content (7.07 g 100 g− 1 FW), while AT11 had the minimum carbohydrates content (2.21 g 100 g− 1 FW). The selected lines were predominantly varied with regard to energy . The accession AT7 demonstrated the maximum energy . Alternatively, the accession AT11 exerted the minimum energy . AT7 and AT11 demonstrated the maximum ash content (5.62 and 5.44 g 100 g− 1), respectively), while the minimum ash content was noted in AT3 (2.86 g 100 g− 1). Digestible fiber predominantly varied regarding accessions (7.26 to 10.03 g 100 g− 1 FW). The advance accession AT3 demonstrated the maximum digestible fiber content (10.03 g 100 g− 1 FW) followed by AT7. Conversely, AT11 had the minimum digestible fiber content (7.26 g 100 g− 1 FW).The selected lines demonstrated significant variations in carbohydrates content regarding accessions (2.21 to 7.07 g 100 g− 1) followed by AT3, AT7, and AT11 including mean content of potassium of 5.81 mg g− 1. The maximum content of calcium was noticed in AT3 (2.48 mg g− 1) followed by AT11 and AT15. In contrast, the minimum content of calcium was noticed in AT7 (1.87 mg g− 1). In this investigation, the selected amaranth demonstrated significant variability in magnesium content regarding accessions (3.26 to 3.62 mg g− 1 FW). AT15 demonstrated the maximum content of magnesium (3.62 mg g− 1 FW). Conversely, AT7 demonstrated the minimum content of magnesium (3.26 mg g− 1 FW) that had statistical similarity to AT3, AT11.Mineral elements of selected amaranth are put in Fig. − 1). The maximum iron content was obtained from AT15 (17.26 μg g− 1 FW), while the minimum iron content was noted in AT7 (10.96 μg g− 1 FW). It revealed from our study that significant variability in manganese content regarding accessions was noticed in amaranth (6.68 μg g− 1 FW and 10.54 μg g− 1 FW). Manganese content was the maximum in AT15 (10.54 μg g− 1 FW), while the minimum manganese content was recorded in AT11 and AT3 (6.68 and 6.86 μg g− 1 FW). The copper demonstrated no notable and significant variability in amaranth genotypes (1.09 to 2.18 μg g− 1 FW). AT15 demonstrated the maximum copper content (2.18 μg g− 1 FW), while AT7 exerted the minimum copper content (1.09 μg g− 1 FW). The zinc of amaranth differed significantly and markedly (6.98 μg g− 1 FW in AT3 to 12.25 μg g− 1 FW in AT15).The iron content demonstrated significant variations regarding accessions (10.96 to 17.26 μg ga was noted regarding the selected amaranth (305.85 to 634.75 μg g− 1 FW). AT7 demonstrated the maximum chlorophyll a (634.75 μg g− 1 FW) followed by AT11 and AT15. Alternatively, the minimum chlorophyll a (305.85 μg g− 1 FW) was noted in AT3. Conversely, the selected amaranth demonstrated minimum differences in chlorophyll b (228.26 to 266.38 μg g− 1 FW). The maximum chlorophyll b was recorded in AT7 (266.38 μg g− 1 FW) followed by AT11. In contrast, AT6 demonstrated the minimum chlorophyll b (228.26 μg g− 1 FW). The significant variability in chlorophyll ab was recorded in amaranth genotypes (534.11 to 901.13 μg g− 1 FW). The advance line AT7 demonstrated the maximum chlorophyll ab (901.13 μg g− 1 FW) followed by AT11 and AT15, while AT3 demonstrated the minimum chlorophyll ab (534.11 μg g− 1 FW).Phytopigments of the selected amaranth are put in Fig. − 1 FW). AT3 demonstrated the maximum content of betacyanins (537.26 ng g− 1 FW) followed by AT11 and AT15. In contrast, AT7 demonstrated the minimum content of betacyanins (407.97 ng g− 1 FW). The selected lines demonstrated significant variations in betaxanthins regarding accessions (428.45 to 588.75 ng g− 1 FW). AT3 demonstrated the maximum content of betaxanthins (588.75 ng g− 1 FW) followed by AT11 and AT15. Conversely, the minimum content of betaxanthins was noted in AT7 (428.45 ng g− 1 FW). The selected amaranth demonstrated significant variations in betalains content regarding accessions (836.42 to 1126.01 ng g− 1 FW). The AT3 demonstrated the maximum betalains (1126.01 ng g− 1 FW) followed by AT11 and AT15. While the AT7 demonstrated minimum betalains (836.42 ng g− 1 FW). Likewise, betalains, carotenoids showed significant variability in amaranth genotypes (32.75 to 82.85 mg 100 g− 1 FW). The maximum carotenoids were recorded in AT15 (82.85 mg 100 g− 1 FW). Whereas, AT7 demonstrated the minimum carotenoids (32.75 mg 100 g− 1 FW).The selected amaranth demonstrated significant variability in betacyanin content regarding accessions (407.97 to 537.26 ng g− 1 in AT7 to 65.87 mg 100 g− 1 in AT15). The high content of β-carotene.β-Carotene, ascorbic acid, total polyphenols (TP), total flavonoids (TF), and radical quenching capacity of amaranth are put in Fig. − 1 FW). Ascorbic acid was the maximum in AT11 (105.78 mg 100 g− 1 FW) and the minimum in AT3 (18.54 mg 100 g− 1 FW). Marked and significant variability was recorded in gallic acid equivalent (GE) TP in the accessions (28.65 μg g− 1 to 38.23 μg g− 1 FW). AT15 demonstrated the maximum TP content (38.23 μg g− 1) followed by AT7. While AT3 demonstrated the minimum TP (28.65 μg g− 1) that had statistical similarity with AT11. The selected amaranth demonstrated rutin equivalent (RE) TF with significant variability regarding accessions (146.58 μg g− 1 to 166.81 μg g− 1 DW). AT15 demonstrated the maximum TF (166.81 μg g− 1) followed by AT7 and AT11, whereas AT3 had the minimum TF (146.58 μg g− 1). The selected amaranth demonstrated high DPPH and ABTS+ radical quenching ability. AT15 demonstrated the maximum Trolox equivalent (TE) DPPH and ABTS+ radical quenching ability  followed by AT7 . Alternatively, the minimum DPPH and ABTS+ radical quenching ability were recorded in AT3  followed by AT11 .was noticed in AT11. The lines demonstrated significant variability in ascorbic acid  were compared with respective peaks and standard masses of these flavonols, flavones, flavanols, and flavanones. Nine flavonoids were detected in amaranth genotypes together with six flavonols, like kaempferol, rutin, hyperoside, myricetin, isoquercetin, and quercetin, one flavanol, like catechin, one flavone like apigenin, and one flavanone, such as naringenin. It is the first effort in which we identified one flavonols such as myricetin, one flavanol, such as catechin, one flavone i. e.,The data of Table apigenin, and one flavanone, like naringenin in amaranth genotypes. Figure − 1 FW, respectively  of the previous study [A. tricolor [A. blitum [A. spinosus weedy amaranth [A. blitum [A. blitum [The ANOVA has shown that amaranth demonstrated huge variations across the traits studied. Huge variations across the traits studied were also noticed in ndriacus , rice [5ndriacus –71, maizndriacus –74, and ndriacus , 76. Acrndriacus  and A. ttricolor . Leaves icolor 1.6% of thetricolor  and sweetricolor . The selicolor 1.6% of thetricolor . The moitricolor , green atricolor , weedy atricolor , stem amtricolor , and A. . blitum . The car. blitum , green a. blitum , A. spinamaranth , and A. . blitum , while t. blitum . The dig. blitum , green a. blitum , stem am. blitum , and A. . blitum , while t. blitum .− 1) and magnesium (3.62 mg g− 1), and Ca (2.48 mg g− 1) in the amaranth genotypes (based on fresh weight). Ample magnesium, calcium, and potassium were noted in several species of amaranth [− 1), manganese (10.54 μg g− 1), zinc (12.25 μg g− 1), and notable copper (2.18 μg g− 1) (based on fresh weight) were recorded in amaranth genotypes. Similarly, adequate iron, manganese, copper, and zinc were noticed in different amaranths [A. spinosus weedy amaranth [It revealed that we noted ample potassium . Conversely, the selected amaranth demonstrated minimum differences in chlorophyll b. Significant and outstanding variability in chlorophyll ab were recorded in the amaranth genotypes. We observed adequate chlorophyll a (634.75 μg g− 1 FW), chlorophyll ab (901.13 μg g− 1 FW), and chlorophyll b (266.38 μg g− 1 FW) in the amaranth genotypes which was pronounced than the chlorophylls of green and red amaranth [. Chlorophyll a, chlorophyll ab, and chlorophyll b in this study were much pronounced than chlorophyll a, chlorophyll ab, and chlorophyll b of red amaranth [A. blitum [a (634.75 μg g− 1 FW), chlorophyll ab (901.13 μg g− 1 FW), betacyanins (537.26 ng g− 1 FW), chlorophyll b (266.38 μg g− 1 FW), betaxanthins (588.75 ng g− 1 FW), betalains (1126.01 ng g− 1 FW) and carotenoids (82.85 mg 100 g− 1 FW) in amaranth genotypes which were corroborated with chlorophyll a, betacyanins, chlorophyll b, betalains, chlorophyll ab, betaxanthins, and carotenoids of green and red amaranth [. Betacyanins, betalains, and betaxanthins in the amaranth were much pronounced than betacyanins, betalains, and betaxanthins of red amaranth [A. blitum [A. blitum [The significant variability in chlorophyll amaranth . Chloropamaranth , green aamaranth , weedy aamaranth , stem amamaranth , and A. . blitum . The linamaranth . Betacyaamaranth , green aamaranth  stem amaamaranth , and A. . blitum  and corr. blitum  while ca. blitum , stem am. blitum , and A. + radical quenching ability. The similarity in the trend of radical quenching ability in DPPH and ABTS+ methods validated the quantification of two different methods of antioxidant capacities. The lines exhibited outstanding β-carotene and ascorbic acid (65.87 and 105.78 mg 100 g− 1 FW) which was pronounced than red amaranth [. TP (38.23 μg g− 1 FW), TF (166.81 μg g− 1 DW), radical quenching ability in DPPH (40.75 μg g− 1 DW), and radical quenching ability in ABTS+ (76.98 μg g− 1 DW) obtained in amaranth was pronounced than the contents of these compounds in green and red amaranth [A. blitum [+ recorded in the advance lines was pronounced than red amaranth [A. blitum [The significant variability was recorded in the β-carotene of the selected amaranth. The amaranth demonstrated significant variability in ascorbic acid. Marked and significant variability was obtained in gallic acid equivalent (GE) TP in amaranth genotypes. The lines demonstrated rutin equivalent (RE) TF with significant variability regarding accessions. The selected amaranth demonstrated significant variations in DPPH and ABTSamaranth . TP  [A. cruentus seeds and sprouts contained three flavonoids namely, rutin, isovitexin, and vitexin [Nine flavonoids were detected in the genotypes together with six flavonols, like kaempferol, rutin, hyperoside, myricetin, isoquercetin, and quercetin, one flavanol i. e., catechin, one flavone i. e., apigenin, and one flavanone, such as naringenin. For the first time, we identified one flavonol such as myricetin, one flavanol i. e., catechin, one flavone i. e., apigenin, and one flavanone, like naringenin in the amaranth genotypes. In green and red amaranth, three flavonols such as isoquercetin, hyperoside, and rutin were previously reported , 87. Thrdriacus) . The A.  vitexin . The fou vitexin . The eda vitexin . Pronoun+ and DPPH of the selected genotypes demonstrated interesting results. All phytopigments positively and significantly correlated among TF, TP, and radical quenching ability in ABTS+ and DPPH. It indicated that the increment in TF, TP, and radical quenching ability in DPPH and ABTS+ were directly related to the increment of chlorophylls, betacyanins, betaxanthins, betalains, and carotenoids or vice versa. It meant all studied phytopigments had good radical quenching ability. Similarly, ascorbic acid demonstrated a positive and significant interrelationship with TF, TP, and radical quenching ability in ABTS+ and DPPH, while it exhibited negative and insignificant correlations across all phytopigments. The earlier work of amaranth also reported a related trend [+ and DPPH. The significant positive interrelationship of β-carotene, ascorbic acid, TP, and TF and radical quenching ability in ABTS+ and DPPH signify that β-carotene, ascorbic acid, TP, and TF had a powerful activity of antioxidants. The capacity of antioxidants validation of amaranth through differential methods confirmed the significance in the associations between radical quenching ability in ABTS+ and DPPH. Studied phytopigments and phytochemicals including β-carotene, ascorbic acid, TP, and TF demonstrated powerful activity of antioxidants, as these showed the significant associations with radical quenching ability in ABTS+ and DPPH. All studied phytopigments, β-carotene, ascorbic acid, TP, and TF played a vital role in the antioxidant potentiality of the amaranth lines as these compounds had a powerful activity of antioxidants.The correlation of phytopigments, β-carotene, ascorbic acid, TF, TP, and radical quenching ability in ABTSed trend , 26. A pThe selected amaranth contained adequate nutraceuticals, phytopigments, phytochemicals including radical quenching ability. Across nine identified flavonoids, naringenin, apigenin, catechin, myricetin were newly reported in drought-tolerant vegetable amaranth. Based on this study, pharmacologists could investigate the suitability of these crops in medicinal drug discovery. Across the accessions, AT7 and AT15 had abundant phytochemicals and radical quenching ability including considerable nutraceuticals and phytopigments in comparison to the accessions AT3 and AT11. The accessions AT7 and AT15 might be directly utilized as nutraceuticals, phytopigments, phytochemicals enrich high yielding commercial cultivars. AT7 and AT15 could also be utilized to extract juice as a source of potential nutritional value, antioxidant phytopigments, β-carotene, antioxidants, flavonoids, phenolics, and ascorbic acid in the diet to accomplishing nutritional and antioxidant sufficiency in the globe. These lines might significantly contribute to the promotion of health benefits and feeding the community across the globe deficit in nutraceuticals and antioxidants. Identified flavonoid compounds in amaranth genotypes open the new route for details pharmacological study.


SORL1 is strongly associated with both sporadic and familial forms of Alzheimer’s disease (AD), but a lack of information about alternatively spliced transcripts currently limits our understanding of the role of SORL1 in AD. Here, we describe a SORL1 transcript (SORL1-38b) characterized by inclusion of a novel exon (E38b) that encodes a truncated protein. We identified E38b-containing transcripts in several brain regions, with the highest expression in the cerebellum and showed that SORL1-38b is largely located in neuronal dendrites, which is in contrast to the somatic distribution of transcripts encoding the full-length SORLA protein (SORL1-fl). SORL1-38b transcript levels were significantly reduced in AD cerebellum in three independent cohorts of postmortem brains, whereas no changes were observed for SORL1-fl. A trend of lower 38b transcript level in cerebellum was found for individuals carrying the risk variant at rs2282649 (known as SNP24), although not reaching statistical significance. These findings suggest synaptic functions for SORL1-38b in the brain, uncovering novel aspects of SORL1 that can be further explored in AD research.The online version contains supplementary material available at 10.1186/s40478-021-01140-7. SORL1 gene encodes the protein SORLA and is associated with Alzheimer’s disease (AD)  Promix (Amersham Biosciences) for 40 min at 37 °C. Subsequently, cells were washed with phosphate-buffered saline (PBS), and both media and lysates were collected at different chase points. Cell lysates were immunoprecipitated at 4 °C overnight with GammaBind G-Sepharose beads (Amersham Biosciences) previously labeled with anti-sol-SorLA diluted in HyClone medium. Beads were then washed with PBS and proteins were resolved by standard SDS-PAGE and fluorography.Cells were incubated in methionine- and cysteine-free medium  supplemented with glutamax and 2% dialyzed FBS (Gibco) for 15 min at 37 °C, prior to biolabeling with [Transfected cells were lysed in lysis buffer containing protease inhibitor , and media were collected. Proteins were fractioned by 4–12% SDS-gel electrophoresis, and transferred to nitrocellulose membranes (Amersham). Membranes were blocked in blocking buffer , 2% skimmed milk powder, 2% Tween 20) for 1 h at RT, and incubated at 4 °C overnight with mouse anti-LR11 (1:500), and rabbit pAb-38b (1:1000) primary antibodies. Incubation with HRP-conjugated anti-mouse (1:1500) or anti-rabbit (1:1500) secondary antibodies was performed at RT for 1 h. Proteins were detected with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) and visualized with LAS-1000 .SORL1-38b  and SORL1-fl transcripts (E38E39). In parallel, sections were hybridized with a PPIB probe  as positive control. Chromogenic detection of specific hybridization was assessed with Fast Red dye , and Fast Red-Green . Nuclei counterstaining was made by incubation with Mayer’s hematoxylin for 2 min at RT. Tissues were rinsed in deionized water and mounted with VectaMount permanent medium . Slices were imaged with a Zeiss Apotome brightfield camera (Zeiss Apotome). Number of transcripts was manually quantified by counting punctate dots observed in PCs in the entire sections. Data were then plotted as mean number of transcripts per counting group . For quantification of somatic and extra-somatic transcripts after BaseScope treatment, 25 random fields were chosen at 20X magnification, counting three PCs in each field in order to compare homogenous tissue areas.From NBB, paraffin embedded human cerebellum, hippocampus and entorhinal cortex were cut to 5 μm thick sections using a microtome. ISH was carried out by the BaseScope and BaseScope Duplex technologies (Advanced Cell Diagnostics) following manufacturer’s instructions. Tissue hybridization was performed for 2 h at 40 °C  with specific probes designed to span exon-exon junctions for recognition of Two published datasets were queried for the presence of RNA-seq reads spanning the exon 38b splice junctions. We downloaded RNA sequencing alignment files generated from the cerebellum  along wiSORL1-38b, and three housekeeping genes: HPRT1, TFRC, and TXNL1. Four replicate measures were taken per sample for each of the four Taqman assays (16 measures per sample) on 384 well reaction plates using the QuantStudio™ 7 Flex Real-Time PCR system and analysis software .The cDNA synthesis and qPCR analysis on the Mayo samples were conducted at Mayo Clinic. RNA was isolated from 50 cerebellum samples from the Mayo Clinic brain bank using TRIzol Reagent (Ambion Life Technology) followed by DNase RNA cleanup step using RNeasy (Qiagen). The quantity and quality of RNA samples were determined by the Agilent 2100 Bioanalyzer using Agilent RNA 6000 Nano Chip. All samples had an RNA integrity number (RIN) of ≥ 6.6, with an equivalent mean RIN for both diagnosis groups . cDNA synthesis was performed with 1–2 μg of RNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems cat. no. 4387406) according to the manufacturer’s instructions. The samples were tested with a custom Taqman probe for detection of SORL1-38b: E38b-Fw: 5′-GCGGTGACTAGTCGTGGAAT and E38b-Rev: 5′-TGCTCTTCCAACATCCCTTC. PCR products were amplified with the following conditions: 50 °C for 2 min, 95 °C for 20 s, 40 cycles of amplification . Three housekeeping genes were used as controls: HPRT1, CYC1 and RPL13.The cDNA synthesis and qPCR analysis on the UW samples were conducted at the University of Washington. From each sample, 1 μg total cerebellum RNA was converted to cDNA . SYBR Green qPCR was performed using Real-Time PCR Detection System (Biorad) with the following primers for Each sample was run in triplicates, and water was included as negative control.SORL1-38b due to Ct values outside the dynamic range for the assay. Primers for PCR of amplicon containing SNP24 was FW: 5′-CTTTAGCTCATTCAGTATTCTTACTGTATG, and Rev: 5′-GAGCATTTCTTCTAATGCAGACATAC.Briefly, genomic DNA was prepared from cerebella samples from UW Neuropathology Core. DNA was genotyped for the presence of risk (T) or protective (C) allele at position rs2282649 by Sanger Sequencing (Genewiz). In total, 67 samples were genotyped and we observed 7 T/T carriers (10.4%), 29 C/C carriers (43.3%), and 31 C/T carriers (46.3%). To obtain groups of equal sample size, we chose 7 samples of each genotype  for 38b transcript analysis. One sample from the T/T genotype group failed qPCR of p value below 0.05 was considered significant, indicated as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).An unpaired student's t test was performed in Graphpad Prism 5.0 to compare differences and determine statistical significance between AD and non-AD samples. A SORL1-38b transcript levels between AD and non-AD samples in the Mayo and UW cohorts was performed in R version 3.6.1. First, a linear regression between deltaCT and diagnosis was performed, with RIN (only available for Mayo cohort), age at death, gender and plate ID as covariates. As no significant association was found for any of the covariates, a Wilcoxon rank sum test was subsequently used to compare AD versus non-AD samples. A meta-analysis of the two cohorts was performed using the metap R package (version 1.3). (Dewey M (2020). metap: meta-analysis of significance values).Comparison of normalized Comparison of the risk allele frequency between AD and non-AD samples for SNP24 in the Mayo cohort was performed using Fisher’s exact test. This test was not performed in the UW cohort, as samples were chosen based on genotype, so allele frequencies were not representative for AD cases and controls.https://adknowledgeportal.synapse.org, 10.7303/syn2580853). The AD Knowledge Portal is a platform for accessing data, analyses, and tools generated by the Accelerating Medicines Partnership (AMP-AD) Target Discovery Program and other National Institute on Aging (NIA)-supported programs to enable open-science practices and accelerate translational learning. The data, analyses and tools are shared early in the research cycle without a publication embargo on secondary use. Data is available for general research use according to the following requirements for data access and data attribution . For access to content described in this manuscript see: https://www.synapse.org/#!Synapse:syn11714444. Genotypes were extracted from the available VCF files using vcftools version 0.1.17.Whole genome-sequence data for 35 of the Mayo Clinic samples is available via the AD Knowledge Portal , indicating differential expression of the alternatively spliced transcript    as well as many cells in the granule cell layer (GCL), the probe for SORL1-38b showed a restricted signal mainly within the Purkinje cell layer and clearly absent from the GCL . While the signal for SORL1-fl was predominantly (89%) located in the soma of PCs, SORL1-38b was only partially present in the PC soma (46%) with the majority of labelled transcripts located in the molecular layer where the dendritic tree of PCs extends  or skipping exon 39 (E38bE40)  in these brain regions  not only validated the expression of SORL1-38b transcripts), we experienced trouble with a background signal in the GCL that confounded the data. We therefore took an alternative approach to support a difference in cellular localization between truncated and SORLA-fl isoforms. Using an antibody that binds the cytoplasmic tail of SORLA, we found that SORLA-fl is almost exclusively located in the PC soma, whereas two different antibodies (IgGs LR11 and 5387), binding to epitopes in the extracellular receptor domain and therefore unable to distinguish between SORLA-fl and SORLA-38b, gave an additional strong signal in the dendritic tree of PCs that was only rarely seen in pictures from immunostainings using the tail antibody , accompanied by a significant reduction in the number of PCs corresponding to groups I  and II  with no cells assigned to group III  were negative (group 0) by the E38E38b ISH analysis in non-AD cerebella, and the remaining 53.4% positive PCs distributed equally between groups I-II-III . However, in cerebella from AD patients, 93.0% of all PCs showed a lack of SORL1-fl transcripts. In line with previous reports that showed robust SORLA expression in AD cerebellum [SORL1-fl levels between AD and non-demented controls , and that none of the four groups were affected by AD    and no change in SORL1-fl . A meta analysis of the two cohorts  found a mean 2.1-fold decrease of SORL1-38b in AD (pmeta = 0.00035) , supporting a trend for association of this variant with AD in this small sample. Because samples from the UW cohort were selected for qPCR based on their SNP24 genotype, allele frequencies are not representative for AD cases and controls, and they were not included in the association analysis with disease status.In the Mayo subset (12 AD and 23 non-AD) the SNP24 allele frequency of the risk allele  was higher among AD samples (41.7%) compared to non-AD samples (19.6%) (SORL1-38b levels in carriers of the risk genotype T/T of SNP24 (rs2282649)   Fig. d, e (pmeus) Fig. f. To detSORL1 locus, genetic variants in SORL1 as well as up- and downstream were downloaded from the AMP-AD knowledge portal for the 35 Mayo samples. Apart from a small cluster of SNPs in the SORL1 promoter, which was to be expected, no other strong eQTL could be identified for E38b-transcript levels within the SORL1 locus  gene holding a PSC, after introduction of a novel exon, is able to escape NMD and be locally translated in neuronal terminals [SORL1-38b may be prevented by trafficking the transcript to dendrites where the truncated SORLA-38b protein is produced.Translation of transcripts including E38b leads to production of a truncated SORLA isoform lacking four 3Fn-domains as well as transmembrane and cytoplasmic domains that are all present in SORLA-fl. This suggests different functions of the two isoforms, in line with multiple studies reporting that locally synthesized proteins are most often structurally and functionally distinct from proteins present at the same cellular site but being transported to their destination [erminals . SimilarSORLA-fl is known to regulate sorting of a number of cargo proteins including APP and the Amyloid-β peptide , 9, in aSORL1-38b expression . This observation is in line with our present findings where only a subset of neurons expresses the alternatively spliced isoform. We suggest future studies should apply protocols for differentiation into PCs [The exact function of the shorter SORLA-38b isoform is not clear, but it is very tempting to speculate a role related to synaptic plasticity, which is the most frequent function associated with localized protein synthesis in dendrites . The stuinto PCs , 48 to iCNTN4 (encoding Contactin-4) introduces a novel stop codon and generates a truncated protein with deletion of several 3Fn-domains [SORLA has previously been speculated to act as an adhesion protein, because 3Fn-domains are often present in adhesion molecules, including protocadherins, immunoglobulin superfamily and contactins , 10. Int-domains  similarl-domains . Local sSORL1-38b we have established several methods including the development of a specific antibody, the application of E38b-specific probes for ISH and the optimization of qPCR assays. Altogether, these made the identification of an otherwise hardly detectable splice isoform possible, providing valuable information about SORL1 transcriptomic profile.In conclusion, for the characterization of the lowly expressed SORL1, a few limitations need to be mentioned. First, the low expression level of SORL1-38b challenged the detection of this transcript in other brain regions than the cerebellum. Thus, the implementation of more sensitive techniques, as well as the optimization of established protocols, will aim at confirming our data in non-cerebellar areas. Second, the small sample size of the cerebellum cohorts limited the analysis of SORL1-38b expression in relation to the risk T/T (SNP24) genotype. Future studies including larger cohorts will help clarifying how SORL1-38b associates with SNP24.Although the study revealed novel findings about SORL1 gene. Our results highlight two major novel findings in SORL1 research: the dendritic localization of SORL1-38b transcripts and the specific reduction of E38b levels in cerebellum from AD patients despite SORL1-fl transcripts being unaltered. Altogether, these data add another layer of complexity to the understanding of SORL1 biology.Here, we provide the first detailed characterization of an alternatively spliced transcript of the Additional file 1. Figure S1: SORLA-38b is retained intracellularly in HEK cells. a Substitutions of cysteine to alanine at position 1 and 13 of the unique tail encoded by E38b do not alter the secretion of SORLA-38b in the medium. Non transf non transfected cells. b WB analysis of lysates and media from CHO cells transiently transfected with SORLA-fl, SORLA variants where stop mutations were introduced at the boundary following each 3Fn domain, or a SORLA variant lacking the cytoplasmic tail (SORLA-ΔCD). Notably, constructs lacking the 3Fn5 are most strongly retained intracellularly (indicated with arrowheads).Additional file 2. Figure S2: Analysis of the 3′ end of SORL1-38b. Agarose gel showing 3′ RACE products for SORL1-fl and SORL1-38b. Bands indicated with arrows have been sequenced.Additional file 3. Figure S3: Control of RNA quality in non-AD and AD cerebella. Samples hybridization with PPIB probe showed no distinct difference in the signal from non-AD compared to AD cerebella.Additional file 4. Figure S4: Analysis of genetic variants affecting the expression of E38b. a Representation of the boundaries of the 3′ end locus in SORL1 including localization of E38b. b eQTL mapping of SORL1 gene did not show significant association with E38b expression (SNP24: rs2282649).


Santini et al. demonstrated favorable prognosis with the resumption of anti‐PD‐(L)1 although statistical power is not enough. In the study by Pollack et al., 31% had partial responses, 23% had stable disease, and 46% had progressive disease among patients underwent resumption of ICIs for disease progression after discontinuation.et al., 32.5% had a partial response, 37.5% had stable disease, and 22.5% had progressive disease during the resumption of ICIs.With regard to the efficacy of ICIs resumption, Santini Thus, re‐challenge of ICIs shows expected tumor response in exchange for higher risk of irAEs, which can rarely be lethal. In line with these previous reports, this case report demonstrated controllable irAE after resumption of nivolumab although objective response was not obtained. Since there is no established evidence supporting the usefulness of resumption of ICIs after irAEs, resumption of ICIs should be determined after adequate discussions with patients.The authors declare no conflict of interest.