RHO-P347S). This was associated with a reduction of apoptotic photoreceptor cells and with a better preservation of photoreceptor marker expression. Transcriptome analysis showed that miR-204 shifts expression profiles of transgenic retinas toward those of healthy retinas by the downregulation of microglia activation and photoreceptor cell death. Delivery of miR-204 exerted neuroprotective effects also in a mouse model of Leber congenital amaurosis, due to mutations of the Aipl1 gene. Our study highlights the mutation-independent therapeutic potential of AAV-miR204 in slowing down retinal degeneration in IRDs and unveils the previously unreported role of this miRNA in attenuating microglia activation and photoreceptor cell death.Inherited retinal diseases (IRDs) represent a frequent cause of genetic blindness. Their high genetic heterogeneity hinders the application of gene-specific therapies to the vast majority of patients. We recently demonstrated that the microRNA miR-204 is essential for retinal function, although the underlying molecular mechanisms remain poorly understood. Here, we investigated the therapeutic potential of miR-204 in IRDs. We subretinally delivered an adeno-associated viral (AAV) vector carrying the miR-204 precursor to two genetically different IRD mouse models. The administration of AAV-miR-204 preserved retinal function in a mouse model for a dominant form of retinitis pigmentosa ( Inherited retinal diseases (IRDs), including among others, retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), cone and cone-rod dystrophies, and macular dystrophies, are the most important causes of vision impairment in the working-age population . These conditions display high genetic heterogeneity, with more than 200 causative genes. Degeneration and death of photoreceptor cells (PRs) are the common landmarks of most IRDs, although the underlying molecular and cellular events are still poorly understood. PR death induces a cascade of reactive processes, such as inflammation and microglia activation, that exacerbate disease progression.,,The lack of effective treatments for IRDs remains a major challenge in ophthalmology. Proof-of-concept studies and clinical trials have recently focused on gene-specific therapeutic strategies (GSTSs),MicroRNAs (miRNAs) are short noncoding RNAs that control fundamental biological processes by targeting networks of functionally correlated genes.,,,We previously identified miR-204 and its closely related paralog miR-211 among the subset of miRNAs that are significantly expressed in the eye.To deliver miR-204 in mouse retinas, we generated recombinant AAV2/8 vectors expressing the precursor sequence of the murine miR-204 (pre-miR-204) under the control of the constitutive cytomegalovirus (CMV) promoterTo confirm effective processing in the retina, particularly in PRs, we injected subretinally the AAV.CMV.miR204 vector in eyes of wild-type mice (n = 3) at postnatal day (PN)30. As controls, the contralateral eyes were injected with the AAV.CMV.EGFP. Three weeks later, we observed proper processing of the exogenously provided miR-204 by quantifying the expression level of the mature form of miR-204 in the photoreceptor layers of the transduced area (collected by laser capture microdissection [LCM]) B.9 genome copies [GCs] of AAV.CMV.miR204 and 1 × 108 GC of AAV.CMV.EGFP per eye) in wild-type mice at PN14 (n = 8) and an equal viral load  of the AAV.CMV.EGFP control in the contralateral eyes  gene harboring the proline347-to-serine mutation.RHO-P347S up to 2–3 months of postnatal life.RHO-P347S mice at PN4 and compared ERG responses at PN30 between miR204-treated eyes (n = 21) and contralateral eyes (n = 22) injected with an equal viral load of control vector   IRD, namely, the transgenic R204MUT) A and 2B.47S mice B. Theref47S mice A and 2B.47S mice B. The ER47S mice C and 2D.RHO-P347S eyes compared to controls, both at PN30 (We did not observe evident differences in the outer nuclear layer (ONL) thickness of the injected eyes by IF assays . However at PN30 E and PN6 at PN30 . We also at PN30  and S4 a at PN30 F.9 GC AAV.CMV.miR211, 1 × 108 GC AAV.CMV.EGFP per eye), a miR-204 paralog, using the same procedure  and PN60 (n = 13) in AAV-miR204-treated eyes versus controls  and analyzed at PN60  mice at PN4 with the AAV.CMV.miR204 vector mix  in one eye and the AAV.CMV.miR204MUT control mix  in the contralateral one. Total RNA was isolated from retinas at PN12, a stage with no detectable signs of PR degeneration in RHO-P347S mice.To dissect the molecular mechanisms underlying the protective effects of AAV-miR-204 administration to the retina, we carried out a comparative transcriptomic analysis. We injected wild-type (C57BL/6) (n = 3) and In wild-type mice, 152 genes were differentially expressed (DE) between the two treatments  were among the upregulated genes, in agreement with the preservation of PR morphology and the stronger staining for PR markers observed by IF at PN30  mice. A total of 4,211 genes  were DE (FDR < 0.1)  and those obtained upon miR-204 delivery in RHO-P347S (n = 420) (RHO-P347S retinas (compared to the control-injected ones)  and dystrophic (R < 0.1) . The DEGn = 420) B. Sixty-n = 420) . Interesed ones) B were sied ones) . On the ed ones) B.RHO-P347S mice shifts transcriptomic profiles toward those of a healthy retina by downregulating pathways that are induced upon degeneration. More specifically, the pathways of innate immunity, inflammation, and cell death are main effectors through which miR-204 confers neuroprotection in IRDs.Taken together, these data indicate that AAV-miR-204 delivery in RHO-P347S mice (injected at PN4 with the AAV.CMV.miR204 vector) by double immunostaining for anti-ionized calcium-binding adaptor molecule 1 (Iba1) and either CD68 or major histocompatibility complex class II (MHCII) at PN30  for direct targets of miR-204 involved in this process. Luciferase assays showed that miR-204 can bind to the 3′ UTR of Xaf1 C, a miR-47S mice . Xaf1 is retinas F and S5CRHO promoterRHO-P347S transgenic mice at PN4 and analyzed retinal function at PN30 (n = 19) and PN60 (n = 11) by ERG (9 GC AAV.RHO.miR204 and 1 × 108 GC AAV.RHO.EGFP per eye) compared to the contralateral, RHO.EGFP-injected (1.1 × 109 GC) controls at PN30  by ERG . We obta at PN30 A. The in at PN30 B; albeitAdministration of AAV.RHO.miR204 delivery at later stages (PN14) did not lead to a statistically significant improvement of a- and b-wave amplitudes at PN30 (n = 17) or at PN60 (n = 5) .We then sought to determine whether the photoreceptor-specific delivery of miR-204 had an impact on PR viability, PR marker expression, and microglia activation, as observed upon CMV-mediated delivery. By IF analysis and TUNEL assay, we did not detect evident differences in the expression of PR markers  and in the extent of cell death between RHO.miR204-injected and control eyes (data not shown). Similarly, we did not observe any significant change in microglia activation following RHO.miR204 delivery (by immunostaining with anti-CD68 and anti-Iba1), thus suggesting that the early effect on immune responses observed in CMV.miR204-treated eyes is likely to be due to the direct effect of miR-204 on innate immune effector cells.RHO-P347S model, which, however, is lower compared to that achieved by a more widespread retinal delivery of miR-204 using the CMV promoter.Taken together, these results indicate that the photoreceptor-specific delivery of miR-204 leads to a significant functional recovery in the Aipl1 homozygous null mice.AIPL1 gene are responsible for a severe form of autosomal recessive IRD (LCA) in humans.Aipl1−/− mice, rod and cone PRs degenerate quickly, leading to a notable reduction of ONL thickness and PR OS,Aipl1−/− mice (n = 11) at PN4, using the above-described scheme. We did not record any improvement of retinal function among injected eyes at PN30 , most likely due to the severity of degeneration in this animal model (data not shown). This notwithstanding, at PN21, we observed a significant increase in the number of preserved PR rows at the ONL and in the density of PR nuclei in miR204-treated eyes compared to controls  are appropriate to demonstrate effects of novel treatments but are not ideal for long-term assessments of the effect of miR-204 delivery on the ONL morphology. Alternative animal models (even from other organisms) that allow extended follow-up periods  will be necessary to address fully the discrepancy between the preservation of visual function and the apparent lack of morphological protection observed after miR-204 delivery.A single subretinal injection of an AAV vector expressing miR-204 preserves PR marker expression and viability, ultimately recovering visual function in 47S mice . The imp47S mice . The sevProtective effects were also obtained by interventions at later time points , when fully differentiated retinas exhibit signs of PR degeneration . These iOur results indicate that miR-204 delivery to IRD mouse models acts on multiple correlated processes associated with innate immunity, inflammatory responses, and cell death. We believe that miR-204 has a primary effect on these pathways, because they were significantly modulated already before the onset of degeneration and were also impacted in wild-type retinas. The latter observation may be, in part, due to the effects of miR-204 in alleviating damage associated with subretinal injection. Based on the gathered experimental evidence, we propose that miR-204 exerts its neuroprotective effect in IRDs through at least two mechanisms.RHO-P347S mice . Therefore, the dampening of microglial reactivity observed upon AAV.CMV.miR204 delivery is most likely due to a direct role of this miRNA on microglia activation rather than secondary to events occurring in PRs . In view of translational applications in additional (pre)clinical models, the risk-benefit balance between a cell-targeted approach and the use of a ubiquitous promoter would need to be carefully assessed.The data obtained with the use of the The described delivery route , vehicle , and disease stage at intervention  are compatible with the development of a clinical protocol for human translational purposes .Recombinant AAV vectors containing the murine precursor sequence of miR-204 (or miR-211) under the CMV promoter were constructed by a two-step cloning protocol. Initially, the cassette containing the precursor of miR-204 (or miR-211) was amplified from mouse genomic DNA using the following set of oligonucleotides: 5′-ATAAGAATGCGGCCGCCTGTTCAGGACTTGGCTAAG-3′ and 5′-CGCGGATCCAACATGGGGTTGTTAATCTG-3′ for miR-204; 5′-ATAAGAATGCGGCCGCTCTGACCATGCAATCACAG-3′ and 5′-CGCGGATCCAATGGATCAGGGTGGCATC-3′ for miR-211. The obtained amplimers were subcloned in the TOPO TA Cloning Vector (Invitrogen) and released following digestion with Not I and BamH I. The fragment was then cloned into the Not I–BamH I sites of the pAAV2.1-CMV-EGFP plasmidGGAAAGGGGTCATCCTATGCCTG-3′, 5′-GGATGACCCCTTTCCGTCCACGAGTCACATG-3′ and 5′-GGAAGGCCCCTTTCAGTTCAATTGTCATCAC-3′, 5′-ATTGAACTGAAAGGGGCCTTCCCAGCCTCC-3′. The external flanking primers, used in combination with the internal mutagenic primers, were the ones described above for the amplification of the miR-204 precursor.The control vector bearing the precursor of miR-204 with a mutated seed sequence was generated by purine-pyrimidine changes in both arms of the precursor to maintain the same complementarity and hairpin structure A. This wRHO promoter sequence.RHO promoter was released from the pAAV2.1-RHO-EGFP plasmidThe vector bearing the precursor miR-204 under the control of a photoreceptor-specific promoter (pAAV.RHO.miR204) was generated by exchanging the CMV promoter of pAAV.CMV.miR204 with the human Recombinant AAV2/8 viruses were produced by the TIGEM Vector Core as reported.RHO-P347S background, pups were obtained by crossing the RHO-P347S transgenicAipl1−/− background, pups were obtained by crosses between homozygous Aipl1−/− mice.For subretinal injections in the 9 viral GCs.Studies were conducted in accordance with the institutional guidelines for animal research and approved by the Italian Ministry of Health . Mice were maintained under specific pathogen-free (SPF)-like conditions at the TIGEM Animal Facility. Surgical procedures were performed under anesthesia, and all efforts were made to minimize suffering. Viral vectors were delivered subretinally in the dorsal retinal areas via a trans-scleral transchoroidal approach.−4 to 20 cd⋅s/m2, generated through a Ganzfeld stimulator . To minimize the noise, three different responses evoked by light were averaged for each luminance step . Electrophysiological signals were recorded with gold-plated electrodes inserted under the lower eyelids in contact with the cornea. Electrodes in each eye were referred to a needle electrode inserted subcutaneously at the level of the corresponding frontal region. The different electrodes were connected to a two-channel amplifier. Amplitudes of a- and b-waves were plotted as a function of increasing light intensities. After completion of responses obtained in dark-adapted conditions (scotopic), the recording session proceeded to dissect the cone pathway mediating the light response (photopic). To this end, ERG responses to light of 20 cd⋅s/m2 were recorded in the presence of a continuous background light (set at 50 cd⋅s/m2). For each group, the mean a- and b-wave amplitude was plotted as a function of luminance (transfer curve) under scotopic and photopic conditions.Electrophysiological recordings were performed as previously described.Immunostaining for retinal and microglial markers was performed as reported.The primary antibodies used were anti-human cone arrestin , anti-Opn1mw , anti-Opn1sw , anti-Rhodopsin , anti-GFAP , anti-glutamine synthetase , anti-Iba1 , anti-MHCII , and anti-CD68 .Enucleated eyeballs were fixed overnight by immersion in 4% PFA. Prior to enucleation, cautery of the sclerae was used to orient the eyes, with respect to the injection site, at inclusion. Eyes were infiltrated with 30% sucrose for cryopreservation and embedded in tissue-freezing medium  in pairs  to facilitate comparative analysis. For each eye, 10 μm-thick serial sections were cut along the horizontal plane. Sections were progressively distributed on 10 slides (each slide contained sections representative of the whole eye at different levels) and stained with DAPI for nuclei visualization .In Situ Cell Death Detection Kit, TMR red; Roche-Merck KGaA, Darmstadt, Germany).Apoptotic nuclei in frozen retinal sections were detected by the TUNEL kit, according to the manufacturer’s instructions (GSE121785). Data analysis was performed as reported.Total RNA was extracted from mouse retinas using the miRNeasy kit . RNA-seq library preparation and sequencing were performed as previously described.http://www.webgestalt.org/GOView/).GOEAhttp://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index; TargetScan,http://www.targetscan.org/) and coexpression-based tools for systematic miRNA target recognition  were used to annotate miR-204-predicted targets.Sequence-based miRNA target-prediction databases  was performed. All values are expressed as means ± SEM.For ERG experiments, unpaired Student’s t test was performed against control-treated eyes. For experiments, a one-way ANOVA, followed by Tukey Frozen retinal sections on polyethylene naphthalate (PEN)-membrane slices were microdissected using a Laser MicroDissection (LMD) 6500 microscope. Slices were fixed in cold 75% ethanol (EtOH) in diethyl pyrocarbonate (DEPC)-treated water (2 min), washed twice in DEPC-treated water (30 s), and stained in Meyer’s hematoxylin . Following hematoxylin staining of nuclei, the slices were washed twice in DEPC water (30 s), dehydrated in a series of EtOH concentrations , and dried on air (15 min). The laser parameters used for the microdissection were the following: Power 60, Aperture 7, Speed 7, Specimen Balance 46, and Offset 25. The pencil function was used to border the photoreceptor layers. Microdissected layers from each eye were pooled, and RNA was extracted, as described above.−ddCt method.qRT-PCR-based detection of mature miR-204, miR-124, and small nucleolar RNA 234 (sno234) was performed using the TaqMan miRNA assays . All reactions were performed in triplicate. The qRT-PCR results, recorded as threshold cycle numbers (Ct), were normalized to the sno234 reference, and the relative fold change of expression was calculated using the 2M.K., E.M.S., and S.B. conceived the study, designed the experiments, analyzed the data, and wrote the manuscript. M.K., I.G., E.M., M.P., I.C., and E.M.S. carried out experimental work. R.D.C. and A.C. analyzed transcriptomic data. E.M. and E.M.S. performed subretinal injection and visual tests in mice. S.C. provided critical expertise for studies in the mouse. S.B. and E.M.S. provided funding for this study. All authors discussed the results and had the opportunity to comment on the manuscript.The authors declare no competing interests.


Amaranthus palmeri  especially those with resistance to glyphosate and acetolactate synthase (ALS) inhibiting herbicides. While investigating the basis of high fomesafen-resistance in A. palmeri, we identified a new amino acid substitution of glycine to alanine in the catalytic domain of PPO2 at position 399 (G399A) . G399 is highly conserved in the PPO protein family across eukaryotic species. Through combined molecular, computational, and biochemical approaches, we established that PPO2 with G399A mutation has reduced affinity for several PPO-inhibiting herbicides, possibly due to steric hindrance induced by the mutation. This is the first report of a PPO2 amino acid substitution at G399 position in a field-selected weed population of A. palmeri. The mutant A. palmeri PPO2 showed high-level in vitro resistance to different PPO inhibitors relative to the wild type. The G399A mutation is very likely to confer resistance to other weed species under selection imposed by the extensive agricultural use of PPO-inhibiting herbicides.Protoporphyrinogen oxidase (PPO)-inhibiting herbicides are used to control weeds in a variety of crops. These herbicides inhibit heme and photosynthesis in plants. PPO-inhibiting herbicides are used to control  Weeds have been the major cause of crop yield losses since the dawn of agriculture. Herbicides are the primary tools used to manage weedy species. Weeds continue to be a growing threat to food security because of resistance to herbicides . The useProtoporphyrinogen IX oxidase (PPO) is one of the potent herbicide targets . It is aPPO genes encode organelle-specific isoforms of PPO, namely, PPO1  and PPO2   . In someroduced) . PPO conroduced)  when ecoroduced) .A. palmeri  in the PPO2 enzyme of A. tuberculatus  were used for sequencing the PPO2 gene. Four plants , were confirmed by gene sequencing to contain the mutation leading to alanine in 399 position (G399A), and were isolated to generate F1 populations. Two male and two female A. palmeri plants that were heterozygous and homozygous for the G399A mutation, respectively, were allowed to hybridize to produce R39P and R43P F1 populations, respectively. Plants were grown in the greenhouse maintained at 32/25 ± 3°C day/night temperature with supplemental light for 14 h. Seeds from SS, MIS-D , R39P, and R43P populations were used for bioassays in the greenhouse. Herbicide treatments were applied with a laboratory sprayer equipped with a 110° flat-fan spray nozzle  delivering 187 L ha-1 of herbicide solution at 310 kPa. The nozzle was set at 51 cm above the plant canopy. Plants were returned to the greenhouse immediately after herbicide treatment. The herbicides were applied when the plants were 5- to 7-cm tall.The resistant Palmer amaranth population designated as MIS-D was used in this study. MIS-D field population was confirmed resistant to glyphosate, PPO- and ALS-inhibiting herbicides . The susPPO2 were amplified using 1F (5′-GG GGTACCCGGGTAAACTGATCTTATGTTAATTC-3′) and 3R (5′- GGAATTCGAGCTCGCATGCTTACGCGGTCTTCTCAT CATC-3′) primer pair, purified, and sent for sequencing using internal primers. The PCR was conducted in a 40-μL volume that consisted of 2 μL (60 ng) of cDNA, 0.5 μM of each primer, and 20 μL of EmeraldAmp MAX PCR Master Mix (Takara). The PCR was run with the following profile: 98°C for 30 s; 40 cycles of 98°C for 1 min, 58°C for 1 min and 72°C for 2 min; followed by a final extension step for 10 min at 72°C. Overlapping internal primers used for sequencing are as follows: Apx2-1F (5′- GTAATTCAATCCATTAC CCAC CTT 3′), Apx2-1R (5′-TTCCATA CGTCGGGAAATGT-3′), Apx2-2F (5′-TGTTGGAACCATTTCTCTGG-3′), Apx2-2R (5′-GGGGATAAGAACTCCGA AGC-3′), Apx2-3F (5′-GATG CTGTGGTTGTCACTGC-3′), and Apx2-3R (5′-TTACGCGG TCTTCTCATCCAT-3′). The PCR product purified from agarose gel using Pure-Link Quick Gel Extraction Kit (Invitrogen) was sent to Eurofins Genomics for sequencing. The resulting overlapping fragments from all individual R and S plants were assembled into one sequence using Sequencher 5.4.6 (Gene Codes Corporation) to obtain the complete coding region of the PPO2 gene. The nucleotide sequences were translated into open reading frames using the online ExPASy translation tool.Some plants from MIS-D in the bioassay experiment were assigned ID numbers and ∼25 mg of leaf tissues were collected before spraying fomesafen. After evaluation of plant response, the leaf tissues collected from survivors were labeled as R while those from plants, which did not survive the herbicide application were labeled as S. Total RNA was isolated using the Plant RNeasy Mini Kit (Qiagen), treated with DNAse (Thermo Fisher Scientific), and converted to cDNA using Access RT-PCR System (Promega). Full-length A. palmeri PPO2 protein, S-PPO2 and R-PPO2, were built using the homology modeling workflow in YASARA  using protein data bank (PDB) entry 1SEZ of tobacco PPO2 as template. The S-PPO2 model used a modified version of the resistant PPO sequence in which position 399 was changed back to the wild-type glycine (G399 model). All other sequence positions were as found in the resistant sequence. The R-PPO2 model used the unmodified resistant PPO sequence (A399 model). Default settings and standard protein preparation steps were used. Fomesafen was initially modeled into the binding-site of the wild type S-PPO2 using the docking scripts and tools of YASARA. To gain insight into the effect of the G399A on fomesafen binding, the predicted binding model was then transferred into the R-PPO2 model, where G399 was changed to A399. The changes in protein-ligand interactions due to the G399A mutation were then examined and interpreted.Two structural models of PPO2 sequence information obtained from the S and R plants, we developed a CAPS marker for detecting the mutation at 399 amino acid position. We found the mutation from wild type codon GGT to GCT at 399 position. RsaI restriction endonuclease recognizes the sequence GTˆAC, so a G to C mutation eliminates the restriction site. Genomic DNA was extracted from all R plants along with a few S plants and a 691-bp fragment was amplified using the primers, RsaIF (5′-CGTACCCCTTTCCGTTATGA-3′) and RsaIR (5′-CCGGGAAGATCTT TTTCCAT-3′). The amplicon was then subjected to RsaI digestion. Upon digestion, the wild-type amplicon would generate a 505-bp and 185-bp band, whereas the homozygous mutant amplicon would generate the intact 691-bp band . Four days after planting, seedlings were thinned to five per pot. When seedlings were 5-7-cm tall, fomesafen  was applied at 16.5 (0.062X), 33 (0.125X), 66 (0.25X), 132 (0.5X), 197.5 (0.75X), 263 (1X), 527 (2X), 1053 (4X), and 2107 (8X) g ai ha-1, where 1X represents the recommended dose. The SS population was sprayed with nine doses as well: 1, 2, 4, 8, 16.5, 33, 66, 132, and 263 g ai ha-1, corresponding to a range of 1/256 to 1X of the recommended dose. Herbicide treatments included a non-ionic surfactant  at 0.5% v/v. A non-treated control was included with each population. The overall effects of fomesafen  were assessed visually on a scale of 0 (no control) to 100% (complete kill) relative to the non-treated control at 21 days after treatment (DAT). The shoot biomass was harvested, dried at 60°C for 72 h, and weighed. Data were analyzed by fitting a non-linear, three-parameter log-logistic regression model using the “drc” package in R 3.5.1  was calculated. The resistance factor was determined by comparing ED50 values between the R and SS accessions.Seeds from Palmer amaranth populations, MIS-D, R R 3.5.1 . The mod39P, was investigated using twice the recommended dose of each PPO herbicide. Seeds from R39P and SS populations were planted in cellular trays in the greenhouse. Herbicides representing each PPO herbicide family were applied to 5-cm tall seedlings. Twenty-five seedlings from each population were treated with acifluorfen (1120 g ai ha-1), carfentrazone (560 g ai ha-1), flumioxazin (141 g ai ha-1), fomesafen (528 g ai ha-1), lactofen (448 g ai ha-1), and saflufenacil (49 g ai ha-1). The herbicides were applied with the respective recommended adjuvants. Carfentrazone and flumioxazin treatments included 0.25% non-ionic surfactant (NIS) (v/v), whereas acifluorfen included 0.125% NIS. Lactofen was applied with 0.5% crop oil concentrate (v/v), whereas saflufenacil was sprayed with 1% methylated seed oil, and 1% ammonium sulfate (w/v). Herbicides were applied in a laboratory spray chamber fitted with 800067 flat fan nozzles calibrated to deliver 187 L ha-1 at 269 kPa. At 21 DAT, injury, and mortality were evaluated visually.The MIS-D population is cross-resistant to the 1X dose of various foliar-applied PPO herbicides . In thisA. tuberculatus backbone. The glycine at 399 position in A. palmeri PPO2 corresponds to G398 in A. tuberculatus PPO2, but the said mutation is referred as G399A in this experiment  to avoid confusion. Recombinant expressions of five other possible G399 substitutions  based on a single nucleotide base change at position one or two of the G399 codon were also investigated. In addition, in vitro inhibition of the wild type and previously known PPO2 mutations conferring PPO resistance  were studied. The PPO2 gene from A. tuberculatus was synthesized de novo and subcloned into pRSetB plasmid  using BamHI and HindIII restriction sites. The construct contained an N-terminal hexa-histidine tag to facilitate protein purification. The plasmid construct was transformed into E. coli strain BL21(DE3)pLysS  and was selected in LB agar plates supplemented with 100 μg mL-1 ampicillin and 34 μg mL-1 chloramphenicol. For pre-cultures, 3 mL of LB medium containing ampicillin and chloramphenicol was inoculated using a single colony picked directly from the agar plates, and then cultivated at 37°C with shaking at 200 rpm for 6 h (pre-culture). From this culture, 20 μL was inoculated into a 100-mL flask containing 20 mL LB medium and the same antibiotics. The culture was incubated overnight in a shaker . An aliquot (100 μL) of the overnight culture was transferred into a 250-mL flask containing 100 mL ZYM-5052 autoinduction medium (preparation procedure is described in -1) and chloramphenicol (34 μg mL-1). The main culture was incubated at 37°C for 5 h with shaking at 200 rpm, then for an additional 21 h at 25°C with shaking.Effects of G399A substitution were studied using g for 30 min at 4°C. For every gram of pellet, 10 mL of PPO lysis buffer , pH 7.5, 20 mg mL-1 lysozyme, 30 unit mL-1 DNase I) containing protease inhibitors  was added. The suspension was sonicated  in an ice-bath to avoid overheating the samples. The cell debris was removed by centrifugation at 38,000 × g for 30 min at 4°C and the supernatant was added with 2 mL of 5 M NaCl. To purify the enzyme, a 500-μL-bed volume of HisPur Ni-NTA resin  was placed in a column and equilibrated with a buffer , 0.1 mM EDTA, pH 8.0). The supernatant, which contained the soluble recombinant protein, was passed through the resin. Non-specifically bound proteins were eluted using 5.6-mL wash buffer , adjusted to pH 7.5) and the target protein was eluted in 1 mL of elution buffer . The purified enzyme was quantified using Scandrop nano-volume spectrophotometer . The protein and the cell debris (2.5 μg) was electrophoresed using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis to check protein quality and solubility.At the end of cultivation, the cells were harvested by centrifugation at 6000 x in vitro PPO inhibitor dose-response assay. PPO inhibitors, purchased as Pestanal standards  were dissolved in 80% DMSO. Inhibitors evaluated included diphenylether, pyrimidinedione, triazolinone, N-phenylphthalimide, phenylpyrazole, and thiadiazole herbicides. Ten PPO inhibitor concentrations (10 μL) ranging from 0.00005 M to 5.12 × 10-12 M were added to the reaction mixture. The samples were incubated for 30 min at room temperature. Protoporphyrinogen IX (3.24 μM) was then added and the fluorescence was recorded for 30.25 min (33 cycles of 55 s cycle-1) using a microplate reader . The substrate protoporphyrinogen IX was prepared from protoporphyrin using sodium amalgam reduction  were estimated using non-linear regression procedures (three-parameter or four-parameter log-logistic function). For each inhibitor, resistance factors were calculated by dividing the IC50 values of mutant with the sensitive PPO2.Protoporphyrinogen IX oxidase was assayed using the fluorescence assay with excitation and emission wavelength of 405- and 630 nm, respectively. The assay was conducted in a 187-μL reaction mixture containing100 mM Tris–HCl, 1 mM EDTA, 5 mM DTT, 0.0085% Tween 80, and 15 μL of enzyme in resuspension buffer . The concentration of enzyme differed for each mutant depending on the activity of the enzyme in the presence of saturating concentration of substrate and absence of PPO inhibitor. Maximum fluorometric readings were recorded for the amount of each enzyme. These values were used to calculate the enzyme activity (fluorescence units/minute) compared to the WT. Recombinant protein that showed enzyme activity in the absence of PPO inhibitor was subjected to PPO2 of five resistant (R) plants and three fomesafen-sensitive (S) plants was sequenced using cDNA. The full-length cDNA of 1608 bp codes for the dual targeting signal peptide, the membrane-binding domain, the FAD-binding domain, and the substrate-binding domain of mature mitochondrial PPO2 protein . The sequences we obtained were 1,608 bp; therefore, we generated full-length cDNA sequences of PPO2 from R and S plants . Comparison of the cDNA-inferred amino acid sequences of the PPO2 protein revealed three amino acid polymorphisms in R plants; however, only a single change in amino acid, glycine to alanine at position 399 (G399A) , consistently differed between all R and S plants  genomic sequence of PPO2 as reference, we found that the genetic code for G399 resides on exon 15 (Nicotiana tabacum (tobacco) PPO2 (PPO1 gene in the same R and S plants and did not find any polymorphisms among R plants from MIS-D population.Using the grain amaranth ( exon 15 . The cor exon 15  based onco) PPO2 . The G39co) PPO2 . An examco) PPO2 . We founN. tabacum PPO2 generated by A. palmeri PPO2-fomesafen complex. In particular, the homology modeling workflow implemented in YASARA was used to derive models for both S-PPO2 and R-PPO2. The binding geometry of fomesafen in the binding-site of the S-PPO2 model was then predicted using in-house docking tools and transferred into the R-PPO2 model. The resulting visualization  group of A399 in R-PPO2 (highlighted pink). Although it comprises just a single heavy atom, this methyl group is oriented directly into a buried part of the PPO binding site, making the binding pocket smaller. This creates close steric contacts  of fomesafen. The SS population was controlled 100% with 8 g ai ha-1 (1/32X) (50) of MIS-D, R43P, and R39P ranged from 123 to 187 g ai ha-1, whereas the ED50 for SS was 12 g ai ha-1. Thus, the levels of resistance to fomesafen in MIS-D, R39P, and R43P were 11-, 12-, and 16-fold, respectively, relative to the SS population  manifests higher resistance, when compared to S plants  . The amppulation . The G39S plants . Also, tIn vitro enzyme activity assay of the wild type and recombinant PPO2 proteins with known resistance-conferring mutations ΔG210, R128L, and the novel mutation G339A using A. tuberculatus PPO2 gene. Constructs were made to generate recombinant protein with ΔG210, R128L, and G339A mutations; other possible exchanges at G399 position; and wild type PPO2. Four G399 protein variants  were soluble, but lacked enzyme activity; no enzyme activity was detected even after adding large amounts of enzyme to the assay mix. G399S had negligible activity, necessitating the addition of a high amount of enzyme to the assay mix before any measurable activity was detected. Among these recombinant variants, only G399A was active. Compared to ΔG210 and R128L, G399A had a very low activity  were calculated after subjecting the recombinant protein to increasing concentrations of diphenylether, pyrimidinedione, triazolinone, N-phenyl-phthalimide, phenylpyrazole, and thiadiazole herbicides  crop in a crop rotation system. Cross-resistance to various families of PPO-inhibiting herbicides suggests that G399A mutation endows broad resistance to PPO-inhibiting herbicides.The field population, MIS-D, was resistant to the 1X dose of carfentrazone, flumioxazin, fomesafen, lactofen, fluthiacet-methyl, and pyraflufen-ethyl. The 1X dose of acifluorfen and saflufenacil caused >90% mortality in MIS-D while the other PPO-inhibiting herbicides caused 31–80% mortality . The numn, MIS-D , reflectAmaranthus species are among the first few weeds to evolve resistance against PPO-inhibiting herbicides  PPO2 variant from A. palmeri with fomesafen was validated by homology modeling and computational docking  with G399A mutation in vitro experiment. Thus, evaluation of fitness penalties associated with the new G399A mutation in the presence and absence of selection would be informative for the development of better control strategies for management of resistance to PPO-inhibiting herbicides. Nevertheless, the continued use of the same PPO inhibitor favors the R plants and would perpetuate the resistant alleles. One more cycle of purification in our experiment increased the resistance level of the next generation.The G399A mutation is less prevalent in the field than the other known mutations among PPO-resistant In vitro data with G399A, ΔG210, and R128L PPO2 variant showed different degrees of cross-resistance among and between the different classes of PPO inhibitors due to the difference in chemical structure that influences the binding and orientation of the inhibitor in the enzyme catalytic site. PPO herbicides compete with the substrate, protoporphyrinogen IX, of PPO2 enzyme by mimicking parts of the substrate. Different inhibitors have different mimicking modes depending on its structure. Herbicides belonging to the same chemical family have the same base structures and usually behave similarly in the way they interact with the enzyme. It is apparent that diphenylethers are particularly highly affected by G399A, ΔG210, and R128L mutations. This is probably because diphenylethers were primary selectors for the evolution of PPO-inhibitor-resistant weeds.in vitro assay revealed that G399A mutation endowed broad cross-resistance to diphenylether, pyrimidinedione, triazolinone, N-phenylphthalimide, phenylpyrazole, thiadiazole, and oxadiazole PPO herbicide families. The position of G399 is critical to the herbicide-binding site, thus the replacement of G399 with alanine weakens the affinity of PPO2 enzyme to different PPO inhibitors. According to the protein structural models derived here, the methyl group of alanine protrudes into the buried part of the PPO-binding site causing the pocket to constrict. Thus, some of the ring structures and side chains of amino acids and those of various PPO-inhibiting herbicides would clash. The drop in frequency of fomesafen-resistant plants in the F1 population relative to the original field population is because of the double dose used on the F1 population (39P to fomesafen (12-fold) turned out to be similar to that of the original population, MIS-D (11-fold) (-1. Fifty percent of R39P could be killed with less than the 1X dose (143 g ai ha-1) of fomesafen. Therefore, treating the F1 population with 2X dose of fomesafen reduced the number of survivors compared to the original population, which was treated with only 1X dose (Both whole plant bioassay and pulation . The res11-fold) . The 1X  1X dose . Except The cross-resistance pattern to various PPO herbicide families and the resistance levels are complex. There is indication that besides zygosity and level of purification of a population, non-target genes contribute to PPO resistance in some populations, including MIS-D. All the 5 R lines we used for sequencing carried the G399A mutation. Also, all survivors from MIS-D population contained only this mutation, but these plants exhibit different levels of resistance in the screening experiments (data not shown). Non-target site mechanisms involve many genes and can co-exist with target-site mutation. This can modify the resistance level and makes the inheritance pattern messy. Follow-up research is needed to sort out the involvement of non-target-site-resistance mechanisms in the evolution of Palmer amaranth resistance to PPO inhibitors.The ΔG210 also caused broad resistance to PPO inhibitors although G210 is not located in the catalytic site. Previous studies showed that the G210 plays a key role in stabilizing the α-8 helix of PPO2. Deletion of G210 unravels the last turn of the helix and results in about 50% enlargement of the active site cavity , therebyin vitro assays. R128 is in the pocket between the active site and the entryway of the substrate, and is crucial for stabilizing the substrate in the catalytic domain  under the following accession numbers: MK408971, MK408972, MK408973, MK408974, MK408975, MK408976, MK408977, and MK408978.PPO2 sequence data from this article can be found in GenBank (in vitro assays and analyzed the data. IC and TM performed computational modeling of the novel mutation. RS-P participated in field sampling of Palmer amaranth populations with NR-B, conducted the resistance bioassays, produced the F1 populations, performed the herbicide dose-response assays, and participated in the manuscript preparation. AL and MN performed the herbicide assays and helped in genotyping. RA conceived the enzyme activity assay study and helped in coordination overall. AP provided the technical input in addressing comments from reviewers, pertaining to mutant PPO enzyme activity, providing details on methodology and supplemental data. NR-B conceived the whole Palmer amaranth adaptation project, obtained funding for the project, organized and led the collection of Palmer amaranth samples and field histories, conceptualized the phenotyping and molecular biology experiments, directed the research implementation, and participated in the manuscript preparation and revisions.GR participated in the design of molecular biology experiments and coordination, performed the molecular experiments, analyzed the data, and wrote the manuscript. RS-P, RA, and MK designed and performed RA, MK, IC, TM, and AP were employed by BASF Company. The remaining 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.


PyPI.The pdb-tools are a collection of Python scripts for working with molecular structure data in the Protein Data Bank (PDB) format. They allow users to edit, convert, and validate PDB files, from the command-line, in a simple but efficient manner. The pdb-tools are implemented in Python, without any external dependencies, and are freely available under the open-source Apache License at https://github.com/haddocking/pdb-tools/ and on The PDB format encodes 44 possible different record types in a human-readable flat-text format, with each record having a number of fields with stringent spacing rules in a total of 80 characters. This strictness and complexity of the PDB format make manual editing difficult. As a result, researchers use molecular viewers, e.g. PyMOL3 and VMD4, to perform basic editing tasks, such as selecting chains from a structure. More advanced edits, such as changing chain identifiers, deleting specific atoms, or renumbering residues require expertise with scripting languages, whose syntax varies from viewer to viewer. Other edits, such as selecting atomic positions based on occupancy values, are even more challenging to accomplish in molecular viewers, if possible at all. The alternative is for researchers to become proficient in a programming language, such as Python, and use one of its structural bioinformatics libraries, such as BioPython5 or ProDy6.Obtaining and analyzing three-dimensional structures of biological macromolecules, such as proteins or nucleic acids, is often a key step towards understanding their biological function. Of the many file formats used in structural biology to store three-dimensional coordinate data, the Protein Data Bank (PDB) format remains one of the most widely adopted, despite the introduction of a new standard - the mmCIF format - in recent years2 . The tools make use of Python generator expressions to improve performance and memory usage. This feature, along with support for streaming input and output data, allows users to create complex pipelines by chaining together different tools .The pdb-tools toolkit is implemented inAs a result, using ’pdb_reres’ to renumber the residues of a structure with 64,606 atoms takes ~0.17 seconds (on an Intel i7-7500U CPU) and ~10 MB of memory. Merging eight copies of the same structure using ’pdb_merge’ took ~0.74 seconds and ~18 MB of memory. These performance indicators make pdb-tools particularly useful when combined with shell scripting for batch processing entire collections of PDB files.To aid the maintenance and development of pdb-tools, we wrote approximately 250 separate unit tests that provide 81% coverage for the tools and their possible usage options. In addition, each commit is checked for coding style against the PEP8 style guide, using flake8. Besides hosting the source code on GitHub, we use setuptools to package and distribute pdb-tools through PyPI. Further, we use semantic versioning to indicate compatibility between releases with the same major version. The code is tested continuously using Travis CI and AppVeyor webhooks on GitHub, which monitor incoming pull requests and any commits to the master branch. We test on virtual instances of Windows 10 and Linux 16.04 LTS, under Python 2.7, 3.6, and 3.7. Nevertheless, the pdb-tools should run on most UNIX derivatives and Windows versions, provided a supported version of Python in installed.Concerning documentation, each tool contains a self-contained help string, describing its purpose, command-line interface, and usage examples. This help text is available by running the tool without any argument. Additional documentation on the usage and installation of pdb-tools is available online on the project’s web-page and in the README file accompanying the source code.Finally, we develop pdb-tools as a community open-source project on GitHub and encourage contributions of any kind. To date, the project has more than 30 forks and has been starred by over a dozen users on GitHub. To help and promote these contributions, and based on our experience with building pdb-tools collaboratively, we have documents setting coding best practices, contribution guidelines, and a code of conduct for developers on our GitHub repository. In addition, we make use of and encourage users to contribute to our issue tracker, which documents our discussions and development decisions publicly.The pdb-tools follow a ’one tool, one job’ design philosophy, where each tool performs a simple operation on an input file, while more complex operations can be achieved by chaining different tools together. Over the years, the pdb-tools have been used in both research and educational contexts, some of which we highlight below.Molecular viewers, such as PyMOL or VMD, are the tools of choice for making selections of specific chains or residue ranges in PDB files. When handling more than one structure, programming libraries such as Biopython or Prody are better suited for the task. The selection tools included in pdb-tools offer a simple solution to make complex selections.$ pdb_fetch 1ctf | pdb_keepcoord | pdb_reres > 1ctf.pdb$ pdb_selres -1:30 1ctf.pdb | pdb_selatom -CA > 1ctf_CA.pdb$ head 1ctf_CA.pdbATOM       2  CA  GLU A   1      17.706 17.982 -14.905 1.00 16.74       CATOM      11  CA  PHE A   2      17.509 14.262 -14.184 1.00 13.24       C... [truncated]parallel utility to distribute processing over several cores.Importantly, users can use shell scripting to efficiently process large collections of structures. Those using UNIX-based systems can also make use of the$ for pdbf in $( ls *.pdb )$ do$   pdb_selchain -A $pdbf | pdb_delelement -H | pdb_tidy > ${pdbf}_A_noH.pdb$ done8, whose web server provides a user-friendly interface that oversaw over 200.000 job submissions to date. The simplest of submissions requires two structures, in PDB format, that cannot have more than one chain, cannot have overlapping residue numbers, and cannot have alternate locations for any atom. Often, users submitting jobs to the HADDOCK web server are faced with error messages because of such formatting requirements. Using pdb-tools, producing a HADDOCK-compliant PDB file is straightforward, as we demonstrate below.HADDOCK is an integrative modeling software for protein interactions$ pdb_fetch 1brs | pdb_keepcoord > 1brs.pdb  # 6 chains$ pdb_selchain -A,D 1brs.pdb | pdb_delhetatm | pdb_selaltloc > 1brs_AD.pdb$ pdb_selchain -A 1brs_AD.pdb | pdb_tidy > 1brs_A.pdb$ pdb_selchain -D 1brs_AD.pdb | pdb_tidy > 1brs_D.pdb-multi option.Although PDB files may include SEQRES records that store the sequence of the construct used for structure determination, the final structure does not necessarily include all the amino acids or nucleotides. This discrepancy is particularly important when building alignments for homology modeling. To this end, we include in pdb-tools a PDB to FASTA converter that extracts the sequence of the structure directly from the ATOM/HETATM records. The user can specify if the entire structure should be exported as a single FASTA sequence record, or divided by chain, using the$ pdb_fetch 1brs | pdb_tofasta>PDB|ABCDEFINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIRAQVINTFDGVADY... [truncated]$$ pdb_fetch 1brs | pdb_tofasta -multi>PDB|AVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR>PDB|BAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR... [truncated]Due to several limitations with the PDB file format, the Protein Data Bank recently introduced the new PDBx/mmCIF format. To support this decision, we include in pdb-tools converters to and from the mmCIF format. These converters allow users to implicitly use pdb-tools on mmCIF files. However, large structures that cannot abide by the PDB format specification (too many chains or residues or atoms) cannot be converted.$ pdb_fromcif 1brs.cif | pdb_selchain -A | pdb_tocif > 1brs_A.cifThe pdb-tools provide a command-line interface to edit three-dimensional molecular structures in the PDB format. Since it is written in Python, without any third-party dependencies, the toolkit is available on Linux, Mac OS, and Windows and supported on both Python 2 and 3. The source code is hosted on GitHub and licensed under the open-source Apache License version 2.0, to encourage contributions from the community, and available for installation through PyPI and SBGrid. As such, the pdb-tools are an efficient, portable, and extremely flexible toolkit for both starting and experienced researchers in structural biology.https://haddocking.github.io/pdb-tools/,https://pypi.org/project/pdb-tools/, andhttps://sbgrid.org/software/titles/pdb-toolspdb-tools is available from:https://github.com/haddocking/pdb-tools,https://github.com/haddocking/pdb-tools/releases/tag/2.0.0-rc1Source code available from:http://doi.org/10.5281/zenodo.21680709Archived source code as at time of publication:License: Apache License, version 2.0 While there are many, often complex, tools available to manipulate pdb files, pdb-tools stands out as a light-weight set of command line tools for common use cases. The requirement for each tool to perform one task makes these tools easy to use in shell scripts and to combine into a pipeline for batch processing a set of PDB structures. On the software side, this is a well-designed open source tool kit available in GitHub that follows best software development practices, including documentation, checking for consistent coding style, high test coverage, using continuous integration, semantic versioning, and deployment on PyPi. A liberal open source license encourages community contributions. The modular design makes this project easy to maintain and expand compared to many of the legacy tools still in use. Recommendation: List all, not just a subset, of the available tools with an example on the documentation page. That way, a Google search will find those tools and examples are helpful for new users.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this work, Rodrigues et al, present to the readers a collection of python based tools to edit, convert and validate PDB files, in a very simple and efficient way. The authors clearly describe in the methods section the procedure used to implement the pdb-tools toolkit, and they even provide some simple use cases where the toolkit can be applied. I have installed the toolkit in my workstation and I did not have any kind of problem. I have tested several of the available functionalities and I have found it to be very useful and easy-to-use on day-to-day work (as the authors clearly state in the article). This type of contribution software is very useful for any scientist (with beginner or advanced skills) dealing with structure data in PDB format.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


This paper presents a simple and economical method for preparing durable anti-static functionalized inkjet prints by using P[St-BA-F6]—novel antistatic agents synthesized by an oxidative polymerization of styrene, butyl acrylate, and allyl alcohol polyether F6. The P[St-BA-F6] was characterized by gel permeation chromatography and Fourier transformation infrared spectroscopy. One bath pretreatment solution containing P[St-BA-F6] and pentaerythritol tetraacrylate (PETA) were applied to polyester fabrics before inkjet printing, in order to enhance the color yield and the anti-static properties. The pretreatment conditions, including the concentrations of P[St-BA-F6], curing temperature, and time, were optimized based on inkjet printed polyester fabrics. SEM (scanning electron microscope), XPS (X-ray photoelectron spectroscopy), XRD (X-ray diffractometer), TG (thermogravimetric), and DSC  examined the fabrics. The results showed that the treated PET fabrics exhibited good applied performances, such as higher color yield, better dry rubbing fastness, lower electrostatic voltage, and durable anti-static properties, even after washing 10 times. These results can be attributed to alcohol polythene group (F6) and allyl group (PETA). PETA can be cross-linked with P[St-BA-F6] and PET fiber. The thermal stability of the treated fabric was lower than that of the untreated fabric, owing to the presence of resin film on the fiber surface. Inkjet printing technology shows many environmental advantages over the conventional dyeing and printing process, which enables the cost-effective short-run for production. The pretreatment process is very important for inkjet printing process. It is because that the diffusion behavior of disperse dye ink and the weave density on untreated polyester ) fabric exerts significant impact on the printing accuracy and quality ,2.2 plasma surface-treatment to improve the color strength and the pigment adhesion of the treated surfaces  composite was prepared by an oxidative polymerization of butyl acrylate (BA), styrene (St), and allyl alcohol polyether F6 (F6) to expand its potential applications in pretreatment before inkjet printing for antistatic PET fabric. In addition, a cross-linking agent of pentaerythritol tetraacrylate (PETA) was applied in pretreatment solution to improve the durability of P[St-BA-F6] resin film.2) was purchased in the market. Commercial samples of disperse red ink (D2551 Red) and CYMK inks  were purchased from DuPont Co., Wilmington, DE, USA. Commercial grade BA, St, and F6 were purchased from Jiangsu Haian Petroleum Chemical Co., Haian, China. Commercial grade emulsifying agent AES , emulsifying agent TO-7 , NaOH, and ammonium persulfate were purchased in the market. Commercial grade Sodium carboxymethyl cellulose (CMC) was supplied by Group Chemical Reagent Co., Shanghai, China. Pentaerythritol tetraacrylate (PETA) was purchased from Suzhou Changchunteng Import and Export Co., Suzhou, China.A scoured PET fabric , emulsifier TO-7 (1.0 g), ammonium persulfate (1.0 g), BA (10.0 g), St (3.0 g), and deionized water (33.0 g) were mixed by mechanical stirrer, namely emulsion solutions A (50.0 g). The requisite amounts of emulsion solutions A (33.0 g), F6 (20.0 g) were mixed by a mechanical stirrer, namely emulsion solutions B (53.0 g). The requisite amounts of deionized water (29.0 g) and ammonium persulfate (1.0 g) were mixed in a four-necked round-bottomed flask. The emulsion solutions A (17.0 g) that was placed in a dropping funnel was slowly released into the system at a temperature between 75–80 °C over a period of 30–40 min. Afterwards, emulsion solution B (53.0 g) was slowly dropped into the system at a temperature of 80–85 °C over a period of 90–100 min, with a mixture of shell monomers consisting of BA, St, and F6. Subsequently, the mixture was allowed to react for further 2 h. The core-shell latex particles of P[St-BA-F6] emulsion was obtained . The coaThe latex particles size data of latex particles were obtained from dynamic light scattering on a Malvern particle size .GPC (gel permeation chromatography) analysis was performed in THF (tetrahydrofuran)  while using a Viscotek TDA302  with a WL. M GPC solvent/sample module.−1 by KBr pellets technique and a resolution of 4 cm−1 was used to analyze the chemical structure of the dried latex particles.FTIR (Fourier transform infrared spectroscopy) analyses were obtained from Nicolet 5700  within the frequency range of 650–4000 cm65 and 10° observer).The following measurements were made in order to evaluate the aimed the printing PET fabric. Color yield measurements of the printed PET fabric were obtained from spectrophotometer . The spectrophotometer was set to exclude specular reflection and a large aperture .The rubbing fastness test was performed according to the standard (ISO 105-171 X12:2016) using a Model 670 type friction fastness machine .Electrostatic properties measurements of the sample were examined while using the electrostatic attenuation meter . Electrostatic voltage and static half period were obtained from the electrostatic attenuation meter. Fabric samples were conditioned in a standard atmospheric condition (35% ± 5% relative humidity and 20 ± 2 °C) for 24 h prior to the measurement.The PET fabric was washed 10 times with simulated domestic washing according to the AATCC (American Association of Textile Chemists and Colorists) Test method 135 under the condition of normal washing cycle at 27 ± 3 °C, followed by tumble drying process, in order to determine the durability of the anti-static printed PET fabric.Scanning electron microscopy  was used to examine the surface morphology of the PET fabric treated and untreated by P[St-BA-F6]. The magnification of the SEM is 1800 times.X-ray photoelectron spectroscopy  was used to examine surface chemical composition of fabric surface, using Al Ka radiation (hν = 1486.6 eV) operated at 14.0 kV and 200 W.2 atmosphere and a temperature of 50–600 °C at a heating rate of 10 °C/min. and a flow rate of 100 mL/min.Thermogravimetric analysis (TG) was conducted on PET fabric using a thermogravimetric analyzer . The TG tests were performed in N2 atmosphere on PET fabric. The experiments were conducted in a Perkin-Elmer PE8500 DSC calorimeter . DSC tests were performed in a temperature of −10 to +270 °C at a heating rate of 10 °C/min. and flow rate of 100 mL/min. The sample was heated at 270 °C for 5 min, cooled at a rate of 2 °C/min. to −10 °C during the crystallization process.The differential scanning calorimetry (DSC) experiments were conducted in a flowing NThe X-ray diffraction (XRD) experiments were conducted on PET fabric using X-ray diffraction . The samples were recorded with X-ray diffractometer in angle range 0°–60°.−1, which are assigned to the C=C stretching of the benzene ring. The peaks at 702 and 619 cm−1 are the C-H benzene ring stretching of St  composite was successfully fabricated through the oxidative polymerization of BA, St, and F6. ng of St ,27. The ng of BA . The peacohol F6 . The peaWe use experimental parameter, such as curing fixation temperature, curing fixation time, and P[St-BA-F6] concentration, in order to obtain durable anti-static properties of PET fabric using P[St-BA-F6] pretreatment.In comparison with untreated PET fabric , electroIn comparison with the untreated PET fabric , the K/SThe K/S of treated PET fabric using cyan, magenta, yellow, and black inks was clearly higher than that of the untreated PET fabric, it can be calculated that K/S increased by 25.30%, 33.25%, 40.47%, and 7.47%, respectively. The higher dry rubbing fastness grade was achieved with treated PET fabric using cyan, magenta, and black inks than that with untreated PET fabric. The wet rubbing fastness grade found to be the same for all of the CMYK inks on treated and untreated PET fabric.When the DSC scanning form 270 °C to −10 °C, the untreated PET fabrics enthalpy values, re-crystallization temperature were −50.65 J/g, 208.3 °C, respectively. The treated PET fabrics also did not exhibit any significant difference in enthalpy values (−50.14 J/g) and re-crystallization temperature (205.6 °C). This indicates that the initial reaction proceeded of re-crystallization more easily on the treated P[St-BA-F6] PET fabrics, it had lower enthalpy values, onset temperate, and peak temperate. In this study, the nanoscale P[St-BA-F6] core-shell particles that were prepared by way of seeded emulsion polymerization. The addition of P[St-BA-F6] enhanced the color yield and anti-static properties of polyester fabrics. The obtained latex has a mean size of 34 nm and an average of number molecular weight of 1794.5 with narrow size distribution. The FTIR analysis confirmed the successful fabrication of P[St-BA-F6] core-shell particles while using butyl acrylate, styrene, and ally alcohol polyethylene F6. The results showed that one-bath pretreatment of polyester fabrics with a P[St-BA-F6] and pentaerythritol tetracycline (PETA) is effective in improving the K/S value and the anti-static properties of the disperse dye inkjet Prints. The optimal pretreatment conditions are as follows: P[St-BA-F6] concentration is 30 g/L, curing fixation temperature is 190 °C, and treatment time was for 45 s.It was demonstrated that the P[St-BA-F6] treated PET fabrics exhibited higher K/S values, better dry rubbing fastness, and durable anti-static properties, even after 10 times washings. These results can be attributed to the alcohol polythene group (F6) and allyl group (PETA). The SEM measurements indicated that treated polyester fabric was coated with a thin resin film. XPS measurements indicated that PETA can be cross-linked with P[St-BA-F6] and PET fiber. XRD measurements showed the marginal influence on the crystalline structure of PET fabrics. The TG and DSC measurements showed the thermal stability of treated fabric decreased owing to existing resin film. The initial reaction proceeded of crystal melt was more difficult, but the initial reaction proceeded of re-crystallization was more easily compared with the untreated fabrics.


C-C chemokine receptor 5 (CCR5) has attracted wide concern for its critical role in the progression of human immunodeficiency virus type 1 (HIV-1) infection. Several studies have demonstrated that CCR5 affects the processes of tumor cell migration, invasion, and metastasis. The aim of this study was to illustrate the association between the polymorphisms of the CCR5 promoter and the development of cervical cancer.CCR5 promoter using a sequencing method.336 women with cervical intraepithelial neoplasia (CIN), 488 women with cervical cancer (CC), and 682 healthy controls were recruited to detect polymorphisms in the CCR5 promoter; the frequencies of the minor alleles of rs1799987 was significantly higher in the CIN group than that in the control group (P = 0.007); and the genotypic frequencies of rs2734648, rs1799987, rs1799988 and rs1800023 were significantly different between the CIN group and the control group (P < 0.008). The inheritance model analysis showed that rs2734648, rs1799987, rs1799988 and rs1800023 significantly increased the susceptibility to CIN in a recessive genetic model (P < 0.008). The haplotype constructed by the major alleles of these 6 SNPs (rs2227010-rs1799987-rs1799988-rs2734648-rs1800023-rs1800024: A-G-A-C-A-T) was highly protective against CIN . In addition, transcription prediction showed that mutation of these 6 SNPs might alternate the binding of particular transcription factors.Six loci with polymorphism were found in the CCR5 promoter polymorphisms were significantly associated with cervical intraepithelial neoplasia by altering the expression of CCR5 on the cell surface in a Chinese Han population.The  C-C motif chemokine receptor type 5 (CCR5), a transmembrane G-coupled cell-surface chemokine receptor, binds to five kinds of CC-chemokines: human macrophage inflammatory protein-1α (MIP-1α), MIP-1β, RANTES , monocyte chemotactic protein 2 (MCP-2) and MCP-4 .CCR5 gene located on 3p21.31, is composed of four exons and two introns. The promoter region has been described previously , with a value of 1 representing perfect disequilibrium. The D’ value over 0.8 indicated the existence of different loci in the LD. The differences in the haplotypes between the case and control groups were determined with the χ2-test. Power-analysis was performed using Power and Sample Size Calculations (version 3.1.2) [P value less than 0.008.All polymorphic loci were tested for deviation from the Hardy-Weinberg equilibrium in the control group with a threshold of 0.05. Association tests for allelic and genotypic frequencies, as well as dominant and recessive inheritance models of these SNPs among CIN, CC and control groups using the χn 3.1.2) . SignifiP > 0.05).There were 336 CIN patients and 488 CC patients compared with 682 normal women. The mean age was 48.122 ± 9.578 (ranged from 22 to 75) years in CIN group, 48.180 ± 9.655 (ranged from 24 to 73) years in the CC group, and 48.934 ± 7.390 (ranged from 33 to 79) years in the control group. No significant differences in age were found in the pairwise comparisons of CIN, CC, and control group , there are 9 identical single-nucleotide polymorphism (SNP) loci in the promoter of P > 0.05). The distribution of allelic and genotypic frequencies of these 6 SNPs among CIN, CC and control group are presented in Tables P = 0.007, OR = 1.289, 95%CI: 1.071–1.551, Table P < 0.008, Table All these 6 SNPs with polymorphism were in the Hardy-Weinberg equilibrium (HWE) in the control group (Our study had a power over 80% to detect and odds ratios (ORs) of 1.403 for rs2227010, 1.971 for rs2734648, 1.963 for rs1799987, 1.898 for rs1799988, 1.906 for rs1800023, and 1.661 for rs1800024, considering the sample size of cases and controls and assuming the minor allele frequencies of each SNP in the Han Chinese population of Yunnan.CCR5 promoter and cervical cancer development. The results showed that rs2734648, rs1799987, rs1799988 and rs1800023 significantly increased the susceptibility to CIN in the recessive inheritance model  was calculated, and the D’ value of these 6 SNPs was over 0.8, which indicated that these 6 SNPs of CCR5 promoter region were in LD.P = 1.42E-03). All 4 kinds of haplotypes showed no differences between the CC and control groups, or between the CIN and CC groups.Then, we constructed the haplotype of these 6 SNPs (rs2227010-rs1799987-rs1799988-rs2734648-rs1800023-rs1800024) and pairwise compared the haplotype frequencies among CIN, CC and control groups. Table http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) was used to predict the potential binding sites of transcription factors in the CCR5 promoter region [PROMO version 8.3  polymorphisms with cervical carcinoma and found that the promoter variations of CCR5 might be associated with development of cervical lesions, cervical intraepithelial neoplasia.Tumor development and progression depend heavily on the presence of angiogenesis factors including chemokines and their receptors . There hCCR5 promoters have been previously described: a weak upstream promoter called PU or P2, and a stronger downstream promoter called PD or P1 [CCR5 on cell surface is highly variable, and CCR5 promoter polymorphisms may alter the CCR5 cell surface expression, which might consequently influence the disease progression. Previously, several SNPs in the CCR5 promoter have been shown to affect CCR5 expression. Salkowitz et al found that rs1799987 (−2459G > A) located in the P1 was related to a different expression level of CCR5 on CD14+ monocytes, and individuals carrying homozygous rs1799987-G exhibited lower CCR5 density on CD14+ monocytes [CCR5 promoter polymorphisms correlate with HIV disease progression possibly by regulating CCR5 cell surface expression and CD4+ T cell apoptosis in HIV patients. In this study, rs1799987-AA significantly increased the risk of susceptibility to CIN, which indicated that rs1799987-AA might be a risk factor for cervical lesions development, possibly by influencing the expression level of CCR5.Two PD or P1 , 27. Theonocytes . Mummidionocytes  reportedCCR5 promoter haplogroups constructed by -2733A/G, −2554G/T, −2459G/A, − 2135 T/C, −2132C/T, −2086A/G, −1835C/T and + 554(Δ32), have been characterized and described before, and CCR5 haplogroups have been named as CCR5-HHA (A-G-G-T-C-A-C-Δ32wild), −HHB (A-T-G-T-C-A-C-Δ32wild), −HHC (A-T-G-T-C-G-C-Δ32wild), −HHD (A-T-G-T-T-A-C-Δ32wild), −HHE (A-G-A-C-C-A-C-Δ32wild), −HHF (A-G-A-C-C-A-T-Δ32wild), −HHG*1 (G-G-A-C-C-A-C-Δ32wild) and -HHG*2 (G-G-A-C-C-A-C-Δ32mutant) [CCR5 promoter haplogroup was demonstrated to correlate with transcriptional activity [CCR5 promoter, and only SNP rs2227010 was not comprised in the defined CCR5 haplogroups. Our results showed that haplotype H1: A-T-G-T-G-C, which was similar to CCR5-HHC, was the most-common haplotype in our participants and consisted of the major alleles of these 6 SNPs revealing a protective effect against CIN; and a tendency of susceptibility to CIN appeared in H2 (similar to CCR5-HHE), H3 (similar to CCR5-HHF) and H4 (similar to CCR5-HHA), although these haplotypes did not show statistical association with CIN. The haplotype analysis results coincided with the results of our allelic and genotypic analysis and indicated that the CCR5 promoter haplotype was associated with cervical lesions development. However the correlation between haplotype of 6 SNPs in this study and expression of CCR5 need to test and verify.2mutant) , 31. A sactivity . CCR5-HHCCR5 promoter and try to find the possible mechanism of association of SNPs to development of cervical lesions. The computer algorithms suggested that there are many binding sites of distinct nuclear and transcription factors in the CCR5 promoter, and the 6 SNPs studied were located in the binding region of specific transcription factors  could bind to four nuclear factor complexes (NF4-NF7), but the affinities for NF5-NF4 complexes bound to the -2554 T were significantly greater than the binding affinities to -2554G [CCR5 translation and CCR5 expression level on cell surface and, finally, affect the development of cervical lesions. By the way, our results revealed that the susceptibility of SNPs in CCR5 promoter arising more in recessive inheritance model, such as rs2734648, rs1799987, rs1799988 and rs1800023. That is to say individuals carried two mutations in both alleles raise the susceptibility to CIN. This can be understood as the efficiency of nuclear factors binding, CCR5 translation and expression could be affected more when two mutations in both alleles of SNP.In 2016, Che et al indicated that CCR5 mRNA and protein expression levels were extremely higher in cervical cancer tissue than in matched normal control tissue, and downregulation of CCR5 inhibited cervical cancer growth and invasion . Sales e4 site rs34648 couo -2554G  also fou4 site rs34648 cou4 site rs34648 couIn this study, we found the frequencies of the minor alleles of rs2734648 was significantly higher in the CIN group than that in the control group and the frequencies of rs2734648-GG, rs1799987-AA, rs1799988-CC and rs1800023-AA were distinctly increase the susceptibility of CIN. However, rare associations of susceptible to CC or from CIN progressing to CC were found. It is known that persisting high-risk HPV infection, which usually related to the lack of HPV-specific T-cell immunity and some immune-response pathway , 13, is CCR5 promoter are significantly associated with development of cervical intraepithelial neoplasia. These polymorphisms could potentially influence not only the CCR5 gene expression but also the CCR5 mRNA stability and the translation efficiency, which subsequently influence the density of CCR5 on cell-surface and, thus, have an impact on virus infection or disease pathogenesis. However, there might be some other mechanism of cervical cancer development, because our results indicated that polymorphisms in the CCR5 promoter are not associated with progression from CIN to CC. Thus, sample size expansion for association study and the functional study on correlation of CCR5 polymorphisms with cervical intraepithelial neoplasia development, and finding other immunogenetic factors associated with cervical carcinoma will be performed in the future.In conclusion, we demonstrated that SNPs in the


The microenvironment of peripheral nerve regeneration consists of multiple neurotrophic factors, adhesion molecules, and extracellular matrix molecules, secreted by unique glial cells in the peripheral nerve system (PNS)-Schwann cell (SCs). Following peripheral nerve injury (PNI), local IGF-1 production is upregulated in SCs and denervated muscle during axonal sprouting and regeneration. Regulation of IGF-1/IGF-1R signaling is considered as a potentially targeted therapy of PNI. We previously identified a group of novel miRNAs in proximal nerve following rat sciatic nerve transection. The present work focused on the role of miR-129 in regulation of IGF-1 signaling after sciatic nerve injury. The temporal change profile of the miR-129 expression was negatively correlated with the IGF-1 expression in proximal nerve stump and dorsal root ganglion (DRG) following sciatic nerve transection. An increased expression of miR-129 inhibited proliferation and migration of SCs, and axonal outgrowth of DRG neurons, which was inversely promoted by silencing of the miR-129 expression. The IGF-1 was identified as one of the multiple target genes of miR-129, which exerted negative regulation of IGF-1 by translational suppression. Moreover, knockdown of IGF-1 attenuated the promoting effects of miR-129 inhibitor on proliferation and migration of SCs, and neurite outgrowth of DRG neurons. Overall, our data indicated that miR-129 own the potential to regulate the proliferation and migration of SCs by targeting IGF-1, providing further insight into the regulatory role of miRNAs in peripheral nerve regeneration. The present work not only provides new insight into miR-129 regulation of peripheral nerve regeneration by robust phenotypic modulation of neural cells, but also opens a novel therapeutic window for PNI by mediating IGF-1 production. Our results may provide further experimental basis for translation of the molecular therapy into the clinic. Peripheral nerve injury (PNI) initiates a sequential response known as Wallerian degeneration, characterized by axonal degeneration and dedifferentiation of Schwann cells (SCs)2, which secrete trophic factors and provide the Büngner’s bands guiding axonal growth3. In addition, SCs remove axonal and myelin debris, and secrete cytokines and chemokines that recruit immune cells to the degenerating nerve to further eliminate cell debris4, thus speeding up axonal regeneration5. Therefore, cellular reactions to stress conditions generated by axonal injury will affect the regenerative capability and therefore functional recovery1.Axonal regeneration depends on the intrinsic growth capacity of neurons and the reaction of glial cells, which expresses proteins that inhibit or promote axonal regeneration8. The increased expression of insulin-like growth factors (IGFs) in the injured nerve has also been suggested to facilitate axonal regeneration after PNI9. The IGF system includes two ligands, their respective receptors, and a family of binding proteins that together regulate a variety of cellular responses10. Insulin-like growth factor 1 (IGF-1) is a polypeptide hormone with critical roles in peripheral nerve regeneration. Furthermore, IGF-1 exerts trophic effects on several different cell types in the nervous system, including spinal moto-neurons. Exogenous administration of IGF-1 promotes regeneration of motor axons after axonal lesions in the sciatic nerve and also acts on myelinating SCs and oligodendroglia11. A role in the regulation of motor nerve sprouting has been suggested by the observation that IGF-1 increases growth of neurite and branching from embryonic motoneurons and that administration of IGF-1 to adult muscle induces intramuscular nerve branching12. IGF-1 signaling pathways have also been associated with age-related neuronal dysfunction and neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease16. Rab8a regulates IGF-1 secretion in a GDP-bound form dependent manner17.The microenvironment of peripheral nerve regeneration consists of multiple neurotrophic factors, adhesion molecules, and extracellular matrix molecules, secreted by multiple cells including SCs in the peripheral nerve system (PNS). Growth factors play key roles in the regenerative microenvironment following PNI. In neural tissue engineering, different growth factors such as the glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and the vascular endothelial growth factor (VEGF) are the ones most commonly used in combination or individually18. MicroRNAs, as a potential way of target treatment, are post-transcriptional regulators of gene expression that may be crucial to age- and disease-related changes in growth factor functions19. Interestingly, our recent report suggested that the global deregulation of miRNAs in transected sciatic nerve axons and related dorsal root ganglions (DRGs)22 may influence the axonal regeneration. SCs proliferation and migration were specifically regulated by let-7d/mir-98 through targeting NGF in vitro and in vivo. The downregulation of let-7d stimulated SCs to increase NGF production, which further encouraged axon regrowth23. Recently, a functional role of miR-126 has been suggested to be involved in dopamine neuronal cell survival in models of Parkinson’s disease (PD)-associated toxicity19. However, the detailed mechanisms of regulation by microRNAs of IGF-1 dysfunctions in sciatic nerve regeneration are still not well understood.Owing to the very limited half-life of growth factors in vivo, complicated strategies, including novel carrier materials as microspheres and hydrogels, and other techniques as affinity-based delivery have been applied reluctantlyIn the present work, our data provided evidence for a novel mechanism of regulating IGF-1/IGF-1R signaling in neurons and SCs by miR-129 and suggest a functional role of this miRNA, broadly, in injured neurons, and the pathogenesis of PNI.24. All animals were then randomly divided into five groups (n = 6) according to different time points. The L4-6 DRGs and the 5-mm-long proximal stump segment in five different groups were collected at 0, 1, 4, 7, and 14 days after sciatic nerve transection, respectively. All the experimental procedures involving animals were conducted in accordance with Institutional Animal Care guidelines of Nantong University, China, and approved ethically by the Administration Committee of Experimental Animals, Jiangsu Province, China.Male Sprague–Dawley (SD) rats (220–250 g) were subjected to surgical transection of sciatic nerve as previously describedThe harvested L4-6 DRGs and sciatic nerve segment were fixed in 4% paraformaldehyde, embedded in 5% sucrose, and cut on a cryostat into 12-μm-thick sections, which were immunostained with primary antibodies: mouse anti-neurofilament (NF) 200 , rabbit anti-IGF-1 , and mouse anti-S100 β (1:100), followed by reaction with fluorescently-labeled secondary antibodies . Images were taken under fluorescence microscopy .26 with modifications. Briefly, the L4-L6 DRGs were removed from the adult SD rats, and transferred to Ca2+/Mg2+-free Hibernate A , where the axon roots and dural tissue were manually removed. The DRGs were then transferred to 0.1% collagenase type I . Following 1.5 h incubation at 37 °C, the DRGs were dissociated in 0.25% trypsin (Gibco) for an additional 15 min at 37 °C, and mechanically triturated through a pipette into the single cell suspension. To remove SCs, a partial purification step was performed by centrifugation at 900 rpm for 5 min on 15% BSA in PBS solution (Sigma). The obtained DRG neurons were cultured on the coated plates in Neurobasal-A and B-27 minus insulin (Gibco) supplemented with penicillin–streptomycin . The modulatory effects of IGF-1 on neurite outgrowth were treated with IGF-1  for 48 h. For the pre-lesion injury assay, we transected the sciatic nerve, waited 4 days, and then cultured adult DRG neurons for 72 h to evaluate their regeneration capacity.DRG neurons were dissociated and cultured as described previously27. The final cell preparation consisted of 99% SCs, as determined by immunostaining with S100 β, a specific SC marker. Primary culture of SCs was maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (complete medium) at 37 °C under humidified 5% CO2. The cell culture was passaged no more than three times before the following tests. Primary cultured SCs were transfected with miRNA mimic, miRNA inhibitor, or siRNAs , respectively, using Lipofectamine RNAiMAX transfection reagent (Invitrogen), according to the manufacturer’s instructions. A non-related, scrambled miRNA was used as a control, such as miRNA mimic control (NC) and inhibitor control (anti-NC). Mutated miR-129 mimic was used to confirm the specific effect by miR-129.SCs were isolated from sciatic nerves of 1-day-old SD rats and treated to remove the fibroblasts using anti-Thy 1.1 antibody  and rabbit complement  as previously describedTM vector  with the XhoI and NotI sites downstream of the hRluc reporter gene. This vector is based on dual-luciferase technology, with firefly Renilla luciferase (hRluc) used as the primary reporter to monitor mRNA regulation and luciferase (hluc) acting as a control reporter for normalization. Mutant 3′-UTR reporter plasmid was constructed with a QuikChange kit (Stratagene). Primers used to generate wild-type and mutant IGF-1 3′-UTR were as follows: IGF-1 3′-UTR wild-type: AATTCTAGGCGATCGCTCGAGGAGGAGCCTCCCGAGGAACAG, ATTTTATTGCGGCCAGCGGCCGCCCTAATTTTGTCCTTTTGGGCTC; IGF-1 3′-UTR mutant 1: GCAAGGTGCAAAGCTTTTTTTTTTTGTTTTTGAAAAACTTTTTTTTTTTTTTTTTTTTAACAAACACTCCTAAAGACAATGTCGGAATGTTTACTT, AAGTAAACATTCCGACATTGTCTTTAGGAGTGTTTGTTAAAAAAAAAAAAAAAAAAAAGTTTTTCAAAAACAAAAAAAAAAAGCTTTGCACCTTGC; IGF-1′-UTR mutant 2: CAGGACCACTTTTGCAAGGTGCAAAGCTTTTTTAAAAACTTTTTGTTTTTGTTTTTTTTTTTTTTTTTTTTAACAAACACTCCTAAAGACAAT, ATTGTCTTTAGGAGTGTTTGTTAAAAAAAAAAAAAAAAAAAACAAAAACAAAAAGTTTTTAAAAAAGCTTTGCACCTTGCAAAAGTGGTCCTG. The sequences of wild-type and mutant 3′-UTR were confirmed by sequencing. For reporter assays, HEK 293T cells were co-transfected with wild-type (mutant) reporter plasmid and miRNA mimic or scrambled control (NC) by Lipofectamine 2000 (Invitrogen). Firefly and Renilla luciferase activities were measured in cell lysates using the Dual-Luciferase Reporter Assay system. After 36 h incubation, the activity of firefly and Renilla luciferases was measured (Promega). Activity was reported by normalizing Renilla to firefly luciferase activity. Luciferase experiments were repeated three independent times in triplicate. Data are presented as means ± SEM.The whole 3′-UTR sequences of IGF-1 (NM_001082479) were amplified from the genomic DNA with appropriate primers, and sub-cloned into the pmiR-RB-REPORT−ΔΔCt method.Total RNA was isolated from tissues and cells using Trizol reagent (Invitrogen), and cDNA was prepared from total RNA using a Prime-Script RT reagent Kit  according to the manufacturer’s instructions. qRT-PCR was performed with SYBR Premix Ex Taq (TaKaRa) on an ABI system  according to standard protocols. The relative expression of miR-129 was quantified with stem-loop RT primers (Ribobio) according to manufacturer’s instructions and normalized against the U6 level. The sequences of IGF-1 primers are as follows: GACCAAGGGGCTTTTAC, TCAGATCACAGCTCCGG. All reactions were run three independent times in triplicate. The relative expression was calculated using the comparative 2Protein extracts were prepared from nerve tissues. Equal amounts of isolated protein were separated on 10% SDS-PAGE and transferred to PVDF membranes (Millipore), which were blocked with 5% nonfat milk in Tris–HCl buffered saline (TBS) at room temperature and probed with primary antibodies against IGF-1 (Proteintech) and IGF-1 Receptor (Abcam), respectively, and then reacted with an HRP-conjugated species-specific secondary antibody, followed by an enhanced chemiluminescence assay .Primary SCs were transfected with IGF-1 siRNA and negative control, miR-129 mimic and control, miR-129 inhibitor and control, respectively. Afterwards, the medium was replaced with FBS-free DMEM for additional incubation. The medium was then taken out and filtered through a 0.22 μm filter (Millipore) to furnish the supernatant. The protein level of IGF-1 in the medium was measured with the IGF-1 ELISA Kit (R&D Systems) according to the manufacturer’s instructions. The data were measured and averaged from three independent cultures, each comprising triplicate wells.5 cells/ml onto poly-L-lysine-coated 96-well plates. At the indicated time points after cell transfection, 50 mM EdU was applied to the cell culture which was then incubated for an additional 24 h. The cells were fixed with 4% paraformaldehyde in PBS for 30 min. After labeling, the cells were analyzed using a Cell-Light EdU DNA Cell Proliferation Kit (Ribobio) according to the manufacturer’s protocol. SC proliferation was expressed as the ratio of EdU-positive cells to total cells, which was determined using images of randomly selected fields obtained on a DMR fluorescence microscope (Leica Microsystems). Assays were performed three times using triplicate wells.SCs were plated at a density of 1 × 105 cells/ml) were resuspended in DMEM and transferred to the top chambers of each transwell to allow their migration in a humidified 5% CO2 incubator at 37 °C with 500 μl complete medium being pipetted into the lower chambers. The upper surface of each membrane was cleaned with a cotton swab at the indicated time point. Cells adhering to the bottom surface of each membrane were stained with 0.1% crystal violet and then counted under a DMR inverted microscope (Leica Microsystems). Assays were performed three times using triplicate wells.The migration ability of SCs was examined using 6.5 mm transwell chambers with 8 μm pores . The bottom surface of each membrane was coated with 10 μg/ml fibronectin. 100 μl Primary SCs , DRG neurons were isolated, and fixed with 4% paraformaldehyde to undergo immunocytochemistry with anti-β-Tubulin III antibody (Sigma) to observe axon outgrowth. For parallel experiment on SC migration, DRG neurons were transfected with miR-129 mimic and control, miR-129 inhibitor and control, respectively. Then SCs with IGF-1R knocking down previously were transferred to the top chambers of each transwell, and were co-cultured with transfected DRG neurons.n = 6 each) to receive injection of a mixture of Matrigel  with purified IGF-1 protein  or PBS vehicle only, and miR-129 inhibitor  or corresponding control (Ribobio) both at a volume ratio of 1:1, respectively. The injection was performed from the opposite opening of the silicone tube into the tube lumen using a precooled micropipette, followed by anastomosis of the tube to the distal nerve stump at the junction. The injection was done as slowly as possible to prevent the formation of air bubbles. Afterwards, the surgical incision was closed in a routine fashion, and animals were housed in large cages. At 10 days after surgery, rats were killed and the silicone tube, together with regenerated nerves, was harvested for cutting into sections, which were subjected to immunohistochemistry anti-NF200 and anti-S100β (both from Sigma), respectively. To assess the axons regeneration or the migration of SCs within the nerve gap, the edge of both proximal and distal nerve stump was labeled. Axons or SCs were identified as NF200- or S100β-positive cells, respectively. Then the length of regenerative axons from the proximal or SCs migration from the distal nerve stump was measured.Adult male SD rats were anesthetized before the sciatic nerve was exposed through an incision on the left hind limb and transected to create a gap. A silicone tube (i.d. 1.0 mm) was implanted to bridge the nerve gap with the proximal nerve stump anastomosed to the tube at the junction. The rats were randomly divided into four groups . Statistical significance was accepted at p value < 0.05.Data are presented as means ± SEM. The Student’s 12. Double immunofluorescence staining for S100β (a SCs marker) and NF200 (a neuron marker) were used to determine the cellular localization of IGF-1. The results showed that IGF-1 expressed in DRG neurons and SCs of the sciatic nerve  and a peak value occurred at 7 days after sciatic nerve transection Fig. .Western blot analysis showed that protein expression of IGF-1 was not significantly increased at 1 day after sciatic nerve transection, but was extensively increased at 7 and 14 days compared to that at 0 h, with a peak value at 7 days Fig. . The pro22 from mRNA and miRNA microarray assays in DRG tissue and the proximal nerve stump following sciatic nerve injury. It indicated that four miRNAs, including miR-129, miR-18a, miR-206, and miR-340-5p, might regulate the important nerve regeneration-associated gene IGF-1. To verify which one/ones of them were exact regulators, the wild-type and mutant 3′-UTR of IGF-1 sub-cloned into the luciferase reporter vector including single target site mutant (mut 1 and mut 2) and double target site mutant (mut 1&2) were constructed and inserted into the reporter plasmid  was injected into the silicone tube. Immunostaining with anti-NF200 or anti-S100β showing that both IGF-1 and miR-129 inhibitor significantly promoted axon outgrowth and SCs migration at 10 days after nerve injury, respectively Fig. , which d29. The function of miRNAs can be definitely influenced by miRNA agomirs or antagomirs that cause effective and precise upregulation or downregulation30. miRNA is easier to deliver into the target cells and less likely to induce high immune response and toxicity than other plasmid DNA-based gene therapy and protein-based drug molecules31. This study focused on the possible regulatory role of miR-129 targeting IGF-1 during peripheral nerve regeneration. The expressions of miR-129 in the injured sciatic nerve changed over time following sciatic nerve transection, which suggested that miR-129 might be involved in peripheral nerve regeneration.Growing interests of PNI have fueled exploration of therapeutic approaches that promote spontaneous nerve regeneration. The IGF system is an appealing target for the treatment of PNI based on its potent effects on neuronal development, physiology, and survival. Moreover, miRNAs have the profound potential as therapeutic agents due to their unique properties32. During the axonal regeneration, IGF-1 has been shown to play a critical role in SCs by promoting their survival, proliferation, maturation, and differentiation to myelinating phenotypes35. IGF-1 has been also reported to increase the survival and axonal outgrowth of neurons38. Our work firstly investigated the target regulation of miR-129 in affecting regenerative microenvironment to further regulate the proliferation and migration of primary SCs, and neurite outgrowth of DRG neurons.SCs are unique glial cells in the PNS and may secrete multiple neurotrophic factors, adhesion molecules, extracellular matrix molecules to form the microenvironment of peripheral nerve regeneration, guiding, and supporting axonal regeneration39. Although the favorable therapeutic impact of miRNA has been recognized, several challenges should be considered, including limitation of efficient miRNA delivery to specific cell types, tissues, and organs41. Since miRNA targets multiple mRNAs via incompletely matching with their 3′UTRs, miRNAs can also induce the off-target silencing31. We previously reported applications of miRNAs in treatment of PNI43. Briefly, a regenerative microenvironment in vivo was provided by bridging the rat sciatic nerve gap using the silicone tube, which was added with the mixture of Matrigel and miRNA agomir/antagomir. Further progress will be made in miRNA-therapeutics to regulate many aspects of human disease by preclinical and clinical development since the number of miRNAs targeting the IGF-1 signaling pathway increases29. To investigate the regulatory mechanisms of IGF-1, our data from a dual-luciferase reporter assay confirmed that IGF-1 was an exact target gene of miR-129 through directly binding to IGF-1 3′-UTR. Moreover, miR-129 was shown to inhibit IGF-1 expression due to IGF-1 mRNA degradation. Compared to that at 0 h (control), the differential expression levels of IGF-1 at mRNA and protein were more dramatic in the proximal nerve segment than that in DRGs at 4, 7, and 14 days following sciatic nerve injury. It indicates that SCs at the nerve injury site are the main source of IGF-1 and benefit to the regenerative microenvironment. Similarly, the decrease of miR-129 was less dramatic in DRGs than that in the proximal nerve segment. Collectively, IGF-1 was shown to be negatively regulated by miR-129 at post-transcriptional level. It indicates that the regulation of injury-induced secretion of IGF-1 by miR-129 plays crucial roles in the regenerative microenvironment following PNI. Exogenous addition of IGF-1 could significantly increase the neurite outgrowth of DRG neurons, whereas IGF-1-neutralizing antibody reduced the effect. Overexpression of miR-129 inhibited the neurite outgrowth, suggesting that miR-129/IGF-1 axis could regulate neurite outgrowth directly based on its autocrine effect. miR-129/IGF-1 axis is also important in the pre-lesion experiment and in vivo model, which would provide more clinical values regarding the application of IGF-1 or anti-miR-129 in patients with peripheral neural injury.Target therapy by miRNAs provides promising beneficial opportunities with several advantages over traditional small-molecule drugsFurthermore, IGF-1 knockdown prevented an increase in proliferation and migration of SCs by miR-129 inhibitors, which indicated that IGF-1 knockdown could recapitulate the suppressing effects of miR-129 on SCs phenotypic modulation. This interesting finding provided further evidence that IGF-1 was a functional mediator of miR-129.44, and Rab8a regulates IGF-1 secretion in a GDP-bound form dependent manner17. Moreover, what are the up-regulators of miR-129 following PNI? Following PNI, hypoxia is usually a consequence of lacking blood supply at the injury site. We previously reported the impact of hypoxia on SC behaviors by miRNAs during peripheral nerve regeneration43. In addition, angiotensin II (Ang II) encoded by AGT gene, exerts various pathophysiological effects in vasoconstriction, proinflammatory, growth-promoting, and vascular remodeling events. Ang II increases expression of rat miR-129 in cultured vascular smooth muscle cells45. Human IL17A protein may also increase expression of human miR-129 in cultured human dermal fibroblasts45.Besides the regulatory mechanism we have provided here, our knowledge regarding the regulation of IGF-1 secretion contains a calcium sensor synaptotagmin-10 that regulates activity-dependent IGF-1 secretion in olfactory bulb neuronsIn summary, IGF-1 was up-regulated both in DRGs and the proximal nerve segment following sciatic nerve injury. Dramatic change of IGF-1 in the proximal nerve segment suggested SCs is the main source of IGF-1 at the nerve injury site, which may further promote axonal regeneration. miR-129 negatively regulated IGF-1 by directly targeting its 3′-UTR. miR-129/IGF-1 axis not only regulated neurite outgrowth of DRG neurons directly, but also modified the regenerative microenvironment by regulating SCs proliferation and migration. Upregulation of IGF-1 secretion in SCs at the injury site by miR-129 could promote axonal regeneration indirectly Fig. . Overall