The isolation of high-quality RNA from endocrine pancreas sections represents a considerable challenge largely due to the high ribonuclease levels. Laser capture microdissection (LCM) of mammalian islets, in association with RNA extraction protocols, has emerged as a feasible approach to characterizing their genetic and proteomic profiles. However, a validated protocol to obtain high-quality RNA from LCM-derived human pancreas specimens that is appropriate for next-generation sequencing analysis is still lacking. In this study, we applied four methods  to extract RNA from human islets obtained from both non-diabetic individuals and patients with type 2 diabetes who had undergone partial pancreatectomy, as well as handpicked islets from both non-diabetic and diabetic organ donors. The yield and purity of total RNA were determined by 260/280 absorbance using Nanodrop 100 and the RNA integrity number with a bioanalyzer. The results indicated that among the four methods, the RNeasy MicroKit + Carrier (Qiagen) provides the highest yield and purity. Laser capture microdissection (LCM) has emerged as a widely used technique for the isolation of specific types of cells or a minimum amount of parenchyma  for a vaGiven the limitations of manual islet isolation, the LCM approach is preferable to limit confounding results. Nevertheless, harvesting high-quality RNA from human islets that can be used for transcriptomic analysis is a continuing challenge, mostly due to the high level of intrinsic ribonuclease (RNase) activity in the pancreas ,11. One Although the LCM technique  has imprTissue-Tek OCT medium Isopentane (2-methylbutane) DEPC-treated water Ethanol 100% Ethanol 70% (dilute 100% ethanol with DEPC-treated water to obtain 70% ethanol solution)Xylene SUPERase·IN RNeasy Micro Kit 50 Qiazol lysis reagent, 50 mL RNeasyMinElute Clean-up Kit 50 PicoPure RNA isolation kit RNase-Free DNase Set 50 The following reagents were used:Cryomold Frosted microscope slides Polypropylene Falcon Tube RNaseZap, 250 mL CapSure HS LCM Caps ® Autoclaved Thin-Walled Reaction Tubes GeneAmpTweezers and forcepsPipettes: 20–200 µL and nuclease-free pipette tipsCryostatFume hood® IIe Laser Capture Microdissection System PixCellIncubator Microcentrifuge The following laboratory materials and equipment were required:To compare the efficiency of different RNA extraction protocols in frozen human islet samples, we used LCM to collect a mean of 100 islets from pancreatic surgical specimens, obtained from non-diabetic or diabetic patients who had undergone partial pancreatectomy for an extra-pancreatic tumor. Surgical procedures were performed by the Hepato-Biliary Surgery Unit of the Department of Surgery . All the patients involved in the study provided informed consent for tissue analyses prior to surgery.n = 7) or diabetic (n = 2), according to the American Diabetes Association guidelines [The pancreas samples were collected from the downstream edge of the surgical cut. On the basis of 2 h glycemia after a standard glucose load (75 g) prior to surgery, patients were divided into non-diabetic , including one non-diabetic and one diabetic individual, provided by the Integrated Islet Distribution Program (IIDP), as positive controls.Sample processing was performed in the Endocrinology Department of the Catholic University (Rome) and shipped to the Joslin Diabetes Center (Boston) for sectioning and LCM procedures.2) were quickly placed into a cryomold and covered with an optimal cutting temperature (OCT) compound. The mold was then placed in an isopentane–dry ice bath until the OCT compound became completely solid. Finally, the frozen specimen was stored in foil in a −80 °C freezer until sectioning.To maintain tissue integrity, tissue processing was completed within 60 min of the surgical removal of the tumor. The dissected pancreatic tissue specimens -treated water70% ethanol for 30 s100% ethanol twice for 1 minXylene for 4 minIslet preparations for downstream analyses were obtained from frozen samples by LCM. Prior to the LCM procedure, the sections were dehydrated and cleared in a fume hood by systematically dipping the slides one after the other in the solutions listed below:All the solutions, except xylene, were prepared the day before and stored at 4 °C to ensure optimum performance. The slides were dried in the fume hood for 3–5 min before LCM. We used desiccant cartridges with silica gel (Fisher Scientific 08-594-14B) and a vacuum desiccator (Fischer Scientific F420120000) as alternative methods to more effectively dry the slides, if necessary.LCM was performed using the PixCell II Laser Capture Microdissection System . The power setting for the laser pulse was at 35 mW with a duration of 2.5 ms to limit tissue trauma and provide greater precision. Subsequently, selected populations of cells were cut with the laser and mounted on transparent LCM caps provided by Arcturus. To collect material for RNA analysis, at the end of the section, the thermoplastic film containing the microdissected cells was covered with a GeneAmp microfuge tube filled with 11 µL of RNA extraction buffer (guanidine isothiocyanate and polyethylene glycol octylphenol) plus 0.5 µL of RNase inhibitor 1 U/µL , as previously reported , and incTo avoid contamination and RNA degradation, we noted that it is necessary to accurately follow all the steps, especially during the final collection. In particular, we observed that the use of 100% ethanol and RnaseZap to clean the bench and microscope surfaces, pipettes, and forceps was critical. Other key precautions included using disposable gloves and wearing a mask during all procedures. Faster execution of LCM procedures vastly improved the quality of the RNA. Further, to minimize exocrine contamination, we selectively collected cells from the islet core.After collection of islets by LCM from surgical specimens of pancreatectomized patients (PP islets), the RNA was extracted using four different protocols, whose efficacy was evaluated by comparing Nanodrop and bioanalyzer results.PicoPure Extraction Kit: First, we used the PicoPure extraction kit by Applied Biosystems by Thermo Fisher Scientific, Vilnius, Lithuania), using a 1:1 ratio of extraction buffer (guanidine isothiocianate) to 70% ethanol. We included DNase treatment by incubating extracted RNA with RNase-free DNase reagent  for 15 min. Subsequently, to obtain better RNA quality compared to previous reports  , we addeTo improve both the quantity and the quality of the RNA, we first performed RNA extraction on LCM-collected islets from one non-diabetic and one diabetic subject using Qiazol reagent as lysis cell buffer, which has been reported to produce high-quality RNA from rat pancreas . We usedn = 7) and diabetic (n = 2) subjects; finally, we optimized the process comparing the total amount and the integrity of the RNA extracted from the two samples with three other protocols.Then, to optimize the use of the extremely limited material, we performed LCM on all samples and pooled all material obtained from non-diabetic  was added to each sample to permit dissociation and homogenization of nucleoprotein complexes, followed by transfer of the supernatant to a new tube. Addition of 140 µL of chloroform followed by a 15 min centrifugation step allowed the separation of the colorless aqueous upper phase containing the ribonucleic acid from the pink lower phase rich in organic proteins and included the interphase where DNA was present. Total RNA was precipitated in a gel-like pellet on the sides and bottom of the tube by mixing the aqueous phase with 350 µL of isopropyl alcohol. To avoid DNA contamination, we incubated the extracted material for 15 min with RNase-free DNase reagent . Two washing steps with ethanol were performed to remove contamination, and the extracted RNA was dissolved in 40 µL of DEPC-treated water for downstream analysis.Qiazol/Clean-up: In testing this method, we used the previous protocol that included a purification step using the RNeasy Mini Clean-up kit , according to the protocol suggested by the manufacturer (Qiagen). The use of a mini spin column allowed binding of total RNA to the membrane, while the contaminants were efficiently washed away. The final RNA was dissolved in 14 µL of DEPC-treated water.Microkit/Carrier: In this protocol, we used Qiazol as lysis cell buffer and performed RNA extraction using the RNeasy Microkit 50 . This method was designed for isolation of total RNA (up to 45 µg) from small samples. To improve the recovery of total RNA from small samples, we added 5 μL of a 4 ng/μL working solution of poly-ARNA carrier to the lysate. Subsequently, the first steps of the column-based isolation protocol were the same as the first method tested  and involved Qiazol and chloroform solutions to lyse and homogenize samples. Ethanol was added to reach ideal binding conditions, and the lysate contained in the aqueous phase was transferred into the RNeasy MinElute spin column to allow RNA binding to the silica membrane. DNase and any contaminants were efficiently washed away with ethanol, and pure concentrated RNA was eluted in 14 µL of DEPC-treated water.In all the protocols described above, we added 0.75 μL of RNase inhibitor to the total volume of isolated RNA at the end of each procedure to minimize its degradation.Nanodrop assessment of RNA, extracted from both non-diabetic and diabetic PP islets using the first protocol  and performed before and after the purification experiment, is reported in p = 0.83; after purification p = 0.88)  (We observed a 50% loss of RNA after the purification step in both non-diabetic and diabetic subjects a despite = 0.88) b. The me = 0.92) b.Although we observed relatively good 260/280 ratios, as assessed by Nanodrop after the purification step , the bion = 7 non-diabetic subjects) and one diabetic sample (resulted from pooling of islets extracted from n = 2 diabetic subjects) and OD islets from one individual non-diabetic and one individual diabetic subject are reported in Nanodrop assays for the RNA extraction protocols performed using Qiazol as lysis buffer on PP islets of one non-diabetic sample  on LCM islets of diabetic PPs as well as on handpicked islets of diabetic ODs.Using the Qiazol protocol, we observed a consistent RNA concentration in the LCM samples from diabetic PPs, while the bioanalyzer assessment showed RIN = 1 A. In theThe use of silica membrane columns in the Qiazol/Clean-up protocol resulted in an improvement of RNA quality for diabetic PP islets B, which The Microkit/Carrier protocol showed a consistent improvement of the RNA integrity number in RNA from diabetic PP islets C, while The table summarizing and comparing bioanalyzer results of RNA extracted using the three protocols with Qiazol from islet samples of PP and OD subjects is reported in In this study, we aimed to identify an optimal protocol to recover high-quality RNA from human islets isolated by LCM that can be used for next-generation transcriptomic analysis. Our results suggest that the Microkit/Carrier protocol results in improved RNA quality from islets collected by LCM from both non-diabetic as well as diabetic subjects. All experiments were performed on freshly isolated pancreas samples from living patients undergoing partial pancreatectomy. These are extremely valuable tissues, and we believe that pancreatic surgery provides a rare opportunity to correlate in vivo endocrine and metabolic pathways (before surgery) with ex vivo data generated after collecting the pancreatic samples Previous reports on cell culture and peripheral blood mononuclear cells (PBMCs) have reported that silica gel column technology using the RNeasy Mini-/Microkit is superior in its ability to extract high-quality RNA compared to protocols using the traditional guanidine isothiocyanate extraction method , which iDespite some limitations, such as the tedious nature of the process and costs, laser capture microdissection (LCM) has several advantages, e.g., minimizing contamination from neighboring cells and avoiding changes due to hypoxia-induced stress that occurs during the normal islet isolation process. Therefore, LCM remains a suitable approach to extract RNA/protein for studies aimed at gene and protein expression in targeted cells. Furthermore, the LCM technique guarantees isolation of high-quality RNA from specific cells from frozen samples. This is especially important when considering formalin-fixed tissues that typically provide low yields and poor quality of extractable DNA and RNA , in partGene and protein expression using DNA microarrays, high-throughput sequencing, or proteomics analyses comparing various physiological or experimental conditions represents an essential tool in diverse biological fields to decipher mechanisms and to identify novel signaling pathways. In the islet biology field, RNA sequencing technologies have a high likelihood of identifying novel islet cell-specific promoters or splice forms as well as long non-coding RNAs, which cannot be detected by array analysis. Successful next-generation sequencing analyses require RNA samples free of contamination and degradation, easily obtained for sundry tissues using conventional protocols for RNA preparation. However, RNA isolation from pancreas samples continues to represent a challenge, given the high levels of endogenous RNases, DNases, and proteases that can quickly induce cell autolysis upon dissection. Consequently, classical RNA extraction protocols based on the phenol guanidine thiocyanate method yield RNAs of insufficient quality for subsequent sequencing analysis. In a published protocol for LCM of human islets from surgical specimens, the mean value of the RNA integrity number obtained was 5.8 , while iIn our experience, Nanodrop results showed no correlation with effective RNA purity, and we emphasize the importance of bioanalyzer assays to reliably evaluate the yield and quality of RNA. Indeed, it is known that 260/280 absorbance could be affected by variables such as pH, phenol or alcohol contamination, or the presence of proteins ,23. MoreUnlike previous studies wherein gene expression is evaluated in beta cell samples enriched by cell fluorescence, our approach used RNA from beta cells that continue to be in the islet microenvironment until extraction and therefore reflects interactions between the different endocrine cell types that is lacking in the former method.In summary, our comparative studies indicate that the Microkit/Carrier protocol provides a relatively better yield in both the quality and the quantity of RNA extracted from human pancreatic samples compared to the other methods, and we propose this approach for studying differences in gene expression between groups.


In this publication we report the creation of a new unc-11 allele that is endogenously-tagged with GFP just upstream of the stop codon. Moreover, we demonstrate that the UNC-11::GFP fusion protein functions like wild type with an expression pattern similar to UNC-11 antibody staining described previously. PICALM facilitates recycling of Vesicle Associated Membrane Proteins (VAMPs) from the plasma membrane  and efficient clathrin-coated endocytosis . unc-11 function in C. elegans is likely conservedasSNB-2 (VAMP2) and the glutamate receptor, GLR-1, both accumulate at the plasma membrane in unc-11(e47) null mutants . Mutant phenotypes outside the worm nervous system have not been described.UNC-11is anortholog of mammalian PICALM (phosphatidylinositol binding clathrin assembly protein) and its neuron-specific paralog, AP180  with the CRISPR knock-in strain, unc-11(vg103) and the null mutant, unc-11(e47). We found no obvious differences between unc-11(+) and unc-11(vg103). Average thrash duration during a 45 second interval was 0.251 seconds  for unc-11(+) and 0.248 seconds  for unc-11(vg103). By contrast, the average thrash duration for unc-11(e47) was significantly higher at 0.566 seconds with a striking increase in variability .To ask if the addition of GFP at the C-terminus of UNC-11 compromises s system . We compet al. 1999) with some noted exceptions. Similar to UNC-11 antibody staining, UNC-11::GFP expression appeared pan-neuronal and was also observed within coelomocytes .Plasmid construction was done in the context of a course entitled Advanced Molecular Techniques at California State University, East Bay. This strain will be made available at the CGC. Given the inclusion of an AID tag and GFP, this strain will be useful for cell-specific protein degradation using auxin-inducible (AID) or GFP nanobody::ZIF-1 degradation methods  and videos were captured at 30 frames per second using an iPhone 7. To open the images in FIJI, the .MOV files were converted to an uncompressed AVI format using ff.WORKS software. The duration of each thrash was measured in FIJI by logging each frame number at which the worm completed a single body bend in one direction. Thrash duration (in seconds) was then calculated as the number of frames per body bend divided by 30 frames per second. Approximately, 1450 frames (~45 seconds) of video were reviewed for each animal. Body bends that did not fall below 150 degrees were not counted as a true body bend. When a body bend angle was suspected to be greater than 150 degrees, the angle ruler in Fiji (Image J) was used. Brief intervals where thrashing occurred along the Z-axis or the worm left the field of view were not included. The data was graphed using Plots of Differences available at https://huygens.science.uva.nl/PlotsOfDifferences/ .Confocal Microscopy:unc-11(vg103) animals were anesthetized with sodium azide (10 mM) and imaged using a Leica SP8 confocal. To visually distinguish true GFP signal from intestinal autofluorescence for Figures 1A and 1D, Z-stacks were captured using a sequential scanning protocol: Seq 1: The 488 laser was set at 2.4%. PMT1 (green) collected wavelengths from 494 to 544 nm (capturing GFP and autofluorescence) while PMT2 (blue) collected wavelengths from 650 to 798 nm (autofluorescence only). Seq 2: The 552 laser was set at 1.0 % and PMT1 (blue) collected wavelengths from 598 to 709 nm (autofluorescence only). Adjustments were made to brightness and contrast of the blue channels to bring out the autofluorescence then the green and blue channels were combined and relevant focal planes were subject to maximum projection. The other images did not emit detectable autofluorescence and so the blue channel images were not included in the Z projection.CTATAATCCAAAgtttaagagctatgctggaa and caagacatctcgcaatagga. The unc-11 target sequence is in all caps and the position of the unc-11 STOP codon is in bold. The GFP::SEC repair template (pDQ1023) was created by Gibson Assembly of pJW1583  digested with AvrII and SpeI and two unc-11 homology arms (HA) amplified by PCR (Q5 DNA polymerase – NEB). Primers used to amplify the 5’HA and 3’HA homology arms from wild type genomic DNA include: ACGACGGCCAGTCGCCGGCATGCGATGGGTTAGTTTATCG and CCTGAGGCTCCCGATGCTCCTAATCCAAATGGATCGGCTG (5’HA); AGGATGACGATGACAAGAGATAGTCTAGTCGTCTATTTCTC and TATGACCATTTATCGATTTCGCAATAGAGATAAAGTTGCA (3’HA). unc-11(vg103[unc-11::gfp]) (DQ3188) was created as described by Dickinson et al. 2015. Successful knock-in was verified by PCR amplification and sequencing of the junction fragments and by GFP expression.The sgRNA/Cas9 plasmid (pDQ1022) was created by Q5 SDM (NEB) using pJW1219 as template (a plasmid containing sgRNA(F+E) and Peft-3::Cas9; Addgene # 61250) and the following primer pair: CGACTAGA


Developing a deeper knowledge about the impact of DNA and RNA epigenetic mutations on sperm production and fertilization performance is essential for selecting best quality samples in Assisted Reproductive Technologies (ART). Indeed, sperm RNAs adenine and guanine are likely to be methylated in low quality RNA sperm samples and their study requires the employment of techniques able to isolate high quality nucleic acids. UV resonance Raman spectroscopy represents a valuable tool that is able to monitor peculiar molecular modifications occurring predominantly in nucleic acids, being less sensitive to the presence of other biological compounds. In this work, we used an UV Resonance Raman (UVRR) setup coupled to a synchrotron radiation source tuned at 250 nm, in order to enhance sperm RNAs adenine and guanine vibrational signals, reducing also the impact of a fluorescence background typically occurring at lower energies. Despite that our protocol should be further optimized and further analyses are requested, our results support the concept that UVRR can be applied for setting inexpensive tools to be employed for semen quality assessment in ART. The detection of specific changes in DNA/RNA sequences is a main challenge both in biology and medicine, with the possibility of revealing cellular states and disease conditions. The applicability of related new methods and concepts to reproductive medicine field is desired, particularly to improve diagnosis and male gamete quality assessment ,2.Infertility is a disease of the male or female reproductive system that affects millions of people of reproductive age worldwide. Men infertility affects more than 12% of the whole population and it is considered a multifactorial phenomenon ,4. It isDespite the role of RNAs and their epigenetic modifications is widely studied in relation to zygote fertilization, metabolic and neurodevelopmental inheritance of the offspring and to embryonic development ,16,17,18In this picture, tailored and inexpensive approaches like Raman spectroscopy would be highly valuable in the field. As very recently reported by Dai and colleagues, Raman spectroscopy has been listed as a novel and promising technique able to measure DNA modifications such as fragmentation . In factIn the present paper, we describe the experimental strategies adopted to exploit the applicability of UV Resonance Raman set-up coupled with a synchrotron radiation source for prototype RNA analysis. We tuned the synchrotron radiation source to a 250 nm-radiation wavelength in order to investigate the quality of RNA extracted from spermatozoa (derived from 5 patients), compared to RNA isolated from commercial immortalized cell lines, willing to reach the lowest detection limit for RNA and possibly enhancing the spectral regions that could be affected by nucleotide modifications. Additionally, the use of the 250 nm-radiation source is actually a good compromise between the reduction of the fluorescence contribution dominating the Raman spectra at lower energies, where adenine vibrations are resonantly enhanced  and the relatively good enhancement of adenine peaks occur at 250 nm .RNA samples were extracted from five patients, whose characteristics are reported in In In order to enhance adenine and guanine contributions and also to avoid the spectral fluorescence background affecting the UVRR spectrum of the nitrogenous bases as previously observed in Reference  and furt−1, while the rest of the UVRR spectra are distorted by fluorescence contributions.2SO4 aqueous solution (0.2 M). After a proper spectral normalization of the SO42− vibrational peak, located at 981 cm−1, it was possible to get a correct estimation of the contribution of each single nitrogenous base contributes to the overall RNA spectral profile  (see N), which appear broader and formed by other sub-peaks. In order to clean up the UVRR spectra of patients 2 to 5 and identify which is the contaminant responsible for those spectral contributions, we subtracted the spectrum of sumN from the UVRR spectra of the patients (see As clearly shown in d 5) see A. In facents see B.−1. Interestingly, those bands could be associated with the presence of proteins and, in particular, to the chemical vibrations of amide III, tyrosine and tryptophan residues and amide I contributions, respectively [In ectively ,32,33. Because of that, in Consequently, we observed that when the RNA concentration is more than 100–120 ng/µL and the 260/280 ratio is higher than 1.8, RNA vibrational bands are those maximally enhanced by the incoming energy, and the proteins’ contribution (if present) is really negligeable (non-resonantly enhanced). At RNA concentrations below this threshold, if the 260/280 ratio is lower than 1.8, the spectral proteins’ contribution becomes relevant.Apart from the presence of biological contaminants, the UVRR spectra collected have been obtained after a careful optimization of the measurement protocols. For our scope and due to the Raman setting, we found out that the use of a RNAse inhibitor is mandatory to prevent the gradual enzymatic degradation of the RNA during the measurement, which leads to a lower signal-to-noise ratio. In fact, in In medicine, the damages and methylations of nitrogen bases are now of increasing interest when specifically affecting RNA, and the translational potential of Raman spectroscopy is in this field is still poorly investigated. We already reported that UV Resonance Raman spectroscopy represents a valuable tool for studying DNA damages  and its Our Raman measurements were performed not only taking into account both the enzymatic degradation, but also the potential photodegradation induced by the incoming radiation. Thus, a preliminary optimization of the radiation power from the synchrotron source and of the rotating sample holder has been made prior to starting.As already mentioned, the Raman spectra of nucleotides and RNA measured using a 266 nm-radiation source are dominated by a strong fluorescence background that can be reduced by choosing a higher-energy radiation wavelength such as 250 nm. Noteworthily, working at 250 nm represents the best choice in order to enhance also the vibrational modes of adenine as observed from the nucleotides spectra, which is one of the most abundant methylated nucleotides in spermatozoa .N spectrum from the UVRR spectra of the patients highlights that those with a RNA concentration lower than 100–120 ng/µL  contains contributions arisen from the presence of proteins-related bands and, in particular, amide I, amide III and aromatic amino acids ones . The use of the 250 nm-radiation source and a proper data reduction allows the analysis of RNA samples without the interference of a strong fluorescence background, while satisfactorily enhancing adenine and guanine vibrational signals. Perhaps Raman spectroscopy has been already indicated as a potential non-invasive technique to measure DNA modifications such as DNA fragmentation in spermatozoa, the present research is the first, to our knowledge, that put the technical basis for future analyses of RNA modifications in sperm. We successfully proved that clear UVRR spectra of the RNA can be obtained only by adding an RNAse inhibitor in the final eluates. This in fact reduces the possibility of chemical degradation, ensuring long-term stability of the nucleic acids throughout the measurements without being an element of spectroscopical disturbance. The spectra are affected by a component deriving from the protein contamination of the samples, and that reassemble mostly misfolded molecules. Despite the presence of proteins and low RNA concentration, that are expectable in clinical samples, the resonant condition and the synchrotron source allowed to successfully identify a spectral region where RNA modifications or methylation should be detectable. Further real analyses are however necessary, possibly in sets of samples with proper molecular characterization, to better demonstrate the potential clinical applicability of this vibrational approach.Five male Caucasian infertile patients were enrolled at the assisted medical procreation center of the Institute for Maternal and Child Health IRCCS Burlo Garofolo . The semen samples were collected through masturbation, and after liquefaction, they are observed under an optical microscope to determine the concentration and the motility . All theFive hundred microliters of each semen sample were used for the analysis. After two washing in physiologic solution, the sperm pellet was lysed in Trizol reagent  and the RNA was extracted following manufacturer’s instruction and eluted in 20 μL of distilled water.The RNA was also isolated from VeroE6 , and 3T3 J2  cell lines. After culturing them for 4 days in T75 flasks (about four-million cells) they were washed in phosphate buffer saline solution, and then detached from the bottom of the flask with a cellular scratcher. After centrifugation, the RNA was extracted from the pellet with the protocol employed for the sperm and resuspended in 50 μL of distilled water. The RNA concentration was measured with a Nanodrop instrument .−1. Final spectra were obtained by averaging 15 spectra of 15 min, for a total integration time of 13,500 s. To obtain the sample spectra, we removed the spectra of the substrate and of the RNAse inhibitor from the samples’ ones.Ten microliters of extracted RNA solution from semen samples and model cell lines were measured by drop-casting onto a rotating horizontally-disposed sample holder. Measurements were carried out at Elettra Synchrotron Radiation facility, using an experimental setup described elsewhere . A 250 n


Besides, the silica-induced increase in the levels of serum ceruloplasmin and HYP was also inhibited. Results of metabolomics indicated that arctiin and arctigenin could regulate the abnormal metabolic pathways associated with the development of silicosis, which involve pantothenate and CoA biosynthesis, cysteine and methionine metabolism, linoleic acid metabolism, and arginine and proline metabolism successively. Furthermore, the analysis of metabolomics, together with network topological analysis in different phases of silicosis, revealed that urine myristic acid, serum 4-hydroxyproline, and L-arginine could be regarded as diagnosis biomarkers in the early phase and formation of pulmonary fibrosis in the latter phases of silicosis. Arctiin and arctigenin could downregulate the increased levels of myristic acid in the early phase and serum 4-hydroxyproline in the latter phase of silicosis. Interestingly, the integration of TLR-4/NLRP3/TGF-β signaling and metabolomics verified the importance of macrophage polarization in the silicosis fibrosis process. To the best of our knowledge, this is the first study reporting that arctiin and arctigenin both can ameliorate silicosis effectively, and the former is a little stronger than its aglycone arctigenin because of its high oral bioavailability, low toxicity, and multimolecular active metabolites as determined by AdmetSAR and molecular docking analysis.Silicosis remains one of the most serious diseases worldwide, with no effective drug for its treatment. Our research results have indicated that arctiin and arctigenin could increase the mitochondrial membrane potential, which in turn reduces the production of reactive oxygen species (ROS), blocks the polarization of macrophages, and inhibits the differentiation of myofibroblasts to reduce oxidative stress, inflammation, and fibrosis. Further, our study revealed that arctiin and arctigenin suppressed the activation of NLRP3 inflammasome through the TLR-4/Myd88/NF- Silicosis is an occupational disease characterized by chronic lung inflammation, progressive pulmonary fibrosis, and systemic immune dysfunction –3. The iMetabolomics can comprehensively reflect changes in the metabolites of organisms in different states. The measurement of metabolite levels and variations in biofluids can offer many insights into disease processes and response to therapeutic intervention . Urine iFructus Arctii, the dried fruit of Arctium lappa L., has the function of clearing away heat and nourishing the lung as recorded in China's Pharmacopoeia [Fructus Arctii as stipulated in Chinese Pharmacopoeia [β-induced epithelial-mesenchymal transition (EMT) in human lung cancer cells, and attenuate PQ-induced EMT and pulmonary fibrosis [Fructus Arctii is much lower than that of arctiin. In our previous research, we found that arctiin could be metabolized into arctigenin in the intestinal tract [acopoeia . Arctiinacopoeia . Researcacopoeia , 15, antacopoeia , and neuacopoeia , 18. Recfibrosis –21. Arctfibrosis –26. Howeal tract , 28, andHerein, urine metabolomics was applied to explore the progress of silicosis with the aim at finding stable bioindices for the early diagnosis of silicosis and investigating the metabolic pathways in different phases of silicosis. Besides, the effects of arctiin and arctigenin on the progress of silicosis were compared to determine the more suitable compound for treatment. Network pharmacology and molecular docking were used to predict and analyze potential mechanisms for providing a theoretical basis for the development and utilization of arctiin and its metabolites.2 suspension (4000 U/mL penicillin sodium), and the model rats were evaluated via serum hydroxyproline and histopathological analysis. As described in Adult male SPF level Wistar rats (weighing approximately 180–220 g) were obtained from the Liaoning Changsheng Biotechnology Co. Ltd., Liaoning, China (license key: SCXK (Liao) 20150001), housed in a specific pathogen-free environment, and maintained under controlled conditions  with free access to standard food and water. Animal research was approved by the Animal Ethical and Welfare Committee of Liaoning University of Traditional Chinese Medicine, and the experimental procedures were conducted according to the Guide for Care and Use of Laboratory Animals of Liaoning University of Traditional Chinese Medicine. The rats were modeled by tracheal intubation with 0.5 mL of 80 mg/mL SiOμm for histological evaluation via hematoxylin and eosin (H&E) and Masson's staining. The levels of hydroxyproline, ceruloplasmin, and lysozyme in the serum and several proinflammatory and profibrosis cytokines in lung tissues, including TNF-α, IL-1β, NF-κB, and TGF-β, were determined by ELISA, and the expression levels of TLR-4, Myd88, NF-κB p65, NLRP3, ASC, cleaved caspase-1, and α-SMA were assayed by Western blotting, aiming at evaluating the mechanism of arctiin and arctigenin against silicosis. Further details have been provided in Supplemental Materials.Mental state, body weight, fur luster, respiratory rate, urine volume, dietary amount, and the amount of drinking water were monitored every week according to the methods specified in Supplemental Materials. Lung, spleen, and thymus tissues were collected and weighed to calculate their coefficient after drawing blood samples. Following this, the right part of the lung was used for the determination of bioindices and the left part was fixed in 4% paraformaldehyde solution, embedded in paraffin, and sectioned at a thickness of 5 https://www.metaboanalyst.ca/).The normalized data were then subjected to Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) in a Simca-p 14.1 workstation. Metabolites with VIP > 1 and P < 0.05 were identified as differential metabolites, and compound validation was conducted to verify the accuracy and eliminate unreliable differential metabolites. The metabolic pathways were identified by analyzing and enriching differential metabolites screened using the Pathway Analysis module in MetaboAnalyst 4.0. The MetScape plugin in Cytoscape 3.7.1 software was used for network construction of different periods and analysis of the topology of each network. Euclidean distance was used to evaluate the similarity between different periods by network topology and to determine the important metabolites in various periods [To explore the endogenous changes in the progression of silicosis, metabolomics was analyzed by HPLC-QTOF-MS , and data were preprocessed using MetaboAnalyst 4.0 ( periods . A Venn μg/mL) was assayed by MTT. The cells were then seeded in a 6-well plate at 1 × 106 cells per well and incubated in DMEM containing 10% FBS for 24 h. The cells of the arctiin group (ARC) and the arctigenin group (ARG) were treated with 1 μM arctiin or arctigenin and silica (50 μg/mL) together for 24 h. The mitochondrial membrane potential and reactive oxygen species were detected using the assay kit, and the expression levels of iNOS, Arg-1, TLR-4, NLRP3, cleaved caspase-1, and TGF-β were evaluated. Further details are shown in Supplemental Materials.To verify the effects of arctiin and arctigenin against inflammation, RAW264.7 murine macrophage cells were obtained from the Cell Bank of the Chinese Academy of Sciences . The cytotoxicity of arctiin and arctigenin toward RAW264.7 cells exposed to silica (50 5 cells per well and incubated in DMEM containing 10% FBS for 24 h. The cells were then treated with TGF-β1 (10 ng/mL) for 48 h. The cells were incubated with arctiin and arctigenin at 1 μM. Following this, the expression level of α-SMA was measured by Western blotting. Additional details have been provided in Supplemental Materials.To verify the effects of arctiin and arctigenin against fibrosis, primary mouse lung fibroblasts (PLFs) were isolated as described in Supplemental Materials. The cytotoxicity of arctiin and arctigenin on PLFs was assayed by MTT. The cells were seeded in a 6-well plate at 1.5 × 10http://lsp.nwu.edu.cn/tcmsp.php), STITCH (http://stitch.embl.de/), and SwissTargetPrediction server (http://www.swisstargetprediction.ch/), with stunted Homo sapiens as organism. CTD (https://ctdbase.org/) and GeneCards (https://www.genecards.org/) databases were used to find relevant target genes of pulmonary fibrosis. The compound-disease-target gene networks were constructed using the merge function of Cytoscape 3.7.1 software. Subsequently, the PPI network was established by STRING (https://string-db.org/) to select the closely connected genes for biological function analysis through FunRich software. The DAVID database (https://david.ncifcrf.gov/tools.jsp) was used to enrich the KEGG and GO pathway analyses of closely connected genes, and the enriched pathways were visualized on the OmicShare cloud platform (http://www.omicshare.com/forum/).Potential targets of arctiin and arctigenin were predicted by using TCMSP (βRI complexed with a 2-aminoimidazole inhibitor (PDB ID: 3faa) was downloaded from the Protein Data Bank (PDB), USA (https://www.rcsb.org/). Protein preparation was conducted using Discovery Studio 3.0 ; the ions, water molecules, and all internal ligands were removed; the missing atoms were inserted; and cocrystallization of small molecules was optimized before minimization of the target protein was conducted. The SDF documents of ligand compounds were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/), the initial molecular structure was optimized using Discovery Studio 3.0, and the molecular docking study was conducted using CDOCKER from Discovery Studio 3.0.For the preparation of the protein and the ligand, the crystal structure of TGF-http://lmmd.ecust.edu.cn/admetsar2/) was used to predict the oral bioavailability according to the Morgan fingerprint and random forest methods.AdmetSAR2.0  was used for statistical analysis of the differences between groups. Data were analyzed statistically and have been expressed as mean ± standard deviation. IBM SPSS Statistics var. 17.0  was used to evaluate data. A value of After rats were exposed to silica for 36 days, the surfaces of lung tissues were uneven locally, exhibiting a hard texture, poor elasticity, and increased volume, with a scattering of small white nodules , a type of innate immunity pattern recognition receptor, can be triggered by endogenous danger signals released by macrophages, and the expression of TLR-4 can promote fibrosis formation , 37. TheκB , 39. In nd TGF-β ; and alsnd TGF-β –44. The nd TGF-β . Numeround TGF-β –48. Howeroblasts –52, controblasts . α-SMA i protein , while t protein . Compare Figures .μM and no cytotoxicity at 1 μM in RAW 264.7 cells exposed to silica  and Arg-1 were macrophage polarization markers of M1 and M2 macrophages, respectively. The NLRP3 inflammasome could mediate M1 macrophage polarization to promote the production of IL-1β and upregulate the expression of IL-4 to stimulate M2 macrophage polarization [β signaling.Based on the abovementioned research results of metabolomics and signal transduction, we found that oxidative stress, inflammation, and enhanced innate immune responses were the main reactions after silica was phagocytosed by macrophages in the early phase. NLRP3 inflammasome was activated through the TLR-4/Myd88/NF-rization –62. Arctd value, representing dissimilarity, according to the Euclidean Distance Formula. This means that the smaller the d values are, the more similar the networks are. The metabolic networks in different periods of silicosis are displayed in Figures P < 0.05 in metabolomics. Subsequently, ROC analysis was conducted, and the diagnostic value of metabolites was evaluated with the AUC cut-off of 0.9. The AUC of myristic acid was up to 0.953, indicating a high diagnosis value in the early phase, and it reduced to 0.875 and even 0.766 gradually with the development of silicosis (Figures 11 metabolites were coexisting in urine during different periods in the MOD group compared with the CON group. The levels of myristic acid, uracil, mevalonic acid, 3-hydroxybutyric acid, pantothenic acid, epinephrine, and succinic acid semialdehyde were downregulated, whereas those of 3,6-octadienoylglycine, 1-methyluric acid, 4-hydroxyproline, and 1-methylhistidine were upregulated in a time-dependent manner . Myristi Figures . Besides Figures , and thein vivo accumulation with long-term administration [Myristic acid is a type of saturated fatty acid . Proteinstration , 67. ResThe metabolic networks are displayed in Based on the abovementioned results, myristic acid in urine had a higher diagnostic value in the early phase, and L-arginine and 4-hydroxyproline in the serum had higher diagnostic values in the final phase of silicosis. Therefore, the regulatory effects of arctiin and arctigenin on these high-value metabolites could evaluate their efficacy. The results showed that arctiin and arctigenin could both modify myristic acid levels in the early phase with similar strength, and arctigenin had a better effect on myristic acid in the middle phase than arctiin, whereas arctiin had a better effect than arctigenin in the final phase of silicosis. Further, the efficacy of a combined medication with arctiin and positive drugs was better than that of arctiin alone, showing a synergistic effect. There was no obvious regulatory effect of arctiin and arctigenin on L-arginine in the serum, but the efficacy was increased with combined medication using arctiin and positive drugs. Arctiin had a better effect on serum 4-hydroxyproline than arctigenin Tables .β-alanine metabolism, arachidonic acid metabolism, butanoate metabolism, arginine and proline metabolism, alanine, aspartic acid and glutamic acid metabolism, and the metabolism of glycine, serine, and threonine in the final phase of silicosis (Tables Compared with the MOD group, the arctiin groups showed significant differences in 11 of 56 urine metabolites in the early phase, 8 of 58 urine metabolites in the middle phase, and 15 of 52 urine metabolites and 16 of 46 serum metabolites in the final phase. In contrast, there were 16 of 56 urine metabolites in the early phase, 11 of 58 urine metabolites in the middle phase, and 22 of 52 urine metabolites and 24 of 46 serum metabolites in the final phase that showed significant differences after treatment with arctigenin. It can be clearly concluded that arctigenin regulated more metabolites than arctiin. The pathways of these abovementioned metabolites indicated that arctiin could regulate the biosynthesis of hormones in the early phase; taurine and hypotaurine metabolism in the middle phase; and taurine and hypotaurine metabolism, arginine and proline metabolism, glycerophospholipid metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis in the final phase of silicosis, whereas arctigenin could regulate arginine and proline metabolism and synthesis and degradation of ketone bodies in the early phase; linoleic acid metabolism in the middle phase; and linoleic acid metabolism, pantothenate and CoA biosynthesis, s Tables . Above aThe network pharmacology of arctiin and its 15 metabolites was performed, and pathway enrichment results of network pharmacology showed the TNF signaling pathway, the NOD-like receptor signaling pathway, Toll-like receptor signaling, the MAPK signaling pathway, and 90 related pathways, indicating that arctiin and its metabolites play important roles in the treatment of silicosis. Biological function analysis further indicated that the regulatory effect of arctiin and its metabolites on silicosis was associated with biological processes such as the lipopolysaccharide-mediated signaling pathway; positive regulation of the ERK1 and ERK2 cascade; cellular response to mechanical stimulus; and molecular functions such as interleukin-1 receptor binding, MAP kinase activity, and ATP binding Figure , providiin vitro; however, arctiin was better than arctigenin for the treatment of silicosis being associated with the toxicity of arctigenin. Arctigenin was mostly regarded as the direct effective compound of arctiin in Fructus Arctii, but its oral bioavailability is lower because of its lignan structure and insolubility in water, and it is usually administered in the form of an injection [Fructus Arctii [″-dihydroxyl-enterolactone showed better binding with TGF-βRI according to the XP Gscore in molecular docking studies (Table Arctiin and arctigenin could inhibit the conversion of fibroblasts to myofibroblasts and reduce the accumulation of extracellular matrix njection . Arctiins Arctii . Our ress Arctii , 72. Thees Table . A previβ signaling pathway, and arctiin and arctigenin could both attenuate the development of silicosis and protect the lungs from injury by blocking the polarization of macrophages and inhibiting the differentiation of myofibroblasts by regulating TLR-4/NLRP3/TGF-β signal transduction. Our research for the first time reveals that both arctiin and arctigenin are effective in silicosis, with the former showing better druggability.To the best of our knowledge, this is the first study identifying urine myristic acid and serum L-arginine and 4-hydroxyproline as biomarkers for silicosis diagnosis. The mechanism underlying the development of silicosis is closely associated with inflammation, oxidative stress, and lipid and amino acid synthesis and degradation. Arctiin and arctigenin could ameliorate oxidative stress, immune-related inflammatory reaction, and fibrosis through pantothenate and CoA biosynthesis, cysteine and methionine metabolism, taurine and hypotaurine metabolism, linoleic acid metabolism, and arginine and proline metabolism successively. The importance of macrophage polarization in fibrosis was verified by integrated arginine and proline metabolism and the TLR-4/NLRP3/TGF-


We focus on biological ligation methods, such as enzymatic ligation from the bacteriophage T4 and ribozyme method. In addition, we summarize the current challenges in the design, synthesis, application, and production of circRNAs, and propose possible solutions in the future. CircRNA is expected to play an essential role in basic research and medical applications.Circular RNA (circRNA) is a unique type of noncoding RNA molecule. Compared with traditional linear RNA, circRNA is a covalently closed circle produced by a process called backsplicing. CircRNA is abundant in many cells and has rich functions in cells, such as acting as miRNA sponge, protein sponge, protein scaffold, and mRNA regulator. With the continuous development of circRNA study, circRNA has also played an important role in medical applications, including circRNA vaccines and gene therapy. In this review, we illustrate the synthesis of circRNAs  EcircRNin vivo , such as and Sry . Besides and Sry  and scaf and Sry . In addi and Sry  and the  and Sry . As biomosophila . Moreoveosophila .6-methyladenosine (m6A) is also proved to promote the extensive translation of circRNAs  reaction  or 1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide to link DNA-RNA hybrids , T4 RNA ligase 1 (T4 Rnl 1), and T4 RNA ligase 2 (T4 Rnl 2). It is worth noting that linear RNA precursor needs a 3′-OH on the acceptor substrate and a 5′ monophosphate on the donor substrate for enzymatic ligation . If the T4 Dnl ligation reaction includes inactivated kinase reaction and hybridized reaction. T4 Dnl can help ligate double-stranded duplexes, such as DNA/RNA hybrids  Figure . TherefoT4 Rnl 1 is a more common ligase for RNA ligation. T4 Rnl 1 catalyzes the nucleophilic attack of the 3′-OH terminus onto the activated 5′-terminus to form a covalent 5′, 3′-phosphodiester bond  and prodT4 Rnl 2 can also be used for RNA ligation . SimilarIn a word, T4 Dnl and T4 Rnl 1 are suitable for RNA ligations without complex secondary structures. T4 Rnl 2 is more suitable for linear RNA precursor with the ligation junction in a double-stranded region. Therefore, different T4 ligases need to be selected according to the secondary structure of the linear RNA precursor. However, all these enzymatic ligation methods cannot realize large RNA molecules ligation, and cannot totally avoid intermolecular end-joining side reactions. These problems still need to be solved .2+ as cofactors and shows great potential for protein synthesis. This method realized RNA ligation through a regular group I intron self-splicing reaction, including two transesterifications at defined splice sites  method . PIE metce sites . PIE met in vivo , which b in vivo . Based oin vitro  can produce circRNAs through rolling circle reaction and the self-splicing reaction from circular single-strand DNA template . The linin vitro . This mein vitro . Freezinin vitro . The disin vitro . Besidesin vitro. HPR method can efficiently produce small circRNAs but suffers from unstable product and exogenous HPR sequences. Among these ribozyme methods, PIE method shows the greatest potential for circRNAs production. At present, PIE method has been widely used in basic research and industrial production (In a word, PIE method using group I introns is suitable for most circRNAs production at present, which is still limited by the secondary structure of linear RNA precursor. Group II introns method can enable more accurate linear RNA precursor ligation, but the mechanism remains unclear oduction .in vitro, there are still many challenges and opportunities. CircRNA is still a hot area of research, and there are many difficulties to be solved (Although there are many ways to synthesize circRNAs e solved .Although PIE method has realized accurate linear RNA precursor ligation , the strIn terms of enzyme design, although existing natural enzymes from the bacteriophage T4 are capable of RNA cyclization, there is still much room for improvement. It is necessary to improve the cyclization efficiency of the ligases by mutation or rational design, especially for large RNA molecules. The critical point of mutation and rational design is to improve the binding efficiency of ligase to substrate RNA and the selectivity of cyclic products.Reducing side reactions during circRNAs synthesis is also a big challenge. At present, the main side reaction is the intermolecular end-joining reaction, which exists in most circRNA synthesis methods and cannot be completely avoided. Therefore, it is necessary to optimize the cyclization reaction conditions and control the linear RNA precursor concentration to reduce the occurrence of such side reactions. In addition, immobilizing the ligase with hydrogels or other materials may realize the compartmentalization of the reaction and the cyclization of a single RNA substrate.With the growing application of circRNAs, the synthesis of circRNAs with chemically modified or unnatural nucleotides is also difficult to be solved. Since circRNA does not have a terminal structure, the modification of circRNA is more difficult than linear RNA. One solution is to use unnatural nucleotides for the synthesis of linear RNA precursors, which can embed unnatural nucleotides into the final circRNA to improve the stability of circRNA and diversify its functions. In addition, the incorporation of chemically modified groups or unnatural nucleotides during the ligation reaction is also a possible solution.Although circRNAs have been proved to hold the potential to become a novel vaccine , the comFaced with the growing demand for circRNAs, large quantities of circRNAs need to be produced. Generally, increasing the yield of circRNAs requires increasing the concentration of the ligation reaction components, such as linear RNA precursors. However, an increase in the concentration of linear RNA precursors will lead to an increase in side reactions. These side reactions will decrease the yield of target circRNAs and increase the difficulty of product purification. Therefore, further optimization of current reaction components and conditions may be required based on scaled-up production systems. This problem may also be solved by new-type reactors, such as microreactors. At the same time, the cost of current circRNAs production is still a concern for researchers. The main reason is the cost of raw materials, such as expensive nucleotides. One potential solution is to use cells to synthesize nucleotides or directly synthesize linear RNA precursors. However, how to efficiently purify these raw materials produced by cells still needs further research.In a word, circRNA is still a hot area of current research and holds great potentials. With the continuous efforts in this field, circRNAs will play an essential role in basic research and medical applications, and become an important part of human health in the near future.


In the last 20 years, an enormous body of evidence has demonstrated critical roles of a unique subset of CD4 T cells, so-called regulatory T cells (Tregs), in maintaining immunological self-tolerance by actively suppressing aberrant immune responses. The multiple functional properties of Tregs target a variety of immune cells through a range of potent suppressive mechanisms. Moreover, it has also become clear that rather than being a homogeneous population, Tregs can undergo functional specialization in response to different environmental clues, allowing them to control local inflammation in a given tissue microenvironment. While numerous studies have established Foxp3 act as a master molecular regulator that orchestrates a transcriptional program that governs the differentiation and function of Tregs, mounting evidence has also demonstrated a crucial role of post-transcriptional mechanisms in controlling multiple aspects of Treg biology.Frontiers in Immunology have addressed post-transcriptional and post-translational mechanisms of regulating Treg development, homeostasis, activation, lineage stability, and suppressive function. In the mini-review “Effects of Non-Coding RNA on Regulatory T Cells and Implications for Treatment of Immunological Diseases”, Luo and Wang provide a comprehensive overview of individual non-coding RNAs that control Treg homeostasis and suppressive function. Non-coding RNAs, including microRNAs and long non-coding RNAs, were once considered “junk” genetic material, but extensive efforts in the last decade have revealed that key non-coding RNAs are indispensable in the generation and function of Tregs. To this end, Kunze-Schumacher and Krueger provide a broad overview of how microRNAs influence Treg development and function in their mini-review, “The Role of MicroRNAs in Development and Function of Regulatory T Cells – Lessons for a Better Understanding of MicroRNA Biology”. The network of microRNAs and their targets are extremely complex. Kunze-Schumacher and Krueger discuss miRNA-target relationships in detail, including context-dependent function and the roles of individual targets vs. complex co-targeting networks.A series of recent contributions in RNA modification, also called “RNA epigenetics” is another attractive area of research on post-transcriptional regulation. Among these modifications, RNA methylation is particularly common and abundant. With the identification of the important “writer”, “eraser” and “reader” enzymes that reversibly mediate methylation, the functions of RNA methylation in T cells, including Tregs, are now being elucidated. In their mini-review entitled “Multiple Functions of RNA Methylation in T Cells: A Review”, Chao et al. provide a detailed summary of recent progress in uncovering the effects of four major forms of RNA methylation  on T cell function.Finally, two articles have provided insights on post-translational regulation in Tregs. In original research entitled “Mouse Double Minute 2 Homolog-Mediated Ubiquitination Facilitates Forkhead Box P3 Stability and Positively Modulates Human Regulatory T Cell Function”, Wang et al. demonstrated that mouse double minute 2 homolog (MDM2), an E3 ubiquitin ligase of p53, also interacts with Foxp3 and unexpectedly stabilizes Foxp3 expression. Dong et al. focus on post-translational regulation of Foxp3 in their review entitled “Post-Translational Regulations of Foxp3 in Treg cells and Their Therapeutic Applications” and provide detailed lists of Foxp3 binding patterns and post-translational modifications and their potential function in regulating Foxp3 function and plasticity.This series of Research Topics provides an extensive update on post-transcriptional and post-translational regulation in Tregs. A deeper understanding of these molecular and cellular mechanisms that control Treg development and function would undoubtedly provide unique opportunities to treat not only various autoimmune diseases but also other immunological disorders.XZ and L-FL contributed equally to this work. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Caenorhabditis elegans (C. elegans). Here we report on the use of co-CRISPR ‘marker’ genes: worms in which co-CRISPR events have occurred have overt, visible phenotypes which facilitates the selection of worms that harbour CRISPR events in the target gene. Mutation in the co-CRISPR gene is then removed by outcrossing to wild type but this can be challenging if the CRISPR and co-CRISPR gene are hard to segregate. However, segregating away the co-CRISPR modified gene can be less challenging if the worms selected appear wild type and are selected from a jackpot brood. These are broods in which a high proportion of the progeny of a single injected worm display the co-CRISPR phenotype suggesting high CRISPR efficiency. This can deliver worms that harbour the desired mutation in the target gene locus without the co-CRISPR mutation. We have successfully generated a discrete mutation in the C. elegans nlg-1 gene using this method. However, in the process of sequencing to authenticate editing in the nlg-1 gene we discovered genomic rearrangements that arise at the co-CRISPR gene unc-58 that by visual observation were phenotypically silent but nonetheless resulted in a significant reduction in motility scored by thrashing behaviour. This highlights that careful consideration of the hidden consequences of co-CRISPR mediated genetic changes should be taken before downstream analysis of gene function. Given this, we suggest sequencing of co-CRISPR genes following CRISPR procedures that utilise phenotypic selection as part of the pipeline.Multiple advances have been made to increase the efficiency of CRISPR/Cas9 editing using the model genetic organism  Originally identified as a mechanism of bacterial immunity , CRISPR/C. elegans is an attractive model organism for the application of CRISPR/Cas9 because their genetic tractability and short life cycle allows for the rapid generation of progeny with cloned genetic identity [C. elegans means CRISPR mediated modifications can be investigated at a molecular and behavioural level [C. elegans genomes [identity . Systemsal level –11. Furt genomes , CRISPR/ genomes –9.C. elegans genes [dpy-10 C. elegans gene. Transplantation of the necessary motifs for efficient CRISPR/Cas9 therefore renders the gene of interest more amenable to editing and hence enhances the scope of CRISPR in C. elegans [Despite its advantages, the efficiency of CRISPR/Cas9 can be limited by a number of factors. For example, the interaction of the single guide RNA (sgRNA) Cas9 complex with target DNA requires Cas9 to bind to a protospacer adjacent motif (PAM) . It has ns genes  thus lim elegans .C. elegans is the use of co-CRISPR genes [dpy-10, rol-6, sqt-1 and unc-58 [dpy-10 result in dumpy, roller or dumpy roller phenotypes, depending on the mutant genotype. Mutations in rol-6 and sqt-1 result in roller phenotypes causing the worms to move in a circular pattern [unc-58 encodes a two-pore domain potassium channel (K2P) in C. elegans [unc-58 is to act as a leak channel that is important in setting the resting membrane potential [unc-58(e665) fourth transmembrane helix results in a hypercontracted and uncoordinated phenotype which leaves the worm immobile [+ to Na+ [unc-58 locus. In particular, the switch from immobile to motile provides an obvious measure of reversion from GOF to WT. The easily recognisable phenotype of unc-58 GOF mutants has led to its successful widespread use in technologies that are aided by phenotypic markers [Another technique used to increase the efficiency of the CRISPR process in PR genes ,17. A cod unc-58 ,17. Muta pattern . unc-58  elegans  which pl elegans ,20. A protential . A gain-immobile ,23. Inte+ to Na+ . This pr markers –26 and t markers ,16,27.Easily recognisable co-CRISPR phenotypes can also be used to identify so called jackpot broods. Jackpot broods are a population of progeny derived from a single parent, injected with Cas9, sgRNAs and repair templates to facilitate CRISPR at both a target gene and co-CRISPR gene, in which a high percentage of the population displays the co-CRISPR marker phenotype . A largeC. elegans. The method encompasses a two-step procedure which uses a d10 sequence to facilitate the generation of an edit in a precise location within the gene of interest and uses co-CRISPR marker genes for rapid phenotypic screening [C. elegans nlg-1 gene. Simultaneously, the co-CRISPR gene unc-58 was edited to generate a GOF mutation that results in an uncoordinated phenotypic marker for visual screening of CRISPR efficiency. Following this, in a second round of CRISPR editing, the d10 sequence in nlg-1 was replaced by a repair template containing the sequence for the desired R451C mutation. Again, simultaneously another co-CRISPR gene, dpy-10, was edited to generate dpy-10(cn64) GOF mutant for the purpose of phenotypic screening.Recently, a method of CRISPR/Cas9 has been developed that combines multiple techniques to increase the efficiency of CRISPR and allows for highly targeted editing in creening . We utilcreening , in ordecreening . This fiunc-58. The resulting mutants did not display the hypercontraction characteristic of the unc-58 GOF but rather resulted in a subtle loss-of-function (LOF) not discernible by visual inspection of gross locomotion. We use this to suggest that the phenotypic appearance of C. elegans is not a robust way to determine the genotype of co-CRISPR genes. Given this, we suggest routine sequencing of co-CRISPR genes following CRISPR procedures that utilise phenotypic selection before downstream analysis.In this study, we describe an unexpected CRISPR mediated mutation that occurred within the co-CRISPR gene C. elegans strains were maintained using standard conditions [nlg-1(ok259) X (x6 outcrossed), CB665 unc-58(e665) X, TN64 dpy-10(cn64) II, provided by Caenorhabditis Genetics Centre (CGC). XA3780 nlg-1(qa3780) X (x2 outcrossed), XA3773 unc-58(qa3788) X; nlg-1(qa3780) X (x1 outcrossed), XA3788 unc-58(qa3788) X (x2 outcrossed), generated in this study. JIP1154 unc-58(bln223) X, with a deletion in unc-58 from exon 1a to 10 (3.8kb), provided by Thomas Boulin.All nditions . Strainsnlg-1 and unc-58 or dpy-10 for the first and second round of CRISPR, respectively. The design of the sgRNAs and repair templates to edit unc-58, dpy-10 and nlg-1 genes were as previously reported [TM–Integrated DNA Technologies). All injection reagents were diluted in molecular grade water to a final concentration of 50ng/μl in the CRISPR mix.The previously reported method by El Mouridi et al was followed for CRSIPR/Cas9 editing . L4+1 dareported ,16. sgRNnlg-1 primers, forward 5’- ATGAGTATACAGATTGGGAAAATCCC-3’ and reverse 5’- ACTGTTTGGTTGCTCTTGGCTCCAAG-3’, were used to amplify the CRISPR targeted region of nlg-1 using a single worm PCR protocol [nlg-1. The primers used for sequencing of the target regions of unc-58 and dpy-10 were: forward 5’- GACTCGGAGATATCGTTGTGACTG-3’, reverse 5’- CGCGGAGTTCGTTATCCAGGAAG-3’ and forward 5’- ACTAATTCAGAGTCATCATCTCGCC-3’, reverse 5’- CATCAATTCCCTTAAGTCCTGGTGG-3’ respectively.protocol . A BanI unc-58(qa3788); nlg-1(qa3780) double CRISPR mutant strain was backcrossed with N2 males once to generate heterozygous F1 progeny. These progeny were cloned by picking a single F1 onto individual plates before being left for 2–3 days to self-fertilise and F2 progeny were screened using a single worm PCR protocol [unc-58 and nlg-1 PCR amplicons were performed using restriction enzymes BsiWI and BstBI respectively to screen for the presence or absence of CRISPR mediated edits. Restriction enzymes were supplied by New England BioLabs (NEB) and used according to manufacturer instructions.protocol  and restE. coli at OD600 of 0.8 was gently spotted on to the middle of an unseeded plate the day prior to the assay. Seven age synchronised L4+1 day old hermaphrodites were gently picked onto the centre of the bacterial lawn on the assay plate. At 2 and 24 hours food leaving events were counted visually using a Nikon SMZ800 microscope (X10 magnification) during 30 minute observations. A food leaving event was defined as when the whole of the worm’s body exited the bacterial lawn. N2 animals were used as a paired control, run in parallel with the strain under investigation and the investigator was blind to the genotypes being observed.Food leaving assays were carried out as previously described . BrieflyUsing a 24 well plate, a single worm was picked per well containing 500μl of M9 with 0.1% bovine serum albumin and left for 5 minutes before thrashing was measured. For each worm, thrashing events were visually counted under a Nikon SMZ800 microscope (X30 magnification) for a period of 30 seconds. This was repeated three consecutive times and the mean was calculated. Each thrash was defined as a complete movement through the midpoint of the worm’s body and back. N2 animals were used as a paired control, run in parallel with the strain under investigation and the investigator was blind to the genotypes.UNC-58, isoform b protein sequence was downloaded from WormBase version WS278. The wild-type or CRISPR mutant protein sequence was entered into the membrane topology prediction tool TMHMM (v.2.0) ,33 to deunc-58 and dpy-10 to facilitate the generation of a precise edit in C. elegans nlg-1 gene. 84 N2 animals were injected with plasmids encoding Cas9 and a sgRNA and repair template designed to transplant a d10 sequence [nlg-1. In addition, the CRISPR injection mix included a sgRNA and repair template designed to generate a GOF mutation in the co-CRISPR gene unc-58  and approximately 3.5mb apart. Identifying the nlg-1 edit in an unmarked worm can be beneficial because it avoids the need for backcrossing to separate two chromosomally linked edits. This resulted in the identification of a single nlg-1 d10 entry strain in an unmarked F1 worm from a jackpot brood which displayed no obvious unc-58 marked phenotype. Consistent with other studies, the lack of unc-58 marked phenotype was used to assume that the nlg-1 d10 entry strain was WT for the unc-58 co-CRISPR loci [We used co-CRISPR genes sequence  into thee unc-58 . This way edited . 2 of thg-1 gene . We alsoSPR loci ,27,29.nlg-1 d10 entry strain, were injected with plasmids encoding Cas9, a sgRNA and a repair template designed to replace the nlg-1 d10 sequence with the R451C mutation of interest. In addition, the CRISPR injection mix included a sgRNA and repair template designed to generate a GOF mutation in the co-CRISPR gene dpy-10 ;nlg-1(qa3780) double mutant strain. Genetic crosses, combined with PCR amplification, enzymatic digest and sequencing allowed the identification of a single strain that retained the nlg-1 CRISPR mutation and had lost the unc-58 CRISPR mutation  null mutant have the same phenotype, suggesting that the CRISPR mediated mutation results in loss of function to the unc-58 gene. These data also show that the loss of function to unc-58 in the unc-58;nlg-1 double mutant results in a reduced thrashing rate which is not displayed in the nlg-1 CRISPR mutant.Given the functional importance of the UNC-58 potassium channel subunit for locomotion , we want control . In comp control . Furtherhenotype . Taken tnlg-1 gene we unexpectedly created a loss of function mutation within the unc-58 gene as a consequence of targeting it with reagents designed to generate a gain-of-function phenotype. Analysis of the unc-58 CRISPR mutant demonstrates that it has a predicted protein sequence that would result in a severe loss of channel function. We show that this loss of function mutation in unc-58 results in a subtle, but significant effect on motility consistent with its established role in regulation of neuronal membrane potential [Overall, in the process of CRISPR editing the otential .nlg-1 and utilised the unc-58 GOF phenotype to facilitate the screening for CRISPR events. The efficiency of d10 transplantation into the gene of interest, nlg-1, was lower than that previously reported [dpy-10 sgRNA [dpy-10 marked progeny. However, within the pool of marked progeny the edit efficiency at nlg-1 was approximately 1%, which is lower than that reported in other genes [We demonstrate the use of a previously described method of CRISPR/Cas9 that relies upon the use of co-CRISPR genes  to facilreported . The hig10 sgRNA  meant ther genes . This liunc-58 as a co-CRISPR gene led to the creation of a novel unc-58 mutant strain and we have provided evidence that the unc-58 CRISPR strain has the same phenotype as an unc-58 null mutant suggesting it is LOF. K2P LOF mutants do not display an easily recognisable phenotype, unlike GOF mutants and are therefore comparitively less well studied [unc-58 is expressed widely in interneurons and motor neurons [Our use of  studied . unc-58  neurons . It is h neurons  and the  neurons . To our unc-58 mutants display a less severe phenotype because there is functional redundancy with other K2P channels and this may compensate for the effect of the loss of UNC-58 on resting membrane potential [twk-7 null has moderately enhanced motility [unc-58 is required for normal thrashing behaviour demonstrates that K2P LOF mutants can display locomotory phenotypes, albeit less severe than for GOF mutants. This is consistent with K2Ps playing an important role in co-ordinating normal functioning of the nervous system and highlights how recombination events within this gene could impact downstream analysis of complex integrative behaviours that are dependent upon the worms being able to execute locomotory behaviour.In comparison, it has been suggested that loss-of-function otential . Of the motility . This, cunc-58 CRISPR LOF mutant the amino acid sequence is mutated in the second half of the fourth transmembrane domain and the majority of the intracellular C-terminus is deleted. This suggests that these domains are likely to play a cruicial role in unc-58 channel functioning. In mammalian K2Ps the C-terminus is important for channel gating [C. elegans K2Ps [C. elegans.We show that in the l gating . Studiesans K2Ps  however unc-58 as a co-CRISPR gene have been previously discussed [unc-58, other more favourable co-CRISPR genes, such as dpy-10 and sqt-1 produce easily recognisable phenotypes in response to both GOF and LOF [dpy-10 can result in dumpy, roller or dumpy roller phenotypes depending on the mutant genotype [unc-58 continues to be used in co-CRISPR approaches [unc-58 as a co-CRISPR gene, relying soley on the gross phenotypic appearance of the worm is not a robust way to assume genotype. Similarly, to unc-58, the loss of function of rol-6 has a very subtle phenotype compared to the wild-type . Therefore, we suggest that sequencing of co-CRISPR genes, particularly unc-58, is important following their use and combining the use of co-CRISPR markers that are dependent upon GOF modifications with approaches that promote HDR  would be further beneficial.The caveats of using iscussed . Unlike  and LOF . For exagenotype . Despiteproaches ,27,29. WC. elegans that is generated by a GOF mutation in unc-58 provides an excellent marker for screening for CRISPR events in target genes of interest. In using this approach we unexpectidely generated a novel unc-58 mutation and provided evidence that this LOF impaired motility in liquid. This will facilitate the understanding of K2Ps in C. elegans and the behaviours they regulate. However, it is clear that if utilised to generate mutants for investigation of more complex behaviours or readouts that depend on neuronal exciatbility, the spurious re-arrangements at the unc-58 marker locus can generate more subtle but not inconsequential LOF mutations.In conclusion, the visibly obvious hypercontraction in the viable adult unc-58 in co-CRISPR strategies destined to target gene loci that impact discrete determinants of integrative behaviour, we conclude that this requires routine sequencing of the co-CRISPR gene to avoid potential confounds of unpredicted CRISPR events.Based on our experience of using S1 Figunc-58 CRISPR mutation are indicated. DNA sequences are shown in the 5’ to 3’ orientation.The TGC codon that encodes the R451C mutation is indicated. The wild-type GT NTs that were deleted as part of the (TIF)Click here for additional data file.


Penicillium digitatum is the most severe pathogen that infects citrus fruits during storage. It can cause fruit rot and bring significant economic losses. The continuous use of fungicides has resulted in the emergence of drug-resistant strains. Consequently, there is a need to develop naturally and efficiently antifungal fungicides. Natural antimicrobial agents such as clove oil, cinnamon oil, and thyme oil can be extracted from different plant parts. They exhibited broad-spectrum antimicrobial properties and have great potential in the food industry. Here, we exploit a novel cinnamaldehyde (CA), eugenol (EUG), or carvacrol (CAR) combination antifungal therapy and formulate it into nanoemulsion form to overcome lower solubility and instability of essential oil. In this study, the antifungal activity evaluation and transcriptional profile of Penicillium digitatum exposed to compound nanoemulsion were evaluated. Results showed that compound nanoemulsion had a striking inhibitory effect on P. digitatum in a dose-dependent manner. According to RNA-seq analysis, there were 2,169 differentially expressed genes (DEGs) between control and nanoemulsion-treated samples, including 1,028 downregulated and 1,141 upregulated genes. Gene Ontology (GO) analysis indicated that the DEGs were mainly involved in intracellular organelle parts of cell component: cellular respiration, proton transmembrane transport of biological process, and guanyl nucleotide-binding molecular function. KEGG analysis revealed that metabolic pathway, biosynthesis of secondary metabolites, and glyoxylate and dicarboxylate metabolism were the most highly enriched pathways for these DEGs. Taken together, we can conclude the promising antifungal activity of nanoemulsion with multiple action sites against P. digitatum. These outcomes would deepen our knowledge of the inhibitory mechanism from molecular aspects and exploit naturally, efficiently, and harmlessly antifungal agents in the citrus postharvest industry. P. digitatum, accounting for more than 80% of the total crop losses  has been widely used to study eukaryotic transcriptomes –24. RNA-P. digitatum was determined with an RNA-Seq approach. P. digitatum transcriptome and the DEGs between nanoemulsion-treated and untreated samples were obtained, which provide a theoretical reference for improving the prevention and control effect of citrus postharvest diseases.In this study, the antifungal mechanism of a combination of eugenol, cinnamaldehyde, and carvacrol nanoemulsion on ® T18 digital, IKA, Staufen, Germany), and finally passed through a microfluidizer  to obtain a stock solution of 100 mg/ml. P. digitatum was isolated from green mold-infected citrus in our laboratory and maintained on PDA medium at 4°C. Fungal conidia from a 7-day-old culture were washed with sterilized water, filtered through four layers of gauze, and finally adjusted to a suspension of 1 × 107 conidia/mL by hemocytometer.Cinnamaldehyde, citral, and eugenol were purchased from Aladdin Co., Ltd. . First, a combination of cinnamaldehyde, citral, and eugenol in a ratio of 1:1:1 was dissolved in 2% Tween-80, then blended with a high-shear mixer  / (control colony diameter) × 100%. There are three replicates of each concentration, and the experiment was conducted twice.The antifungal activity of compound nanoemulsion against n method  with som7 conidia/ml) was added to 150 ml liquid culture medium  and cultured in an incubation shaker at 140 rpm for 48 h. Then, the mycelia were centrifuged at 3,000 × g for 20 min, followed by washing with phosphate buffer (pH 7.0) three times and resuspended in 100 ml PBS (pH 7.0). Afterward, the stock nanoemulsion was added to the flask (D-T) to the final concentration of 0.125 mg/mL, which is the minimum inhibitory concentration on P. digitatum, and then kept in an incubation shaker for 12 h; there was not nanoemulsion added, which was taken as the control (D-C). Finally, the mycelia, which removed phosphate buffer, were rapidly frozen in liquid nitrogen and kept in a −80°C refrigerator. Each treatment was performed three times.About 1.5 ml spore suspension , Qubit® RNA Assay Kit in Qubit® 2.0 Fluorometer , RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system  respectively. Each group were conducted for triple biological replicates.Total RNA preparation, quality control, cDNA libraries construction, and RNA-seq were conducted by Shanghai Applied Protein Technology (APT) Co., Ltd. The TRIzol reagent was used  to isolate total RNA according to the instruction. One percent agarose gel electrophoresis was used to evaluate RNA degradation and contamination. RNA purity, concentration, and integrity were checked using the NanoPhotometer® UltraTM RNA Library Prep Kit for Illumina®  following the manufacturer's recommendations, and each sample was to attribute sequences by adding index codes. The divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X) were used to fragment. The fragments were used to synthesize the first-strand cDNA with random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). The second-strand cDNA was transformed from the first-strand cDNA using RNase H and DNA polymerase I. Fragments of preferential lengths of about 250~300 bp were purified using the AMPure XP system , end-repaired, and adapter-ligated. Then, 3 μl of USER Enzyme  was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers, and index (X) primer. Finally, PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system.The input material is 3 μg RNA of each sample for the RNA preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads by using NEBNextde novo assemble processed reads (k = 25). The seed k-mers were extended to both ends to form a contig. The overlapped contigs are clustered to form components, and each component becomes a set of possible representations of variable splicing isoform or homologous genes. Each component has a corresponding de Bruijn graph. The de Bruijn graph is simplified, and the path with continuous nodes is combined to form a more extended sequence, and the best path is found to obtain the transcriptional sequence.After filtration of the lower-quality reads, obtained clean reads were used for the following analysis. The clean reads were mapped to the reference genome and assembled according to the previous method . The Trinity program was used to ed reads . The seqP-value < 0.05 were considered significantly enriched by DEGs. We used the clusterProfiler package (version 3.4.4) to annotate the pathways of DEGs in KEGG pathways.First, we were blasting the optimal transcript against the NCBI non-redundant protein sequences database (NR) and Swiss-Prot to obtain unigenes. To further annotate the unigenes, the Blast2GO program with Blast2GO default parameters was used to obtain Gene Ontology (GO) annotations. GO enrichment analyses of DEGs were implemented by the cluster profile package (version 3.4.4), in which gene length bias was corrected. GO terms with corrected P-values, which are based on the negative binomial distribution model, false discovery rate was calculated by Benjamini and Hochberg's approach. Genes with an adjusted P-value < 0.05 were assigned as differentially expressed.The FeatureCounts software (version 1.5.0-p3) was used to count the reads numbers mapped to each gene. Expression levels of unigenes were normalized and calculated as the values of fragments per kilobase of transcripts per million mapped fragments (FPKM). To select DEGs by DESeq2 package (version 1.16.1), DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting ® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) , according to the manufacturer's instructions. The qRT-PCR assay was performed on CFX Real-Time PCR Detection Systems . The actin gene was obtained as an internal reference .After the treatment with nanoemulsion, the mycelia were collected and used to determine the membrane lipid peroxidation-related parameters. The malondialdehyde (MDA) content was determined by following the previous method with minor modifications . Accordig et al. , using aP. digitatum, and bovine serum protein was used as the standard curve. P. digitatum was incubated in a shaker at 27°C, 180 rpm for 48 h. The exact amounts of mycelia were suspended in 10 mL phosphoric acid buffer (pH 7.0), and nanoemulsion was added to make the final concentration of 0.125 mg/mL. The culture was kept in a shaker at 27°C, 180 rpm, for 0, 2, 4, 6, and 12 h. The mycelium was frozen by liquid nitrogen, and then the mycelia were grounded into a paste with 5 mL distilled water and centrifuged at 4°C, 12,000 rpm for 15 min. About 1.0 mL of supernatant was taken, and 5 mL of Coomassie Bright Blue solution was added. The solution was shaken thoroughly, then let stand for 5 min. The OD value was measured at 595 nm with distilled water as blank control. According to the standard curve, the soluble protein content of mycelia was calculated, and the result was expressed as the mass of soluble protein per gram of mycelia (mg/g). Each treatment has three biological replicates, and the experiment was conducted twice.Coomassie Bright Blue method was used to determine the change in soluble protein content in the mycelium of p < 0.05).Data were expressed as the mean ± SD, which was conducted triplicate, and were performed using SPSS 22.0 . One-way ANOVA and Duncan's multiple range test were used to evaluate the significance  on the 7th day, the control did not show an inhibitory activity. These results indicated that nanoemulsion could inhibit the mycelia growth of P. digitatum in a dose-dependent manner.As illustrated in P. digitatum generated 46.9 and 42.7 million raw reads, respectively. After filtering the adaptor sequences, 46.7 and 42.5 million clean reads were obtained. The GC content of each sample is above 50%, indicating that the base content is stable and there was no AT or GC separation; the Q30 base percentage of all samples is higher than 95%, which means obtained clean reads were accurate and can be used for subsequent analysis.The quality inspection was conducted on all samples to meet the requirements of sequencing database construction. From the sequencing data shown in P. digitatum after nanoemulsion treatment: 1,141 genes showed upregulation tendency, whereas 1,028 genes were downregulated . Therein, the significantly abundant DEGs (216) were enriched in the metabolic pathway (ko01100), 95 DEGs were enriched in the biosynthesis of secondary metabolites (ko01110), 18 DEGs were enriched in glyoxylate and dicarboxylate metabolism (ko00630), and 15 and 14 DEGs were enriched in DNA replication (ko03030) and fatty acid degradation (ko00071), respectively. P-value < 0.05). The results indicated that the downregulated pathways involved mainly belonged to the DNA replication, proteasome, chloroalkane, and chloroalkene degradation. The upregulated pathways involved are ABC transporters, alpha-linolenic acid metabolism, metabolic pathways, and biosynthesis of unsaturated fatty acids.P-value < 0.05), which is indicated by P-value < 0.05). The results indicated that the upregulated GO terms were cellular respiration, energy derivation by the oxidation of organic compounds, proton transmembrane transport of biological process; proton transmembrane transporter activity, monovalent inorganic cation transmembrane transporter activity and cytochrome-c oxidase activity of molecular function; mitochondrion, mitochondrial envelope, mitochondrial part of the cellular component of a cellular component. The downregulated GO terms were chromosome organization, cellular component organization, organelle organization of biological process; structural constituent of the cytoskeleton, structural molecular activity, purine nucleoside binding of molecular function, intracellular organelle, organelle, and intracellular organelle part of a cellular component.The most enriched GO in 2169 was DEGs  encoding ergosterol biosynthesis was downregulated by 2.62-, 3.57-, and 2.71-fold, respectively. Previous research reported that ERG3 gene downregulation caused P. digitatum cells to lose the capacity to convert lanosterol to ergosterol  showed upregulated tendency after peptide MAF-1A treatment, involved in oxidative stress response , amino acid synthesis, proteasome, glyoxylate and dicarboxylate metabolism, ribosomes biogenesis, and mitogen-activated protein kinases were notably affected by nanoemulsion treatment. The results indicated that nanoemulsion triggered gene expression variation and induced multiple pathways involvement. A deep understanding of the transcriptomic view mechanism further demonstrated the applicability of nanoemulsion as a natural origination, environment-friendly, and safe approach to combat against the green mold of citrus.In conclusion, through RNA-seq technology, we analyzed the effect of nanoemulsion on the http://www.ncbi.nlm.nih.gov/bioproject/745208.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: SubmissionID: SUB9953553; BioProject ID: PRJNA745208; CW and JC involved in conceptualization, resources, supervision, and project administration. RY, XC, and CC involved in methodology. RY and XC involved in software and formal analysis. RY and CC involved in validation. RY, XC, and QH involved in investigation. RY and CW involved in data curation and visualization. RY involved in writing the original draft preparation. CW and KR involved in writing the review and editing. CW involved in funding acquisition. All authors have read and agreed to the published version of the manuscript.This work was supported by the National Natural Science Foundation of China (31760598) and the Natural Science Foundation of Jiangxi Province of China (20181BCB24005).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The current SARS-CoV-2 pandemic underscores the importance of understanding the evolution of RNA genomes. While RNA is subject to the formation of similar lesions as DNA, the evolutionary and physiological impacts RNA lesions have on viral genomes are yet to be characterized. Lesions that may drive the evolution of RNA genomes can induce breaks that are repaired by recombination or can cause base substitution mutagenesis, also known as base editing. Over the past decade or so, base editing mutagenesis of DNA genomes has been subject to many studies, revealing that exposure of ssDNA is subject to hypermutation that is involved in the etiology of cancer. However, base editing of RNA genomes has not been studied to the same extent. Recently hypermutation of single-stranded RNA viral genomes have also been documented though its role in evolution and population dynamics. Here, we will summarize the current knowledge of key mechanisms and causes of RNA genome instability covering areas from the RNA world theory to the SARS-CoV-2 pandemic of today. We will also highlight the key questions that remain as it pertains to RNA genome instability, mutations accumulation, and experimental strategies for addressing these questions. Nothing in Biology Makes Sense Except in the Light of Evolution” [One of the favorite aphorisms in modern biology is the title of the Theodosius Dobzhansky’s essay “olution” . In his olution” —that intIn today’s biological systems, the genetic material making up the genome is primarily DNA. In contrast, a plethora of viruses that infect cellular hosts throughout all kingdoms rely upon RNA as their primary genetic material. Whichever the genetic material of the virus genome, there is the requirement that the genome remains stable to allow for the transmission of viable genetic material to progeny, and to prevent the extinction of species ,4,5,6. HTo date, there has been numerous studies into the stability/instabilities of DNA genomes, but the same level of research has not been performed for RNA. This disparity is important to note because RNA genomes are predicted to be a vital key to evolution, as prior to the last universal common ancestor (LUCA) the RNA world theory predicts the existence of protocells that used RNA as a genetic material. Further, RNA was also proposed to be used as an enzyme to mediate all metabolic functions (as proteins had not yet evolved) ,16,17,18The modes of replication of modern viruses are known  arrived at where we are today. Though, we do not know the intermediate steps between the protocells to today, it is still possible that remnants of these protocells remain as viral pathogens. Generally, there are three classes of RNA viruses, and the key feature for separating the classes is based on the state the viral RNA genome  is present ,29. The With these viruses being maintained differently requires different modes of replication (reviewed in ). SpecifBoth APOBECs and ADARs are part of the innate immunity and act as an antiviral by inducing base substitutions in viral genomes ,34,35,36Replication of RNA genomes in viruses is rarely perfect, often there is the introduction of errors  discuss as well Replicative RNA recombination begins when the viral RdRp stalls during replication of the viral genome followed by the dissociation of the newly synthesized RNA molecule from the template that subsequently binds to another template where it is used as a primer to begin synthesis A, reviewNon-replicative RNA recombination is a much rarer form of RNA recombination, occurring independent of RdRp, where two molecules are joined at their ends to create a chimeric molecule B 54,55,,61,62,63RNA viral evolution has resulted in a diverse population of virus types that inevitably contain different combinations of genes within each virus, but present in all classes of viruses is RdRp. ,68. ThouIt was argued that the low fidelity of the RdRp drives the evolution of RNA viruses ,72. ThisWith such a high mutation rate observed in viruses, there is a selection for smaller genomes because a larger genome would have more opportunities for the acquisition of a deleterious mutation resulting in “error catastrophe” ,85,86. CFlexiviridae plant viruses do encode for an AlkB sequence, gained through horizontal gene transfer, that when transcribed reverses alkylated bases within RNA and DNA, with a preference for RNA [Viral genomic RNAs as well as cellular RNAs are the subject to environmental and endogenous lesions. These lesions can result in RNA breakage or can block RNA, where the broken RNA genomes would be either lost or participate in recombination like events which can in turn produce rearranged genomes see . Like DN for RNA ,104. ThiFlaviviridae family of viruses indicating an essential role for pseudouridine in viruses [There have been 170 different RNA modifications identified in RNA across all species, with 60 of those being identified in eukaryotes . Many of viruses . The phy viruses ,105, whi viruses . N5-meth viruses  along wi viruses . Lastly, viruses . Though,So far, only two kinds of enzymatic RNA edits—cytidine to uridine (C to U) by APOBEC cytidine deaminases  and adenThe introduction of base substitutions by APOBEC or ADAR is a common anti-viral strategy as excessive numbers of mutations is likely to result in inviable viral genomes ,34,35,36https://www.gisaid.org/, accessed on 15 April 2021). However, since viral genomes are small, each genome would contain a small number, or even no mutations, so a mutation catalogue representative of a population (or a species) could only be built from sequences of multiple genomes. Building such a catalog must be done under the consideration of important factors to allow meaningful statistical evaluation of mutational spectra and signatures [A similar strategy that was applied to human cancers can now be applied to several RNA viruses, especially because of the accumulation of extensive sequencing data for multiple viruses, most notably including SARS-CoV-2, which was a subject of a gigantic genome resequencing effort across the world   DNA polymerases to successfully accomplish genome duplication. TLS polymerases are often error-prone and results in mutagenesis, while the lack of TLS can lead to genome rearrangements or to replication failure ,160. HowRecent studies indicated that adenosine deaminases ADAR and cytidine deaminases APOBEC are the prevailing sources of base substitutions in several human RNA viruses including SARS-CoV-2, summarized in Defining diagnostic mutational signatures can develop into a multiprong scalable approach to understanding sources and mechanism of mutagenesis in RNA viruses. Mutational signatures turned to be a productive approach for another set of unstable genomes—human cancer. This could be a pure agnostic analysis of large datasets of genome instability catalogs ,121,163,