Nucleic acid extraction from biological samples is an important step for hepatitis C virus (HCV) diagnosis. However, such extractions are mostly based on silica-based column methodologies, which may limit their application for on-site diagnosis. A simple, rapid, and field-deployable method for RNA extraction is still needed. In this study, we evaluated the efficacy of four simple RNA extraction methods for the detection of HCV in plasma samples: a silica-membrane-based method, a magnetic-beads-based method, boiling with diethyl pyrocarbonate (DEPC)-treated distilled water, and using a commercial lysis buffer. HCV RNA was detected using both real-time reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Using real-time RT-PCR, extracted RNA from the silica-membrane-based and magnetic-beads-based methods had a 100% detection rate for RNA extraction from plasma. Using RT-LAMP, extracted RNA from the silica-membrane-based method showed a 66% detection rate, while the magnetic-beads-based method had a 62% detection rate. In summary, magnetic-beads-based extraction can be used as an alternative RNA extraction method for on-site HCV detection. Boiling with DEPC-treated distilled water was not appropriate for low HCV load samples, and boiling with a lysis buffer was not recommended. Hepatitis C virus (HCV) is a leading cause of chronic liver disease, cirrhosis, and liver cancer. The current oral direct-acting antiviral (DAA) combinations can cure 95–99% patients , but theg for 1 min, and the supernatant was collected; and (4) boiling with lysis buffer, which was conducted as above, but 100 µL plasma was mixed 1:1 with a commercial lysis buffer . The yield and purity of RNA extracted by the four methods were measured using a NanoDrop™ 2000/2000c Spectrophotometer. For RNA detection by real-time RT-PCR, four µL of viral nucleic acid extract was amplified with 400 nM each of HCV forward and reverse primers, as well as 100 nM of HCV probe [6 IU/mL in 1× phosphate buffer saline were prepared. Aliquots of each dilution were extracted by the four extraction methods. All RNA extracts were tested for HCV RNA by both real time RT-PCR and RT-LAMP. Each reaction was performed in triplicate.This study used leftover plasma samples of 50 HCV-infected individuals who had undergone HCV viral load testing and genotyping for clinical care at the Faculty of Associated Medical Sciences, Chiang Mai University. HCV viral load was initially measured using a commercial real-time RT-PCR assay (COBAS AmpliPrep/COBAS TaqMan HCV Test). The study was approved by the Human Experimentation Committee  and the Institutional Biosafety Committee  of the Research Institute for Health Sciences, Chiang Mai University, Thailand. Viral RNA was extracted from plasma samples using four different methods: (1) NucleoSpin RNA Virus  and (2) NucleoMag Virus kit , following the manufacturer’s recommendations; (3) boiling with water, in which 100 µL plasma was mixed 1:1 with diethyl pyrocarbonate (DEPC)-treated distilled water  and boiled at 95 °C for 10 min [6 IU/mL. The dilutions that were used were 105, 104, and 103 IU/mL. The four extraction methods showed considerably variable quantities of RNA. Boiling plasma samples with a commercial lysis buffer provided the highest yield of RNA, with a concentration of 695.37 ng/µL, and boiling with DEPC-treated distilled water produced the lowest yield of 0 ng/µL. The purity ranged from 0.14 to 3.46 and was the highest with the silica-membrane-based extraction method (Ct) and by RT-LAMP at the 106 concentration. The numbers of triplicates detected by both techniques decreased when initial concentrations were 105 or lower. RNA extracts obtained after boiling in DEPC-treated distilled water were less well detected at higher Ct than the silica-membrane and magnetic-bead RNA extracts. Though boiling with a commercial lysis buffer gave the highest RNA yield, none of the RNA extracts yielded a signal in real-time RT-PCR, regardless of the initial HCV RNA concentration. The results from RT-LAMP could not be interpreted, as the color turned from purple to sky blue immediately after adding the extracted RNA to the reaction mix. Thus, boiling with a commercial lysis buffer was not further evaluated.The efficacy of the four extraction methods was evaluated using serial dilutions of plasma with HCV viral load of 10n method . AnalytiCt values of RNA extracted from the three different extraction methods showed an excellent correlation between the Ct values of samples extracted with the silica-membrane-based and magnetic-beads-based methods , Our study showed variable sensitivities of molecular HCV detection in plasma samples depending on the RNA extraction method used. The silica-membrane-based extraction method is a simple bind–wash–elute process. As noted, the eluates of this kit contain both viral nucleic acids and carrier RNA, which may exceed the amount of authentic nucleic acids from the virus when quantified by the photometric method. However, the extracted RNA from this method is suitable for HCV molecular diagnosis by real-time RT-PCR, and by RT-LAMP when using samples with a high viral load. Previous studies also showed that this method was suitable for RNA isolation from plasma and ready for use in subsequent reactions for viral detection ,8. The sCt values from HCV samples extracted by boiling were slightly higher than those of RNA extracted by silica-membrane-based and magnetic beads-based extraction methods and did not strongly correlate. Although the boiling method could be used as a cost-effective alternative to expensive extraction methods, it can only be used when samples have a high viral load. Boiling samples with a commercial lysis buffer provided the highest RNA concentration but a quite low purity of RNA. This might be due to the RNA carrier contained in the lysis buffer and the absence of an additional step to remove the lysis buffer components or purification prior to amplification. We were unable to detect any HCV RNA in all RNA extracts from this method, and the results of RT-LAMP could not be interpreted. This may be due to an effect of inhibitors created or released by boiling. Thus, boiling plasma samples with a commercial lysis buffer cannot be used for RNA extraction in viral detection. However, some studies suggested that boiling techniques could become a suitable DNA extraction method when LAMP assays are used for detection, but this may require specific reagents, such as Triton X-100 or Chelex buffer [The magnetic-beads-based extraction method is based on the reversible adsorption of nucleic acids to paramagnetic beads and uses only a magnetic separator. It does not need a centrifugation step but requires careful pipetting to remove the solution from the beads. In this method, 200 µL plasma sample was used, compared to only 100–150 µL of sample in the other three methods, which may lead to an increase in the RNA extraction capacity. Previous studies also recommended the magnetic bead technology for viral RNA extraction from serum or plasma . Our resx buffer ,15. The In summary, this study demonstrated that the magnetic-beads-based extraction method can be used as an alternative method of plasma RNA extraction for HCV detection. This method is simple, rapid, and inexpensive, and it does not require a centrifugation process, which makes it suitable for on-site diagnosis or in the field when combined with the RT-LAMP technique. This approach may improve the accessibility of HCV testing and help to identify HCV-infected individuals in need of treatment.


These mutations were suggested to affect the ability of RNAP to transcribe damaged DNA and to decrease the stability of transcription complexes, thus facilitating their dislodging during DNA replication and repair, although this was not explicitly demonstrated. Here, we obtained nine mutations of this class located around the DNA/RNA binding cleft of Escherichia coli RNAP and analyzed their transcription properties in vitro. We found that these mutations decreased promoter complex stability to varying degrees, and all decreased the activity of rRNA promoters. However, they did not have strong effects on elongation complex stability. Some mutations were shown to stimulate transcriptional pauses or decrease intrinsic RNA cleavage by RNAP, but none altered the ability of RNAP to transcribe DNA templates containing damaged nucleotides. Thus, we conclude that the suppressor phenotypes of the mutations are unlikely to result from direct effects on DNA lesion recognition by RNAP but may be primarily explained by changes in transcription initiation. Further analysis of the effects of these mutations on the genomic distribution of RNAP and its interactions with regulatory factors will be essential for understanding their diverse phenotypes in vivo.Bacterial RNA polymerase (RNAP) coordinates transcription with DNA repair and replication. Many RNAP mutations have pleiotropic phenotypes with profound effects on transcription-coupled processes. One class of RNAP mutations ( Transcription—the process of RNA synthesis on the genomic DNA template by RNA polymerase (RNAP)—is a pivotal step in gene expression. However, transcribing RNAP poses a major obstacle for other cellular machineries acting on the genomic DNA, first of all, during DNA replication , 2, 3. Rin vitro and in vivo, and transcription complexes stalled on damaged DNA are a major threat to DNA replication. At the same time, transcribing RNAP acts as a sensor of DNA damage in the template strand, and stalled TECs recruit repair factors to DNA lesions during transcription-coupled DNA repair (TCR)  represent a bigger challenge for the replisome in comparison with active TECs or promoter complexes , 17, 18.ir (TCR) , 27, 28.Escherichia coli strains with defects in DNA repair and stringent response revealed a class of rpo∗ mutations in the β and β′ subunits of RNAP that restored the viability of these strains under DNA damaging conditions and could also suppress mutations in the RuvABC resolvase  (A). The R1148H, H1244Q, and G1260D substitutions in the β subunit decreased t1/2 of promoter complexes 2- to 2.5-fold, while the remaining three RNAPs, K215E, Δ312-314, and K789Q in the β′ subunit had only minor effects on promoter complex stability  A. The R1rrnB P1 promoter. The plasmid also contained a second promoter responsible for synthesis of RNA I from the replication origin, which forms stable complexes with RNAP  (B).We further tested the activity of the mutant RNAPs on rRNA promoters, which form highly unstable promoter complexes , using aith RNAP . It was l cases) B.It can therefore be concluded that various suppressor mutations decrease the stability of promoter complexes to varying degrees, but all have a common effect on the activity of rRNA promoters and possibly of other promoters that form unstable complexes with RNAP.E. coli RNAP proposed that they might destabilize TECs formed by the mutant RNAPs thus helping the replisome and repair factors to displace them from the DNA template (see Introduction). To reveal possible effects of these mutations on transcription elongation, we compared the TEC stabilities, the rates of RNA synthesis, and the efficiency of intrinsic transcription termination by the WT and mutant RNAP variants.Previous studies of suppressor mutations in C). It was found that the majority of the complexes formed by WT E. coli RNAP remained bound to the sorbent during the course of experiment. Similarly, the analyzed RNAP substitutions did not result in dramatic changes in the TEC stability, with the major fraction of RNA remaining bound to the TEC within 30 min. Therefore, the mutations do not have great destabilizing effects on the TEC, at least in the absence of additional factors.We first tested the stabilities of TECs formed by the mutant RNAPs obtained after transcription initiation and stalled at the +20 template position by nucleotide deprivation. The TECs were bound to affinity resin and incubated at high ionic strength conditions to induce RNA dissociation, and the fraction of stably bound–labeled RNA transcripts was measured over time C. It wasrpoB gene lacking strong pause-inducing signals . Fo. ForpoB  signals . Further signals . An exce signals  and S4. If the suppressor mutations destabilized the TEC, it could be expected that they would increase the efficiency of intrinsic transcription termination, by stimulating RNAP dissociation. To determine whether this was the case, we compared the efficiency of intrinsic transcription termination by the WT and mutant RNAPs on the λ tR2 terminator . It was -10(C/T)-1G+1 signal, surrounded by additional less conserved motifs . At the same time, for three RNAPs, the pause duration was increased about 2-fold (H447P and H1244Q) or 3.5-fold (T563P) relative to the WT enzyme (p < 0.05)  prompted us to investigate the effects of the suppressor mutations on the recognition of site-specific pausing signals by RNAP. We first tested whether any of the mutations can change the duration of the elemental transcriptional pause revealed in genome-wide studies of RNAP pausing , 40, 41.d motifs . We assed motifs  and S2. d motifs A and 1/25) A and .Figure 5B). Most analyzed RNAP mutations, including T563P that stimulated consP pausing, did not strongly affect hisP pausing (≤1.5-fold changes in the pause t1/2) . A similar stimulatory effect of this mutation on hisP pausing was observed on a DNA template containing the native hisP signal during promoter-dependent transcription , by assembling the TEC on a synthetic template containing the hisP sequence. Formation of the upstream RNA hairpin required for stabilization of the pause was mimicked by the addition of a short RNA oligonucleotide complementary to the RNA transcript in the complex  and S2 , 43. TheFig. 5cription . TherefoThe process of RNAP backtracking during the elongation step of transcription plays important roles in the regulation of gene expression and in the maintenance of genome stability. Backtracked complexes are reactivated by RNA cleavage in the active site of RNAP, which can be stimulated by the secondary channel factors GreA and GreB , 45. RNAA). Furthermore, we tested the effects of the mutations on RNA extension in a mismatched TEC containing a noncomplementary nucleotide at the RNA 3′-end . The mutant RNAPs also did not differ from the WT enzyme in the pattern of RNA extension . Therefore, the mutations do not change the fidelity of RNAP in these reactions.We first analyzed possible effects of the mutations on the ability of RNAP to misincorporate a noncomplementary nucleotide in a TEC containing fully complementary RNA transcript   and S2. Fig. S62+ ions in the absence of nucleotides   . Thus, se <0.05) .Figure 6During initial characterization of the suppressor mutations in RNAP, it was proposed that they may change the ability of RNAP to transcribe damaged DNA and potentially affect the stability of transcription complexes stalled at DNA lesions , 30. TheA). O6-meG had a similar effect on RNA extension, with a stronger pause observed upstream of the lesion and a weaker pause opposite the lesion    . The kinFig. 7Fig. 7B) . εA stroFig. 7C) , 51, 53.E. coli RNAP . Genetic screenings identified  strains , 30; thene ppGpp , 30, 32.and UvrD . Recentlin vitro  or upon in vitro . It was aged DNA . Here, wA hybrid , in a sein vitro (rpo∗ mutations indeed decrease promoter complex stability on the moderately stable T7A1 promoter (A) . This is the single common property of the rpo∗ mutants that we have studied, which may possibly explain their common phenotypes observed in vivo (see below).All stringent RNAP mutations were proposed to decrease promoter complex stability but only a few of them were directly studied in vitro , 34, 35.promoter A 56). T. Tin vitoter (A)  and confoter (A) . Previoupromoter , and thepromoter , 58. Thearvation , 34, 35.promoter B. This irpo∗ mutations were proposed to decrease stability of TECs . A similar result was obtained previously in another study of H1244Q RNAP  and purified as described previously  was incubated with a PCR fragment (25 nM) containing the T7A1 promoter in transcription buffer  for 10 min at 37 °C. Heparin was added to 100 μg/ml, the samples were incubated for increasing time intervals, NTP substrates were added , and the reaction was stopped after 5 min at 37 °C by addition of an equal volume of stop-solution . Analysis of RNAP activity on the rrnB P1 promoter was performed using a pTZ19-derived plasmid containing the rrnB P1 promoter placed 88 nt upstream of the hisT terminator; the length of the control RNA I transcript encoded by the ori region in this plasmid is 108 to 110 nt (70 factor) was incubated with plasmid DNA (25 nM) in transcription buffer containing 150 mM KCl for 10 min at 37 °C. NTP substrates were added , and the reaction was performed for 10 min at 37 °C.The schematics of all R promoter and a 500 bp fragment of the rpoB gene (32P-UTP) for 7 min at 37 °C to obtain TECs stalled at the +26 template position. The samples were transferred to 20 °C, and all four NTPs were added , and the reaction was performed for increasing time intervals. DksA  and ppGpp  were added prior to NTP addition, when indicated.To measure the rate of transcription elongation, promoter complexes were formed on linear DNA containing the λPpoB gene , 79. Tra32P-CTP) for 5 min at 37 °C to obtain TECs stalled at the +20 template position. Ni-NTA-agarose equilibrated in the transcription buffer was added to the samples (20 μl per 100 μl of the reaction volume), and the samples were stirred for 5 min at 37 °C. 4 M KCl was added to the final concentration of 1 M, the samples were incubated for increasing time intervals at 37 °C, 20 μl aliquots were removed, washed with 1 ml of the buffer containing 1 M KCl, then with 1 ml of the buffer containing 40 mM KCl, resuspended in 20 μl of the same buffer and 20 μl of the stop solution.For analysis of the TEC stability, transcription was initiated on a linear DNA fragment containing the T7A1 promoter using an incomplete NTP set , and the reaction was stopped after 10 min. To determine the efficiency of transcription termination, the ratio of terminated (101 nt) to the sum of terminated and run-off (150 nt) products was calculated. Analysis of hisP pausing after promoter-dependent transcription initiation was performed in a similar way, using a linear DNA template containing the WT hisP pause sequence fused to the λ PR promoter. TECs stalled at +26 template position were obtained after transcription initiation with an incomplete set of nucleotides , then all four NTPs were added , and RNA synthesis was performed for increasing time intervals at 37 °C.For analysis of intrinsic transcription termination, transcription was performed on a linear DNA fragment containing the T7A1 promoter and λ tR2 terminator. Promoter complexes were obtained at 37 °C as described above, NTPs were added  or cognate CTP (1 mM) were added together with MgCl2 (10 mM) at 20 °C to initiate RNA extension, and the reactions were stopped after increasing time intervals. For analysis of intrinsic RNA cleavage, MgCl2 (10 mM) was added to the rA-dG TEC at 37 °C in the absence of NTP substrates, and the kinetics of RNA cleavage was monitored over time.Analysis of nucleotide misincorporation and mismatch extension was performed in fully complementary (rA-dT) and mismatched (rA-dG) TECs, respectively . The TEC32-labeled RNA was mixed with the template and nontemplate DNA oligonucleotides  in the transcription buffer at 65 °C and slowly cooled down to 25 °C. The samples were diluted to 10 nM RNA concentration, core RNAP was added to 25 nM, the samples were incubated for 10 min at 25 °C, NTPs were added to 100 μM, and the reactions were stopped after increasing time intervals. When indicated, DksA (2 μM) and ppGpp (200 μM) were added 5 min prior to NTP addition.For analysis of translesion transcription, TECs were reconstituted using DNA oligonucleotides containing modified nucleotides downstream of the RNA 3′-end  as descrmax×e x p(-kobs × t), where A is the RNAP activity measured at a given time point, Amax is the projected maximal activity at zero time point, kobs is the observed rate constant for promoter complex dissociation, and t1/2=ln2/kobs. The activities of WT and mutant RNAPs on the rrnB P1 promoter relative to the RNA I promoter were obtained by dividing the amounts of corresponding RNA products synthesized by each RNAP. The efficiencies of pausing in the consP and hisP TECs were calculated as the ratio of the paused RNA to the sum of the paused and read-through RNAs. The rates of the pause decay and the pause half-life times were calculated by fitting the data to the single-exponential equation P = Pmax × exp(-kobs × t) + B, where P is the pausing efficiency, Pmax is the projected pausing at zero time point, kobs is the observed rate constant for the pause decay, and B is the remaining fraction of permanently paused TECs; t1/2=ln2/kobs. To calculate the rates of intrinsic RNA cleavage, the efficiencies of RNA cleavage were calculated for each time point by dividing the amounts of the 13 nt cleavage product by the sum of 13 nt and 15 nt RNAs, and the data were fitted to the single-exponential equation C = Cmax × (1 – exp(–kobs × t)) + B, where C is the fraction of cleaved RNA, Cmax is the maximal cleavage, B is a background level of cleavage at zero time point, and kobs is the observed rate of the reaction. Statistical significance of the observed differences in the reaction rates was analyzed using Student's unpaired t test.In all cases, RNA products were analyzed by 10%, 15%, or 23% PAGE and quantified with a Typhoon 9500 scanner using ImageQuant Software . The half-life times of T7A1 promoter complexes were calculated by fitting the data to the single-exponential equation A = AAll experimental data are available from the corresponding authors upon request.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Background: Obtaining high quality RNA from skin biopsies is complex due the physical composition and high content of nucleases of this tissue. This becomes particularly challenging when using compromised skin samples with necrotic, inflammed or damaged areas, such as those from patients suffering skin conditions, which affect more than 900 million people annually. We evaluated the impact of the biopsy size and tissue preservation method on the quality and quantity of RNA extracts.Methods: Skin lesion biopsies were obtained from patients with cutaneous leishmaniasis (CL). Biopsy specimens of 2 mm (n = 10) and 3 mm (n = 59) were preserved in Allprotect® reagent, and 4 mm biopsies in OCT (n = 54). Quality parameters were evaluated using Nanodrop and Bioanalyzer. The informativeness of the extracted samples for downstream analyses was evaluated using RT-qPCR and RNA-Seq.Results: The success rate, based on quality parameters of RNA extraction from tissue biopsies stored in OCT and 2 mm biopsies stored in Allprotect®, was 56% (30/54) and 30% (3/10), respectively. For 3 mm skin biopsies stored in Allprotect® was 93% (55/59). RNA preparations from 3 mm-Allprotect® biopsies had an average RIN of 7.2 ± 0.7, and their integrity was not impacted by sample storage time (up to 200 days at -20°C). RNA products were appropriate for qRT-PCR and RNA-seq. Based on these results, we propose a standardized method for RNA extraction from disrupted skin samples. This protocol was validated with lesion biopsies from CL patients (n = 30), having a success rate of 100%.Conclusions: Our results indicate that a biopsy size of 3 mm in diameter and preservation in Allprotect® for up to 200 days at -20°C, are best to obtain high quality RNA preparations from ulcerated skin lesion biopsy samples. Although the skin is one of the most accessible clinical samples, it is a defiant tissue for RNA/DNA-based research because it contains a high level of nucleases, rendering nucleic acids highly susceptible to degradation. Due to the stability of the hyaluronic acid-collagen matrix, skin disruption and homogenization require mechanical and chemical forces that can damage nucleic acids. Therefore, obtaining high-quality nucleic acids becomes particularly challenging from tissues such as ulcerated skin, and thus downstream analyses are often minimally informative or biased by the method used for DNA and RNA preparations.The skin is the principal anatomical barrier for protection against environmental exposure to microorganisms, xenobiotics, and other insults that could damage the host. Among these are a number of neglected tropical skin diseases, of which cutaneous leishmaniasis (CL) contributes to more than one million cases annually. We aimed to provide a standardized protocol for obtaining good quality RNA from inflamed skin tissue, and to describe the most appropriate conditions for sample collection, storage, and RNA extraction from human samples.Tissue samples with necrotic or damaged areas due to infectious diseases or trauma, or with substantial inflammatory infiltrate and/or edema, are prone to high DNA/RNA degradation. This is the case of dermal lesion samples from patients with skin conditions caused by chronic non-communicable or infectious diseases, which affect over a billion people globally each yearEthics approval: Study protocols, consent forms, and all procedures were approved by Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM) Institutional Review Board for the ethical conduct of research involving human subjects, in compliance with national  and international  guidelines. All participants provided written informed consent.Samples: This study was based on a secondary analysis of data from skin biopsy samples from patients with parasitological confirmation of CL recruited as part of two projects carried out during different periods at CIDEIM in the cities of Cali and Tumaco, Colombia. The samples were divided into two groups according to the reagent used for tissue storage. The first group (Group 1) included 54 lesion biopsies from 27 CL patients enrolled between the years 2009–2015. Lesion biopsies were obtained from patients before initiation of antileishmanial treatment with meglumine antimoniate (n = 27) and at the end of treatment (n = 27). Group 2 included 69 biopsies of CL lesions from 24 patients enrolled between the years 2016–2019. Sequential biopsies from the same lesion of each patient were obtained before (n = 24), in the middle (n = 24), and at the end of treatment (n = 21). Group 3 was defined as a validation group, which included 30 lesion biopsies obtained before treatment from 30 CL patients. Skin biopsies were taken under local anesthetic, and one sample was taken at a time. The punch biopsy was obtained considering the following ratio: 1/3 of healthy skin and 2/3 of the edge of the lesion (the indurated edge did not include necrotic tissue) (tissue) .Tissue processing: Overall summary of skin biopsy collection, preservation, disruption, homogenization, and RNA extraction for the groups ( groups .Group 1: Punch biopsies of 4 mm were frozen in Tissue-Tek™ OCT Compound (Sakura Finetek Europe B.V # 4583) and cryopreserved in liquid nitrogen until processing. For evaluation of gene expression, 20 tissue sections of 10 µm each were obtained before RNA extraction and stored back in liquid nitrogen. The sections were removed from the nitrogen tank and immediately homogenized by strong vortexing for 1 min with RLT buffer . Due to the high protein content of the skin, the sections were subjected to enzymatic denaturation with proteinase K  for 10 minutes at 55°C. RNA was extracted using the RNeasy Mini Kit, following the manufacturer's protocol and using DNAse digestion  for the removal of contaminating genomic DNA. RNA was eluted from the RNAeasy column in 14 µL of RNase-free water (Qiagen) and stored at -80°C until further use. For cDNA synthesis, RNA (100 ng) was transcribed with the High-capacity cDNA reverse transcription kit . Gene expression was measured in real-time with the CFX96 Real-Time System (RRID:SCR_018064)  using Taqman Universal PCR master mix  and Taqman gene expression assays forGAPDH gene (Hs99999905_m1) (Thermofisher Scientific), with the following protocol: denaturation at 95°C for 10 minutes, followed by 49 cycles of 95°C for 15 seconds, and 60°C for 1 minute, or using PCR arrays  that included the GAPDH gene (PPH00150F) and RT² SYBR green qPCR mastermix , with the following protocol: denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. Ct values were analyzed in theCFX ManagerTM 3.1 software #1845000 (Bio-Rad) (RRID:SCR_017251).Group 2: Skin biopsies of 2 mm (n = 10) and 3 mm (n = 59) were taken and immediately stored in 1 mL Allprotect® (Qiagen). Samples were equilibrated overnight at 4°C and then stored at -20°C until processing. The tissue sample was cut into smaller pieces with sterile scalpel blades, followed by tissue disruption and homogenization using a manual glass tissue grinder on an ice bath containing Trizol® for no more than 5 minutes (https://dx.doi.org/10.17504/protocols.io.x54v9yn7zg3e/v1). The lysate was centrifuged together with chloroform solution at 13000 rpm for 15 minutes, and the aqueous phase was mixed with ethanol and passed through RNA purification columns , following the manufacturer's instructions. RNA was eluted from the RNAeasy column in 25 µL of RNase-free water (Qiagen) and stored at -80°C until it was used for analysis. cDNA was synthesized using the RT first-strand synthesis kit . RT-qPCR reactions were run on a CFX96 Real-Time System (RRID:SCR_018064) (Bio-Rad) using Taqman Universal PCR master mix  and Taqman gene expression assays forGAPDH gene (Hs99999905_m1) (Thermofisher Scientific), with the following protocol: denaturation at 95°C for 10 minutes, followed by 49 cycles of 95°C for 15 seconds, and 60°C for 1 minute or using custom made PCR arrays  that included the GAPDH gene (PPH00150F) and RT² SYBR green qPCR mastermix , with the following protocol: denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. Ct values were analyzed in the CFX ManagerTM 3.1 software #1845000 (Bio-Rad) (RRID:SCR_017251).Group 3 : Skin biopsies of 3 mm (n = 30) were taken and immediately stored in 1 mL Allprotect® (Qiagen). Samples were processed with the RNA extraction protocol described for group 2, and five samples were used for RNAseq.RNA quality assessment: The quantity and purity of the extracted RNA was evaluated in a Nanodrop ND-1000 spectrophotometer (RRID:SCR_016517). The acceptable ranges of absorbance ratios at 260/280 and 260/230 were 1.8–2.0 and 1.8–2.2, respectively. For the samples stored in OCT, the RNA integrity was evaluated by the presence of two intact ribosomal bands (18S and 28S) in agarose gel electrophoresis (1.3%). For samples stored in Allprotect®, this was assessed using an Agilent 2100 Bioanalyzer (RRID:SCR_018043) ; an RNA integrity number (RIN) value of ≥7 was defined as good quality RNA.RNA-Seq: Construction of cDNA libraries was performed using the Illumina TruSeq® Stranded mRNA kit V2. The quality evaluation and quantification of the libraries were carried out using the Agilent 2100 Bioanalyzer (RRID:SCR_018043) . The samples were sent to the University of Maryland Bioscience and Biotechnology Research Institute for pair-end 100bp Illumina HiSeq RNA-seq. The quality control of the sequences and the assembly were performed using standard pipelines developed for CL lesion transcriptomic analyses. Bioinformatic deconvolution of transcriptomes (n=5) was performed usingxCell.Statistical analyses: The Kolmogorov-Smirnov and Shapiro-Wilk tests were used to determine the parametric or nonparametric distribution of the data. One-way analysis of variance (ANOVA) or Kruskal-Wallis test followed by multiple-comparison tests were employed for group comparisons. For nonparametric data, comparisons were analyzed using the Wilcoxon signed-rank test. Statistical significance was defined at ap-value < 0.05. All data were analyzed usingGraphPad Prism 6 software (RRID:SCR_002798), .. Based on a comparative analysis, we then propose a standardized method for preservation, processing, and RNA extraction of ulcerated skin lesion samples.RNA quality is essential for obtaining meaningful and reliable results from gene expression data, even more when samples are irreplaceable such as those collected from human participants of clinical studies. Here, we report the quality characteristics of RNA samples obtained from skin biopsies from ulcerated cutaneous lesions from CL patients, which were preserved and processed using different commercially available protocolsThe size of the skin biopsy rather than the sample preservation method determines the quantity of RNA extracted from punch biopsy samples. Obtaining high-quality RNA in sufficient quantity from small skin biopsy samples is challenging due to the alteration of the tissue and the activation of endogenous RNases, which makes the skin one of the most difficult tissues from which to obtain intact RNA. Initially, large amounts of frozen skin tissue were used (>100 mg) and downsized to less than 10 mg of tissue per sample. Previous studies in mice have shown that the minimum size of a skin biopsy to consistently obtain sufficient RNA for transcriptomics experimentation was a 1.5 mm skin punch biopsy. However, these results were based on intact skin samples. In our study, we address one additional difficulty: how to obtain good quality and good quantity RNA samples for downstream gene expression analyses from compromised human skin tissue such as those from CL patients whose lesions are highly inflamed and ulcerated.p <0.0001). This shows that the amount of tissue, rather than the preservation medium, is the principal factor affecting the RNA quantity in skin biopsy extracts and that lesion skin biopsies with a size < 3 mm should not be used for samples which include areas of high inflammatory infiltrate, edema or necrosis.RNA concentrations obtained from processing the entire 2 mm biopsies stored in Allprotect®, and the 20 sections of 10 µm each of the 4 mm biopsies stored in OCT , were comparable  and togeRNA samples from 3 mm skin biopsies stored in Allprotect®outperform in terms of sample purity and RNA integrity. RNA samples without protein or solvent contamination should have a 260/280 absorbance ratio of 1.8–2.0 and 260/230 of 2.0–2.2, respectively. Tissues preserved in OCT and Allprotect® (3 mm) had good and comparable 260/280 ratios: 2.04 and 2.06, respectively. Biopsies of 2 mm stored in Allprotect® had a significantly lower 260/280 ratio of 1.64 (of 1.64 . Only 3 of 1.64 . Accordip = 0.82)  , indicat= 0.82) .. However, some RNA degradation has been documented in tissues preserved for one week in Allprotect® at 8°C, and protein degradation after two weeks. In a comparative study using pig skin, biopsies were immediately stored as freeze-dry biopsies, or stored in Allprotect®, QIAzol lysis reagent, or RLT lysis buffer containing beta-mercaptoethanol. All four strategies resulted in RNA with high RIN values (≥ 8.5); however, samples in Allprotect® and QIAzol had lower OD 260/230 indices, which we also observed for human ulcerated skin samples preserved in Allprotect®.Several commercial RNA stabilization reagents are available for tissue preservation, minimizing the requirement of immediate sample processing and freezing. These systems allow the collection and shipment of samples from rural areas to central laboratories without the requirement of complex shipping conditions, such as liquid nitrogen tanks. Skin tissue preservation in Allprotect® offers the advantage of avoiding intermediate tissue freezing compared to OCT. Other studies have also demonstrated effective stabilization of tissue from primary colorectal tumor samples, breast cancer, and colorectal liver metastases with Allprotect®, resulting in high-quality isolated DNA, RNA, and proteins when compared to samples preserved in liquid nitrogenEffects of RNA preparations on downstream applications. The success rate of RNA extraction was defined according to the number of samples that met the quality parameters (integrity and purity thresholds) over the total number of samples corresponding to their study group . A new cohort of 30 CL patients from which 3 mm lesion biopsy samples were obtained, was evaluated as a validation cohort for the suggested protocol, achieving a success rate of 100% (30/30), based on the integrity parameters defined above. The average concentration of RNA was 527.5 ± 368.2 ng/µL, the 260/280 and 260/230 indexes were 2.05 ± 0.04 and 2.18 ± 0.09, respectively, and RIN 7.1 ± 1.L (V). panamensis (L.V. panamensis (v36) genomes using HISAT2.2.1 (RRID:SCR_015530). On average, 93.8 ± 2.1% of the total number of reads aligned to the human genome, while 1.2 ± 1.5% toL. V. panamensis.As a proof-of-principle of the adequacy of 3 mm skin biopsy samples preserved in Allprotect® for in-depth RNA-based processes, cDNA libraries were constructed from RNA samples from lesions of 5 CL patients infected withamensis . cDNA liamensis . The num, and compared this to previously published immune cell characterization of CL lesions. Past characterization studies of the cell populations present in lesions of CL patients, have been based on immunohistochemistry and flow cytometry, which are restricted to the study of a small group of cells. Through deconvolution analysis of RNA-Seq data, we pursued an alternative method to overcome this limitation. xCell performs cell-type enrichment analysis from gene expression data for 64 immune cells and stromal types, generating a score that refers to the estimate of the cell population. Bioinformatic results show that CL lesions were characterized by the presence of structural skin cells , and an important infiltrate of immune cells which included B cells (memory and plasma cells), lymphocyte subpopulations CD4+ , CD8+ , followed by macrophages and dendritic cells , lymphocyte subpopulations CD4+ , and CD8+ . These subpopulations have also been reported in targetted phenotypic analyses of CL lesions.We performed a deconvolution analysis to infer the immune cell composition of the tissues using xCellc cells . A immunIn conclusion, we present a validated methodology to obtain good quality RNA from ulcerated skin samples for use in translational research studies.We acknowledge that imbalanced sample sizes between groups may impact the overall statistical power for comparisons between groups. The 2mm-Allprotect® group had only 10 samples compared to the 59 samples of the 3mm-Allprotect®. This was due to the prospective recruitment strategy of study participants, initiated with collection of 2mm biopsy samples. Upon preliminary analyses the performance of 2mm biopsies was low, leading us to switch to 3mm biopsy samples. Analysis of the first 9 3mm-Allprotect® biopsies evidenced a considerable improvement in the parameters of RNA concentration, purity and integrity. Therefore, in the interests of protection of study subjects and sample integrity we stopped sample procurement with 2mm-Allprotect® (see raw data in: data availability).Written informed consent for publication of the patients’ details and their images was obtained from the patients. The revised manuscript can be accepted.Is the rationale for developing the new method (or application) clearly explained?PartlyIs the description of the method technically sound?YesAre the conclusions about the method and its performance adequately supported by the findings presented in the article?YesIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?YesAre sufficient details provided to allow replication of the method development and its use by others?YesReviewer Expertise:Bacterial infectionsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors validated a successful method to obtain good quality RNA from ulcerated skin samples for use in translational research studies. Their results showed a biopsy size of 3 mm in diameter and preservation in Allprotect® for up to 200 days at -20°C, are best to obtain high quality RNA preparations from ulcerated skin lesion biopsy samples. The manuscript is reasonably well written and presented. It is a pity that the authors haven't evaluated the quality of the tissue stored in OCT media. So, the RNA quality for medium storage can't be compared. This needs to be added into the limitation of the study. Another limitation is that the samples size of 2mm-Allprotect® biopsies is small, only 10 samples compared with 59 samples of 3mm-Allprotect® biopsies. The discussion also needs to address why the small biopsy size affects RNA quality.Is the rationale for developing the new method (or application) clearly explained?PartlyIs the description of the method technically sound?YesAre the conclusions about the method and its performance adequately supported by the findings presented in the article?YesIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?YesAre sufficient details provided to allow replication of the method development and its use by others?YesReviewer Expertise:Bacterial infectionsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. In the current manuscript, the authors aimed to optimize the quality of RNA isolated from damaged skin using biopsies of varied sizes preserved in different ways.  The authors successfully showed that both size and preservation methods ultimately interfere with the quality and amount of RNA obtained from cutaneous lesions. They showed that 3mm biopsies preserved in Allprotect offered the best results. The authors also validated their standardized method using an extra 30 samples, proposing an efficient preservation method for skin biopsies that will be reserved for gene expression assays.  The manuscript is overall very organized and well-written.Is the rationale for developing the new method (or application) clearly explained?YesIs the description of the method technically sound?YesAre the conclusions about the method and its performance adequately supported by the findings presented in the article?YesIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?YesAre sufficient details provided to allow replication of the method development and its use by others?YesReviewer Expertise:Transcriptomics, molecular biology and immunology.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


A recent report on the detection of the ncRNA Signal Recognition Particle gene in the nuclear ribosomal internal transcribed spacer region of several species of three genera of ectomycorrhizal basidiomycetes prompted a more thorough bioinformatics search for additional ncRNA genes in the full fungal ribosomal operon. This study reports on the detection of three ncRNA genes hitherto not known from the fungal ribosomal region: nuclear RNase P RNA, RNase MRP RNA, and a possible snoRNA U14 in a total of five species of Auricularia and Inocybe. We verified their presence through resequencing of independent specimens. Two completed Auricularia genomes were found to lack these ncRNAs elsewhere than in the ribosomal operon, suggesting that these are functional genes. It seems clear that ncRNA genes play a larger role in fungal ribosomal genetics than hitherto thought.Non-coding RNA ( A range of functions has been ascribed to the various groups of ncRNAs known to date, including important roles in translation, gene regulation, and chromosome inactivation , and they constitute the RNA component of the respective ribonucleoprotein (RNP) particles  RNAs in the nuclear ribosomal ITS1 region of 11 species in three ectomycorrhizal genera of the Basidiomycota: Astraeus, Russula, and Lactarius. Indirect evidence furthermore suggested that these SRP RNAs could be functional. Queries in the GenBank  region of a set of Auricularia and Inocybe species, and we submit that ncRNAs are elements that can no longer be disregarded in the context of fungal ribosomal biology.The recent trend of employing various high-throughput sequencing technologies to generate longer stretches of the fungal ribosomal operon than just the ITS region  offers aInocybe, we also sequenced the full ribosomal operon of four Inocybe species: Inocybecincinnata, Inocybeflocculosa, Inocybeleiocephala, and Inocybephaeocystidiosa. We queried these, too, for any new ncRNAs. We considered only highly significant matches that passed the Rfam E-value threshold applied for each gene. We then double-checked all matches by detailed manual examination of all conserved motifs as well as the secondary structure to filter out any partial or spurious candidates.Since ncRNAs are conserved primarily on the secondary structure level rather than the primary sequence level, we queried GenBank for fungal ribosomal ncRNAs using the secondary structure covariance models from the Rfam database  and DumencRNA matches belonging to four different ncRNA genes in the IGS1 region of several Auricularia species from After quality control filtering, our GenBank query produced more than 30 highly significant Auricularia and four species of Inocybe for sequencing of the full ribosomal operon. The fungal DNA was extracted using a DNA plant Mini Kit (Qiagen) and subsequently amplified using the primers NS1rc and RCA95rc or Fun-rOP-F/Fun-rOP-R as detailed in Auricularia and Inocybe consensus sequences were queried for the presence of ncRNAs as detailed above.We targeted two species of Auricularia specimens may be functional – rather than pseudogenes – presented itself through the draft genome assemblies of Auriculariaheimuer  in the IGS1 region of several Auricularia species in GenBank  in the IGS1 of Inocybecincinnata, Inocybeflocculosa, and Inocybeleiocephala; however, Inocybephaeocystidiosa was found to lack all of the above ncRNAs in the ribosomal operon  was found to contain a ribosomal stretch comprising the expected rRNA genes nuclear small-subunit (nSSU), 5.8S in the ITS region, nuclear large-subunit (nLSU), and 5S but also the RNase P, MRP, and SRP genes, plus a putative U14/SNORD14 ncRNA copy. The result was the same for Auriculariacornea. No other copies of these ncRNAs were found in any other part of the genome assembly. Auriculariasubglabra strain TFB-10046 SS5 (AFVO01) produced a similar result: we could not identify any of the ncRNAs in the genome assembly. Unfortunately, for this species the rDNA region was not included in the assembly or available elsewhere, but it seems probable that these ubiquitous ncRNAs would be located in the same region as in A.heimuer. The same result was obtained for Auriculariaauricula-judae strain B14-8 (NCVV01). However, in the genome assembly of Auriculariapolytricha strain MG66 (QFEN01), these ncRNAs were found on different contigs and none of these contigs contained any rRNA, implying those are the functional copies should additional ones exist also in the rDNA region. Interestingly, the A.polytricha strains AP112 and APFJ do have these ncRNAs in the IGS1 region, but there is no genome assembly available for either AP112 or APFJ.One of the contigs of the draft genome of ncRNA other than the standard SSU, 5.8S, LSU, and 5S genes – namely the SRP RNA – in the ITS region of fungi. We expand on those findings by highlighting not only the SRP RNA gene but also an additional three ncRNA genes – nuclear RNase P RNA, RNase MRP RNA, and a possible U14 – in the IGS region of several Auricularia species. We verified the presence of these IGS ncRNAs through DNA sequencing of conspecific specimens. We furthermore used sequencing to recover the SRP RNA and a putative copy of the U14 gene in the IGS region of three Inocybe species. Our findings suggest that the SRP RNA results of SRP RNA gene seems to have been independently and repeatedly incorporated into the ribosomal operon of numerous fungi. This certainly warrants further investigation.SRP RNA, we also found strong evidence for two other ncRNAs in the IGS1 of both Auricularia specimens we sequenced – Auriculariadelicata (MFLU16-2118) and Auriculariacornea (MFLU16-2108) – namely RNase P RNA and RNase MRP RNA. These genes correspond to important components for the maturation of tRNAs and rRNAs, respectively  basidiomycete genus Auricularia. This suggests that fungal nutritional mode may not determine or require the presence of these ncRNAs in the ribosomal operon, something that would be interesting to pursue in light of further data. It should nevertheless be pointed out that all five fungal genera from which ribosomal operon ncRNAs have been reported - Astraeus, Russula, Lactarius, Inocybe, and Auricularia – belong to the class Agaricomycetes of the Basidiomycota. The significance of this is unclear, but even a cursory glance at the finer levels of the Basidiomycota phylogeny shows that multiple independent gains/losses are needed to explain the observed ncRNA distribution. The fact that a single fungal class has seen a multitude of these events, whereas no other fungal class seems to have seen even a single one, certainly calls for an explanation.Interestingly, whereas our former study found Agaricomycetes fungi at that. The ribosomal operon is routinely excluded from many genome assemblies due to assembly difficulties  RNA – in the internal transcribed spacer (ITS) region of three ectomycorrhizal genera of basidiomycetes. There are indications that these are functional genes rather than pseudogenes. The occurrence of these non-coding RNAs and their distribution in the fungal tree of life calls for further research attention but also caution in, e.g., multiple sequence alignment-based phylogenetic inference efforts involving the ribosomal regions of these fungi.This study reports on the detection of three non-coding RNA genes hitherto not known from the fungal ribosomal region: nuclear RNase P RNA, RNase MRP RNA, and a possible snoRNA U14 in a total of five species of


A non-canonical DNA/RNA structure, G-quadruplex (G4), is a unique structure formed by two or more guanine quartets, which associate through Hoogsteen hydrogen bonding leading to form a square planar arrangement. A set of RNA-binding proteins specifically recognize G4 structures and play certain unique physiological roles. These G4-binding proteins form ribonucleoprotein (RNP) through a physicochemical phenomenon called liquid-liquid phase separation (LLPS). G4-containing RNP granules are identified in both prokaryotes and eukaryotes, but extensive studies have been performed in eukaryotes. We have been involved in analyses of the roles of G4-containing RNAs recognized by two G4-RNA-binding proteins, TDP-43 and FUS, which both are the amyotrophic lateral sclerosis (ALS) causative gene products. These RNA-binding proteins play the essential roles in both G4 recognition and LLPS, but they also carry the risk of agglutination. The biological significance of G4-binding proteins is controlled through unique 3D structure of G4, of which the risk of conformational stability is influenced by environmental conditions such as monovalent metals and guanine oxidation. The G4 e genome . Such G4n genome . Some ofn genome   gene, TDP-43, FUS, hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), hnRNP A2/B1, hnRNPA3, EWSR1 (Ewing’s sarcoma RNA binding protein 1), and TIA1 (T cell-restricted intracellular antigen-1)  and MALAT1 (metastasis associated in lung adenocarcinoma transcript 1/NEAT2) contain abundant G4 motifs  . Surpristigen-1) . For ins4 motifs . More in4 motifs . RNP gra4 motifs . RNP gra4 motifs . Dysregu4 motifs .The G4-binding proteins often carry one or more intrinsically disordered regions (IDRs) . In geneIntrinsically disordered proteins drive protein clustering and subsequent LLPS using their IDRs . These GThe best characterized ALS-linked G4-RNA binding proteins, TDP-43 and FUS, share structural and functional similarities, including participation in ALS pathogenesis . RNP graThe effects of amino acid substitution mutations from ALS patients are common to these two proteins, and TDP-43 and FUS mutant proteins show reduced interaction with G4.  Figure . ALS-linThe assembly of RNP granules by G4-RNA and G4-RNA binding proteins control many biological processes, including mRNA transport from soma to distal neurites for local translation. However, there are several risks associated with the molecular properties of G4-RNA and G4-RNA binding proteins that lead to perturbation of the interplay between G4-RNA and G4-RNA binding protein including the pathogenesis of ALS. Several factors are shown below :ALS is characterized by the accumulation of protein aggregates in motor neurons . HoweverOxidative stress is a risk factor of neurodegeneration and also a therapeutic target . Mutatio+ is known as a factor that interferes with the uptake of Na+ and K+, which stabilizes the G-quadruplex structure in vitro. Although Na+ and K+ are the physiologically relevant monovalent ions in the context of G4 structures, however, Li+ with smaller ionic radius than Na+ and K+ causes destabilization of G4 conformation (+ has not been observed, administration of lithium to ALS patients resulted in almost 70% of adverse events, including death (G-quartets are stabilized in the presence of cations, but different cations result in different structural properties . Li+ is ormation . Althougng death .G4-associated regulations are involved in varieties of biological systems, including replication, recombination, telomere regulation, transcriptional regulation, RNA processing, RNA stability, translation, RNA transport, and stress responses. Although many of them have long been overlooked without experimental confirmation, but the advent of a new age arrived to reveal molecular details underlying the interaction of G4-RNAs and G4-binding proteins. The dysregulation of G4-RNAs and G4-binding proteins has been identified as neurodegenerative diseases in human. Risk factors in G4 and G4 binding proteins are involved in the pathogenic mechanism of ALS (for instance, E. coli. It may provide a platform for analyzing the formation of complex structures in G4.As an extension of this line research, we have started to identify the role of G4 in the most well-characterized prokaryote


Heterogeneous nuclear ribonucleoproteins (hnRNP) function in RNA processing, have RNA-recognition motifs (RRMs) and intrinsically disordered, low-complexity domains (LCDs). While RRMs are drivers of RNA binding, there is only limited knowledge about the RNA interaction by the LCD of some hnRNPs. Here, we show that the LCD of hnRNPA2 interacts with RNA via an embedded Tyr/Gly-rich region which is a disordered RNA-binding motif. RNA binding is maintained upon mutating tyrosine residues to phenylalanines, but abrogated by mutating to alanines, thus we term the RNA-binding region ‘F/YGG motif’. The F/YGG motif can bind a broad range of structured (e.g. tRNA) and disordered (e.g. polyA) RNAs, but not rRNA. As the F/YGG otif can also interact with DNA, we consider it a general nucleic acid-binding motif. hnRNPA2 LCD can form dense droplets, by liquid–liquid phase separation (LLPS). Their formation is inhibited by RNA binding, which is mitigated by salt and 1,6-hexanediol, suggesting that both electrostatic and hydrophobic interactions feature in the F/YGG motif. The D290V mutant also binds RNA, which interferes with both LLPS and aggregation thereof. We found homologous regions in a broad range of RNA- and DNA-binding proteins in the human proteome, suggesting that the F/YGG motif is a general nucleic acid-interaction motif. The concept of the ‘RNA world’ rests on the idea that early evolution was dominated by RNA molecules capable of genetic information storage as well as its replication via catalytic RNA enzymes, ribozymes . In a maThe very broad range of protein–RNA interactions encoded by RBPs play key roles in basic RNA-related processes of the cell, such as the assembly of ribosomes, regulation of transcription and mRNA splicing, RNA editing or signal sensing, with such intriguing examples as bacterial immunity relying on the CRISPR/Cas system . ImportaDue to diverse functions and regulatory roles, RNA recognition by RBPs has been extensively studied. Most of our insight into the underlying details derives from studying and structurally characterizing specific RNA-binding regions  of RBPs. About 17 such ‘canonical’ RBDs, such as RNA-recognition motif (RRM), hnRNP K homology domain (KH), DEAD box helicase domain, double-stranded RNA-binding domain (dsRBD), and cold-shock domain (CSD), are known . In chemA highly exciting field where RNA binding by IDRs and canonical RBD(s), might be of particular relevance is liquid–liquid phase separation (LLPS), in particular by the family of heteronuclear ribonucleoproteins (hnRNPs), such as TDP-43, FUS, hnRNPA1 and hnRNPA2 . These pMotivated by these results and the general existence of RNA-binding motifs in IDRs, we have scrutinized hnRNPA2, a protein involved in basic cellular pathways, like RNA processing and the formation of RNA transport granules , and alshttp://n2t.net/addgene:98657; RRID:Addgene_98,657), and hnRNPA2 LCD MBP were a gift from Prof. Dr. N. Fawzy Y[G/S] domain and, as stated above, to the FG domain. The [G/S]Y[G/S] sequence element has been identified as important for LLPS and aggregation. The Tyr can be changed to Phe, highlighting the importance for aromatic stacking. In the case of the F/YGG motif however, it is one (or more) glycine(s) neighbouring the aromatic residue, never a serine. However, while there can be an easy evolutionary switch from glycine to serine (only one nucleotide change), this switch is not observed even in distant hnRNPA2 homologues. Thus, while the modes of action of these three motifs are probably very related, they are not interchangeable, but three distinct motifs ,64.−33, respectively, one-tailed paired t-test) .It is also important to note the role of phosphorylation as a key mechanism of the aforementioned regulatory circuits. How phosphorylation suppresses the LLPS of the hnRNP protein family is a highly studied question in the field . InteresBy addressing the effect of various nucleic acids on the LLPS hnRNPA2 LCD, we found that all types of RNA, both folded and unfolded, and independent of sequence, had a similar influence, namely increasing the dilute phase concentration, and decreasing the number and size of droplets. The only exception is rRNA, which had no influence on LLPS. Of possible relevance, it has been seen before that as riboparticles mature towards rRNA, they become less phase separation prone , and rRNIn general, RNA has been observed to play crucial roles in both promoting and inhibiting LLPS in a concentration-dependent manner, with higher RNA concentrations generally inhibiting LLPS . FurtherAs about half of RNA-binding proteins have no identified RNA-binding domains, and much of such regions fall into disordered regions, the F/YGG repeat motif may be an important addition to the growing list of RNA-binding motifs . Furtherin vivo, RBPs typically have modular architecture containing multiple RNA-interacting regions [An interesting aspect of this RNA-binding IDR domain may derive from the fact that hnRNPA2 also contains other, canonical RNA-binding domains, RRMs. As affinity and specificity of a single RBDs is often not sufficient to provide selective binding  regions , enablin regions ; this isWith regard to mechanistic details of the interaction of F/YGG motif and RNA, protein–RNA interactions most often rely on electrostatic attraction and hydrogen bonds, whereas hydrophobic interactions between RNA bases and hydrophobic side provide further stabilization. In certain cases, hydrophobic interactions may account for 50% of the protein–RNA interface, in the form of pi-pi stacking . It has It is to be noted that the excessive presence of Gly and aromatic residues could also have a dangerous edge. Namely, it has been shown that Gly is intrinsically destabilizing in beta sheets. However, pairing of Gly with Phe or Tyr allows the presence of Gly in beta sheets to be thermodynamically favourable. Therefore, the excessive presence of Gly + aromatic residues could lead to beta sheet aggregation. Indeed, LARK domains (Low-complexity Aromatic-Rich Kinked Segments) are highly aggregation prone, and enriched in similar residues . ConclusIts occurrence in a broad range of proteins raises interesting structural, molecular and functional question we plan to scrutinize in the near future. To conclude, we show the F/YGG motif as a novel, important disordered DNA- and RNA-interaction domain, as exemplified by hnRNPA2 LCD.Click here for additional data file.


Several functions have been attributed to tRFs and tiRs since their initial characterizations, spanning all aspects of regulation of the Central Dogma: from nascent RNA silencing, to post-transcriptional gene silencing, and finally, to translational regulation. The length distribution, sequence diversity, and multifaceted functions of tRFs and tiRs positions them as attractive new models for small RNA therapeutics. In this review, we will discuss the principles of tRF biogenesis and function in order to highlight their therapeutic potential. The widespread introduction of small RNA sequencing technologies has uncovered a wide variety of non-microRNA (miRNA) small RNAs (nmsRNA) . tRNA frInitial characterizations focused primarily on tRFs having a miRNA-like mechanism, due to the similar size distribution between miRNAs and tRFs . To datetRNA derived small RNAs are divided into two major classes based on length: shorter tRFs and longer tRNA halves . The twoThe discrete length distributions of tRFs suggest specific cleavage by cellular ribonucleases. However, with the exception of tRF-1s, a general biogenesis mechanism for highly expressed tRFs remains to be elucidated. tRF-1s were one of the first tRFs to be characterized . tRF-1s tiR biogenesis may also have more than one mechanism. Initially, tiRs were found to be upregulated under various stress conditions, giving them the name stress-induced tRNA halves. The RNase IV enzyme, angiogenin (ANG), was identified to be the enzyme responsible for tiR production under stress conditions and the 5′-terminal oligoguanylate (TOG) motif was found to be present in these tiRs . HoweverIn order to highlight the potential of tRFs to enhance therapeutic small RNA development, it is important to understand their endogenous cellular roles. The first report of a biological function for tRFs was reported for tRF-1001 . This tRThe lack of miRNA-like functions for tRF-1s was also established in another study . Transfevia mimic transfection, shRNA, or parental tRNA mediated overexpression led to repression of a luciferase reporter. Although these data clearly support a role for tRF-1 mediated repression, it is unclear if the effects are mediated by the SSB/La-DICER1 regulatory axis, as the parental tRNA overexpression followed by luciferase reporter was not conducted in SSB/La or DICER1 deficient cells.In the one case that tRF-1s are associated with miRNA-like repression, the tRF-1 is generated by DICER1 cleavage. DICER1 cleavage of tRNAs can occur when the precursor tRNA maturation protein, SSB/La, is depleted . Under tUnlike tRF-1s, tRF-3s have been shown to readily enter RISC and repress gene expression in a base-pairing specific manner. As alluded to previously, overexpression of parental tRNAs leads to a direct upregulation of tRF expression. Parental tRNA overexpression leads to tRF-3001a, -3003a, and -3009a generation and entry into AGO for efficient repression of luciferase reporters . HaussecThe difference between the ability of tRF-1s and tRF-3s to repress target gene expression is unclear. The answer may lie in the inherent differences between the two classes: 1) different biogenesis enzymes ; 2) tRF-via base-pairing in the context of respiratory syncytial virus (RSV) infection   . Lipid n .Delivery of small RNAs to tissues may also be enhanced by encapsulation in extracellular vesicles . In suppAnother challenge for tissue delivery is oligonucleotide stability in circulation . Much ofThe second challenge of delivery, endosomal escape, is still a major unresolved bottleneck in the oligonucleotide therapeutics field . Small mAlthough tRNA based therapies are outside the scope of this review, it is a burgeoning field of development because nonsense suppressor tRNAs can prevent the formation of truncated, dysfunctional proteins . It is cIt is clear that tRFs/tiRs have important roles in biology in all domains of life. Mechanistically, tRFs/tiRs function differently, even between and within class distinctions. General trends in tRF/tiR’s role in regulating nascent gene silencing, post-transcriptional gene silencing, and mRNA translation make this class of small RNAs one of the most versatile classes discovered to date. Small RNA therapeutics based on basic knowledge about miRNA function are experiencing a renaissance, with several RNAi based therapeutics on the market. Perhaps tRFs and tiR based therapeutics will follow suit. In order for tRFs/tiRs to reach the clinic, several challenges must be addressed. For example, it is clear that tRF-5s and tiR-5s can behave as protein synthesis inhibitors. What is less clear is whether there is an RNA modification or RNA sequence code that is important for tRF-5 and tiR-5 mediated protein synthesis inhibition. For example, PUS7 mediated pseudouridylation is important for protein synthesis inhibition in stem cells . Since tAnother challenge is understanding the role that tRFs play in nascent RNA silencing and post-transcriptional gene silencing. Do all tRFs follow the same rules  as miRNAs when it comes to post-transcriptional gene silencing? What role do tRF modifications play in nascent RNA silencing and post-transcriptional gene silencing? Since most tRF target prediction tools are built on miRNA-based assumptions and rules or more general complementary pairing , are we


Neisseria gonorrhoeae causes the sexually transmitted infection (STI) gonorrhea, which afflicts over 80 million people each year. No vaccine is available to prevent gonorrhea. The pathogen alters the expression and antigenic presentation of key surface molecules, making the identification of suitable vaccine targets difficult. The human host utilizes metal-binding proteins to limit free essential transition metal ions available to invading pathogens, limiting their infective potential, a process called nutritional immunity. To overcome this, N. gonorrhoeae employs outer membrane TonB-dependent transporters (TdTs) that bind host nutritional immunity proteins and strip them of their metal cargo. The TdTs are well conserved, and some play key roles in establishing infections, making them promising vaccine targets. One TdT, TdfJ, recognizes human S100A7, a zinc-binding protein that inhibits the proliferation of other pathogens via zinc sequestration. N. gonorrhoeae uses TdfJ to strip and internalize zinc from S100A7. TdfJ contains a conserved α-helix finger in extracellular loop 3; a similar α-helix in loop 3 of another gonococcal TdT, TbpA, plays a critical role in the interaction between TbpA and human transferrin. Therefore, we hypothesized that the TdfJ loop 3 helix (L3H) participates in interactions with S100A7. We determined the affinity between wild-type TdfJ and S100A7 and then generated a series of mutations in the TdfJ L3H. Our study revealed that mutagenesis of key residues within the L3H reduced S100A7 binding and zinc piracy by the gonococcus, with profound effects seen with substitutions at residues K261 and R262. Taken together, these data suggest a key role for the TdfJ L3H in subverting host metal restriction. Neisseria gonorrhoeae causes the common sexually transmitted infection (STI) gonorrhea, a global disease with serious public health consequences. Gonorrhea infected an estimated 87 million people worldwide in 2016, and the Centers for Disease Control and Prevention (CDC) reported 616,392 cases of gonorrhea in the United States in 2019 . The TdTs are highly conserved across gonococcal isolates and play key roles in overcoming nutritional immunity, the process by which the human host restricts essential nutrients to handicap invading pathogens  to TdfJ in amino acid sequence. The crystal structure of ZnuD (PDB accession numbers 4RDR and 4RDT) shows that extracellular loop 3 contains two zinc-sensing regions, which are enriched in histidine, aspartate, and glutamate. Furthermore, this region undergoes considerable rearrangement in a substrate-dependent manner. When zinc is absent, the region of loop 3 between the zinc-sensing clusters adopts an α-helical configuration that extends far from the TdT barrel. Conversely, in the presence of zinc, this region is remodeled into a pair of flexible β-strands, exposing zinc in the peripheral binding site to a newly available, high-affinity site buried deeper in the barrel   . A schemtdfJ mutants grown with and without IPTG to detect TdfJ production  and identified those that recognized either folded surface-exposed TdfJ, Western-blotted TdfJ, or both (tdfJ knockout (KO) strain was not detected above background levels. The capacity for recognizing TdfJ on the gonococcal cell surface made the mAbs a useful tool for assessing the global fold of mutated forms of TdfJ. We immobilized gonococci producing these mutated proteins on a nitrocellulose membrane and probed their surface with mAbs for comparison to the pattern for the WT -labeled S100A7 (S100A7-HRP) . We founTo ensure that the binding defects seen in the ELISA were not due to protein misfolding or instability induced during purification, we utilized the TdfJ-specific mAbs to probe purified TdfJ mutant proteins. The His-tagged TdfJ mutants were seeded onto a Ni-NTA-coated ELISA plate and subsequently probed with the same set of mAbs as the ones that were used in the surface exposure test . NotablyDK calculation could be made. While the data could likely be fit to a more complex model with more variables, the inability to fit the data to the simpler model was attributed to the much lower sensitivity of the data as a result of the very small range of the response in the sensorgrams, and we concluded that an alternate model was not merited.We next focused on the mutants that were most defective in our previous binding assays, namely, ΔL3H and K261P/R262P. While both mutants showed similar binding deficiencies, we chose to utilize only K261P/R262P for affinity calculations as the point mutant was a better representative overall for TdfJ than ΔL3H, which contained a large deletion. We performed SPR as described above for the WT protein, using His-tagged K261P/R262P TdfJ as the sensor chip ligand and a 1 to 500 nM analyte series of S100A7 . These e4 instead of Zn-S100A7, suggesting that baseline growth defects were not present in the TdfJ mutants and that zinc utilization in general was not compromised when a suitable zinc source was present. When samples were fully restricted for zinc by the addition of the zinc-specific chelator N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), no growth was observed for any strain. However, when the TPEN-treated samples were also supplemented with Zn-S100A7, gonococci producing TdfJ variants (plus IPTG) exhibited various capacities for growth, consistent with their binding phenotypes. Unsurprisingly, WT TdfJ facilitated the most growth, while the ΔL3H and K261P/R262P samples reached optical densities similar to those of their TPEN-only counterparts, suggesting effectively no capacity for Zn-S100A7 use. Likewise, R262P, Q265P, L266P, and Q265P/L266P fell between the two extremes, consistent with our findings in the binding assays.As a consequence of their impaired binding, we next investigated whether other known TdfJ-S100A7 interaction phenotypes were also impacted by mutagenesis. We previously reported that gonococci producing functional TdfJ are able to utilize Zn-loaded S100A7 (Zn-S100A7) as a sole zinc source in metal-depleted medium and that growth in the presence of Zn-S100A7 leads to zinc accumulation within gonococci in a TdfJ-dependent way . To deteFinally, we assessed whether TdfJ mutation affected zinc accumulation within the gonococci when Zn-S100A7 was present. We grew cultures in zinc-restricted medium supplemented with Zn-S100A7 and harvested the cell pellets. After the removal of exogenous metals, the cell pellets were analyzed for their zinc content by inductively coupled plasma mass spectrometry (ICP-MS) . These eN. gonorrhoeae, continue to emerge. During this pathogen’s life cycle within the human host, it is confronted by host efforts to limit the availability of critical nutrients such as iron and zinc, a concept termed “nutritional immunity,” in order to starve out the infection. In response, the gonococcus deploys eight TdTs to its outer membrane, which serve the critical function of overcoming host nutritional immunity efforts by binding host factors and pirating their metal cargo , suggesting that the helical structure itself is the predominant conserved property, but further investigation of other TdT-ligand pairs would be needed to validate this.The importance of an α-helix in a TdT extracellular loop is not unique to TdfJ. The crystal structure of meningococcal TbpA in complex with transferrin shows such a helix finger in proximity to the transferrin C-lobe, where it is thought to disrupt iron coordination . As mentN. gonorrhoeae strain, FA1090, with an inactivated transferrin receptor system is unable to cause infection in human males  and found that upon immunizing factor H transgenic mice, a nonbinding mutant elicited more bactericidal antibodies and more factor H-blocking antibodies than what was seen for WT fHbp  agar with Kellogg’s supplement I (3)3 (GCB agar plates) at 36°C with 5% CO2. Where appropriate, GCB plates were supplemented with 1 mM IPTG. For metal-restricted growth, gonococcal cells were inoculated to a final optical density at 600 nm (OD600) of 0.09 in 5 mL of defined medium that was pretreated with Chelex-100 resin (Bio-Rad) (Chelex-treated defined medium [CDM]) in beveled sidearm flasks. Cultures were grown at 36°C with 5% CO2, with shaking at 225 rpm.Strains of lement I  and 12.4tdfJ mutants, mutated tdfJ gene sequences were submitted to either Bio Basic Inc. or Genewiz Inc. for de novo synthesis and subsequently cloned into pVCU234 between the XmaI and XhoI sites. The XmaI site was reconstructed during this cloning step, leaving 6 nucleotides between the plasmid’s ribosome-binding site and the tdfJ start codon. For gonococcal transformations, plasmids were first linearized with PciI and then used to transform a piliated population of gonococcal strain MCV928 (tdfJ gene(s) was verified by PCR.All strains and plasmids utilized in this study are summarized in n MCV928 . TransfotdfJ was amplified from the complementation plasmids by PCR, and fragments were subsequently cloned via the In-Fusion system (Clontech) into pET-20bHT  cleavage site. Clones were used to transform Escherichia coli cells, which were recovered on 100 μg/mL carbenicillin and verified by PCR.To generate expression systems for recombinant TdfJ, the mature coding region of WT and mutant ET-20bHT  between E. coli C41(DE3). For purification, a starter culture of the appropriate plasmid was prepared in Luria-Bertani (LB) medium supplemented with 100 μg/mL carbenicillin and subcultured into Terrific broth (TB) with the same antibiotic. Expression cultures were grown without the inducer for approximately 48 h at 20°C, and cell pellets were harvested by centrifugation . Cell pellets were resuspended in lysis buffer  using a Dounce homogenizer, with 10 mL of buffer used per g of cell paste. Cell suspensions were then mechanically lysed via two passages through an Emulsiflex C3 instrument (Avestin Inc.) with a homogenizing pressure of ~17,500 lb/in2. Insoluble material was removed via centrifugation , and the supernatant was collected and mixed with 1% Triton X-100 for 1 h at room temperature. The supernatant was then centrifuged at 160,000 × g for 1 h to pellet the membranes, and the remaining supernatant was discarded. The membranes were resuspended in a minimal volume of membrane buffer  and allowed to mix at 4°C overnight. Undissolved membranes were then pelleted via centrifugation , and the remaining solubilized material was mixed with Ni-NTA resin for 2 to 3 h at 4°C. The resin was collected in a chromatography column and then washed with 10 column volumes (CV) each of wash buffer  containing 0, 50, 100, 200, and 300 mM imidazole. TdfJ eluted cleanly at 300 mM. The eluted protein was then dialyzed overnight at 4°C into phosphate-buffered saline (PBS) (pH 8.0) plus 0.25% Elugent and, if necessary, concentrated using a 50,000-Da-cutoff centrifugal filter. Protein was then aliquoted and flash-frozen at −80°C.Human S100A7 was produced from a pET-22b expression vector provided by Joachim Grötzinger (pET-22b-pso) using the protocol described previously by Grötzinger and coworkers . His-tag2O) prior to use. For binding experiments, NTA-coated sensor chips were first cleaned via successive injections of 10 mM HCl (150 μL/min) and 350 mM EDTA (100 μL/min), followed by surface activation with an injection of 40 mM NiCl2 (20 μL/min). For ligand immobilization, WT or K261P/R262P TdfJ containing a 10× N-terminal His tag was injected into flow channel 2 at a concentration of 89 μg/mL (20 μL/min), and the remaining NTA groups were blocked via the addition of 50 μg/mL His-tagged streptavidin into both channels 1 and 2 (20 μL/min), allowing channel 1 to serve as a reference for nonspecific interactions. S100A7 was injected (20 μL/min) into both channels in successive steps using the following concentrations: 1, 10, 50, 100, and 500 nM. No chip regeneration step was found to be necessary. Sensorgrams were exported to TraceDrawer, where readouts for each concentration were aligned and bubble peaks were removed. For affinity analysis, sensorgrams from multiple, independent experiments were combined, aligned, and used to generate a single binding curve interval, which was then analyzed using a single-site fit model and the EC50 analysis setting. In total, 13 data points were used in the WT affinity model, and 10 were used in the attempted fit of the K261P/R262P mutant.All SPR experiments were performed using an OpenSPR XT instrument (Nicoya), and trace analysis was conducted using TraceDrawer (Ridgeview). All reagents were prepared according to the manufacturer’s instructions, using degassed buffers. TdfJ and S100A7 were buffer exchanged into degassed PBS (pH 8.0) prior to experiments, and all dilutions were performed using the same buffer. His-tagged streptavidin was reconstituted in degassed distilled water  bovine serum albumin (BSA) or 5% (wt/vol) nonfat dry milk dissolved in Tris-buffered saline plus 0.05% Tween 20 (TBST). For S100A7 binding assays, blots were returned to the dot blotter and probed with horseradish peroxidase (HRP)-labeled S100A7 (S100A7-HRP) dissolved in blocker at the following concentrations: 0.4, 0.2, 0.1, 0.05, 0.025, and 0.0125 μM. After probing for 1 h at room temperature, the probe was removed via a vacuum, and the blot was washed three times for 10 min using TBST. The HRP signal was developed using the 4-Chloro-1-naphthol and 3,3′-Diaminobenzidine mixture (CN/DAB) substrate (Thermo Fisher). For monoclonal antibody probing during mAb characterization, dried and blocked blots were returned to the blotter and probed with a hybridoma supernatant containing the mAbs diluted in blocker, as follows: 1:10 → 1:50 → 1:100 for 4-2E2, 4-2A2, 4-3F11, and 4-5E11 and 1:10 → 1:20 → 1:40 for 1-2B11 and 1-8H4. Probing was performed at 4°C overnight. For mutant surface exposure and folding analyses, dried and blocked blots were probed with 4-2A2 , 4-2E2 , 4-3F11 , 4-5E11 , 1-2B11 , and 1-8H4 , also at 4°C overnight. Following primary probes, blots were washed three times with TBST and then probed for 1 h at room temperature with either alkaline phosphatase (AP)- or HRP-labeled anti-mouse IgG secondary antibodies . Blots were washed again, and the signal was developed with the CN/DAB substrate for HRP and nitroblue tetrazolium (NBT)–5-bromo-4-chloro-3-indolylphosphate (BCIP) for AP.Gonococcal strains were grown on GCB agar plates with or without 1 mM IPTG before being resuspended in PBS. Cell suspensions were standardized to an OD2SO4. Coloration was quantified by reading the absorbance at 450 nm using a Cytation5 plate reader (BioTek). For S100A7 binding assays, 100 μL of His-tagged TdfJ (WT and mutants) was seeded into the Ni-NTA plates at 1 μM and allowed to bind for 1 h at room temperature. Wells were blocked as described above, 100 μL of 1 nM S100A7-HRP in blocker was then added to the wells, and the wells were probed for 1 h. Liquid was again siphoned away, and the wells were washed three times with 200 μL TBST. The HRP signal was developed as described above, and all A450 values were standardized as a percentage of wild-type binding.For conformational testing with the mAbs, 100 μL of His-tagged TdfJ (WT and mutants) or TdfH was seeded into the wells of a Ni-NTA-coated ELISA plate at 1 μM. Proteins were allowed to bind for 2 h at room temperature or overnight at 4°C. Protein was siphoned off by a vacuum, and wells were blocked with 200 μL of 5% (wt/vol) BSA in TBST for 1 h at room temperature. After blocking, the wells were probed with 100 μL of the mAb  supernatant diluted in blocker  for 2 h at room temperature. Liquid was again siphoned off, and the wells were washed three times with 200 μL TBST for 10 min each. HRP-labeled anti-mouse IgG was diluted in blocker , and 100 μL was added to the wells for secondary probing for 1 h at room temperature. After siphoning and three more washes, the HRP signal was developed by the addition of 100 μL of the 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate, and the reaction was neutralized with 100 μL of 180 mM H600 of 0.02 in the same medium and added to a 96-well plate, where they were supplemented with either 5 μM ZnSO4 as a positive control, 5 μM TPEN as a negative control, or 5 μM TPEN plus 5 μM Zn-loaded S100A7 to test S100A7 utilization. A total of 1 mM IPTG was added where appropriate. Cultures were grown for 12 h (36°C with 5% CO2 at 225 rpm) in a Cytation5 plate reader (BioTek), with the OD600 being measured every 30 min to track growth.Gonococcal growth assays under zinc-restricted conditions were performed as previously described . In shorg for 10 min). Pellets were washed twice with buffer containing 10 mM HEPES plus 1 mM EDTA and then once more with 10 mM HEPES only. Cell pellets were frozen at −20°C before being sent to the Plasma Chemistry Laboratory at the University of Georgia Center for Applied Isotope Studies for ICP-MS analysis. Data are reported as micrograms of zinc per gram of cell pellet.Gonococcal cultures were grown as described above in CDM until exponential phase. At this point, cultures were back-diluted to one-half their original density and supplemented with 1 μM Zn-loaded S100A7 and 1 mM IPTG where appropriate. Cultures were grown for 4 h before cell pellets were harvested by centrifugation  and resuspending cells in 2× Laemmli solubilizing buffer before storage at −20°C. Immediately preceding use, samples were thawed, mixed with 5% β-mercaptoethanol, and boiled for 5 min. Protein samples were separated on a precast 4-to-20% gradient polyacrylamide gel before transfer to nitrocellulose. The blots were stained with Ponceau S to verify equal protein sample loading. To detect TdfJ, the blots were first blocked in 5% (wt/vol) nonfat dry milk dissolved in TBST. The blots were then probed with either TdfJ peptide-specific guinea pig polyclonal antiserum (1:200 in blocker) or the mAb hybridoma supernatant (1:100 in blocker) for 2 h at room temperature. The generation of the TdfJ peptide antiserum was described previously . The blo


Glioblastoma is a malignant tumor, and its prognosis is as poor as 1.5 to 2 years. Most cases recur within one year even under the standard treatment. The majority of recurrences are local, and in rare cases, metastasize mostly within the centra nervous system. Extradural metastasis of glioma is exceedingly rare. Here, we present a case of vertebral metastasis of glioblastoma.We present a 21-year-old man post total resection of the right parietal glioblastoma, diagnosed with lumbar metastasis. He originally presented with impaired consciousness and left hemiplegia and underwent gross total resection of the tumor. Given the diagnosis of glioblastoma, he was treated with radiotherapy combined with concurrent and adjuvant temozolomide. Six months after tumor resection, the patient presented with severe back pain, and was diagnosed as metastatic glioblastoma on the first lumbar vertebrae. Posterior decompression with fixation and postoperative radiotherapy were conducted. He went on to receive temozolomide and bevacizumab. However, at 3 months after the diagnosis of lumbar metastasis, further disease progression was noted, and his care was transitioned to best supportive care. Comparison on copy number status between primary and metastatic lesions on methylation array analysis revealed more enhanced chromosomal instability including 7p loss, 7q gain and 8 gain in the metastatic lesion.Based upon the literature review and our case, younger age of initial presentation, multiple surgical interventions, and long overall survival seem to be the risk factors of vertebral metastasis. As the prognosis of glioblastoma improves over time, its vertebral metastasis is seemingly more common. Therefore, extradural metastasis should be kept in mind in the treatment of glioblastoma. Further, detailed genomic analysis on multiple paired specimens is mandated to elucidate the molecular mechanisms of vertebral metastasis. Here, wThis study was reported in agreement with principles of the CARE guidelines . Written22.12/day) per the Stupp regimen was conducted. Intensity Modulated Radiation Therapy (IMRT) of 60Gy was conducted at the edematous area surrounding the tumor, and IMRT of 50Gy at the tumor removal site. Four cycles of temozolomide (150mg/m2/day) were administered during the maintenance phase. At the same time, bevacizumab (10mg/kg) was administered twice, two weeks apart.A 21-year-old man post total resection of the right parietal glioblastoma was diagnosed with lumber metastasis. At the age of 20, without any significant family history or past medical history, he presented to a local emergency department with impaired consciousness and left hemiplegia. Computed tomography (CT) showed intracranial hemorrhage in the right parietal lobe, and hematoma evacuation was conducted. Dilated veins were observed around the hematoma and the patient was diagnosed with intracranial hemorrhage from venous hemangioma. Five months after the operation, he began to complain of headache, and magnetic resonance imaging (MRI) showed an enhanced lesion in the right hemisphere which was rapidly increasing in size. Partial removal of the mass and external decompression was conducted. Then he was referred to our institute for resection of the remaining mass and adjuvant therapy. Gross total resection of the tumor, placement of carmustine wafer in the resection cavity and cranioplasty were conducted at our institute Figure 12/day) and two more administrations of bevacizumab . Three months after the diagnosis of lumbar metastasis, however, disease progression with multiple metastasis to the lymph nodes, the lungs, and the liver was found, prompting the transition to best supportive care.At 6 months after the resection of tumor at our institute (14 months after the first operation), the patient presented with severe back pain. CT showed an osteolytic mass on the body of the first lumbar vertebrae and MRI showed an enhanced lesion constricting the spinal canal Figure 22.2This study was approved by the Ethics Committee of the University of Tokyo (#G10028). DNA was extracted from FFPE tissue samples and analyzed using the Illumina Infinium Human Methylation EPIC Bead Chip array according to the manufacturer’s instructions. All DNA methylation analyses were performed using R version 4.1.0. Methylation values were calculated as β values. The following filtering criteria were applied: removal of probes overlapping with single-nucleotide polymorphisms, those mapped to chromosomes X and Y, or the Illumina control probes. Copy number alterations were calculated using signal data from the methylation array using the conumee Bioconductor package version 1.26.0.Primary and metastatic samples had overall copy number status in common such as chromosome 1p loss, 16 loss and 22q loss; however, the metastatic sample had more prominent copy number alterations, including newly acquired 4 gain, 7p loss, 7q gain, 8 gain, 13q gain, 19q gain, and 20 loss Figure 43In this case illustration, we present an extremely rare case of glioblastoma which metastasized extracranially to the lumbar vertebra during the course of treatment. Glioblastoma was not believed to metastasize outside of the CNS until Davis first reported glioma meningeal metastasis in 1928 , 6. ExtrVertebral metastasis is exceedingly rare; to the best of our knowledge, there has been 59 cases reported in the literature , 8–58. MConsidering the biological obstacles that prevent glioblastomas from infiltrating outside of the CNS, it can be speculated that deposition of tumor cells into the blood stream or excision of the dura due to surgical interventions may attribute to extracranial metastasis. As for the 59 reported cases, the mean number of surgeries conducted prior to vertebral metastasis was 1.5 . In our case, the patient first presented with intracranial hemorrhage and the tumor was hypervascular enough to be misdiagnosed for venous hemangioma, allowing tumor cells to be feasibly deposited into the blood stream. Also, multiple surgeries including external decompression were conducted causing defects of the dura and the skull, which could have facilitated the extradural infiltration of glioblastoma cells. Based upon the literature and our case, younger age of initial presentation of glioblastoma and multiple surgical interventions seem to be the risk factors of vertebral metastasis of glioblastoma, though we still lack further statistical evaluation.Little has been known about the genetic and epigenetic changes seen in the extracranial metastasis of glioblastoma. Copy number alterations have been known to be enhanced at the metastatic site compared with the primary site, in parallel with mutational burden, in systemic cancers such as lung, hepatocellular and urothelial malignancies among others –61. ThisDue to the improvement in the prognosis of the disease, vertebral metastasis is suspected to be encountered more commonly. Therefore, extradural metastasis of glioblastoma must be included in differential diagnoses in treating patients with glioblastoma. Further studies with detailed genomic analysis on multiple paired tumor specimens are warranted to unravel the molecular mechanisms of vertebral metastasis.The data analyzed in this study is subject to the following licenses/restrictions: This is a case report. All the information is stored in the medical chart. Requests to access these datasets should be directed to STan, stanaka@m.u-tokyo.ac.jp.The studies involving human participants were reviewed and approved by The University of Tokyo. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual and the patient’s legal guardian for the publication of any potentially identifiable images or data included in this article.AM and STan contributed to conception and design of the study. AM organized the database. AM and HT performed the statistical analysis. AM and STan wrote the first draft of the manuscript. HT wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.