No other significant differences between Groups for any mfERG parameters were found. All Sd-OCT measurements, apart from the inner retina macular volume in the central 1 mm, were significantly reduced in MS-ON-P patients compared to Controls. The functional impairment in the MS-ON-P Group was associated but not correlated with structural changes of the outer and inner retinal layers in corresponding retinal Areas and Sectors. Our results suggest that in MS, exclusively after ON with poor recovery of BCVA, the neurodegenerative process can induce dysfunctional mechanisms involving photoreceptors and bipolar cells of the fovea and of the more central nasal macular area.The involvement of macular preganglionic elements’ function, during the neurodegenerative process of multiple sclerosis (MS), is controversial. In this case-control observational and retrospective study, we assessed multifocal electroretinogram (mfERG) responses from 41 healthy Controls, 41 relapsing-remitting MS patients without optic neuritis (ON) (MS-noON Group) and 47 MS patients with ON: 27 with full recovery of high-contrast best corrected visual acuity (BCVA) (MS-ON-G Group) and 20 with poor recovery (between 0.2 and 1 LogMAR) of BCVA, (MS-ON-P Group). In the latter Group, Sd-OCT macular volumes and thicknesses of whole and inner and outer retina were measured. MfERG N1 and P1 implicit times (ITs), and N1-P1 response amplitude densities (RADs), were measured from concentric rings (R) with increasing foveal eccentricity: 0–5° (R1), 5–10° (R2), 10–15° (R3), 15–20° (R4), 20–25° (R5), and from retinal sectors  between 0–15° and 0–25°. In the MS-ON-P Group, mean mfERG RADs detected from R1 (0–5°) and from the central nasal sector (0–15°) were significantly reduced ( Multiple sclerosis (MS) is a neurodegenerative disease, characterized by chronic demyelination of the central nervous system, which can result in visual system involvement including retrobulbar optic neuritis (ON) .The ON event is followed by secondary neurodegenerative processes for retrograde trans-synaptic degeneration  that invAt the present, it is a debated topic to understand whether the neurodegenerative mechanisms occurring in MS, could involve retinal structures beyond the IML towards the preganglionic elements  located in the outer and in middle retinal (O-MR) layers.The function of preganglionic elements can be assessed by electroretinogram (ERG) recordings  that, wiIn MS patients, the Ff-ERG cone a- and b- waves’ amplitudes have been found reduced ,16,17,18Regarding the mfERG responses in MS, contrasting data have been reported in the recent literature: in fact, mfERG signals have been found either abnormal ,20,21 orAll this contrasting electrophysiological evidence led us to consider that there are no conclusive findings on whether there is or not an O-MR layers dysfunction or functional expression of the extended neurodegenerative process beyond IML in MS.Therefore, to add information to the debated topic of preganglionic functional involvement or sparing from neurodegeneration, the aim of our work was to assess the function of preganglionic elements in MS patients with the absence or presence of a history of ON, followed by good or poor recovery of the best corrected visual acuity (BCVA).We attempted to determine whether an O-MR dysfunction could be detected in the central macular area, or whether it might affect more peripheral retinal regions. In addition, we investigated whether the possible O-MR involvement could be observed in specific sectors (Superior (S), Nasal (N), Inferior (I), Temporal (T)) of the central macular region (0 to 15 degrees) and/or in more eccentric retinal areas within the vascular arcades (0 to 25 degrees).In addition, a morphological involvement of the outer macular layers in MS patients with history of ON was described , but witAll research procedures described in this work adhered to the tenets of Declaration of Helsinki. The study protocol (CEC/795/14) was approved by the local Ethical Committee  and upon recruitment, informed consent after full explanation of the procedure was obtained from each subject enrolled in the study.Eighty-eight relapsing remitting (RR) MS patients were enrolled at the Visual Neurophysiology and Neurophthalmology Research Unit, IRCCS- Fondazione Bietti referred by the Multiple Sclerosis center of the Tor Vergata University Hospital in Rome, between September 2016 and 20 October 2020.n = 342) based on the following demographic and clinical characteristics:Age between 28 and 45 years;Diagnosis of RR MS according to validated 2010 McDonald criteria ;MS disease duration (MS-DD), estimated as the number of years from onset to the most recent assessment of disability, ranging from 5 and 15 years;Expanded Disability Status Scale (EDSS), as ten-point disease severity derived from nine ratings for individual neurological domains , rangingTreatment with disease-modifying therapies (DMT) currently approved for preventing MS relapses. DMT considered in our study were Interferon-β-1a, Interferon-β-1b, Peginterferon beta-1a, Glatiramer acetate, Natalizumab, Dimethyl fumarate and Teriflunomide ;Absence of ON, or a single episode of ON without recurrence, that elapsed from the onset of the disease at least 12 months (ranging from 13 to 20 months) before the inclusion in the study. For MS patients with ON, this criterion was chosen, since it is known that the retrograde degeneration following ON occurs over a period of 6 months . When anBased on the ophthalmological examination, other inclusion criteria were: mean refractive error (when present) between −3.00 and +3.00 spherical equivalent; intraocular pressure less than 18 mmHg, absence of glaucoma or other diseases involving cornea, lens , uvea, retina; BCVA between 0.0 and 1.0 LogMAR of the Early Treatment of Diabetic Retinopathy (ETDRS) charts; absence of central visual field defects and ability to maintain a stable fixation that allowed performing multifocal ERG (see below); absence of other systemic diseases  that may influence the retinal function.In order to obtain homogeneous MS Groups  the MS patients were selected form a large cohort , providing 41 normal eyes, with BCVA of 0.0 LogMAR (mean 0.0 ± 0.0), served as Controls.The selected MS patients were divided into two Groups for age, MS-DD, EDSS and for previous history of presence or absence of ON.The 41 MS patients  were without history of unilateral or bilateral clinical signs of ON  and high-contrast BCVA of 0.0 LogMAR (mean 0.0 ± 0.0). When both eyes met the inclusion criteria, only one eye was randomly chosen for the study. Therefore, we considered 40 eyes from 40 MS patients without ON (MS-noON Group).The 47 MS patients  were with previous history of unilateral or bilateral ON . They were further divided in to two Groups on the basis of the recovery of BCVA after ON:The 27 MS patients  were with previous history of a single unilateral or bilateral ON and with “good” recovery of high-contrast BCVA  after ON. Therefore, we considered 27 eyes from 27 MS patients with ON (MS-ON-G Group);The 20 MS patients  were with previous history of a single unilateral or bilateral ON with “poor” recovery of high-contrast BCVA  after ON, and reduced P-ERG amplitude with respect to our normative data collected in healthy subjects . TherefoBased on the previous mentioned inclusion criteria, the MS Groups with or without ON were homogeneous for age, MS-DD, EDSS and the MS Groups with ON were homogeneous for number of ON and for the time elapsed from ON. All groups were similar for male/female ratio  and white (200 cd/m2) according to a binary m sequence. This resulted in a contrast of 99%. The luminance of the monitor screen and the central fixation cross (used as target) was 100 cd/m2. The visual stimulation was performed by correcting BCVA for the distance of the visual stimuli. The m-sequence had 213-1 elements, and total recording time was approximately 8 min. Total recording time was divided into sixteen segments. Between segments, the subject was allowed to rest for a few seconds. Focusing lenses were used when necessary. To maintain a stable fixation, a small red cross target (0.5 degree) was placed in the center of the stimulation field. At every mfERG reported that he/she could clearly perceive the fixation target. The eye’s position was continuously monitored by an in-built video system to track fixation losses.The mfERG was recorded by using a modified version of Espion system  according to our previously published method ,30,31 foMfERGs were binocularly recorded in the presence of pupils that were maximally pharmacologically dilated with 1% tropicamide to a diameter of 7–8 mm. Pupil diameter was measured by an observer (LuB) by means of a millimeter ruler and a magnifying lens and stored for each tested eye. The cornea was anaesthetized with Benoxinate eye drops 0.4%. MfERGs were recorded between an active Dawson–Trick–Litzkow (DTL) contact electrode and a reference electrode (Ag/AgCl skin electrode placed on the correspondent outer canthi). A small Ag/AgCl skin ground electrode was placed at the centre of the forehead. Interelectrode resistance was <3 KOhms. After automatic rejection of artefacts and post-acquisition processing done by the in-built Espion software, the first-order kernel response was examined. MfERG responses with a signal to noise ≥3 were accepted for the analysis.2 (ηV/degree2).In the analysis of mfERG responses, we considered, for each obtained averaged response, the implicit times (ITs) of the first negative peak (N1) and the first positive peak (P1) measured in milliseconds (msec) and the N1-P1 peak-to-peak response amplitude density (RAD) measured in nanoVolt/degreeRing analysis: the averaged response obtained from five concentric annular retinal areas (rings) centered on the fovea: from 0 to 5 degrees , from 5 to 10 degrees , from 10 to 15 degrees , from 15 to 20 degrees  and from 20 to 25 degrees  .Sector analysis 1: the averaged bioelectrical response obtained from the central macular region up to 15 degrees (0–15 degrees) sectioning it in four sectors: superior (S1-S), nasal (S1-N), inferior (S1-I) and temporal (S1-T) with respect to the fovea. In each sector, we included also the responses obtained from the more central macular area (0–5 degrees) .Sector analysis 2: the averaged bioelectrical response obtained from the retinal area from the fovea up to 25 degrees (0–25 degrees) sectioning it in four sectors: S2-S, S2-N, S2-I and S2-T with respect to the fovea. In each sector, we included also the responses obtained from the more central macular area (0–5 degrees) .We considered three possible retinal topographies to explore the bioelectrical responses derived from specific retinal areas. Data were analyzed as follows:In all MS-ON-P patients, the macular morphology was evaluated by the RTVue-100 Sd-OCT device, following our recently published method .(1)the 1 mm central area (2)the middle 1–3 mm ring ,(3)the external 3–6 mm ring ,(4)the whole 6 mm area .Segmentation analysis was performed in order to measure the macular volume (MV) and macular thickness (MT) of whole, inner and outer retinal layers  from concentric areas corresponding to the ETDRS topographical map:We also performed a sectorial segmentation analysis of the S, T, I and N sectors within 6 mm .This allowed to compare the electrophysiological data to the morphological ones from corresponding localized retinal areas .Therefore, we considered WR, IR and OR MVs and MTs measured in Area 1 corresponding to mfERG R1, in Area 2 corresponding to mfERG R2, in Area 3 corresponding to mfERG R3 and in Area 1 + Area 2 + Area 3, corresponding to mfERG R1 + R2 + R3. Accordingly, we also compared WR, IR and OR MV and MT values from S, T, I, N sectors to the corresponding mfERG data from Sector analysis 1 (see above).We assumed a Gaussian distribution of our data. The normal distribution was assessed by using the Kolmogorov-Smirnov test.The differences of age, MS-DD, EDSS between MS-noON, MS-ON-G and MS-ON-P Groups were evaluated by the one-way analysis of variance (ANOVA). The differences of the number of ON and the time elapsed from the ON between MS-ON-G and MS-ON-P Groups were evaluated by the ANOVA.Considering each different mfERG retinal topography , the differences of mfERG N1 and P1 IT and N1-P1 RAD mean values between Controls, MS-noON, MS-ON-G and MS-ON-P Groups were evaluated by ANOVA. In addition, mean values of segmented MV and MT from all Areas and Sectors within 6 mm detected in MS-ON-P Group were compared to those of Controls by ANOVA. In MS-ON-P Group, Pearson’s test was used to linearly correlate the values of BCVA with those of mfERG parameters and to correlate the individual mfERG values with the segmented MV and MT ones from corresponding retinal Areas and Sectors.p = 0.05/number of comparison: 0.05/6 = 0.0082. Therefore, we rounded up to a p-value lower than 0.01 to be considered as statistically significant. Minitab 17 (version 1) software was used for statistical analysis.Since for each considered mfERG and OCT parameter, a multiple comparison between Groups  was performed, the value of statistically significance was calculated by: In Examples of averaged mfERG recordings from five rings , obtained in representative Control (#7), MS-noON (#34), MS-ON-G (#22) and MS-ON-P (#12) eyes, are presented in In p > 0.01) differences between all Groups were found.On average, when we considered the mean values of N1 and P1 IT obtained in the central retinal areas  and in the more peripheral retinal areas , not statistically significant (p > 0.01) different with respect to those of Controls. In MS-ON-G Group, the mean values of N1-P1 RAD were not significantly (p > 0.01) different when compared to those of Control and MS-noON Groups; by contrast, in MS-ON-P Group, the mean values of N1-P1 RAD were significantly (p < 0.01) reduced with respect to the ones from Control, MS-noON and MS-ON-G Groups; the reduction of the individual N1-P1 RADs were not significantly correlated (p > 0.01) with the corresponding values of BCVA.The mean values of N1-P1 RAD obtained in the most central retinal areas  in MS-noON Group were not statistically (p > 0.01) different with respect to those of Controls, and not statistically significant (p > 0.01) differences were found between MS Groups.In MS-noON, MS-ON-G and MS-ON-P Groups, the mean values of N1-P1 RAD obtained in the other areas  were not statistically (Examples of averaged mfERG recordings from four sectors superior (S1-S), temporal (S1-T), inferior (S1-I) and nasal (S1-N) within 15 degrees of foveal eccentricity, obtained in representative Control (#7), MS-noON (#34), MS-ON-G (#22) and MS-ON-P (#12) eyes, are presented in Sector analysis 1 reports the averaged values of N1 and P1 IT and of N1-P1 RAD obtained from four macular areas enclosed between 0 and 15 degrees with respect to the fovea on the basis of the retinal topography: superior (S1-S), temporal (S1-T), inferior (S1-I), nasal (S1-N). The bioelectrical responses obtained from the central 0–5 degrees were enclosed in the sector analysis 1.In p > 0.01) differences between all Groups were found.On average, when we considered the mean values of N1 and P1 IT obtained in the central sectors  not statistically significant (p > 0.01) different with respect to those of Controls.The mean values of N1-P1 RAD obtained in these sectors in MS-noON Group were not statistically (p > 0.01) different when compared to those of Control and MS-noON Groups. By contrast, in MS-ON-P Group, while mean values of N1-P1 RAD detected in S1-I, S1-T and S1-S were not significantly (p > 0.01) reduced with respect to Control, MS-noON and MS-ON-G ones, a significant (p < 0.01) reduction of N1-P1 RADs in the S1-N sector was observed as compared to Controls, MS-noON and MS-ON-G Groups.In MS-ON-G Group, the mean values of N1-P1 RAD from all four sectors were not significant (p > 0.01) with the corresponding values of BCVA.The individual reduced N1-P1 RAD values from S1-N sector in MS-ON-P eyes were not significantly correlated  within 25 degrees of foveal eccentricity in representative Control (#7), MS-noON (#34), MS-ON-G (#22) and MS-ON-P (#12) eyes are presented in Sector analysis 2 reports the averaged values of N1 and P1 IT and of N1-P1 RAD obtained from four retinal areas from 0 to 25 degrees based on the retinal topography: superior (S2-S), temporal (S2-T), inferior (S2-I), nasal (S2-N), with respect to the fovea. The bioelectrical responses obtained from the central 0–5 degrees were enclosed in the sector analysis 2.The mean values of N1 and P1 IT and of N1-P1 RAD detected in the 4 sectors  in Control, MS-ON and MS-noON Groups and the relative statistical analysis between Groups are reported in p > 0.01) different when compared with those of Controls. In MS-ON-G and MS-ON-P Groups the mean values of N1-P1 RAD from all four sectors were not significantly (p > 0.01) different when compared to those of Control and MS-noON Groups. Furthermore, not statistically significant differences (p > 0.01) were found when mean N1-P1 RADs were compared between MS-ON-G and MS-ON-P Groups in all sectors.On average, the mean values of N1 and P1 IT and of N1-P1 RAD detected in all sectors  in MS-noON, MS-ON-G and MS-ON-P Groups were not statistically  and MS-ON-P (#12) eyes are presented in p < 0.01) between these Groups for all structural values but the IR MV from Area 1.In p < 0.01) reduction of all morphological parameters from each Sector in MS-ON-P with respect to Controls.In p > 0.01) linear correlations between the reduced mfERG RADs from Ring 1 with the reduction of OR MV and MT from Area 1. No other significant correlations between mfERG parameters from other Rings with Sd-OCT values from corresponding retinal areas were found.In MS-ON-P patients, we found not significant  or more peripheral retina within the arcades (0 to 25 degrees), topographically distinguished in rings or sectors. Our results apply to MS Groups with or without ON highly homogeneous for age, MS-DD, EDSS, and when present for number of ON and for the time elapsed from ON to the BCVA and mfERG assessment, differently from all previous reported studies in the literature ,20,21,22In addition, since a morphological impairment of macular OR has been described in MS-ON patients  and in op < 0.01) differences in MS-ON-P Group compared to Controls, MS-noON and MS-ON-G only when analyzing responses from Ring 1 (0–5 degrees) and from the S1-N sector, which covers the 0–15 central degrees area. In all other examined central or peripheral rings or sectors, we did not find any significant difference in the values of N1-P1 RAD between Groups. Our results indicate that photoreceptors and bipolar cells of the central fovea, as well as of the more central nasal macular sector within 15 degrees, are functionally impaired in MS only in occurrence of ON and when full recovery of BCVA is not achieved. These results do not apply either to MS-noON nor MS-ON-G Groups, thus confirming that the preganglionic element dysfunction is independent from the event of ON in itself.Our mfERG findings showed not statistically significant differences of N1 and P1 IT values in all Groups  in any considered central circular areas  or sectors  and more peripheral circular areas  or sectors  either when responses were compared to Controls or with MS Groups. As for N1-P1 RAD values, we found statistically significant , we found, in agreement with our previous work , a signiThese observed reduced R1 RAD values let us consider that when BCVA recovery after ON is poor, the wiring of retinal circuitry in the fovea, where the cones and the RGCs have the highest density ,38, can All these findings could have different explanations. First, the absence of a perfect anatomical overlapping between the stratified measurements by mfERG and Sd-OCT assessments. For instance, when segmenting IR and OR layers, our RTVue-100 device software automatically divides the inner and outer neurosensory retinas at the boundary between the inner nuclear layer (INL) and the outer plexiform layer (OPL). The OR encloses the OPL, the outer nuclear layer, and the photoreceptor layer. The IR examines the retinal nerve fiber layer (RNFL), the ganglion cells/inner plexiform layer (GC/IPL), and the INL. On the other hand, the mfERG system allows us to record the bioelectrical activity driven mainly by cones and bipolar cells, specifically mfERG response amplitude values are more correlated with photoreceptors activity whereas peak timing is more associated with the contribution to the signal by bipolar cells . Thus, iBy contrast, since in the more peripheral areas (Area 2 and Area 3) we detected normal mfERG responses (ITs and RADs), but reduced WR, IR and OR MVs and MTs, it should be hypothesized that this morphological involvement is not sufficient to induce functional changes, as suggested by the lack of correlation between mfERG and Sd-OCT data, as reported in In these patients, the observed macular functional changes were not significantly related with the reduced BCVA, as well as we recently reported  that theOur findings of abnormal mfERG responses specifically in the S1 nasal sector links with Boquete et al.  findingsMoreover, all sectorial WR, IR and OR MVs and MTs in MS-ON-P eyes were significantly reduced as compared to Controls. To our knowledge, no previous reports described similar investigations on Sd-OCT macular sectors. These morphological findings can suggest that in MS-ON-P eyes there is not a prevalent structural involvement of one macular sector with respect to others.Nevertheless, this morphological impairment cannot influence the functional condition of the S, T and I sectors (that was not significantly different from Controls), as suggested by the lack of correlation between mfERG and Sd-OCT data see . In addiThe biological mechanisms underlying the reduction of RADs in our selected group of MS-ON-P, with no previous or present signs of retinal inflammation, can only be hypothesized.One hypothesis is that in a sub-set of MS-ON patients, a dysfunction of photoreceptors and inhibitory bipolar cells (leading to reduced mfERG RADs) is due to trans-synaptic retrograde degeneration distal to IML. Indeed, the injury that involves the IML  couA second hypothesis that can explain the reduction of mfERG RADs in MS-ON-P patients is a process related to autoimmunity. For instance, in some MS patients with autoantibodies against the retinal protein α-enolase, a reduction of ERG responses has been found . In addiOf course, all previous electrophysiological studies done by recording Ff-ERG or flicker ERG in MS eyes, and almost unanimously finding subnormal cone-driven bipolar cell function ,22,51, aIn conclusion, in our study we detected an absence of mfERG abnormalities in MS patients without and with ON followed by full recovery of BCVA. Thus, our results suggest that in MS the function of preganglionic elements located in the O-MR layers is not modified by the occurrence of ON itself. By contrast, the MS neurodegenerative processes could induce a dysfunction of the preganglionic elements of the fovea and the retinal nasal sector after an event of ON followed by permanent impairment of visual acuity (poor recovery of BCVA after ON). This functional impairment was associated, but not correlated, with OR and IR structural changes. In order to better understand the role of middle retinal elements in this process, further studies on both experimental ,48 and c


Rheinheimera sp. D18, was isolated from the sea water of an industrial maricultural system in the Yellow Sea, China. Whole-genome sequencing of D18 revealed the presence of a novel 25.8 kb antibiotic resistance island, designated GEI-D18A, which carries several antibiotic resistance genes (ARGs), including aadA1, aacA3, tetR, tet(B), catA, dfrA37, and three sul1 genes. Besides, integrase, transposase, resolvase, and recombinase encoding genes were also identified in GEI-D18A. The transferability of GEI-D18A was confirmed by mating experiments between Rheinheimera sp. D18 and Escherichia coli 25DN, and efflux pump inhibitor assays also suggested that tet(B) in GEI-D18A was responsible for tetracycline resistance in both D18 and the transconjugant. This study represents the first characterization of a mobilizable antibiotic resistance island in a species of Rheinheimera and provides evidence that Rheinheimera spp. could be important reservoirs and vehicles for ARGs in the Yellow Sea area.Aquatic environments act as reservoirs of antimicrobial-resistant bacteria and antimicrobial resistance (AMR) genes, and the dissemination of antibiotic resistance from these environments is of increasing concern. In this study, a multidrug-resistant bacterial strain, identified as  Then, one of the single colonies was picked and streaked onto LB solid medium containing tetracycline in order to obtain a pure culture. After further purification, a single colony was selected and grown as a pure culture, which was named strain D18. To identify the species of strain D18, a partial fragment of 16S rDNA was amplified with the primer pair 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), and then DNA sequencing was performed for preliminary identification.Rheinheimera species  were selected from the NCBI FTP site. Moreover, the 16S rRNA gene sequence from Gallaecimonas pentaromativorans CEE 131 was included as an outgroup in phylogenetic analysis. The phylogenetic tree was inferred by using the maximum likelihood method based on the Tamura-Nei model  server . The quality of the genomic DNA sample was analyzed using a NanoDrop spectrophotometer . The genome of D18 was sequenced and assembled with a PacBio RS II platform and an Illumina NextSeq platform at BGI Co., Ltd. . Genome annotation was performed using the Prokaryotic Genome Annotation Pipeline (PGAP) on NCBI) server  and usin) server . Genomic) server . To dete) server . IS tran) server . The int) server . Sequencsoftware  was usedRheinheimera sp. D18 to other bacteria by horizontal gene transfer (HGT), mating experiments between D18 and the sodium azide-resistant E. coli 25DN strain (derived from E. coli ATCC 25922) were performed as previously described , and cultured on LB solid medium containing tetracycline, sodium azide and X-Gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid) to screen the transconjugants. D18 (donor) and E. coli 25DN (recipient) are inhibited by sodium azide and tetracycline, respectively, and only the transconjugants of E. coli can survive on the selective medium and degrade X-Gluc to blue compounds. The episomal form of GEI-D18A in Rheinheimera sp. D18, and GEI-D18A in the transconjugants were verified by PCR  guidelines  and verapamil were used as efflux pump inhibitors at final concentration of 0.1 and 80 mg/L, respectively . E. coliRheinheimera (Rheinheimera (class Gammaproteobacteria) as a separate subline adjacent to Rheinheimera baltica. Rheinheimera sp. D18 exhibited 96.84% 16S rDNA nucleotide sequence identity with the R. baltica type strain OSBAC5.Strain D18 was isolated from the seawater of an industrial mariculture system in the Yellow Sea, China. The phylogenetic tree based on the 16S rRNA gene sequence shows that strain D18 is within a subcluster of the genus nheimera . D18 posRheinheimera sp. D18 was sequenced, and the general features of the complete genome sequence were analyzed ]; sulfonamides ; chloramphenicol/florfenicol ; aminoglycoside ; macrolides (macA); penicillin (lpoA); fosmidomycin (hdN1), and antibiotic efflux pump encoding genes. The identification of numerous ARGs partially illuminated the basis for the extreme multidrug resistance trait of strain D18. It is worth noting that strain D18 had extremely high resistance to tetracycline (MIC ≥ 160 mg/L). Notably, only one known tetracycline resistance gene, tet(B), which encodes the tetracycline resistance major facilitator superfamily (MFS) efflux pump (E0Z06_RS15040), and the tetR gene, encoding the TetR-family transcriptional regulator, were predicted in the genome of D18. We hypothesize that TetB efflux pump is major factor in the high level of tetracycline resistance of Rheinheimera sp. D18.Dozens of ARGs were identified in the D18 genome , includiRheinheimera sp. D18 genome, but not other sequenced strains of the genus Rheinheimera. Sequence examination further suggested that the integration of GEI-D18A occurred within the intergenic sequence between E0Z06_RS15010 and E0Z06_RS15195 and that it resulted in the 12-bp direct repeat (DR) GATCTGCANNAA flanking GEI-D18A. Thirty-six ORFs were identified in GEI-D18A by genomic annotation, including night ARGs associated with tetracycline [tetR and tet(B)], sulfonamide (three sul1 genes), aminoglycoside (aadA1 and aacA3), trimethoprim (dfrA37) and chloramphenicol/florfenicol (catA) resistance. In addition, the three transposase encoding genes E0Z06_RS15145, E0Z06_RS15170 and E0Z06_RS15180; two integrase encoding genes, E0Z06_RS15085 and E0Z06_RS15165; one DDE-type integrase/transposase/recombinase encoding gene E0Z06_RS15015; one recombinase encoding gene E0Z06_RS15195; one resolvase encoding gene E0Z06_RS15010; and two ATP-binding protein TniB (involved in transposition) encoding genes, E0Z06_RS15020 and E0Z06_RS15105, were identified in GEI-D18A, suggesting that GEI-D18A can potentially transfer into other bacteria via horizontal gene transfer. Further sequence comparisons revealed that partial genes in GEI-D18A were highly similar with those of Escherichia coli AR436, Escherichia coli plasmid NR1 and Shewanella xiamenensis T17 plasmid pSx1  were carried out. The transconjugants were selected on tetracycline plates, and results revealed that the transconjugation frequency between D18 and 25DN was 6.4 × 10–7 CFU/donor. One of the transconjugants was isolated and named 25D18-T2. MIC testing revealed that the MIC for tetracycline in the transconjugant 25D18-T2 was 72 mg/L, much weaker than that of the donor strain D18 (160 mg/L) but much higher than the MIC for the recipient strain 25DN  and ain 25DN . In addi sp. D18 , indicatRheinheimera sp. D18 revealed the presence of only one tetracycline efflux pump encoding gene, tet(B). The efflux pump TetB belongs to the major facilitator superfamily , reduced the MIC for tetracycline from 160 to 50 mg/L for strain D18. CCCP also reduced the MIC for tetracycline from 72 to 24 mg/L for strain 25D18-T2 (tet(B) gene in GEI-D18A.Resistance to tetracycline can be associated with efflux pumps, ribosomal protection systems and/or other mechanisms . The whoerfamily . TetB iserfamily . In this25D18-T2 . As showIn addition to plasmid-mediated transformation and conjugation, and phage-mediated transduction, GEIs are an important means of bacterial HGT . GEIs arRheinheimera sp. D18 harbors only a circular chromosome, and no plasmids or phages were identified. Nevertheless, mating experiments revealed that tetracycline, florfenicol, amikacin, and sulfonamide resistance genes in Rheinheimera sp. D18 could be transferred to E. coli 25DN via HGT. Further analysis of the D18 genome sequence identified a novel antibiotic resistance island, which we named GEI-D18A. In addition to ARGs, GEI-D18A also contains genes encoding transposases, recombinases, resolvase, and transposition proteins  flanking GEI-D18A as E. coli 25DN harbors dozens of this above consensus sequence in genome. In addition, GEI-D18A may also be transferred from Rheinheimera sp. D18 to E. coli 25DN through other mechanisms that remain to be investigated.Antibiotic resistance genes can be transferred between bacterial strains by plasmids or phage. However, proteins , whereasproteins  in the Dproteins . Since tmolecule . Then, tsul1 gene is usually located in a class 1 integron, which is considered to be the main mobile genetic element to transfer sul1 among strains in the Yellow Sea  encoding genes. Mating assays demonstrated that GEI-D18A could be transferred from Rheinheimera sp. D18 to E. coli 25DN, and we propose that this transfer may have occurred via a type IV secretion system. This study identifies the first mobilizable antibiotic resistance island in a Rheinheimera species. However, the mechanisms by which GEI-D18A can be transferred to other bacterial strains remain to be investigated.In conclusion, the whole-genome sequencing of the multidrug-resistant Rheinheimera sp. D18 was deposited in GenBank under accession No. CP037745. The 16S rDNA sequence of strain D18 was submitted to GenBank and was assigned accession No. MN726625.The datasets generated for this study can be found in the whole genome sequence of GC designed the work and revised the manuscript. JF executed the experiments, manuscript preparation and submission. PZ and ML carried out the interpretation of data. CZ contributed to the experimental design. GZ helped in data analysis. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Klebsiella pneumoniae is a nosocomial pathogen, pointed out by the World Helth Organisation (WHO) as “critical” regarding the highly limited options of treatment. Lipopolysaccharide  and capsular polysaccharide (K-antigen) are its virulence factors and surface antigens, determining O- and K-serotypes and encoded by O- or K-loci. They are promising targets for antibody-based therapies (vaccines and passive immunization) as an alternative to antibiotics. To make such immunotherapy effective, knowledge about O/K-antigen structures, drift, and distribution among clinical isolates is needed. At present, the structural analysis of O-antigens is efficiently supported by bioinformatics. O- and K-loci-based genotyping by polymerase chain reaction (PCR) or whole genome sequencing WGS has been proposed as a diagnostic tool, including the Kaptive tool available in the public domain. We analyzed discrepancies for O2 serotyping between Kaptive-based predictions (O2 variant 2 serotype) and the actual phenotype (O2 variant 1) for two K. pneumoniae clinical isolates. Identified length discrepancies from the reference O-locus resulted from insertion sequences (ISs) within rfb regions of the O-loci. In silico analysis of 8130 O1 and O2 genomes available in public databases indicated a broader distribution of ISs in rfbs that may influence the actual O-antigen structure. Our results show that current high-throughput genotyping algorithms need to be further refined to consider the effects of ISs on the LPS O-serotype. Klebsiella pneumoniae is a Gram-negative bacterium which is part of the human microbiota; however, it is also a frequent cause of nosocomial and community-acquired infections in newborns, the elderly, and immunocompromised patients -α-D-Galp- or {→3)-β-D-Galf-(1→3)-[α-D-Galp-(1→2)]-α-D-Galp-(1→} (O2aeh) disaccharides, respectively [f-(1→3)-α-D-Galp-(1→} and {→3)-α-D-Galp-(1→3)-β-D-Galp- ,20,21. Tpe) [rfb a. The gmectively ,20,21. OK. pneumoniae (strains BIDMC 7B and ABC152), in which Kaptive-based O-serotype prediction and O-antigen structural analysis reveal different O-serotypes. Molecular characterization was performed to explain the genotype–phenotype discrepancies as a result of insertion sequences (ISs) within their rfb regions. Further, large-scale in silico analysis of 8130 K. pneumoniae genomes available in public databases was performed, in order to assess the prevalence of such insertions in rfb of K. pneumoniae O1 and O2 genomes.In this paper, we describe two clinical isolates of K. pneumoniae BIDMC 7B and ABC152 clinical isolates were characterized by nuclear magnetic resonance (NMR) spectroscopy on the native isolated LPS. LPS was extracted from bacteria with yields of 0.32% and 0.70% for BIDMC 7B and ABC152, respectively, and then analyzed by the high-resolution magic angle spinning (HR-MAS) 1H, 13C NMR spectroscopy  and PCM-27 (O2v2) isolates -α-D-Galp-. The comparison of 1H, 13C HSQC-DEPT spectra of BIDMC 7B -β-D-Galing unit . The saming unit . The lacBIDMC 7B e and ABCBIDMC 7B .rfb clusters demonstrated an increased size (by 777 bp) when compared to the reference sequences in the Klebsiella O-locus primary reference database in Kaptive. The gmlABC genes showed 100%, 90.79%, and 97.33% identity, respectively, to those in the Kaptive reference database.Kaptive-based O-serotyping was performed with whole-genome sequences of both strains . ContrargmlB genes from BIDMC 7B, ABC152, and from the O1/O2v2 reference strains K. pneumoniae NTUH-K2044 and 441 are shown in gmlBs in both strains by an identical IS element, ISR1, whereas other genes in the rfb locus were intact, in comparison to the reference strains. These results indicated that the ISR1 disruption completely inactivated the GmlB glycosyltransferase gene, resulting in biosynthesis of the O2v1 instead of O2v2 structure, and thus, being the likely reason for the discrepancy between the actual O-antigen phenotype and Kaptive-based predictions.Molecular analysis of BIDMC 7B and ABC152 was performed to explain the discrepancies observed between the O-antigen phenotype and the Kaptive-predicted O-serotype. The alignment of the rfb loci of K. pneumoniae, 8130 genome sequences available in the public domain were analyzed (rfb region (≥ 400 bp), of which 49 genomes were of sufficient quality for further analysis  were identified in several genes of these loci; namely, gmlBC, kfoC, wbbMNO, glf, wzm, and wzt , one of which was excluded due to the low quality of reads  disruption of the studied isolates BIDMC 7B and ABC152 was found in four other genomes.Among the 839 Kaptive-identified O1v2 isolates, significant length discrepancies (≥700 bp) occurred in the of reads . Selecte ISKpn14 . Two andrfb loci of the available K. pneumoniae O2v2 and O2v1 genomes, clonality (MLST) and phylogenetic analyses were performed on the isolates using the ABC152 strain as a reference  or wbbM:ISR1  disruptions, ST258 with double gmlB:ISKpn26 (nt 453) plus wbbO:ISKpn26 (nt 490) disruptions, or ST34 with gmlC:IS903B . In some cases, an additional IS insertion likely marked on-going diversification within a lineage, such as wbbO:IS903B (nt 130) in ST34 with gmlC:IS903B . An interesting case was the gmlB:ISR1 (nt 818) disruption in the study isolates BIDMC 7B and ABC152, which was observed also in four others. BIDMC 7B plus the four others formed a closely related cluster of ST258 isolates. ABC152 was of a non-related ST147, suggesting a horizontal transfer and recombination event. A similar case was represented by the disruption wbbO:ISKpn26 , present in two ST258 and ST512 close relatives, as well as a non-related ST17 isolate. Based on these results, it may be assumed that IS disruptions within the rfb loci in K. pneumoniae O2v2 and O2v1 genomes might have occurred at least ≈35 and ≈10 times, respectively , followed by the O2a, O3, and O5 O-genotypes [K. pneumoniae surface polysaccharide loci, and demonstrated its utility using more than 500 K. pneumoniae genomes [As the molecular genetics of the isolates ,14,24. FK. pneumoniae O1/O2v1 and O1/O2v2 serotypes based on two steps: First, Kaptive recognizes the serotype by searching for the D-galactan-II-encoding genes  characteristics of the O1 serotype. Second, the O1 and O2 serotypes are distinguished by the analysis of genes found in the rfb cluster. Finally, these are reported as variant v1 or v2 [rfb region.Kaptive Web-supported differentiation between v1 or v2 . As the K. pneumoniae clinical isolates BIDMC 7B and ABC152, the actual phenotype of which was O2v1, whereas Kaptive predicted O2v2. However, the tool provided an alert about “length disruption” within the rfb region and recommended further analyses. In the case of BIDMC 7B and ABC152 isolates, the structural analysis by the HR-MAS NMR spectroscopy proved the O-antigen structure to be O2v1 . Other identified cases also suggested O-antigen biosynthesis failure, including O1v2 isolates  revealed ISs in the isolates . RegardiBC genes . Detailerfb region; however, the frequency of such events is hard to evaluate. Although there are no previous data on IS disruptions within the O-locus, in general, these elements are common in K. pneumoniae genomes [K. pneumoniae isolate of another ST [Kpn26 [Kpn26 was commonly found in these (≈50%). These data further emphasized the impact of ISs on the evolution of K. pneumoniae ST258; however, one must also consider the over-representation of ST258 genomes in public databases, resulting from the high clinical and epidemiological relevance of these organisms.Sequence analysis of isolates from the database confirmed the occurrence of many IS insertions in the  genomes . The hypother ST ,31. SeveT [Kpn26 ,32. In oK. pneumoniae strains have at least one IS in their genome, where transposition of these elements within the genome causes rearrangements and may create new genotypes [rfb cluster genes may be the protection of bacteria against the host immune system. Although the presence of ISs in gmlABC genes may cause the phenotype–genotype discrepancy discussed above, the disruption of the genes determining D-galactan I elements may abolish the O-antigen synthesis, as in the case of the wzm or wzt genes, coding for ABC transporters [rfb and/or wbbYZ operon can either inhibit the expression of the O-antigen on the surface of the bacterial cell [K. pneumoniae isolates could determine the consequences of ISs in the rfb region and their effects on bacterial antigenicity and host interactions. Such changes can significantly affect the ability of bacteria to survive during antibacterial therapies; for example, by changing the surface antigens and virulence in regard to reactivity with the complement, antibodies, or phage resistance [Salmonella species, the O-antigen composition of which affects the host–pathogen interactions during infection. Strains belonging to one serovar can have a different repertoire of O-antigen-modifying genes. Moreover, their expression is different depending on the phase variations. The gtrABC operon acquired by horizontal gene transfer is such a set of genes for modification of the Salmonella O-antigen. These genes encode proteins showing functional homology to the glycosyltransferases encoding by the gmlABC genes cluster in K. pneumoniae [According to Adams et al., 94% of enotypes . One consporters ,16,25. Iial cell   was obtained through BEI Resources, NIAID, NIH: “Klebsiella pneumoniae, strain BIDMC 7B, NR-41923”, as a reagent bought as a part of the Klebsicure-Eurostars project (no. E!7563). Strain ABC152 (urine isolate) was recovered from the Abu Dhabi Hospital (UAE) in 2013, kindly provided by Agnes Pal-Sonnevend and Tibor Pal from the United Arab Emirates University. Both strains were selected for a previous large-scale serotyping study (unpublished results), due to the inconsistency between the lack of LPS reactivity with O2v2-specific monoclonal antibody [gmlABC genes. Bacteria were grown on Trypcase Soy Agar plates. For semi-preparative scale LPS preparation, the strains were cultured in Luria–Bertani (LB) broth in 500 mL flasks with shaking (at 37 °C), inactivated overnight with 3% formalin at 22 °C, then harvested by centrifugation, washed with water, and freeze-dried.antibody  and PCR K. pneumoniae PCM-27 and Kp26 O-specific polysaccharides were obtained from the Laboratory of Microbial Immunochemistry and Vaccines in the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, PAS  and isolated as previously described [escribed .K. pneumoniae strains BIDMC 7B and ABC152 were isolated by hot phenol/water extraction [LPS of traction  and puritraction , followe2H2O, processed and analyzed as described previously [2H2O and placed into the ZrO2 rotor. Acetone was used as an internal reference (δH/δC 2.225/31.05 ppm) for both O-PS and LPS spectra [1H, 13C HSQC-DEPT, 1H, 1H COSY, and TOCSY) were assigned with the use of NMRFAM-SPARKY  [All NMR spectra were obtained at 298 K using an Avance III 600 MHz  spectrometer equipped with a PH HR MAS probe  or 5 mm QCI cryoprobe with z-gradients . NMR spectra of isolated O-PSs were obtained in eviously . For hig spectra . The proin, USA) .Genomic DNA of the BIDMC 7B and ABC152 strains were extracted from overnight cultures with the Genomic Mini kit .Libraries were prepared using the Nextera XT DNA Library Preparation Kit  and sequenced on an Illumina MiSeq platform .https://github.com/katholt/Kaptive) for in silico O-serotyping [rfb gene clusters were compared with those of the reference strains K. pneumoniae NTUH-K2044 (serotype O1v2) and K. pneumoniae 441 (O1/O2v2)  using the CLC Main Workbench version 20 software . All K. pneumoniae whole-genome sequences available from GenBank  were downloaded and screened using Kaptive for the O1v2- and O2v2-predicted serotype isolates, and for the rfb region length discrepancies among these . Genomes of all such isolates were typed by 7-loci MLST with Multi-Locus Sequence Typing 2.0 (http://cge.cbs.dtu.dk/services/MLST) [http://www-is.biotoul.fr) [rfb length discrepancies were aligned against the strain ABC152, inferring SNP-based phylogeny with Parsnp v.1.2. [https://itol.embl.de) [The obtained reads were trimmed by Trimmomatic v0.39 . The genrotyping . Sequences/MLST) . Identifp v.1.2. . The phyThe sequence of the BIDMC 7B strain is available in GenBank under accession number JCNG00000000.1. The sequence of the ABC152 strain was submitted to the GenBank database under accession number JACENF000000000.


Both MicroRNAs and HMGB1 took part in pathological process of myocardial I/R injury though several signaling pathways. We hypothesized that mircoRNA451 (miR-451), a group of small non-coding RNAs, could improve this injury by inhibiting HMGB1. Male SD rats were randomly distributed into 5 groups and subjected to I/R process. After 24 hours of reperfusion injury, the serum content of CK and LDH, the content of MDA in tissue and activity of SOD were detected; The infarcted areas were defined by TTC staining and Evans Blue; TUNEL staining and cleaved-Caspase 3 were used to test apoptosis; HMGB1 was detected by real-time fluorescence quantitative PCR and Western Blotting. Compared with the I/R and I/R+Ad-GFP group, upregulation of miR-451 could reduce the infarcted areas, cardiomyocytes apoptosis index, expression of cleaved-caspase 3 and content of CK and LDH significantly(P<0.05); Meanwhile, upregulation of miR-451 could also obviously inhibit HMGB1, the increase of MDA and the decrease of SOD (P<0.05). So this study revealed that upregulation of miR-451 could prevent myocardial I/R injury by suppressing HMGB1. Cardiovascular disease is a major cause of human death. Myocardial Ischemia Reperfusion Injury (MIRI) can cause heart failure, re-infarction, arrhythmia and other complications, which pose a serious threat to the life and health of patients , 2. MIRIMicroRNAs, containing 18 to 25 nucleotides, are endogenous noncoding RNAs, which participate in post-transcriptional regulation of protein expression, lead to the target mRNAs translational degradation or repression , 5. IncrHMGB1 is a non-histone nucleoprotein containing two lysine-rich DNA binding regions and an unusual C-terminal acid tail. It consists mainly of aspartic acid and glutamate residues and is released passively by apoptotic, necrotic and innate immune cells. Previous studies have found that HMGB1 can promote autophagy and inflammation . HMGB1 cRecent studies suggested that miR-451 was downregulated in ex and vivo I/R rat hearts significantly, which further involved in the prevention of cell death, simulated I/R-triggered in human cardiomyocyte , 19. Mea5’-TTTGGGAATGGCGAGGAAACCGTTACCATTACTGAGTTTAGTAATGGTAATGGTTCTCTTGCTGCTCCCACA-3’) and asmiR-451(Forward primer: 5’-CCGGAACTCAGTAATGGTAACGGTTTTTTTTG-3’. Reverse primer: 5’-AATTCAAAAAAAACCGTTACCATTACTGAGTT-3’). Recombinant adenoviruses were propagated on HEK293 cells and purified by using Adeno-X™ system as previously described.An sequence containing pre-miR-451 and asmiR-451 was constructed and amplified by PCR using the following primers: After cloned into an pDC316- siRNA shuttle vector and co-transfected HEK393 cells with pBHG lox ΔE1,3 Cre vector in an Ad-Max system, we harvested the recombinant adenovirus containing pre-miR-451 were divided into five groups randomly:10 pfu/rat) and other steps are the same as I/R; 4) Ad-miR-451 and I/R group(N = 15): Rats were injected Ad-miR-451  and other steps are the same as I/R; 5) Ad-asmiR-451 and I/R group(N = 15): rats were injected Ad-asmiR-451  and other steps are the same as I/R.1) Sham group: Rats underwent surgical thoracotomy to expose the heart and needle insertion below the anterior descending branch, but did not induce ischemia; 2) Ischemia reperfusion injury group : Three days before surgery, PBS(150μl) was injected into 6 different anterior wall of rat left ventricle, then the left anterior descending branch was occlused for 30 minutes and reperfused for 24 hours; 3) Ad-GFP and I/R group(N = 15): Rats were injected Ad-GFP  to anesthetize by Intraperitoneal injection. Mechanical ventilation was via tracheal intubation, Cut the skin, bluntly separate through the third intercostal space to expose the precordial area, inelastic 6–0 thread was passed under it 2mm below the left atrial appendage. Then, a small section of silicone tube is placed over the vessel and ligated with the vessel to occlude the LAD. Then chest cage was closed in layers for 30min. For reperfusion, the knot was released for a 24h. The success of I/R model was verified by visual measurement of myocardial tissue below ligation point color change and electrocardiogram monitoring. For ischemia period, the ST-segment elevated in Leads-II and ischemia region turned into cyanosis on surface. For the reperfusion, the elevated ST segment set down and the surface of ischemia region turned back red.After 24h reperfusion, re-anesthetize rats and open the thoracic cavity to expose the anterior cardiac region, then LAD was reoccluded at the same site. External jugular vein was injected by 1mL of 1% Evans blue dye to identify the location that was not supplied by LAD. To determine the infarct size of the heart, ventricular slices (2 mm) were incubated in 1% TTC , the TTC unstained area is the infarct area. Infarct size is expressed as a percentage of IAR (% IAR).Apoptosis in heart was measured by TUNEL staining. The apoptosis index was determined by randomly counting TUNEL-positive myocytes in 10 fields.Total RNA of cardiac tissues was extracted by Trizol reagent . For the quantification of miR-451, RNA was reverse transcribed into corresponding cDNA with oligo(dt) from total RNA. HMGB1 expression and miR-451 content were measured by RT-PCR analysis using a SYBR Green -based qPCR kit .As mentioned before, quantitative immunoblotting was performed with HMGB1 and cleaved caspase 3 antibodies . GAPDH aThe injury of myocardium was evaluated by the serum concentration of CK and LDH. Protocols accorded to the instructions of manufacturer. .The lipid superoxide level and indexes of oxygen free radical in cardiac tissue represent oxidative stress damage. MDA and SOD were tested followed the protocol.SPSS 17.0 was performed. All data are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with Sidak correction was applied for multiple comparisons. p<0.05was defined statistical significance.24 h reperfusion later, treatment with Ad-miR-451 could decrease the infarcted size caused by I/R compared with that in I/R group and I/R+ Ad-GFP group (P<0.05). Nevertheless, compared with I/R group and I/R+ Ad-GFP group, the infarcted area was not increased in Ad-asmiR-451+I/R group .24h after reperfusion, compared with SO group, CK and LDH contents in I/R group and I/R+ Ad-GFP group increased significantly (P<0.05). The intervention of Ad-miR-451 significantly inhibited in CK and LDH contents caused by reperfusion injury (P<0.05). Whereas, compared with I/R group and I/R+ Ad-GFP group, there was no significant difference in the content of CK and LDH in I/R+Ad-asmiR-451 group .Compared with the SO group, MDA levels were significantly increased in the I/R and I/R+Ad-GFP groups at 24 h after reperfusion, while SOD levels were significantly lower (P<0.05). Treatment with Ad-miR-451 could significantly inhibit this change. (both P<0.05). However, there was no difference for MDA and SOD levels in the I/R+Ad-asmiR-451 group compared with the I/R and I/R+Ad-GFP groups .Cardiomyocytes apoptosis was determined by the TUNEL assay and the protein expression level of cleaved- caspase3 contained in myocardium. Ad-miR-451 decreased I/R-induced cardiomyocytes apoptosis and the expression level of cleaved-caspase3 (P<0.05). However, Ad-asmiR-451 failed to increase I/R-induced cardiomyocytes apoptosis but resulted in increased expression level of cleaved- caspase3 (P<0.05) . Western blotting analysis suggested that the HMGB1 protein level had similar results with qRT-PCR. However, compared with in I/R and I/R+ Ad-GFP group, the HMGB1 mRNA and protein levels were not altered obviously in I/R+Ad-asmiR-451 group Figs .In this study, we have attempted to identify the regulation mechanism of HMGB1 in the field of miRNAs. We used in vivo models to show that miR-451 attenuated the inflammatory status, oxidant stress injury and cell apoptosis. The results of this study showed that miR-451 expression decreases in I/R, meanwhile HMGB1 expression increased . The expHMGB1 is known as an early mediator of organ damage and inflammation during myocardium I/R injury , 21. ConIncreasing evidences suggested that miRNAs play crucial roles in various heart diseases by degradation or translational inhibition of their target genes. Several ischemia and reperfusion related miRNAs including miR-320, miR-29, the muscle-specific miR-1, miR-133 and miR-21 as well as their target genes including HSP60, HSP 70, Bcl-2, HSP 20, and PDCD4 have been identified and validated , 27. TheThere are some limitations in this study. We did not measure the different regions  of the heart separately, which may result in different results due to different infarct areas of different samples. At present, there are few studies on the effect of directly inhibiting or increasing HMGB1 on the infarct size, which may become a potential research direction.In general, our results confirm that miR-451 plays a significant role in improving myocardial I/R injury by inhibiting HMGB1. Although this relationship is one of the mechanisms underlying miR-451 protected against ischemia and oxidative stress, collective evidences suggested that miR-451 may act as a potential drug for myocardium reperfusion injury.S1 File(RAR)Click here for additional data file.


It is of utmost importance to investigate novel therapies for cancer, as it is a major cause of death. In recent years, immunotherapies, especially those against immune checkpoints, have been developed and brought significant improvement in cancer management. However, on the other hand, immune checkpoints blockade (ICB) by monoclonal antiboties may cause common and severe adverse reactions (ADRs), the cause of which remains largely undetermined. We hypothesize that ICB-agents may induce adverse reactions through off-target protein interactions, similar to the ADR-causing off-target effects of small molecules. In this study, we propose a hybrid phenotype mining approach which integrates molecular level information and provides new mechanistic insights for ICB-associated ADRs.We trained a conditional random fields model on the TAC 2017 benchmark training data, then used it to extract all drug-centric phenotypes for the five anti-PD-1/PD-L1 drugs from the drug labels of the DailyMed database. Proteins with structure similar to the drugs were obtained by using BlastP, and the gene targets of drugs were obtained from the STRING database. The target-centric phenotypes were extracted from the human phenotype ontology database. Finally, a screening module was designed to investigate off-target proteins, by making use of gene ontology analysis and pathway analysis.Eventually, through the cross-analysis of the drug and target gene phenotypes, the off-target effect caused by the mutation of gene BTK was found, and the candidate side-effect off-target site was analyzed.This research provided a hybrid method of biomedical natural language processing and bioinformatics to investigate the off-target-based mechanism of ICB treatment. The method can also be applied for the investigation of ADRs related to other large molecule drugs. Immunotherapy has been very popular in cancer treatment recently, owing to the breakthroughs made in the past few decades, including investigation of mechanisms and related immune response . MechaniImmunotherapy has cured many people’s immune-related diseases, brought new medical tools to humans, and promoted new developments in medicine. However, while curing the disease, it also brings many side-effect diseases to people. Therefore, studying the side-effect mechanism of immunomonoclonal drugs can better provide more powerful evidence for drug modification and production.The binding of PD-1 to its ligand PD-L1 is critical for the homeostatic regulation of the immune system. The primary role of the PD-1/PD-L1 signaling process is to suppress autonomic T cells and accelerate programmed cell death of T cells for the prevention of human immune diseases. After antigen receptor and cytokine signaling, PD-1 is expressed on mature CD4+ and CD8+ T cells, killer T cells, and some cell subsets of B cells . Howeverγ, Bcl-xL and IL-2) expression by PI3K activation of T cell intracellular AKT pathway. The T cell proliferation initiates an anti-tumor response, as shown in Fig. In a tumor-free environment, PD-L1 expression is restricted in normal tissues . BindingThe PD-1/PD-L1 inhibitor targets the epitope on the PD-1/PD-L1 molecule with high affinity and specificity. By blocking their interaction pathways, T cells can “recognize” cancer cells and initiate immune responses .The PD-1/PD-L1 inhibitor drugs that have been approved by the FDA are mainly: 1. Anti-PD-1 antibodies: Nivolumab developed by BMS, Pembrolizumab of Merck, 2. Anti-PD-L1 antibody: Atezolizumab, Pfizer of Roche /Avelumab from Merck, Germany, Durvalumab from AstraZeneca, etc , 13  with medical informatics. First, we targeted at five PD-1/PD-L1 inhibitor drugs, and extract all the adverse drug reaction (ADR) from DailyMed drug labes by using conditional random fields (CRF) . Second,Five drugs against PD-1/PD-L1 are listed at Table The N-terminal of an lgG antibody, approximately 120 amino acids, is a complementarity determining region (CDR). It was recently found that the heavy chain CDR2 of Avelumab binds to a specific part of PD-L1 and prevents PD-1 from being linked to PD-L1, thereby blocking the channel effect of PD-1/PD-L1 . Both AtTaking Pembrolizumab as an example, the amino acid sequences of the heavy and light chains are shown in the Fig. Heavy Chain Sequence QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGI NPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPC SRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR VESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKLight Chain SequenceEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLL IYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEChttps://dailymed.nlm.nih.gov/dailymed/) in XML format.As training data for phenotype extraction, we downloaded the gold dataset of the TAC 2017 shared task on adverse reaction extraction , which ci)HPO. Human phenotype ontology (HPO) contains standardized vocabulary for phenotypic abnormalities in human diseases .ii)UniproKB. We used the local BlastP  and protiii)STRING. Protein-protein interaction (PPI) analysis helps to study the molecular mechanisms of disease and discover new drug targets. In this paper, antibody-producing proteins may be obtained from the STRING database.Several multi-Omics data sets were used in this research.Though the exact physiological process of immune-related side effects is unclear, it is thought to be closely related to maintaining immune homeostasis. Since most immune-related adverse reactions resolve within weeks to months after the onset of immunosuppressive therapy, one of the most important clinical practice is to restore the safety of immune checkpoint blockade after the adverse events have subsided. Because the study protocol often requires that if there is a serious adverse reaction related to the immune system, the immune checkpoint blockade treatment must be permanently stopped, so the prospective data from clinical trials is limited . TherefoFigure Pre-processing module. This module downloads the anti-PD-1/PD-L1 drug from the Drugbank database, and the XML description file from the DailyMed database. In addition, it downloads the protein sequence of the antibody drug from the Drugbank database.Extraction module of drug-centric phenotypes. As shown in the left module section in Fig. Step-1: Formatting drug label texts. The BIEO scheme is used as the entity tagging format. ADRs in the results are marked as B-AdverseReaction, I-AdverseReaction, E-AdverseReaction, where B-type means begin, I-type means Inside, E-type means End, and O indicates Outside. For instance, if the word “pruritis” is an ADR entity, it is marked as “B-AdverseReaction”; if a phrase “injection site hemorrhage” is an ADR entity, the phrase is marked as “B-AdverseReaction I-AdverseReaction E-AdverseReaction”.Step-2: Setting up feature function. DISO (Disorders) is a semantic group in the UMLS [the UMLS  through the UMLS . This seStep-3: NER of phenotype terms with CRF. Gold standard annotated text from the TAC shared task is first used to learn a CRF model. That model is then applied to the unannotated DailyMed drug label texts to detect drug side effects. The training data is randomly divided according to 7: 3. During the training model, 7 copies use the actual training model and 3 copies use the development set to adjust the parameters. After the model is optimized, all data is applied to the DailyMed drug label texts to detect side effect mentions.Extraction module of target-centric phenotypes. As shown in the right module section in Fig. Step-1: Sequence similarity computation. We used the native BlastP and protein database UniprotKB to obtain homologous sequences of the five drugs. The parameters were set to: Indentities >30% and E-value >10−e4.Step-2: PPI extension for searching target with function relevance. We entered the protein name in the BlastP result into the STRING database, setting the confidence parameter to 0.7 and the maximum number of display nodes to 500.Step-3: Target-centric phenotypes extraction with HPO query. We downloaded the HPO database and compared the previous protein interaction results with the HPO database. We thus obtained the side effect phenotype of each drug, and predicted the most likely drug off-target site.Screening module for off-target proteins. A Gene Ontology (GO) analysis is performed using the obtained gene list information. We analyzed the signal pathways involved in each gene to screen for genes related to T cell proliferation activation, immune regulation, such as AKT and PI3K pathways, and matched the genes by cross-checking. Eventually, we performed literature search to find evidence supporting their relevance.In this section, the results of the proposed hybrid phenotype mining method are given. Specifically, drug- and target-centric phenotype mining results were obtained and comprehensively integrated to unveil a cluster of possible target proteins of five drugs.The precision (proportion of correctly predicted entities), recall  and F-score  obtained on the training data for each entity type are shown in Table After training, the model was used to extract all adverse reactions from the drug labels of the five targeted drugs. The extraction results are shown in Fig. i)BlastP results. We run BlastP to obtain proteins with similar structures of five target drugs. In this process, a total of 2,540 protein sequences were found before screening, and 848 sequences remained after screening. In the pre-screening results, some proteins will have different positions to match the drug protein sequence, so the number of proteins before screening will be repeated. For instance, P0DOX2, P0DOX3, P0DOX4, P0DOX5, P0DOX6, P0DOX7, P0DOX8 are immune one strand of globulin that do not have its corresponding gene name, so the number of proteins after screening is reduced. Although the five drugs were reduced before and after screening, the reduction trend was about the same, with Pembrolizumab decreasing the most, i.e., 70%. The statistic summary of the protein after BlastP is shown in Fig. ii)STRING results. We use STRING to obtain proteins that have similar structure with BlastP screened proteins. Thus an expanded candidate off-target protein set is obtained. The statistic summary is shown in Fig. iii)HPO results. HPO terms were extracted from the drug labels of the five targeted drugs. As can be seen from Fig. The results for the target-centric phenotypes are shown in Fig. i)Side effect phenotype cross matching results. We use embedding similarity strategy [strategy  to matchii)GO analysis results. Gene ontology analysis aims to investigate the function of the filtered proteins. By filtering the genes with function in the signal pathways related to T cell proliferation activation, immune regulation, only three genes remains, which are AKT1, ACTG2, and BTK. The results of drug/gene/phenotype link are shown in Table γ is also involved in immunosuppressive signaling, helping tumors to escape. Bruton’s tyrosine kinase (BTK) is involved in the activation process of B cells and is involved in immune regulation. However, the other two genes did not find relevant side effects, and there may be few articles reporting the corresponding side effects of the gene, so no relevant literature was found.In the result of GO analysis, ACTG2 is closely related to T cell activation and immune process, and may be associated with drug off-target. AKT1 is associated with inflammatory factor-mediated diseases, in which IFN- In this section, the process of phenotype mining is discussed, and a candidate side-effect off-target site is proposed and analyzed accordingly.The screening process of BTK discovery was represented in Fig. First, the module of drug-centric phenotype extraction started with five PD-1/PD-L1 inhibitors, searched the DailyMed database for related side-effect phenotypes, and used a CRF for sequence labeling to mine 905 drug-centric side effects. The precision of that process was 88% on the training data, so a moderate amount of noise is expected. This noise should however be reduced by the intersection with target-centric phenotype extraction. Conversely, recall was 68%, meaning that about one third of the drug side effects are likely to be missed.Second, in the module of target-centric phenotype extraction, a total of 848 amino acid sequences were found to be similar with sequences of the five PD-1/PD-L1 inhibitors by using blastP. Subsequently, a total of 134 target proteins of drug were found in the STRING database. Finally, the 134 proteins were compared with the HPO database, and a total of 2,071 target-centric side-effect phenotypes were found.Third, in the screening module for off-target proteins, the phenotypes of the first two steps were cross-matched, and a total of 110 target-centered target proteins were found. For more accurate screening, we performed GO analysis and found genes related to AKT and PI3K pathways. A total of three were found: ACTG2, AKT1 and BTK. Since only BTK related literature reports were found during the literature search, only one gene related to PD-1/PD-L1 off-target was found.As introduced in the result section, the proposed hybrid phenotype extraction method found a related side-effect off-target site, BTK, and we hypothesized that the off-target site caused a cellulitis side effect due to mutation of BTK gene.While tracing back the side effect of Avelumab and Pembrolizumab in Table Literature research unveiled the fact that BTK is associated with B cell activation. If B cells are not activated, the efficiency of immunization is greatly reduced, and it also affects the PI3K pathway, which affects autoimmunity and causes adverse immune reactions. As shown in Fig. In detail, the gene encoding BTK is mutated to form XLA. The defect of BTK leads to the development of B cells in the development of differentiation. Therefore, patients are basically lack of antibody-producing plasma cells, and are susceptible to each The effects of infectious diseases such as Helicobacter. H. cinaedi, formerly known as Campylobacter, in patients with weak immune function, diseases caused by H. cinaedi include repeated fever, bacteremia, arthritis, osteomyelitis, cellulitis, abdominal abscess and diarrhea .In conclusioin, the literature evidence of cellulitis is consistent with the above hypothesis.Although PD-1/PD-L1 targeted therapy has proven the enormous power of cancer immunotherapy, in recent studies, anti-PD-1 drugs have no therapeutic effect on non-Hodgkin’s lymphoma, but have a tendency to deteriorate. PD-1 signaling in a mouse model prevents the proliferation of cancerous T cells, and in these mice, anti-PD-1 treatment can aggravate the disease by re-activating cancer cells for continued proliferation . AlthougPD-1/PD-L1 is not classified according to tumor type but based on biomarkers , PD-L1 eHowever, due to the complexity of cancer immunization, the mechanism is still unknown by which PD-1/PD-L1 targeted therapy improves the survival time of cancer patients, so the molecular and cellular mechanisms that clarify the immunosuppressive function of each immunological checkpoint molecule are helpful.


H-naphthopyrans, two colored isomers TC (transoid-cis) and TT (transoid-trans) are formed. In terms of optimized photo-switchable materials, synthetic efforts are nowadays evolving toward developing 3H-naphthopyran derivatives that would not be able to photoproduce the long-living transoid-trans, TT, photoproduct. The substitution with a methoxy group at position 10 results in significant reduction of the TT isomer formation yield. The TC photophysics responsible for TT suppression were revealed here using a combination of multi-scale time resolved absorption UV-vis spectroscopy and ab initio calculations. The substitution changes the TC excited-state potential energy landscape, the bicycle-pedal isomerization path is favored over the rotation around a single double bond. The bicycle-pedal path is aborted in halfway to TT formation due to S1→S0 internal conversion populating back the TC species in the ground electronic state. This is validated by a shorter TC S1 state lifetime for methoxy derivative in comparison to that of the parent-unsubstituted compound (0.47 ± 0.05 ps vs. 0.87 ± 0.09 ps) in cyclohexane.In the photochromic reactions of 3 H-naphthopyran (see H)chromene) , is located in the region close to the diagonal of bicycle-pedal mechanism should prevail over the twisting motion in the OCH3 molecule, for the energy reasons.For OCHrivative a, the vi1BP and S1TW minima were also determined for the unsubstituted parent compound. In this case, the S1TW excited-state global minimum  is more energetically favored. It is by 0.05 eV more stable than that of the corresponding S1BP form . As a consequence, the position of the deeper twist-type S1TW global minimum on S1-PES suggests that the twisting motion prevails over the bicycle-pedal, for the model of unsubstituted compound. Furthermore in agreement with recently reported experiments chromene) was purchased from TCI .CF-OCHocedures . CF-H  was used to perform photochromic reaction in a 1 cm × 1 cm fused silica cuvette placed in a temperature-controlled cuvette holder  with stirring . White-light of 150 W xenon lamp  with intensity reduced to a small level was used as a probing beam. 150 ms were used for accumulation of each white-light continuum spectrum. An average of 60 initial spectra before sample UV-irradiation was used to calculate UV LED  equipped with the mentioned above temperature-controlled cuvette holder, stirring and UV LED source was used.2) at 365 nm of CF solution in a quartz cell 2 mm thick with stirring.Femtosecond UV-vis transient absorption spectra were obtained using a commercially available system  . PhotostThe instrument response function (IRF) was about 200 fs (FWHM) estimated from a stimulated Raman scattering observed in pure solvent. The energy of laser pulses at 475 nm was 1 μJ. The data set was composed of a series of 280 spectra acquired for pump-probe delay ranging from -1 to 50 ps. Each transient absorption time see . The obtThe accuracies of the obtained time-constants derived from analysis of transient absorption results were following: ± 5% (UV-vis data in time window over tens of seconds) and ± 10% (ultrafast UV-vis data).3 and H derivatives of 3H-naphthopyran and its isomers in their closed-shell ground state (S0) were obtained with the MP2 method [1) equilibrium geometries were determined with the second-order algebraic diagrammatic construction ADC(2) method [3 and H molecular system. All calculations were performed using TURBOMOLE program package [The equilibrium geometries of the OCH2 method  without ) method ,42,43. T) method . The ver) method ,46. The  package .S1 of TC-OCH3 derivative is shorter (0.47 ± 0.05 ps) than that of TC-H (0.87 ± 0.09 ps) due to more effective radiationless S1→S0 internal conversion channel, populating back the TC form. Theoretical studies show two possible variants of photoisomerization reaction: (i) single-twist and (ii) bicycle-pedal motions. As demonstrated by the calculations, in photoexcited TC-OCH3 the bicycle-pedal motion is energetically more favored than the single-twist. The bicycle-pedal motion leads to a minimum in S1BP which is located in a close proximity of S0-PES facilitating S1→S0 internal conversion, which through further structural evolution leads toward the relaxed TC minimum. The bicycle-pedal channel is less probable in H counterpart, which favors population of the single-twisted form S1TW. The excited-state decay of S1TW leads to the ground state at the geometry close-lying to the S0-state isomerization barrier, thus the energy gradient can drive the molecule toward TT or back to the TC minimum. Our studies have shown that the choice of appropriate substituent or polar solvent can favor the bicycle-pedal TC→TT isomerization path, which can be aborted leading to the decrease in TT formation yield. Altogether, the bicycle-pedal path is seen as photostabilizing channel for TC form and opens the way to design new efficient naphthopyrans free from TT formation.The experiments in cyclohexane show TT photoreaction yield four times lower for methoxy derivative than for the parent unsubstituted compound. Ultrafast experiments demonstrated that the excited state lifetime τ


Glioblastoma multiforme (GB) is the most malignant primary brain tumor in humans, with an overall survival of approximatively 15 months. The molecular heterogeneity of GB, as well as its rapid progression, invasiveness and the occurrence of drug-resistant cancer stem cells, limits the efficacy of the current treatments. In order to develop an innovative therapeutic strategy, it is mandatory to identify and characterize new molecular players responsible for the GB malignant phenotype. In this study, the RNA-binding ubiquitin ligase MEX3A was selected from a gene expression analysis performed on publicly available datasets, to assess its biological and still-unknown activity in GB tumorigenesis. We find that MEX3A is strongly up-regulated in GB specimens, and this correlates with very low protein levels of RIG-I, a tumor suppressor involved in differentiation, apoptosis and innate immune response. We demonstrate that MEX3A binds RIG-I and induces its ubiquitylation and proteasome-dependent degradation. Further, the genetic depletion of MEX3A leads to an increase of RIG-I protein levels and results in the suppression of GB cell growth. Our findings unveil a novel molecular mechanism involved in GB tumorigenesis and suggest MEX3A and RIG-I as promising therapeutic targets in GB. Glioblastoma multiforme (GB) is the most frequent and malignant primary tumor of the central nervous system , with a In the recent years, several genetic and epigenetic aberrations in molecular pathways  ,7,8,9 haUbiquitylation is a post-translational modification that controls a wide range of cellular functions  and the most important physiological processes ,11. UbiqDeregulation or mutations of E3-ubiquitin ligases have been associated with several human tumors; for this reason, they are considered to be promising targets for novel anticancer therapies ,13,14. AIn this regard, we evaluated the expression levels of catalytic E3-ubiquitin ligase complex components  and F-boCaenorhabditis elegans MEX3, a translational repressor involved in germline totipotency and in the specification of posterior blastomere identity during embryogenesis [MEX3A belongs to an evolutionary conserved RNA-binding ubiquitin ligase protein family composed of four members (MEX3A-D). These proteins are the homologs of ogenesis . Human Mogenesis . MEX3 progenesis ,23,24,25ogenesis ,23,24,26ogenesis . Interesogenesis .RIG-I is a critical cytosolic pattern recognition receptor (PRR) that acts as RNA sensor to activate innate antiviral immunity and interferon (IFN) production . HoweverIn this study, we observed that MEX3A is up-regulated in GB and correlates with low RIG-I protein levels. We demonstrated that MEX3A interacts with RIG-I and impairs its protein stability by inducing its ubiquitylation and proteasome-dependent degradation. Interestingly, the genetic depletion of MEX3A results in the impairment of GB cell proliferation, providing new insights on GB tumor biology and identification of potential and innovative therapeutic approaches.http://gepia.cancer-pku.cn/)   A. We foc tissues . This ev.amc.nl) . These d.amc.nl)  by assesAt present, ubiquitylation substrates of MEX3A have not been identified. We first hypothesized that this E3-ubiquitin ligase could be involved in the regulation of RIG-I, a tumor suppressor that controls differentiation, apoptosis and tumorigenesis processes. This idea was supported by the evidence that RIG-I stability and activity is modulated by the E3-ligases RNF122  and MEX3RIG-I in GB. As shown in RIG-I in GB specimens compared to noncancerous brain tissues.Given the double function of MEX3A as RNA-binding protein and E3-ubiquitin ligase , we firsConversely, an opposite trend of MEX3A and RIG-I protein levels was observed in GB samples. Indeed, RIG-I protein levels were strongly decreased in GB tissues, in which high MEX3A protein levels were found; an inverse trend of the protein expression was observed in the corresponding paratumor samples B,C. ThesOverall these data suggested MEX3A as a potential regulator of RIG-I protein.To investigate if MEX3A is involved in the regulation of RIG-I, we evaluated the effect of its ectopic expression on RIG-I protein levels. As shown in Interestingly, MEX3A showed a robust effect on the reduction of RIG-I protein expression A,B, withRIG-I mRNA levels was observed (We further confirmed the effect of MEX3A on RIG-I protein stability by cycloheximide (CHX) assay. Half-life of RIG-I was significantly decreased in presence of ectopic expression of MEX3A, suggesting the role of this E3 ligase in the degradation of RIG-I E,F. Accoobserved . TogetheTo elucidate the molecular mechanisms through which MEX3A controls RIG-I stability, we tested the ability of MEX3A to interact with RIG-I. To verify this hypothesis, we carried out reciprocal co-immunoprecipitation (Co-IP) experiments in HEK293T cells transfected with Myc-MEX3A and GFP-RIG-I expression plasmids. The immunoblot analysis revealed that MEX3A binds RIG-I A,B. The Next, we verified whether MEX3A controls RIG-I protein levels by inducing its ubiquitin-dependent degradation. To this aim, we performed an in vivo ubiquitylation assay upon ectopic expression of MEX3A. Increasing amounts of MEX3A promoted the poly-ubiquitylation of RIG-I and its subsequent degradation by the proteasome, as indicated by the accumulation of RIG-I ubiquitylated forms and its protein levels in cells treated with the proteasome-inhibitor MG132 F,G.Overall, these results indicated that MEX3A interacts with RIG-I and mediates its ubiquitylation, thereby inducing RIG-I proteasome-dependent degradation.To investigate the biological relevance of MEX3A function in GB, we analyzed the effect of its genetic depletion on the proliferation ability of two GB cell lines. We observed a decrease in cell growth over time and a significant reduction of BrdU uptake in A-172 cells silenced for MEX3A (shMEX3A) compared to the control cells (shCTR) A–D.This was consistent with increased levels of the cleaved PARP protein E,F. Of nThese data suggest the inhibition of MEX3A as a potential therapeutic approach for the treatment of GB.GB is the most common malignant primary brain tumor, driven by complex signaling pathways and particularly difficult to treat ,35. AlthAt present, it is clearly demonstrated that post-translational modifications play a crucial role in the control of normal cellular processes, but also in cancer initiation and progression ,38,39,40Here, we described for the first time that the RNA-binding MEX3A affects GB proliferation, acting as an E3-ubiquitin ligase. So far, little is known about the role of this protein in carcinogenesis ,23,24,26We found that MEX3A is strongly up-regulated in GB specimens compared to the normal brain tissues and regulates the protein stability of RIG-I. The multifunctional protein RIG-I is an important PRR involved in the activation of antiviral innate immunity and interferon response pathway , and knoIn particular, Glas and colleagues ,33,41 suRIG-I activity is significantly modulated through ubiquitylation processes mediated by several E3 ligases ,45,46,47In our study, we found higher expression levels of MEX3A and RNF122, compared to other E3 ligases, and lower baseline protein levels of RIG-I in GB specimens than in paratumor tissues. This evidence suggests the prevalence of degradative rather than regulative processes on the control of RIG-I in GB.We demonstrated that MEX3A interacts with ubiquitylates RIG-I, promoting its proteasome-dependent degradation and altering its stability. We showed that the gene silencing of MEX3A increases protein levels of RIG-I and strongly inhibits the proliferation of human GB cells .In this scenario, the stimulation of RIG-I, as well as the design of MEX3A inhibitors or small molecules able to counteract the binding between MEX3A and RIG-I might represent a novel strategy for cancer immunotherapy, by improving the dual function of RIG-I as cell death inducer and immune response activator . TargetiTM) and human glioblastoma A-172 cells  were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum . Human glioblastoma multiforme T98G and human glioblastoma-astrocytoma U-87, (purchased from ATCC) cells were maintained in Eagle’s minimum essential medium .HEK293T . Mycoplasma contamination in cell cultures was routinely monitored by using a PCR detection kit .TM Gold transfection reagent  in accordance with the manufacturer’s protocol. Transient transfections were performed by using DreamFectLentiviral particles were generated in HEK293 cells transfected with packaging and envelope plasmids (pCMV-dR8.74 and VSV-G/pMDG2), pGFP-pLKO.1 plasmids , TL308061C (#2), Origene, Rockville, MD, USA). A-172 and T98G cells were infected with complete medium of HEK293 cells containing lentiviral particles and polybrene  for 72 h.The pCMV6-Entry-Myc-DDK-RNF122 (RC210868), pCMV6-Entry-Myc-DDK-MEX3A (RC215359), pCMV6-Entry-Myc-DDK-MEX3C (RC221125), pCMV6-AC-GFP-DDX58 (RG217615), non-effective Scrambled shRNA Cassette in pGFP-C-shLenti Vector , pGFP-C-shLenti-MEX3A (TL308061B (#1) and TL308061C (#2)) were purchased from Origene .Mouse anti-RIG-I D-12 , anti-RIG-I HRP conjugated D-12 , mouse anti-HA-probe F-7 HRP  and mouse anti-β-Actin C4  were purchased from Santa Cruz Biotechnology . Anti-Flag M2 HRP  was purchased from Sigma-Aldrich. Rabbit anti-RIG-I  and Rabbit anti-PARP (95426S) were purchased from Cell Signaling . Rabbit anti-MEX3A  was purchased from Abcam . HRP-conjugated secondary antibodies were purchased from Bethyl Laboratories .Where indicated, cells were treated with MG132  for 4 h, Cycloheximide  at indicated time.For Immunoblot analysis, cells were lysed in a solution containing RIPA buffer  supplemented with protease and phosphatase inhibitors. The lysates were incubated on ice and then centrifuged at 13,000 g for 30 min, at 4 °C. After centrifugation, a determined amount of supernatant was resuspended in sample loading buffer, boiled for 5 min, resolved in SDS-PAGE and then subjected to immunoblot analysis. For Co-immunoprecipitation (Co-IP) assays, cell pellets were lysed with Triton Buffer , supplemented with protease and phosphatase inhibitors. An established amount of the whole-cell protein extracts was immunoprecipitated overnight at 4 °C, with rotation with specific primary antibodies (2 µg/mg of protein lysate) or IgG used as a control  and then incubated with Protein agarose beads (Santa Cruz Biotechnology) for 1 h, at 4 °C, with rotation. The immunoprecipitates were then washed five times with the lysis buffer described above, resuspended in sample loading buffer, boiled for 5 min, resolved in SDS-PAGE and then subjected to immunoblot analysis.Clinical samples for immunoblot analysis or Co-IP assay were mechanically disrupted in RIPA buffer or Triton buffer, respectively, supplemented with protease and phosphatase inhibitors. The lysates were sonicated for 10′’ at 100 W amplitude in ice and then centrifuged at 13,000 g for 30 min at 4 °C. The lysates were assayed with the procedures described above.HEK293T were transfected with equal amount of GFP-RIG-I and HA-Ub and with Myc-MEX3A in 1:20, 1:10 and 1:5 ratio compared to GFP-RIG-I amount. After 24 h from transfection, cells were treated with MG132 (50 µM) or DMSO as control, for 4 h. Cells were lysed in a solution containing RIPA buffer, as described above. Lysates were subjected to immunoprecipitation with mouse anti-RIG-I (Santa Cruz Biotechnology), overnight, at 4 °C, with rotation. The immunoprecipitated proteins were then washed five times with the RIPA lysis buffer, resuspended in sample loading buffer, boiled for 5 min, resolved in SDS-PAGE and then subjected to immunoblot analysis. Polyubiquitylated forms were detected by using mouse anti-HA from Santa Cruz Biotechnology.Trypan blue count was performed in GB cell lines, silenced by infection with lentiviral particles encoding either control shRNA (shCTR) or MEX3A shRNAs (shMEX3A#1 and shMEX3A#2) to assess the growth rate and cell viability. BrdU incorporation assay  was used to determine cell proliferation in A-172 and T98G cells. First, cells were infected with lentiviral particles encoding either short hairpin RNA targeting human MEX3A or a control non-targeting sequence (shCTR) for 72 h, pulsed 24 h with BrdU and then fixed and permeabilized with 4% paraformaldehyde and 0.2% Triton X-100 (Sigma Aldrich). Dako Fluorescent mounting  was used as mounting medium, and nuclei were counterstained with Hoechst reagent; BrdU detection was performed according to the manufacturer’s instructions. At least 500 nuclei were counted in triplicate, and the number of BrdU-positive nuclei was recorded.® S3 experiments, infected A-172 and T98G cells were seeded in 96-well plates  in complete medium and incubated overnight. The day after seeding, T98G cells were treated with IncuCyte® NucLight Rapid Red Reagent . Plates were transferred into the IncuCyte® S3 Live Cell Analysis Systems and incubated at physiological condition , over 72 h. Images were collected every 2 h, and proliferation was evaluated as number of NucLight positive cells for T98G, or as cell confluence for A-172, which does not incorporate NucLight reagent. The experiments were performed in triplicate, and data were analyzed by using the IncuCyte software package .For IncuCyteMEX3A, RNF122, MEX3C and RIG-I mRNA expression was performed by using the ViiATM 7 Real-Time PCR System (Life Technologies). Standard qPCR thermal cycler parameters were used to amplify a reaction mixture containing cDNA template, SensiFAST SYBR Lo-ROX Kit  and primer probe. Then, mRNA quantification was performed by using SDS version 2.3 software, each sample was amplified in triplicate, and the average of the three threshold cycles was used to calculate the number of transcripts. Data were normalized with the endogenous housekeeping genes (GAPDH and HPRT) and expressed as the fold change respect to the control sample value.Total RNA was isolated from GB and paratumoral biopsies, using TRIzol reagent , and reverse-transcribed with a SensiFAST cDNA Synthesis Kit . Quantitative real-time PCR (qRT-PCR) analysis of hMEX3A For 5′- ATCGTGGGCAGG CAAGGCT-3′hMEX3A Rev 5′- GCTGCTGAGATGATTT CCC-3′ hRNF122 For 5′- CCAACAAGTCCTGCTCGATG-3′hRNF122 Rev 5′- ACTGCGCAGGTCTGCCCATA-3′hMEX3C For 5′ –TGAACGGGGAGCAGGCG-3′hMEX3C Rev 5′- TGACTTGGACGGTGGTTTGA-3′hRIG-I For 5′- TGTGCTCCTACAGGTTGTGGA-3GhRIG-I Rev 5I- CACTGGGATCTGATTCGCAAA A-3nhGAPDH For 5′- TCCCATCACCATCTTCCAGG-3′hGAPDH Rev 5′- ATGAGTCCTTCCACGATACC-3′hHPRT For 5′- ATTATGCTGAGGATTTGGAAAGGG-3′hHPRT Rev 5′- GCCTCCCATCTCCTTCATCAC-3′The following primers were used to amplify the indicated genes:Tissues used for immunohistochemical staining were fixed in formalin and paraffin-embedded (FFPE). FFPE slides were incubated with monoclonal antibodies against RIG-I or MEX3A . The day after, the slides were incubated for 20 min, with secondary antibodies coupled with peroxidase (Dako). Bound peroxidase was detected by diaminobenzidine (DAB) solution  and EnVision FLEX Substrate buffer containing peroxide (Dako). Cell quantification was performed on collected sections, using the imaging software NIS-Elements BR 4.00.05 . Images were captured by HistoFAXS software , at 20× magnification.3 cells/well were seeded into a 96-well ImageLock tissue culture plate  and incubated at 37 °C with 5% CO2 incubator, for 36 h, until they reached 100% confluence. Wounds were made by the 96-well WoundMaker (Essen BioScience). The wounded cells were washed twice to remove the detached cells, and then incubated at 37 °C, in complete medium, with or without Thymidine 5 mg/mL , to block the proliferation. Images of the wounds were automatically acquired within the incubator by IncuCyte zoom software (Essen BioScience). The wound image updates were taken at 2 h intervals for the duration of the experiment. Data were analyzed with respect to wound confluence and calculated by using the IncuCyte software package (Essen BioScience).A-172 and T98G cells were infected with lentiviral particles encoding short hairpin RNA targeting MEX3A (shMEX3A) or a control non-targeting sequence (shCTR). Then, 20 × 10p-values were determined by two-tailed Student’s t-test, and statistical significance was set at p < 0.05. Results are expressed as mean ± SD from an appropriate number of experiments .Statistical analysis was performed by using the StatView 4.1 software . For all experiments, In this work, we identified new molecular players involved in GB tumorigenesis. The RNA-binding ubiquitin ligase MEX3A was found up-regulated in GB specimens, and this correlates with low protein levels of the tumor-suppressor RIG-I. We showed that MEX3A binds and ubiquitylates RIG-I, inducing its proteasome degradation. Genetic depletion of MEX3A results in the increase of RIG-I protein levels and the inhibition of GB cell growth. Our study unveils a new molecular mechanism involved in GB tumorigenesis and suggests that targeting RIG-I and MEX3A could pave the road for new therapeutic strategies for GB.