RNA-based tools are frequently used to modulate gene expression in living cells. However, the stability and effectiveness of such RNA-based tools is limited by cellular nuclease activity. One way to increase RNA’s resistance to nucleases is to replace its D-ribose backbone with L-ribose isomers. This modification changes chirality of an entire RNA molecule to L-form giving it more chance of survival when introduced into cells. Recently, we have described the activity of left-handed hammerhead ribozyme  that can specifically hydrolyse RNA with the opposite chirality at a predetermined location. To understand the structural background of the RNA specific cleavage in a heterochiral complex, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy as well as performed molecular modelling and dynamics simulations of homo- and heterochiral RNA complexes. The active ribozyme-target heterochiral complex showed a mixed chirality as well as low field imino proton NMR signals. We modelled the 3D structures of the oligoribonucleotides with their ribozyme counterparts of reciprocal chirality. L- or D-ribozyme formed a stable, homochiral helix 2, and two short double heterochiral helixes 1 and 3 of D- or L-RNA strand thorough irregular Watson–Crick base pairs. The formation of the heterochiral complexes is supported by the result of simulation molecular dynamics. These new observations suggest that L-catalytic nucleic acids can be used as tools in translational biology and diagnostics. A fundamental feature of the proteins and nucleic acids is their homochirality. These natural biopolymers are composed of only one optically active ‘isomer’: L- amino acids or D-nucleotides. Although the origin of homochirality in biological macromolecules is not fully understood, it is assumed to be essential to ensure the high specificity and fidelity of the recognition mechanism between them. The stereospecificity of enzyme (protein) or ribozyme (nucleic acids) catalysed reactions is the foundation of the homochiral world.5dA and D-poly(rU) have been suggested, but no hybridization of L-dU oligomer and D-poly(dA) was observed [in vitro that selected L-DNA aptamers bind to a D-RNA target [Changing chirality is a strategy to overcome the low chemical stability of the phosphodiester bond of D-RNA in order to build more effective RNA-based tools. It is known that mirror-image ribonucleic acids consisting of L-ribose instead of D-ribose are resistant to hydrolysis with ribonucleases. Also L-DNA, where D-deoxyribose is substituted with L-deoxyribose, are resistant to nucleases ,2. Shortobserved . Also noobserved . A few yobserved . RecentlA target , but WatA target ,12.The intense interest in enantiomeric nucleic acids observed in the last 40 years has dampened recently because the finding that L- and D- oligonucleotides do not form a regular double helix with Watson–Crick base pairs .Recently, for the first time, some of us have shown that mirror image hammerhead ribozymes (L-ribozyme) and L-DNAzyme cleave the complementary D-RNA substrate of reciprocal chirality at the predetermined site ,15. On tChemically synthesized RNA oligonucleotides were from IBA (Germany) or ChemGenes (U.S.A.). The 14-nt synthetic D- and L-RNA oligomers with the sequence 5′CUUCAAGUCCGCCA3′ were labelled with fluorescein at the 5′ end. The D-HH (D-Rz), L-HH (L-Rz) hammerhead ribozymes were 33-nt long with the sequence 5′UGGCGCUGAUGAGGCCGAAAGGCCGAAACUUGA3′. Nucleotides of L33 at positions 1-6 and 29-33 for parallel complex were methylated at 2′OH. D-anti HH Ribozyme 5′UGUCAGCUGAUGAGGCCGAAAGGCCGAACCAGCGG3′ was synthesized and labelled with FAM.2 concentrations. Hydrolysis products were separated with 20% polyacrylamide gel electrophoresis (PAGE) in the presence of 7 M urea in 0.09 M Tris-borate buffer at pH 8.3. The gels were exposed to a Fuji Film FLA 5100 phosphoimager using the manufacturer’s (Fuji) BCIP/NBT Liquid Substrate System (Sigma), and bands were quantified using the ImageQuant software from Molecular Dynamics.The activity of the D- and the L-hammerhead ribozymes with labelled L- and D-RNA targets were measured in 10 μl reaction volumes containing 50 mM Tris-HCl at pH 7.5 at 37°C. The RNA substrate and L- or D-Rz ribozymes were denatured for 2 min at 73°C and cooled to 25°C at a rate of 1°C/min. The reactions were done at 0 and 25 mM MgClCD spectra were recorded on a JASCO J-815 spectropolarimeter. The L- and D-oligonucleotides were dissolved in 50 mM NaCl, 20 mM Tris-HCl pH 7.5 buffer to achieve a concentration of 3.0 μM. All samples were denatured at 90°C for 5 min and then slowly cooled to room temperature before data collection . The spe2. Equimolar amounts D-RNA and L-Rz (L33) was dissolved in 150 mM NaCl and 10 mM sodium phosphate pH 6.8. The solvent was H2O/D2O . The samples were annealed by heating at 90°C for 5 min and then slowly cooled to room temperature. The 3 mm thin wall tubes were used with a final sample volume of 200 μl. The water signal was suppressed by excitation sculpting with a gradient pulse. Spectra were processed and prepared with TopSpin 3.2 Bruker Software.All NMR spectra were acquired on a Bruker AVANCE III 700 MHz spectrometer, equipped with a QCI CryoProbe. D-RNA, L-Rz and their equimolar amounts (3 μM) complex 3 were dissolved in buffer 150 mM NaCl, 50 mM Tris-HCl pH 7.5 with 10 mM MgClhttp://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). We incorporated the three-way junction motif covering the catalytic center of the ribozyme (PDB: 3ZP8). RNAfold generates up to ten structure models and we selected only the model with the lowest total energy. Visualizations of the molecular models were done in PyMOL (http://www.pymol.org). Finally, the linker was removed, and the structure was stored. The heterochiral model L/D we got from D/D structure with the X-Plor program ver. 2.21 [The 3D structures of homochiral and heterochiral complexes of RNA hammerhead (Rz) ribozyme (33-mer) and RNA oligonucleotide (14-mer) were constructed based on the 3D crystal structure of the catalytic center of hammerhead ribozyme (3ZP8) with RNA Composer . The 14 er. 2.21 . The parer. 2.21 . FurtherGeometry of the D/D and D/L duplex models were of heterochiral complex. Correctness of the centre chirality was evaluated by a data processing validated with RCSB MAXIT (MAcromolecularEXchange Input Tool), covering geometry of covalent bond and chirality centres. The two other models L/L and L/D were obtained by changes of values of the Z axis from D/D and D/L, respectively.The fours starting structures (models) of DL, LD, LL and DD were built using SYBYL and UCSF Chimera ,22. In ttleap module of Amber 18 to prepare the topologies and coordinates for simulations. We utilized the AMBER ff99bsc0χOL3 force field for D-RNA and its modified version for L-RNA [OL3 to account for L-ribonucleotides involved corrections of dihedral angles (γ and χ). Each of the structures was solvated with TIP3P water molecules in a truncated octahedral box with minimal distance of 10 Å between solute and the box border [+ cations and NaCl excess salt was added to obtain the concentration of 150 mM by using Joung and Cheatham ion parameters [2 applied to solute atoms and finally without any restraints. Then, at the heating stage, the temperature was gradually raised to 300 K within 80 ps followed by 20 ps of equilibration while solute atoms restrained with harmonic potential of 15 kcal/(mol·Å2). Next, the restraints were gradually released in four rounds  of 100 ps each. After that, two sets of MD simulations were carried out: one set of entirely unrestrained simulations and a second set with small harmonic potential of 0.5 kcal/ mol·Å2 applied to base heavy atoms of residues involved in Watson–Crick hydrogen bonding between heterochiral strands, to preserve these bonds and to allow the sugar-phospate backbone to adjust to it. The long-range electrostatics interactions were calculated using the particle mesh Ewald method, periodic boundary conditions, and the non-bonded cut-off set to 9 Å. The covalent bonds were constrained using SHAKE, and the integration time step was set to 2 fs. The Langevin thermostat with collision frequency 2.0 ps −1 was used to control the temperature and the Berendsen barostat was used to enforce a constant pressure simulation. MD simulations were performed with the pmemd module of AMBER 18 on CUDA Device (Ge Force RTX2080). The data generated from MD were analysed using the CPPTRAJ module of Amber Tools [MD simulations were performed with the AMBER18 package . We usedor L-RNA ,25. The x border . The sysrameters . The soler Tools .Recently, we have shown that L-hammerhead ribozyme  hydrolysed a D-RNA target . The che2  and migrates slowly . One can2 . One shoTo get a deeper insight into the conformation of the heterochiral complexes, we measured the CD spectra of L/D-ribozymes with L/D-RNA substrates . The L- 1H NMR spectra. They show spectra of single-stranded D-RNA or L-RNA . For the complex with parallel stems new imino-proton signals in the region of 13–14.5 ppm were also observed: however, their pattern and intensity were different than those obtained for anti-parallel heterochiral catalytic complex . It suggests that the nature of the hydrogen bond interactions is different for the parallel and anti-parallel complex.To confirm the formation of heterochiral complexes and evaluate the nature of hydrogen bonding interactions in these complexes, we recorded the or L-RNA  without or L-RNA . This isHaving experimental data on the specific hydrolysis of RNA with catalytic nucleic acids (zymes) in a heterochiral manner  as well To understand how this complex is self-assembled, we developed a new approach to find the possible configuration of L-nucleosides. We started with the right-handed homochiral complex of D-RNA/D-ribozyme, called the D/D RNA model A. The moZ-coordinates of the right-handed (D-RNA/D-Rz) homochiral complex multiplied by -1 leads into the left-handed homochiral L-RNA/L-Rz complex , one can clearly see that Watson-Crick type base pairs within the heterochiral complex were very similar to those of the homochiral complex with some irregularities  and the chi angle values of the calculated heterochiral complex. The violin plot analysis of the molecular dynamics simulation of the D-RNA/L-Rz complex with positional restraints shows that all nucleotides of the L-Rz adopt C2′-endo sugar pucker (S-type) with pseudororation phase values (P) of approximately 1800 . This suormation . These rThe molecular structures can be chiral, and have mirror-image isomers of the opposite chirality. These isomers are called enantiomers or optical isomers, because they rotate plane-polarized light either to the right (D) or to the left (L). The building blocks of the life are homochiral, which means that naturally occurring amino acids, protein, sugars, and nucleic acids exist as only one chiral form. If these building blocks had mixed chirality, molecular chaos would ensue, and life would not be possible.In principle, mirror-image or looking-glass versions of chiral molecules should work in the same way as original ones, but they are resistant to attack by viruses or enzymes that have not evolved in a glass-looking world. That suggestion opens the door to a new dimension of mirror-image molecular biology with broad applications in diagnostics and therapeutics. During last 40 years there have been many studies showing the formation of complexes of two strands of opposite chriality; however, the conclusions drawn from their structure are not clear .In this work, we were interested in the structure of mirror image catalytic RNAs (L-RNA) and its interaction with RNA of reciprocal chirality (D-RNA). We concentrated on chemistry of the ribozyme because a specific RNA  hydrolysis is based on two important premises: (i) the tertiary structure of the active center should allow the substrate to adopt a precise conformation and formation of a transient active structure, and (ii) the active site of both the homochiral and heterochiral complexes should be very similar to perform the same specific catalysis.The D-hammerhead ribozyme activity against various D-RNAs (D-targets) has been studied recently ,15,30–32+2 ions, specific hydrolysis takes place. One can also see that L-HHRz binds to D-anti-HHRz . After binding to the substrate to L-Rz, no change of the intensity of the CD signals was detected indicating that in this case the heterochiral complex did not form C.Analysis of NMR spectra fully confirmed the formation of heterochiral L-Rz/D-RNA complex. New imino signals in the range of 13.5–14.5 ppm were observed in NMR spectrum when L-Rz and L-RNA/D-RNA was annealed .We have also consider parallel complex formation of D-RNA target with L-HH ribozyme. However NMR spectra of such complex  did not show down-field imine protons , and therefore did not support a parallel helices 1 and 3.Our 3D structure of the heterochiral models evidently shows that Watson-Crick base pairs between the ribozyme and substrate can be form although their geometry are slightly different C,D.It is interesting that using standard, well-known modeling tools and simple mathematical operation one can get antiparallel heterochiral Watson–Crick base pairs, that have geometry that is very similar with the canonical Watson–Crick base pairs C,D. As o5)A with D-poly (U), [To obtain deeper structural insight into the heterochiral complexes, we studied the molecular dynamics simulations of the complexes. These results suggest that the Watson-Crick like type pairing formed by strands of reversed chirality in helixes 1 and 3, are induced and stabilized by the homochiral helix 2 of the ribozyme. Such interactions maintain the active center of the catalytic RNA. Pairing between flanking L-nucleosides of ribozyme and D-RNA substrate is possible due to changes within sugar puckers and syn-anti conformation changes, which occur regularly every second nucleoside . Our resoly (U), . That oboly (U), .Click here for additional data file.


ΔC120, MΔC170, MΔC190, and MΔC210, and M C-terminal plasmid of pCAGGS-HA-MC190 and C-terminal deletion mutant plasmid of pCAGGS-MΔN143-182 were constructed. By using immunoprecipitation, immunofluorescence, and virus-like particle (VLP) germination experiments, we found that N was encapsulated into M-mediated VLP through N and M interaction. Moreover, the C-terminus of the M played a key role in the interaction between M and N. The C-terminus of the M encapsulated the N into the VLP. We finally determined that the 143-182 amino acids in the M were the functional regions that encapsulated the N into the M-mediated VLP. Our findings confirmed the interaction between M and N and for the first time clarified that the 143-182 amino acid region in M was the functional region that interacted with N, which provides a molecular basis for exploring effective anti-HPIV3 targets.Human parainfluenza virus type 3 (HPIV3) is the main pathogen that causes respiratory infections in infants, young children, and the elderly. Currently, there are no vaccines and effective anti-infective drugs. Studying the replication and proliferation mechanism of HPIV3 is helpful for exploring the targets of anti-HPIV3 infection. Matrix protein (M) and nucleocapsid protein (N) are two key structural proteins of HPIV3 that exert important functions in HPIV3 proliferation. Herein, we aim to clarify the functional domains of M and N interaction. HPIV3 M and N expression plasmids of pCAGGS-HA-M and pCAGGS-N-Myc/Flag, M C-terminal truncation mutant plasmids of pCAGGSHA-M Human parainfluenza virus (HPIV) belongs to paramyxovirus, which is a single- and negative-stranded and enveloped RNA virus. HPIV is a common pathogen of acute viral respiratory infections, which mainly causes upper respiratory tract and lower respiratory tract infections in children , 2. In aThe HPIV3 genome is about 15 kb and encodes 6 main structural proteins, including matrix protein (M), nucleocapsid protein (N), phosphoprotein (P), RNA polymerase (L), hemagglutinin and neuraminidase (HN), and fusion protein (F) , 9. HPIVStudies have shown that the M of enveloped viruses is released outside the cell by germination and exists in the form of a virus-like particle (VLP). When the VLP germinates, it uses the lipid bilayer membrane of the host cell to encapsulate some viral structural proteins. VLP is very similar to the real virus in morphology and structure but does not contain the genome of the virus and is neither proliferative nor infectious , 15. In In this study, to clarify the functional domains of M and N interaction, we first constructed expression plasmids of HPIV3 M and N, namely, pCAGGS-HA-M, pCAGGS-N-Myc, and pCAGGS-N-Flag. The M was expressed in eukaryotic cells to form VLP and coexpressed with N. The interaction between N and M during the formation of HPIV3 VLP was analyzed by VLP germination experiment, coimmunoprecipitation, and immunofluorescence. In addition, by further constructing M truncation mutants and deletion mutants, the interaction domains of M with N during the formation of VLPs were identified. For the first time, a completely new functional domain of M interacting with N was elucidated, which provides a molecular basis for further research on the molecular mechanism underlying the interaction between N and M and the role of M in the assembly and germination of HPIV3 RNP complex.ΔC120, MΔC170, MΔC190, and MΔC210, respectively, encoding the C-terminal truncated mutant M with a deletion of 120, 170, 190, and 210 amino acids; pCAGGS-HA-MC120, MC170, and MC190, respectively, encoding M mutants with only C-terminal 120, 170, and 190 amino acids; and pCAGGS-HA-MΔN143-182, encoding a mutant M in which the 143-182 amino acids were lacking. The plasmid pocus-HPIV3 containing the HPIV3 genome  was used as a template for PCR amplification of the gene fragments of N or M mutants. The amplified fragments and plasmid pCAGGS-MCS were cut with EcoRI and NheI, respectively, and the digested fragments were cloned into pCAGGS-MCS. All constructed plasmids were verified by Sanger sequencing.We designed and constructed the following plasmids: pCAGGS-N-Myc and pCAGGS-N-Flag, respectively, encoding the N fused with Myc and Flag tags at C-terminal; pCAGGS-HA-M, encoding the M fused to the HA tag at the N-terminal; pCAGGSHA-M2. When cell confluence reached 40%-50% for 293T cells and 50%-60% for HeLa cells, the cells were transfected with the plasmids with calcium phosphate reagent (Beyotime) for 293T cells and Lipo 3000 (Invitrogen) for HeLa cells.The 293T cells and HeLa cells  were cultured with DMEM (HyClone) containing 16% fetal bovine serum (EveryGreen) and 1% penicillin-streptomycin (Gibco) and placed at 37°C, 5% COAt 48 h after transfection, 293T cells were collected and lysed with precooled TNE solution (50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 2 mM EDTA (pH 8.0), 0.1% 2-mercaptoethanol, and protease inhibitor cocktail) for 30 min. After the lysate was centrifuged at 13,000 rpm, 4°C for 30 min, the supernatant was collected. Protein concentration was quantified by using the BCA Protein Assay Kit . Then, protein electrophoresis was performed with 12% or 15% SDS-PAGE gel. The protein on the gel was transferred to a nitrocellulose membrane, and the membrane was blocked with 5% skimmed milk in PBST (1/1000 of Tween-20 dissolved in PBS) at room temperature for 30 min. Next, the corresponding primary antibody  diluted with PBST was added and incubated at room temperature for 1 h. After washing the membrane 3 times with PBST, the corresponding secondary antibody  diluted with PBST was then incubated at room temperature for 45 min. After washing the membrane three more times, a developing substrate  was added for color development.μl TNE solution for WB analysis. The ability of M or its mutants to encapsulate N into VLP was defined as the germination index. In detail, germination index = the level of N in VLP/level of M or M mutant in VLP. The relative germination index was normalized to the germination index of M+N coexpression and was calculated as the ratio of (M mutants+N) group/(M+N) group.The VLP germination experiment  was usedμl protein (A+G) agarose beads (Beyotime) overnight at 4°C. The next day, the sample was centrifuged at 8000 rpm for 10 seconds and then washed 5 times. Then, the samples were subjected to WB analysis.At 48 h after transfection, 293T cells were collected and lysed as described above. The lysates were incubated with anti-Myc (Santa Cruz) or anti-HA (Sigma) antibodies at 4°C for 1 h. After brief centrifugation, the samples were incubated with 40 After 24 h of transfection in the HeLa cells, the cells were washed three times with PBS and then fixed with 4% paraformaldehyde at room temperature for 20 min. After washing three times, the cells were permeated with 0.2% Triton X-100 at room temperature for 20 min. After washing again, the cells were blocked with 3% bovine serum albumin solution for 30 min at room temperature. The N was stained with rabbit anti-Flag primary antibody (Proteintech) and AF488-conjugated goat anti-rabbit fluorescent secondary antibody (Thermo Fisher). The M was stained with mouse anti-HA primary antibody and AF568-conjugated goat anti-mouse fluorescent secondary antibody (Thermo Fisher). Finally, the cells were observed under a fluorescence microscope .t-test. A p value < 0.05 was considered as statically significant.All experiments were performed at least three times. The data were shown as mean ± SD. The statistical analysis was performed using Student's In order to verify whether there is an interaction between M and N, Myc-labeled N and HA-labeled M were coexpressed in 293T cells, and the interaction between M and N was detected by Co-IP. It was found that Myc-N protein was coprecipitated with HA-M protein . SimilarTo explore the interaction between N and M in the germination of HPIV3 progeny virus, N and M were coexpressed in 293T cells, and M and N expressed alone were used as control, respectively. The results showed that N was detected in VLP of cells with coexpression of N and M, but not in VLP of cells with N or M expression alone . These rΔC120, MΔC170, MΔC190, and MΔC210. Then, a VLP germination experiment was performed. In detail, compared with wild-type M, the ability of mutant MΔC120 or MΔC170 to encapsulate N into VLP was significantly reduced . Therefore, we only used MC190 for subsequent experiments. After cotransfection of plasmids pCAGGS-HA-MC190, pCAGGS-N-Myc, pCAGGS-HA-Md-type M , which md-type M . This reTo detect the interaction between MC190 and N, we coexpressed HA-MC190 and N-Myc and performed a VLP germination experiment. The results showed that although the content of MC190 in VLP was less than that of wild-type M, the ability of MC190 to encapsulate N into VLP was comparable to that of wild-type M Figures . The Co-ΔC120 and MΔC170 retained part of the ability to bind to N, while MΔC190 and MΔC210 had completely lost their ability to bind to N. Therefore, we speculate that MΔC171-190  or MΔC171-210  play an important role in N and M interactions. To verify this hypothesis, we cotransfected HA-MΔN143-182 and N-Myc and tested VLP germination. As expected, although MΔN143-182 mediated the production of a sufficient amount of VLP like wild-type M, it could not encapsulate the N into the VLP (ΔC190 and MΔC210, the ability of MΔN143-182 to bind to the N was almost completely lost (As described above, M the VLP . As in tely lost . These rM and N are two structural proteins encoded by the HPIV3 genome. Recent studies have shown that they have important functions in the proliferation of HPIV3 –24. HPIVIn this study, we verified the interaction between the M and the N of HPIV3 through VLP germination experiment, immunoprecipitation, and immunofluorescence colocalization. We speculate that the interaction between N and M exists in the process of replication and assembly of HPIV3 progeny. Moreover, the interaction between N and M may start from the replication of the HPIV3 progeny virus, which exists during the assembly and germination of the progeny virus. The RNA and other structural proteins of the progeny virus may be assembled into a complete virus through the action of N and M. The complete viruses will be released from the cells as the M germinates.ΔC120 and MΔC170 to encapsulate N into VLP was significantly reduced, while MΔC190 and MΔC210 completely lost the ability to encapsulate N into VLP. In addition, the C-terminal region of the M, namely, MC190, compensated for the ability of the C-terminal truncation mutant to bind to the N. One possible reason is that MC190 and the C-terminal truncation mutant are structurally and functionally complementary, performing functions equivalent to the wild-type M. Another possibility is that MC190 alone contains the functional domain where M interacts with N. We finally confirmed the latter speculation and further clarified that the key region of the M that interacted with the N was located at the C-terminus of the M.We found that when the C-terminal truncated mutant of M was expressed alone, its ability to produce VLP was greatly lost compared to wild-type M, but when it was coexpressed with N, these mutants restored the ability to generate VLP in varying degrees. The recovery of the mutant VLP's germination ability is probably due to the increased expression level of N in the lysate, suggesting that the N may be able to increase the solubility of the M mutant in the lysate. However, although the VLP germination ability of the M truncation mutants was restored to some extent in the presence of N, their ability to encapsulate N into VLP was reduced compared to the wild-type M. Among them, the ability of MA previous report indicates that the M L305 is a key residue, which plays an important role in the interaction of N and M, and the mutant L305A cannot interact with N nor can it encapsulate N into the VLP . M consiIn addition, in the N-M interaction, M and N have their own functional sites or regions involved. Although we have preliminarily determined the functional region in the M, the domain responsible for the N-M interaction in the N is still unknown. It has been reported that the C-terminus of paramyxovirus N may be involved in its interaction with P and M , 29. WheIn this study, the new functional regions we identified in the M played a key role in the N-M interaction. These results provide a molecular basis for further revealing the interaction of virus proteins during virus proliferation and for identifying effective antiviral targets.


To explore the role of all-trans retinoic acid (ATRA) in renal ischemia/reperfusion injury of diabetic rats. Sixty adult male rats were randomly divided into 6 groups, including sham group (S group), ischemia-reperfusion group (I/R group), ischemia-reperfusion+ATRA group (A group), diabetic group (D group), diabetic ischemia-reperfusion group (DI/R group), diabetic ischemia-reperfusion +ATRA group (DA group). The levels of creatinine (Cr), cystatin C (Cys-C) and β2-microglobulin (β2-MG) were measured. Morphology of renal tissue was observed under light microscope. DJ-1, Nrf2, HO-1 and caspase-3 were detected by western blot. DJ-1, Nrf2, HO-1 and caspase-3 in I/R group, D group and DI/R group was higher than that in S group. Compared with I/R group, Nrf2 and HO-1 in A group was decreased, but caspase-3 was increased. However, Nrf2 in DA group was higher than that in DI/R group, HO-1 and caspase-3 in DA group were lower than that in DI/R group. Compared with group S, Cr, Cys-C and β2-MG in I/R group, A group, D group, and DI/R group were higher. Whereas the levels of Cr, Cys-C, β2-MG and renal injury score in DA group were lower than those in DI/R group. ATRA has a protective effect on renal ischemia-reperfusion injury in diabetic rats, maybe relating to DJ/Nrf2 pathway. During perioperative period, renal ischemia-reperfusion injury easily occurs in diabetic patients, which aggravates diabetic nephropathy. Due to surgery, especially the complex process of kidney surgery, stress, shock and prolonged stimulation resulted in renal insufficiency2.Diabetes mellitus, as a chronic metabolic disorder, is characterized by an abnormal insulin secretion and action resulting from interaction of hereditary and environmental factors. As one of the common complications of diabetes, diabetic nephropathy has an increasing gradually incidence. The pathogenesis of diabetic nephropathy is associated with the interaction of hyperglycemia, advanced glycation end-product, increased po-lyol pathway and oxidative stress4. In addition, ATRA can affect the progression of diabetic nephropathy without causing any significant side effects and has a therapeutic effect on diabetic nephropathy5.All-trans retinoic acid (ATRA) is a potent derivative of vitamin A. Studies have shown that ATRA can effectively prevent the progression of diabetes in rats. Pancreatic β-cells, acinar and ductal cells gradually restore their normal appearance under ATRA treatment. What’s more, insulin messenger RNA and serum indices almost normalize, which improves the histological changes of the pancreas and the serum indices in diabetic ratset al.6have demonstrated that DJ-1/Nrf2 pathway was involved in the pathogenesis of diabetic nephropathy in rats, which played a protective role on diabetic nephropathy. Nrf2/HO-1 pathway is one of the most important endogenous antioxidant pathway8. Upregulation of Nrf2 protein levels can protect the kidneys from I/R-associated oxidative damage9. Meanwhile, ATRA is an inhibitor of Nrf210. This study aimed to investigate whether ATRA had a protective effect on renal ischemia- reperfusion in diabetic rats and whether it was related to DJ-1/Nrf2 pathway.Sun Sixty adult SD male rats, weighing 200-220g, were purchased from the Animal Experimental Center of Wuhan University. Sixty SD Rats were fed 5 days after adaptation and were randomly assigned to six groups: sham group (S group), ischemia-reperfusion group (I/R group), ischemia-reperfusion +ATRA group (A group), diabetic group (D group), diabetic ischemia reperfusion group (DI/R group), diabetic ischemia reperfusion +ATRA group (DA group), every group n =10. ATRA was purchased from Sigma (R2625-100MG), and the rats were intraperitoneally injected with 1mg/mL of ATRA at a dose of 1mL per day.11. Normal rats received an equivalent volume of citrate buffer only. The fasting blood glucose (FBG) level was monitored every week.The rats were fasted overnight 12 hours before STZ injection. Then the rats received a single intraperitoneal injection of STZ  at a dose of 60 mg/kg freshly dissolved in 0.1 M citrate buffer (ph =4.5). After 3 days of diabetes induction, tail vein blood glucose samples were collected and measured with OneTouch glucometer . Rats with a FBG level above16.7 mmol/L were considered diabetic and were selectedThe rats were anesthetized with pentobarbital sodium (50 mg/kg) by intraperitoneal injection. The abdomen was opened, and the bilateral renal pedicles were occluded lasting for 30 min, followed by 24 h of reperfusion.Blood samples were collected by cardiac puncture, centrifuged at 3,000 rpm, for 10 min at 4°C. Serum was separated and stored at −20 °C. Levels of Cr, Cys-C and β2-MG were measured using ELISA assay kits  according to the manufacturer’s instructions.et al.13using the following scoring system: 0), normal histology; 1), tubular cell swelling, brush border loss and nuclear condensation, with up to one-third of the tubular profile exhibiting nuclear loss; 2), same as for score 1, but more than one-third and less than two-thirds of the tubular profile displaying nuclear loss; and 3), same as for score 1, but more than two-thirds of the tubular profile exhibiting nuclear loss.The left kidney was cut into sections and fixed in 4% formaldehyde, embedded in paraffin and sectioned into 4 μm slices for light microscopy , followed by staining with hematoxylin and eosin (HE). Histological assessment of tubular necrosis was semi-quantitatively determined using a method modified from McWhinnie 15. Briefly, equal amounts of protein (50 µg) were separated by 12% SDS-PAGE at 100 V for 3 h. After electrophoresis, proteins were transferred onto polyvinylidenedifluoride membranes at 200 mA for 70 min. The membranes were incubated with the primary antibodies . After washing three times in TBS-T, membranes were incubated with anti-rabbit immunoglobulin G conjugated to horseradish peroxidase at a dilution of 1: 2,000 in TBS-T containing 5 % skimmed milk for 2 h at room temperature. The immunoreactive bands were visualized by enhanced chemiluminescence  and captured on X-ray film. Blots were stained with an anti-β-actin antibody, and the protein levels were normalized with respect to β-actin band density.Western blot analysis was performed as previously describedAll data are expressed as the mean ±standard deviation (SD). Statistical analysis was performed using GraphPad Prism software version 7.0 . Analysis of variance (ANOVA) test and Student’s t test were used to evaluate statistical significance. The level of significance was set at P<0.05 for all statistical tests.P<0.05). This increase was significantly enhanced in I/R group and DI/R group compared to I/R group and D group individually respectively (P<0.05). Histological alterations were obviously improved in specimens from DA group compared to DI/R group (P<0.05).Edema, tubular cell necrosis and cytoplasmic vacuoles were observed in histological specimens in I/R group, A group, D group and DI/R group, but were absent in S group. Compared with the renal histological evaluation score of kidneys obtained from S group, I/R group and D group exhibited a clearly increase in renal histologic evaluation score . And those blood indices were increased furtherly in A group and DI/R group compared to I/R group and D group individually (P<0.05). Administration of ATRA visibly reduced the Cr, Cys-C and β2-MG levels compared with DI/R group rats (P<0.05).The serum levels of Cr, Cys-C and β2-MG in I/R group and D group were significantly higher than in S group .Compared with sham group, the expression of DJ-1, Nrf2, HO-1 and caspase-3 in I/R group and D group were increased remarkably. And administration of ATRA significantly decreased the expression of Nrf2 and HO-1 in A group contrasted with I/R group. Moreover, the expression of DJ-1 and caspase-3 in A group strengthened much more than in I/R group. In DI/R group, the expression of DJ-1, Nrf2, HO-1 and caspase-3 also were enhanced in contrast with D group. However, the expression of Nrf2 and HO-1 in DA group was heightened compared with DI/R group, and the caspase-3 expression in DA group reduced perceptibly , P<0.05.The present study found that ATRA inhibited the expression of Nrf2 leading to a decreasing of HO-1 in A group, but the expression of caspase-3, the serum levels of Cr, Cys-C and β2-MG and the renal histological evaluation score of kidneys were increased in A group more than those in I/R group. It demonstrated that ATRA aggravated kidney damage caused by renal ischemia-reperfusion. However, when ATRA was administrated in diabetic rats with ischemia-reperfusion, the expression of Nrf2 strengthened while the expression of caspase-3, the serum levels of Cr, Cys-C and β2-MG and the renal histological evaluation score of kidneys was decreased in comparison with DI/R group. Thus, ATRA not only improved the histological alterations and the blood indices, but also ameliorated kidney function and mitigated kidney damage.18. ATRA improves the histological changes of the pancreas of diabetic rats resulting in a significant decrease in blood glucose and total peroxides, elevated pancreatic insulin mRNA, and serum insulin4. ATRA shows to limit glomerular cell proliferation and kidney damage by reducing renal TGF-β1 and TGF receptor II expression in nephropathy rat models16. ATRA reduces the incidence of T1DM by protecting islet β-cells, upregulating the levels of the pancreatic β-cell factor IL-4, reducing the cell toxicity-associated levels of IFN-γ in β-cells, and maintaining the Th1/Th2 balance3. What’s more, ATRA exhibits an anti-oxidant property by blocking lipid peroxidation in streptozotocin-induced diabetic rats19. It has been suggested that oxidative stress, as a key factor, is involved in the pathogenesis and progression of diabetic nephropathy (DN)22. DJ-1/Nrf2 pathway has been found to perform a protective effect on diabetic nephropathy23, which is one of the most important endogenous antioxidant pathway8. In this study, the expression of DJ-1/Nrf2/HO-1 in DA group was upregulated, and histological alterations clearly improved edema, as tubular cell necrosis and cytoplasmic vacuoles almost disappeared. These data suggested that ATRA exerted protective effects in STZ-induced diabetic rats with renal ischemia-reperfusion injury.ATRA exerts beneficial effects on nephropathy25. Nrf2 is known as the “master regulator” of antioxidant/anti-inflammatory/cytoprotective responses28. Under normal conditions, Nrf2 is retained in the cytoplasm, being bound to KEAP1, which targets it for ubiquination and proteosomal destruction29. In response to oxidative stress, critical KEAP1 cysteine residues are disrupted, Nrf2 is thus freed from ubiquination, and enhanced nuclear translocation results. In concert with a small Maf protein, and with export of the nuclear repressor Bach1, Nrf2 binds to antioxidant response elements (AREs) in the promoter regions of diverse cytoprotective genes. However, ATRA is an inhibitor of transcription factor Nrf2 by interfering with recruitment of Nrf2 to the ARE, whereas nuclear levels of Nrf2 are not affected by ATRA10. In this study, the expression of Nrf2/HO-1 in I/R group was decreased, but the expression of caspase-3 was increased, which is related to apoptosis30. Otherwise, the kidney injury scores and the blood indices in I/R group were worsen. These showed that ATRA indeed limited the expression of Nrf2, leading to increased apoptosis and aggravated kidney injury.In the renal ischemia-reperfusion rats model, increased oxidative stress also participates in the pathogenesis of renal I/R and deteriorates kidney injuryTaken together, ATRA impacts the kidney histopathology and function in renal ischemia-reperfusion rat model by downregulating Nrf2 protein expression. However, in STZ-induced diabetic rats with renal ischemia-reperfusion injury, due to many renal protective factors of ATRA as above stated, the effect of inhibiting Nrf2 does not perform a central role. Conversely, accompanying with the expression of DJ-1 upregulated, Nrf2 protein expression also increased so that more Nrf2 exerted an anti-oxidant effect. The deficiency of this study is that it does not explore the relationship between ATRA and the DJ-1/Nrf2 pathway, and that the mechanism of ATRA renal protective in STZ-induced diabetic rats with renal ischemia-reperfusion injury is still unclear.This study suggested that ATRA played a protective effect on renal ischemia-reperfusion injury in diabetic rats, which might relate to the DJ/Nrf2 pathway. Our results might provide a potential therapeutic strategy against diabetic renal ischemia-reperfusion injury.


They are commonly overexpressed or dysregulated in cancer as a consequence of inactivation or mutations in the retinoblastoma protein. Therefore, one or more of the activating E2Fs  have been recognized as antitumor targets. The combination of a peptide targeting transcription of E2F-1, 2, and 3a, with cisplatin, and especially with pemetrexed, showed enhanced antitumor activity in-vivo studies, the peptide was encapsulated in PEGylated liposomes (PL-PEP) to increase half-life and stability. Methods: Prostate cancer (DU145 and PC3), breast cancer , lymphoma (CCRF-CEM), and non-small cell lung cancer (NSCLC) cell lines  were treated with D-Arg PEP in combination with cisplatin or pemetrexed. Western blot analysis was performed on the NSCLC for E2F-1, pRb, thymidylate synthase, and thymidine kinase. The H2009 cell line was selected for an in-vivo study. Results: When the PEP was combined with cisplatin and tested against solid tumor cell lines and the CCRF-CEM leukemia cell line, there was a modest synergistic effect. A marked synergistic effect was seen when the combination of pemetrexed and the PEP was tested against the adenocarcinoma lung cancer cell lines. The addition of the PEP to pemetrexed enhanced the antitumor effects of pemetrexed in a xenograft of the H2009 in mice. Conclusions: The D-Arg PEP in combination with cisplatin caused synergistic cell kill against prostate, breast, lung cancers, and the CCRF-CEM cell line. Marked synergy resulted when the D-Arg PEP was used in combination with pemetrexed against the lung adenocarcinoma cell lines. A xenograft study using the PL-PEP in combination with pemetrexed showed enhanced anti-tumor effects compared to each drug alone.Background: We tested the antitumor effects of a modified E2F peptide substituting D-Arg for L-Arg, conjugated to penetratin (PEP) against solid tumor cell lines and the CCRF-leukemia cell line, alone and in combination with pemetrexed or with cisplatin. For  The E2F family members are critical to many cellular processes, including development, proliferation, DNA repair, and differentiation ,2. All fA central player in E2F regulation is the tumor suppressor retinoblastoma protein (pRB) and family members. When under-phosphorylated, RB binds to the activating E2Fs at promoter regions and inhibits their transcriptional activity ,7,8,9. IGiven the role of the E2Fs in cell cycle regulation and proliferation, there have been many attempts to downregulate E2F in order to inhibit tumor cell growth. For example, knockdown of E2F-1 by siRNA in castrate-resistant prostate cells lacking pRB decreased proliferation . HoweverUsing phage display, we identified a novel peptide that inhibits transcription of E2F-1, 2, and 3a, and as a consequence their target genes, including TS, TK, and DHFR. When coupled to a penetratin to enhance uptake, the penetratin E2F peptide (PEP) was shown to be cytotoxic to a subset of tumors that overexpress E2F . Moreovein-vitro to treat prostate, breast, and lymphoma and with pemetrexed in NSCLC cells, and pemetrexed were used with PL-PEP in mice bearing the H2009 NSCLC tumor to determine in-vivo efficacy.We hypothesized that downregulation of enzymes involved in DNA synthesis as a consequence of E2F inhibition, particularly TS and TK, would sensitize tumor cells to combinations of the D-Arg PEP with agents targeting these enzymes, e.g., pemetrexed, which targets TS . As E2F Platinum compounds, e.g., cisplatin and carboplatin, have been used for the treatment of non-small cell lung cancer (NSCLC) and breast cancer in the clinic for decades. Cisplatin causes DNA damage and forms irreversible adducts with DNA, causing damage to replication and transcription processes . E2F-1 fThe D-Arg PEP in combination with cisplatin against breast cancer cell lines  and the lymphoma cell, CCRF-CEM also showed a modest synergistic anti-cancer effect , when tr50) of the cells to the individual compounds and combination indices to determine synergy, additivity, or antagonism [As treatment of cells with the PEP downregulated TS and TK due to inhibition of E2F-1 expression , it was tagonism . Despitein-vivo study, as the H2009 cell line showed high levels of E2F-1, thymidylate synthase, and thymidine kinase expression. The binding of retinoblastoma protein (Rb) with the E2F-1 acts as a transcriptional repressor for the E2F targeting genes, and the cells arrest in G1 phase of the cell cycle. The loss of Rb by the deletion or mutation or phosphorylation of Rb by cyclins and cyclin-D kinases (cdks) leads to release of free E2F-1 which causes transcriptional activation of DNA synthesis genes, and thus the cells proceeds from G1 to S-phase of the cell cycle. Thus, phosphorylation of pRB due likely to CDK 4, 6, and 2 in three of the four cell lines resulted in high levels of E2F-1 as shown by Westerns  was incubated in saline . Less thin-vitro) with IC50 of 105 µM  were obtained from the Taconic Biosciences, Inc. .2. Cisplatin and pemetrexed were obtained from the Rutgers Cancer Institute of New Jersey (CINJ) pharmacy. The L- and D-Arg PEP were synthesized by Bio Basic Inc., Ontario, Canada, and validated by the Rutgers Chemistry Core as previously described [Prostate cancer (DU145 and PC3), breast cancer , lymphoma (CCRF-CEM), and non-small cell lung cancer cell lines  were cultured in RPMI 1640 medium  with 10% FBS  and 1% penicillin/streptomycin  in an atmosphere of 5% COescribed . MTS tet® Aqueous One Solution cell proliferation assay was added to each well and incubated for 2 to 3 h. Absorbance was measured at 490 nm as per the manufacturer’s protocol, to determine the cell viability.Five thousand cells were plated in a 96-well plate in 180 µL of respective media containing 10% FBS. After 24 h, 20 µL of either D-Arg PEP or cisplatin or pemetrexed  was added and incubated for 48 h. Twenty µL of the MTS tetrazolium Promega CellTitre 96TM Series cell viability analyzer . The 50% inhibitory concentration  was determined using the nonlinear regression curve fit of the graphs drawn by GraphPad Prism 4 software . All experiments were performed in triplicate, and all experiments were repeated at least three times.Five thousand cells (suspension) were plated in a 12-well plate in RPMI 1640 media supplemented with 10% FBS. After 24 h, the drug was added and incubated for 48 h. To assess cell viability, the cells with or without drug treatment were collected and cell viability was determined using the Vi-CELL50s). After 48 h after adding the drugs, cell viability was assessed by the MTS assay or Vi-CELL. For drug combination experiments, the cell viability assays were performed, and the results were analyzed for synergistic, additive, or antagonistic effects using the combination index (CI) method developed by Chou and Talalay by using Compusyn v1.0 software . Combination indices, CI < 1, CI = 1, and CI > 1 indicate synergism, additive effects, and antagonism, respectively.The cells were treated with either a single or combination of the drugs in a fixed ratio . Next day, cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature (RT), washed three times with PBS, and permeabilized in 1% Triton X-100 at 37 °C for 30 min, followed by 10 min at RT and again washed three times with PBS. After blocking with 1% bovine serum albumin (BSA), washed cells were incubated with gamma H2AX (phospho-Ser139) monoclonal primary antibody  at a dilution of 1:5000 overnight at 4 °C. The cells were incubated with goat anti-rabbit IgG-FITC  at 1:2500 dilution for 2 h in the dark at room temperature and washed 3 times with the PBS. The coverslips were mounted on microscopic glass slides and sealed with the microscopic fluid on outer edges. The images were captured using a Nikon TE2000-U Fluorescence Microscope-AV (from the CINJ core facility) with 10× magnification.Cells were treated with ICThe expression of pRb, E2F-1, TS, and TK was checked by Western blot analysis using a standard protocol. Anti-E2F-1(sc-251), anti-TS(sc-33679), anti-TK(sc-56967), anti-vinculin (sc-73614), and secondary antibody were from Santa Cruz Biotechnologies, USA. Anti-pRb (9308S) was from the Cell Signaling Technology, Danvers, MA, USA.® R-210/R-215 , and rehydrated with 0.9% NaCl to final lipid concentration 20 mM. The lipid mole ratio for this formulation was 51:44:5 EPC:Chol:DSPE-PEG, respectively. Liposomes were stored at room temperature for 1 h, followed by extrusion through polycarbonate membranes 200 nm and 100 nm using the extruder device . The size distribution and zeta potential of liposomes were measured using Malvern ZetaSizer NanoSeries  according to the manufacturer’s recommendations. Measurements were performed at 25 °C. The sizes and zeta potential were measured five times, and average values were calculated. The average size of particles was 160 ± 10 nm; zeta potential was −10 ± 2 mV.Egg phosphatidylcholine (EPC), cholesterol, and 1,2,-distearoyl-sn-glycero-3-phosphoethanolamine-N-aminopolyethelenglycol—Mw—2000 ammonium salt (DSPE-PEG) were purchased from Avanti Polar Lipids, Alabaster, AL. PEGylated liposomes were prepared as previously described ,32. Brie® R-210/R-215 , and rehydrated with 0.9% NaCl to final lipid concentration 20 mM. D-Arg PEP concentration was 10 mg/mL. In order to measure D-Arg PEP content in the liposomes, the aliquots of PEGylated liposomes containing D-Arg PEP were disrupted in isopropanol in a ratio of 10:90 (NLC/isopropanol), and the concentration D-Arg PEP was determined by high-performance liquid chromatography (HPLC) using a symmetry C18 column 150 mm × 4.6 mm  operated at room temperature. The mobile phase consisted of 0.15 M NaCl, 20% acetonitrile solution; the flow rate was set to 0.4 mL/min, wavelength 280 nm. The chromatographic apparatus consisted of a Model 1525 pump , a Model 717 Plus auto-injector , and a Model 2487 variable wavelength UV detector  connected to the Millennium software. The average loading efficiency of liposomes was close to 95%.D-Arg PEP was dissolved with lipids in chloroform, evaporated to a thin film layer using rotary evaporator RotavaporD-Arg PEP release experiments were performed in phosphate-buffered saline at pH 7.4 and temperature-equilibrated 25% human serum supplemented with RPMI nutrient media at 37 °C. The liposomes were put into dialysis tubing with a molecular weight cutoff of 10–12 kDa and dialyzed against 100 mL release buffers at 37 °C. At predetermined time intervals , samples were withdrawn from the release medium and assayed for D-Arg PEP by high-performance liquid chromatography as described above. Prior to final analysis, samples containing plasma were extracted by protein precipitation with 6% aqueous trichloroacetic acid, while samples without plasma were analyzed directly.6 H2009 cells with matrigel  were injected subcutaneously in the abdominal right flanks of nude mice . Once the tumor was palpable (around 2 weeks from cell injection), mice were randomized into four groups. One group of mice were injected with PL-PEP (80 mg/kg) every other day (3 times per week) and the other group with pemetrexed (250 mg/kg) twice a week. The control mice group received saline. The combination group received PL-PEP 80 mg/kg three times per week and pemetrexed 250 mg/kg twice a week. The mice were treated with either a single or combination of drugs for around 3 weeks. Tumor size and body weight were measured twice a week and tumor volume was calculated using (L)(W)2/2. Data were plotted and SEM was calculated.5 × 10Tumor samples extracted from the H2009 xenograft study at 7 days after last treatment were studied for immunohistochemistry. Samples were fixed in 4% formalin and paraffin embedded. Immunohistochemistry was performed on 4 µm sections with antibodies to Ki67 ((M3062) from Spring Bioscience Corp., Pleasanton, USA), TS (sc-33679), and TK (sc-56967) from Santa Cruz Biotechnologies, USA. Sections were developed and stained with hematoxylin and eosin using standard methods. All histological preparations and immunostaining were conducted by the Rutgers Cancer Institute of New Jersey Biospecimen Repository and Histopathology Core.in-vitro experiments were performed three times, and each experiment was done in triplicate. Statistical analysis was performed using GraphPad Prism software. In all cases, ANOVA followed by two-tailed, unpaired Student’s t-tests were performed to analyze statistical differences between groups; p-values of < 0.05 were considered statistically significant.All in-vitro and in-vivo.The D-Arg PEP targeting activator family members of transcription factor E2F— namely, E2F-1, E2F-2, and E2F-3a—showed a synergistic anti-cancer effect when used in combination with chemotherapeutic drug cisplatin against the prostate cancer, breast cancer, and lymphoma cell lines. However, marked synergy was observed when used in combination with chemotherapeutic drug pemetrexed, a potent thymidylate synthase inhibitor, against the non-small cell lung cancer both


Inactivation was evaluated by the infection ratio of Madin–Darby canine kidney (MDCK) cells or chicken embryonated eggs. Irradiation by the 260 nm UV-LED showed the highest inactivation among all treatments. Because the irradiation-induced inactivation effects strongly correlated with damage to viral RNA, we calculated the correlation coefficient (RAE) between the irradiant spectrum and absorption of viral RNA. The RAE scores strongly correlated with the inactivation by the UV-LEDs and LP-UV lamp. To increase the RAE score, we combined three different peak WL UV-LEDs (hybrid UV-LED). The hybrid UV-LED (RAE = 86.3) significantly inactivated both H1N1 and H6N2 subtypes to a greater extent than 260 nm (RAE = 68.6) or 270 nm (RAE = 42.2) UV-LEDs. The RAE score is an important factor for increasing the virucidal effects of UV-LED irradiation.Influenza A viruses (IAVs) pose a serious global threat to humans and their livestock. This study aimed to determine the ideal irradiation by ultraviolet-light emitting diodes (UV-LEDs) for IAV disinfection. We irradiated the IAV H1N1 subtype with 4.8 mJ/cm Influenza viruses are enveloped viruses whose genome consists of segmented negative-sense single-strand RNA segments. There are four types of influenza viruses, including A, B, C and D, and types A and B show epidemic spread. Influenza A viruses (IAVs) are encircled by the M1 matrix protein and a host-derived lipid bilayer envelope in which the virus surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), as well as the M2 matrix protein, are embedded . FollowiIAVs exhibit zoonotic potential because novel IAV mutants frequently emerge and are able to cross species barriers. Therefore, IAV infections are a globally important issue not only in humans but also in their livestock. Avian influenza (AI) viruses are classified by their pathogenic strength based on the feasibility of infection in chickens . The crip > 0.05). Meanwhile, Li Q.G., et al. . The reactions were heated at 95 °C for 30 seconds followed by 40 cycles of a 10-second denaturing step at 95 °C, 20-second annealing step at 60 °C, and 15-second extension step at 72 °C.The damage to viral RNA by UV irradiation was measured by reverse transcription (RT)-quantitative real-time polymerase chain reaction (qPCR) as previously reported ,24,25. Tg for 20 min followed by filtration through 0.45 ìmö pore filters. The viruses were then purified by centrifugation  through PBS containing 20% sucrose, as previously reported [RAE to investigate the relationship between the normalized absorbance spectrum of viral RNA and the normalized emission spectrum. RAE was calculated using the following formula:A(λ) is the normalized viral RNA absorption spectrum, and S(λ) is the normalized emission spectrum of the UV-LED or LP-UV lamp. The wavelength of the spectrum was used from 245 nm (b) to 320 nm (B) for the calculation. For the calculation of RAE of the 365 nm UV-LED, the maximum wavelength ranged up to 400 nm and was normalized by those of the other UV-LEDs. We used MATLAB R2017b software  for the computation of RAE.We were interested in investigating the relationship between the absorbance spectrum of viral RNA and the UV-LED emission spectra. After propagating the virus in 10-day-old chicken embryonated eggs for 48 h at 37 °C, allantoic fluids were precleared by centrifugation at 3300× reported . Virus pp < 0.05 was considered statistically significant. Spearman’s rank correlation test was used for the analysis of the association between two variables. For comparisons between two correlations, the correlation coefficient values were transformed into z scores by Fisher’s r to z transformation and analyzed for statistical significance by determining the observed z test statistic.Statistical analysis of differences was performed using ANOVA with Bonferroni’s multiple comparison tests using Statview 5.0 software . The Student’s t-test was used for paired data where appropriate. 2) and duration (2 sec) and infected MDCK cells and embryonated chicken eggs with these virus suspensions. Irradiation by 290–365 nm UV-LEDs did not affect the infection ratio, whereas the 260–280 nm UV-LEDs and LP-UV lamp decreased the ratio in both MDCK cells and embryonated chicken eggs (2) by 260–280 nm UV-LEDs showed an inactivation effect on the IAV H1N1 subtype in both host organisms. The lowering effect of UV irradiations on the infection ratio was correlated with those peak WLs, but the lowering effect on the infection ratio by the 260 nm UV-LED was significantly lower than that by the LP-UV lamp.To determine the effects of UV irradiations on the IAV H1N1 subtype, we irradiated virus suspensions using UV-LEDs or an LP-UV lamp with an equal fluence rate  and embryonated chicken eggs (R = 0.9102). These results suggest that the inactivation effects of irradiation by the UV-LEDs and LP-UV lamp are dependent on the damage of viral RNA, and we hypothesized that the wider range of emission spectra of UV-LEDs than that of the LP-UV lamp was an important factor for the inactivation of IAVs and induction of RNA damage.To measure the viral RNA damage induced by LED irradiation, we purified viral RNA from viral suspensions with or without UV-LEDs and LP-UV lamp irradiation at 4.8 mJ/cm section ,24,25. NRAE between the spectrum and emission spectrum of the UV-LEDs and LP-UV lamp  was lower than that of the 260 nm UV-LED (RAE = 68.56) and 270 nm UV-LED (RAE = 42.24)  b. The codiations . From thRAE, we combined three different UV-LEDs (hybrid LED), including MO-2257-U270 (2 (0.8 mW/cm2 each UV-LED) (RAE score of the hybrid UV-LED (RAE = 86.3) was higher than those of the other UV irradiations in this study  a,b. The is study b.p < 0.05) or a moderate trend toward significantly (p = 0.068) higher inactivation effects on both H1N1 and H6N2 subtypes than the 258 nm, 260 nm or 270 nm UV-LED (p < 0.05) or a moderate trend toward significantly (p = 0.067) higher inactivation effect on H1N1 subtype than the 258 nm, 260 nm, or 270 nm UV-LED, but the effect on H6N2 was not different between the hybrid LED and 258 nm LED  was higher than that of the LP-UV lamp (RAE = 11.1), and the RAE of UV irradiations was strongly correlated to the viral inactivation effect by UV irradiations  by combining three different UV-LEDs  repair, such as recA- and lexA-mediated SOS responses. [E. coli by the suppression of DNA repair using combined 265/365 nm UV-LED irradiations. However, some reports showed that the combination of 265/280 nm UV-LEDs had no synergistic effect on E. coli [RAE = 0.253) showed a minimal contribution to the increased RAE score  in this study, and generated CPDs in herpes simplex virus 1 by dot-blot analysis using anti-CPDs antibody (data not shown). Petit-Frère C, et al. [We showed the loss of infectivity by UV-LED irradiations was due to damage to viral RNA, confirmed by RT-PCR, as previously reported in poliovirus-1 and bacteriophages ,24,25. D, et al.  reported, et al. . Some st, et al. ,37, but , et al. . TherefoWe compared the viral inactivation effects of different UV-LEDs on various IAV subtypes, including H1N1 and avian H6N2, infected into MDCK cells and embryonated chicken eggs. In infected MDCK cells, both H1N1 and H6N2 subtypes were equally inactivated by UV-LED irradiations c,d. SimiRAE score should be an important factor for increasing the virucidal effect by UV-LED irradiation. However, we calculated the score using a range of 254–365 nm as the peak WL of the UV-LEDs and LP-UV lamp, but we should improve the RAE score by generating more data using lower range WLs of the UV irradiations. Our study may contribute to preventing the spread of IAVs and avian flu viruses. Because the external quantum efficiencies of AlGaN-based UV-LEDs remain <10%, the development of higher quantum efficiencies is necessary for the application of viral inactivation in the near future.In this study, we propose that the


Escherichia coli (STEC) strains, including the serogroups of O157 and most of the top six non-O157 serotypes, are frequently associated with foodborne outbreaks. Therefore, they have been extensively studied using next-generation sequencing technology. However, related information regarding STEC O45 strains is scarce. In this study, three environmental E. coli O45:H16 strains  and one clinical E. coli O45:H2 strain (SJ7) were sequenced and used to characterize virulence factors using two reference E. coli O45:H2 strains of clinical origin. Subsequently, whole-genome-based phylogenetic analysis was conducted for the six STEC O45 strains and nine other reference STEC genomes, in order to evaluate their evolutionary relationship. The results show that one locus of enterocyte effacement pathogenicity island was found in all three STEC O45:H2 strains, but not in the STEC O45:H16 strains. Additionally, E. coli O45:H2 strains were evolutionarily close to E. coli O103:H2 strains, sharing high homology in terms of virulence factors, such as Stx prophages, but were distinct from E. coli O45:H16 strains. The findings show that E. coli O45:H2 may be as virulent as E. coli O103:H2, which is frequently associated with severe illness and can provide genomic evidence to facilitate STEC surveillance.Many Shiga toxin-producing  Escherichia coli (STEC), as one of the major foodborne pathogens, can produce Shiga toxins and cause severe human disease, such as diarrhea, hemorrhagic colitis, and hemolytic–uremic syndrome (HUS) [E. coli O157 and the “top six” non-O157 serogroups, including O26, O45, O103, O111, O121, and O145, are the primary STEC pathotypes frequently associated with foodborne outbreaks around the world [E. coli O45:H2-associated outbreaks were reported to have caused 18 illnesses in the United States, and contaminated smoked game meat and goats were implicated as sources of contamination in these outbreaks [Shiga toxin-producing me (HUS) ,2,3. STEme (HUS) . E. colihe world . Subsequutbreaks .E. coli O104:H4 strain associated with a large outbreak in Germany in 2011 was a clone of the E. coli O104:H4 (HUSEC041) strain isolated from the German outbreak in 2001 [E. coli O145:H28 outbreak strains (RM13714 and RM13716), linked to a U.S. lettuce-associated outbreak and a Belgium ice cream-associated outbreak, respectively, shared a common lineage with five different STEC O157:H7 strains, including the E. coli Sakai strain implicated in a Japanese outbreak [E. coli O26:H11 and E. coli O111:H11 strains [The extensive research into STEC has utilized the advantages conferred by whole-genome sequencing technology and genomic analyses in the characterization of genetic information and the virulence factors associated with the diversity and evolution of STEC pathogens ,11,12. B in 2001 . Anotherstx genes, were located on the Stx prophage sequences and could be transferred to other susceptible strains by phage transduction [− strains (95JB1 and 95NR1) shared high genomic homology; however, the strain 95NR1 contained two additional Stx2 prophages that contributed to a higher HUS morbidity than 95JB1 [espB, espD, espA, tir, and eae, were virulence factors of STEC strains in charge of the production of proteins associated with intimate adherence to intestinal epithelial cells and the formation of attaching-and-effacing (A/E) lesions [E. coli strains through specific insert loci, including selC, pheU, and pheV in the bacterial chromosome [Other studies have demonstrated that the pathogenicity evolution of most STEC strains was highly associated with the acquisition of mobile virulence elements, such as Stx prophages, virulence plasmids, and pathogenetic genomic islands ,14,17. Ssduction ,19. For an 95JB1 . In addi lesions ,22. Severomosome ,24. Moreromosome . Thereforomosome .E. coli O157 and the “top six” non-O157 serogroups of STEC, including O26, O45, O103, O111, O121, and O145, to have zero tolerance in raw ground beef and tenderized beef [Due to the close association with foodborne outbreaks and human illness, the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) has declared zed beef . Many stzed beef ,29,30. HE. coli O45:H16 strains  and one clinical E. coli O45:H2 strain (SJ7) were subject to whole-genome sequencing in the present study. The E. coli O45:H2 strain SJ7 was obtained from the Centers for Disease Control and Prevention (CDC), and was originally derived from a patient’s stool. The E. coli O45:H16 strain RM11911 was isolated from a standing water sample, and two E. coli O45:H16 strains (RM13745 and RM13752) were isolated from cattle feces in California. All strains were stored in the culture collections of the Produce Safety and Microbiology Research Unit in the USDA, Agricultural Research Service (ARS), Western Regional Research Center (WRRC). All strains were stored at −80 °C and grown in 10 mL of tryptic soy broth  in a sterile 15-mL conical tube at 37 °C with shaking at 175 rpm before use.Three environmental https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Nucleotides) [https://www.ncbi.nlm.nih.gov/genome/annotation_prok/) and deposited in GenBank. The circular maps were generated using the CGview server [E. coli O45:H2 (SJ7) and three E. coli O45:H16 strains  are included in Genomic DNA was extracted from the cultures grown to the mid-exponential-phase in 10 mL TSB using a Quick-DNA Miniprep Plus Kit , according to the manufacturer’s instructions. The DNA library was constructed using a SMARTbell Express Template Prep kit 2.0  and subsequently sequenced on a Pacific Biosciences Sequel II with v1 reagents  by SNPsaurus  . An avereotides) . The resw server . The accE. coli O45:H2 strains (FWSEC0003 and 2011C-4251) were obtained from the NCBI database to facilitate the genomic analyses of virulence and antibiotic resistance genes of STEC O45 strains. E. coli clinical O45:H2 (FWSEC0003) was previously isolated from human feces in Canada [E. coli O45:H2 strain (2011C-4251) was isolated from samples of human feces obtained in the United States [https://github.com/tseemann/abricate). The setting with a minimum of both 95% nucleotide sequence identity and coverage was used to screen for these genes.Two reference n Canada , and thed States . The vird States . The pred States  and AbriThe identification of prophage and prophage-like sequences from each bacterial genome was conducted using the PHASTER web server . GenomicE. coli strains were analyzed and phylogenetic networks were constructed via a complete linkage method using BioNumerics 7.6 . The GenBank accession numbers of 11 reference bacterial genomes obtained from the public database are indicated in Six STEC O45 strains, including four strains sequenced in this study and two reference strains from the NCBI database, as well as nine different STEC strains of clinical and outbreak origin  were subE. coli O103:H2 strains (12009 and 2015C-3163) of clinical origin were subject to comparative genomics analysis with STEC O45:H2 on the virulence genes and mobile genetic elements to evaluate their genetic relatedness using the BLAST Ring Image Generator (BRIG) with a minimum nucleotide sequence identity of 50% [Based on the results of wg-MLST, two reference y of 50% . The cruy of 50% . The aliy of 50% ,44.E. coli O45:H16 strains RM11911, RM13745, and RM13752 had genome sizes of 5,310,338, 5,264,698, and 5,264,517 bp, containing a total of 5194, 5264, and 5264 regions of coding DNA sequence (CDS), respectively; 91% of which were annotated with known functions  encoding hemolysin, afa family  encoding adhesins, cdt family  encoding cytolethal distending toxin B, espP encoding extracellular serine protease, cnf1 encoding cytotoxic necrotizing factor 1, and iha encoding the IrgA homolog adhesin , with a genome size of 99,009 bp that contained the virulence genes, including hyl operon and cnf1. No antibiotic resistance genes were found in any of these plasmids.The RM11911 strain contained one large circular plasmid, pRM11911, which was 175,089 bp in length, with an average G+C content of 47.6%. The plasmid pRM11911 also harbored several virulence factors, including the  adhesin . The RM1E. coli O45:H2 strain SJ7, isolated from a patient stool, contained a 5,444,105 bp chromosome which was approximately 130–180 kb smaller than the genomes of the three E. coli O45:H16 strains , ehxA (encoding enterhemolysin), and etpD (encoding type II secretion protein). The second-largest plasmid, pSJ7-2, was 53,543 bp in length with 49.2% GC content, and contained no virulence genes. Neither plasmids harbored antibiotic resistance genes.SJ7 had two plasmids: pSJ7-1 and pSJ7-2 . PlasmidE. coli O45:H2 strains (FWSEC0003 and 2011C-4251) were obtained from the NCBI database to facilitate the genomic characterization of STEC O45:H2 strains , and the virulence genes etpD and stcE were found in the smaller plasmid. Furthermore, no antibiotic resistance genes were found in the plasmids of these two strains  which contained the virulence genes  strains .E. coli O45 strains  sequenced in this study, two reference E. coli O45 strains (FWSEC0003 and 2011C-4251), and nine reference STEC strains with different serotypes, was investigated through whole-genome multilocus typing analysis. The results show that these STEC strains grouped into five clusters in the phylogenetic tree (E. coli O145:H28 (RM13514) and E. coli O157:H7 (Sakai), grouped into cluster 1, while a clinical E. coli O103:H25 (2013C-3264) and an outbreak-associated E. coli O121:H19 (16-9255) strain were located in cluster 2. Additionally, cluster 3 contained a clinical E. coli O103:H11 strain (2013C-4225) and two outbreak strains of E. coli O26:H11 (11368) and E. coli O104:H4 (2011C-3493). Cluster 4 was composed of three E. coli O45:H2 strains  and two clinical E. coli O103:H2 strains (12009 and 2015C-3163), whereas three environmental E. coli O45:H16 strains  grouped into cluster 5. Additionally, the results showed that the three clinical E. coli O45:H2 strains shared a common evolutionary lineage with two E. coli O103:H2 strains (cluster 4), but differed from the environmental E. coli O45:H16 strain (cluster 5) and other STEC strains of clinical and outbreak origin  reference strains and two E. coli O103:H2 (12009 and 2015C-3163) reference strains were compared against the E. coli O45:H2 strain (SJ7) using BLASTn, and the results show that the genomic backbones of these strains shared more than 95% identity covering over 95% of the SJ7 chromosome  present in the genome of SJ7 was absent in both genomes of FWSEC0003 and 2011C-4251. Additionally, both FWSEC0003 and 2011C-4251 contained a prophage sequence sharing a low nucleotide sequence similarity to prophage_7 located in similar regions in the chromosome of SJ7. Another prophage sequence (prophage_6) in SJ7 shared low homology to the counterpart prophage sequence in 2011C-4251. On the other hand, genomic heterology of the mobile genetic elements was also observed for the E. coli O45:H2 strain (SJ7) and two E. coli. O103:H2 (12009 and 2015C-3163) strains. The prophage_1 and prophage_17 sequences found in SJ7 were absent in similar regions in the chromosomes of 2015C-3163 and 12009, respectively. Additionally, there was a region of low homology located at the sequence of genomic island_7 in SJ7 compared to the counterpart in both E. coli O103:H2 strains. Minor variations in specific regions, such as genomic island_1, prophage_7, and prophage_10, in E. coli O45:H2 (SJ7) were observed in comparison to both E. coli O103:H2 strains. These results indicate that the presence of different mobile genetic elements, and prophages in particular, was primarily attributed to the heterology of the closely related E. coli O45:H2 and E. coli O103:H2 strains.Due to the close genetic relatedness, the comparative genomics analysis was further performed on the backbones and mobile genetic elements of the three romosome . FurtherE. coli O45:H2 and E. coli O103:H2 were analyzed, and the results show that all virulence genes of each strain, except for gad, were located on different prophages and genomic islands (E. coli O45:H2 and E. coli O103:H2 were predicted and compared to investigate their genetic similarity (E. coli O103:H2 (12009) was classified as being alone in cluster 1. The Stx1a prophages from two E. coli O45:H2 strains (SJ7 and 2011C-4251) and two E. coli O103:H2 strains (12009 and 2015C-3163) were grouped into cluster 2. The Stx1a prophage and Stx2a prophage from E. coli O45:H2 (FWSEC0003) and E. coli O45:H2 (2011C-4251), respectively, were classified into cluster 3. Based on the phylogenetic results, the high sequence homology between the Stx1a prophage of E. coli O45:H2 strain (SJ7) and the two E. coli O103:H2 strains likely indicates that E. coli O45:H2 might exhibit a similar Stx prophage-associated pathogenicity to E. coli O103:H2.The virulence factors of  islands . Stx promilarity . The phyE. coli O45:H2 and E. coli O103:H2 strains  in the highly conserved region (E. coli O103:H2 (2015C-3163), contained the LEE pathogenicity islands harboring extra virulence genes, nleB and efa1, which were located in the non-homologous region of the LEE pathogenicity island. The comparative genomics of LEE pathogenicity islands from the selected strains also showed that the E. coli O103:H2 strain (12009), associated with a diarrhea patient from a previous foodborne infection in Japan, and the three clinical E. coli O45:H2 strains  shared a high nucleotide sequence identity, demonstrating that these E. coli O45:H2 strains could harbor similar human pathogenesis, causing diarrhea.Additionally, one of the essential virulence factors of STEC strains, the LEE pathogenicity island, was also present among the genomes of these  strains . LEE patd region . All strE. coli strains. A number of studies have utilized whole-genome sequencing technology to predict the parallel evolution of distinct STEC strains, including the O26, O111, O103, O145, and O157 serogroups [Due to the advancement of next-generation sequencing technology, the high accuracy of complete bacterial genome information has dramatically facilitated the genomic characterization of STEC pathogens and investigation of the evolutionary relationship between different pathogenic E. coli O45:H16 strains  contained similar virulence factors, most of which were located on similar prophage sequences. All three E. coli O45:H16 strains contained the same Stx1a prophage  with 100% nucleotide sequence homology  were shown to contain the crucial virulence factors, eae and tir, that are found in most STEC O103:H2 strains and are involved in the formation of A/E lesions [E. coli O45:H2 and E. coli O103:H2 used in this study were found to contain non-LEE-encoded type III translocated virulence genes, which are related to bacterial colonization and the development of HUS [The current results of wg-MLST analysis showed that three STEC O45:H2 strains  and two STEC O103:H2 strains (12009 and 2015C-3163) have a close evolutionary relationship . Moreove and HUS ,51. In p lesions . Additiot of HUS ,54.E. coli O145:H28 (RM13514) and E. coli O157:H7 (Sakai) shared a close phylogenetic relationship, which is in agreement with the findings of a previous study demonstrating that STEC O145:H28 and O157:H7 strains evolved from a common evolutionary lineage [E. coli O45:H2 and E. coli O103:H2 strains fell on the sequences corresponding to prophages and genomic islands which were not associated with virulence factors  and E. coli O103:H2 strains used in this study likely implies that these strains are susceptible to accepting exogenous genes, rather than E. coli O45:H16 strains. Additionally, other prophages predicted from all of the STEC O45 and O103 strains in this study also contained several virulence factors, including non-LEE encoded type III translocated effectors , serine protease autotransporters (espI), and increased serum survival gene (iss). Similar results were also found in a previous study, in which the genes encoding type III secretion in E. coli were located on a vast prophage region, which acted as a crucible for the evolution of pathogenicity in numerous E. coli species [E. coli O45:H2 and E. coli O103:H2 strains, but were absent in the E. coli O45:H16 LEE-negative strains. This phenomenon will need to be investigated in future studies.Prophages represent an essential member of mobile genetic elements and are highly distributed in the genomes of different  strains . Many st of STEC ,67,68,69rophages . Therefo species . InteresE. coli O45:H16 strains were phylogenetically distinct from the clinical E. coli O45:H2 strains, whereas both clinical E. coli O45:H2 and E. coli O103:H2 shared a common evolutionary ancestor. Furthermore, most of the crucial virulence factors from E. coli O45:H2 and E. coli O103:H2 shared high nucleotide sequence similarity and were located on mobile genetic elements, such as prophages, genomic islands, or plasmids, which is strongly associated with the pathogenicity evolution of these STEC strains. The findings of this study provide better insights into the genomic characterization, evolutionary relatedness, and virulence evolution of STEC O45 strains and indicate that E. coli O45:H2 strains still pose potential threats to public health and should, therefore, be included in epidemiological surveillance.This is the first study to report the genomic characterization of STEC O45 strains of different origins. The whole-genome-based phylogenetic analysis revealed that the environmental


Mycobacterium avium subspecies paratuberculosis (MAP). An accumulative effect by insensitive diagnostic tools, a long subclinical stage of infection, and lack of effective vaccines have made the control of JD difficult. Currently lacking in the model systems of JD are undefined correlates of protection and the sources of inflammation due to JD. As an alternative to commonly studied immune responses, such as the Th1/Th2 paradigm, a non-classical Th17 immune response to MAP has been suggested. Indeed MAP antigens induce mRNAs encoding the Th17-associated cytokines IL-17A, IL-17F, IL-22, IL-23, IL-27, and IFNγ in CD3+ T cell cultures as determined by RT-qPCR. Although not as robust as when cultured with monocyte-derived macrophages (MDMs), MAP is able to stimulate the upregulation of these cytokines from sorted CD3+ T cells in the absence of antigen-presenting cells (APCs). CD4+ and CD8+ T cells are the main contributors of IL-17A and IL-22 in the absence of APCs. However, MAP-stimulated MDMs are the main contributor of IL-23. In vivo, JD+ cows have more circulating IL-23 than JD– cows, suggesting that this proinflammatory cytokine may be important in the etiology of JD. Our data in this study continue to suggest that Th17-like cells and associated cytokines may indeed play an important role in the immune responses to MAP infection and the development or control of JD.The gastrointestinal disease of ruminants is clinically known as Johne's disease (JD) and is caused by  Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent for the clinical onset of Johne's disease (JD) in ruminants. A MAP infection of the ileum leads to chronic diarrhea and reduces the ability of an animal to absorb nutrients due to inflammation and disruption of the intestinal lining. Clinical JD leads to early culling, reduced milk production, and/or premature death. The cumulative effects of JD are a rising concern to both the animal welfare and the dairy industry. Dairy operations infected with MAP may have risen by ~23% from 2007 to 2013 (68 to 91%) according to the National Animal Health Monitoring System and more recent studies  . The pla−ΔΔCt values.Total RNA was extracted from cultures containing CD3+ using RNeasy Mini Kit (Qiagen) as per the manufacturer's instruction. Due to the lower culture sizes of the T cell isotypes, Arcturus PicoPure RNA isolation kit (Applied Biosystems) was used to isolate total RNA from these samples as per the manufacturer's instruction, with DNA digestion using RNase free DNase set (Qiagen). RNA was quantified using a NanoDrop and samples within the 260/280 range of 1.90–2.20 continued for analysis. The samples outside of the range were excluded. Synthesis of cDNA followed isolation using a high-capacity cDNA Reverse Transcription kit (Applied Biosystems) following the manufacturer's instruction. RT-qPCR was performed in triplicate for all genes. Prior studies in our lab demonstrated that Peptidylprolyl isomerase A (PPIA) and TATA-box binding protein (TBP) were ideal housekeeping candidates for our genes . PPIA waThe plasma samples were diluted 1:2 and analyzed for circulating IL-23 using pre-made plates and solutions (MyBioSource) following the manufacturer's recommended protocol. All ELISA analytics were performed at 450 nm using Molecular Devices Spectra Max M5 and SoftMax Pro 6.5.1 analytical software.p ≤ 0.05 . All statistics were computed using Prism GraphPad statistical analysis software. The samples were subject to Grubb's outlier test to find the extreme outliers and it is mentioned in the text if any extreme outliers were excluded in the results.The IL-23 plasma cytokine levels and cultures with CD3+ T cells were analyzed using Kruskal–Wallis and Dunn's multiple-comparison tests. Isolated T cell subtype culture medians were compared using Wilcoxon–Mann–Whitney tests. Statistical significance was set at n = 8) significantly increased the mRNA encoding the Th17-promoting cytokine IL-23 when compared to the unstimulated control cultures  was able to significantly upregulate IL-23 mRNA when compared to the unstimulated control cultures (p < 0.05), but to a lesser extent than the live MAP . IL-17A mRNA increased 118-fold in MAP-treated cultures relative to the control untreated cultures . PPDj also tended to increase IL-17A and IL-22 mRNA levels  in cocultures relative to untreated cells; however, these differences were not statistically significant in this test. All analyses were completed using Kruskal–Wallis and Dunn's multiple-comparison tests. One extreme outlier was identified for IL-23 within the PPDj group by Grubb's outlier test and is not included in this analysis. A smaller set of MDM plus CD3+ T cell cocultures were rinsed to remove any unbound CD3+ T cells before RNA isolation. When compared to their respective unstimulated controls, the MAP-stimulated MDM cultures with remaining bound CD3+ T cells demonstrated a mean 440-fold increase of IL-17A mRNA relative to the untreated cells  or PPDj- (n = 6) stimulated cultures when compared to their respective untreated control cultures  and IL-22 (n = 6) mRNA, but these differences were not significant . One extreme outlier was identified in all PPDj-stimulated groups by Grubb's outlier test, and these values are not included in this analysis.B cells have the capacity to be antigen-presenting cells . In lieus n = 7; . Howevern = 8) when compared to the control cultures . When compared to MAP-stimulated MDM plus CD3+ T cell and sIgM+ B cell plus CD3+ T cell cocultures, the average fold change of IL-17A mRNA in MAP-stimulated CD3+-only cultures relative to that of the controls was lower  but was similar to the IL-22 mRNA levels seen in sIgM+ B cell plus CD3+ cell cocultures  increased the expression of mRNA-encoding IL-17A with a median 23-fold increase compared to the unstimulated CD4+ T cells  upregulated IL-17A mRNA with a median 130-fold increase compared to the non-stimulated cells  compared to the non-stimulated cells  tended to express more (median 79-fold) IL-22 mRNA relative to the unstimulated CD8+ cells  or IL-22 mRNA , but not in CD8+ T cells  or γδ T cells  compared to the respective controls. Grubb's test identified one extreme outlier in the γδ T cell IL-23 assays that was not included in the results.Although γδ T cells are able to produce IL-17A in the absence of APCs , CD4+ anp = 0.02; n = 10; p = 0.0006; n = 10; p = 0.0135; n = 10; r = −0.59), but this is only nearly significant with the sample size we currently have   . There w < 0.05) .Th17 cells and cytokines are likely playing a major role in MAP infection and Johne's disease . While iMAP is known to upregulate IL-6, IL-23, IL-1β, and TGF-β mRNA expression as early as 1 h post-infection in MAP-infected MDMs , indicatIn experimentally and naturally infected JD+ cows, the B cell counts are increased, including activated and memory B cells , 49. B cIn the current study, we cultured CD3+ T cells without appreciable traditional APCs present. Indeed CD3+ T cells were able to increase IL-17A (19-fold), IL-22 (9.4-fold), and IL-23 (2.2-fold) mRNA levels in the absence of traditional APCs. Macrophages are a major supplier of IL-23 . As seenWe sought to determine which CD3+T cell subtypes were responsible for producing IL-17A, IL-22, and perhaps IL-23. To this end, we sorted T cell subgroups representing CD4+, CD8+, and TCR1+ (γδ) T cells. These sorted T cell subtypes were cultured with MAP, but without added MDM cells, and analyzed to determine which subtypes were contributing to the production of Th17 cytokines during MAP stimulation. We found that αβ T cells were the main producers of IL-17A and IL-22 mRNAs, with CD4+ cells producing only small amounts of IL-23 mRNA. The CD8+ cells exhibited a larger increase in IL-17A mRNA (129-fold) than either CD4+ cells (23-fold) or γδ T cells (1.55-fold) relative to the unstimulated cells. However, CD4+ cells produced more IL-22 mRNA in response to MAP (182-fold) than either CD8+ (79-fold) or γδ T cells (6.67-fold) relative to the unstimulated cells. These results clearly indicate that T cells can directly respond to MAP with production of Th17-associated cytokine mRNA without significant APCs. Our study is not the first to show the CD4+ cells expressing a Th17 phenotype without the direction or activation of APCs . Other sM. tuberculosis or M. bovis upregulate IL-17A, IL-22, and IL-23 expression. These infected tissues also exhibit increased levels of IL-17F as well as the Th17 inhibitors IL-27 and IFNγ  deficient (M. tuberculosis and increased mycobacterial killing (Mycobacterium leprae infection (MAP induces CD3+ T cells to adopt a Th17-like phenotype even in the absence of APCs as evidenced by the enhanced production of IL-17A, IL-17F, IL-22, and IL-23. However, the continued or robust expression of Th17 cytokines IL-17A and IL-22 during early infection is reliant on the presence of IL-23, which could be supplied by MAP-infected macrophages. MAP-stimulated expression of cytokine mRNA follows similar patterns seen by other mycobacterial infections, including significant increases in IL-22 mRNA and increased expression of mRNA-encoding IFNγ and IL-27 . sIgM+ Beficient . Neutral killing , 59. IL-nfection . It is pThe datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by Michigan State University Animal Use and Care Committee.JD helped plan all experiments and performed all cell sorting and isolation. JD also performed all data analysis and supervised HC, CA, and NL. HC, CA, and NL performed Q-RT-PCR studies, assisted in blood collection and processing, assisted in cell sorting, and helped with cell cultures. PC was the overall project supervisor, reviewed all data, worked with JD to plan all experiments and assisted in preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pseudomonas putida (P. putida) KT2440 is a paradigmatic environmental-bacterium that possesses significant potential in synthetic biology, metabolic engineering and biodegradation applications. However, most genome editing methods of P. putida KT2440 depend on heterologous repair proteins and the provision of donor DNA templates, which is laborious and inefficient. In this report, an efficient cytosine base editing system was established by using cytidine deaminase (APOBEC1), enhanced specificity Cas9 nickase (eSpCas9ppD10A) and the uracil DNA glycosylase inhibitor (UGI). This constructed base editor converts C-G into T-A in the absence of DNA strands breaks and donor DNA templates. By introducing a premature stop codon in target spacers, we successfully applied this system for gene inactivation with an efficiency of 25–100% in various Pseudomonas species, including P. putida KT2440, P. aeruginosa PAO1, P. fluorescens Pf-5 and P. entomophila L48. We engineered an eSpCas9ppD10A-NG variant with a NG protospacer adjacent motif to expand base editing candidate sites. By modifying the APOBEC1 domain, we successfully narrowed the editable window to increase gene inactivation efficiency in cytidine-rich spacers. Additionally, multiplex base editing in double and triple loci was achieved with mutation efficiencies of 90–100% and 25–35%, respectively. Taken together, the establishment of a fast, convenient and universal base editing system will accelerate the pace of future research undertaken with P. putida KT2440 and other Pseudomonas species. Pseudomonas spp. are well-known gram-negative environmental bacteria, which contain more than 200 species  KT2440 is used as a chassis for synthetic biology, metabolic engineering and biocatalysis   , and the provision of donor DNA templates or single-stranded DNA is also not straightforward   and heteand upp)  have incand Ssr)  are usedapproach  that hasrganisms , includinas spp. . Neverthtforward . Additiotforward .in vivo is potentially affected by the endogenous base excision repair enzyme uracil N-glycosylase (UNG) . The potential editable window is located between position −18 and −13 upstream of the PAM sequence, and base editing efficiencies are influenced by nucleotides neighboring C bases and the size of the editable window (The CRISPR-assisted cytidine deaminase system is a newly developed base editing approach that uses cytidine deaminase (APOBEC1 or AID) with a catalytically impaired Cas9 to enable C:G to T:A mutations . In the se (UNG) . To circhia coli , Clostrierinckii , Klebsieneumonia , Corynebutamicum , and somnas spp. . Howevere window .P. putida KT2440  in a KT2440 . This sya KT2440  compriseA/R1060A ], a cytiA/T1337R ] into eSGG to NG . To obtaGG to NG ] with a GG to NG . MultiplEscherichia coli DH5α was used for cloning and maintenance. Luria Broth (LB) medium was used for cell growth. King’s medium  was used for shaking flask fermentation. P. putida KT2440, P. entomophila L48 and P. fluorescens Pf-5 were grown at 30°C, while P. aeruginosa PAO1 and Escherichia coli were cultivated at 37°C. Antibiotics were added as the following concentrations: gentamicin (Gm), 50 μg/mL (E. coli), 100 μg/mL  and 30 μg/mL (P. aeruginosa PAO1); kanamycin (Km), 50 μg/mL (E. coli) and 100 μg/mL (P. putida KT2440).All of the strains used in this study are listed in Phanta Max Super-Fidelity DNA Polymerase was used for DNA amplification, and Green Taq mix was applied to colony PCR. The One Step Cloning Kit was used for seamless cloning. All of these reagents were purchased from Vazyme Biotech Co., Ltd .Pseudomonas putida KT2440. Gene SpCas9 was amplified from pCAS-RK2T  in pSEVA6BE. By the combination of the backbone T1 terminator-sgRNA cassette-cytosine base editor Module 6-T0 terminator from pSEVA6BE and the fragment RSF1010 replicon-kanamycin resistance marker from pVLT33 using primers 62-1F/1R and 62-2F/2R, pSEVA2BE was constructed by seamless cloning.By replacing eSpCas9ppsacB was cloned from pCAS-RK2T, and inserted into the plasmids pSEVA2BE, pSEVA6BE-YE1, pSEVA6BE-NG and pSEVA6BE via seamless cloning using primers Sac-1F/1R and Sac-2F/2R, which generated pSEVA2BE-S, pSEVA6BE-YE1-S, pSEVA6BE-NG-S, and pSEVA6BE-S, respectively.The sequence of counter-selection marker Pseudomonas according to a previous electroporation method (P. aeruginosa PAO1 at 37°C) for 2 h. Next, the recovered cells were concentrated and then plated onto LB agar containing antibiotics to select recombinants. The individual transformant was inoculated into a 5 mL LB tube together with the addition of antibiotics and inducers (6 mg/mL L-arabinose or 2 mg/mL m-toluic acid) and then cultivated at 30°C (P. aeruginosa PAO1 at 37°C) for 24 h. After the cultivation process, the base editing cells were streaked onto the selective plates and cultured until the appearance of visible colonies. Colony PCR was used to amplify the target PCR products and base editing results were confirmed by DNA sequencing.pSEVA6BE, pSEVA6BE-NG, pSEVA6BE-YE1 or other derivative plasmids was transformed into n method . After eP. putida KT2440 by electroporation for achieving multiplex base editing. In the coelectroporation recovery process, the cultivation time was extended to 3 h. To enhance the editing efficiency in multiple loci by one-plasmid or two-plasmid system, individual transformants colonies were cultured for 36 h.In particular, pSEVA6BE-PobA (or pSEVA6BE-PobA-TrpE) and pSEVA2BE-QuiC were co-transformed into Pseudomonas strains were inoculated into a 5 mL LB tube containing 10 g/L sucrose and 5 g/L glucose, and then cultivated at the optimum growth temperature for 24 h. Next, the cultivated cells were streaked onto LB agar and plasmid-curing was identified by colony PCR using primers C9-F/R.To facilitate the loss of sacB-containing plasmids in cells, the mutant P. putida KT2440 strains were inoculated into LB medium without adding antibiotics. The cultivated strains were used as seed cultures and then transferred into 250 mL flasks containing 50 mL King’s medium under agitation rate of 250 rpm at 30°C for 66 h. The titer of PCA in P. putida KT2440 fermentation cultures was analyzed by HPLC  with a reverse phase C18 column . The flowing phase comprised 70% solvent A (water with 0.1% formic acid) and 30% solvent B (methanol). The optimal ultraviolet absorbance for PCA was set to 270 nm. The temperature of column oven was controlled at 40°C and the flowing rate was adjusted to 0.5 mL/min.Protocatechuic acid (PCA) was purchased from Aladdin Chemistry  and used as standards. The edited D10A nickase (a codon non-optimized SpCas9D10A or a codon-optimized and enhanced specificity eSpCas9ppD10A) were evaluated to establish an efficient cytidine base editing system in P. putida KT2440. Using the plasmid pSEVA-gRNAF (ttgA gene in KT2440) was used as the target site and inserted into pSEVA-gRNAF to give pSEVA-TtgA.Six expression combinations  of APOBEVA-gRNAF  as the tD10A nickase expression modules, Module 1 (containing SpCas9D10A) and Module 3 (containing eSpCas9ppD10A). Both of these modules were under the control of the strong constitutive promoter Pbs  and PA2018 (encoding resistance-nodulation-cell division multidrug efflux transporter) were selected as target sites for investigation of knock-out efficiency in P. aeruginosa PAO1. After base editing, DNA sequencing  and PFL_0556 (encoding flagellar motor protein MotB) were designed as target base editing sites, respectively. As shown in PSEEN1196 and PSEEN1668 genes in P. entomophila L48, respectively. Base editing results  with NG PAM in hexR using the online tool gBIG, and pSEVA6BE derivative plasmids were used as control. The results of HexR-2 and HexR-3 showed the conversion of C to T was present at the target sites with a CG or AG PAM  and the target nucleotides (cytidines) must be distributed within the editable window. To expand the cytosine base editing candidate sites, the plasmid pSEVA6BE-NG containing an eSpCas9ppA/T1337R ] were inpSEVA6BE . To asser AG PAM . Gene inr AG PAM . The appP. putida KT2440 was selected as the target site for gene inactivation by the cytosine base editor. To achieve gene inactivation of hexR, the desired cytidine mutation should occur at position 7 by mutating CAG to TAG. However, DNA sequencing .The CRISPR-assisted cytosine base editor exhibits a wide editable window width mainly from position 3 to position 8 , countinquencing  showed tP. putida KT2440, we observed that the stop codon TAA was introduced successfully into five out of eight tested colonies , ten randomly picked colonies in each system were used as DNA templates for colony PCR. The editing efficiency of the double-locus was 100 and 90% for the one-plasmid system and two-plasmid system  was selected as the third target site for multiple base editing. The TrpE editing cassette was inserted into pSEVA6BE-PobA-QuiC-2 and pSEVA6BE-PobA by using the same construction strategy to generate pSEVA6BE-PobA-QuiC-2-TrpE and pSEVA6BE-PobA-TrpE. After transformation of the one-plasmid (pSEVA6BE-PobA-QuiC-2-TrpE) or two-plasmid system (pSEVA6BE-PobA-TrpE and pSEVA2BE-QuiC-2) into P. putida KT2440 cells, the transformant colonies were identified, inoculated and cultivated in accordance with the previous cytosine base editing procedure. The simultaneous triple-locus gene inactivation efficiency was 25% (2/10 + 3/10) and 35% (4/10 + 3/10) for the one-plasmid and two-plasmid systems, respectively . The genes encoding protocatechuate 3,4-dioxygenase subunit beta (pcaH) and pyruvate kinase (pykA) (D-arabinoheptulosonate-7-phosphate (DAHP) synthase isozyme AroF-2   were sele (pykA) . The inte AroF-2 . CassettP. putida KT2440 strain KT2440::AroF2-PJ23119 (containing a strong constitutive promoter PJ23119 inserted in the upstream region of AroF-2) was used as the starting strain for the production of PCA. We obtained the mutant strain KT2440::AroF2-PJ23119ΔpcaHΔpykA after double-locus base editing using pSEVA6BE-NG-pcaHpykA-S. After curing of the double-locus plasmid, pSEVA6BE-NG-AF1- S was transformed into KT2440::AroF2-PJ23119ΔpcaHΔpykA for achieving point mutations. The base editing results  High efficiency base editing was confined mainly to TC and CC motifs, with moderate efficiency toward the AC motif and no editing of the GC motif. (ii) The requirement of a NGG PAM reduces the candidate sites in target loci.During our study, D10A. By utilizing the pSEVA6BE-NG system, we introduced a premature stop codon into the HexR gene with an efficiency of 100% , HexR-5 (targeting the PP_1021 gene) and HexR-4 (targeting the PP_1021 gene), which contains a GC motif in the editable window .JS, L-RY, and J-PW designed the experiments. JS, L-BL, and T-XL performed the experiments. JS analyzed the results and wrote the manuscript. All the 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.